KR20220124057A - Polyelectrolyte multilayer Nanoparticles (PEMN) containing active ingredients, their use and their preparation - Google Patents

Abstract

The present invention relates to: PEMN, which contains an active component, a positive ionic compound, and a negative ionic compound, forms PEMN by electrostatic interaction between the same, and the active component is encapsulated in the formed PEMN structure; uses thereof; and preparation thereof. In the present invention made as described above, PEMN containing pharmacologically active components can be provided, and therapeutic proteins, peptides, antibodies, nanoparticles, and nucleic acids can be provided.

Description

Multilayer Polyelectrolyte Nanoparticles Containing Active Ingredients, Uses Thereof, and Manufacturing Methods Thereof

The present invention homogenizes a polyelectrolyte complex (PEC) or polyelectrolyte multilayer (PEM) comprising an active ingredient, a cationic compound and an anionic compound, and is formed by an electrostatic interaction between them, and the active ingredient is encapsulated Polyelectrolyte Multilayer Nanoparticles (PEMN), the use thereof, and a method for manufacturing the same.

A variety of diseases today are treatments involving the injection of peptide-, protein-, and nucleic acid-based drugs, particularly those involving transfection of nucleic acids into cells or tissues. requires

The full therapeutic potential of peptide, protein, and nucleic acid-based drugs is often not fully exploited due to their limited ability to cross the plasma membrane of mammalian cells, resulting in poor cellular access and inadequate therapeutic effects. Today, these difficulties represent significant challenges for biomedical advancement and the commercial success of many biopharmaceuticals.

Nucleic acid-based drugs are nucleic acids such as deoxyribonucleic acid, ribonucleic acid or backbone, polynucleotide derivatives in which sugars or bases are chemically modified or terminally modified, for example, RNA, DNA, RNA- Guided genome editing system, siRNA (short interfering RNA), aptamer, antisense oligodeoxynucleotide (ODN), antisense RNA, ribozyme, DNAzyme, etc. Nucleic acids, that is, anionic There are a wide variety of substances, such as drugs, and different delivery systems and production methods are required depending on the type of nucleic acid to be delivered. Also, as a nucleic acid delivery agent, formulations with increased stability and production yield and a production method thereof are required.

In particular, mRNA (messenger RNA) is a material before protein synthesis and contains genetic information. Since mRNA is accessible to various therapeutic agents, it can be used as a vaccine for prevention or treatment, and missing proteins can also be synthesized through mRNA.

For several diseases or disorders, gene therapeutic approaches have been developed as specialized forms of such treatment. While gene therapy generally involves the injection of one or more nucleic acids or genes into individual cells and tissues to additionally treat diseases such as genetic diseases, such treatments use transfection of nucleic acids or genes into cells or tissues, such as Treatment replaces the defective mutant trait with a functional one.

Moving or inserting one or more of these nucleic acids or genes into individual cells still represents a major challenge today, and especially in the field of gene therapy, the excellent therapeutic effect of absolutely nucleic acid-based drugs is definitely not. need.

Today's transfection reagents typically include peptides, various polymers, and lipids, as well as nano- and microparticles. Such transfection agents have typically only been successfully used in in vitro reactions.

The development of methods capable of delivering therapeutic genes to selected cells with appropriate gene expression presents an important challenge towards gene therapy. However, gene delivery and particularly successful transfection of nucleic acids into cells or tissues is not straightforward and generally depends on many factors.

For successful delivery, for example, delivery of a nucleic acid or gene into a cell or tissue must overcome many obstacles. An ideal nucleic acid delivery method satisfies three important characteristics. (1) It is necessary to protect the target nucleic acid from degradation by nucleases in the intracellular matrix (matrices). (2) The target nucleic acid must be able to cross the plasma membrane. (3) There shall be no adverse effect.

This goal can be achieved using a combination of various compounds or vectors. In particular, there are several compounds or vectors that overcome some of these obstacles.

In the treatment using anionic drugs including nucleic acids, safe and efficient drug delivery technology has been studied for a long time, and various delivery systems and delivery technologies have been developed. The delivery system is largely divided into a viral delivery agent using adenovirus or retrovirus, etc. and a non-viral delivery agent using cationic lipids and cationic polymers. In the case of a viral carrier, it is known that there are many problems in commercialization because it is exposed to risks such as non-specific immune reaction and the production process is complicated.

Over the past decade, research on nanoparticles (NPs) applied in the medical field has increased significantly, from inorganic to organic systems. In the case of inorganic nanoparticles, the commonly reported systems correspond to metal nanoparticles (silver, gold, zinc oxide, iron oxide, etc.). (I) metal nanostructures (II) metal oxide nanostructures and (III) metal nanoalloys are classified into three groups that can be produced by chemical, physical, and biosynthetic methods. On the other hand, there are various types of organic NPs, with dendrimers, self-assembled systems (micelles and liposomes) and polymer nanoparticles being the most used and reported systems.

With respect to polymeric NPs, they are commonly used in biomedical fields due to their properties of compatibility, biodegradability and low cytotoxicity, and natural polymers such as chitosan, modified starch and natural gums are very common. However, the development of polymer NPs is not limited to biopolymer systems because they can also be obtained from biocompatible synthetic materials. It is mainly used for polylactide (PLA), polycaprolactone (PCL), polyglycolic acid (PGA) and acrylate-based polymers. Similarly, polymer NPs are classified according to their structure, and the most common ones are nanospheres, nanocapsules, polymer nanoconjugates, polyelectrolyte complex (PEC), and multilayer polyelectrolyte complex (PEM).

PEC particles are often polydisperse systems with diameters between 10 nm and 1 μm. Particle size is usually measured by indirect techniques that maintain the integrity of nanoparticles in solution, mainly static and dynamic light scattering. Imaging techniques are often used to support the results of light scattering, but in this case the size of the PEC particles can be affected during sample preparation and analysis.

As a general model, the polyelectrolyte complex (PEC) is formed mainly based on the electrostatic attraction between oppositely charged polyions. However, the increase in entropy when counterions are released from the polyelectrolyte is the main driving force for PEC particle self-assembly.

With respect to PEC, they can be obtained in several ways, one of the most used being the traditional polyelectrolytic complexation process (bottom-up method). This technique is based on spontaneous interpolymeric aggregation given by electrostatic attraction when cationic and anionic polyelectrolytes are mixed in aqueous solution. The preparation of this polymer electrolytic composite is in principle an easy method to carry out.

However, bottom-up PEC and PEM fabrication methods may be vice versa as they depend on several internal and external variables. The internal variables are the polymer's chemical properties, molecular weight, and fractional charge. External variables depend on many dissolution medium conditions (temperature, pH, or ionic strength) as well as many process conditions such as polyelectrolyte ratio, mixture volume, rate and order of addition and stirring rate during polyelectrolyte complexation. These factors lead to the fact that it is particularly difficult to obtain polyelectrolyte multilayer nanoparticles (PECN) with a nanometer size. Therefore, standardization and extension of PEC using bottom-up methods is a great challenge to perform difficult experiments.

In the present invention, a multilayer polyelectrolyte multilayer (PEM) is prepared by layer-by-layer deposition of an anionic compound and a cationic compound, and a technique of homogenizing it to provide polyelectrolyte multilayer nanoparticles (PEMN) completed.

In the present invention, with a focus on biomaterials and delivery strategies, for clinical use of nucleic acid-based therapeutics, research on delivery vehicles that can facilitate the clinical use of nucleic acid-based therapeutics, nucleic acid for protein therapy, gene editing and vaccination An application of delivery-based delivery was developed.

The active ingredient, the cationic compound and the anionic compound can easily form a polyelectrolyte complex (PEC) or a polyelectrolyte multilayer (PEM). In order to maximize the biocompatibility and efficacy of the PEC and PEM, it is not known which type of polymer with which type of properties can form which polyelectrolyte multilayer nanoparticles (PEMN) under which type of conditions. didn't

The active ingredient may be selected from metals, small molecule drugs, proteins, growth factors, peptides, antibodies, nanoparticles and nucleic acids. Preferably, the pharmacologically active ingredient may be an anionic nucleic acid.

In view of this, an object to be solved by the present invention is to provide a novel technique for forming a PEMN encapsulated in a nucleic acid in a nanometer size.

The present invention is to provide a carrier that considers the biodegradability and biocompatibility of the constituents, the biostability of the nucleic acid as an active ingredient, repeated administration, and an effective payload of the PEMN as a pharmaceutical product.

The present invention mainly relates to the interaction of nucleic acids, cationic compounds, in particular protamine (PS), chitosan, cell penetrating peptides (CPP) or derivatives thereof, and anionic compounds, in particular human serum albumin, hyaluronic acid or derivatives of these polysaccharides. It is intended to provide a nucleic acid PEMN that depends on it.

The present invention can provide a nucleic acid PEMN that mainly depends on the interaction of a nucleic acid, a cationic compound, and an anionic compound. We want to provide a way to do that.

The object to be solved by the present invention is to provide a novel technology suitable for the preparation of PEMN containing nucleic acids, cationic compounds and anionic compounds in order to secure the in vivo stability of the active ingredient nucleic acid and the non-toxicity and biodegradability of the carrier. will provide

It is an object of the present invention to solve a variety of additional problems including the above problems, and a more efficient composite particle containing a nucleic acid and a disease prevention and aggravation prevention effect using the same, in particular, a high The purpose is to provide treatment results.

However, these problems are exemplary, and the scope of the present invention is not limited thereto.

Among the components of the composition according to the present invention, PEC and PEM prepared by an active ingredient, a cationic compound and an anionic compound, and PEMN prepared by homogenizing them, the active ingredient may be encapsulated in the PEMN structure.

The active ingredient may be selected from metals, small molecule drugs, proteins, growth factors, peptides, antibodies, nanoparticles and nucleic acids. Preferably, the pharmacologically active ingredient may be an anionic nucleic acid.

Nucleic acids include deoxyribonucleic acid, ribonucleic acid or backbone, nucleic acids such as polynucleotide derivatives in which sugars or bases are chemically modified or terminated, for example, RNA, DNA, RNA-guided genome editing system, siRNA (short interfering) RNA), aptamer, antisense ODN (antisense oligodeoxynucleotide), antisense RNA, ribozyme, and nucleic acid such as DNAzyme, that is, as an anionic drug, there are a wide variety of substances and delivery A different delivery system and production method are required depending on the type of nucleic acid to be desired, and an object of the present invention is to provide a formulation with increased stability and production yield as a nucleic acid delivery agent and a production method thereof.

A method of preparing PEC by mixing the active ingredient anionic nucleic acid and a cationic polymer and electrostatic interaction between the core particles and the prepared core particles and the anionic polymer by mixing the electrostatic interaction therebetween; and

A method for preparing PEC by mixing an active ingredient, a cationic polymer, and an anionic polymer by electrostatic interaction therebetween; and

A method of producing PEM characterized by any one selected from the group comprising; a method for producing PEM by alternately manufacturing and eluting active ingredients, cationic polymers and anionic polymers on a substrate by various layer-by-layer deposition techniques including inkjet would like to provide

An object of the present invention is to provide a PEMN, a use thereof, and a manufacturing method thereof, comprising the step of preparing PEMN (Polyelectrolyte multilayer nanoparticles) by homogenizing the prepared PEC and PEM.

Nucleic acids, cationic compounds, and anionic compounds each have several types of compounds, and one type or two or more types may be selected and used from each group to meet the purpose of the present invention.

In the present invention, in the PEC formed by the core particle composed of a nucleic acid and a cationic compound and an additional anionic compound, in an aqueous environment, the anionic compound forms the outer wall of the multiparticulate, and the cationic compound forms the inner wall of the PEC, It is a structure in which a nucleic acid and a cationic compound complex are encapsulated in the formed composite particle.

An object of the present invention is to provide a PEMN and a method for manufacturing the same, characterized in that any one or more selected from the group including shaking and ultrasonication is performed in the PEC manufacturing step.

The PEMN has a particle size of 50 to 400 nanometers and a polydispersity index (PDI) of 0.3 or less.

The step of preparing the PEMN is to provide a PEMN characterized by any one or more selected from the group consisting of extrusion homogenization, upstream pressure homogenization, high pressure homogenization and ultra-high pressure homogenization, and a method for manufacturing the same.

The upstream pressure may be 20 to 60 MPa, the high pressure may be up to 150 to 200 MPa, and the ultra-high pressure may be up to 350 to 400 MPa;

An object of the present invention is to provide a PEMN and a method for preparing the same, characterized in that the active ingredient in the PEMN releases a nucleic acid at a constant rate.

Among the polymers used in nucleic acid-based therapies, the present invention, in order to highlight the applicable properties that are very suitable for nucleic acid delivery, the present invention is biodegradable and biocompatible cations such as Protamine (PS), chitosan and cell penetrating peptide (CPP). Anionic compounds such as hyaluronic acid and albumin were selected.

Cationic compounds such as PS, chitosan and CPP can electrostatically interact with nucleic acids to form stably positively charged polyplexes, while neutral and anionic compounds often favor more efficient interactions with nucleic acids. It requires the presence of other ingredients. Anionic compounds are mucoadhesive polymers, but have opposite charges, greatly affecting their use in nucleic acid delivery. This important difference stems from the chemical structure, which is provided by a positively charged guanidino group in the basic amino acids of protamine, particularly in the arginine monomer, or a negatively charged carboxyl group in the glucuronic acid unit of hyaluronic acid. can do.

It can be said that the advantages of the nucleic acid intracellular carrier proposed in the present invention are clearly achieved by the cationic compound and complemented by the anionic compound. First, the efficient physical interaction of cationic compounds with negatively charged nucleic acids protects them from nuclease degradation. Second, they provide a net positive charge, enhancing the interaction of the complex with the anionic surface of the cell. Nevertheless, association with anionic polysaccharides such as albumin and hyaluronic acid in the carrier of the present invention can improve bioavailability by avoiding nucleic acid excretion through the glomerular capillary wall. Indeed, anionic material is "unfilterable" due to the repulsive forces established by the negative charge of the glomerular membrane.

In the PEMN of the present invention, cationic compounds and derivatives thereof may be used to obtain different types of carriers, and anionic compounds and derivatives thereof may be used to obtain different types of carriers. Nevertheless, the present invention seeks to provide a nucleic acid carrier that mainly relies on the interaction of nucleic acids, in particular cationic compounds such as PS, CPP, chitosan, and anionic compounds such as hyaluronic acid, albumin or derivatives of these polysaccharides.

In view of this, an object to be solved by the present invention is to provide a novel technology suitable for the preparation of PEMNs containing cationic compounds, anionic compounds and nucleic acids.

The present invention has developed a PEMN that prepares nucleus and core particles with nucleic acids and cationic polypeptides as active ingredients, and forms an outer layer with anionic polysaccharides.

The present inventors have found that the degree of cationization is important as a property of a cationic compound capable of forming a core particle including a nucleic acid in order to prepare PEMN, and thus completed the present invention.

The nucleic acid and the cationic compound complex maintains the encapsulated state in the PEMN structure formed by the anionic compound, thereby improving stability in blood or body fluid.

The cationic compound is the biodegradable and biocompatible polymeric carrier molecule, for example, sufficient transfection of nucleic acids for gene therapy or other therapeutic applications without loss of activity, particularly in vitro as well as in vivo. for sufficient aggregation of nucleic acids for the purpose of sufficient transfection of the nucleic acids into other cell lines. The PEMN carrier molecule is furthermore not toxic to cells and allows for sufficient release of the nucleic acid delivery agent.

The present invention additionally employs biodegradable and biocompatible anionic compounds that provide improved stability of the nucleic acid delivery and prevent recognition of PEMN by the immune system, in particular hydrophilic polymer chains (e.g., The reversible addition of PEG-monomer) may confer improved resistance to aggregation.

The cationic polypeptides such as PS and CPP constituting the PEMN of the present invention have a cation binding sequence with a defined chain length that provides strong condensation of nucleic acid delivery and complex stability, albeit with fairly small non-toxic monomer units. (cationic binding sequence) is formed. Under the reduced state of the cytosol (e.g., cytosolic GSH), the complexes are rapidly degraded into their monomers, which are further degraded (e.g., oligopeptides) or secreted (e.g., PEG) . This aids in the deliberation of the nucleic acid transporter within the cell matrix. Because it is degraded into small oligopeptides in the cell matrix, the toxicity known to be observed with high molecular weight oligopeptides, for example, high molecular weight oligoarginine is not observed.

The PEG-coating of the anionic compound constituting the PEMN of the present invention coats the carrier with a hydrophilic coating at its end, which can prevent salt-mediated aggregation and unwanted interactions with serum contents. . Within the cytosol, this coating is easily removed under the reducing conditions of the cell. Again, this effect enhances the induction of nucleic acid delivery in the cell matrix.

The particle size of the PEMN of the present invention may be 10-1000 nm, more specifically 10-200 nm.

In addition, the surface charge of the PEMN of the present invention may be -50 to 50 mV, more specifically -40 to 40 mV.

The PEMs of the present invention correspond to polyelectrolytes, multilayer polyelectrolytes, nanogels, hydrogels and polymer micelles. When the particle size and surface charge of the PEMN of the present invention are maintained at the above levels, it is preferable in terms of stability of the PEMN structure, content of components, absorption of nucleic acids in the body, and convenience of sterilization as a pharmaceutical composition.

According to another aspect of the present invention, there is provided a composition for treating cancer comprising the composition for drug delivery as an active ingredient.

In the present invention made as described above, it is possible to provide PEMN containing a pharmacologically active ingredient, and to provide therapeutic proteins, peptides, antibodies, nanoparticles and nucleic acids, and extracellular matrix degrading enzymes or degrading enzymes according to an embodiment PEMN containing a nucleic acid encoding an inhibitory substance prevents or aggravates ECM-related diseases or, in particular, destroys cancer cells by decomposing the extracellular matrix that interferes with the proximity of immune cells, nucleic acid drugs and anticancer compounds by herpes around tumor tissue. Since it can be selectively removed, it can be usefully used in the treatment of cancer.

1 shows the amplified PCR product was digested using EcoRI/BamHI enzymes, then cloned using the pUC19 vector linearized with EcoRI/BamHI enzymes and T4 DNA ligase, and the reaction solution was transformed into E. coli, and in the selected strain It is the result of electrophoresis after separating the plasmid and treating with EcoRI/BamHI restriction enzyme,
2 is a transcript obtained by in vitro transcription of a PCR product obtained by amplifying a pUC19 vector into which DNA having <T7 promoter - 5'UTR - target protein ORF DNA - muag - A64 - C30 - histone-SL> is inserted as a template. It is the result of electrophoresis with different amounts,
3 is a nucleic acid prepared only with natural nucleotides, a nucleic acid containing a modified nucleotide, and a modified nucleic acid with Cholesterol added to the 3' end of the cell culture medium for various times and then RT-PCR to confirm the in vitro stability;
4 is a PCS measurement result of particle size according to the amount of PS in nucleic acid/PS basic particle-DW and nucleic acid/PS basic particle-CM, PDI grade: (a) PDI ≤ 0.1, (b) PDI ≤ 0.15, (c) PDI ≤ 0.2. Without classification, PDI > 0.2,
5 is a result of confirming the nucleic acid not encapsulated in the composite particle according to the ratio of the nucleic acid;
Figure 6 is the result of confirming the unsealed state of the nucleic acid in the preparation of core particles 2.0 according to the assembly route PC, DC and MP using the nucleic acid, the cationic compound protamine (PS), and the cationic compound DOTAP,
7 shows PCS measurements of formulations containing 30 μg nucleic acid, 90 μg PS and 0 to 2,000 μg HSA, and PDI grades: (a) PDI ≤ 0.1, (b) PDI ≤ 0.15, (c) PDI ≤ 0.2. Without classification, PDI > 0.2,
8 is a result of analyzing the particle size change according to the sonication and extruder methods using PCS after reacting nucleic acids, PS and HA sequentially by the sonication & extruder method according to the weight ratio;
Figure 9 is the result of examining the drug release profile over time in the [nucleic acid / PS]:HA addition molar ratio of PEMN 1:1.5 mass ratio,
10 is a result of DLS analysis of PEMN prepared by ultra-high pressure homogenization;
11 is a result of synthesizing and purifying an HA-DAB-aptamer conjugate using HA and aptamer-SH;
12 is a result of analysis of HA-DAB-aptamer conjugates synthesized with two different cross linkers through agarose electrophoresis (a). HA-ss-aptamer is a disulfide bond between HA and aptamer (-ss- ), and HA-aptamer means a conjugate (control group) connected by a general covalent bond rather than a disulfide bond, and FIGS. By adding FBS to the HA-DAB-aptamer conjugate and checking the amount of remaining aptamer through agarose electrophoresis by time (b to d), HA-aptamer conjugate means HA-DAB-aptamer conjugate, ,
Figure 13 shows the amount of multiparticulates from 293T cells transfected with the target protein nucleic acid:PS:HSA multiparticle (a) and the secretion of each protein over time (b) with Human sTNFR2 ELISA Kit (MyBioSource, USA). ), G-CSF Human Instant ELISA™ Kit (Invitrogen, USA), EPO Quantikine ELISA™ Kit (R&D Systems, USA), anti-RSV-F antibody ELISA™ kit (ab94968, abcam, USA) or Human Anti- It is the result of quantification with Respiratory syncytial virus IgG ELISA™ Kit (RSV) (ab108765, abcam, USA),
Figure 14 is the result of quantification according to the amount (a) and time (b) dp of the multiparticulate after transfection of various amounts of HYAL 1, PH20 or sPH20 nucleic acid / PS / HA complex particles into cells,
15 shows cells transfected with sPH20 multiparticulates were cultured for 6 hours, washed, and then, 0.5 mg/mL bovine aggrecan (Sigma-Aldrich, USA) was added and incubated for 24 hours, after which , after removing the medium and fixing it with 2% glutaraldehyde in PBS (pH 7.4) and replacing it with a suspension with 10 ⁹ /mL mouse RBCs, the cells were replaced with a camera scanner and imaging program (DiagnostiC Instruments, Inc., USA). It is the result of imaging with a combined phase contrast microscope,
16 shows the expression level of the target protein according to the amount of particles (a) and time elapsed (b) when human skin fibroblasts transfected with various amounts of MMP1, MMP 8 or MMP 13 nucleic acid:PS]/HA complex particles are cultured. is the result of quantification,
17 shows the target protein according to the amount of particles (a) and time elapsed (b) when cells transfected with various amounts of TIMP 1, TIMP 2, TIMP 3 or TIMP 4 nucleic acid:PS]/HA multiparticle 1.1 are cultured; is the result of quantifying the expression level of
18 is a result of transfecting multiparticulates having TCA into MDCK-ASBT cells and MDCK cells and quantifying the amount of TIMP1 expression;
19 shows almost no expression in [MMP1 nucleic acid:PS]/HA PEMNE 1.1 transfected EGFR+ lung cancer cell line A549 cells, colorectal cancer cell line SW48 and skin cancer cell line A431, EGFR- breast cancer cell line, MDA-MB-453, colon This is the result of examining the expression level of MMP1 in the cancer cell line HT-29 and the neuroblastoma cell line SK-N-MC (a), EGFR+ lung cancer cell line A549 cells transfected with [MMP1 nucleic acid:PS]/HA-Apt_EGFR complex particles, large intestine The expression level of MMP1 was relatively high in the cancer cell line SW48 and the skin cancer cell line A431, and the expression level of MMP1 was investigated in the EGFR- breast cancer cell line, MDA-MB-453, colorectal cancer cell line HT-29, and the neuroblastoma cell line SK-N-MC. result (b).

A protein generally used in the present invention and a protein to be expressed in the present invention are referred to as target proteins, and the two terms are used almost synonymously, and in general, a protein includes one or more peptides or polypeptides. In general, proteins fold into three-dimensional shapes that may be required for proteins to exert biological functions.

Peptide: Generally, a peptide or polypeptide is a polymer of amino acid monomers linked by peptide bonds. It generally contains less than 50 monomer units. However, the term peptide is not a waiver for molecules having more than 50 monomer units. Long peptides, also called polypeptides, generally have 50 to 600 monomer units.

Fragment or part of protein: In the present invention, fragment or part of protein can be generally understood as a peptide corresponding to a continuous part of the amino acid sequence of a protein, preferably from about 6 to about 20 or more amino acids. a portion having a length, e.g., 8, 9, or 10, (or 11 , or even 12 amino acids), or fragments processed and presented by MHC class II molecules, preferably about 13 or more amino acids, for example 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids in length, wherein these fragments may be selected from any portion of the amino acid sequence. These fragments can generally be recognized by T cells in the form of a complex consisting of a peptide fragment and an MHC molecule, ie the fragments are generally not recognized in their native form. Fragments or portions of proteins as defined herein may also include epitopes or functional sites of these proteins. A fragment or portion of a protein is an antigen, in particular an immunogen, for example an antigenic determinant (also called an 'epitope') or has antigenic characteristics and elicits an adaptive immune response. Accordingly, fragments of proteins or peptides may comprise at least one epitope of these proteins or peptides. Furthermore, a domain of a protein may be understood to include fragments of a protein, such as an extracellular domain, an intracellular domain or a transmembrane domain and short or truncated versions of a protein. .

Chimeric Enzyme: A chimeric enzyme refers to an enzyme that is not a natural enzyme found in nature. Thus, a chimeric enzyme may comprise catalytic domains derived from different sources (eg, different enzymes) or catalytic domains derived from the same source (eg, the same enzyme), arranged in a manner different from that found in nature. Chimeric enzymes include monomeric (ie single unit) enzymes as well as oligomeric (ie multiple unit) enzymes, particularly hetero-oligomeric enzymes. Monomeric enzymes refer to single-unit enzymes consisting of only one polypeptide chain.

In the present invention, in a nucleotide, nucleoside or polynucleotide (e.g., nucleic acids of the invention, e.g., mRNA molecule), "modified" or suitably "modified" means A, G, U or C refers to modifications to ribonucleotides. Generally, in the present invention, these terms are not intended to refer to ribonucleotide modifications within the naturally occurring 5'-terminal mRNA cap moiety. In polypeptides, "modification" refers to a modification compared to a moiety, a canonical set of 20 amino acids.

Where the nucleic acid is an mRNA, the coding region, flanking regions and/or terminal regions may contain one, two, or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, a modified polynucleotide introduced into a cell may exhibit reduced degradation in the cell as compared to an unmodified polynucleotide.

Polynucleotides may contain any useful modifications to sugars, nucleobases, or internucleoside linkages (eg, to linking phosphate/to phosphodiester linkages/to phosphodiester backbones). . For example, a major groove face of a polynucleotide, or a major groove face of a nucleobase, may include one or more modifications. One or more atoms of the pyridine nucleobase (e.g., on the major groove face) are optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro). In certain embodiments, modifications (eg, one or more modifications) are present on each sugar and each internucleoside linkage. Modifications according to the present invention are ribonucleic acid (RNA) to deoxyribonucleic acids (DNA) modifications, for example 2'H of 2'OH of ribofuranicil ring, threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNAs), or a hybrid thereof. Further variations are described herein.

As used herein, "genome" encompasses chromosomal DNA present in the nucleus as well as organ DNA present in cell subcomponents (eg, mitochondria, plastids).

"Polynucleotide", "nucleic acid sequence", "nucleotide sequence" or "nucleic acid fragment" are interchangeably referred to as single-stranded or double-stranded RNA or DNA, optionally comprising synthetic, non-natural or modified nucleotide bases. It is used to refer to a polymer that has been Nucleotides (generally found in the 5'-monophosphate form) are referred to by shorthand names as follows: "A" = adenylate or deoxyadenylate (RNA or DNA, respectively), "C" = cytidylate or deoxycytidylate, "G" = guanylate or deoxyguanylate, "U" = uridilate, "T" = deoxythymidylate, "R" = purine (A or G), "Y" = pyrimidine (C or T), "K" = G or T, "H" = A or C or T, "I" = inosine, "N" = any nucleotide.

"Polypeptide", "peptide", "amino acid sequence" and "protein" are used herein interchangeably to refer to a polymer of amino acid residues. These terms apply to natural amino acid polymers as well as amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding natural amino acid. The terms "polypeptide", "peptide", "amino acid sequence" and "protein" also include, but are not limited to, glycosylation, lipid binding, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-hydration. modifications such as ribosylation.

It may be desirable to introduce a modified nucleic acid molecule into a cell so that it is degraded intracellularly. For example, degradation of the modified nucleic acid molecule may be desirable where precise timing of protein production is desired. Accordingly, the present invention provides a modified nucleic acid molecule containing a degradation domain, capable of acting in a direct manner within a cell. Binding of a major groove interaction, e.g., binding of a partner to a polynucleotide (e.g., when a modified nucleotide reduces binding affinity for a major groove interaction partner, compared to an unmodified nucleotide) It provides polynucleotides containing nucleosides or nucleotides capable of interfering with Polynucleotides can be used with other agents [e.g., RNAi-inducing agents, RNAi agents, siRNA, shRNA, miRNA, antisense RNA, ribozyme, catalytic DNA, tRNA, RNA inducing triple helix formation, aptamers, vectors, etc.] may be optionally included. Polynucleotides may comprise one or more messenger RNAs (mRNAs) having one or more modified nucleosides or nucleotides (ie, modified mRNA molecules).

Nucleic Acid Molecules: A nucleic acid molecule is a molecule comprising, preferably consisting of, a nucleic acid component. The term nucleic acid molecule refers to DNA or RNA. It is used synonymously with the term "polynucleotide". Nucleic acid molecules are polymers comprising or consisting of nucleotide monomers and are covalently linked to each other by phosphodiester bonds of sugar/phosphate-backbone.

DNA: DNA is a generic abbreviation for deoxyribonucleic acid. It is a nucleic acid molecule, i.e. a polymer made up of nucleotides. These nucleotides are usually deoxyadenosine-monophosphate, deoxythymidine-monophosphate, deoxyguanosine-monophosphate and deoxycytidine-monophosphate monomers composed of a sugar moiety, a base component and a phosphate component. , polymerized by a unique backbone structure. The backbone structure is generally formed by a phosphodiester bond between the sugar moiety of the first nucleotide proximal to the monomer, i.e., deoxyribose and the second phosphate moiety. The specific order of the monomers, i.e., the order of the bases linked to the sugar/phosphate-backbone, is referred to as the DNA-sequence. DNA can be single-stranded or double-stranded. In the double stranded form, the nucleotides of the first strand generally hybridize with the nucleotides of the second strand, eg, A/T-base pairs and G/C-base pairs.

Gene: A gene refers to a specific nucleic acid sequence for producing one or more cellular products and/or for achieving one or more intercellular or intracellular functions. More specifically, the term relates to a nucleic acid section (typically DNA, but RNA in the case of RNA viruses) comprising a nucleic acid encoding a particular protein or functional or structural RNA molecule.

Expression is used in its most general sense and includes the production of RNA or the production of RNA and proteins. This also includes partial expression of nucleic acids. In addition, expression may be transient or stable. In the context of nucleic acids, the term "expression" or "translation" relates to the process by which one strand of messenger RNA directs the assembly of amino acid sequences within the liposome of a cell to make a peptide or protein.

RNA, mRNA: RNA is a general abbreviation for ribonucleic acid. It is a nucleic acid molecule, that is, a polymer made up of nucleotides. These nucleotides are usually adenosine-monophosphate, uridine-monophosphate, guanosine-monophosphate and cytidine-monophosphate monomers linked together along the so-called backbone. The backbone is formed by a phosphodiester linkage between the first sugar proximal to the monomer, namely ribose, and the second phosphate component. A specific chain of monomers is called an RNA-sequence. Ordinarily RNA may be obtainable by transcription of a DNA-sequence, for example, inside a cell. In an advancing cell, transcription is usually carried out inside the nucleus or mitochondria.

mRNA refers to messenger-RNA and typically relates to a transcript produced using a DNA template and encoding a peptide or protein. Typically, an mRNA comprises a 5'UTR, a protein coding region, a 3'UTR, and a poly(A) sequence. mRNA can be produced by in vitro transcription from a DNA template. In vitro transcription methodologies are well known to those skilled in the art. For example, a variety of in vitro transcription kits are available on the market. According to the present invention, mRNA can also be modified by stabilizing modification and capping.

In vivo, transcription of DNA usually results in so-called premature RNA, which must be processed into so-called messenger-RNA, usually abbreviated as mRNA. For example, processing of immature RNA in eukaryotic organisms involves a variety of different post-transcriptional modifications including splicing, 5' capping, polyadenylation, and export from the nucleus or mitochondria. includes The sum of these processes is called the maturation of the RNA. Mature messenger RNA usually provides a nucleotide sequence that can be translated into the amino acid sequence of a particular peptide or protein. Generally, a mature mRNA comprises a 5' cap, optionally a 5'UTR, an open reading frame, optionally a 3'UTR and a poly(A) sequence. In addition to messenger RNA, several non-coding types of RNA exist, which may be involved in the regulation of transcription and/or translation. The term "RNA" also includes other encoding RNA molecules, such as viral RNA, retroviral RNA and replica RNA.

Sequence of Nucleic Acid Molecules: The sequence of a nucleic acid molecule is generally understood to be a specific and discrete order, ie, a sequence of its nucleotides. The sequence of a protein or peptide is generally understood to be a sequence, ie a sequence of its amino acids.

Stabilized Nucleic Acid Molecules: Stabilized nucleic acid molecules are more stable to disintegration or degradation by, for example, environmental factors or enzymatic degradation such as exo- or endonucleases, than unmodified nucleic acid molecules, A DNA or RNA molecule that has been so chemically modified. In the present invention, the stabilized nucleic acid molecule is stabilized in cells such as prokaryotic or eukaryotic cells, mammalian cells such as human cells. The stabilizing effect can also be exerted outside the cell, for example, in a buffer solution, etc., for example, in a process for preparing a pharmaceutical composition comprising a stabilized nucleic acid molecule.

"Transient transfection" or transformation means that a nucleic acid molecule or protein is introduced into a cell so that the exogenous gene performs its function without being stably inherited. In the case of transient transformation, no exogenous nucleic acid sequence is inserted into the genome.

Transfection: Transfection refers to the introduction of a nucleic acid molecule, DNA or RNA (eg mRNA) molecule into a cell, preferably a eukaryotic cell. In the present invention, transformation includes any method known to a person skilled in the art for introducing a nucleic acid molecule into a cell, a eukaryotic cell such as a mammalian cell. Such methods include, for example, electroporation, for example cationic lipid and/or liposome-based lipofection, calcium phosphate precipitation, nanoparticle-based transformation, virus-based transformation, or transformation based on cationic compounds such as DEAE-dextran or polyethyleneimine. The introduction is non-viral.

Carrier/Polymer Carrier: A carrier may generally be a compound that facilitates transport or complexation of another compound (cargo). A polymeric carrier is generally a carrier that forms a polymer. A carrier may be associated with its cargo by covalent or non-covalent interactions. A carrier is capable of transporting a nucleic acid, eg, RNA or DNA, into a target cell. The carrier may be a cationic component.

Vehicle: A vehicle is generally understood to be a material suitable for storing, transporting, and/or administering a compound, such as a pharmaceutically active compound. For example, it may be a physiologically acceptable lipid suitable for storage, transport and/or administration of a pharmaceutically active compound.

Pharmaceutically effective amount: In the present invention, a pharmaceutically effective amount is generally used to replace a pathological level of an expressed peptide or protein, or to replace a missing gene product, e.g., a pathology. It is understood to be in an amount sufficient to induce a pharmaceutical effect such as an immune response in the case of a hostile situation.

Genetic vaccination: Genetic vaccination can generally be understood as vaccination by administration of a nucleic acid molecule encoding an antigen or immunogen or fragment thereof. The nucleic acid molecule may be administered to an individual's body or to an isolated cell of an individual. Upon transformation of a particular cell of the body, or upon transformation of an isolated cell, an antigen or immunogen can be expressed by those cells and subsequently presented to the immune system, eliciting an adaptation, i.e., an antigen-specific immune response. can be Thus, genetic vaccination generally involves a) transformation of a nucleic acid, RNA molecule into a subject, into a patient, or in cells of a patient in vivo/ex vivo or in vitro. administration to isolated cells of a subject, usually a patient; b) transcription and/or translation of the introduced nucleic acid molecule; and optionally c) re-administration of the isolated, transformed cells to the subject, the patient, if the nucleic acid was not directly administered to the patient.

I. Nanoparticles containing active ingredients

The active ingredient may be selected from metals, small molecule drugs, proteins, growth factors, peptides, antibodies, nanoparticles and nucleic acids. Preferably, the pharmacologically active ingredient may be an anionic nucleic acid.

In the context of the present invention, the nucleic acid of the nanoparticles is DNA, preferably but not limited thereto, for example, genomic DNA, single-stranded DNA molecules, double-stranded DNA molecules. -stranded DNA molecules), coding DNA, DNA primers, DNA probes, pDNA, may be an appropriate nucleic acid selected from immunostimulating DNA, PNA (peptide nucleic acid) or It can be selected from these, and is RNA, preferably, but not limited to, coding RNA, messenger RNA (mRNA), siRNA, shRNA, antisense RNA, or riboswitch ( riboswitches), immunostimulating RNA (isRNA), ribozymes, or aptamers.

In addition, the nucleic acid may be ribosomal RNA (rRNA), transfer RNA (tRNA), messenger RNA (mRNA), or viral RNA (viral RNA, vRNA). Preferably, the nucleic acid is RNA, more preferably coding RNA.

The nucleic acid may be a linear single-stranded RNA or mRNA. mRNAs typically contain several structural elements, e.g., an upstream ribosome binding site followed by a coding region, an optional 5'-UTR, poly-A-tail (and/or poly-C-tail) RNA containing an optional 3'-UTR that may be followed by mRNA may appear as mono-, di-, or even multicistronic RNA, i.e. one, two or more (same or different) proteins or peptides as defined herein. RNA that carries the coding sequence of In di-, or even multicistronic mRNA, such coding sequences may be separated by at least one internal ribosome entry site (IRES) sequence.

In addition, the nucleic acid of the biodegradable polymer carrier delivery complex may be a single-stranded or double-stranded nucleic acid due to the non-covalent association of two single-stranded nucleic acids (molecules).

or partially double-stranded or partially single-stranded nucleic acids, at least partially complementary, all of which are partially double-stranded or partially single-stranded nucleic acids are generally made up of a longer or shorter single-stranded nucleic acid or are of length is made by the same two single-stranded nucleic acids, one single-stranded nucleic acid is partially complementary to the other single-stranded nucleic acid and thus both form a double-stranded nucleic acid molecule at this portion. That is, it forms a partially double-stranded or partially single-stranded nucleic acid (molecule).

Preferably, the nucleic acid (molecule) may be a single-stranded nucleic acid molecule. Furthermore, the nucleic acid (molecule) may be a circular or linear nucleic acid molecule, preferably a linear nucleic acid molecule.

Transfection of nucleic acid carriers faces extracellular and intracellular barriers. Nucleic acid transporters are exposed to serum nucleases and phagocytic uptake, which significantly reduces their biological half-life. In addition, the negative charge of the cell membrane as well as the extracellular matrix (ECM) prevents the nucleic acid transporter from reaching its target and acting.

Among the many barriers to function, nucleic acids must cross the cell membrane to reach the cytoplasm. Cell membranes are dynamic and powerful barriers to intracellular transport. Various ion pumps and ion channels help maintain the negative potential. It maintains a negative charge in the cytoplasmic space by regulating the balance of most essential metal ions (eg, K+, Na+, Ca2+ and Mg2+) across the cell membrane (-40 to -80 mV).

Cell membranes create a strong barrier to highly negatively charged nucleic acid molecules. Unsaturated lipids, particularly cis-double bonded lipids, increase cell membrane fluidity by introducing defects into the membrane structure. The other major component of the bilayer includes sterols (about 30% of total lipids). Cholesterol, the major sterol in the lipid bilayer, helps to maintain a balance between fluidization and condensation of the lipid bilayer by creating bilayer defects.

Transfection of nucleic acids is performed using viral or non-viral vectors. For successful delivery, these viral or non-viral vectors must overcome the obstacles mentioned above. The most successful gene therapy strategies today rely on the use of viral vectors such as adenoviruses, adeno-associated viruses, retroviruses and herpes viruses. Viral vectors can mediate gene transduction with high efficiency and the potential for long-term gene expression, satisfying two out of three hypotheses. However, the acute immune response, immunogenicity, and insertional mutagenesis revealed in gene therapy clinical trials raise serious concerns about the safety of some commonly used viral vectors.

A solution to this problem may be found in the use of non-viral vectors. Although non-viral vectors are not as efficient as viral vectors, many non-viral vectors offer a more stable alternative to gene therapy.

Methods of non-viral gene delivery are being studied using physical (carrier-free gene delivery) and chemical (gene delivery based on synthetic vectors) approaches. Physical approaches usually include needle injection, electroporation, gene gun, ultrasound and hydrodynamic delivery, using physical forces that penetrate cell membranes, and promotes gene introduction into cells. Chemical approaches are generally used as carriers for transport of gene of interest into cells synthesized or naturally occurring compounds (cationic lipids, cationic polymers, lipid-polymer hybrid systems). is used as a carrier.

Although significant advances have been made in basic science and in various non-viral gene delivery systems, the majority of non-viral approaches are still less efficient than viral vectors, particularly in in vivo gene delivery.

In order to achieve steric stability of the nucleic acid of the present invention and to improve the BD/PK properties of the carrier, polyethylene glycol conjugated (PEGylated) lipids approved by the FDA for human use in other pharmaceuticals may be used. The entire particle surface may be covered with PEG to minimize any possible immune response and activation of the complement system.

Polyelectrolyte complex (PEC) is a complex in which macromolecules are bound together mainly by electrostatic interaction, formed spontaneously when two polymers of opposite charges are mixed. Also, "nanoplex" is widely used in the literature to denote a complex formed between a positively charged nanoparticle and a nucleic acid, DNA or RNA. When the positively charged component is based on a cationic polypeptide such as chitosan, a nanoplex can also be defined as a polyplex.

PECs are predominantly spherical, contain a stoichiometric mixture of cationic and anionic polymers, and consist of a neutral core surrounded by an excess of polyelectrolyte that allows the formation of a charge stabilizing shell.

Since the self-assembly of PEC is a reversible process, its integrity may be compromised in the presence of other charged species due to electrostatic shielding and polyion exchange between the PEC and the medium. Therefore, the stability of PEC can be severely affected in biological media in the presence of charged proteins and high concentrations of small electrolytes. A strategy to overcome this problem is to improve the stability of PEMs by covalently crosslinking complex polyelectrolytes to prevent separation of polymer chains from PEMs. PEM-based nanoparticles are attractive because they are easy to fabricate and cost-effective, and the reversibility of electrostatic interactions is attractive because they provide special tunability to the behavior of the carrier while maintaining the integrity of the composite under appropriate conditions.

One of the most developed methods for nucleic acid delivery is a combination preparation of lipid nanoparticles (LNP). LNP formulations generally contain (1) polymeric materials bearing ionizable or cationic lipids or tertiary or quaternary amines to encapsulate polyanionic nucleic acids; (2) cationic lipids similar to those of cell membranes (eg, 1,2-dioreoyl-sn-glycero-3-phosphoethanolamine [DOPE]); (3) cholesterol, which stabilizes the lipid bilayer of LNPs; and (4) polyethylene glycol (PEG)-lipids to form a hydration layer on nanoparticles to improve colloidal stability and reduce protein absorption.

The most representative particle manufactured by encapsulation is lipid nanoparticles, and phospholipids are recognized as one of the most promising carriers of nucleic acids due to their biocompatibility, relative ease of large-scale production, and recent approval for use in clinical trials.

Phospholipids are amphiphilic molecules that, when dispersed in water, form spontaneous bilayer structures and trap the dispersed hydrophilic load within the aqueous core of the molding structure. Therefore, chemical modification of the head groups of these molecules with cationic polypeptides facilitates the capture of the negatively charged nucleic acids prepared in the present invention through electrostatic interaction, making them a promising component of a transporter.

The nucleic acid delivery system of the present invention is aimed at improving the pharmacokinetics of the nucleic acid delivery system in terms of plasma stability and circulation time as well as in terms of cell entry.

The nucleic acid constituting the nucleic acid of the present invention is a strongly negatively charged polymer, and the cationic polymer plays a very important role in the process of preparing the particles. Cationic polypeptides offer several advantages, including the ability to promote complex formation with negatively charged nucleic acids through electrostatic interactions, cellular uptake, and proton sponge-mediated endosomal escape. However, a disadvantage of cationic polymers is that they are highly toxic due to changes in cell membrane integrity and high immunogenicity.

A further promising approach in gene therapy is the use of cationic polymers. Cationic polymers have been found to be useful in transfection of nucleic acids because they form tight complexes and condense negatively charged nucleic acids. Therefore, many cationic polymers are being studied as carriers for gene delivery in vitro and in vivo.

A cationic compound generally refers to a charged molecule, which is generally 1 to 9, less than 9 (eg 5 to 9), or less than 8 (eg 5 to 8), or less than 7 ( For example, it has a positive charge (cation) of a physiological pH value of 5 to 7), for example 7.3 to 7.4. Thus, the cationic component can be any positively charged compound or polymer, a cationic peptide or protein that is positively charged under physiological conditions, in particular in vivo physiological conditions. A 'cationic peptide or protein' may contain at least one positively charged amino acid or one or more positively charged amino acids, for example selected from Arg, His, Lys or Orn. Thus, 'polycationic' compounds are also within the range that exhibit more than one positive charge under given conditions.

Such cationic polymers include polyethylenimine (PEI), polyamidoamine dendrimers, polypropylamine dendrimers, polyallylamine, cationic dextran, chitosan, cationic proteins and cationic peptides.

Although most cationic polymers have the function of condensing DNA into small particles and promoting cellular uptake by endocytosis through charge-charge reactions with anionic localization on the cell surface, transfection activity (transfection) There is a marked difference between activity and toxicity.

Cationic polymers exhibit better transfection capacity at higher molecular weights due to stronger complexation of negatively charged nucleic acid carriers. However, the toxicity of the cationic polymer increases as the molecular weight increases. Polyethylenimine (PEI) is probably the most active and most studied polymer in the field of gene delivery, but its greatest weakness as a transfection reagent is related to its non-biodegradable properties and toxicity. .

One unusual approach in gene therapy is the use of cationic lipids. However, although many cationic lipids show excellent transfection activity in cell culture, most perform poorly in the presence of serum and only a few have activity in vivo. When lipoplexes are exposed to overwhelming amounts of negative charge and amphiphilic proteins and polysaccharides present in blood, mucus epithelial lining fluid or tissue matrix, their size, surface charge and lipid Significant changes occur in structure.

Once administered in vivo, lipoplexes react with negatively charged blood components and tend to form large aggregates, which are either adsorbed to the surface of circulating red blood cells or deposited in a thick mucus layer. It can become entrapped or embolize the microvasculature, preventing the lipoplex from reaching its intended target cells at a distal location.

Moreover, toxicity associated with gene transfer by lipoplexes involves the induction of inflammatory cytokines in particular. In humans, negative inflammatory reactions of varying degrees, including flu-like symptoms, have been reported among patients receiving lipoplex. Therefore, it remains a question whether Lipoplex can be safely used by everyone.

Moreover, although polyplexes made by polymers with high molecular weight exhibit improved stability under physiological conditions, such polymers can interfere with vector unpacking. For example, very rapidly DNA dissociation from complexes formed with poly L-lysine (PLL) results in clearly enhanced short-term gene expression. At least 6 to 8 cationic amino acids in length are required to pack DNA into structures that are active for receptor-mediated gene transport. However, polyplexes made of short polycations are not stable under physiological conditions and generally aggregate rapidly in physiological saline.

To overcome this negative aspect, linear reducing polycondensation (reducible) produced by oxidative polycondensation of Cys-Lys10-Cys peptide that can be cleaved by the intracellular environment to promote the release of nucleic acids A novel type of synthetic vector based on polycation (RPC) was also developed. It has been shown that polyplexes made by RPC can be destabilized by a reducing state that allows for efficient release of DNA and mRNA.

The cationic peptide protamine has been shown to protect mRNA from degradation by serum RNases. However, only mRNA complexed with protamine showed limited protein expression and efficacy in cancer vaccine models due to the strong binding force between protamine and mRNA. To address this issue, we developed an RNAtive vaccine platform in which protamine-formulated RNA acts only as an immune activator, not as an expression vector. CureVac has developed an RNActive vaccine platform based on a protamine-made RNA complex that acts as a TLR 7/8 agonist to induce a Th1 T cell response, wherein the nucleotide-modified mRNA functions as an antigen producer. This vaccine platform has resulted in favorable immune activation against cancer and infectious diseases in both preclinical animal models and patients.

Cellular transport of nucleic acids is also possible by physical assembly or chemical ligation of cell penetrating peptides (CPPs), which are also expressed as protein-transduction domains (PTDs). CPP corresponds to a short peptide with a sequence of about 10 to 30 amino acids, which can cross the plasma membrane of mammalian cells and thus offers unprecedented possibilities for cell drug transport.

Almost all of these peptides contain a series of cationic amino acids in combination with sequences that are in the α-helix form at low pH. When the pH is continuously lowered in vivo due to the proton pump, the conformational change of the peptide is usually initiated rapidly.

This helix motif mediates insertion into the membrane of the endosome leading to the release of the contents into the cytoplasm (cytoplaa). The cleaved HIV-1 Tat protein basic domain crosses the plasma membrane and accumulates in the cell nucleus. Despite these advantages, major obstacles to CPP-mediated drug transport often involve rapid metabolic clearance of peptides when encountering or crossing the enzymatic barriers of epithelia and endothelia. think that

Cellular delivery peptides (CPPs) have been studied for their potential as vectors for intracellular delivery of nucleic acid delivery. Although their internalization mechanism is not fully understood, it is hypothesized that CPPs may promote clustering of negatively charged glycosaminoglycans at the cell surface. This causes macropinocytosis and lateral diffusion or directly disrupts the lipid bilayer. CPPs with arginine-rich amphiphilic RALA sequence repeats have been reported to function as mRNA vectors for dendritic cells and induce T cell immunity.

In conclusion, the metabolic stability of CPPs represents an important biopharmaceutical factor for their cell bioavailability. However, on the one hand, they are stable enough to transport their transport to the target before they are metabolically cleaved, while on the other hand, they can accumulate and be completely eliminated from the tissue before they can reach toxic levels. CPPs were not previously available.

In addition to the barrier of cell membranes, nucleic acids are subjected to degradation by extracellular ribonucleases, which are abundantly present in the skin and blood. In order to protect nucleic acids from degradation by nucleases and to protect their negative charge, amine-containing substances are generally used as non-viral vectors.

The nucleic acid multiparticulate should have the ability to fully protect the nucleic acid from enzymatic degradation and should be of an appropriate size to allow extravasation and retention of the target site while preventing renal removal. It should also possess appropriate surface properties that allow efficient entry by cells while preventing serum protein interactions and avoiding phagolysosomes. Another desirable property that facilitates clinical application is the easy functionalization of targeting ligands to improve tissue specificity, biocompatibility and reduce toxicity. Nucleic acid transporters also face the problem of leaving the endosome in time to deliver the nucleic acid to the protein synthesis site located in the cytoplasm.

The backbone of the nucleic acid constituting the nucleic acid complex particle of the present invention is a nucleic acid and should be protected from nucleolytic enzymes to ensure stability in blood, and the ideal delivery system of the present invention should be able to achieve the above object.

In the present invention, chemical modification of nucleic acids, protection of nucleic acid ends, etc. can be performed, and a method for manufacturing particles by encapsulating the nucleic acid with a polymer material can be provided.

Reducible polycation (RPC) reduced the toxicity of polycations to a level comparable to that of peptides with low molecular weight. This approach requires additional endosomolytic drug chloroquine or cationic lipid DOTAP to improve transfection efficiency to an appropriate level. As a result, reducing polycations (RPCs) may contain histidine residues, which are known to have endosome buffering capacity.

Histidine-rich reducing polycations can enable sufficient cytoplasmic transport of various nucleic acids, including plasmid DNA, mRNA, and siRNA molecules, without the endosomal disruption drug chloroquine. However, much research is needed on whether it is possible to directly apply histidine-rich reducing polycations in vivo. Since the binding of serum proteins to the polyplexes limits cellular uptake, transfection was performed without serum to avoid compromising the ability of histidine residues to enhance gene transduction.

Other approaches to technology for delivering delivery molecules into cells, such as gene therapy, involve peptide ligands. Peptide ligands may be short sequences separated from larger proteins representing essential amino acids required for receptor recognition, such as the EGF peptide used in target cancer cells. Other peptide ligands may include ligands used to target the lectin-like oxidized LDL receptor (LOX-1).

Up-regulation of LOX-1 in epithelial cells is associated with disorders such as hypertension and atherosclerosis. However, peptide ligands cannot be linked to their delivery molecules by complexation or adhesion and require covalent bonding, making them unsuitable for many gene therapy approaches, and generally use crosslinkers with intracellular cytotoxic effects. for example.

It is capable of condensing and stabilizing nucleic acids for the purposes of gene therapy and other therapeutic applications and exhibiting effective transfection activity, particularly in terms of the combination of effective release of nucleic acid delivery and low or no toxicity in vivo. Appropriate methods or carriers should be developed.

There is still a strong need in the art to provide carriers for the purpose of gene delivery, and this strong need is that they are stable enough to deliver their delivery to the target before they are metabolically cleaved, on the other hand they It must be removed from the tissue before it can accumulate or reach toxic levels.

The therapeutic success of nucleic acid-based agents depends on overcoming many challenges associated with nucleic acid delivery in vivo. The development of safe and effective nucleic acids is crucial for the successful application of nucleic acid-based therapies. Nanostructured carriers offer unique advantages such as protection from premature degradation and improved interaction with the biological environment. It also enhances tissue uptake, prolongs nucleic acid retention time, enhances cellular internalization, and enables nucleic acid to migrate into the cytoplasm of target cells.

Systemically delivered LNPs that deliver nucleic acids face multiple barriers to delivery. The mononuclear phagocytosis system (MPS), particularly in the liver and spleen, is a frequent destination for injected nanoparticles because of their intrinsic role in the body's protective function against nanoscale infectious agents. The kidneys filter out naked nucleic acids or any nanoparticles with a hydrodynamic diameter of less than 5.5 nm.

Most LNPs are about 100 nm in diameter and are large enough not to exit the MPS and reach other organs of interest. The liver, the main organ of MPS, has a porous vasculature and contains phagocytes such as Kupffer cells that carry cationic LNPs. Also, large cationic LNPs do not leak out of the capillaries in the lungs and cannot be filtered into the bloodstream by the kidneys. Transmitters may accumulate in the liver, lungs, or other organs. Thus, clearance and biodegradability of the delivery system components are one consideration when developing nucleic acid delivery materials.

Ester groups are the most commonly used functional groups to enhance the biodegradability of biomaterials, but the in vivo degradation of other ester bonds may depend on the overall chemistry of the molecule and formulation. In certain tissues, the presence of ester functional groups can accelerate degradation of LNPs, potentially limiting protein expression. For example, OF-Deg-Lin was able to reach liver cells but selectively induced protein expression in the spleen. It was hypothesized that this could be due to the rapid degradation of LNP by liver enzymes. Rational design of degradable lipids or polymers based on polyester or polycarbonate can better control the degradation of the carrier.

It has proven more difficult to target specific organs or tissues other than the liver with LNPs. Some LNPs accumulate or are absorbed in the liver in an apolipoprotein E-dependent manner. In the case of APE, it has been found that the core structure of the tertiary amino alcohol of the polymer may play an important role in targeting specific organs. There are limitations to these targeting strategies, and rationally designed targeting approaches, such as the incorporation of ligands into tissue-specific receptors, typically from high-throughput screening to find successful targeting solutions for specific tissues, are alternative strategies.

Recently, in vitro active targeting of sialic acid-terminated glycoproteins on the surface of dendritic cells was reported. A 30-amino acid synthetic peptide with glutamic acid-alanine-leucine-alanine (GALA) is a complex conjugated to a polyplex carrying EGFP-nucleic acid. However, any opsonization of LNPs forming a protein corona can be detrimental to active targeting strategies due to masking of the targeting ligand. Therefore, new strategies targeting specific tissues need to be studied to solve clinical challenges.

In the field of delivering nucleic acid-based therapeutics to cells, the final endpoint is endosomal escape. ASO and siRNA delivery to the liver can be addressed by ASGPR-targeted GalNAc-siRNA conjugates, and mRNA and CRISPR delivery by LNPs appear to be promising, but for the delivery of effective and clinically prepared nucleic acid-based therapeutics to the whole body other than the liver. The approach is still at the final endpoint stage.

Although the exact reason is unknown, ASGPR has properties that are very suitable for the delivery of macromolecular drugs to hepatocytes. ASGPR binds to blood asialoglycoproteins and attracts them to the clathrin-coated endosome, where they enter the lysosome for degradation. ASGPR-bound asi-aloglycoproteins are not thought to escape into the cytoplasm.

However, hepatocytes express numerous ASGPRs on the cell surface, which cycle remarkably fast, at rates of every 10 to 15 minutes. A minimum of 5,000 siRNAs are required for a maximal RNAi response. GalNAc binding of ASGPR rarely causes membrane instability, which allows millions of siRNA molecules to enter the ASGPR endosome every 15 minutes, with escape efficiencies lower than 0.01%, resulting in over 5,000 molecules per day via GalNAc delivery. can transmit Unfortunately, no other ligand-receptor system exists that can express the receptor at such a rapid rate or cycle it rapidly into the endosome.

In fact, most cell surface receptors are expressed in the 10,000-100,000 range (or less), and caveolin and clathrin-mediated cell membrane entrapment are typically rotated every 90 min. Theoretically, a receptor-targeted delivery method could bring more than ~100,000 siRNAs to the cell's endosomes every 2 hours. However, if the endosomal escape efficiency is estimated to be less than 0.01%, it becomes difficult to reach the required threshold of at least 5,000. Currently, several research groups are working on solutions to the problem of endosomal escape.

The classic approach is to use chloroquine, a low-molecular endosomal degradation factor. Chloroquine (chloroquine) passively diffuses through the cell membrane and becomes a proton when the pH falls and is trapped inside the endosome, and the concentration in the endosome rapidly increases. Chloroquine inserts a hydrophobic motif into the lipid bilayer and dissolves endosomes at a certain concentration. However, at the concentration showing the effect, these endosome degrading factors non-specifically dissolve endosomes containing siRNA compounds as well as endosomes in other cells, thereby causing toxicity. As a result, these small molecule endosome degrading factors have received attention in the development stage, but present a potential toxicity risk for clinical use.

As an alternative endosomal escape approach, a two-molecule dynamic polyconjugate (DPC) system was developed as a method of limiting the degradation to endosomes containing nucleic acid therapeutics by conjugating endosomal degrading peptides or molecules directly to nucleic acids. siRNA binds to cholesterol to form a large aggregate structure (low-density lipoprotein) in the blood, moves to the liver, and is absorbed by hepatocytes by entrapment of the cell membrane. The second molecule is a derivative of the membrane-dissolving pore-forming melittin peptide (from bee venom). Melittin was synthesized with pH sensitivity and conjugated to GalNAc. At low pH of hepatocyte endosomes, melittin is activated and degrades endosomes to release siRNA into the cytoplasm. However, due to toxicity caused by melittin, it was decided to discontinue all three clinical programs that depended on melittin with the FDA. This point shows that solving the endosome escape problem is a difficult task.

II. Polyelectrolyte complex (PEC)

As a general model, the polyelectrolyte complex (PEC) is formed mainly based on the electrostatic attraction between oppositely charged polyions. However, the increase in entropy when counterions are released from the polyelectrolyte is the main driving force for PEC particle self-assembly.

PEC particles are often polydisperse systems with diameters between 10 nm and 1 μm. Particle size is usually measured by indirect techniques that maintain the integrity of nanoparticles in solution, mainly static and dynamic light scattering. Imaging techniques are often used to support the results of light scattering, but in this case the size of the PEC particles can be affected during sample preparation and analysis.

Self-assembly of PEC particles is a reversible process in which nanoparticles coexist in equilibrium with free polyions in solution. This dynamic nature of PEC particles intrinsically compromises their integrity upon dilution and addition of other charged compounds, leading to electrostatic shielding and multiple ion exchange from these nanomaterials. Consequently, the stability of PEC particles may be severely compromised in biological environments where high concentrations of small electrolytes and charged proteins are found. However, the PEC particles can be easily stabilized through crosslinking of the polyelectrolyte components, and in this way exchange with the free polymer in solution can be minimized. On the other hand, structural damage due to physiological electrolytes to PEC particles could be beneficial, for example, by increasing the permeability of these nanomaterials to cause release of preloaded cargo.

Finally, only certain polyelectrolytes mixed under appropriate conditions form dispersed and stable PEC particles. Several factors have to be optimized during the creation of these nanomaterials, especially the molecular weight, concentration and charge density of the polyelectrolyte, the pH and ionic strength of the (aqueous) medium, or the relative ratio and mixing order of the two polyelectrolytes. These parameters are usually interdependent and should be optimized in parallel. Often stable colloidal PEC particles are produced with high dilution and low ionic strength, and can be produced with more than one of the polyions, ideally with a significant mismatch in molecular weight with weak ionic groups on one of the polymers. Under different conditions, coacervates separate from solution and coalesce into high-density materials known as compact polyelectrolyte complexes, which also show great potential in some biomedical applications, such as synthetic cartilage and enzyme-activated biocomposites.

① Charge density, molecular weight and topology

The binding properties of multiple ions to charged compounds are strongly dependent on the density and number of charged groups in the polyelectrolyte structure. Due to the cooperative nature of polyelectrolyte bonding, polymers with higher charge densities show stronger bonding to charged compounds.

The simplest way to increase the charge density is to increase the molecular weight of the polymer electrolyte. Given the partially entangled morphology of the polymer in solution, the higher the molecular weight, the larger the polyelectrolyte core. The stability of PEC particles is affected by the number of charged groups per polymer chain (ie multivalent) and can form complexes that are stable beyond 'upper critical length' polyions. Nevertheless, significant differences in the molecular weight of polyions are often required for the colloidal stability of PEC particles.

Alternatively, the charge density can be "diluted" by adding neutral monomers to the polyelectrolyte. The destabilizing effect of NaCl on the PEC particles is greatly reduced with higher polyelectrolyte content. Nanoparticles made from pure polyelectrolyte are not affected by NaCl concentrations that are twice as high as those that cause the particles with weak charge density to expand. This result is because the direct correlation between the charge density and the binding force of the polyelectrolyte is strengthened.

The topology of the polyelectrolyte can have a significant impact on the charge density. Branched and dendritic polyelectrolytes are known to have increased binding affinity for charged surfaces compared to their linear analogues. This effect is due to the high spatial density of the monomers in the branched structure, which increases the binding force. As such, branched polyelectrolytes can electrostatically cross-link multiple counter-poly ion chains to create a stiffer polymer network. Under the same conditions, linear poly(ethylene imine) (PEI) formed polyplexes of 20-50 nm and left uncomplicated DNA fragments, while branched PEIs of similar molecular weight formed fully condensed DNA and much larger complexes (100 -200 nm). This is probably due to the condensation due to the higher bonding force than the linear PEI.

Although challenging due to their low charge density, PEC particles can be successfully prepared as small molecules containing specific motifs that aid in the nucleation process. Small cationic detergents with DNA self-assemble into lipid polyplexes or lipoplexes. There are dimeric detergents that can condense DNA despite low charge densities through hydrophobic interactions between aliphatic chains. After initial aggregation of C14CO with plasmid DNA, oxidative dimerization of this detergent to (C14CO)2 reduces the critical micelle concentration, increasing the packing and stability of the complex.

② Polyelectrolyte concentration

We rationalize the mechanism of PEC particle self-assembly into sequential nucleation of polyions into primary molecular complexes and then aggregate into colloidal complexes. Debye length increases as the nanoparticles grow larger, which explains this behavior, creating a stronger repulsive static between the "secondary" complexes. The size of the PEC particles can be fine-tuned with a combination of polyion length and concentration.

Often during assembly of PEC particles, high concentrations of the starting polyelectrolyte form micron-sized complexes with poor colloidal stability. As a result, dispersed PEC nanoparticles can only form in the polyelectrolyte mixture below the critical concentration, above which the system aggregates or precipitates (ca. <1 mg/mL). Concentration limitations during PEC particle preparation can be a limitation when it is necessary to prepare concentrated dispersions of these complexes. Nevertheless, the PEC particles can be centrifuged and redispersed, and the polydispersity of the complex can be purified through successive centrifugation cycles to ultimately achieve a single-mode size distribution without affecting the initial size.

③ pH

From a biomedical point of view, polyelectrolytes can be classified as weak or strong, whether they are partially or fully ionized at physiological pH. Consequently, unlike strong polyelectrolytes, the charge density of weak polyions is highly dependent on the pH (4.5–7.4) of tissues and organelles in the body. Changes within this pH range can lead to significant changes in polyelectrolyte binding and PEC particle stability due to loss or increase in valence (ie, charge per molecule). A good example of the pH-responsive behavior of multi-ion complexes is that at low pH (2.0–2.5), PAA is only deprotonated to a small extent, resulting in a more swollen and looser complex as a result of the lower charge density. However, at higher pH (3.5–5.5), the PAA chains become more ionized, increasing the number of electrostatic cross-links to form more complexes with PAHs.

Thus, weak polyions can alter the degree of (de)protonation and charge density with changes in biological pH. In addition, different chemical environments and stericities lead to different pKa values for the same monomer at different locations in the polyelectrolyte. This is especially the case for branched polymers, which translates into a large buffering capacity. As such, changes in pH found in various physiological settings were used to trigger release of drug and DNA from PEC particles by ionization changes of complex polyions. A classic example of this behavior is the so-called 'proton sponge' effect displayed by PEI, which allows polyplexes to exit acidic endosomes after cellular uptake). For example, branched PEI (B-PEI) has a wide buffering range of about pH 2 to 10, regardless of molecular weight. It can thus buffer the entry of protons into the endosome, ultimately rupturing the organelle, increasing the osmotic pressure releasing the polyplex into the cytoplasm.

Other relevant examples of pH-responsive PEC particles include enzymatic and antimicrobial delivery, which are based on swelling upon nanoparticle degradation or polyion (de)protonation, respectively. Reversible pH-responsive PEC particles can be prepared by mixing a weak polyacid (PAA) and a strong polybasic (PDADMA) in PDADMA-co-PAA. Mixing this copolymer with PSS at pH 4.1 formed PEC particles, which were dissolved at pH >5.7 due to deprotonation of PAA in the copolymer and subsequent repulsion between PSS and this partially anionic copolymer. When the solution was acidified again, the PIC particles were modified at pH 4.5-5.5, demonstrating the reversibility of the process.

Similarly, the pH chosen for self-assembly of the two polyions affects the charge density and thus may affect the final properties of the resulting nanoparticles. There is a mixing ratio dependent effect of generating more positive or negative PEC particles at more acidic or basic pH, respectively. This result is consistent with higher (de)protonation of polyions at higher pH values.

④ Ionic strength

The addition of a small electrolyte can aid the self-assembly of PEC particles by shielding the intramolecular charge repulsion of polyions, thus increasing the flexibility and self-assembly ability of the polymer. On the other hand, high salt concentration can lead to strong electrostatic screening between polyelectrolytes and inhibit the nucleation of PEC particles]. Therefore, successful assembly of PEC particles requires careful selection of the ionic strength of the medium.

In general, the size of the PIC particles decreased when the polyions were mixed in the presence of a higher concentration of NaCl, probably due to the better packing of the more flexible polyelectrolyte. The size difference found between the copolymers of water decreases at 10 mM NaCl to ultimately form particles of the same size at 100 mM NaCl. Overall, polyions with stronger charge density (i.e., lower NMVA content) were more resistant to NaCl during PEC particle preparation. In addition, particles prepared in pure water expand from 100 nm in diameter up to 1 μm when exposed to NaCl after assembly. This post-assembly effect of NaCl penetrates inside the core of the composite to neutralize the electrostatic cross-linking between the polyelectrolyte and subsequently allows the preformed composite to expand. The combination of swelling and subsequent aggregation of the expanded complex rapidly increases in size after addition of NaCl (less than about 10 min). Again, copolymers with higher charge densities (i.e., PDADMA content) exhibited improved resistance to the swelling effect due to NaCl due to higher binding force, and in some cases, swelling compared to less charged copolymers. It may be necessary to double the NaCl concentration to

Physiological electrolytes can also cause swelling and impair the function and stability of PEC particles in the biological environment. If flame-resistant particles are desired, the bound polyelectrolyte chains can be covalently cross-linked to hold the complexes together even after the weak electrostatic forces have been screened with salts. Nevertheless, the expansion of PEC particles with small electrolytes was exploited to release the entrapped drug within the complex as a result of increased permeability.

⑤ mixing ratio

It is the ratio of positive and negative charges in the mixed polymer electrolyte. The mixing ratio affects not only the final size and charge of the PEC particles, but also the biological activity. Since the degree of ionization of a polyelectrolyte is pH dependent, the 'effective' mixing ratio is different for the same polyion mixture at different pH values. Mixing ratio is expressed as the ratio or ratio of acidic and basic monomers in solution, regardless of pH (e.g. 'N:P' ratio is usually used to indicate the 'N' number of amine monomers complexed with 'P' phosphate. used in polyplex formulations).

Non-stoichiometric mixing ratios are required to provide the colloidal PEC particles with an excessively stabilizing shell of polyions. Based on this, the absolute value and fluctuation of the ζ potential of the PEC particles can be predicted from the change in the mixing ratio as long as other variables such as pH, ionic strength and polyion structure are kept constant.

Given the strong dependence of numerous PEC particle systems on mixing ratios, ratio ranges should always be evaluated in order to draw consistent and representative conclusions of PEC particle behavior. The range of mixing ratios generally evaluated varies for mixtures that are highly positively charged in polyplexes (from N:P up to 50), but other systems have values closer to equimolarity, e.g. 0.2- 1.8 or 0.5-2.0 in n-/n+).

The formation of PEC particles from a mixture of chitosan and dextran sulfate is twice the size above and below equimolarity. First, the absolute charge of the complex is the charge of the excess polyion. Second, the size of nanoparticles is distributed in three ranges of [NH3 +]/[SO4-] ratio: more than 10 (420 nm), 10 to 1 (250 nm), and less than 1 (200 nm). These three particle sizes are due to the different nucleation mechanisms occurring in excess of the two polymers. At near-neutral mixing ratios, unstable complexes form and aggregate. Also in excess of polyions (more than 5 times), more than 70% of this polymer remains uncomplicated in solution. Comparing this with other examples, the mixing ratio has the same effect on the ζ potential of different PEC particles, but the effect on size is not consistent and is affected by other variables (e.g. molecular weight and polyion strength).

⑥ Mixing order

The order in which the polyelectrolyte was mixed together revealed what the excess ions were at the beginning of the assembly process. It is very important to note that when polyanions are added to polycations for the preparation of non-stoichiometric PEC particles, for example at [n +/n-] = 2, there is always an excess of positive charge in the complex. The repulsive forces between them help maintain colloidal stability. However, if the same polymer mixture is prepared by adding polycations (in excess) to polyanions, the negative charge of the initial coacervate will reach an intermediate value through addition of polycations. As a result, if the polyelectrolyte is mixed in the wrong order, neutral particles may agglomerate. Therefore, the scarce poly ions may be added to the excess ions, resulting in different nucleation kinetics depending on the properties of the polyelectrolyte.

The effect of the order of addition becomes apparent when titrating the mixing ratio of the polyelectrolyte. For example, the mixing order of the polyelectrolyte not only affects the size of the nanoparticles, but also changes the size up to a factor of two, as well as the stability of these complexes to NaCl. This is probably due to the different packing of the polymer. showed somewhat different results, finding strong dependence or almost negligible effects on the final form of the complex. Discrepancies between studies should be attributed to the influence of other variables (e.g. molecular weight and multiple ionic strength), so each case should be evaluated individually.

⑦ PEC particle crosslinking

PEC nanoparticles, particularly low molecular weight polymers, partially ionized polyions, or nanoparticles prepared when exposed to high salt concentrations, equilibrate with free polyions in solution. This equilibrium can be displaced towards the release of polyions from the nanoparticles in a situation that weakens the electrostatic bonding. As a result, PEC particles can degrade when diluted or exposed to other charged compounds.

Alternatively, the stability of the PEC particles can be greatly improved by covalently crosslinking the composite polyions to prevent dissociation of the polymer chains from the nanoparticles. In essence, crosslinking works by 'polymerizing' the polyelectrolyte chains, greatly increasing the overall molecular weight of the polymer, which in turn increases the binding affinity. To this end, the polyelectrolyte can be crosslinked either during or after particle self-assembly, depending on whether the polyelectrolyte has crosslinking groups in its structure or whether a third component is added to the system that crosslinks the pre-assembled composite.

Illustrative examples of PEC particles after crosslinking are nanoparticles from their core and/or crosslinked poly(acrylic acid) (PAA) and poly(l-lysine) (PLL). The amines or carboxylic acids found in the shells of these PEC particles were cross-linked using genipin or cystamine, respectively. The PLL chain of the core is crosslinked by silica deposition using orthosililic acid as a precursor. These cross-linked particles can withstand up to 150-fold dilution.

Crosslinking after assembly of PEC particles has the advantage that it does not require the functionalization of polymers with crosslinking groups, and therefore is applicable to almost any polyelectrolyte with suitable functional groups (eg amines or carboxylic acids if described). Given the wide application of polyamines in gene delivery, other amine crosslinking agents, including glutaraldehyde, citric acid, carboximidates, and N-hydroxysuccinimide esters, are applicable for polyplex fabrication.

Alternatively, the functionalized polyelectrolyte cross-links when the PEC particles are formed. By far the most used group for functionalizing and crosslinking polyions in PEC particles is thiols with mild reaction conditions and reversible crosslinking with disulfide bridges. For example, one can make disulfide crosslinked polyplexes from plasmid DNA and PLL decorated with cysteine residues. Upon exposure to reducing conditions, the disulfide bridges of these polyplexes are broken, resulting in a sudden decrease in the apparent molecular weight of the PLL, initiating the degradation of nanoparticles and release of DNA.

Crosslinking short polyions through labile crosslinking agents can produce stimuli-responsive PEC particles, which can be degraded in the presence of agents that degrade these labile linkers, thus resulting in abrupt charge density and bonding between polyions. It can cause a loss of affinity. Thus, the reducing conditions found in the cytoplasm were utilized to trigger intracellular drug and nucleic acid delivery through the reduction of disulfide cross-linking vehicles.

By combining two different labile linkers, we developed a dual-stimulatory PEC particle that degrades in the presence of an enzyme and a reducing agent. In this system, PEI formed a complex with a short anionic peptide containing two cysteine residues, allowing oxidative crosslinking (P1SH, Fig. 10), giving the PEC particles the same redox properties as in the previous example. Furthermore, the amino acid sequence of these peptides can be specifically cleaved by bacterial elastase (LasB), thus providing another point of cleavage of the cross-linked peptide chain and ultimately the PEC particle itself.

The cross-link integrity of these particles is paramount to ensure particle stability, but at the same time unstable cross-links can be designed into sacrificial groups that cause particle degradation and release of cargo upon cleavage.

Ⅲ. Polyelectrolyte Multilayer (PEM)

Both synthetic and natural polyelectrolytes have been used as building blocks for layer-by-layer (LbL)-based nano and micromaterials. The main advantage of using synthetic polymers is the ability to tune several parameters over a wider range (eg ionic strength and assembly pH) and the ability to tune resistance to more physical and chemical stresses. It can also be easily modified chemically.

In vivo, natural tissue consists of cells embedded in an extracellular matrix composed of proteins, polysaccharides and other bioactive molecules such as growth factors. Therefore, a closer step to reproducing the original tissue with which cells interact in vivo is to use extracellular matrix components as components of the film. Natural polymers have shown great potential in the field of biomedical science, given their structural similarity to natural extracellular matrix and their biodegradable properties. Polysaccharides are of particular interest because of their high hydration, high biocompatibility, and often biodegradation. It can also be easily processed into PEM films.

LbL PEMs are being used in biomedical applications including biosensors, drug delivery, biomaterial coatings and tissue engineering. In addition, colloidal templates or support materials can be used to prepare various types of LbL-based objects, including individually coated cells, encapsulated cell aggregates, hollow capsules, or free-standing membranes. In the latter case, the underlying substrate (colloidal template or support material) must be removed, which is a difficult step as defects may be introduced or the roughness may be altered.

Methods for preparing multilayer polyelectrolytes (PEMs) by sequential deposition of oppositely charged polyelectrolytes include user-friendly preparations in the surrounding environment and physiological conditions, the possibility to incorporate various biomolecules, and fine control and production of PEM structures. Due to its robustness, it has received a lot of attention. This nanoscale control can be achieved simply by changing deposition conditions such as pH, ionic strength, polymer functionality and charge density. Polymer concentration also plays an important role in film growth. In particular, combining pH-amplified growth with changes in polyelectrolyte concentration may produce very thick assemblies with minimal deposition steps.

Complexing of transfection reagents with nucleic acids for better stability is helpful in the approach of substrate-mediated transfection. These complexes can be prepared in large quantities by coating, so that they serve to transport active ingredient nucleic acids. The use of PEM is a promising approach in the field of substrate-mediated active substance delivery. The ability to integrate drugs into PEMs is related to nucleic acid release and desired function of this system. A simple technique for PEM deposition is a layer by layer (LbL) technique, in which a polymer electrolyte can be deposited alternately on a substrate. Alternating positively and negatively charged substances consist of electrostatic interactions that stabilize the structure.

Good for cell-biomaterial interactions in many PEM tasks, such as cationic polyelectrolytes such as poly(L-lysine), chitosan, poly(ethylenimine) (PEI) and anionic polyelectrolytes such as alginic acid, hyaluronic acid, polyacrylic acid Among cationic polymers, PEI is widely used for complexing negatively charged nucleic acids because it forms non-covalent bonds with a strong cationic charge density. By complexing nucleic acids, limiting factors of nucleic acid transfection such as degradation by nucleases and stimulation of the immune system by negative charges can be avoided. PEI has nitrogen at every third atom, so it can release protons from the endosome. Due to their buffering capacity, chloride anions flow into the endosome, causing an influx of water and consequently osmotic expansion. This is due to the so-called *?**?*proton sponge effect*?**?* causing endosomes to rupture and release the PEI-nucleic acid complex into the cytoplasm.

Selection of molecular weight, chemical structure, and phosphate/nitrogen (N/P) ratio is important for successful transfection. The N/P ratio is defined as the molar ratio of amine groups in cationic polymers to phosphate groups in DNA or RNA and is important for efficient gene delivery. Ratio is an important measure for cell experiments as it determines the size of the particles for transfection.

Hyaluronic acid (HA) is a negatively charged glycosaminoglycan that occurs naturally as in the extracellular matrix, synovial fluid, umbilical cord, and blood. Biopolymer properties such as biocompatibility, biodegradability and no immunogenicity make hyaluronic acid a suitable material for wound healing, knee osteoarthritis treatment or tissue engineering scaffolds. Important for this is the molecular weight of hyaluronic acid, which is responsible for the properties of the polymer. High molecular weight hyaluronic acid has an anti-inflammatory effect upon tissue damage, while low molecular weight hyaluronic acid activates pro-inflammatory chemokines and cytokines. Although HA is not a typical transfection reagent due to its negative charge, it is frequently used for nucleic acid delivery in conjunction with chitosan or poly-L-arginine. Combining HA with the transfection reagent PEI has the great advantage that HA can reduce the cytotoxic effect of PEI.

1. LbL Assembly Hollow Capsule

Micrometer- and nanometer-sized capsules made of multilayer polyelectrolytes (PEMs) have been the subject of intensive research because of their importance in biotechnology and nanotechnology for applications in chemistry, physics, biology and medicine. Multilayer capsules are prepared by alternately depositing anionic and cationic PE onto a sacrificial colloidal mold and then dissolving the core. Methods for preparing core-shell particles and multilayer hollow capsules via LbL assembly by deposition of LbL on colloidal templates, core dissolution and drug encapsulation into LbL assembly capsules have been systematically and extensively studied.

The advantage of LbL-assembled capsules lies in the diversity of interactions (eg, covalent and non-covalent) between the polyelectrolyte (PE) used to manufacture the capsule and its properties. The multilayer showed selective permeability because it was permeable to low molecular weight compounds and not to larger macromolecules. The size of the multilayer hollow capsule can be easily controlled by changing the size of the sacrificial template. The shell thickness can be changed according to the number of layers and preparation conditions, so that the thickness and shape can be controlled. In particular, these capsules have better encapsulation efficiency and stable zeta potential, mainly due to the chemical properties and assembly conditions of the polymers used. Successful permeability changes for encapsulation and release appear through changes in pH or ionic strength of weak PE systems. The chemical properties of the polymer used, along with the type of core construction, define the mechanical properties of the hollow capsule, such as elastic modulus, toughness, strength and rigidity. Manufacturing conditions such as polymer type (e.g. thicker layer is formed by PE with lower charge density), concentration of polymer solution (higher concentration results in thicker walls), ionic strength and pH also the type of interaction of the capsule, shell thickness and permeability.

Encapsulation of various macromolecules inside hollow capsules is accomplished by tuning the physicochemical properties of polymers to manipulate shell permeability. However, most of the work uses a strong PE such as polystyrene sulfonate (PSS) as one of the polymers, so perturbation of intermolecular forces (e.g. covalent bonding, hydrogen bonding, and electrostatic interactions) occurs to release the payload. . It is essential to protect capsule stability. Due to this, various environmental triggers such as pH, ionic strength, polarity and temperature were observed, which played an important role in manipulating capsule permeability by modulating the shell interaction force.

Easier and more successful encapsulation of different molecules/compounds into capsules shows the potential of multilayer capsules as drug delivery vehicles. They can encapsulate all kinds of substances and/or molecules ranging from enzymes, nucleic acids, peptides, proteins, therapeutics, biomolecules, fluorescent molecules and nanoparticles into hollow pores. This can be achieved in a number of ways by using the material itself as a template, incorporating the material into a core template, or encapsulating the drug in a preformed capsule by using an environmental trigger to control the permeability of the capsule. These elements, along with several features introduced into the shell, are also used to release encapsulated payloads in a controlled way. Incorporation of functions such as organic molecules, NPs, fluorescent dyes, polymers, nanotubes, and other biomolecules into PE multilayers during manufacturing allows easy control of the shell's internal structure, mechanical properties and permeability, allowing the loaded cargo to remain exposed to external triggers. emission can be induced.

Thereafter, the payload may be released under exposure of external triggers such as laser light, ultrasound, magnetic field, enzymatic modification, and mechanical modification. In the latter case, the capsule ruptures irreversibly, releasing the loaded molecules in an explosive or sustained manner. Weak PE exists in an ionized form only in a specific range of pH or ionic strength, so the open and closed state of the multilayer capsule can be easily controlled with various pH or ionic strength, which is a widely used concept in in vivo applications. , polynucleotides, lipids and polysaccharides have been studied a lot because they are biodegradable under normal physiological conditions through enzyme and pH-induced degradation.

To date, robust PE capsules have been widely used in a variety of practical applications, from loading and controlled release of therapeutics following minor changes in environmental properties, inhibition of interchain interactions, to degradation/rearrangement of LbL films under the action of physical factors. is being used Despite the fact that weak PE systems can provide many of these benefits, there are only a few examples. For example, reactivity to immediate environmental changes (eg, pH, ionic strength, and temperature) occurs because weakly charged groups are in physical proximity to each other. The effect of PE density can be particularly pronounced when there are many charged groups in the system. The use of different charge groups for LbL assembly allows the residual charge of the multilayer film to play an important role in manipulating polymer/polymer interactions, giving the LbL system a unique reactivity, allowing for easy engineering of properties.

2. LBL based film

Currently, many technologies such as chemical vapor deposition (CVD), atomic layer deposition, colloidal assembly, and molecular beam epitaxy have been developed for manufacturing nano-thick multilayer thin films. However, the layer-by-layer assembly method is a technology that can be freely adjusted at the nanoscale, does not limit the properties of various materials, and can be made simply without heat treatment or strong stimulation. unique The layer-by-layer lamination method forms a thin film with a thickness of me through the process of stacking materials with mutual attraction such as electrostatic attraction, hydrogen bonding, van der Waals force, and hydrophobic bonding. Nano-thin films manufactured by the layer-by-layer method have no material limitations and are being actively studied in various fields such as cell culture, drug delivery, catalyst, and sensing.

In general, there are a dip-coating method, a spin-coating method, and a spray-coating method for performing a layer-by-layer lamination method. The dip-coating method is a method of coating a functional material on the surface of an object by sequentially putting an object in a solution, and it is a method developed for the first time. In the spin-coating method, a substance dissolved in a solution is adsorbed to the surface of an object by a force rotating at a constant speed to make a nano-thin film. The spray-coating method is a method of coating droplets by spraying a solution from a nozzle to adhere to the surface of an object.

These existing technologies for manufacturing functional nano-thin film technology have advantages and disadvantages. In the case of the dip-coating method, there are almost no restrictions on objects and materials, and no special equipment is required, so the cost is very low. However, since it takes a long time and the object and the solution are highly likely to be contaminated by the intensive contact between the object and the solution after the manufacturing is finished, it is difficult to preserve the properties of the material, so it is difficult to reuse it. The spin-coating method distributes the solution evenly by centrifugal force and can produce a nano-thin film at a faster speed. The disadvantage is that it requires equipment to generate centrifugal force, and it is difficult to manufacture nano thin films on a large area or in large quantities, and expensive equipment to generate greater centrifugal force is required for mass production. In the spray-coating method, a large area can be sprayed using equipment that can spray the solution. However, the pressure when sprayed from the nozzle is dispersed, so the size of the droplets is not constant and an even coating may not be achieved. In addition, these representative existing methods can provide various functionalities such as thickness, material, and roughness vertically at the nano level, but have a common disadvantage in that it is difficult to provide various functionalities horizontally.

In order to maintain the advantages of existing methods and minimize disadvantages such as long manufacturing time, wastage of materials, and difficulty in imparting horizontal functionality, the present invention combines the layer-by-layer stacking method with inkjet printing technology to create a two-dimensional object on the surface of the object. It is possible to manufacture nano-thin films capable of fast time, minimization of material use, and horizontal functionality.

LBL assembly was first described by Decher in the 1990s as an alternative method to LB (Langmuir-Blodgett) assembly for thin film fabrication. LB assembly has been extensively reviewed, allowing the fabrication of multilayers using predominantly amphiphilic molecules with alternating head-to-head/tail-tail configurations. Despite the ability to produce multilayer films, the technique itself is time consuming, requires expensive equipment to perform, and is limited by the types of molecules available. The advent of LBL assemblies has overcome many of these limitations and has resulted in a popular approach to multilayer assemblies.

Using this method, a multilayer polymer electrolyte (PEM) is fabricated by sequentially and alternately adsorbing negatively and positively charged polymers (polyelectrolyte, PE) onto a charged substrate. Each monomer unit of PE has a charge, creating a multi-charged molecule capable of electrostatic interactions. Although less common, other interactions may lead to PEM formation, including hydrogen bonding, coordination chemistry, and hydrophobic interactions. Thus, LBL assembly provides a simple method of PEM preparation without the same material limitations as LB assembly, which can be performed with minimal equipment while maintaining nanoscale control over film preparation.

PE can be divided into a group with a permanent charge in solution (strong PE) and a group whose charge is highly dependent on pH (weak PE). The charge density based on the number of groups that make up the repeating monomer units also affects the possible interactions between PEs. The properties of PE (i.e., charge density, molecular weight, weak vs. strong) have a significant impact on the behavior of the films produced. LBL films are held together mainly by electrostatic interactions. The charge in the film is neutralized either intrinsically or extrinsically. Intrinsic charge compensation refers to the pair of charges between PEs, and the charge neutralized by the external compensation is paired with a counter ion in the surrounding solution. A fixed charge is a charge in the film that cannot pair with the charge of another PE due to the constraints imposed on the film structure (i.e. stereo). They rely on neutralization through external rewards.

Early models of PEM growth show a linear growth system in which the increase in mass and film thickness is proportional to the number of deposited bilayers (deposition steps). Indeed, there are many examples of linear growth films comprising synthetic and natural PE, including poly(sodium styrenesulfonate) (PSS)/poly(aliamine hydrochloride) (PAH) and chitosan (CHI)/cellulose nano whisker films. In this film, distinct lamellar morphology is seen within the film, as each bilayer interacts with only directly adjacent bilayers (above or below) with little interpenetration. However, PEM using PLL and hyaluronan (HA) is a proven film system that does not have a linear growth mechanism. Rather, the film thickness increased exponentially with each deposition cycle. This type of growth is due to a diffusion model in which at least one PE can migrate in and out of the film during build-up.

In general, films grown by this mechanism are composed of natural polymers. However, linear and exponentially growing films are interrelated. The experimental conditions under which the film is constructed (i.e., temperature, pH, ion concentration) play a large role in the resulting interaction strength between the relevant PEs, which in turn influences the growth mechanism, density and strength of a given film. These parameters are as important as the properties of the PE itself (ie, weak vs. strong, molecular mass, 2° interaction) to determine the film behavior. Linearly grown films tend to form high-density film structures in favor of high-charge PE and low ionic strength conditions where intrinsic charge compensation predominates. However, as the ionic strength increases, the growth mechanism can be altered to exhibit exponential behavior. This means that PSS/PAH films composed of 1M NaCl grow exponentially while films composed of 0.15M NaCl grow linearly. The opposite was also observed. By reducing the ionic strength, it is possible to revert the characteristic exponential growth to a linear growth. For example, CHI/HA films grew linearly at 10 ⁻⁴ M NaCl but exponentially at a concentration of 0.15 M NaCl. However, films showing exponential growth tend to be unstable at low ionic strengths and there is a structural impact on deposition under these conditions. For example, CHI/HA films deposited in 10 ⁻⁴ M NaCl cannot form continuous films and remain island-like.

There is a direct method to greatly amplify the exponential growth of multilayers by alternately depositing PEI at high pH and poly(acrylic acid) (PAA) at low pH. Alternating pH changes the degree of ionization of the polyelectrolyte in the multilayer, enhancing the diffusion of PEI into and out of the film, increasing the deposited mass per cycle. The synergistic action of pH-tunable charge density and diffusivity of weak polyelectrolytes provides a novel method for the enhanced growth of multilayers with hierarchical micro- and nanostructured surfaces.

Polyelectrolyte multilayers were constructed by alternately depositing PEI at high pH and PAA at low pH. Measurements of increases in film mass and thickness at different pH values to quantify the effect of pH on the growth rate of multilayers show the amplification of film growth with increasing pH differences in multielectrolyte solutions. Films prepared at pH 7.2 for both PEI and PAA grow linearly with the number of depositions, as expected. Mass and thickness increase nonlinearly when multilayers are prepared in PEI 8.0/PAA 5.0. The nonlinearity of film growth becomes more pronounced as the pH difference increases.

In addition, the exponential growth mechanism of multilayer films depends on the diffusion of PEI molecules into and out of the multilayer film during each deposition cycle. Polyethylene imine (PEI) and poly(acrylic acid) (PAA) are both weak polyelectrolytes whose charge density can change with the pH of the solution. Alternating exposure of the entire film structure to high pH (in PEI solution) and low pH (in PAA solution) enhances the uncompensated concentration for polycations/polyanions within the multilayer. Therefore, the PEI in the high pH electrolyte solution diffuses into the film to compensate for the bulk charge as well as to compensate for the reversal of the surface layer. Upon further contact of the multilayer with PAA at low pH, protonated PEI with uncompensated bulk charge diffuses to the interface and neutralizes it. Since pH affects the charge density of the overall film structure, the uncompensated amount for the polyelectrolyte is controlled by the uncompensated concentration for the bulk charge proportional to the total thickness of the film. Multilayer growth is accelerated due to the synergistic action of the pH-tunable charge density and the diffusivity of the weak polyelectrolyte.

pH-amplified growth of PAA/PEI multilayer films by alternately depositing PEI at high pH and PAA at low pH (a) pH-amplified exponential growth of PAA/PEI negatively charged, PAA-terminated film starts with (b) Immersion of the film in a high pH PEI solution deprotonates the multilayer film, making the charge negative. This diffuses PEI into the film and (c) does not compensate for polycations remaining in the film after rinsing. (d) Immersion of the multilayer film in a low pH PAA solution protonates the multilayer. This causes PEI to diffuse out of the film and uncorrected PEI to react with the PAA at the film/solution interface. (e) Finally, a negatively charged PAA-terminated surface is generated. Alternating exposure of the complete film structure to high pH (in PEI solution) and low pH (in PAA solution) enhances the uncompensated concentration of polycations/polyanions within the multilayer, amplifying growth per deposition cycle.

The LBL film is created through an iterative procedure in which the substrate is immersed in a polyelectrolyte solution of opposite charge with an intermediate rinse step to prevent cross-contamination between solutions. Although many studies have focused on the influence of various conditional parameters such as pH, temperature and ionic strength, no work has been done to determine the effect. In addition to the intermediate rinse steps for film growth, there are many cases of linear to exponential transitions as a result of the experimental conditions, but few cases of the opposite in the early stages of deposition (within the first 10–15 layers) have been reported. The integrity and continuity of the film are not affected.

Multilayer DNA coatings with different cations have been developed and used as polymers for use as implant coatings for biomedical applications. 300-bp DNA can be combined with poly (allylamine hydrochloride) (PAH; MW ~ 70 000 D) or poly-D-lysine (PDL; MW 30,000-70 000 D) to form substrate [PDL/DNA] 5 or substrate [PAH/ DNA] to form 5 layers. That is, the coating always starts with the polycationic polymer in contact with the substrate and ends with the top layer of DNA. The DNA content of the coating is about 3 μg/cm ² per bilayer and is independent of the substrate (quartz, silicon, titanium). This titanium coating enhances the formation of precipitates of hydroxyapatite carbonate in Simulated Body Fluid (SBF). PAH-containing coatings provide full hydroxyapatite coverage, whereas uncoated substrates and PDL-containing coatings only produce dot-like deposits. In both cases, preformed coatings from hydroxyapatite and implant surface treatment to enhance hydroxyapatite deposition under physiological conditions are widely used to enhance the biological performance of implant surfaces for bone contact. These coatings have the ability to bind bones. Similarly, a bi-layer system with a bisureido surfactant as the base layer and 300 bp DNA as the top layer on ultra-high molecular weight polyethylene and polycaprolactone, widely used in biomedicine, increases crystallization.

For biomedical applications, cp-titanium was coated with a multilayer film of a cationic polymer and poly-L-lysine (PLL), a fish sperm DNA. A system using PLL as the top coating was found to enhance bovine serum albumin (BSA) adsorption and reduce the particle size of hydroxyapatite crystals when exposed to SBF. All coating systems enhanced the deposition on calcium phosphate during SBF exposure.

Salmon testis DNA 300bp or 7000bp and protamine sulfate were bound to the titanium surface, bound to a 10-layer coating, starting with protamine immobilized on Ti and ending with the topmost layer. The LbL approach was applied to the surface engineering of poly(D,L-lactic acid) films using 2000bp salmon DNA and chitosan as anionic and cationic polymers. Starting with the initial PEI layer for coupling, a minimum of 5 bilayers were required for full substrate coverage. This 5-ply layer was completely decomposed in 5000 U/mL of lysozyme solution at 37°C within ˜30 hours.

LbL multilayer coatings using DNA as polyanionic component not only allow immobilization of biologically active molecules, but also can tune the surface charge and surface energy of the surface of biomaterials. This is of high interest as negatively charged surfaces and surfaces with enhanced surface energy generated by other approaches have been shown to improve the healing process and osseointegration. It is worth mentioning that the polyvalent cationic component is mainly used as a means of DNA immobilization.

3. Free standing film

LbL PEMs are being used in biomedical applications including biosensors, drug delivery, biomaterial coatings and tissue engineering. In addition, colloidal templates or support materials can be used to prepare various types of LbL-based objects, including individually coated cells, encapsulated cell aggregates, hollow capsules, or free-standing membranes. In the latter case, the underlying substrate (colloidal template or support material) must be removed, which is a difficult step as defects may be introduced or the roughness may be altered.

To date, the development of free standing films has mostly focused on synthetic or nanocomposite films that are transparent, mechanically robust and/or electrically conductive. Thermal properties were also studied.

To date, studies on free-standing (FS) films made entirely of polysaccharides are still lacking. The difficulty of the separation step arises from the lack of cohesion and manipulation of these membranes. We succeeded in fabricating an ultra-thin FS chitosan/alginate (CHI/ALG) membrane of 2.9 nm per layer pair, which should be supported by a water-soluble poly(vinyl alcohol) membrane for manipulation, and used it to repair visceral pleural defects in dogs. For the separation step of the polysaccharide film, they studied various experimental conditions by changing the number of layer pairs, deposition time and polymer concentration, and built CHI/Hyaluronan (CHI/HA) films on low-energy substrates. Only one experimental condition was able to produce a separable and robust ~3.5 μm thick membrane. An additional crosslinking step was required to make this membrane stable in physiological buffers.

To produce thick polysaccharide membranes (in the tens of μm range) that can be used for drug delivery and tissue engineering. Biocompatible (CHI/ALG) FS membranes made of 25-200 layer pairs, with corresponding dry thicknesses of 4-35 μm, were prepared by separation from the underlying inert substrate without any post-treatment steps. CHI and ALG were chosen for their abundance and variety. CHI is a cationic polysaccharide obtained from the N-deacetylation of chitin, an abundant polysaccharide found in crustacean shells. ALG is extracted from algae. It is a very versatile and adaptable material with tunable physicochemical properties. CHI and ALG are already widely used in biomedical applications and are typical components of wound dressing materials. ALG is also used for cell encapsulation.

FS membranes made of PEM films were isolated from the basal inert substrate and were stable in physiological buffers without any post-treatment steps. It also allowed penetration of the model drug.

Ⅵ. Multilayer Polyelectrolyte Nanoparticles

The present invention homogenizes a polyelectrolyte complex (PEC) and a polyelectrolyte multilayer (PEM) comprising an active ingredient, a cationic compound, and an anionic compound and formed by electrostatic interaction between them, and It relates to polyelectrolyte multilayer nanoparticles (PEMN) encapsulated in minutes.

The pharmacologically active ingredient may be selected from metals, small molecule drugs, proteins, growth factors, peptides, antibodies, nanoparticles and nucleic acids. Preferably, the pharmacologically active ingredient may be an anionic nucleic acid.

Over the past decade, research on nanoparticles (NPs) applied in the medical field has increased significantly, from inorganic to organic systems. In the case of inorganic nanoparticles, the commonly reported systems correspond to metal nanoparticles (silver, gold, zinc oxide, iron oxide, etc.). (I) metal nanostructures (II) metal oxide nanostructures and (III) metal nanoalloys are classified into three groups that can be produced by chemical, physical, and biosynthetic methods. On the other hand, there are various types of organic NPs, with dendrimers, self-assembled systems (micelles and liposomes) and polymer nanoparticles being the most used and reported systems.

With respect to polymeric NPs, they are commonly used in biomedical fields due to their properties of compatibility, biodegradability and low cytotoxicity, and natural polymers such as chitosan, modified starch and natural gums are very common. However, the development of polymer NPs is not limited to biopolymer systems because they can also be obtained from biocompatible synthetic materials. It is mainly used for polylactide (PLA), polycaprolactone (PCL), polyglycolic acid (PGA) and acrylate-based polymers. Similarly, polymer NPs are classified according to their structures, and the most common ones are nanospheres, nanocapsules, polymer nanoconjugates, and polyelectrolyte complexes (PECs).

Regarding PEC, they can be obtained in several ways, one of the most used is the traditional polyelectrolytic complexation process (bottom-up method). This technique is based on spontaneous interpolymeric aggregation given by electrostatic attraction when cationic and anionic polyelectrolytes are mixed in aqueous solution. The preparation of this polymer electrolytic composite is in principle an easy-to-perform methodology. However, it can also be vice versa, as it depends on several internal and external variables. The internal variables are the polymer's chemical properties, molecular weight, and fractional charge. External variables depend on many dissolution medium conditions (temperature, pH, or ionic strength) as well as many process conditions such as polyelectrolyte ratio, mixture volume, rate and order of addition and stirring rate during polyelectrolyte complexation. These factors lead to the fact that it is difficult to obtain polyelectrolyte composites, especially in the nanometer size. Therefore, standardization and extension of PEC obtained through bottom-up methods is a great challenge to perform difficult experiments.

On the other hand, other methods (top-down method) that can be used to obtain polyelectrolyte multilayer nanoparticles (PEMN) include sonication treatment, extrusion and high pressure homogenization (HPH: It is a means to homogenize PEC and PEM by applying high energy by High Pressure Homogenization) and Ultra High Pressure Homogenization (UHPH).

However, in this top-down method, chemical degradation of the material by the excess energy applied during the homogenization process is one of the several disadvantages of this method. Nevertheless, the greatest advantage of this technique is that the implemented conditions can be easily reproduced and scalable at the industrial level. Therefore, the NP technology developed in this way can be easily transferred to the industrial site. In addition, one of the main problems encountered in traditional bottom-up polyelectrolytic complexation is the PEC system with large particle size and high polydispersity. This can be improved by supplementing the existing top-down PEMN manufacturing method with a top-down homogenization method using high energy including sonication treatment, extrusion, high pressure (HPH) and ultra high pressure (UHPH).

Ultrasonic treatment and/or extrusion through a screen membrane at high pressure, high pressure and superpressure can be used to produce a homogeneous PEMN suspension from the PEC and PEM prepared in the present invention. did. With sonication in PEMN formation, large PEMs are broken, the PEMs can entangle and/or disperse, the surface can be activated or functionalized, and the PEM can be emulsified.

In this processing step, high-power ultrasound has a strong effect. When sonicating suspensions at high intensity, sound waves propagating into the liquid medium frequently alternate high-pressure (compression) and low-pressure (lean) cycles with frequency. During the low-pressure cycle, high-intensity ultrasound creates small vacuum bubbles or voids in the liquid. When the bubble reaches a volume where it can no longer absorb energy, it collapses violently during the high-pressure cycle. Larger particles can be disrupted by surface erosion (via cavitation collapse of the surrounding liquid) or particle size reduction (due to collisions between particles or collapse of cavitation bubbles formed on the surface). This results in rapid acceleration of diffusion, mass transfer processes and solid-state reactions due to crystal size and structure changes.

Polyelectrolyte multilayer nanoparticles (PEMN) comprising an active ingredient, a cationic compound and an anionic compound according to the present invention, preferably, a layer of a complex comprising a nucleic acid having a negative charge and a cationic compound through an electrostatic interaction and an anionic polymer It is a composite particle including an outer layer.

In the present invention, PEMN comprises the steps of (a) an anionic nucleic acid, a cationic compound, and an anionic compound as active ingredients, and preparing PEC and PEM by electrostatic interaction between them; and

(b) homogenizing the prepared PEM to prepare a PEMN in which the nucleic acid is encapsulated.

The preparation of PEC and PEM of the present invention is performed by adding the anionic compound to a suspension solution in which the anionic nucleic acid and the cationic compound are continuously vortexed in the reaction solution for 30 seconds and continuously vortexed for 30 seconds or / and ultrasonic waves After the reaction was completed by treatment, the first method of stabilizing at room temperature for 1 hour was used.

The particles prepared by self-assembly by mixing the selected nucleic acid, cationic compound and anionic compound were referred to as PEM. One or more types of the nucleic acid, cationic compound, and anionic compound may be selected for each, and PEM may be prepared by mixing the selected compound.

The second method was used in which the anionic nucleic acid, the cationic compound and the anionic compound were continuously vortexed and/or sonicated for 30 seconds in the reaction solution to complete the reaction and then stabilized at room temperature for 1 hour.

A third method for preparing an eluted membrane was used by layer-by-layer technology in which anionic nucleic acids, cationic compounds and anionic compounds were alternately stacked on a selected substrate.

The PEMN prepared in the present invention may be a polymer-based nanoparticle or a hydrogel. The present invention is not limited thereto.

In the present invention, (a) a suspension solution containing core particles prepared by electrostatic interaction between the anionic nucleic acid and a cationic compound as an active ingredient in a mixed suspension solution, and an anionic compound by mixing the anionic compound in the suspension solution PEM formed by electrostatic interaction between particles and the anionic compound;

(b) PEM formed by electrostatic interaction between anionic nucleic acids, cationic compounds and anionic compounds as active ingredients in a mixed suspension solution; and

(c) the active ingredient anionic nucleic acid, cationic compound, and PEM prepared and eluted by a layer by layer method of alternately depositing an anionic compound on a substrate; may include.

The PEM may include a polyelectrolyte, a multilayer polyelectrolyte, a nanogel, a hydrogel, and a polymer micelle.

In the PEM manufacturing step, any one or more selected from the group including shaking and ultrasonic treatment may be performed.

Here, the nucleic acid, the cationic compound, or the anionic compound may be one or more types selected from each. Each ratio may be different depending on the purpose.

In the present invention, first, nuclear and core particles are prepared from nucleic acids and cationic compounds as active ingredients.

In the present invention, the cationic compound is a polypeptide or a peptide. allyl-based amine polymers, or cationic polysaccharides. By using these cationic compounds, the stability of the core particles can be improved.

In the present invention, the cationic compound forms a core particle with a nucleic acid. The present invention also relates to a method for producing the above-mentioned composite particles. That is, the production method of the present invention is characterized in that the nucleic acid and the cationic compound having a cationization degree of 0.2 or more are mixed in an aqueous solvent. According to the present invention, the above-described core particles can be easily produced.

The present invention is characterized in that an aqueous solution of a nucleic acid (A) and an aqueous solution of a cationic compound (B) are separately prepared, and these aqueous solutions are mixed with each other. Accordingly, it is possible to prevent aggregation of the core particles by separately preparing aqueous solutions of (A) and (B).

In addition, the concentration of the nucleic acid in the aqueous solution (A) described above is preferably 1.7 mM or less. In addition, the concentration of the total cationic compound in the aqueous solution (B) described above is preferably 1.7 mM or less. By setting the concentration of the polymer in the aqueous solutions (A) and (B) in the ranges described above, it is possible to produce core particles that are difficult to aggregate.

The method for producing a PEM containing the nucleic acid according to the present invention focuses on the characteristic that the nucleic acid itself has an anion, coating the nucleic acid with a positively charged molecule to prepare a core particle, and additionally coating an anionic polysaccharide with a secondary coating. A method for making a PEM is provided.

Preferably, the nucleic acid PEM of the present invention may be a multiparticulate by mixing a nucleic acid, a cationic compound and an anionic compound.

More specifically, the cationic compound forms a condensed PEM by electrostatic interaction with the nucleic acid.

The nucleic acid is composed of n nucleotides and has n-1 negative charges per molecule or 1.8×10 ²⁵ negative charges per mole.

Protamine (PS), a representative material in the cationic compound, has a molecular weight of 5.8 kDa and is rich in basic amino acids, particularly arginine (70-80%), so that the cation per mole is 1.26 ± 10 ²⁵ . Thus, upon complexing with the complex, PS neutralizes the negative charge of the complex phosphate group. These materials, commonly called core particles, are made up of polymers of biological macromolecules containing groups that can dissociate in water and interact to form charged components.

It is also possible to provide a method in which the charge ratio of the nucleic acid to the cationic compound of the present invention is 1:0.4 or more, that is, the nucleic acid is 1 and the PS is 0.4 or more.

The binding between the core particles aggregating due to the electrostatic interaction of the nucleic acid and PS is determined according to each charge of the nucleic acid and PS, and in the particle of the present invention, the weight ratio of the nucleic acid and PS (μg/mL) is 30 :15 to 150, preferably 30:15 to 90, more preferably 30:75.

Human serum albumin (HSA), hyaluronic acid, amphipathic biopolymers, negatively charged nanoparticles or nucleic acids and CaCl ₂ for smooth release of nucleic acids by electrostatic interaction of nucleic acids and cationic compounds constituting the core particles Any one selected from the salt to be used may be used.

In the core particle presented in the present invention, the nucleic acid necessarily further contains a cationic compound to form an ionic complex. That is, the ionic complex formed by the negatively charged nucleic acid encapsulated in the cationic compound due to the electrostatic interaction with the cationic compound is located in the core portion of the particle. The layer of core particles formed by the contact of the nucleic acid with the cationic compound has a strong positive charge, so there is a problem in that adhesion to cells is increased and it is difficult to pass through the tissue barrier.

In the present invention, in order to solve this problem, the anionic compound having good biocompatibility and having a negative charge is secondarily coated on the outer surface of the cationic compound layer to form an anionic compound layer to manufacture PEM.

The anionic compound layer coating step is a step of adding an anionic compound to coat the anionic compound layer on the outer surface of the cationic molecular layer by electrostatic attraction.

In addition, one or more ends of the nucleic acid may be modified with one or more selected from the group consisting of cholesterol, tocopherol and fatty acids having 10 to 24 carbon atoms. The cholesterol, tocopherol and fatty acids having 10 to 24 carbon atoms include cholesterol, tocopherols and their respective analogs, derivatives, and metabolites.

In the present invention, the nucleic acid may be included in an amount of 0.05 to 10% by weight, more specifically 0.1 to 5% by weight, and even more specifically 0.5 to 3% by weight, based on the weight of the total composition. If the content of the nucleic acid is less than 0.05% by weight based on the weight of the total composition, there may be side effects due to the carrier because the amount of the carrier used is too large compared to the drug, and if it exceeds 10% by weight, the size of the nanoparticles is too large As it increases, the stability of nanoparticles is reduced, and there is a risk that the loss rate during filter sterilization may increase.

The cationic compound is bound to the nucleic acid by electrostatic interaction to form a complex, and the complex is encapsulated in the nanoparticle structure of the anionic compound. Accordingly, the cationic compound includes all types of compounds capable of forming a complex by electrostatic interaction with nucleic acids, and may be, for example, cationic lipids or cationic polymers.

The PEM preparation is characterized in that an aqueous solution containing a nucleic acid, an aqueous solution containing a cationic compound, and an aqueous solution containing an anionic compound are separately prepared, and these aqueous solutions are mixed with each other, and the mixture is dispersed with an ultrasonicator. It may include making

As the ultrasonic crusher, it is preferable to use a bath type ultrasonicator. Bath type sonicators are known to provide mild sonicating of 20 to 40 Watts/Liter and provide a very non-uniform distribution, whereas probe type ultrasonicators provide approximately 20,000 W/L. can be added This means that the probe-type ultrasonic grinder is about 1,000 times superior to the bath-type ultrasonic grinder due to the concentrated and uniform ultrasonic intensity input.

In general, nucleic acids have been known to be impossible or difficult to sonicate due to their instability, but as in the present invention, it is possible to use a sonicator only by encapsulating nucleic acids in polymer nanoparticles as in the present invention, making it more convenient and efficient to manufacture a delivery system. It is possible.

The present invention also relates to a method for producing a PEM-containing composition containing an active ingredient prepared by the above-described production method. That is, the method for preparing the PEM-containing composition of the present invention includes the mixing step of the first or second method, and is characterized in that the pH of the composition after mixing may be different from the pH of the aqueous solvent.

The PEM prepared by the above-described manufacturing method does not disappear even when the pH is changed thereafter. Therefore, according to the present invention, a PEM-containing composition can be easily prepared.

In addition, the targeting molecule may be covalently linked to the anionic molecule. The composite particles can be used as an active carrier. The targeting molecule may be a cell surface-binding compound, antibody or peptide, aptamer, or a combination thereof.

In addition, the present invention also relates to a PEM comprising a cationic compound and a nucleic acid, wherein the PEM is prepared from one or two or more selected from the group consisting of non-amplifying RNA and self-amplifying mRNA. You may.

It is characterized in that the particle diameter of the composite particle of the present invention is 500 nm or less.

After the step of removing the water-miscible organic solvent, it may further include a step of freeze-drying by adding a freeze-drying aid.

The manufacturing method may further include a step of sterilizing the aqueous polymer nanoparticle solution with a sterilizing filter before the freeze-drying.

The freeze-drying aid used in the present invention is added to help the freeze-dried composition maintain a cake shape, or to help the anionic compound composition melt uniformly in a fast time in the process of reconstitution after freeze-drying. Specifically, it may be at least one selected from the group consisting of lactose, mannitol, sorbitol and sucrose. The content of the freeze-drying adjuvant may be 1 to 90 wt%, more specifically 10 to 60 wt%, based on the total dry weight of the freeze-dried composition.

The nanoparticles in the composition in which the nucleic acid and the cationic compound complex are encapsulated in the anionic compound nanoparticle structure, prepared through the preparation method according to the present invention, are stable in blood, and the size can be specifically 10 to 200 nm. and, more specifically, may be 10 to 150 nm.

According to another embodiment, the present invention also provides a pharmaceutical composition, wherein the pharmaceutical composition comprises a nucleic acid delivery agent as defined herein and a general formula biodegradable polymer carrier delivery complex, optionally with a pharmaceutically acceptable carrier and/or vehicle (vehicle).

As a first important element, the biodegradable pharmaceutical composition comprises a nucleic acid delivery agent and a polymer carrier delivery complex made of biodegradable polymer carrier molecules.

As a second important element, the biodegradable pharmaceutical composition may comprise at least one additional pharmaceutically active ingredient. In this regard, the pharmaceutically active component is a compound that has a therapeutic effect in healing, ameliorate, or prevent a specific symptom, preferably in cancer disease, autoimmune disease, allergy or infectious disease. is a compound with

The biodegradable pharmaceutical composition can be used for humans and also for veterinary purposes, preferably for humans, and can be used as a general pharmaceutical composition or as a vaccine.

As defined above, the biodegradable polymer carrier carrier complex itself is a non-specific innate immune response that allows the biodegradable polymer carrier carrier complex to be used as an immunostimulatory agent (without the addition of other pharmaceutically active components). causes If treated with other pharmaceutically active components, preferably particularly immunogenic components, preferably antigens, the nucleic acids of the invention may aid in the specific adaptive immune response elicited by the other pharmaceutically active components. The giver acts as an adjuvant.

According to another particularly preferred embodiment, the biodegradable pharmaceutical composition (or biodegradable polymer carrier delivery complex) may be provided or used as a vaccine.

If mRNA is used as a nucleic acid molecule of a nucleic acid delivery system and a biodegradable polymer carrier delivery complex, the biodegradable vaccine is generally constructed as a biodegradable pharmaceutical composition and preferably the immune response of the immune system of the patient to be treated, for example, Helps or elicits an innate immune response. Furthermore or alternatively, preferably, if the nucleic acid of the nucleic acid carrier and the biodegradable polymer carrier carrier complex encodes any of the above-mentioned antigens or proteins that elicit an adaptive immune response, the biodegradable vaccine is capable of generating an adaptive immune response. can cause

Other additives that may be included in the biodegradable vaccine include emulsifiers, such as Tween; wetting agents, such as sodium lauryl sulfate; coloring agents; taste-imparting agents, pharmaceutical carriers; tablet-forming agents; stabilizers; antioxidants; It may contain preservatives.

Furthermore, the present invention provides several applications and uses of biodegradable polymer carrier molecules, including nucleic acid delivery and biodegradable carrier delivery complexes made of biodegradable polymer carrier molecules, pharmaceutical compositions comprising them, or kits comprising them. ) is provided.

According to one embodiment, the present invention is preferably a gene therapy or a medicament for the treatment of a disease as defined herein, a biodegradable polymer carrier molecule, a nucleic acid delivery agent, and a biodegradable polymer made by the biodegradable polymer carrier molecule. It relates to a first medical use of a sex polymer carrier vehicle complex, or a kit comprising the same.

The drug may be present in the form of a pharmaceutical composition or in the form of an adjuvant or vaccine, which is a specific form of the pharmaceutical composition. In the context of the present invention, a pharmaceutical composition generally comprises a biodegradable polymer carrier molecule or a nucleic acid delivery agent and a biodegradable polymer carrier delivery complex made of a biodegradable polymer carrier molecule, optionally including other ingredients, for example For example, it preferably comprises a biodegradable nucleic acid as defined herein and optionally a pharmaceutically acceptable carrier and/or vehicle.

Each of the anionic nucleic acid group, the cationic compound group and the anionic compound group may be a single type, but is not limited thereto. Two types of compounds may be selected from each group to achieve the object of the present invention.

Preferably, the nucleic acid PEM of the present invention may be a multiparticulate by mixing a nucleic acid, a cationic compound and an anionic compound.

The ratio of nucleic acid to cationic compound, ie the net charge of the particle, is important. Therefore, in the present invention, a nucleic acid complex particle-based delivery platform using cationic compounds and anionic compounds was developed.

In the present invention, a number of novel nucleic acid multiparticulates have been developed to facilitate nucleic acid delivery, increase protein translation and protect nucleic acids from RNases. The present invention seeks to overcome concerns about rapid extracellular nucleic acid degradation and aggregation with serum proteins to present a possibility for systemic administration of nucleic acids.

Another important issue is the biodistribution of nucleic acid carriers after systemic delivery. Certain cationic fat nanoparticles were delivered via intravenous injection, primarily to the liver, which is not ideal for dendritic cell activation. That is, while positively charged particles mainly target the lungs, negatively charged particles are delivered to secondary lymphoid tissues and bone marrow to activate an immune response.

In the present invention, the polyanionic nucleic acid was prepared as a cationic polymer to prepare basic particles and coated with the anionic polymer to ultimately prepare a negatively charged PEM.

The PEM of the present invention may include any one or more selected from the group consisting of an imaging material and a neutral or cationic polypeptide. Alternatively, an imaging material and cholesterol, which is a neutral molecule, may be attached to the nucleic acid. The multiparticulate can simultaneously possess the protein encoding and pharmaceutical function by the contained pharmaceutical active ingredient, the imaging function by the image material, and the protection function of the nucleic acid drug by cholesterol.

Since the nucleic acid itself is defenseless against an enzyme that decomposes nucleic acid composed of nucleotides in the human body, it is difficult to fully deliver its efficacy into the target tissue, and there is a problem in that it is discharged by the renal filtration function. Therefore, the present invention provides a method for overcoming the above problems in order to develop the complex into an excellent drug as follows.

A detailed look at the composition and manufacturing method of the PEMN of the present invention is as follows.

cationic compound

The core particle of the present invention is a complex in which the nucleic acid and the cationic compound are condensed by electrostatic interaction. When the nucleic acid having a (-) charge and a cationic compound having a (+) charge are mixed with each other, they may be condensed. More specifically, a molecule having a strong positive charge may reduce the particle size by condensing or compressing the negatively charged nucleic acid while surrounding the nucleic acid.

In the present invention, the expression "cationic compound" denotes a polymer exhibiting cationicity at least in water, and also includes amphoteric polymers exhibiting cationicity as a whole.

In the present invention, a polymer having a cationization degree of 0.2 or more is used. By using the cationic compound, it is possible to easily form the anionic compound and the composite particles.

On the other hand, from the viewpoint of forming multiparticulates with the anionic compound, there is no specific upper limit on the degree of cationization of the cationic compound. As a reference, cationic compounds having a degree of cationization of 2 or less can be used.

In this regard, the expression "cationization degree" denotes a value obtainable by calculation according to the following formula.

｛ (number of cationic functional groups contained in polymer) - (number of anionic functional groups contained in polymer) ｝/ total amount of monomers constituting the polymer

The cationic compound used in the present invention may be a natural product or a synthetic compound as long as it has a cationic functional group.

Examples of the cationic functional group contained in the cationic compound include an amino group, a guanidino group and an imino group. The cationic compound of the present invention contains these groups in the side chain or main chain of the polymer.

The cationic compound may include cationic polypeptides, cationic polysaccharides, cationic lipids, cationic surfactants, cationic molecules and synthetic polymers covalently bound to bile acids or bile acid derivatives.

Preferred cationic polypeptides are protamine, polylysine and polyarinine;

cationic polysaccharides such as chitosan, polyben, glycol chitosan;

cationic polymers such as polyethyleneimine (PEI);

Cationic lipids, e.g. DOTMA: [1-(2,3-sioleyloxy)propyl)]-N,N,N-trimethylammonium chlorite ([1-(2,3-sioleyloxy)propyl)] -N,N,N-trimethylammonium chloride), DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DODAP, DOPE: Dioleyl phosphatidylethanol-amine , DOSPA, DODAB, DOIC, DMEPC, DOGS: Dioctadecylamidoglicylspermin, DIMRI: Dimyristo-oxypropyl dimethyl hydroxyethyl ammonium bromide, DOTAP : dioleoyloxy-3-(trimethylammonio)propane, DC-6-14: O,O-ditetradecanoyl-N-(α-trimethylammonioacetyl) Diethanolamine chloride (O,O-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride), CLIP1: rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammonium Chloride, CLIP6: rac-[2(2,3-dihexadecyloxypropyl-oxymethyloxy)ethyl]trimethylammonium, CLIP9:rac-[2(2,3-dihexadecyloxypropyl-oxysuccinyl) oxy)ethyl]-trimethylammonium, oligofectamine, etc., or

cationic or polycationic compounds, for example modified polyamino acids such as β-amino acid-polymers or reversed polyamides, PVP (poly(N-ethyl-4-vinylpyridinium bromide)), etc. Modified polyethylene, modified acrylates such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), etc., modified amidoamines, diamines and modified 1,4-butanediol such as pAMAM (poly(amidoamine)), etc. Modified polybetaaminoesters (PBAE), such as diacrylate-co-5-amino-1-pentanol polymers, dendrimers such as polypropylamine dendrimers or dendrimers based on pAMAM, PEI:poly(ethyleneimine), poly Polyimine(s) such as (propyleneimine), polyallylamine, cyclodextrin-based polymers, dextran-based polymers, sugar backbone-based polymers such as chitosan, silane backbone-based polymers such as PMOXA-PDMS copolymers, etc.; a combination of one or more cationic blocks (eg one selected from the cationic compounds mentioned above) and one or more hydrophilic or hydrophobic blocks (eg polyethylene glycol); It may include a block polymer consisting of, etc.

Multiparticulates containing cationic compounds have the added advantage of improving transfection efficiency by formulating a smaller uniform particle size. Cationic compounds tend to condense and package negatively charged nucleic acids. Poly-L-lysine (PLL) was the first cationic polymer investigated for DNA transfection. In addition, PEI (poly-ethylenimine), a novel branched cationic polymer with the highest cationic charge density, was synthesized. PEI is a highly branched polymer that can undergo protonation due to charged amino groups. The higher transfection efficiency of PEI is due to the buffering capacity of several amino groups on PEI that can dissipate protons pumped by the vesicular ATPase proton pump present in endosomes. This 'proton-sponge effect' of PEI leads to the influx of chloride ions and water into endosomes, which ultimately leads to osmotic expansion and endosome destruction. However, PEI-based formulations are cytotoxic because they bind to serum proteins and red blood cells due to their high positive charge, resulting in plasma membrane disruption. Moreover, cell lines treated with PEI polymer show autophagy, necrosis and apoptosis.

To solve the aforementioned problems, the next-generation cation-based polymers, poly[(2-dimethylamino)ethylmethacrylate] (pDMAEMA), polyamidoamine (PAMAM) dendrimers, biodegradable poly(β-amino esters) (PBAE) Polymers have been developed. Because of the tertiary amine end groups, pDMAEMA and PBAE help endosomes escape and have excellent transfection efficiency. PBAE is less toxic than PEI, but caution still needs to be taken considering the surface charge density. Therefore, to increase transfection efficiency and reduce non-specific binding, a new generation of poly (amino-co-ester) (PACE)-based polymers have been developed and optimized for nucleic acid delivery.

The basic foundation of PACE polymers is based on the presence of a branched amino group with a cleavable ester moiety that aids in endosomal disruption and can be readily hydrolyzed under biological conditions. PACE-based cationic polymers show minimal cytotoxicity compared to other types of cationic polymers. High molecular weight (MW) PACE polymers were synthesized using more hydrophobic moieties to reduce cationic charge and reduce systemic cytotoxicity. High MW PACE has excellent transfection efficiency due to significant condensation of DNA and formation of stable DNA complexes.

In the present invention, the type of cationic polypeptide is not particularly limited, and the ratio of basic amino acid residues to all amino acid residues is preferably 70% or more, more preferably 80% or more, still more preferably 90% or more. , more preferably 99% or more of basic RNA is preferably used.

In particular, a basic cationic polypeptide composed only of basic amino acid residues can be preferably mentioned.

As the basic cationic polypeptide, RNA composed of arginine and lysine can be mentioned particularly suitably.

Specifically, polylysine or polyarginine is preferably used.

In addition, as the cationic polypeptide, a cationic polypeptide composed of a single amino acid residue, or a cationic polypeptide composed of two or more amino acid residues can be used.

The cationic compound of the present invention is a molecule having a weight average molecular weight of 100 to 100,000, and may be, for example, selected from the group consisting of cationic polypeptides, cationic polysaccharides, cationic lipids, cationic surfactants, and synthetic polymers.

The cationic polypeptide may be selected from the group consisting of protamine (PS), cell penetrating peptide (CPP), poly-L-lysine (PLL: poly-L-lysine) and polyamidoamine (PAMAM: polyamidoamine), and , preferably PS.

A candidate for the cationic polypeptide is protamine, a polycationic peptide with various applications. In particular, it is used for drug delivery and has been reported to have the effect of delaying the absorption of an injected insulin suspension. Protamine has a molecular weight of 5.8 kDa and is rich in basic amino acids, especially arginine (70-80%), so it has 1.26 ± 10 ²⁵ cations per mole. Thus, upon forming a complex with DNA, protamine neutralizes the negative charge of the DNA phosphate group. These substances, commonly called Poly-Electrolyte Complexes (PECs), are composed of polymers of biological macromolecules containing groups that can dissociate in water and interact to form charged components. PS is abundantly present in the sperm nucleus of fish including salmon among animal testes, and is known as a protein involved in the expression of genetic information through association or dissociation with DNA like histones. Usually, the molecular weight of PS extracted from the sperm nucleus of fish is about 4,000 to 10,000, and 70% or more of the constituent amino acids are arginine, but the present invention is not limited thereto.

According to the present invention, the PS of the present invention may be one or two or more proteins selected from the group consisting of Protamine P1, Protamine 1, and Protamine 2).

The PS of the present invention includes both PS itself and a pharmaceutically acceptable salt thereof. Specifically, the salt of PS of the present invention encompasses a salt form with an acidic substance including hydrochloride or sulfate. PS can be dissolved in water without salt formation, and since the salt form is also soluble in water, it can be used in any form.

In order to obtain a denser 'particle' structure, it is also possible to use a form modified by treating PS with acrylamide (expressed as mPS) and a form modified by treatment with an acrylate group on HA (denoted as mHA), in which case the final gel is It can also be constructed by photocrosslinking through UV irradiation.

Poly-L-lysine (PLL: poly-L-lysine) is linked by a biodegradable peptide bond, so it can form a complex and an ionic complex and can be delivered into the cell. Because there is a limit, it can be used by attaching an endosome-destroying material or an endosome fusion peptide, including CPP and pH-dependent CPP.

Currently, the most commonly used nucleic acid stabilizing material is polyethylenimine (PEI), which has a positive charge, but has a limitation in its use due to toxicity in the body. On the other hand, PS is non-cytotoxic and has the advantage of allowing nucleic acids to be safely released into the cytoplasm by promoting penetration into negatively charged cell membranes (endosomal membranes) and endosomal escape of nucleic acids.

Cell penetrating peptides (CPPs) are used extensively to transport nucleic acids into cells. CPP increases the stability and bioavailability of nucleic acids in cells. Thus, the use of CPPs to deliver nucleic acids can have significant therapeutic advantages. A common method for binding negatively charged nucleic acids to positively charged peptides is non-covalent electrostatic interactions.

In the cell-penetrating peptide (Cell Penetrating Peptides, CPP), the peptide that activates cell permeability by pH (a) has almost no membrane permeability at a high peptide concentration above the effective concentration (EC) and a physiological pH range (pH 7.0), (b) In the acidic pH range (pH5.5) of endosomes and low peptide concentrations below the effective concentration (EC), macromolecular-sized defects can be efficiently formed in the membrane, and a domain that increases cell membrane permeability and a pH-dependent membrane Peptides composed of active domains are presented in <Table 1>.

Cell Penetrating Peptides (CPP) that activates cell permeability by pH type amino acid sequence note Melittin GIGAVLKVLTGLPALISWIKRKRQQ (SEQ ID NO: 1) Membrane activation over a wide pH range LP GWWLALALALALALALALASWIKRKRQQ (SEQ ID NO: 2) Hylin a1 IFGAILPLALGALKNLIK (SEQ ID NO: 3) LPE3-1 GWWLALAEAEAEALALASWIKRKRQQ (SEQ ID NO: 4) at acidic pH
membrane active pHD24 GIGAVLKVLATGLPALISWIKAAQQL (SEQ ID NO: 5)

According to the present invention, the present invention provides a nucleic acid/PS core particle comprising DNA and RNA.

According to the present invention, the nucleic acid / PS core particle is 1:0.4 nucleic acid and PS

It is manufactured by mixing in a mass ratio of 1:5, has a size of 200 nm or less, and has a positive charge characteristic.

The weight ratio of the nucleic acid and PS may be 1:0.2-5, preferably 1:0.4-3.0. In an environment with a w/w ratio of 1:2.5 or less, it is further condensed, and when PS is added at a ratio of 1:5 or more, the particles may become microscopic or larger.

Although the particles of the present invention remain stable in high-purity water due to their physicochemical stability, the addition of salts to the colloidal system can result in rapid agglomeration of the particles. Another disadvantage of these first-generation nucleic acid/PS particles, so-called 'particles', may be the low release of the nucleic acid from the particle.

To overcome the two problems, biodegradable, non-toxic macromolecules, negatively charged nanoparticles or salts were investigated to prevent aggregation of core particles. In addition, intracellular dissociation between nucleic acids and PS can be enhanced with amphiphilic biopolymers, negatively charged nanoparticles and salts.

The negatively charged nanoparticles are silver (Ag), gold (Au), platinum (Pt), copper (Cu), tin (Sn), iron (Fe), nickel (Ni), ruthenium (Ru), titanium (Ti) ), tantalum (Ta), niobium (Nb), zirconium (Zr), aluminum (Al), etc. conductive metal elements or metal oxides thereof are synthesized in one or more combinations It can be a nanoparticle selected from the group have.

Human serum albumin (HSA) may be a promising candidate to solve these problems. With a molecular weight of about 65,000 Da and a slightly negative surface charge under physiological conditions, HSA is a non-toxic macromolecule widely used in particulate formulations and other pharmaceuticals. HSA is a protective colloid. These colloids are trapped only in small amounts in the particle matrix. Most of the macromolecules are left in the surrounding medium to inhibit secondary aggregation of the particles. At a high HSA concentration (1000 μg/ml), the particles are completely coated with HSA, so that the stability of the core particles can be prolonged by this protective colloid.

Examples of cationic polysaccharides include chitosan, glycochitosan, cationic cellulose, cationized guar gum and cationized starch.

Among all polysaccharides used in nucleic acid-based therapy with cationic compounds, the present invention has specifically selected chitosan to highlight applicable properties and characteristic features that are well suited for nucleic acid delivery. It provides a positively charged amino group in the glucosamine monomer of chitosan or a negatively charged carboxyl group in the glucuronic acid unit of hyaluronic acid.

Cationic polysaccharides such as chitosan can electrostatically interact with nucleic acids to form stably positively charged polyplexes, and the most efficient intracellular delivery of nucleic acids is clearly achieved by positively charged nanocarriers. This can be explained by two factors. First, efficient physical interaction with negatively charged nucleic acids protects them from nuclease degradation. Second, they provide a net positive charge, enhancing the interaction of the complex with the anionic surface of the cell.

The cationic compound used in the present invention may be included in an amount of 1 to 50% by weight, more specifically 2 to 40% by weight, and even more specifically 3 to 30% by weight, based on the weight of the total composition. . If the content of the cationic lipid is less than 1% by weight based on the weight of the total composition, it is not a sufficient amount to form a complex with the nucleic acid, and if it exceeds 50% by weight, the size of the nanoparticles becomes too large There is a risk that the stability is reduced and the loss rate during filter sterilization may increase.

The cationic compound and the nucleic acid bind through electrostatic interaction to form a complex. As a specific embodiment, the ratio of the charge amount of the nucleic acid (P) and the cationic compound (N) (N/P; the ratio of the cationic charge of the cationic compound to the anionic charge of the nucleic acid) may be 0.5 to 100, and more Specifically, it may be 1 to 50, more specifically 2 to 20. When the ratio (N/P) is less than 0.5, since it is difficult to form a complex including a sufficient amount of nucleic acid, it is advantageous to form a complex including a sufficient amount of an anionic drug when it is 0.5 or more. On the other hand, when the ratio (N/P) exceeds 100, there is a risk of causing toxicity, so it is better to set it to 100 or less.

anionic compound

The PEM of the present invention includes an anionic compound layer coated on the outer surface of the cationic compound layer.

Since the cationic compound layer coating the outer surface of the core particle particle made of the nucleic acid has a strong positive charge, there is a problem in that it is difficult to pass through the tissue barrier. In the present invention, in order to solve this problem, an anionic compound having good biocompatibility and a negative charge was used to coat the outer surface of the cationic compound layer secondarily.

In the present invention, particles may be prepared by mixing a nucleic acid, a cationic compound, and an anionic compound.

In the present invention, a cationic compound is necessarily further included in order to form an ionic complex with the nucleic acid. That is, the negatively charged nucleic acid forms an ionic complex enclosed in the cationic compound due to electrostatic interaction with the cationic compound, and the formed ionic complex is coated with the anionic compound and is located in the core of the composite particle on which the anionic compound layer is formed do.

The anionic compound layer coating step is a step of adding an anionic compound to coat the anionic compound layer on the outer surface of the particle by electrostatic attraction.

The multiparticulate according to the present invention contains an anionic compound as an essential element. The anionic compound is not limited as long as it is harmless to the human body, and examples of the anionic compound are suitably silk protein fibroin, anionic protein including serum albumin, polyglutamic acid and polyaspartic acid anionic poly Amino acids, agar, hyaluronan, chondroitin sulfate, dextran sulfate, heparan sulfate, dermatan sulfate, fucoidan, keratan sulfate, heparin, carrageenan, succinoglucan, gum arabic, xanthan gum, alginic acid, pectin and carboxymethyl cellulose and anionic polysaccharides such as polyacrylic acid and salts thereof. In particular, hyaluronan, polyglutamic acid and salts thereof can be mentioned suitably.

As the anionic compound, commercially available products can be used.

The molecular weight of the anionic compound used in the present invention is not limited, preferably 100 to 100000 kDa, more preferably 200 to 50000 kDa.

Specifically, as anionic polypeptides, proteins including silk protein fibroin, gliadin, and albumin (when ionized in water of pH 7.4 found in the body) are negatively charged.

Albumin offers several advantages that ensure success in future developments of Abraxane and other types of nanomedicine. 1) Albumin is a component of human blood. It is an endogenous protein (35-50 g/L human serum), which is non-immunogenic and ensures biocompatibility for albumin-based nanoparticles. 2) The half-life of albumin in human blood averages about 19 days. Unlike free drugs, albumin provides a long half-life that helps to improve plasma area under the curve (AUC) values, thus maintaining drug concentration over a relatively long time. 3) Albumin is an excellent cryoprotectant for nanopharmaceuticals in solid form, and the dried nanoparticles can be immediately redispersed in the injection solution. These advantages are advantageous for the development of room-temperature-stable nano-medicines by avoiding drug release and deposit formation, which are common problems in the development of clinically translatable nano-medicines.

There are two types of albumin commonly used to construct nanoparticles: human serum albumin (HSA) and bovine serum albumin (BSA). Both types of albumin are serum albumin proteins (human serum and bovine serum, respectively) and share many properties, including high solubility in water, long half-lives in blood, similar molecular weights (65-70 kDa), and similar numbers. Number of amino acid residues (585 amino acids for HSA, 583 amino acids for BSA). There is no noticeable difference in properties in what constitutes a nanomedicine. Therefore, both types of albumins have been widely used to design multifunctional nanoparticles. Technologies developed with alternative albumin can be easily replaced.

Serum albumin, one of the high molecular substances in the living body, has excellent biocompatibility and is a very stable protein with a half-life of 19 days in the blood. Therefore, serum albumin is chemically bound to various low molecular weight chemical drugs, proteins, antibodies, etc., and can be used to improve the in vivo stability of various drugs and effectively deliver the drugs to target cancer sites. For example, representative hydrophobic anticancer drugs such as doxorubicin (Drug Delivery 6 (1999) 1-7) or methotrexate (MTX; Anticancer Drugs 8 (1997) 835-844) are chemically bound to albumin to target cancer Studies have been reported to improve the drug and improve the stability of the drug in blood. In addition, with respect to the use of albumin as a drug carrier, based on the high targetability of albumin to cancer tissue, fluorescent or radioactive isotope-labeled albumin was injected into small animals transplanted with cancer, and then imaged. It has been reported that positive albumin selectively accumulates in cancer tissues (Cancer Research 46 (1986) 6387-6392), which results in the enhanced permeability and retention (EPR) effect occurring in loose blood vessels around cancer tissues. It can be seen that this is indicated by As such, the superiority of albumin as an anticancer drug carrier is known, but albumin itself lacks binding force to be used as an siRNA carrier having a strong negative charge. Therefore, while maintaining the unique properties of albumin, modifications are required to increase the binding force.

As salts forming hyaluronan salts and polyglutamate salts, sodium, potassium, calcium, ammonium, magnesium, basic amino acid salts and the like can be mentioned.

More specifically, when hyaluronan or a salt thereof is used as the anionic polysaccharide, hyaluronan or a salt thereof having a molecular weight of preferably 100 to 2000 kDa, more preferably 200 to 1500 kDa may be used.

In addition, when polyglutamic acid or a salt thereof is used as the anionic compound, polyglutamic acid or a salt thereof having a molecular weight of preferably 500 to 30000 kDa, more preferably 1000 to 20000 kDa, and still more preferably 1500 to 15000 kDa is used. can

The degree of anionization of the anionic compound is not particularly limited, and the lower limit is preferably 0.1 or more, more preferably 0.2 or more, from the viewpoint of promoting the formation of multiparticulates with the cationic polypeptide.

On the other hand, from the viewpoint of forming multiparticulates with the cationic polypeptide, there is no particular upper limit on the degree of anionization of the anionic polysaccharide. As a standard, anionic polysaccharides having an anionization degree of 2 or less can be used.

In this regard, the expression "degree of anionization" denotes a value obtainable by calculation according to the following formula.

{(Number of anionic functional groups contained in polymer) - (Number of cationic functional groups contained in polymer) ｝/ Total amount of monomers constituting the polymer

The anionic polysaccharide of the present invention is heparin, hyaluronic acid, chondroitin sulfate, carboxy methyl cellulose, pectin, carbopols, polyacrylates (Polyacrylates) and anionic lipids.

The anionic lipid may be selected from the group consisting of phosphatidylserine, phosphatidic acid, phosphatidylcholine and phosphatidylglycerol.

The anionic polysaccharide has a negative charge and a molecule having biosynthesis can be used without limitation.

Heparin has a structure including a hydroxyl group, a carboxyl group and an amino group. Unfractionated heparin, molecular weight heparin, low molecular weight heparin, heparin fragment, recombinant heparin, heparin analog, heparan sulfate, sulfonated polysaccharide having heparin activity, etc. can be used. have.

The hyaluronic acid (HA) is a large complex oligosaccharide composed of up to 50,000 pairs of disaccharide glucuronic acid-β(1-3) N-acetylglucose-amine β(1-4) as basic units. It is found in vivo as a major component of the extracellular matrix. Its tertiary structure is in the form of a random coil with a diameter of about 50 nm.

Hyaluronic acid (HA) has been used to treat diseases and disorders in the human body either systemically or locally because of its ability to target active substances to diseased or diseased sites. It is known that HA can be seen in the damaged carotid artery (for the intact contralateral artery) and colorectal cancer in experimental animals and is maintained in the skin of these animals. The weight average molecular weight of the anionic polysaccharide usable in the present invention may be in the range of 5 to 90 K.

The anionic polysaccharide layer such as hyaluronic acid is coated on the outer surface of the cationic polypeptide layer by electrostatic attraction. The weight ratio of the core particles to the hyaluronic acid may be in the range of 1:0.5 to 2 (core particles: HA).

Hyaluronic acid of the present invention is one of the complex polysaccharides composed of amino acids and uronic acid, and is a natural molecular substance without cytotoxicity. Among the hyaluronic acid receptors present in the body, the CD44 receptor is overexpressed in most cancer cells and can be used as a target for therapeutic agents that selectively target cancer.

The anionic compound layer forms the outermost layer, and thus, the composite particle also exhibits a negative charge on the outside thereof. In the case of normal skin, it is known that the electrostatic properties of the particle surface slightly affect the skin permeation, but the cationic properties increase this. However, in the case of skin, the structure of fatty acids in sebaceous lipids and epidermal phospholipids is damaged, and the decrease in acidity of the skin as a result of this is a (-) property as in the present invention. It facilitates the penetration of strong anionic composite particles.

The drug composition of the present invention is composed of a nucleic acid (core), a cationic compound layer (intermediate layer) and an anionic compound layer (outermost layer), which are pharmaceutically active ingredients, and the particle size is 50 to 500 nm, preferably 100 to 400 nm, more preferably For example, it may be 150-300 nm. The pharmaceutical composition of the present invention has a reduced particle size than the composite hydrogel as a pharmaceutically active ingredient provided as a core.

Since the present invention further comprises an outer layer and a hyaluronic acid layer of the core particle consisting of the nucleic acid and the cationic polypeptide, it is possible to protect the nucleic acid from enzymes that degrade the nucleic acid in vivo.

In addition, the present invention reduces the particle size to a nano size by condensing a nucleic acid as a pharmaceutical active ingredient having a micro size or a nano size equivalent thereto, and coating the outermost layer with hyaluronic acid having a negative charge As a result, it is possible to provide particles that easily pass through the skin barrier.

natural polysaccharides

Natural polysaccharides are presented in <Table 2>, animals (eg chitosan, hyaluronic acid), plants (eg starch, cellulose, cyclodextrin, pectin), algae (eg alginate) or bacteria (eg dextran) can be obtained from It has a wide range of applications, from the agricultural and food industries to the cosmetic, pharmaceutical and biomedical sectors. Polysaccharides are formed by monosaccharides forming neutral or charged, linear or branched and carbohydrate structures with varying degrees of hydrophilicity.

The widespread use of natural polysaccharides in pharmaceuticals for drug delivery is their biocompatibility, biodegradability and low toxicity. It has also been used in combination with synthetic materials as a strategy to mitigate the latter toxicity. Their structural diversity makes it possible to select the most suitable polysaccharides in each case according to the needs of the final application, and more importantly, their functional properties can be tuned and optimized by chemical modifications to their functional groups.

Many natural polysaccharides have biological adhesion properties due to the presence of specific functional groups, making them useful starting materials for nanocarrier designs with improved retention times, especially for intestinal and lung applications. The presence of such groups also allows for certain modifications of the polymer structure to enhance its delivery or targeting ability. In addition, natural polysaccharides are neutral (eg, cellulose, pullulan, starch, dextran), negatively (eg, hyaluronic acid, alginate, chondroitin sulfate), or positively charged (eg, chitosan). ) can be

Types of polysaccharides Animal Polysaccharides Chitin
Chitosan
Glycosamminoglycans Vegetal Polysaccharides Land Vegetal Marine Vegetal Cellulose
Pectins
Galactomannans
Acacia gum
Starch Alginate (Phaeophyceae brown seaweed)
Agar (Rhodophyceae red seaweed)
Carragenans (Rhodophyceae red seaweed)
-
- Microorganis/Fungi Alginate (Pseudomonas aeruginosa and Azotobacter vinelandii)
Dextran (Leuconostoc spp. and Lactobacillus spp.)
Gellan (Pseudomonas elodea)
Pullulan (Aureobasidium pullulans)
Scleroglucan (Sclerotium glucanicum) Xanthomonas campestris
Xanthan (Xanthomonas campestris)

Among all polysaccharides used in nucleic acid-based therapy, we specifically selected hyaluronic acid to highlight applicable properties and characteristic features that are well suited for nucleic acid delivery. Although hyaluronic acid is a mucoadhesive polymer, it has an opposite charge, greatly affecting its use in nucleic acid delivery. This important difference stems from the chemical structure, which provides a negatively charged carboxyl group in the glucuronic acid unit of hyaluronic acid.

On the other hand, association of nucleic acid expression cassettes with anionic polysaccharides such as hyaluronic acid can improve bioavailability by avoiding nucleic acid excretion through the glomerular capillary wall. Indeed, negatively charged species are "unfilterable" due to the repulsive forces established by the negative charge of the glomerular membrane.

Modified Ionic Molecules

<Modified Cationic Molecules>

According to the present invention, the nucleic acid and the cationic polypeptide are bound by electrostatic interaction, and the bile acid or its derivative covalently bound to the cationic polypeptide is located on the surface of the particle in a form having a hydrophilic ionic group. You may.

A bile acid or bile acid derivative comprises one or more cationic polypeptides and complexes covalently bound, wherein the covalently bound bile acid or bile acid derivative has an ionic group.

The particles of the present invention are formed by an electrostatic interaction between a cationic polypeptide and a complex to which a bile acid or derivative thereof is covalently bound, or an anionic polysaccharide to which a bile acid or a derivative thereof is covalently bound, an electrostatic interaction between a cationic polypeptide and a nucleic acid It is formed by action, and it can induce cellular internalization of nucleic acids more safely.

That is, the core particle of the present invention is a particle in which a bile acid is surface-modified, in which a cationic polypeptide and a nucleic acid are present in the core of the particle, and a bile acid having a hydrophilic ionic group is located on the surface of the particle.

In order to deliver the nucleic acid to a target cell, when the particles on which the bile acid of the present invention is located on the surface are used, the complex is well delivered through the intestinal mucosa, and at the same time, it is very safe in the gastrointestinal tract due to the ionic complex present in the core of the particle. From this, it can be seen that the particles for delivery of the complex according to the present invention are very suitable for oral administration.

In the present invention, the bile acid or its derivative has a hydrophilic ionic group in addition to a functional group capable of covalently bonding with an ionic molecule. The ionic molecule is deoxycholic acid (DCA), urodeoxycholic acid (taurodeoxycholic acid), taurocholic acid (TCA: taurocholic acid), glycocholic acid (glycocholic acid) and glycochenodeoxycholine acid (glycochenodeoxycholic acid).

The bile acid is absorbed with a high efficiency of more than 95% through the ileum of the small intestine and is absorbed into the liver, which is called 'enterohepatic circulation'. Since it is specifically recognized by ASBT (apical sodium-dependent bile acid transporter) present in the distal intestinal region, it can act as a targeting ligand in oral delivery.

That is, the bile acid, which is absorbed through the ileum, is located on the surface of the nucleic acid delivery particle, thereby increasing target intracellular absorption of the nucleic acid delivery particle through the intestinal lining, which is the biggest problem of the existing oral administration method through ASBT. serves to make Due to this, the particles for nucleic acid delivery of the present invention can be used orally because they are easily absorbed through the ileum for absorbing bile acids, thereby increasing the bioavailability of the drug.

In the present invention, the degree of covalent bonding of the bile acid or its derivative may vary depending on the number of functional groups of the ionic molecule. Preferably, the bile acid or its derivative and the ionic molecule may be covalently bonded in a molar ratio of 1:1 to 1:300.

Preferably, the bile acid or its derivative and chitosan may be covalently bonded in a molar ratio of 1:1 to 1:100.

Preferably, the bile acid or its derivative and heparin may be covalently bonded in a molar ratio of 1:1 to 1:30.

Preferably, the bile acid or its derivative and hyaluronic acid may be covalently bonded in a molar ratio of 1:1 to 1:200.

In the present invention, the nucleic acid particles can be absorbed regardless of the particle size, preferably 500 nm or less, more preferably 100 to 300 nm.

In the present invention, the ionic molecule to which the nucleic acid and the bile acid or the bile acid derivative are ionically bonded may be bound in a mass ratio of 1:0.5 to 1:70, preferably 1:0.5 to 1:5.

In the present invention, the bile acid or bile acid derivative ion-coupled complex and the cationic polypeptide may be mixed in a mass ratio of 1:0.5 to 1:10, preferably 1:0.5 to 1:5. Since there is a limit to the binding of the cationic polypeptide and delivery is possible only in the range where it can have an optimal strong binding ability, the formation of an ionic complex due to the electrostatic interaction of the cationic polypeptide and the nucleic acid within the above range well done

<Modified Anion Molecules>

In addition, the present invention includes a nucleic acid/cationic polypeptide particle comprising one or more anionic polysaccharides and nucleic acids covalently bound to a bile acid or bile acid derivative, wherein the covalently bound bile acid or bile acid derivative is a nucleic acid having an ionic group It provides a pharmaceutical composition comprising particles as an active ingredient.

Taurocholic acid of the present invention is a type of bile acid, secreted in the body and known as a substance that helps the absorption of lipids or vitamins during the digestive process, and is characterized by being reabsorbed by more than 90% through enteric circulation. Most of the taurocholic acid is absorbed by ASBT (Apical Sodium Dependent Bile Acid Transporter) at the end of the small intestine during digestion. Therefore, when taurocholic acid is combined with hyaluronic acid to prepare particles, the oral absorption rate in the small intestine can be improved, and delivery through the enteric circulation is improved.

According to one embodiment of the present invention, the present invention provides a hyaluronic acid-taurocholic acid conjugate.

According to another embodiment of the present invention, in the hyaluronic acid-taurocholic acid conjugate of the present invention, hyaluronic acid and taurocholic acid are added in a molar ratio of 1:1 to 1:100 (feed mole ratio, hyaluronic acid: taurocholine). acid), and has the characteristic of having a negative charge.

If the hyaluronic acid-taurocholic acid conjugate has a positive charge, there is a disadvantage in that it cannot bind to a nucleic acid/PS conjugate having a positive charge.

1:100 범위의 첨가몰비(feed mole ratio, 히알루론산:타우로콜린산)로 혼합하여 제조할 수 있다. 바람직하게는 상기 히알루론산-타우로콜린산 결합체는 히알루론산과 타우로콜린산이 1:1~1:50 범위의 첨가몰비로 혼합하여 제조할 수 있으며 보다 바람직하게는 1:10 범위의 첨가몰비로 혼합하여 제조할 수 있다.According to the present invention, the hyaluronic acid-taurocholic acid conjugate is 1:1 with hyaluronic acid and taurocholic acid.

1:100 addition molar ratio (feed mole ratio, hyaluronic acid: taurocholic acid) can be prepared by mixing. Preferably, the hyaluronic acid-taurocholic acid conjugate can be prepared by mixing hyaluronic acid and taurocholic acid in an addition molar ratio in the range of 1:1 to 1:50, and more preferably in an addition molar ratio in the range of 1:10. It can be prepared by mixing.

If it is prepared by mixing the hyaluronic acid and taurocholic acid in an addition molar ratio of less than 1:1, it is not efficient to coat the complex/PS, and the hyaluronic acid and taurocholic acid are added in a molar ratio exceeding 1:100. When prepared by mixing, it is not economically efficient because only a hyaluronic acid-taurocholic acid conjugate having a similar binding ratio is made.

The complex particle for oral administration of the present invention has a form in which the nucleic acid/PS is coated with a hyaluronic acid-taurocholic acid conjugate.

In detail, the complex particle for oral administration of the present invention has a form in which the nucleic acid / PS is located and the hyaluronic acid-taurocholic acid conjugate is coated on the surface of the nucleic acid / PS, has a negative charge property, and the diameter of the particle It is 250 nm or less, and the encapsulation rate of the nucleic acid/PS protein is 90% or more.

If the diameter of the nucleic acid / PS particle exceeds 100 nm, the size of the nucleic acid / PS / HA-TCA composite particle prepared by the coating of the following HA-TCA conjugate exceeds 250 nm, so the advantage as a particle may disappear. /PS It is preferable to manufacture so that the diameter of the core particle is 100 nm or less. In addition, when the charge characteristic of the nucleic acid PS core particle is negatively charged, it is difficult to bind (coat) with the negatively charged HA-TCA conjugate, so it is preferable to prepare the nucleic acid PS core particle to have a positive charge.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition comprising a pharmaceutically effective amount of the nucleic acid PS / HA-TCA multiparticulate.

PEMC manufacturing

A detailed look at the manufacturing method of the PEMN of the present invention is as follows.

In the step of preparing the PEMN, any one or more selected from the group comprising extrusion homogenization, upstream pressure homogenization, high pressure homogenization, ultra-high pressure homogenization and nanostructures may be used. .

The upstream pressure may be 20 to 60 MPa, the high pressure may be up to 150 to 200 MPa, and the ultrahigh pressure may be up to 350 to 400 MPa.

1) Extrusion homogenization

The present invention was practiced using high extrusion pressure. Extrusion done under higher pressure will have a higher flow rate than other identical extrusions done under lower pressure, will be less prone to clogging and fouling, will allow the membrane to withstand a high degree of fouling or clogging during the production process, and will PEM will be produced. The pressure that can be used is limited only by the extrusion equipment used and the resistance of the membrane. Pressures greater than about 400 psi are used.

The methods and apparatus of the present invention can be used to make PEMs of any desired average diameter. In general, as described above, membranes are selected that have an average pore diameter comparable to the desired average PEM diameter. The average PEM size is reduced as described herein, for example, by extruding the extruded PEM one or more additional times, using multiple layers of membranes, using thinner membranes, increasing the extrusion pressure, or treating the PEM as described herein. can do it The size of the PEM may be determined using any technique known in the art.

PEM produced using the method or apparatus of the present invention may be further processed using any processing technique. To reduce the degree of contamination, the membrane is passed through the membrane in alternating anterior and posterior directions. The average diameter of the extruded PEM can be further reduced by sonication using high frequency. Conditions leading to efficient PEM synthesis using intermittent high-frequency fracturing water can be screened by Dynamic Light Scattering (DLS) evaluation.

The methods and devices of the present invention may be practiced using any number of stacked membranes. One of the techniques in the art to which this invention pertains is that extrusion through a higher number of stacked membranes results in a lower flow rate than other similar extrusions through a smaller number of stacked membranes, resulting in a PEM with a smaller average diameter. know that The number of membranes used for stacking is limited only by the resistance of the extrusion apparatus. In a preferred embodiment, the stacked stack consists of between 2 and 10 films. In a most preferred embodiment, the stacked pile consists of between 2 and 5 membranes. In another preferred embodiment, the stacked films are substantially identical. In another preferred embodiment, at least one membrane in the stack is different from at least one of the other membranes in the pile. The difference lies in some properties that affect extrusion. As described herein, differences can be made, for example, in the composition, coating, pore size, pore density, pore angle, pore shape, or membrane size of the membrane.

Extrusion may be effected by multiple passes through a single membrane or stacked membranes. If an embodiment using a stacked membrane is used for extrusion, multiple passes may be unnecessary to obtain a PEM of the desired diameter. In particular, a step-down method is used. In the step-down method, several passes of the suspension are made through a membrane of decreasing pore diameter. In a particularly preferred embodiment of the step-down method, first through a membrane having a pore diameter of about 0.4 μm, second through a membrane having a pore diameter of about 0.2 μm, if necessary a third, fourth, and fifth Second and sixth, it is carried out through a membrane having a pore diameter of about 0.1 μm.

The membrane may be treated with a flushing agent. The membrane may be treated with a flush agent prior to extrusion. The membrane may be treated with the flush agent after at least one pass and prior to at least one pass through the membrane. A flushing agent can be any kind of substance or composition that removes material from clogged or contaminated membrane pores or prevents clogging, contamination or creating a 'sieving effect'. The flushing agent consists of organic alcohol. The flushing agent consists of ethanol.

Any extrusion apparatus capable of receiving suitable membranes and withstanding high extrusion pressures may be used in practicing the methods and apparatus of the presently claimed invention. The extrusion apparatus of the present invention and apparatus useful for practicing the method of the present invention consist of a hydrophilic, curved orifice or hydrophilic, curved screen membrane. The membrane is a polyester (PETE) membrane.

The extruder consists of a cover and a collecting vessel thereon, the cover being operably attached to the first face of the membrane by a pressure and liquid resistant seal, and the collecting vessel being the two sides of the membrane to receive the extruded suspension as it forms. placed on the second side. The device may additionally consist of a membrane pedestal or instrument. The extrusion device is arranged so that the aqueous suspension can be extruded through the membrane in alternating forward and backward directions. The extruder uses a tangential flow.

Commercially available devices suitable for suitable membranes and which can be used in the present invention are described in THE MINI-EXTRUDER ^™ , Cat. No. 610000 (AVANTI R Polar Lipids, Inc., Alabaster AL), Subbarao, et al., 1991, Biochim. Biophys. Acta, 1063: 147-54, Liposome Extruder, Part No. ER-1 (Eastern Scientific, Rockville MD), EMULSIFLEX R-C50 Extruder, Cat. No. EFC50EX (Avestin, Inc., Ottowa, Ontario, Canada), LIPOSOFAST™, (Avestin, Inc.), LIPEX™ Extruders (Northern Lipids Inc., Vancouver, British Columbia, Canada).

The extrusion equipment must be capable of withstanding high extrusion pressures. As a general rule, greater pressure results in improved performance, eg increased flow rates, reduced membrane fouling and clogging, and a faster reduction in the size of the extruded PEM. As a minimum, the extrusion equipment must be able to withstand extrusion pressures greater than about 400 psi. Membrane pedestal holders or covers that optimize the effective surface area may also be used if they can withstand the extrusion pressure it is subjected to. The cartridge holder device offers the advantage of minimizing the manipulation of the membrane and greater efficiency in stacking the membrane. In addition, the cartridge holder device provides an overall improved efficiency in production, for example during exchanging a clogged or soiled cartridge holder device, a clogged or soiled membrane may be exchanged while the product flow is diverted to a new cartridge holder device. .

2) pressure homogenization

The first application of homogenization for the stabilization of food and dairy emulsions was presented by Auguste Gaulin at the Paris World's Fair in 1900 and is an invention for "milk mixing" using pressures of up to 30 MPa. Since then, conventional homogenization has extended the pressure range to 50 MPa. HPH, also known as dynamic HPH, has been frequently highlighted for its potential for food pasteurization. The latest ultra-high pressure homogenizers enable pressures 10-15 times higher than conventional pressures and implement a pressure range of 300 to 400 MPa, which is called UHPH. UHPH technology is based on the same principle as conventional homogenization, but its action is pressure drop, torsion and shear stress, turbulent flow, cavitation, shock wave, temperature increase caused by the application of dynamic high pressure. It may be associated with emerging physical technologies as a result of several mechanisms, such as

It is possible to use a homogenization treatment, in particular ultra-high pressure homogenization (UHPH), also called dynamic high pressure. UHPH is a new technology recently studied in food, cosmetic and pharmaceutical fields, which fragments the particles of dispersions or emulsions, creates fine and stable emulsions, modifies the viscous properties of fluids due to particle size reduction, and facilitates metabolite extraction. doing it To achieve inactivation of microorganisms, enzymes or some viruses. In fact, UHPH is a continuous process that offers the added benefit of significantly reducing the microbial load, at least on par with pasteurization. Depending on the pressure level, this technique is called high pressure homogenization (HPH, up to 150 to 200 MPa) or ultra-high pressure homogenization (UHPH, up to 350 to 400 MPa). For comparison, a standard homogenizer operates with an upstream pressure of 20 to 60 MPa, as in the dairy industry. UHPH has benefited from the latest developments in high pressure (HP) technology, namely HP reinforcement, the concept of high pressure resistant materials (stainless steel, ceramics, seals). Sophisticated homogenizing valves with a needle and seat and needle made of ceramic or coated with artificial diamond developed specifically for UHPH equipment are currently being studied to withstand pressures from 350 to 400 MPa.

The PEMN prepared in the present invention, quasi-electric light scattering (QELS), also known as Dynamic Light Scattering (DLS), is described in Bloomfield, 1981, A nn. Rev. Biophys. Bioeng. 10: 421-50. In a preferred embodiment, the PEM has an average diameter between about 50 and 400 nm. In a more preferred embodiment, the average diameter is between about 50 and 150 nm. In an even more preferred embodiment, the average diameter is between about 100 and 150 nm.

The polydispersity index (polydispersity index) of the PEMN prepared in the present invention may be 0.01 to 0.5. For example, the polydispersity index of the PEMN may be 0.01 to 0.45, 0.01 to 0.4, 0.01 to 0.35, 0.01 to 0.3, 0.01 to 0.2, 0.01 to 0.19. In the present specification, the polydispersity index indicates the degree of polydispersity, and a smaller number indicates monodispersity. Monodispersity means that the particle size is uniform.

The PEMN prepared in the present invention may have a particle size of 50 to 400 nanometers and a polydispersity index (PDI) of 0.3 or less.

The PEMN prepared in the present invention can release the active ingredient nucleic acid at a constant rate.

The PEMN prepared in the present invention may be an active drug carrier by a compound constituting the outermost layer or a ligand bound thereto.

The PEMN prepared in the present invention can be used for injection, cream, and oral mucosal administration.

The PEMN prepared in the present invention may further include any one or more selected from the group including ionic salts, small molecule drugs, polymer drugs, polymer drugs including drugs, and polymers including fluorescent materials.

The polymer drug may be any one or more selected from the group comprising proteins, antibodies, and nucleic acids.

It is possible to treat a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the PEMN, thereby treating the disease.

Hereinafter, the present invention will be described in more detail through the following examples. However, these Examples are for illustrative purposes of the present invention, and the scope of the present invention is not limited only to these Examples.

Example 1. Design and Preparation of Nucleic Acid Templates

The nucleic acid of the present invention proposes an RNA having a structure capable of protein synthesis in a cell. The target protein of the nucleic acid used in the present invention is a therapeutic protein (sTNFR2, EPO, G-CSF), an extracellular matrix degrading enzyme (HYAL 1, Hyaluronidase PH20, MMP1, MMP 8, MMP 13), an extracellular matrix degrading enzyme ORFs encoding inhibitors (TIMP 1, TIMP 2, TIMP 3 and TIMP 4) and proteins of interest for vaccination (RAV-G and RSV-F proteins). In particular, a nucleic acid capable of stably expressing various proteins including the target protein, in which the expression cassette is linear, nuclear-independent, and capable of cytoplasmic expression was prepared as follows.

1-1. Design of Nucleic Acid Templates

The nucleic acid is a non-amplifying mRNA (non-amplifying mRNA) construct; and self-amplifying mRNA (mRNA) constructs. The unamplified mRNA construct comprises (a) an open reading frame (ORF) encoding the target polypeptide: (b) a 5' UTR; (c) 3' UTR; and (d) a 5' cap structure or IRES; The self-amplifying mRNA construct comprises (a) an mRNA construct for expressing RNA dependent RNA polymerase (RdRp); and (b) an mRNA replicon capable of being replicated by said RdRp and comprising an ORF encoding said polypeptide of interest, wherein said two kinds of components are in the same single RNA cis-replicon RNA constructs or trans-replicon RNA constructs in two different RNAs each.

In order to prepare the nucleic acid of the present invention, a target protein DNA having a T7 promoter capable of producing a target protein ORF is designed and manufactured, and then recombined in a pUC19 cloning vector, and the target protein DNA having a T7 promoter obtained from the recombinant pUC19 vector is used as a template. After in vitro transcription, it can be prepared by adding a 5'Cap structure to the transcript.

The target protein DNA of the present invention was prepared in the order of <T7 promoter - 5'UTR - target protein DNA - muag - A64 - C30 - histone-SL> as a DNA fragment capable of efficiently synthesizing the target protein.

The target protein DNA of the present invention is a DNA fragment capable of efficiently synthesizing the target protein orf, and a DNA fragment consisting of <T7 promoter followed by 5'UTR, ORF encoding the target protein, 3'UTR> was designed.

The target protein DNA of the present invention is located downstream of the T7 promoter and 5'UTR in the pUC19 vector, muag (mutated alpha-globin-3'UTR), A64 poly(A) sequence, poly(C) sequence (C30) and Histone stem-loop sequence (histone-SL) was placed upstream of the sequence.

The unit elements constituting the target protein DNA fragment are partially presented in <Table 3>.

The nucleotide sequence composing the target protein DNA fragment backbone DNA or amino acid sequences T7 promoter TAATACGACT CACTATAGGG (SEQ ID NO: 6) hAg-Kozak ATTCTTCTGG TCCCCACAGA CTCAGAGAGA ACCCGCCACC (SEQ ID NO: 7) Sec ATGAGAGTGA CCGCCCCCAG AACCCTGATC CTGCTGCTGT CTGGCGCCCT GGCCCTGACA GAGACATGGG CCGGAAGCGG ATCC (SEQ ID NO: 8)
(MRVTAPRTLILLLSGALALTE TWAGSGS; SEQ ID NO: 9) muag - A64 - C30 - Histone S GCCCGATGGG CCTCCCAACG GGCCCTCCTC CCCTCCTTGC ACCGAGATTA ATAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAATGCA TCCCCCCCCC CCCCCCCCCC CCCCCCCCCC CCAAAGGCTC TTTTCAGAGC CACCA (SEQ ID NO:10) 2 BgUTR CCGAGAGCTC GUCGGCCAAC AAAAGGTTCU TGCCCAAGTC CAACA CAAAC TGGGGGATAT TATGAAGGGC CTTGAGCATC TGGATTCTGC CTAATAAAAA ACATTTACAT TGCTGCGTCG AGAGCTCGCT TCTTGCTGTC CAATTTCTAT TAAAGGTTCC TTTGTTCCCT AAGTCCAACT ACTAAACTGG GGGATATTAT GAAGGGCCTT GAGCATCTGG ATTCTGCCTA ATAAAAAACA TTTACATTGC TGCGTC (서열번호 11) A30LA70 GAGACCTGGT CCAGAGTCGC TAGCAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAGCATAT GACTAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAA (SEQ ID NO: 12) (G4S)3-linker GGAGGCGGTG GTAGTGGAGG TGGCGGGTCC GGTGGAGGTG GAAGC (SEQ ID NO: 13)
(GGGGSGGGGGSGGGGS; SEQ ID NO: 14)

The 5'UTR of the present invention may be mouse ribosomal protein Raji 35A (mRPL35A), and its base sequence is SEQ ID NO: 15.

GGGCCATCTT GGCGCCTGTG GAGGCCTGCT GGGAACAGGA CTTCTAACAG CAAGTAAGCT TGAGGATG (SEQ ID NO: 15)

The 5'UTR element is 5'-UTR of human ribosamal protein Large 32 lacking the 5' terminal oligopyrimidine tract GGCGCTGCCTACGGAGGTGGCAGCCATCTCCTTCTCGGCATC (SEQ ID NO: 16)

preferably comprises or consists of a nucleic acid sequence derived from the human hydroxysteroid (17-beta) dehydrogenase 4 gene (HSD17B4).

GGGTCCCGCA GTCGGCGTCC AGCGGCTCTG CTTGTTCGTG TGTGTGTCGT TGCAGGCCTT ATTCAAGCTT ACCATG (SEQ ID NO: 17)

Preferably, the 5'UTR is hAg-Kozak, SEQ ID NO:7

The 5'UTR includes a Kozak consensus sequence, and the Kozak consensus sequence may be ACCAUGG (SEQ ID NO: 18).

3'UTR may be any one selected from the group consisting of muag-A64-C30-histone-SL and 2BgUTR-A30LA70. If the target protein is a fusion protein, the linker is (G4S)3-linker and. Sec can be used for the purpose of secreting the target protein to the outside of the cell.

wherein 3'UTR is the sequence 5'-GCCCG a TGGG CCTCCCAACG GGCCCTCCTC CCCTCCTTGC ACCG-3' (SEQ ID NO: 19; muag; underlined nucleotides are mutated compared to wild-type sequence) (alpha-) globin mutated UTR, poly (A) The tail is 64 adenine nucleotides, the poly(C) sequence is 30 cytosine nucleotides, and the histone stem-loop is an RNA sequence consisting of (5'-CAAAGGCUCU UUUCAGAGCC ACCA-3' SEQ ID NO: 20).

A64: GGACTAGTAG ATCTAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAA (SEQ ID NO: 21)

C30: TGCATCCCCC CCCCCCCCCC CCCCCCCCCC CCCCCTCTA G (SEQ ID NO: 22)

The target protein used in the present invention is a therapeutic protein comprising sTNFR2, EPO, G-CSF and E3 ligase; ECM degradation related proteins including hyaluronidase, MMP and TIMP; And vaccine-related proteins including, but not limited to, G protein (RAV-G) of rabies virus and RSV-F protein of RSV long strain were used.

<Therapeutic protein>

sTNFR2

The therapeutic protein of the present invention is a TNFR2 protein or an extracellular region fragment thereof. Tumor necrosis factor (TNF) is a pleiotropic cytokine, in particular TNF-α plays an important role in the inflammatory response and immune system. TNFR1 (molecular weight, 55 kD; TNF-R55) and TNFR2 (molecular weight, 75 kD; TNF-R75) are known as receptors that bind to TNF, wherein the affinity of TNF-α for TNFR2 is the affinity for TNFR1. It is known to be several times higher than

In the present invention, the TNFR2 protein is encoded by 1386 bp DNA (NM_001066.3, this protein or its extracellular region fragment (sTNFR2) has a high binding affinity for TNF-α and autoimmune by inhibiting the action of TNF-α. It can exhibit a particularly excellent effect in the treatment of diseases.

Preferably, the sTNFR2 may be an amino acid sequence polypeptide of SEQ ID NO: 23 including 1 to 256 from the N-terminus of the TNFR2 protein.

Also preferably, the sTNFR2 cDNA may be a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 24.

sTNFR2 : transmembrane sequence fragment consisting of 1 to 256 amino acids of TNFR2

MAPVAVWAAL AVGLELWAAA HALPAQVAFT PYAPEPGSTC RLREYYDQTA QMCCSKCSPG QHAKVFCTKT SDTVCDSCED STYTQLWNWV PEMLSCGSRC SSDQVETQAC TREQNRICTC RPGWYCALSK QEGCRLCAPL RKCRPGFGVA RPGTETSDVV CKPCAPGTFS NTTSSTDICR PHQICNVVAI PGNASMDAVC TSTSPTRSMA PGAVHLPQPV STRSQHTQPT PEPSTAPSTS FLLPMGPSPP AEGSTG (서열번호 23)

sTNFR2 cDNA ; cDNA corresponding to sTNFR2 (768 bp)

ATGGCGCCC GTCGCCGTCT GGGCCGCGCT GGCCGTCGGA CTGGAGCTCT GGGCTGCGGC GCACGCCTTG CCCGCCCAGG TGGCATTTAC ACCCTACGCC CCGGAGCCCG GGAGCACATG CCGGCTCAGA GAATACTATG ACCAGACAGC TCAGATGTGC TGCAGCAAAT GCTCGCCGGG CCAACATGCA AAAGTCTTCT GTACCAAGAC CTCGGACACC GTGTGTGACT CCTGTGAGGA CAGCACATAC ACCCAGCTCT GGAACTGGGT TCCCGAGTGC TTGAGCTGTG GCTCCCGCTG TAGCTCTGAC CAGGTGGAAA CTCAAGCCTG CACTCGGGAA CAGAACCGCA TCTGCACCTG CAGGCCCGGC TGGTACTGCG CGCTGAGCAA GCAGGAGGGG TGCCGGCTGT GCGCGCCGCT GCGCAAGTGC CGCCCGGGCT TCGGCGTGGC CAGACCAGGA ACTGAAACAT CAGACGTGGT GTGCAAGCCC TGTGCCCCGG SEQUENCE GGACGTTCTC CAACACGACT TCATCCACGG ATATTTGCAG GCCCCACCAG ATCTGTAACG TGGTGGCCAT CCCTGGGAAT GCAAGCATGG ATGCAGTCTG CACGTCCACG TCCCCCACCC GGAGTATGGC CCCAGGGGCA GTACACTTAC CCAGCCTG CCCAGCCAGT GTCCACACAC ACCCAGCCACTACCAGC GGCC GC GAGCCAGT GTCCAC GG CG

EPO

For the preparation of the EPO nucleic acid of the present invention, the nucleotide sequence of the murine EPO cDNA is as follows.

EPO cDNA

ATG GGCG TGCCCGAGCG GCCGACCCTG CTCCTGCTGC TCAGCCTGCT GCTCATCCCC CTGGGGCTGC CCGTCCTCTG CGCCCCCCCG CGCCTGATCT GCGACTCCCG GGTGCTGGAG CGCTACATCC TCGAGGCCAA GGAGGCGGAG AACGTGACCA TGGGCTGCGC CGAGGGGCCC CGGCTGAGCG AGAACATCAC GGTCCCCGAC ACCAAGGTGA ACTTCTACGC CTGGAAGCGC ATGGAGGTGG AGGAGCAGGC CATCGAGGTC TGGCAGGGCC TGTCCCTCCT GAGCGAGGCC ATCCTGCAGG CGCAGGCCCT CCTGGCCAAC TCCAGCCAGC CCCCGGAGAC ACTGCAGCTC CACATCGACA AGGCCATCTC CGGGCTGCGG AGCCTGACCT CCCTCCTGCG CGTGCTGGGC GCGCAGAAGG AGCTCATGAG CCCGCCCGAC ACGACCCCCC CGGCCCCGCT GCGGACCCTG ACCGTGGACA CGTTCTGCAA GCTCTTCCGC GTCTACGCCA ACTTCCTGCG GGGCAAGCTG AAGCTCTACA CCGGGGAGGT GTGCCGCCGG GGCGACCGCT GA (SEQ ID NO:25)

GM CSF

For the preparation of the GM CSF nucleic acid of the present invention, the nucleotide sequence of GM CSF (CSF2) (NM_000758) is as follows.

GM CSF (CSF2) cDNA (NM_000758)

ATGTGGCTGC AGAGCCTGCT GCTCTTGGGC ACTGTGGCCT GCAGCATCTC TGCACCCGCC CGCTCGCCCA GCCCCAGCAC GCAGCCCTGG GAGCATGTGA ATGCCATCCA GGAGGCCCGG CGTCTCCTGA ACCTGAGTAG AGACACTGCT GCTGAGATGA ATGAAACAGT AGAAGTCATC TCAGAAATGT TTGACCTCCA GGAGCCGACC TGCCTACAGA CCCGCCTGGA GCTGTACAAG CAGGGCCTGC GGGGCAGCCT CACCAAGCTC AAGGGCCCCT TGACCATGAT GGCCAGCCAC TACAAGCAGC ACTGCCCTCC AACCCCGGAA ACTTCCTGTG CAACCCAGAT TATCACCTTT GAAAGTTTCA AAGAGAACCT GAAGGACTTT CTGCTTGTCA TCCCCTTTGA CTGCTGGGAG CCAGTCCAGG AGTGA (서열번호 26)

FBXW7

FBXW7 is a well-characterized SCF belonging to the E3 ligase and promotes the destruction of oncogenic proteins such as C-MyC, transforming protein RhoA (RhoA), transcriptional activator BRG1 (Brg1) and zinc-finger protein GFI-1 (GFI1). do. All of these target proteins promote tumorigenesis. For example, Brg1 promotes cancer metastasis, RhoA is associated with tumorigenesis, and GFI1 promotes cell proliferation as an oncoprotein.

FBXW7 cDNA (1770 nt): NM_001013415.2

ATGTCAAAAC CGGGAAAACC TACTCTAAAC CATGGCTTGG TTCCTGTTGA TCTTAAAAGT GCAAAAGAGC CTCTACCACA TCAAACTGTG ATGAAGATAT TTAGCATTAG CATCATTGCC CAAGGCCTCC CTTTTTGTCG AAGACGGATG AAAAGAAAGT TGGACCATGG TTCTGAGGTC CGCTCTTTTT CTTTGGGAAA GAAACCATGC AAAGTCTCAG AATATACAAG TACCACTGGG CTTGTACCAT GTTCAGCAAC ACCAACAACT TTTGGGGACC TCAGAGCAGC CAATGGCCAA GGGCAACAAC GACGCCGAAT TACATCTGTC CAGCCACCTA CAGGCCTCCA GGAATGGCTA AAAATGTTTC AGAGCTGGAG TGGACCAGAG AAATTGCTTG CTTTAGATGA ACTCATTGAT AGTTGTGAAC CAACACAAGT AAAACATATG ATGCAAGTGA TAGAACCCCA GTTTCAACGA GACTTCATTT CATTGCTCCC TAAAGAGTTG GCACTCTATG TGCTTTCATT CCTGGAACCC AAAGACCTGC TACAAGCAGC TCAGACATGT CGCTACTGGA GAATTTTGGC TGAAGACAAC CTTCTCTGGA GAGAGAAATG CAAAGAAGAG GGGATTGATG AACCATTGCA CATCAAGAGA AGAAAAGTAA TAAAACCAGG TTTCATACAC AGTCCATGGA AAAGTGCATA CATCAGACAG CACAGAATTG ATACTAACTG GAGGCGAGGA GAACTCAAAT CTCCTAAGGT GCTGAAAGGA CATGATGATC ATGTGATCAC ATGCTTACAG TTTTGTGGTA ACCGAATAGT TAGTGGTTCT GATGACAACA CTTTAAAAGT TTGGTCAGCA GTCACAGGCA AATGTCTGAG AACATTAGTG GGACATACAG GTGGAGTATG GTCATCACAA ATGAGAGACA ACATCATCAT TAGTGGATCT ACAGATCGGA CACTCAAAGT GTGGAATGCA GAGACTGGAG AATGTATACA CACCTTATAT GGGCATACTT CCACTGTGCG TTGTATGCAT CTTCATGAAA AAAGAGTTGT TAGCGGTTCT CGAGATGCCA CTCTTAGGGT TTGGGATATT GAGACAGGCC AGTGTTTACA TGTTTTGATG GGTCATGTTG CAGCAGTCCG CTGTGTTCAA TATGATGGCA GGAGGGTTGT TAGTGGAGCA TATGATTTTA TGGTAAAGGT GTGGGATCCA GAGACTGAAA CCTGTCTACA CACGTTGCAG GGGCATACTA ATAGAGTCTA TTCATTACAG TTTGATGGTA TCCATGTGGT GAGTGGATCT CTTGATACAT CAATCCGTGT TTGGGATGTG GAGACAGGGA ATTGCATTCA CACGTTAACA GGGCACCAGT CGTTAACAAG TGGAATGGAA CTCAAAGACA ATATTCTTGT CTCTGGGAAT GCAGATTCTA CAGTTAAAAT CTGGGATATC AAAACAGGAC AGTGTTTACA AACATTGCAA GGTCCCAACA AGCATCAGAG TGCTGTGACC TGTTTACAGT TCAACAAGAA CTTTGTAATT ACCAGCTCAG ATGATGGAAC TGTAAAACTA TGGGACTTGA AAACGGGTGA ATTTATTCGA AACCTAGTCA CATTGGAGAG TGGGGGGAGT GGGGGAGTTG TGTGGCGGAT CAGAGCCTCA AACACAAAGC TGGTGTGTGC AGTTGGGAGT CGGAATGGGA CTGAAGAAAC CAAGCTGCTG GTGCTGGACT TTGATGTGGA CATGAAGTGA (서열번호 27)

<Extracellular matrix degrading enzyme>

hyaluronidase

Hyaluronidase (enzyme that degrades HA) includes HYAL1 (NM_153281), HYAL2 (NM_033158), HYAL3 (NM_003549), HYAL4 (NM_012269) and Hyaluronidase PH20 (SPAM1) (NM_001174044), sPH20, and the like.

PH20 CDNA, SEQ ID NO: 28 is the coding nucleotide sequence for the protein Hyaluronidase PH20 (SPAM1) (NM_001174044) from the start codon (ATG) to position 1467.

Hyaluronidase PH20 (SPAM1) cDNA (NM_001174044)

ATGGGAGTGC TAAAATTCAA GCACATCTTT TTCAGAAGCT TTGTTAAATC AAGTGGAGTA TCCCAGATAG TTTTCACCTT CCTTCTGATT CCATGTTGCT TGACTCTGAA TTTCAGAGCA CCTCCTGTTA TTCCAAATGT GCCTTTCCTC TGGGCCTGGA ATGCCCCAAG TGAATTTTGT CTTGGAAAAT TTGATGAGCC ACTAGATATG AGCCTCTTCT CTTTCATAGG AAGCCCCCGA ATAAACGCCA CCGGGCAAGG TGTTACAATA TTTTATGTTG ATAGACTTGG CTACTATCCT TACATAGATT CAATCACAGG AGTAACTGTG AATGGAGGAA TCCCCCAGAA GATTTCCTTA CAAGACCATC TGGACAAAGC TAAGAAAGAC ATTACATTTT ATATGCCAGT AGACAATTTG GGAATGGCTG TTATTGACTG GGAAGAATGG AGACCCACTT GGGCAAGAAA CTGGAAACCT AAAGATGTTT ACAAGAATAG GTCTATTGAA TTGGTTCAGC AACAAAATGT ACAACTTAGT CTCACAGAGG CCACTGAGAA AGCAAAACAA GAATTTGAAA AGGCAGGGAA GGATTTCCTG GTAGAGACTA TAAAATTGGG AAAATTACTT CGGCCAAATC ACTTGTGGGG TTATTATCTT TTTCCGGATT GTTACAACCA TCACTATAAG AAACCCGGTT ACAATGGAAG TTGCTTCAAT GTAGAAATAA AAAGAAATGA TGATCTCAGC TGGTTGTGGA ATGAAAGCAC TGCTCTTTAC CCATCCATTT ATTTGAACAC TCAGCAGTCT CCTGTAGCTG CTACACTCTA TGTGCGCAAT CGAGTTCGGG AAGCCATCAG AGTTTCCAAA ATACCTGATG CAAAAAGTCC ACTTCCGGTT TTTGCATATA CCCGCATAGT TTTTACTGAT CAAGTTTTGA AATTCCTTTC TCAAGATGAA CTTGTGTATA CATTTGGCGA AACTGTTGCT CTGGGTGCTT CTGGAATTGT AATATGGGGA ACCCTCAGTA TAATGCGAAG TATGAAATCT TGCTTGCTCC TAGACAATTA CATGGAGACT ATACTGAATC CTTACATAAT CAACGTCACA CTAGCAGCCA AAATGTGTAG CCAAGTGCTT TGCCAGGAGC AAGGAGTGTG TATAAGGAAA AACTGGAATT CAAGTGACTA TCTTCACCTC AACCCAGATA ATTTTGCTAT TCAACTTGAG AAAGGTGGAA AGTTCACAGT ACGTGGAAAA CCGACACTTG AAGACCTGGA GCAATTTTCT GAAAAATTTT ATTGCAGCTG TTATAGCACC TTGAGTTGTA AGGAGAAAGC TGATGTAAAA GACACTGATG CTGTTGATGT GTGTATTGCT GATGGTGTCT GTATAGATGC TTTTCTAAAA CCTCCCATGG AGACAGAAGA ACCTCAAATT TTCTACAATG CTTCACCCTC CACACTATCT GCCACAATGT TCATTGTTAG TATTTTGTTT CTTATCATTT CTTCTGTAGC GAGTTTGTGA (SEQ ID NO: 28)

sPH20

sPH20 cDNA is SEQ ID NO: 28, a nucleotide sequence from the transcription start (ATG) to position 1467 of the PH20 cDNA, lacking the hydrophobic tail (nucleotides 1468-1527) of the PH20 protein and adding a stop codon (TAA) at the end. is a protein secreted by cells.

sPH20 cDNA

ATGGGAGTGC TAAAATTCAA GCACATCTTT TTCAGAAGCT TTGTTAAATC AAGTGGAGTA TCCCAGATAG TTTTCACCTT CCTTCTGATT CCATGTTGCT TGACTCTGAA TTTCAGAGCA CCTCCTGTTA TTCCAAATGT GCCTTTCCTC TGGGCCTGGA ATGCCCCAAG TGAATTTTGT CTTGGAAAAT TTGATGAGCC ACTAGATATG AGCCTCTTCT CTTTCATAGG AAGCCCCCGA ATAAACGCCA CCGGGCAAGG TGTTACAATA TTTTATGTTG ATAGACTTGG CTACTATCCT TACATAGATT CAATCACAGG AGTAACTGTG AATGGAGGAA TCCCCCAGAA GATTTCCTTA CAAGACCATC TGGACAAAGC TAAGAAAGAC ATTACATTTT ATATGCCAGT AGACAATTTG GGAATGGCTG TTATTGACTG GGAAGAATGG AGACCCACTT GGGCAAGAAA CTGGAAACCT AAAGATGTTT ACAAGAATAG GTCTATTGAA TTGGTTCAGC AACAAAATGT ACAACTTAGT CTCACAGAGG CCACTGAGAA AGCAAAACAA GAATTTGAAA AGGCAGGGAA GGATTTCCTG GTAGAGACTA TAAAATTGGG AAAATTACTT CGGCCAAATC ACTTGTGGGG TTATTATCTT TTTCCGGATT GTTACAACCA TCACTATAAG AAACCCGGTT ACAATGGAAG TTGCTTCAAT GTAGAAATAA AAAGAAATGA TGATCTCAGC TGGTTGTGGA ATGAAAGCAC TGCTCTTTAC CCATCCATTT ATTTGAACAC TCAGCAGTCT CCTGTAGCTG CTACACTCTA TGTGCGCAAT CGAGTTCGGG AAGCCATCAG AGTTTCCAAA ATACCTGATG CAAAAAGTCC ACTTCCGGTT TTTGCATATA CCCGCATAGT TTTTACTGAT CAAGTTTTGA AATTCCTTTC TCAAGATGAA CTTGTGTATA CATTTGGCGA AACTGTTGCT CTGGGTGCTT CTGGAATTGT AATATGGGGA ACCCTCAGTA TAATGCGAAG TATGAAATCT TGCTTGCTCC TAGACAATTA CATGGAGACT ATACTGAATC CTTACATAAT CAACGTCACA CTAGCAGCCA AAATGTGTAG CCAAGTGCTT TGCCAGGAGC AAGGAGTGTG TATAAGGAAA AACTGGAATT CAAGTGACTA TCTTCACCTC AACCCAGATA ATTTTGCTAT TCAACTTGAG AAAGGTGGAA AGTTCACAGT ACGTGGAAAA CCGACACTTG AAGACCTGGA GCAATTTTCT GAAAAATTTT ATTGCAGCTG TTATAGCACC TTGAGTTGTA AGGAGAAAGC TGATGTAAAA GACACTGATG CTGTTGATGT GTGTATTGCT GATGGTGTCT GTATAGATGC TTTTCTAAAA CCTCCCATGG AGACAGAAGA ACCTCAAATT TTCTACAATG CTTCACCCTC CACACTATCT TAA (SEQ ID NO: 29)

HYAL1 cDNA (NM_153281)

ATGGCAGCCC ACCTGCTTCC CATCTGCGCC CTCTTCCTGA CCTTACTCGA TATGGCCCAA GGCTTTAGGG GCCCCTTGCT ACCCAACCGG CCCTTCACCA CCGTCTGGAA TGCAAACACC CAGTGGTGCC TGGAGAGGCA CGGTGTGGAC GTGGATGTCA GTGTCTTCGA TGTGGTAGCC AACCCAGGGC AGACCTTCCG CGGCCCTGAC ATGACAATTT TCTATAGCTC CCAGCTGGGC ACCTACCCCT ACTACACGCC CACTGGGGAG CCTGTGTTTG GTGGTCTGCC CCAGAATGCC AGCCTGATTG CCCACCTGGC CCGCACATTC CAGGACATCC TGGCTGCCAT ACCTGCTCCT GACTTCTCAG GGCTGGCAGT CATCGACTGG GAGGCATGGC GCCCACGCTG GGCCTTCAAC TGGGACACCA AGGACATTTA CCGGCAGCGC TCACGGGCAC TGGTACAGGC ACAGCACCCT GATTGGCCAG CTCCTCAGGT GGAGGCAGTA GCCCAGGACC AGTTCCAGGG AGCTGCACGG GCCTGGATGG CAGGCACCCT CCAGCTGGGG CGGGCACTGC GTCCTCGCGG CCTCTGGGGC TTCTATGGCT TCCCTGACTG CTACAACTAT GACTTTCTAA GCCCCAACTA CACCGGCCAG TGCCCATCAG GCATCCGTGC CCAAAATGAC CAGCTAGGGT GGCTGTGGGG CCAGAGCCGT GCCCTCTATC CCAGCATCTA CATGCCCGCA GTGCTGGAGG GCACAGGGAA GTCACAGATG TATGTGCAAC ACCGTGTGGC CGAGGCATTC CGTGTGGCTG TGGCTGCTGG TGACCCCAAT CTGCCGGTGC TGCCCTATGT CCAGATCTTC TATGACACGA CAAACCACTT TCTGCCCCTG GATGAGCTGG AGCACAGCCT GGGGGAGAGT GCGGCCCAGG GGGCAGCTGG AGTGGTGCTC TGGGTGAGCT GGGAAAATAC AAGAACCAAG GAATCATGTC AGGCCATCAA GGAGTATATG GACACTACAC TGGGGCCCTT CATCCTGAAC GTGACCAGTG GGGCCCTTCT CTGCAGTCAA GCCCTGTGCT CCGGCCATGG CCGCTGTGTC CGCCGCACCA GCCACCCCAA AGCCCTCCTC CTCCTTAACC CTGCCAGTTT CTCCATCCAG CTCACGCCTG GTGGTGGGCC CCTGAGCCTG CGGGGTGCCC TCTCACTTGA AGATCAGGCA CAGATGGCTG TGGAGTTCAA ATGTCGATGC TACCCTGGCT GGCAGGCACC GTGGTGTGAG CGGAAGAGCA TGTGGTGA (서열번호 30)

MMP can be classified as shown in <Table 1>, collagenase (MMP-1, MMP-8, MMP-13 and MMP-18) gelatinase (MMP-2 and MMP-9), stromelysin ( MMP-3, MMP-10, MMP-11 and MMP-17), matrilysins (MMP-7 and MMP-26), membrane types (MMP-14, MMP-15, MMP-16, MMP-24 and MMP-25) ) and other types (MMP-12, MMP-19, MMP-20, MMP-21, MMP-22, MMP-28, MMP-29), etc.

For the preparation of the MMP nucleic acid of the present invention, the nucleotide sequences of MMP1 (NM_002421) and MMP9 (NM_004994) selected from various types of MMP are as follows.

MMP1 cDNA (NM_002421)

ATGCACAGCT TTCCTCCACT GCTGCTGCTG CTGTTCTGGG GTGTGGTGTC TCACAGCTTC CCAGCGACTC TAGAAACACA AGAGCAAGAT GTGGACTTAG TCCAGAAATA CCTGGAAAAA TACTACAACC TGAAGAATGA TGGGAGGCAA GTTGAAAAGC GGAGAAATAG TGGCCCAGTG GTTGAAAAAT TGAAGCAAAT GCAGGAATTC TTTGGGCTGA AAGTGACTGG GAAACCAGAT GCTGAAACCC TGAAGGTGAT GAAGCAGCCC AGATGTGGAG TGCCTGATGT GGCTCAGTTT GTCCTCACTG AGGGGAACCC TCGCTGGGAG CAAACACATC TGACCTACAG GATTGAAAAT TACACGCCAG ATTTGCCAAG AGCAGATGTG GACCATGCCA TTGAGAAAGC CTTCCAACTC TGGAGTAATG TCACACCTCT GACATTCACC AAGGTCTCTG AGGGTCAAGC AGACATCATG ATATCTTTTG TCAGGGGAGA TCATCGGGAC AACTCTCCTT TTGATGGACC TGGAGGAAAT CTTGCTCATG CTTTTCAACC AGGCCCAGGT ATTGGAGGGG ATGCTCATTT TGATGAAGAT GAAAGGTGGA CCAACAATTT CAGAGAGTAC AACTTACATC GTGTTGCGGC TCATGAACTC GGCCATTCTC TTGGACTCTC CCATTCTACT GATATCGGGG CTTTGATGTA CCCTAGCTAC ACCTTCAGTG GTGATGTTCA GCTAGCTCAG GATGACATTG ATGGCATCCA AGCCATATAT GGACGTTCCC AAAATCCTGT CCAGCCCATC GGCCCACAAA CCCCAAAAGC GTGTGACAGT AAGCTAACCT TTGATGCTAT AACTACGATT CGGGGAGAAG TGATGTTCTT TAAAGACAGA TTCTACATGC GCACAAATCC CTTCTACCCG GAAGTTGAGC TCAATTTCAT TTCTGTTTTC TGGCCACAAC TGCCAAATGG GCTTGAAGCT GCTTACGAAT TTGCCGACAG AGATGAAGTC CGGTTTTTCA AAGGGAATAA GTACTGGGCT GTTCAGGGAC AGAATGTGCT ACACGGATAC CCCAAGGACA TCTACAGCTC CTTTGGCTTC CCTAGAACTG TGAAGCATAT CGATGCTGCT CTTTCTGAGG AAAACACTGG AAAAACCTAC TTCTTTGTTG CTAACAAATA CTGGAGGTAT GATGAATATA AACGATCTAT GGATCCAGGT TATCCCAAAA TGATAGCACA TGACTTTCCT GGAATTGGCC ACAAAGTTGA TGCAGTTTTC ATGAAAGATG GATTTTTCTA TTTCTTTCAT GGAACAAGAC AATACAAATT TGATCCTAAA ACGAAGAGAA TTTTGACTCT CCAGAAAGCT AATAGCTGGT TCAACTGCAG GAAAAATTGA (SEQ ID NO: 31)

MMP9 cDNA (NM_004994)

ATGAGCCTCT GGCAGCCCCT GGTCCTGGTG CTCCTGGTGC TGGGCTGCTG CTTTGCTGCC CCCAGACAGC GCCAGTCCAC CCTTGTGCTC TTCCCTGGAG ACCTGAGAAC CAATCTCACC GACAGGCAGC TGGCAGAGGA ATACCTGTAC CGCTATGGTT ACACTCGGGT GGCAGAGATG CGTGGAGAGT CGAAATCTCT GGGGCCTGCG CTGCTGCTTC TCCAGAAGCA ACTGTCCCTG CCCGAGACCG GTGAGCTGGA TAGCGCCACG CTGAAGGCCA TGCGAACCCC ACGGTGCGGG GTCCCAGACC TGGGCAGATT CCAAACCTTT GAGGGCGACC TCAAGTGGCA CCACCACAAC ATCACCTATT GGATCCAAAA CTACTCGGAA GACTTGCCGC GGGCGGTGAT TGACGACGCC TTTGCCCGCG CCTTCGCACT GTGGAGCGCG GTGACGCCGC TCACCTTCAC TCGCGTGTAC AGCCGGGACG CAGACATCGT CATCCAGTTT GGTGTCGCGG AGCACGGAGA CGGGTATCCC TTCGACGGGA AGGACGGGCT CCTGGCACAC GCCTTTCCTC CTGGCCCCGG CATTCAGGGA GACGCCCATT TCGACGATGA CGAGTTGTGG TCCCTGGGCA AGGGCGTCGT GGTTCCAACT CGGTTTGGAA ACGCAGATGG CGCGGCCTGC CACTTCCCCT TCATCTTCGA GGGCCGCTCC TACTCTGCCT GCACCACCGA CGGTCGCTCC GACGGCTTGC CCTGGTGCAG TACCACGGCC AACTACGACA CCGACGACCG GTTTGGCTTC TGCCCCAGCG AGAGACTCTA CACCCGGGAC GGCAATGCTG ATGGGAAACC CTGCCAGTTT CCATTCATCT TCCAAGGCCA ATCCTACTCC GCCTGCACCA CGGACGGTCG CTCCGACGGC TACCGCTGGT GCGCCACCAC CGCCAACTAC GACCGGGACA AGCTCTTCGG CTTCTGCCCG ACCCGAGCTG ACTCGACGGT GATGGGGGGC AACTCGGCGG GGGAGCTGTG CGTCTTCCCC TTCACTTTCC TGGGTAAGGA GTACTCGACC TGTACCAGCG AGGGCCGCGG AGATGGGCGC CTCTGGTGCG CTACCACCTC GAACTTTGAC AGCGACAAGA AGTGGGGCTT CTGCCCGGAC CAAGGATACA GTTTGTTCCT CGTGGCGGCG CATGAGTTCG GCCACGCGCT GGGCTTAGAT CATTCCTCAG TGCCGGAGGC GCTCATGTAC CCTATGTACC GCTTCACTGA GGGGCCCCCC TTGCATAAGG ACGACGTGAA TGGCATCCGG CACCTCTATG GTCCTCGCCC TGAACCTGAG CCACGGCCTC CAACCACCAC CACACCGCAG CCCACGGCTC CCCCGACGGT CTGCCCCACC GGACCCCCCA CTGTCCACCC CTCAGAGCGC CCCACAGCTG GCCCCACAGG TCCCCCCTCA GCTGGCCCCA CAGGTCCCCC CACTGCTGGC CCTTCTACGG CCACTACTGT GCCTTTGAGT CCGGTGGACG ATGCCTGCAA CGTGAACATC TTCGACGCCA TCGCGGAGAT TGGGAACCAG CTGTATTTGT TCAAGGATGG GAAGTACTGG CGATTCTCTG AGGGCAGGGG GAGCCGGCCG CAGGGCCCCT TCCTTATCGC CGACAAGTGG CCCGCGCTGC CCCGCAAGCT GGACTCGGTC TTTGAGGAGC CGCTCTCCAA GAAGCTTTTC TTCTTCTCTG GGCGCCAGGT GTGGGTGTAC ACAGGCGCGT CGGTGCTGGG CCCGAGGCGT CTGGACAAGC TGGGCCTGG G AGCCGACGTG GCCCAGGTGA CCGGGGCCCT CCGGAGTGGC AGGGGGAAGA TGCTGCTGTT CAGCGGGCGG CGCCTCTGGA GGTTCGACGT GAAGGCGCAG ATGGTGGATC CCCGGAGCGC CAGCGAGGTG GACCGGATGT TCCCCGGGGT GCCTTTGGAC ACGCACGACG TCTTCCAGTA CCGAGAGAAA GCCTATTTCT GCCAGGACCG CTTCTACCGG CGCGTGAGTT CCCGGAGTGA GTTGAACCAG GTGGACCAAG TGGGCTACGT GACCTATGAC ATCCTGCAGT GCCCTGAGGA CTGA (서열번호 32)

<Extracellular matrix degrading enzyme inhibitor: TIMP>

Tissue inhibitors of metalloproteinases (MPs) (TIMPs), four members of the TIMP-1, 2, 3 and 4 family, are endogenous inhibitors of originally secreted metalloproteinases (MPs).

For the preparation of the TIMP nucleic acid of the present invention, the nucleotide sequences of TIMP1 (NM_003254), TIMP2 (NM_003255), TIMP3 (NM_000362) and TIMP4 (NM_003256) are as follows.

TIMP1 cDNA (NM_003254)

ATGGCCCCCT TTGAGCCCCT GGCTTCTGGC ATCCTGTTGT TGCTGTGGCT GATAGCCCCC AGCAGGGCCT GCACCTGTGT CCCACCCCAC CCACAGACGG CCTTCTGCAA TTCCGACCTC GTCATCAGGG CCAAGTTCGT GGGGACACCA GAAGTCAACC AGACCACCTT ATACCAGCGT TATGAGATCA AGATGACCAA GATGTATAAA GGGTTCCAAG CCTTAGGGGA TGCCGCTGAC ATCCGGTTCG TCTACACCCC CGCCATGGAG AGTGTCTGCG GATACTTCCA CAGGTCCCAC AACCGCAGCG AGGAGTTTCT CATTGCTGGA AAACTGCAGG ATGGACTCTT GCACATCACT ACCTGCAGTT TCGTGGCTCC CTGGAACAGC CTGAGCTTAG CTCAGCGCCG GGGCTTCACC AAGACCTACA CTGTTGGCTG TGAGGAATGC ACAGTGTTTC CCTGTTTATC CATCCCCTGC AAACTGCAGA GTGGCACTCA TTGCTTGTGG ACGGACCAGC TCCTCCAAGG CTCTGAAAAG GGCTTCCAGT CCCGTCACCT TGCCTGCCTG CCTCGGGAGC CAGGGCTGTG CACCTGGCAG TCCCTGCGGT CCCAGATAGC CTGA (SEQ ID NO:33)

TIMP2 cDNA (NM_003255)

ATGGGCGCCG CGGCCCGCAC CCTGCGGCTG GCGCTCGGCC TCCTGCTGCT GGCGACGCTG CTTCGCCCGG CCGACGCCTG CAGCTGCTCC CCGGTGCACC CGCAACAGGC GTTTTGCAAT GCAGATGTAG TGATCAGGGC CAAAGCGGTC AGTGAGAAGG AAGTGGACTC TGGAAACGAC ATTTATGGCA ACCCTATCAA GAGGATCCAG TATGAGATCA AGCAGATAAA GATGTTCAAA GGGCCTGAGA AGGATATAGA GTTTATCTAC ACGGCCCCCT CCTCGGCAGT GTGTGGGGTC TCGCTGGACG TTGGAGGAAA GAAGGAATAT CTCATTGCAG GAAAGGCCGA GGGGGACGGC AAGATGCACA TCACCCTCTG TGACTTCATC GTGCCCTGGG ACACCCTGAG CACCACCCAG AAGAAGAGCC TGAACCACAG GTACCAGATG GGCTGCGAGT GCAAGATCAC GCGCTGCCCC ATGATCCCGT GCTACATCTC CTCCCCGGAC GAGTGCCTCT GGATGGACTG GGTCACAGAG AAGAACATCA ACGGGCACCA GGCCAAGTTC TTCGCCTGCA TCAAGAGAAG TGACGGCTCC TGTGCGTGGT ACCGCGGCGC GGCGCCCCCC AAGCAGGAGT TTCTCGACAT CGAGGACCCA TGA (SEQ ID NO:34)

TIMP3 cDNA (NM_000362)

ATGACCCCTT GGCTCGGGCT CATCGTGCTC CTGGGCAGCT GGAGCCTGGG GGACTGGGGC GCCGAGGCGT GCACATGCTC GCCCAGCCAC CCCCAGGACG CCTTCTGCAA CTCCGACATC GTGATCCGGG CCAAGGTGGT GGGGAAGAAG CTGGTAAAGG AGGGGCCCTT CGGCACGCTG GTCTACACCA TCAAGCAGAT GAAGATGTAC CGAGGCTTCA CCAAGATGCC CCATGTGCAG TACATCCATA CGGAAGCTTC CGAGAGTCTC TGTGGCCTTA AGCTGGAGGT CAACAAGTAC CAGTACCTGC TGACAGGTCG CGTCTATGAT GGCAAGATGT ACACGGGGCT GTGCAACTTC GTGGAGAGGT GGGACCAGCT CACCCTCTCC CAGCGCAAGG GGCTGAACTA TCGGTATCAC CTGGGTTGTA ACTGCAAGAT CAAGTCCTGC TACTACCTGC CTTGCTTTGT GACTTCCAAG AACGAGTGTC TCTGGACCGA CATGCTCTCC AATTTCGGTT ACCCTGGCTA CCAGTCCAAA CACTACGCCT GCATCCGGCA GAAGGGCGGC TACTGCAGCT GGTACCGAGG ATGGGCCCCC CCGGATAAAA GCATCATCAA TGCCACAGAC CCCTGA (SEQ ID NO: 35)

TIMP4 cDNA (NM_003256)

ATGCCTGGGA GCCCTCGGCC CGCGCCAAGC TGGGTGCTGT TGCTGCGGCT GCTGGCGTTG CTGCGGCCCC CGGGGCTGGG TGAGGCATGC AGCTGCGCCC CGGCGCACCC TCAGCAGCAC ATCTGCCACT CGGCACTTGT GATTCGGGCC AAAATCTCCA GTGAGAAGGT AGTTCCGGCC AGTGCAGACC CTGCTGACAC TGAAAAAATG CTCCGGTATG AAATCAAACA GATAAAGATG TTCAAAGGGT TTGAGAAAGT CAAGGATGTT CAGTATATCT ATACGCCTTT TGACTCTTCC CTCTGTGGTG TGAAACTAGA AGCCAACAGC CAGAAGCAGT ATCTCTTGAC TGGTCAGGTC CTCAGTGATG GAAAAGTCTT CATCCATCTG TGCAACTACA TCGAGCCCTG GGAGGACCTG TCCTTGGTGC AGAGGGAAAG TCTGAATCAT CACTACCATC TGAACTGTGG CTGCCAAATC ACCACCTGCT ACACAGTACC CTGTACCATC TCGGCCCCTA ACGAGTGCCT CTGGACAGAC TGGCTGTTGG AACGAAAGCT CTATGGTTAC CAGGCTCAGC ATTATGTCTG TATGAAGCAT GTTGACGGCA CCTGCAGCTG GTACCGGGGC CACCTGCCTC TCAGGAAGGA GTTTGTTGAC ATCGTTCAGC CCTGA (SEQ ID NO: 36)

<Vaccination protein>

G protein of rabies virus (RAV-G)

For the preparation of the RAV-G nucleic acid of the present invention, the nucleotide sequence of the rabies virus G protein (RAV-G) is as follows.

RAV-G cDNA

ATGGT GCCCCAGGCC CTGCTCTTCG TCCCGCTGCT GGTGTTCCCC CTCTGCTTCG GCAAGTTCCC CATCTACACC ATCCCCGACA AGCTGGGGCC GTGGAGCCCC ATCGACATCC ACCACCTGTC CTGCCCCAAC AACCTCGTGG TCGAGGACGA GGGCTGCACC AACCTGAGCG GGTTCTCCTA CATGGAGCTG AAGGTGGGCT ACATCAGCGC CATCAAGATG AACGGGTTCA CGTGCACCGG CGTGGTCACC GAGGCGGAGA CCTACACGAA CTTCGTGGGC TACGTGACCA CCACCTTCAA GCGGAAGCAC TTCCGCCCCA CGCCGGACGC CTGCCGGGCC GCCTACAACT GGAAGATGGC CGGGGACCCC CGCTACGAGG AGTCCCTCCA CAACCCCTAC CCCGACTACC ACTGGCTGCG GACCGTCAAG ACCACCAAGG AGAGCCTGGT GATCATCTCC CCGAGCGTGG CGGACCTCGA CCCCTACGAC CGCTCCCTGC ACAGCCGGGT CTTCCCCGGC GGGAACTGCT CCGGCGTGGC CGTGAGCTCC ACGTACTGCA GCACCAACCA CGACTACACC ATCTGGATGC CCGAGAACCC GCGCCTGGGG ATGTCCTGCG ACATCTTCAC CAACAGCCGG GGCAAGCGCG CCTCCAAGGG CAGCGAGACG TGCGGGTTCG TCGACGAGCG GGGCCTCTAC AAGTCCCTGA AGGGGGCCTG CAAGCTGAAG CTCTGCGGCG TGCTGGGCCT GCGCCTCATG GACGGGACCT GGGTGGCGAT GCAGACCAGC AACGAGACCA AGTGGTGCCC CCCCGGCCAG CTGGTCAACC TGCACGACTT CCGGAGCGAC GAGATCGAGC ACCTCGTGGT GGAGGAGCTG GTCAAGAAGC GCGAGGAGTG CCTG GACGCC CTCGAGTCCA TCATGACGAC CAAGAGCGTG TCCTTCCGGC GCCTGAGCCA CCTGCGGAAG CTCGTGCCCG GGTTCGGCAA GGCCTACACC ATCTTCAACA AGACCCTGAT GGAGGCCGAC GCCCACTACA AGTCCGTCCG CACGTGGAAC GAGATCATCC CGAGCAAGGG GTGCCTGCGG GTGGGCGGCC GCTGCCACCC CCACGTCAAC GGGGTGTTCT TCAACGGCAT CATCCTCGGG CCCGACGGCA ACGTGCTGAT CCCCGAGATG CAGTCCAGCC TGCTCCAGCA GCACATGGAG CTGCTGGTCT CCAGCGTGAT CCCGCTCATG CACCCCCTGG CGGACCCCTC CACCGTGTTC AAGAACGGGG ACGAGGCCGA GGACTTCGTC GAGGTGCACC TGCCCGACGT GCACGAGCGG ATCAGCGGCG TCGACCTCGG CCTGCCGAAC TGGGGGAAGT ACGTGCTGCT CTCCGCCGGC GCCCTGACCG CCCTGATGCT GATCATCTTC CTCATGACCT GCTGGCGCCG GGTGAACCGG AGCGAGCCCA CGCAGCACAA CCTGCGCGGG ACCGGCCGGG AGGTCTCCGT GACCCCGCAG AGCGGGAAGA TCATCTCCAG CTGGGAGTCC TACAAGAGAGCG GCGGCGAGAC CGGGCTGTGA GGACTAGTTAGTGA No. 37)

RSV-F protein of RSV long strain

For the preparation of the RSV-F nucleic acid of the present invention, the base sequence of the RSV-F protein of the respiratory syncytial virus (RSV) long strain is as follows.

RSV-F long (GC) cDNA

ATGGAGC TGCCCATCCT CAAGGCCAAC GCCATCACCA CCATCCTGGC GGCCGTGACG TTCTGCTTCG CCAGCTCCCA GAACATCACC GAGGAGTTCT ACCAGAGCAC CTGCTCCGCC GTCAGCAAGG GCTACCTGTC CGCCCTCCGG ACCGGGTGGT ACACGAGCGT GATCACCATC GAGCTGTCCA ACATCAAGGA GAACAAGTGC AACGGCACCG ACGCGAAGGT GAAGCTGATC AACCAGGAGC TCGACAAGTA CAAGAACGCC GTCACCGAGC TGCAGCTGCT CATGCAGAGC ACGACCGCCG CCAACAACCG CGCGCGGCGC GAGCTGCCGC GGTTCATGAA CTACACCCTG AACAACACCA AGAAGACGAA CGTGACCCTC TCCAAGAAGC GCAAGCGGCG CTTCCTGGGG TTCCTGCTCG GCGTGGGGAG CGCCATCGCC TCCGGCATCG CCGTCAGCAA GGTGCTGCAC CTGGAGGGCG AGGTGAACAA GATCAAGTCC GCCCTCCTGA GCACCAACAA GGCGGTCGTG TCCCTGAGCA ACGGGGTGTC CGTCCTCACC AGCAAGGTGC TGGACCTGAA GAACTACATC GACAAGCAGC TCCTGCCCAT CGTGAACAAG CAGTCCTGCC GGATCAGCAA CATCGAGACG GTCATCGAGT TCCAGCAGAA GAACAACCGC CTGCTCGAGA TCACCCGGGA GTTCAGCGTG AACGCCGGCG TGACCACCCC CGTCTCCACG TACATGCTGA CCAACAGCGA GCTGCTCTCC CTGATCAACG ACATGCCCAT CACCAACGAC CAGAAGAAGC TGATGAGCAA CAACGTGCAG ATCGTGCGCC AGCAGTCCTA CAGCATCATG TCCATCATCA AGGAGGAGGT CCTCGCCTAC GTGGTGCAGC TG CCGCTGTA CGGGGTCATC GACACCCCCT GCTGGAAGCT CCACACGAGC CCCCTGTGCA CCACCAACAC CAAGGAGGGC TCCAACATCT GCCTGACGCG GACCGACCGC GGGTGGTACT GCGACAACGC CGGCAGCGTG TCCTTCTTCC CCCAGGCCGA GACCTGCAAG GTCCAGAGCA ACCGGGTGTT CTGCGACACC ATGAACTCCC TCACGCTGCC GAGCGAGGTG AACCTGTGCA ACGTCGACAT CTTCAACCCC AAGTACGACT GCAAGATCAT GACCTCCAAG ACCGACGTGA GCTCCAGCGT GATCACCTCC CTCGGCGCGA TCGTCAGCTG CTACGGGAAG ACGAAGTGCA CCGCCAGCAA CAAGAACCGC GGCATCATCA AGACCTTCTC CAACGGGTGC GACTACGTGA GCAACAAGGG CGTGGACACC GTCTCCGTGG GCAACACCCT GTACTACGTG AACAAGCAGG AGGGGAAGAG CCTGTACGTC AAGGGCGAGC CCATCATCAA CTTCTACGAC CCCCTCGTGT TCCCGTCCGA CGAGTTCGAC GCCAGCATCT CCCAGGTGAA CGAGAAGATC AACCAGAGCC TGGCCTTCAT CCGGAAGTCC GACGAGCTGC TGCACCACGT CAACGCCGGG AAGAGCACGA CCAACATCAT GATCACCACC ATCATCATCG TGATCATCGT GATCCTCCTG TCCCTGATCG CGGTCGGCCT CCTGCTGTAC TGCAAGGCCC GCAGCACGCC CGTGACCCTC TCCAAGGACC AGCTGA (서열번호 38)

1.2. Preparation of target protein nucleic acids

The target protein of the target protein nucleic acid used in the present invention is a therapeutic protein (sTNFR2, EPO, G-CSF), an extracellular matrix degrading enzyme (HYAL 1, Hyaluronidase PH20, MMP1, MMP 8, MMP 13), extracellular matrix decomposition ORFs encoding enzyme inhibitors (TIMP 1, TIMP 2, TIMP 3 and TIMP 4) and proteins of interest for vaccination (RAV-G and RSV-F proteins).

In the present invention, the target protein DNA fragment was synthesized in the order of <T7 promoter - 5'UTR - target protein ORF DNA - muag - A64 - C30 - histone-SL> (TriLink Biotenologies, USA). Amplification using ' -GAATTC TAAT ACGACTCACT ATAGGGGAGA CCACAACGGT TTCCCTCTAG AAAGAACC AT GAACACGATT AACA -3' (SEQ ID NO: 39) and reverse primer 5'- GGATCC TGGTG GCTCTAAAA GAGCCTTTGG-3' (SEQ ID NO: 40). Here the target sequence of T7pol is underlined. Both ends of the primers used include ECoRI/BamHI recognition sequences.

The amplified PCR product was digested using EcoRI/BamHI enzymes, and then cloned using a pUC19 vector linearized with EcoRI/BamHI enzymes and T4 DNA ligase. The reaction solution was transformed into E. coli, and the plasmid was isolated from the selected strain, treated with EcoRI/BamHI restriction enzymes, and then electrophoresed (FIG. 1).

In the present invention, a nucleic acid comprising a target protein ORF including a capped chemically modified nucleotide was prepared as follows.

In order to synthesize the capped target protein mRNA, first, the forward primer SEQ ID NO: 20 and It was amplified using reverse primer SEQ ID NO:21. Here the target sequence of T7pol is underlined. The PCR product was transcribed in vitro as a template (FIG. 2).

Using the PCR product, T7 promoter-target protein DNA as a template, using the HighYield T7 ARCA mRNA Synthesis Kit (m5CTP/Ψ-UTP) (Jena BioSCienCe, USA) to synthesize an expression cassette containing the capped target protein-modified mRNA in vitro. did.

Specifically, a reaction solution containing anti-reverse Cap analog [(ARCA) (3′-O-Me-7mG(5′)ppp(5′)G Cap analog)] and chemically modified nucleotides (6 mM In vitro transcription was performed in ARCA, 1.5 mM GTP, 7.5 mM 5-Methyl-CTP, 7.5 mM Pseudo-UTP, 7.5 mM ATP). DNA nuClease was added to remove the dsDNA template, and the remaining capped target protein-modified mRNA was purified using ethanol precipitation or spin column membrane.

As a control, the capped target protein mRNA synthesis by adding a 5'Cap structure to the RNA transcript obtained from the T7 promoter-target protein DNA template was performed using the Hiscribe ^™ T7 ARCA mRNA Kit (NEB, USA). 5')ppp(5')G; m7GpppG) was added.

Addition and Stability of Cholesterol

The oligo-chol of the present invention was prepared as a structure in which cholesterol (chol) was conjugated with an oligonucleotide consisting of 20 nucleotide sequences and a complementary nucleotide sequence at the 3' end of the nucleic acid (Bioneer, Korea).

An RNA-oligo-chol expression cassette hybrid was prepared by hybridizing the capped nucleic acid or the capped modified nucleic acid prepared in the above section with the oligo-chol prepared below, in which the nucleic acid (RNA) and oligo-chol were complementary to each other.

After mixing 15 pmole of various types of nucleic acids and 15 pmole of oligo-chol, nuclease free water was added to make a total of 5 ul, followed by heating at 95°C for 5 minutes, followed by hybridization at 25°C for 1 hour. The hybridization ratio was 1:1, 1:5, and 1:10 in the molar ratio of nucleic acid and oligo-chol, and as a result of retardation assay using 2% agarose gel, it was confirmed as a single band when hybridized at 1:1.

The stability of the prepared various RNA-expressing cassettes and hybrids prepared with oligo-chol was investigated in the serum, and the remaining hybrids were analyzed by processing various RNA-oligo-chol expression cassettes prepared in the serum solution. did.

As shown in <Fig. 3>, the prepared capped nucleic acid, capped modified nucleic acid, and capped modified nucleic acid with cholesterol added to the three ends were treated for various times in cell culture medium, and then RT-PCR was performed to confirm in vitro stability. .

The prepared capped modified nucleic acid had excellent in vitro safety than the capped nucleic acid, and the capped modified RNA-oligo-chol expression cassette conjugate with cholesterol added to the 3' end had the highest stability.

Addition of video material

The prepared Rhodamine Labeled Oligo and FITC Labeled Oligo are custom-made (Trilink Biotechnologies, USA), and the prepared nucleic acid and the fluorescent material-labeled oligo are mixed in a certain ratio (W/W, 1:1 to 0.1) to prepare Rhodamine. It can be used for the preparation of multiparticulates with B or FITC.

Example 2. Fabrication Design of Multilayer Polyelectrolyte Nanoparticles (PEMN)

The target protein of the nucleic acid used in the present invention is a therapeutic protein (sTNFR2, EPO, G-CSF), an extracellular matrix degrading enzyme (HYAL 1, Hyaluronidase PH20, MMP1, MMP 8, MMP 13), an extracellular matrix degrading enzyme ORFs encoding inhibitors (TIMP 1, TIMP 2, TIMP 3 and TIMP 4) and proteins of interest for vaccination (RAV-G and RSV-F proteins).

The particles prepared by self-assembly by mixing the selected nucleic acid with the cationic compound were referred to as core particles. In the present invention, it was divided into a core particle 1.0 consisting of the following nucleic acid and one type of cationic compound (PS, Protamine) and a core particle 2.0 consisting of a nucleic acid and two types of cationic compound (PS/cationic lipid). One or more types of nucleic acids may also be selected, and one or more selected from each of DNA and RNA may be mixed and used. PEM can be prepared by adding one or more types selected from anionic compounds to the prepared core particles.

The preparation of the core particle or PEM of the present invention is performed by adding the anionic compound to the suspension solution in which the anionic nucleic acid and the cationic compound are continuously vortexed in the reaction solution for 30 seconds and continuously vortexed for 30 seconds or/and After the reaction was completed by ultrasonication, the first method of stabilizing at room temperature for 1 hour was used.

The particles prepared by self-assembly by mixing the selected nucleic acid, cationic compound and anionic compound were referred to as 'PEM'. One or more types of the nucleic acid, cationic compound, and anionic compound may be selected for each, and PEM may be prepared by mixing the selected compound.

The second method was used in which the anionic nucleic acid, the cationic compound and the anionic compound were continuously vortexed and/or sonicated for 30 seconds in the reaction solution to complete the reaction and then stabilized at room temperature for 1 hour.

A third method was used for preparing an eluted film prepared by a free standing layer by layer layering technique in which anionic nucleic acids, cationic compounds and anionic compounds were alternately layered on a selected substrate. Preferably, the anionic compound is initially deposited on the substrate. A cationic compound, an active ingredient anionic nucleic acid, a cationic compound, and an active ingredient anionic nucleic acid are stacked on the stacked anionic layer and repeatedly deposited as a unit, and the outermost layer contains a cationic compound or an anionic compound Selected and manufactured. Then, the PEM prepared by various methods was eluted. After preparing each layer, the substrate with the prepared layer was immersed in a cleaning solution and washed.

Example 3. Preparation of core particles

3.1. Preparation of Core Particle 1.0

Use sterile distilled water (DW), PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4; pH 7.4) or RPMI 1640 cell medium without phenol red to stock 2.5 mg/mL each of nucleic acids and PS. prepared as a solution.

The selected nucleic acid contains n nucleotides, resulting in n-1 negative charges per molecule or 1.8×10 ²⁵ negative charges per mole. The PS has a molecular weight of 5.8 kDa and is rich in basic amino acids, particularly arginine (70-80%), so that the cation per mole is 1.26 × 10 ²⁵ . PS neutralizes the negative charge of the nucleic acid phosphate group. Therefore, theoretically, a nucleic acid core particle can be prepared in which the selected nucleic acid and PS have a charge ratio of 1:0.4 or more, that is, nucleic acid is 1 and PS is 0.4 or more.

When cationic PS was added to the anionic nucleic acid solution, an interaction between PS and nucleic acid occurred, and the mixture of PS and nucleic acid can also spontaneously form particles.

The nucleic acid-based core particle of the present invention is sterile distilled water ( DW) or isotonic solution (CM) to prepare core particles consisting of nucleic acid/PS particles.

In the present invention, particles were prepared by adding nucleic acid to an aqueous solution composed of HSA and PS, and this was referred to as nucleic acid/PS core particle 1.1 or core particle 1.1.

Then, the reaction product was stabilized at room temperature for 1 hour and lyophilized to obtain a nucleic acid/PS core particle 1.1 prepared.

In the present invention, PS-TCA, CS, or CS-TCA can be used instead of PS in the nucleic acid/PS core particle 1.0, and the core particles formed therefrom are collectively referred to as 'core particle 1.0', and in detail, the core particle using PS is When using the core particle 1.1, PS-TCA was 1.1, CS was 1.3, and CS-TCA was 1.4.

For the preparation of PS-TCA or CS-TCA, reference was made to the present inventor's patent application (Korean Patent Application: 10-2019-0007873).

These results are not limited to selected nucleic acids, but have been generally observed for other nucleic acids as well.

Particles may be used with a Phoenix RS-VA10 Vortexer (Phoenix Instrument, Germany) (10 sec, 25 W) and a SpeedMixer™ DAC 150.1 CM 41 (HausChild & Co KG, Germany) (5 min, 3000 rpm).

3.2. Evaluation of Core Particle 1.0

Effect of PS on the size of core particle 1.0

The size of the core particles 1.1 prepared in Example 2.1 was determined by Photon Correlation SPEMtroscopy (PCS) using Malvern Zetasizer 3000 HSA (Malvern Panalytical technologies, UK). The size of the core particle 1.1 was expressed as the mean hydrodynamic diameter (dh), and the width of the size distribution was expressed as the polydispersity index (PDI). The software data analysis for the size distribution calculation of the core particle 1.0 was based on the fitting method of non-negative constrained least-squares (NNLS). Measurement was performed after 1 hour of incubation of the mixture to which the components were added.

According to the research results of the present invention, as a result of PCS measured with Malvern Zetasizer 3000 HSA, an initial complex of nucleic acid and PS was formed in the first step, and when a strongly positively charged PS is additionally added, the nucleic acid/PS particle is self-assembled. formed.

Referring to FIG. 4 , the core particle 1.1-DW sample was a very large particle with a nucleic acid:PS (μg/ml) ratio of 30:15, and a large size distribution (PDI >0.2) was observed. Core particle formation was observed by increasing the PS concentration from a nucleic acid:PS ratio of about 30:45 mass ratio or more. Multiparticulates with a small size distribution and most suitable size can be prepared at a PS concentration in which the nucleic acid:PS (μg/ml) ratio is higher than 30:60 mass ratio.

Referring to FIG. 4, the core particle 1.1-CM sample exhibited a small size distribution (PDI ≤0.2) and a dh of about 50 nm at a low PS concentration, and was stabilized while increasing as the PS concentration increased. At a PS concentration in which the nucleic acid:PS ratio was higher than about 30:60 mass ratio, the core particle 1.0-appearing sample showed comparable dh and size distribution data to the core particle 1.0-DW sample.

The core particle 1.1 of the present invention is an ideal drug in distilled water (DW) under the condition that the weight ratio of the nucleic acid and PS (μg/mL) is 30:15 to 150, preferably 30:15 to 90, more preferably 30:75. It has the properties of a carrier.

As a result of studying the effect of PS concentration on particle size, in order to prepare nucleic acid/PS core particle 1.1 having a stable small monomodal size distribution in isotonic solution, a nucleic acid:PS ratio of at least 30:60 mass ratio is ideal.

The above study results are similar to the theoretical predictions. The physicochemical particle properties and biological activity of the core particle 1.0 are determined by the ratio between the positively charged nitrogen (N) of the cationic compound PS and the negatively charged phosphorus (P) of the mRNA. The N/P ratio given here can be obtained by calculating the weight carrying 1 mol unit of charge.

A nucleic acid of 2000 bases has a negative charge of about 340.5 g/mol. Thus, 1 g of nucleic acid is equal to 2.94 mmol of negative charge. At physiological pH, the phosphate group in the RNA backbone is anionic based on the associated pKa of the phosphate group. The amount of positive charge on PS is determined by the sulphate concentration provided by the manufacturer. PS has a positive charge of 3.6 mmol per gram of substance. Potentiometric titration (supplementation) binds 0.35 mmol at pH 7.2 (preparation conditions). In summary, 1 g material corresponds to 2.94 mmol negatively charged nucleic acid and 3.6 mmol positively charged PS.

These results were not limited to selected mRNAs, but were generally observed for other nucleic acids as well.

suture and release

In the preparation of core particle 1.0, positively charged PS and negatively charged nucleic acid are mixed to form particles according to electrostatic interaction. The formed core particles 1.0 were left at room temperature for 30 minutes for stabilization. As a solvent for mixing PS and nucleic acid, a DEPC-treated RNase-free solvent was used to prevent degradation of the nucleic acid.

The binding of nucleic acids to PS was measured by performing electrophoresis on a 2% agarose gel. Nucleic acids were stained with 0.5 μg/ml of EtBr, and electrophoresis was performed for 20 minutes at 80 Volts using 1x TBE buffer.

In order to confirm the result of nucleic acid encapsulation by PS, the degree of movement of the band in the electrophoresis image of the core particle 1.0 prepared according to the mass ratio of nucleic acid and PS was confirmed. Nucleic acid/PS (μg/ml) ratio required to adsorb nucleic acid is 30:15, 30:30, 30:60, 30:75, 30:90 and 30:150. The nucleic acid is mixed with PS and reacted for 1 hour. After stabilization, centrifugation and electrophoresis of the supernatant were investigated.

Referring to <FIG. 5>, 82% unbound nucleic acid was found at a nucleic acid/PS (μg/ml) ratio of 30:15 mass ratio of the core particle 1.1. At a mass ratio of 30:30, 40% nucleic acid remained in solution. In the 30:75, 30:90 and 30:150 mass ratios, the nucleic acid was quantitatively present in the form of particles. This is because, under the influence of positively charged PS, as the amount of PS added increases, it reacts strongly with the nucleic acid to form a more stable particle form. In the core particle 1.1, it can be seen that the mRNA and PS complex are strongly formed as PS increases at a nucleic acid:PS (㎍/ml) ratio of 1:1 or more. This trend is similar to other previous reports.

The stability of core particle 1.0 and the release of nucleic acid were investigated in PBS at 37°C. After 24 hours, no nucleic acid release occurred at the nucleic acid:PS ratio of 30:90 and 30:150. A small release of nucleic acid was shown at 30:75 condition, and 8.0% of unbound nucleic acid was found. Nucleic acid of the core particle: PS (μg/ml) ratio 30:15 mass ratio ratio 90.0%, and nucleic acid of 63.0% at 30:30 mass ratio were found in the solution.

As the PS increased in the nucleic acid:PS (μg/ml) ratio of 1:1 or higher in the core particle 1.0, mRNA strongly bound to PS and the release degree was very high. This trend is similar to other previous reports.

These results are not limited to selected nucleic acids, but have been generally observed for other nucleic acids as well.

Example 4. Core Particle 2.0

4.1. Preparation of Core Particle 2.0

When the cationic compound PS and DOTAP were used together to prepare core particle 2.0, the concentration of the positive charge generated from PS varied from 15% (low), 45%/55% (medium), and 85% (high). made it

It was prepared by three different assembly routes, starting with the PS/nucleic acid core and adding DOTAP (PC), or vice versa, starting with the DOTA/nucleic acid core and adding PS (DC), and first with the nucleic acid, DOTAP and Core particles 2.0 were prepared by a process (MP) of mixing PS.

Then, the reaction was stabilized at room temperature for 1 hour and lyophilized to obtain nucleic acid/PS/DOTAP core particles 2.0 prepared.

Particles may be used with a Phoenix RS-VA10 Vortexer (Phoenix Instrument, Germany) (10 sec, 25 W) and a SpeedMixer™ DAC 150.1 CM 41 (HausChild & Co KG, Germany) (5 min, 3000 rpm).

4.2. Evaluation of Core Particle 2.0

When the cationic compound PS and DOTAP were used together to prepare core particle 2.0, the concentration of the positive charge generated from PS varied from 15% (low), 45%/55% (medium), and 85% (high). made it

The physicochemical particle properties and biological activity of Core Particle 2 are determined by the ratio between the positively charged nitrogen (N) of the cationic compounds PS and DOTAP and the negatively charged phosphorus (P) of mRNA. The N/P ratio given here can be obtained by calculating the weight carrying 1 mol unit of charge.

A nucleic acid of 2,000 bases has a negative charge of about 340.5 g/mol. Thus, 1 g of nucleic acid is equal to 2.94 mmol of negative charge. At physiological pH, the phosphate group in the RNA backbone is anionic based on the associated pKa of the phosphate group. The amount of positive charge on PS is determined by the sulphate concentration provided by the manufacturer. PS has a positive charge of 3.6 mmol per gram of substance. Potentiometric titration (supplementation) binds 0.35 mmol at pH 7.2 (preparation conditions). The molecular weight of DOTAP is 698.5 g/mol and there is one quaternary amine group per molecule. Therefore, it is ionized regardless of the pH value. The positive charge per g of DOTAP is 1.43 mmol. In summary, 1 g material corresponds to 2.94 mmol negatively charged nucleic acid, 3.6 mmol positively charged PS and 1.43 mmol positively charged DOTAP.

Therefore, all core particles of 2.0 can be ideally prepared at a final N/P ratio of 2. This is similar to the result of studying the effect of PS concentration on particle size in the preparation of core particle 1.0 in Example 2.1. As a result of Example 2.1, in order to prepare nucleic acid/PS core particle 1.0 having a stable small monomodal size distribution in isotonic solution, it was confirmed that the nucleic acid:PS (㎍/ml) ratio was at least 30:60 mass ratio.

For each assembly route, three different N/P ratio compositions of PS and DOTAP were prepared. In the case of 0.3/1 intermediate PC (low) and DC (high), mRNA was only partially bound to the cationic compound. Therefore, for the final PC (low) and DC (high) particles, the interaction of the second added material with the preformed particles and free RNA should be considered, which may lead to a more complex Core Particle 2.0 construction.

Core particles 2.0 with limited size (particle size <300 nm, PDI <0.3) can be obtained regardless of the PS:DOTAP ratio in the assembly route and the selected final N/P ratio of 2. Under these conditions, all core particles of 2.0 are positive zeta and potential > 20 mV. As the amount of PS (low > medium > high) increases, MP core particle 2 decreases in size, while DC core particle 2.0 increases in size, and PC core particle 2.0 tends not to change significantly.

To evaluate the RNA encapsulated in the core particle 2.0, gel retardation assay was performed to evaluate the amount. All core particles 2.0 were confirmed to be loaded with RNA. In the core particle 2.0 synthesized with MP, the amount of RNA loaded twice compared to other assembly pathways (PC/DC) was measured (FIG. 6).

To further differentiate Core Particle 2.0, heparin-triggered RNA release was investigated. Since anionic heparin interacts with the positive charge of DOTAP and PS, heparin competes with anionic mRNA at the site of interaction to release RNA from the cationic compound. The added heparin concentration was increased from 10-fold to 100-fold excess compared to the RNA concentration (% w/w).

Particles with an N/P ratio of 2 composed only of DOTAP/RNA or PS/RNA were evaluated compared to core particles obtained by other assembly routes with similar absolute DOTAP/PS/RNA ratios. For DOTAP/RNA lipoplexes, even a 100-fold excess of heparin could not observe RNA release, whereas for PS/RNA polyplexes, a 10-fold excess was sufficient to release RNA.

The release of 80% RNA of bound RNA in Core Particle 2.0 was clearly different depending on the manufacturing route in Core Particle 2.0. While PC (medium) core particle 2 produced almost no mRNA release, DC (low) showed moderate RNA release, and MP (medium) core particle 2.0 produced the most RNA release. The high RNA release from the MP (medium) core particle is thought to be due to the loading of a larger amount of RNA.

Example 5. HSA Multiparticulates

In the present invention, 'HSA multiparticulate' including nucleic acid/PS/HSA multiparticle 1.1, nucleic acid/PS-TCA/HSA multiparticle 1.2, nucleic acid/CS/HSA multiparticle 1.3 or nucleic acid/CS-TCA/HSA multiparticle 1.4 was collectively called.

5.1. Preparation of HSA composite particles

The target protein of the nucleic acid used in the present invention is a therapeutic protein (sTNFR2, EPO, G-CSF), an extracellular matrix degrading enzyme (HYAL 1, Hyaluronidase PH20, MMP1, MMP 8, MMP 13), an extracellular matrix degrading enzyme ORFs encoding inhibitors (TIMP 1, TIMP 2, TIMP 3 and TIMP 4) and proteins of interest for vaccination (RAV-G and RSV-F proteins).

The HSA multiparticulates of this example used nucleic acids encoding therapeutic proteins and vaccine proteins, but are not limited thereto.

The selected nucleic acid, cationic protein protamine (PS) and anionic protein human serum albumin (HSA) solutions were each prepared in distilled water (DW), PBS buffer (NaCl 137 mM, KCl 2.7 mM, Na2HPO4 10 mM, KH2PO4 1.8 mM; pH 7.4) or It was prepared at a concentration of 2.5 mg/ml using RPMI 1640 cell medium without phenol red.

0-2000 μg of HSA was added to 15-150 μg of PS and then mixed for 5 seconds. Then, the 30 μg nucleic acid prepared above was added to reach a final volume of 1 ml, and vortexed for 5 seconds to prepare particles.

In another method, the order of addition of the three types of compounds may be arbitrarily determined.

Another method may be prepared by mixing the above three types of compounds.

Then, the reactant was left at room temperature for 1 hour and freeze-dried for 2 days to obtain a prepared composite particle, which was referred to as 'nucleic acid/PS/HSA composite particle 1.1 or HSA composite particle 1.1'.

Here, the nucleic acid may be any one type selected from the above various types or a mixture of a plurality of types.

Here, PS-TCA, CS (Chitosan) or CS-TCA may be used instead of PS. For the preparation of PS-TCA or CS-TCA, reference was made to the present inventor's patent application (Korean Patent Application: 10-2019-0007873).

PS and DOTAP can be selected instead of PS and their concentrations can be varied to 15% (low), 45%/55% (medium) and 85% (high) of the positive charge generated from PS. may be

5.2. Evaluation of HSA Multiparticulates

Effect of HSA on particle size

Referring to FIG. 7 , HSA composite particles 1.1-DW was obtained by adding 100 to 500 μg of HSA to 1 ml of distilled water containing 30 μg nucleic acid and 90 μg PS and mixing, dh was in the range of 250 to 280 nm and PDI was ≤ 0.1. In the case of nucleic acid/PS/HSA complex particles 1.1-DW, when HSA of 1000~2000㎍ was increased, dh increased and PDI became more than 0.2 (red bar graph).

The HSA composite particles 1.1-DW formed small particles in the presence of a small amount of HSA, but as the HSA increased, the particles aggregated into a large structure. These results indicate that the high concentration of HSA negatively affects the manufacturing process in DW, resulting in less stable particles.

Referring to FIG. 7 , the stability of HSA complex particle 1.1-isotonic solution was improved when 100-2000 μg of HSA added to 1 ml isotonic solution with 30 μg nucleic acid and 90 μg PS was added and mixed. The dh of these particles decreased from 1-2 mm to 250-400 nm depending on the isotonic solution used. Particles with dh = 245 nm and PDI ≤ 0.15 were formed in isotonic solution of cell medium RPMI 1640 without phenol red. This was referred to as nucleic acid/PS/HSA multiparticulate 1.1-RPMI.

7, the optimized concentration of HSA is about 100 μg/ml in HSA multi-particle 1.1-DW, and about 1000 μg/ml in nucleic acid/PS/HSA multi-particle 1.1-isotonic solution, and the optimized concentration of PS is 15~ It was evaluated in the 150 μg range.

The effect of the HSA on the HSA multiparticulates 1.1 is the HSA multiparticulates 1.2, 1.3. and 1.4 were similarly observed.

These results are not limited to selected nucleic acids, but have been generally observed for other nucleic acids as well.

Example 6. HA PEMN

6.1. Preparation of HA PEMN

The target protein of the nucleic acid used in the present invention is a therapeutic protein (EPO and G-CSF), an extracellular matrix degrading enzyme (HYAL 1, Hyaluronidase PH20, MMP1, MMP 8 and MMP 13), an extracellular matrix degrading enzyme inhibitor (TIMP 1, TIMP 2, TIMP 3 and TIMP 4) and ORFs encoding target proteins for vaccination (RAV-G and RSV-F proteins).

Nucleic acid/PS/HSA complex particles are extracellular matrix degrading enzymes (HYAL 1, Hyaluronidase PH20, MMP1, MMP 8 and MMP 13), extracellular matrix degrading enzyme inhibitors (TIMP 1, TIMP 2, TIMP 3 and TIMP 4) A nucleic acid encoding a nucleic acid was used, but is not limited thereto.

Particles formed by self-assembly by mixing the core particles and anionic polysaccharides or particles formed by ultrasonic spraying while mixing mRNA, cationic polypeptides and anionic polysaccharides were referred to as PEMs. In the present invention, the following nucleic acid/PS/HA PEM 1.1 and nucleic acid/PS/HA-TCA PEM 1.2 are referred to as 'HA PEM'.

For the preparation of the nucleic acid/PS/HA PEM, 1 mg/mL of the anionic polysaccharide hyaluronic acid (HA, 83 kDa) was dissolved in sterile distilled water or isotonic solution for surface modification of the previously prepared core particle 1.0 again to 0.1 % stock solution.

The nucleic acid: PS (μg/ml) ratio of the lyophilized core particles 1.0 prepared at a mass ratio of 30:75 was dissolved in sterile distilled water or isotonic solution at 1 mg/mL to prepare a 0.1% core particle 1.1 solution.

The prepared 0.1% core particle 1.0 solution and 0.1% HA solution were mixed at 1:0.5, 1:1, 1:1.5 or 1:2 feed mole ratio (nmol), then treated for about 5 minutes and freeze-dried. HA PEM-DW was obtained.

Then, a sonicator or an extruder may be used as appropriate to control the size of the particles. Lyophilization gave HA PEMN 1.1-DW.

Alternatively, in the other preparation of the HA PEMN 1.1, the nucleic acid/PS]:HA or [nucleic acid/PS]:HA addition molar ratio (feed mole ratio, nmol) is the composite particle and the HA addition molar ratio 1:0.5, 1:1 , 1:1.5 or 1:2, after mixing nucleic acid, PS and HA for about 5 minutes, a sonicator or extruder can be used appropriately to control the size of the particles, and freeze-dried HA PEMN 1.1 was obtained.

Here, HA-TCA may be used instead of HA. The HA-TCA conjugate prepared with reference to the inventor's patent application (Korean Patent Application: 10-2019-0007873) is obtained by sterilizing the HA-TCA conjugate prepared under the condition that the addition molar ratio of HA and TCA is 1:10 in sterile distilled water or isotonic solution. A 0.1% HA-TCA aqueous solution was prepared by dissolving at 1 mg/mL.

Nucleic acid/PS/HA-TCA HA composite particle 1.2 of the present invention is slowly vortexed at a molar ratio of 1:0.5, 1:1, 1:1.5 or 1:2 addition to the 0.1% core particle 1.1 solution and 0.1% HA-TCA aqueous solution. It was added while stabilizing at room temperature for 1 hour and lyophilized to obtain.

Instead of the PS, the PS and the DOTAP concentration may be used by varying the desired positive charge ratio from PS to 15% (low), 45%/55% (medium), and 85% (high).

Homogenization using an extruder

A control is a PEM prepared from a suspension solution obtained by mixing the active ingredient nucleic acid, cationic compound and anionic compound in an aqueous solution obtained by a general conventional method (bottom-up method, bottom-up method).

The suspension solution containing the PEM prepared in the bottom-up manner was subjected to a preliminary experiment for manufacturing PEMN according to various conditions with a LIPEX ^TM extruder (Northern Lipids, Canada).

The prepared PEM suspension solution was passed through a PORETICS ^TM PETE membrane (Cat.No.T01CP02500) with an average pore size of 0.1㎛ stacked in 5 layers at 200psi using the LIPEX ^TM extruder set as the preliminary experiment 5 times reciprocally. After stabilizing at room temperature for 1 hour, the formed HA PEMN was harvested and analyzed.

6.2. Assessment of HA PEMN

Morphological analysis and encapsulation confirmation

Particle size, polydispersity index (PDI) and zeta potential analysis were determined using a Zetasizer nano ZSP (Malvern Panalytical Ltd, UK) equipped with a red He/Ne laser (633 nm). Particle size and PDI were measured using a dynamic light scattering (DLS) and quartz flow cell (ZEN0023) with a scattering angle of 173° at 20°C, whereas zeta potential was measured using a disposable folded capillary cell (DTS1070). DLS was able to obtain particle size and size distribution parameters by measuring the diffusion of particles resulting from Brownian motion and using the correlation function in the instrument corresponding to the Stokes-Einstein relationship. These correlation functions are fitted to simple exponentials obtained by cumulative analysis and mean size (z-mean diameter) and distribution width estimates (polydispersity index) obtained. Similarly, the correlation function is also suitable for multiple exponents in which the particle size distribution is obtained with non-negative least squares (NNLS) or constrained normalization (CONTIN). In this work, particle size is measured as the z-mean diameter and the PDI ranges from 0 to 1 corresponding to monodisperse and very broad distribution, respectively. All nanoparticles were dispersed in ultrapure water using a ~ 1:100 v/v dilution factor. All measurements were performed in triplicate and were reported as mean ± standard deviation.

Table 4 shows the results of examining the size distribution of HA PEM 1.1 prepared at various ratios. These results show that when the size of the HA PEM 1.0 is in the 200 nm range, the ratio of nucleic acid/PS:HA is 1:0.5 and 1:1.

DLS analysis result of the prepared HA PEM 1.1 addition molar ratio
(feed mole ratio, nmol) RNA:PS One One One One HA One 1.5 2 2.5 Nucleic acid PS/HA size(nm) 184.8±7.1 182.3±14.1 493.2±22.6 1259±147 PDI 0.30±0.07 0.34±0.04 0.30±0.02 0.35±0.09 Complex efficiency
(% vs RNA) 80.9 71.7 100 100

The two types of materials selected for nucleic acid, PS and HA obtained in the above example were mixed in the reaction solution while sonication on an ice pack for 1 hour without heating, and the remaining materials that were not treated in the previous step were added to the resulting complex solution on ice without heating. After the reaction was completed by sonication on the pack for 1 hour, the particles were analyzed using a Zetasizer for HA PEMN 1.1 formed using a LIPEX ^TM extruder (Northern Lipids, Canada) after maintaining at room temperature for 1 hour. Same as 5.

The distribution and size of the formed HA PEMN 1,1 are shown in FIG. 8 .

Result of DLS analysis of HA PEMN 1.1 Ingredients One 2 3 4 RNA (μg) One One One One PS (μg) 0.5 0.5 One 2 HA (μg) 0.5 One 0.5 One Sonication SiZE (nm) 184.8±7.1 182.3±14.1 493.2±22.6 1259±147 PDI 0.30±0.07 0.34±0.04 0.30±0.02 0.35±0.09 Kcps 80.2 56.4 181.4 225.5 Complex efficiency
(% vs RNA) 80.9 71.7 100 100 Extrusion Size 159.6±1.2 163.2±1.3 186.5±9.9 258.3±11.3 PDI 0.17±0.004 0.21±0.008 0.33±0.05 0.22±0.01 Zeta -23.3±0.9 -16.6±4.6 -18.3±3.4 -8.6±0.8 Kcps 40.5 32.6 22.9 39.7

The HA PEMN 1.0 was prepared at a nucleic acid/PS];HA addition molar ratio of 1:1.5, centrifuged, lyophilized, rehydrated with 5% w/w Trehalose solution, and particle size before and after rehydration was investigated. .

Looking at <Table 6>, there is little change in particle size before and after rehydration by lyophilization, and the particle size increases by about 3 to 4% in PBS than in distilled water (DW).

DLS analysis result by rehydration after lyophilization of HA PEMN 1.1 Nucleic Acid/PS/HA Size(nm) 137.0±30.4 PDI 0.15±0.22 Freeze-drying and rehydration DW PBS Size(nm) 140.7±5.4 150.5±19.7 PDI 0.12±0.06 0.17±0.1 CE(%) 96.4 100

These results are not limited to selected nucleic acids, but have been generally observed for other nucleic acids as well.

HA PEMN drug release profile

The stability of HA PEMN 1.1 and the release of nucleic acids were investigated in the supernatant obtained by centrifugation after treatment in PBS at 37°C for 24 hours. The nucleic acid release profile by the HA PEMN 1.1 was obtained by electrophoresis of the supernatant obtained under various conditions using 1x TBE buffer on a 2% agarose gel at 80 Volt conditions for 20 minutes, and then the nucleic acid was 0.5 μg/ml EtBr (Ethidium bromide) was used for staining.

[Nucleic acid/PS]: Almost no mRNA release occurred in the conditions of 1:1.5 and 1:2 addition molar ratio of HA. [Nucleic acid/PS]:HA addition molar ratio of 1;0.5 to 90.0% and 1:1 to 63.0% mRNA was found in the solution. A small release of nucleic acids was shown in the 1:1.5 condition, and 8.0% of unbound nucleic acids were found. It can be determined that, under the influence of positively charged HA, as the amount of HA added increases, it reacts strongly with the nucleic acid to form a more stable particle form.

The drug release profile over time was investigated at a mass ratio of [nucleic acid/PS]:HA addition of 1:1.5 mass ratio of the multiparticulates (FIG. 9).

The results of reading the band intensities of FIG. 9 are shown in <Table 7>, and HA PEMN 1.1 released 100% of the encapsulated level of nucleic acids accumulated for 120 hours in PBS solution, and 50% release between 72 and 96 hours. was observed.

Hourly Release Profiles of Nucleic Acids from HA PEMN 1.1 Incubation Time (hours) 0 4 8 24 48 72 96 120 Band density(%) 20.0 27.9 25.5 36.0 48.0 42.7 41.2 50.2 Release of RNA
(%, normalization) 0 7.9 5.5 17.0 33.8 43.6 65.3 104.9

Example 7. Preparation of PEMN using FS_PEM

7.1. Production of FS_PEM

The target protein of the nucleic acid used in the present invention is a therapeutic protein (EPO, G-CSF), an extracellular matrix degrading enzyme (HYAL 1, Hyaluronidase PH20, MMP1, MMP 8, MMP 13), an extracellular matrix degrading enzyme inhibitor (TIMP 1, TIMP 2, TIMP 3 and TIMP 4) and ORFs encoding target proteins for vaccination (RAV-G and RSV-F proteins).

Free standing LbL-based PEM (FS_PEM) was built on four substrates: silicon wafers (Si), polystyrene (PS), polypropylene (PP), and Teflon (PTFE). It was washed with ethanol prior to film deposition and rinsed thoroughly with water before drying with a stream of nitrogen.

Multielectrolyte solutions were prepared at various concentrations (1, 3 or 5 mg/mL) in 0.15 M NaCl pH pH 7.4 unless otherwise noted. HA has a negative charge because its pKa is about 2.9, and PS has a positive charge because its pKa is 9.2 to 13.3 and is dissolved in aqueous solution. For pH adjustment, pH 5.5 sodium acetate buffer (0.1 m) was prepared in the presence of additional salt (0.15 m NaCl).

All solutions were made with Milli-Q water and filtered through a Corning 0.22 μm cellulose acetate filter device. The solution pH was adjusted with diluted acetic acid. Glassware was rinsed 5 times with distilled water and 5 times with deionized water before use. Unless otherwise specified, deionized water was used for all experiments.

Anionic HA, cationic PS, active ingredient nucleic acid, PS, and HA were sequentially deposited on the washed slide, and this was repeatedly deposited as a unit to prepare PEM. The washed slides were successively immersed in PS and HA (10 mL) solutions for 15 min each, followed by rinsing twice in 0.15 M NaCl pH 4.5 between solutions.

In another preparation method, FS_PEM was produced using a polypropylene polypropylene scaffold that was first washed with ethanol and water. Polyelectrolyte solutions were alternately deposited on the template to form PS and HA bilayers using a dipping robot specially designed for the automated production of multilayer polyelectrolytes.

Between each polyelectrolyte solution deposition step, the slides are immersed in acetate buffer solution. After the deposition was completed, it was checked whether the film was separated from the substrate without a washing treatment step. We were able to produce defect-free FS PEMs that peel off easily from the substrate due to the weak van der Waals interaction between the polyelectrolyte and the substrate.

The (PS/HA) multilayer film made of Si was not robust, and the film made of the PTFE substrate could not be separated. Conversely, PEMs built on PP and PS substrates were able to separate well just by letting them dry. It is easily handled with tweezers and can be cut into any shape with scissors.

Another LbL technique is to deposit a cationic compound, poly (4-vinylpyridiniomethanecarboxylate) (PVPMC), on a selected substrate, and then use a specially designed dipping robot for anionic HA, cationic PS, and active ingredient nucleic acid. , PS, and HA were sequentially deposited, and PEM was prepared by repeatedly depositing them as one unit. The layer-by-layer (LbL) PEM grew linearly as the number of layers increased. The rate of decomposition of PVPMC-based multilayers can be well controlled by changing the concentration of salts in the aqueous solution. The PVPMC-based PEM can completely separate only the PEM within 15 minutes in 0.9% saline.

Another LbL technology uses a paper made of cellulose fiber as a substrate and inkjet printing, and each ink cartridge in the print head contains a solution containing an active ingredient nucleic acid, a cationic electrolyte solution, and an anionic electrolyte. The containing solution was filled, and a color suitable for each was selected and printed on paper. One layer was made with one printing and one rinse (washing once with excess PBS), and this was repeated to form a multi-layered nano-thin film. Paper-based PEM can only be carefully separated from PEM in 0.9% saline.

7.2. Ultra-high pressure homogenization (UHPH)

The target protein of the nucleic acid used in the present invention is a therapeutic protein (EPO, G-CSF), an extracellular matrix degrading enzyme (HYAL 1, Hyaluronidase PH20, MMP1, MMP 8, MMP 13), an extracellular matrix degrading enzyme inhibitor (TIMP 1, TIMP 2, TIMP 3 and TIMP 4) and ORFs encoding target proteins for vaccination (RAV-G and RSV-F proteins).

The control is a PEM prepared from a suspension solution in which the active ingredient nucleic acid, cationic compound and anionic compound are mixed in a general conventional method (bottom-up method, bottom-up method).

The development of PEMN in the PEM prepared in Examples 6.1 and 7.1 was performed in several steps according to the hybridization sequence of the anionic nucleic acid, the cationic polymer and the anionic polymer and their ratio, the UHPH application pressure and the recycle cycle.

In the case of Example 6.1, the anionic nucleic acid solution and the cationic polymer solution were mixed with shaking (~ 300 rpm, 25° C.) to prepare a suspension solution A in which core particles were formed by electrostatic interaction. Then, the anionic polymer was added while shaking the suspension solution A, followed by ultrasonication to prepare a suspension solution B containing PEM formed by the electrostatic interaction between the core particles and the anionic polymer.

In the case of Example 6.1, the anionic nucleic acid solution, the cationic polymer solution, and the anionic polymer solution were respectively added while shaking, followed by sonication to prepare a suspension solution C containing PEM formed by the electrostatic interaction therebetween. .

In the case of Example 7.1, a solution D containing PEM eluted and prepared in a layer-by-layer method on the substrate was prepared.

The prepared suspension solutions B, C, and D were homogenized with UHPH using a Nano DeBEE 2000 Series Homogenizer (BEE International, USA) to prepare PEMN.

The homogenizer has a structure in which a zirconium nozzle (Z8, diameter: 200 μm) and six zirconium reactors (diameter 0.95 mm) are arranged in parallel. The operating conditions used are pressure (68.9, 137.9, 172.4 and 2000 MPa) and number of recycle cycles (1 and 4).

The prepared PEMN suspension was subjected to DLS analysis, and the results are shown in FIG. 10 .

The PEMN suspension prepared above was mixed with ultrapure water obtained from a purification system (Millipore Elix Essential, Merck KGraA, Germany).

In the present invention, the drug release analysis method was prepared by adding FBS to the particles to prepare a reaction solution having a final 50% FBS concentration, and reacted at 37 ˚C, CO ₂ in an incubator for each time period, followed by centrifugation to analyze the supernatant by gel retardation analysis.

In the present invention, freeze-drying is performed by treating the particle solution at -70 ° C. for O/N, operating the freeze dryer in advance to operate the pressure and temperature to the maximum, and then connecting the treated particles (solid) to the freeze dryer to freeze-drying. proceeded. The freeze-drying time was determined according to the amount and was 1 hour/100 μL. After the freeze-drying was completed, the sample was stored at room temperature using a container separated from the freeze-drying machine while maintaining a vacuum state.

Example 8. HA-aptamer composite particles

HA-aptamer synthesis

HA-aptamer conjugates can be prepared by a coupling reaction between the carboxyl group of HA and the amine group of the aptamer using EDC/NHS chemistry. Briefly, 83 kDa HA (1 g, 20 μmol) was dissolved in 200 mL of sterile distilled water under nitrogen for 5 h. EDC (100.4 mg, 0.52 mmol) and NHS (60.7 mg, 0.52 mmol) were added to the HA solution. After 20 minutes, an aptamer-amine group (99.4 mg, 0.52 mmol) was slowly added. The pH was adjusted to 5.5 with 0.1 M HCl and the solution was reacted overnight at room temperature. Unreacted residues and by-products were removed with a dialysis membrane (MW Cut off = 100 kDa) in distilled water (pH 5). The final product was freeze-dried and stored at -20°C.

HA-DAB-aptamer synthesis

First, HA-DAB (1,4-diaminobutane) is 1-ethyl-3-[3-(dimethylamino)-propyl] carbodiimine (Carboxyl group) of HA (100 g) having a molecular weight of 100 kDa at pH 6.0 After activation with 1-ethyl-3-[3-(dimethylamino)-propyl]Carbodiimide, EDC) and 1-hydroxy bentriazole monohydrate (HOBt), 1,4-diaminobutane ( 1,4-diaminobutane, DAB) was added and synthesized. At this time, the reaction time was varied to 1 hour, 6 hours, and 24 hours to obtain compounds having different substitution rates. HA-DAB was prepared by filtration purification of the compound obtained as described above using a filtration tube (Cut off 100 kDa).

Then, HA-DAB-SPDP in which a disulfide bond was introduced into the prepared HA-DAB was prepared. Specifically, the synthesized HA-DAB (20 mg; 4.8 x 10 ^-5 mol) and succinimidyl 3-(2-pyridyldithio) propionate (Succinimidyl 3-(2-pyridyldithio) propionate; SPDP) (5.6 mg; 1.8 x 10 ^-5 mol) was mixed and reacted to introduce a pyridyl group to the HA chain, followed by filtration purification using a filtration tube (MW Cut off = 100 kDa).

Purified HA-DAB-SPDP was treated with TCEP (Tris(2-carboxyethyl)phosphine) and absorbance was measured at 343 nm to calculate the concentration of the pyridyl group (pyrimidyl group concentration: 34 mM).

The EGFR aptamer (Tan Y et al., Acta Pharmacologica Sinica (2013) 34: 1491-1498) used in this Example is a nucleotide sequence having SEQ ID NO: 42 at the N30 region of SEQ ID NO: 41

5'-GCAATGGTAC GGTACTTCC- (N30)-CAAAAG TGCACGCTAC TTTGCTAA-3' (SEQ ID NO: 41)

5'-TGAATGTTGT TTTTTCTCTT TTCTATAGTA-3' (SEQ ID NO: 42)

The EGFR aptamer was organically synthesized as a single-stranded DNA structure in which a thiol group was introduced at the 3*?**?* end (TriLink Biotechnologies, USA).

EGFR aptamer having a thiol group introduced at the 3' end to the prepared HA-DAB-SPDP solution was added in various ratios from 0.2 to 1 times the concentration of the pyrimidyl group to form HA- with disulfide bonds. A DAB-aptamer conjugate was synthesized.

At this time, the synthesized HA-DAB-aptamer can control the number of conjugated aptamers per 100 kDa HA molecule from 2 to 15.

Identification of HA-DAB-aptamer conjugates

In order to confirm the production of the HA-DAB-aptamer conjugate synthesized in Example 7, high performance liquid chromatography (HPLC) was used. The HPLC conditions used a superdex 200 10/300 GL (GE Health) column, the mobile phase was pH 7.0 50 mM phosphate buffer, the flow rate was 0.5 ml/min, and absorbance was measured at 210 nm and 260 nm.

The result is described in FIG. 11, and the compound was separated and purified based on the result. As can be seen from FIG. 11 , it was confirmed that the HA-DAB-aptamer conjugate production rate (reaction rate) was about 64%.

The conjugate production rate (reaction rate) was calculated as follows:

Reaction rate (%) = {(level of aptamer required to form the conjugate)/(level of aptamer added at the beginning of the reaction) }X100

In addition, it was confirmed whether the separated and purified compound was synthesized through agarose gel electrophoresis and whether it was decomposed by a reducing agent (TCEP), and the results are shown in FIGS. 12A to 12D .

12a is a result of analysis of HA-DAB-aptamer conjugates synthesized with two different cross linkers through agarose electrophoresis. Specifically, in Fig. 12a, HA-ss-aptamer means a conjugate including a disulfide bond (-ss-) between HA and an aptamer, and HA-aptamer is a conjugate connected by a general covalent bond rather than a disulfide bond. (control) means. As shown in the two columns on the right, when the reducing agent TCEP is treated, the aptamer is separated from the conjugate containing the disulfide bond and a band appears at the bottom, whereas in the control group, the aptamer is not separated and remains at the top. .

In addition, FIGS. 12b to 12d show the amount of aptamer remaining through agarose electrophoresis by time by adding FBS to the HA-DAB-aptamer conjugate to evaluate the safety of the HA-DAB-aptamer conjugate ( 12b and 12d, HA-aptamer conjugate means HA-DAB-aptamer conjugate). As a result, it can be confirmed that the HA-DAB-aptamer conjugate remains undecomposed for a long time compared to the HA-DAB-aptamer unconjugated aptamer (naked aptamer).

Preparation of HA-aptamer composite particles

The nucleic acid: PS (μg/ml) ratio of the lyophilized core particles 1.1 prepared at a mass ratio of 30:75 was dissolved in sterile distilled water or isotonic solution at 1 mg/mL to prepare a 0.1% core particle 1.1 solution.

By mixing the prepared 0.1% core particle 1.1 solution and 0.1% HA-aptamer solution at 1:0.5, 1:1, 1:1.5 or 1:2 feed mole ratio (nmol) to prepare composite particles, After stabilization for 1 hour, lyophilization was performed to obtain nucleic acid/PS/HA-aptamer multiparticulate-DW.

Alternatively, in the preparation of the nucleic acid/PS/HA-aptamer composite particle of another method, the nucleic acid/PS]:HA-aptamer addition molar ratio (feed mole ratio, nmol) is the composite particle and the HA-aptamer addition molar ratio After mixing the nucleic acid/PS and HA-aptamer according to 1:0.5, 1:1, 1:1.5 or 1:2, use an appropriate ultrasonicator (soniCator) or extruder ( extruder) can be used. By freeze-drying, a nucleic acid/PS/HA-aptamer multiparticulate-DW was obtained.

Instead of the PS, the PS and DOTAP concentrations may be used with various desired ratios of positive charges from PS to 15% (low), 45%/55% (medium), and 85% (high).

Example 9. Biological evaluation of HSA multiparticulates

293T cells (ATCC CRL-3216™, Thermo Fisher, USA) are grown in DMEM + 2 mM Glutamine + 10% Fetal Bovine Serum (FBS) medium to cover >70% of the culture vessel. After washing with fresh medium, 0 ng, 46.875 ng, 93.75 ng, 187.5 ng, 375 ng, 750 ng, or 1500 ng of [sTNFR2 Nucleic Acid:PS]/HSA Multiparticle 1.1, [G-CSF Nucleic Acid:PS]/HSA Multiparticle 1.1 , [EPO Nucleic acid:PS]/HSA multiparticulates 1.1, [RSV-F nucleic acid:PS]/HSA multiparticulates 1.1, or [RAV-G nucleic acid:PS]/HSA multiparticulates 1.1 were added to the medium, incubated for 6 hours and fresh 293T cells were transfected by washing with medium and re-cultured in fresh medium.

The concentration of each protein secreted in the culture medium was measured in triplicate at 0, 6, 12, 24, and 48 hours post-transfection for each concentration of each nucleic acid.

The secretion of each protein from the transfected 293T cells was determined by Human sTNFR2 ELISA Kit (MyBioSource, USA), G-CSF Human Instant ELISA™ Kit (Invitrogen, USA), EPO Quantikine ELISA™ Kit (R&D Systems, USA), Anti-RSV-F antibody ELISA™ kit (ab94968, abcam, USA) or Human Anti-Respiratory syncytial virus IgG ELISA™ Kit (RSV) (ab108765, abcam, USA) was used (Fig. 13).

Example 10. Biological evaluation of HA PEMN 1.1

HYAL 1, Hyaluronidase PH20 and sPH20

PC3 cell line (ATCC CRL-1435™, Thermo Fisher, USA) was cultured in F-12K Medium (Kaighn's ModifiCation of Ham's F-12 Medium) + 2mM Glutamine + 10% Fetal Bovine Serum (FBS) medium in this example. was used.

2ml of the cell culture solution was put in a 40mm cell culture dish, and the cells were inoculated at a concentration of about 1.2x10 ⁵ and then cultured to the extent that >70% of the culture vessel was covered. After washing with fresh medium, 0 ng, 46.875 ng, 93.75 ng, 187.5 ng, 375 ng, 750 ng or 1500 ng of HYAL 1, PH20 or sPH20 nucleic acid/PS/HA PEMN was added to the medium, incubated for 6 hours, and washed with fresh medium. Washed and incubated again in fresh medium. The concentration of sPH20 secreted in the culture medium was quantified using the sPH20 ELISA™ Kit (Invitrogen, USA) three times at 0, 6, 12, 24, and 48 hours after transfection for each concentration of each nucleic acid. (Fig. 14).

The cells transfected with the various doses of HA PEMN were cultured at 37° C. for 6 hours, washed, and then, 0.5 mg/mL bovine aggrecan (Sigma-Aldrich, USA) was added to 0, 1, 2 together. , 8, 16, 24 and 48 hours. Thereafter, the medium was removed and fixed with 2% glutaraldehyde in PBS (pH 7.4) and replaced with a suspension with 10 ⁹ /mL mouse RBCs. Cells were imaged with a phase-contrast microscope combined with a camera scanner and imaging program (DiagnostiC Instruments, Inc., USA) (FIG. 15).

MMP: MMP1, MMP 8 and MMP 13

Human skin fibroblast CCD-986Sk (ATCC　 CRL-1947™) was obtained from 10% FBS, 0.1 mM non-essential amino acids, 1.5 g/L sodium bicarbonate, 1 mM sodium pyrbate under the conditions of 37°C and 5% carbon dioxide. It was cultured in Iscove's Modified Dulbecco's Medium (IMDM) medium (hereinafter abbreviated as cell medium).

2ml of cell culture solution was placed in a 40mm cell culture dish, and human fibroblasts were inoculated at a concentration of about 1.2x10 ⁵ and then cultured to the extent that >70% of the culture vessel was covered. Human dermal fibroblasts are transfected with 0 ng, 46.875 ng, 93.75 ng, 187.5 ng, 375 ng, 750 ng, or 1500 ng of MMP1, MMP 8 or MMP 13 nucleic acid:PS]/HA PEMN. Secreted MMP concentrations in the culture medium are measured three times at 0, 6, 12, 24, and 48 hours post-transfection for each concentration of each nucleic acid. The secretion of MMP1, MMP 8 or MMP 13 from transfected 293T cells was quantified using MMP1, MMP 8 or MMP 13 Human Instant ELISA™ Kit (Invitrogen, USA) (FIG. 16).

2ml of cell culture solution was placed in a 40mm cell culture dish, and human fibroblasts were inoculated at a concentration of about 1.2x10 ⁵ and then cultured at 37°C, 5% CO2 environment for 24 hours. Thereafter, the medium prepared in Example 7 was replaced with a medium containing MMP1 nucleic acid:PS]/HA PEMN 1.1 (10 or 100 μg/ml, respectively) and cultured for 3 days. Thereafter, the culture medium was harvested and centrifuged for 5 minutes at 4° C., 7500 rpm, and the protein change of type 1 procollagen (Procollagen Type IC Peptide EIA Kit, Takara, Shiga, Japan) was confirmed using the ELISA method. In human fibroblast experiments, it was found that the MMP1 nucleic acid:PS]/HA PEMN treatment group inhibited the production of type 1 procollagen protein in a concentration-dependent manner. This effect was superior when compared to the untreated group with MMP1 nucleic acid:PS]/HA PEMN, and compared to the untreated group, 109% or 138% of the treated group at a dose of 10 or 100 μg/ml, respectively. Inhibition of type 1 procollagen protein was performed.

Through the above results, the cells transfected with the MMP1 nucleic acid:PS]/HA PEMN of the present invention increased the expression of MMP-1, a collagen degrading enzyme, and suppressed the production of type 1 procollagen.

By mixing the sPH20 mRNA expression cassette and the MMP1 RNA expression cassette, and preparing PEMN using PS and HA, it is also possible to efficiently decompose ECM of cancer tissue composed of hyaluronic acid and collagen as main components.

TIMP: TIMP 1, TIMP 2, TIMP 3 and TIMP 4

Human skin fibroblast CCD-986Sk (ATCC　 CRL-1947™) was prepared under the conditions of 37°C and 5% carbon dioxide, with 10% FBS, 0.1 mM non-essential amino acids, 1.5 g/L sodium bicarbonate, 1 mM sodium pyrbate. It was cultured in IMDM medium (hereinafter abbreviated as cell medium) to which was added.

2ml of IMDM culture solution was put in a 40mm cell culture dish, and human fibroblasts were inoculated at a concentration of about 1.2x10 ⁵ , and then cultured at 37°C, 5% CO2 environment for 24 hours. 0ng, 46.875ng, 93.75ng, 187.5ng, 375ng, 750ng, or 1500ng of the prepared TIMP 1, TIMP 2, TIMP 3 or TIMP 4 nucleic acid:PS]/HA After replacing with a medium containing PEMN 1.1 and culturing for 6 hours It was replaced with fresh medium and cultured for 3 days. Thereafter, the culture medium was harvested and centrifuged for 5 minutes at 4° C., 7500 rpm environment, and the TIMP concentration secreted in the culture medium using the ELISA method was 0, 6, 12, 24, and 0 after transfection for each concentration of each nucleic acid. Measured three times at 48 hours. The secretion of TIMP 1, TIMP 2, TIMP 3 or TIMP 4 from transfected human fibroblasts was quantified using TIMP 1, TIMP 2, TIMP 3 or TIMP 4 Human Instant ELISA™ Kit (Invitrogen, USA). (Fig. 17).

2ml of DMEM culture solution was placed in a 40mm cell culture dish, and human fibroblasts were inoculated at a concentration of about 1.2x10 ⁵ and then cultured at 37°C, 5% CO2 environment for 24 hours. Thereafter, the medium prepared in Example 1 was replaced with a medium containing TIMP 1 nucleic acid:PS]/HA PEMN (10 and 100 μg/ml, respectively) and cultured for 3 days. Thereafter, the culture medium was harvested and centrifuged for 5 minutes at 4° C., 7500 rpm, and the protein change of type 1 procollagen (Procollagen Type IC Peptide EIA Kit, Takara, Japan) was confirmed using the ELISA method. In human fibroblast experiments, it was found that the TIMP nucleic acid:PS]/HA PEMN treatment group increased the production of type 1 procollagen protein in a concentration-dependent manner. This effect was found to be superior when compared to the untreated group with TIMP nucleic acid:PS]/HA PEMN, and compared to the untreated group, the treated group improved by 109% or 138% at a dose of 10 or 100 μg/ml, respectively. By showing an increase in the production of the type 1 procollagen protein, it was shown that the effect of increasing the production of the type 1 procollagen protein was greater during the TIMP 1 nucleic acid:PS]/HA PEMN treatment.

Through the above results, the TIMP nucleic acid: PS]/HA PEMN of the present invention acts on the MMP signaling system to promote the production of type 1 procollagen and inhibit the expression of MMP-1, a collagen degrading enzyme. Through this, it was confirmed that there was an effect of effectively preventing the generation of wrinkles and improving the generated wrinkles.

Example 11. Cell uptake experiment of HA-TCA PEMN 1.2

To evaluate the degree of cellular uptake by the bile acid receptor of the [TIMP1 nucleic acid: PS]/HA-TCA (Taurocholate) PEMN 1.2, MDCK without ASBT and ASBT-expressing MDCK-ASBT cells (BioIVT, USA) were used. An experiment was performed using MDCK or MDCK-ASBT cells were cultured in Dulbecco's Modified Eagle's Medium containing 10% FBS.

2ml of DMEM culture solution was placed in a 40mm cell culture dish, and the cells were inoculated at a concentration of about 1.2x10 ⁵ and cultured at 37° C., 5% CO2 environment for 24 hours. Thereafter, the medium containing [TIMP1 nucleic acid:PS]/HA PEMN 1.2 prepared in Example 2 was replaced and cultured for 6 hours, and then replaced with a fresh medium and cultured for 3 days.

Thereafter, the culture medium was harvested and centrifuged for 5 minutes at 4 ° C., 7500 rpm. The TIMP1 concentration secreted in the culture medium using the ELISA method was 0, 6, 12, 24, 0, 6, 12, 24, and three measurements at 48 hours. The secretion of TIMP1 from the transfected cells was quantified using the TIMP1 Human Instant ELISA™ Kit (Invitrogen, USA).

The TCA is a type of bile acid, which is a substance capable of enterohepatic circulation by bile acid receptors, and increases the systemic circulation of the drug. There is this. MDCK-ASBT cells were cells in which ASBT receptors were overexpressed, and MDCK cells were used as a control.

Referring to FIG. 18 , it can be confirmed that PEMN having TCA is introduced into MDCK-ASBT cells as time elapses and TIMP1 expression is increased. As a result of the experiment, the expression level of TIMP1 was almost absent in MDCK cells in which ASBT was not expressed. In MDCK-ASBT cells, it was confirmed that the expression level of TIMP1 was clearly higher than that of the control group, and it was confirmed that it increased more and more as time passed.

In addition, the TIMP1 nucleic acid: PS/HA-TCA particle 1.2 was pre-treated with a high concentration of TCA in order to confirm that the 1.2 was absorbed by the ASBT receptor of MDCK-ASBT cells. It was confirmed that the uptake of MDCK-ASBT cells was significantly reduced.

Therefore, it was confirmed that the [nucleic acid:PS]/HA-TCA PEMN of the present invention has very low cytotoxicity and can enter the enterohepatic circulation through the end of the small intestine.

Example 12. Cancer cell targeting assay of HA-aptamer PEMN

The effects of [MMP1 nucleic acid:PS]/HA PEMN 1.1 and [MMP1 nucleic acid:PS]/HA-Apt_EGFR PEMN on MMP1 expression were evaluated according to the expression level of EGFR in the transfected cells. The cell line used in this Example is an EGFR+ cell line, <lung cancer cell line A549, colon cancer cell line SW48, and skin cancer cell line A431>; And <breast cancer cell line MDA-MB-453, colorectal cancer cell line HT-29 and neuroblastoma cell line SK-N-MC> as EGFR- cell lines.

2ml of the culture solution was put in a 40mm cell culture dish, and the cells were inoculated at a concentration of about 1.2x10 ⁵ and then cultured at 37°C, 5% CO2 environment for 24 hours. Thereafter, the medium containing [MMP1 nucleic acid:PS]/HA PEMN 1.1 and [MMP1 nucleic acid:PS]/HA-Apt_EGFR PEMN prepared in Example 2 was replaced and cultured for 6 hours, then replaced with a fresh medium and cultured for 3 days did.

Thereafter, the culture medium was harvested and centrifuged for 5 minutes at 4° C., 7500 rpm. The concentration of TIMP secreted in the culture medium using the ELISA method was 0, 6, 12, 24, 0, 6, 12, 24, and three measurements at 48 hours. The secretion of MMP1 from the transfected cells was quantified using the MMP1 Human Instant ELISA™ KiT (Invitrogen, USA).

Referring to FIG. 19A , [MMP1 nucleic acid:PS]/HA PEMN 1.1 without the EGFR ligand had almost no expression in EGFR+ lung cancer cell line A549 cells, colon cancer cell line SW48 and skin cancer cell line A431, EGFR- breast cancer cell line, MDA-MB The expression level of MMP1 was high in -453, colon cancer cell line HT-29, and neuroblastoma cell line SK-N-MC.

Referring to FIG. 19b , [MMP1 nucleic acid: PS]/HA-Apt_EGFR PEMN having the EGFR ligand had a relatively high MMP1 expression level in EGFR+ lung cancer cell line A549 cells, colon cancer cell line SW48 and skin cancer cell line A431, and EGFR- breast cancer cell line , MDA-MB-453, colorectal cancer cell line HT-29, and neuroblastoma cell line SK-N-MC were hardly measurable.

These cytotoxicity results indicate that MMP1 expression is induced by selectively delivering [MMP1 nucleic acid:PS]/HA-Apt_EGFR PEMN with an EGFR ligand to the cancer cells depending on the presence or absence of an EGFR target molecule. Mean ± standard deviation (n = 5).

These results were not limited to selected mRNAs, but were generally observed for other mRNAs as well.

The foregoing description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

Claims (27)

(a) including an active ingredient, a cationic compound and an anionic compound;
(b) homogenizing the polyelectrolyte complex (PEC) or polyelectrolyte Multilayer (PEM) formed by the electrostatic interaction therebetween,
(c) PEMN characterized in that the active ingredient is encapsulated, its use, and its manufacturing method.
Here, the active ingredient is any one or more selected from the group comprising metals, small molecule drugs, proteins, growth factors, peptides, antibodies, nanoparticles and nucleic acids. The method of claim 1, wherein the nucleic acid is
(a) DNA medicinal products, including antisense, DNA aptamers and gene therapy; and
(b) RNA pharmaceuticals including mRNA, micro RNAs, RNA aptamer, short interfering RNAs, ribozymes, RNA decoys and circular RNAs; . The method of claim 2, wherein the mRNA is
(a) an open reading frame (ORF) encoding the target polypeptide:
(b) a 5'UTR;
(C) 3'UTR; and
(d) a PEMN characterized by an isolated RNA construct encoding a polypeptide, comprising a 5' cap structure or an internal ribosome entry site (IRES), uses thereof, and methods for preparing the same. The method of claim 1, wherein the cationic compound is
Cationic lipids, PACE-based cationic polymers, PEMN characterized in that any one or more selected from the group comprising a cationic polypeptide, and a cationic polysaccharide, its use, and a method for producing the same. 5. The method of claim 4, wherein the cationic polypeptide is
Protamine, cell penetrating peptide (CPP), chimeric CPP, pH dependent CPP, nucleoline, spermine, spermidine, Poly-L-lysine (PLL), basic polypeptide, poly-arginine, transportan, MPG peptide, HIV (human immunity) Human Immunodeficiency Virus (HIV)-binding peptide, Trans-activating Transcriptional aCtivator (tat), HIV-1 tat (HIV), tat-derived peptide derived peptide), oligoarginine, penetratin family member, penetratin, Antennapedia-derived peptide, Drosophila Antennapedia peptide (pAntp) , Islet-1 peptide (pIsl), antimicrobial-derived CPP (antimicrobial-derived CPP), Buforin-2, Bactenecin 7 peptide fragment 15-24 (bactenecin 7 peptide fragment 15 -24, Bac715-24), syncytin B, SynB, SynB(1), vascular endothelial cadherin derived cell penetrating peptide (pVEC), human calcitonin (hCT) -Derived peptide, sweet arrow peptide arrow peptide, SAP), model amphipathic peptide (MAP), KALA, PptG20, Proline-rich peptide, Lologomere, Arginine-riCh peptide , Calcitonin-peptide (Calcitonin-peptide), Fibroblast growth faCtors (FGF), Lactoferrin, histone (histone), VP22 peptide (VP22 peptide), Herpes simplex virus (HSV), Cationic poly containing VP22 (Herpes simplex), protein transduction domain (PTD), lysine-rich peptide, Pep-1, L-oligomer (L-oligomer) PEMN, its use, and its preparation method, characterized in that it contains any one or more selected from peptides. 5. The method of claim 4, wherein the cationic polysaccharide is
(a) chitosan, glycochitosan and artificially derived cationic polysaccharides;
(b) a cationic molecule to which a bile acid or a bile acid derivative is covalently bound; and
(c) cationic polysaccharide derivatives or salts, and combinations thereof; 7. The method of claim 1 to 6, wherein any one cationic compound selected is
PEMN, characterized in that it contains 10-100% (molar ratio) with respect to other selected cationic compounds, use thereof and method for preparing the same. The method of claim 1, wherein the anionic compound is
(a) polysaccharides and derivatives thereof having a functional group selected from a carboxyl group, -OSO3H group, -SO3H group, phosphate group and salts thereof;
(b) polyamino acids containing amino acid residues having negatively charged side chains;
(c) PEG derivatives having carboxyl side chains;
(d) a synthetic polymer having a functional group selected from a carboxyl group, -OSO3H group, -SO3H group, phosphate group and salts thereof;
(e) a carboxyl group, -OSO3H group, -SO3H group, phosphate group and salts thereof; and
(f) a polymer having a functional group selected from an optionally substituted amino group, an ammonium group, or a salt thereof, and a combination thereof; The method of claim 8, wherein the anionic compound is
(a) silk protein fibroin (Silk protein fibroin), gliadin (Gliadin) and anionic proteins including albumin;
(b) anionic polyamino acids including polyglutamic acid and polyaspartic acid;
(c) agar, hyaluronan, chondroitin sulfate, dextran sulfate, heparan sulfate, dermatan sulfate, fucoidan, keratan sulfate, heparin, carrageenan, succinoglucan, gum arabic, xanthan gum, alginic acid, pectin and carboxymethyl anionic polysaccharides including cellulose and variants thereof;
(d) polyacrylic acid; and
(e) artificially derived anionic polysaccharides and salts thereof; PEMN, characterized in that at least one selected from the group comprising a, a use thereof, and a method for producing the same. 10. The method of claim 8 to 9, wherein the anionic compound is
Serum albumin, hyaluronic acid (hyaluronic acid, HA) or a variant thereof; PEMN containing; its use, and a method for producing the same. 11. The PEMN according to any one of claims 1 to 10, wherein the selected one anionic compound comprises 10-100% (molar ratio) relative to the other selected anionic compounds, PEMN, its use, and its preparation method. The method of claim 1, wherein the PEM is
(a) a suspension solution containing a core particle prepared by electrostatic interaction between the active ingredient anionic nucleic acid and a cationic compound in a mixed suspension solution, and an anionic compound mixed with the core particle and the anion PEM formed by electrostatic interactions between sexual compounds;
(b) PEM formed by the electrostatic interaction between active ingredients anionic nucleic acid, cationic compound and anionic compound in a mixed suspension solution; and
(c) PEM which is prepared and eluted by a layer by layer method of alternately depositing active ingredients anionic nucleic acids, cationic compounds and anionic compounds on a substrate; PEMN, characterized in any one selected from the group comprising; Uses and methods of manufacturing the same. [13] The PEMN according to claim 12, wherein the PEM is at least one selected from the group consisting of a polymer electrolyte, a multilayer polyelectrolyte, a nanogel, a hydrogel, and a polymer micelle, a use thereof, and a method for manufacturing the same. [Claim 2] The PEMN according to claim 1, wherein the PEMN has a particle size of 50 to 400 nanometers and a polydispersity index (PDI) of 0.3 or less. 15. The method of claim 14, wherein the PEMN is a PEMN characterized in that the active ingredient nucleic acid is released at a constant rate, its use, and its preparation method. [16] The method according to claim 14 to 15, wherein the PEMN is an active drug delivery system using a compound constituting the outermost layer or a ligand bound thereto, a use thereof, and a method for preparing the same. 17. The method of claim 14 to 16, wherein the PEMN is one selected from the group consisting of injections, creams, and oral mucosal administration, PEMN, its use, and its preparation method. 18. The method of claim 14 to claim 17, wherein the PEMN further comprises at least one selected from the group consisting of ionic salts, small molecule drugs, polymer drugs, polymer drugs including drugs, and polymers including fluorescent substances. Characterized by PEMN, its use and its preparation method. [Claim 19] The method of claim 18, wherein the polymer drug is selected from the group consisting of proteins, antibodies, and nucleic acids. 20. The method according to any one of claims 1 to 19,
A PEMN for treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the PEMN, thereby treating the disease, a use thereof, and a method for preparing the same. (a) comprising an active ingredient, a cationic compound and an anionic compound, and preparing a polyelectrolyte multilayer (PEM) by electrostatic interaction between them; and
(b) homogenizing the prepared PEM to prepare PEMN (Polyelectrolyte multilayer nanoparticles) in which the nucleic acid is encapsulated, PEMN, its use, and its manufacturing method. 22. The method of claim 21, wherein in the PEM manufacturing step, any one or more selected from the group comprising shaking and sonication is performed, PEMN, its use, and its manufacturing method. The method of claim 21, wherein the manufacturing of the PEMN comprises:
PEMN characterized by at least one selected from the group comprising extrusion homogenization, upstream pressure homogenization, high pressure homogenization, ultra-high pressure homogenization and nanostructures, uses thereof, and preparation thereof Way. 24. The method of claim 23, wherein the upstream pressure is 20-60 MPa,
high pressure up to 150~200 MPa,
Ultra-high pressure is up to 350 ~ 400 MPa; PEMN, its use, and its manufacturing method, characterized in that. 25. The PEMN according to any one of claims 1 to 24, wherein the outermost layer of the PEMN is an anionic compound, a use thereof, and a method for preparing the same. 26. The PEMN according to claim 1 to 25, wherein the cationic compound and the anionic compound are biodegradable, the use thereof and the preparation method thereof. A multilayer polymer electrolyte (PEM) manufactured by layer by layer deposition technology,
(a) comprising an active ingredient, a cationic compound and an anionic compound as independent layers,
(b) the outermost layer is composed of any one selected from the group comprising a cationic compound and an anionic compound,
(c) PEM characterized in that the structure encapsulates the active ingredient, its use, and its manufacturing method.
Here, the active ingredient is any one or more selected from the group including metals, small molecule drugs, proteins, growth factors, peptides, antibodies, nanoparticles and nucleic acids.

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