KR20230073629A - Complex comprising MLKL mRNA, and pharmaceutical composition for preventing or treating cancer comprising the same as an active ingredient - Google Patents

Abstract

The present invention relates to nanoparticles containing MLKL mRNA for inducing cancer cell death and a composition for preventing or treating cancer based thereon, and to a pharmaceutical composition mediated by MLKL mRNA capable of inducing physical apoptosis of cancer cells. .

Description

Complex comprising MLKL mRNA, and pharmaceutical composition for preventing or treating cancer comprising the same as an active ingredient}

The present invention relates to an anticancer composition containing MLKL mRNA, and relates to an MLKL mRNA mediated anticancer agent capable of inducing necroptosis of cancer cells. The pharmaceutical composition according to the present invention induces necroptosis at the tumor site by a complex containing MLKL mRNA, and DAMP (Damage-associated molecular pattern) and tumor antigen are released in their original state with immunogenicity, and various It can be used in the treatment of carcinoma. In addition, since it can induce stronger immunogenicity than immunogenic cell death using conventional anticancer treatment, it can be applied to anticancer combination treatment technology using this.

Necroptosis is a programmed form of necrosis or inflammatory cell death mechanism, and since it was first named in 2005, related key factors such as RIPK3 (receptor-interacting protein kinase 3) have been identified since 2009. there is. Necroptosis causes rupture of the cell membrane, which is characterized by a stronger immune response than immunogenic cell death induced by conventional chemotherapy or recently emerging photodynamic therapy. there is Since it does not involve proteolysis or oxidative processes within cells, damage-associated molecular patterns (DAMPs) and tumor antigens flow out in their original state with immunogenicity from the tumor site that loses immunogenicity during the above process, effectively anti-cancer. It is due to the ability to induce an immune response. Due to the above characteristics, it was expected that necroptosis could maximize the therapeutic efficacy by amplifying the responsiveness to conventional anticancer treatment.

However, most tumor cells were found to have markedly suppressed expression of RIPK3 and MLKL (mixed lineage kinase domain-like protein), which are involved in the occurrence of necroptosis, and necroptosis-inducing agents such as TRAIL Tumor necrosis factor cytokines, including zVAD, pan-caspase inhibitors, and MLKL interfering RNA were developed, but their efficacy was limited. In addition, necroptosis of vascular cells can promote tumor metastasis, and necroptosis of T lymphocytes has side effects such as inhibiting the immune activity of surrounding T lymphocytes. Therefore, since current anticancer treatment through necroptosis has limited clinical application, it is necessary to develop a necroptosis-inducing agent that acts selectively on cancer cells and does not depend on the expression level of RIPK3/MLKL.

Accordingly, the present inventors developed a complex containing MLKL mRNA capable of inducing cancer cell-specific necroptosis-like apoptosis, and confirmed the MLKL mRNA-mediated anticancer effect using the complex to complete the present invention.

Accordingly, an object of the present invention is to provide a complex comprising MLKL mRNA, a biocompatible cationic compound and an anionic compound.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, comprising the complex containing MLKL mRNA according to the present invention as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer, comprising the MLKL mRNA-containing complex and immune checkpoint blockers (ICB) according to the present invention as active ingredients.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, comprising nanoparticles containing MLKL mRNA according to the present invention and DPP4 inhibitor (dipeptidyl peptidase 4 inhibitor, DPP4i) as active ingredients.

Another object of the present invention is to provide a pharmaceutical composition for inhibiting cancer metastasis, comprising the complex comprising MLKL mRNA according to the present invention as an active ingredient.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned can be clearly understood by those skilled in the art from the description below. will be.

In order to achieve the object of the present invention as described above, the present invention provides a pharmaceutical composition comprising MLKL mRNA, a cationic compound and an anionic compound.

Among the components of the composition according to the present invention, a pharmaceutical composition prepared by MLKL mRNA, a cationic compound and an anionic compound, and nanoparticles prepared by homogenizing them, the active ingredient MLKL mRNA can be encapsulated inside the nanoparticle structure .

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

There are a wide variety of substances, such as nucleic acids, that is, anionic drugs, and different delivery systems and manufacturing methods are required depending on the type of nucleic acid to be delivered. To provide a formulation with increased stability and production yield as a nucleic acid delivery system and a manufacturing method thereof.

A core particle prepared by electrostatic interaction between active ingredient anionic MLKL mRNA and a cationic compound, and a method of preparing a complex by mixing the prepared core particle and anionic compound and electrostatic interaction between them;

A method of preparing a complex by mixing active ingredients, anionic MLKL mRNA, a cationic compound, and an anionic compound, and electrostatic interaction between them; and

A complex characterized by any one selected from the group comprising: anionic MLKL mRNA as an active ingredient, a cationic compound, and an anionic compound alternately prepared and eluted on a substrate by various layer-by-layer deposition techniques, including inkjet, to prepare a complex; To provide a manufacturing method of.

It is intended to provide nanoparticles, uses thereof, and methods for preparing them, characterized in that they include the step of preparing homogenized nanoparticles by homogenizing the prepared composite.

There are several types of nucleic acids, cationic compounds, and anionic compounds, respectively, and one or two or more types may be selected and used in each group to meet the purpose of the present invention.

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

The anionic polyelectrolyte may bind to a specific receptor.

To provide a composite and a manufacturing method thereof, characterized in that at least one selected from the group consisting of shaking and ultrasonic treatment is performed in the composite manufacturing step.

The composite is intended to provide nanoparticles and a manufacturing method thereof characterized by a particle size of 50 to 400 nanometers and a polydispersity index (PDI) of 0.3 or less.

The step of preparing the nanoparticles is to provide nanoparticles characterized by at least one selected from the group consisting of extrusion homogenization, upstream pressure homogenization, high pressure homogenization, and ultra-high pressure homogenization, and a method for producing 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;

It is intended to provide a nanoparticle and a method for preparing the same, characterized in that the active ingredient nucleic acid is released from the nanoparticle at a constant rate.

Among the compounds used in nucleic acid-based therapy, the inventors of the present invention, in order to highlight the applicable properties that are well suited for nucleic acid delivery, are biodegradable and biocompatible cations such as Protamine (PS), Chitosan and Cell Penetrating Peptides (CPP). Sexual compounds and anionic compounds such as hyaluronic acid and albumin were selected.

Hyaluronic acid can specifically and strongly bind to CD44, which is overexpressed at the tumor site, and albumin to G60.

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

It can be said that the advantages of the intracellular delivery system for nucleic acids proposed in the present invention are clearly achieved by cationic compounds and supplemented by anionic compounds. 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 delivery vehicle of the present invention can improve bioavailability by avoiding nucleic acid excretion through the glomerular capillary wall. In fact, anionic substances are "unfilterable" due to the repulsive force established by the negative charge of the glomerular membrane.

The nanoparticles of the present invention may be used to obtain different types of cationic compounds and their derivatives, and also anionic compounds and their derivatives may be used to obtain different types of carriers. Nevertheless, the present invention mainly relies on the interaction of nucleic acids, especially cationic compounds such as PS, CPP, chitosan and anionic compounds such as hyaluronic acid, albumin or derivatives of these polysaccharides, to provide a nucleic acid delivery system.

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

The present invention has developed nanoparticles in which nuclei and core particles are prepared with nucleic acids and cationic polypeptides as active ingredients, and an outer layer is formed with anionic polysaccharide.

The present inventors have found that the degree of cationization is important as a characteristic of a cationic compound capable of forming core particles containing nucleic acids in order to prepare nanoparticles, and thus completed the present invention.

The complex of the nucleic acid and the cationic compound maintains an encapsulated state in the nanoparticle structure formed by the anionic compound, so that stability in blood or body fluids is improved.

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. It is for sufficient aggregation of nucleic acids for the purpose of sufficient transfection of nucleic acids into other cell lines. The nanoparticle carrier molecule furthermore allows sufficient release of the nucleic acid deliverable without being toxic to cells.

In the present invention, additionally, a biodegradable and biocompatible anionic compound is used, which provides improved stability of the nucleic acid delivery and prevents recognition of the nanoparticles by the immune system, and a hydrophilic polymer chain (e.g., , PEG-monomer) can also impart improved resistance to agglutination.

The cationic polypeptides, such as PS and CPP, constituting the nanoparticles of the present invention, although consisting of fairly small non-toxic monomeric units, have binding sequences of cations with defined chain lengths that provide strong condensation of nucleic acid deliveries and complex stability. (cationic binding sequence) is formed. Under reduced conditions 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 delivery of the nucleic acid transporter into the cell matrix. Since it is degraded into small oligopeptides in the cell matrix, toxicity known to be observed with high molecular weight oligopeptides, such as high molecular weight oligoarginine, is not observed.

The PEG-coating of anionic compounds constituting the nanoparticles of the present invention coats the carrier with a hydrophilic coating at its ends, which can prevent salt-mediated aggregation and unwanted interactions with serum contents. . Within the cell matrix, this coating is readily removed under the reducing conditions of the cell. Again, this effect enhances the induction of the nucleic acid transporter into the cell matrix.

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

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

The nanoparticles of the present invention may include polymer electrolytes, multilayer polymer electrolytes, nanogels, hydrogels and polymeric micelles. When the particle size and surface charge of the nanoparticles of the present invention are maintained at the above levels, it is preferable in terms of the stability of the nanoparticle structure, the content of components, the absorption of nucleic acids in the body, and the convenience of sterilization as a pharmaceutical composition.

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

In another embodiment of the present invention, the nanoparticles containing the MLKL mRNA may induce necroptosis-like cell death in tumors.

In another embodiment of the present invention, the nanoparticles containing MLKL mRNA may induce efflux of DAMP (Damage associated molecular pattern) in tumor cells, but is not limited thereto.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising nanoparticles containing MLKL mRNA according to the present invention as an active ingredient.

Furthermore, the present invention provides a nanoparticle immuno-anticancer composition comprising the nanoparticle containing MLKL mRNA according to the present invention as an active ingredient.

In addition, the present invention provides a method for preventing or treating cancer, comprising administering to a subject a nanoparticle containing MLKL mRNA according to the present invention.

Furthermore, the present invention provides a use of the nanoparticles containing MLKL mRNA according to the present invention for preventing, ameliorating or treating cancer.

In addition, the present invention provides the use of the nanoparticles containing MLKL mRNA according to the present invention for producing a drug used for cancer.

In one embodiment of the present invention, the cancer is lung cancer, gastric cancer, glioma, liver cancer, melanoma, kidney cancer, urothelial cancer, head and neck cancer, Merkel-cell carcinoma, prostate cancer, hematological cancer, breast cancer, It may be a cancer selected from the group consisting of colorectal cancer, colon cancer, rectal cancer, pancreatic cancer, brain cancer, ovarian cancer, bladder cancer, bronchial cancer, skin cancer, cervical cancer, endometrial cancer, esophageal cancer, thyroid cancer, bone cancer, and combinations thereof, but is not limited thereto. no.

In another embodiment of the present invention, the pharmaceutical composition may further include an immune checkpoint inhibitor (ICI) as an active ingredient, but is not limited thereto.

In another embodiment of the present invention, the immune checkpoint inhibitor may be an antibody that specifically binds to one or more proteins selected from the group consisting of CTLA-4, PD-1 and PD-L1, but is not limited thereto. .

In another embodiment of the present invention, the composition may further include a DPP4 inhibitor (dipeptidyl peptidase 4 inhibitor, DPP4i) as an active ingredient, but is not limited thereto.

In another embodiment of the present invention, the DPP4 inhibitor may be a DPP4 protein activity inhibitor, but is not limited thereto.

In another embodiment of the present invention, the activity inhibitor of the DPP4 protein is an antibody against DPP-4, sitagliptin, vildagliptin, saxagliptin, linagliptin ), dutogliptin, gemigliptin, alogliptin, anagliptin, evogliptin, berberine, diprotin and lupiol (Lupeol), but may be one or more selected from the group consisting of, but is not limited thereto.

In addition, the present invention provides a pharmaceutical composition for inhibiting metastasis of cancer, comprising nanoparticles containing MLKL mRNA according to the present invention as an active ingredient.

Moreover, the present invention provides a method for inhibiting cancer metastasis, comprising administering to a subject a nanoparticle containing MLKL mRNA according to the present invention.

In addition, the present invention provides a use of the nanoparticles containing MLKL mRNA according to the present invention for preventing or inhibiting cancer metastasis.

The complex containing MLKL mRNA for inducing necroptosis according to the present invention can induce necroptosis selectively at the tumor site, and thus can be used for tumor treatment. In addition, since it can induce stronger immunogenicity than immunogenic cell death using conventional anticancer treatment, it is expected to be widely applied to anticancer combination treatment technology using this.

Figure 1 is a schematic diagram showing the induction of necroptosis in the tumor site by MLKL mRNA, the release of DAMP and tumor antigen, and the anti-cancer immune response through ICB mRNA.

The present invention relates to a pharmaceutical composition containing MLKL mRNA for inducing necroptosis.

Carrier/Polymer Carrier: A carrier can generally be a compound that facilitates transport or complexation of another cargo. A polymer carrier is a carrier that generally forms a polymer. A carrier may be associated with its cargo by covalent or non-covalent interactions. A carrier can transport 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 substance suitable for storing, transporting, and/or administering a compound, such as a pharmaceutically active compound. For example, it can be a suitable physiologically acceptable lipid for storing, transporting and/or administering a pharmaceutically active compound.

Pharmaceutically effective amount: In the present invention, a pharmaceutically effective amount generally replaces a pathological level of an expressed peptide or protein, or replaces a missing gene product, e.g., a pathologically effective amount. An amount sufficient to induce a pharmacological effect such as an immune response in the case of an adverse situation is understood.

I. Pharmaceutical composition comprising MLKL mRNA

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

In relation to the present invention, the pharmacologically active ingredient is MLKL mRNA for inducing necroptosis.

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

Transfection of nucleic acid delivery vehicles encounters extracellular and intracellular barriers. Nucleic acid carriers are exposed to serum nucleases and phagocytic uptake, resulting in a marked reduction in 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 working.

Among the many barriers to function, nucleic acids must cross the cell membrane to reach the cytosol. The cell membrane is a dynamic and strong barrier to intracellular delivery. Various ion pumps and ion channels help maintain a negative potential. -40 to -80 mV across the cell membrane), maintaining a negative charge in the cytoplasmic space by regulating the balance of most essential metal ions (e.g., K+, Na+, Ca2+ and Mg2+).

Cell membranes create a strong barrier to highly negatively charged nucleic acid molecules. Unsaturated lipids, especially cis-double linked lipids, increase cell membrane fluidity by introducing defects in membrane structure. Other major components of the bilayer include sterols (about 30% of total lipids). Cholesterol, the major sterol in lipid bilayers, helps to maintain the balance between fluidization and condensation of lipid bilayers by creating bilayer defects.

RNA delivery methods have been 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, and use physical force to penetrate cell membranes; And it promotes gene introduction into cells. Chemical approaches generally use synthetic or naturally occurring compounds (cationic lipids, cationic polymers, and lipid-polymer hybrid systems) as carriers to transport the gene of interest into the cell. is used as a carrier.

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

One of the most developed methods for nucleic acid delivery is the co-formulation of lipid nanoparticles (LNPs). LNP formulations generally consist of (1) a polymeric material containing 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-dioleoyl-sn-glycero-3-phosphoethanolamine [DOPE]); (3) cholesterol to stabilize 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 uptake.

The most representative particle produced by encapsulation is lipid nanoparticles. Phospholipids are recognized as one of the most promising carriers for 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 form spontaneous bilayer structures when dispersed in water and trap the dispersed hydrophilic load within the aqueous core of the forming structure. Therefore, chemical modification of the head group of these molecules with cationic polypeptides facilitates entrapment of the negatively charged nucleic acid prepared in the present invention through electrostatic interaction, making it a promising component of delivery systems.

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 cell entry.

A polyelectrolyte complex (PEC) is a complex that forms spontaneously when two polymers of opposite charge are mixed, in which macromolecules are bound together primarily by electrostatic interactions. Also, "nanoplex" is widely used in the literature to refer to 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 mainly spherical and consist of a neutral core containing a stoichiometric mixture of cationic and anionic polymers and surrounded by an excess of polyelectrolyte that allows the formation of a charge-stabilizing shell.

As 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. Thus, 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 covalently cross-link complex polyelectrolytes to prevent separation of the polymer chains from the PEM, thereby enhancing the stability of the PEM. PEM-based nanoparticles are attractive because they are easy and cost-effective to manufacture, and because the reversibility of electrostatic interactions provides extraordinary tuning of the behavior of the carrier while maintaining the integrity of the composite under appropriate conditions.

Nucleic acids constituting the nucleic acids of the present invention are strongly negatively charged polymers, and cationic polymers play a very important role in the process of preparing 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 endosome escape. However, the disadvantages of cationic polymers are high toxicity due to altered cell membrane integrity and high immunogenicity.

A further and more promising approach in gene therapy is the use of cationic polymers. Cationic polymers have been shown 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 in vitro and in vivo gene delivery.

Cationic compounds generally refer to charged molecules, which are generally 1 to 9, less than 9 (eg 5 to 9), or less than 8 (eg 5 to 8), or less than 7 ( It has a positive charge (positive ion) with a physiological pH value of, for example, 5 to 7), for example 7.3 to 7.4. Thus, the cationic component can be any positively charged compound or polymer, cationic peptide or protein that is positively charged under physiological conditions, particularly under physiological conditions in vivo. 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 of exhibiting more than one positive charge under given conditions.

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

Although most cationic polymers have the function of condensing DNA into small particles and facilitating cellular uptake by endocytosis through anion site on the cell surface and charge-charge reaction, they have transfection activity. activity and toxicity are significantly different.

Cationic polymers show better transfection ability with higher molecular weight due to stronger complexation with negatively charged nucleic acid carriers. However, as the molecular weight increases, the toxicity of cationic polymers increases further. Polyethyleneimine (PEI) is probably the most active and most studied polymer in the field of gene delivery, but the biggest weakness of polyethyleneimine as a transfection reagent is related to its non-biodegradable nature and toxicity. .

One unusual approach in gene therapy is the use of cationic lipids. However, although many cationic lipids exhibit good transfection activity in cell culture, most do not perform well in the presence of serum and only a few are active in vivo. Size, surface charge and lipid 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. Significant changes in structure occur.

Lipoplexes, once administered into the body, react with negatively charged blood components and tend to form large aggregates, which may be adsorbed on the surface of circulating red blood cells or deposited in a thick layer of mucus. become entrapped or embolize the microvasculature, preventing lipoplexes from reaching their intended target cells at a distal location.

Moreover, the toxicity associated with gene transfer by lipoplexes includes induction of inflammatory cytokines, among others. In humans, adverse inflammatory reactions of varying degrees, including flu-like symptoms, have been reported among patients receiving Lipoplex. Thus, the question remains whether Lipoplex is safe for everyone to use.

Moreover, although polyplexes made by polymers with high molecular weight show improved stability under physiological conditions, such polymers may interfere with vector unpacking. For example, very rapid DNA dissociation in complexes formed with poly L-lysine (PLL) results in apparently enhanced short-term gene expression. A cationic amino acid length of at least 6 to 8 is required to pack the DNA into the structure active for receptor-mediated gene transport. However, polyplexes made of short polyions are not stable under physiological conditions and aggregates are generally formed rapidly in physiological saline.

To overcome these negative aspects, linear reducible polycations made by oxidative polycondensation of the Cys-Lys10-Cys peptide, which can be cleaved by the intracellular environment, to promote the release of nucleic acids. A new 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 enabling 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. This problem led to the development of an RNAtive vaccine platform in which protamine formulated RNA acts only as an immune activator and not as an expression vector. CureVac has developed an RNAactive vaccine platform based on protamine-made RNA complexes that act as TLR 7/8 agonists to induce Th1 T cell responses, and nucleotide-modified mRNAs function as antigen producers. This vaccine platform produced favorable immune activation against cancer and infectious diseases both in preclinical animal models and in patients.

The therapeutic success of nucleic acid-based agents depends on overcoming many of the 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 residence time, enhances cellular internalization, and enables nucleic acid to move 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 its inherent role in protecting the body against nanosized infectious agents. The kidney filters out naked nucleic acids or any nanoparticles with a hydrodynamic diameter less than 5.5 nm.

Most LNPs are about 100 nm in diameter and are large enough to exit the MPS and not reach other organs of interest. The liver, the main organ of MPS, has a vascular structure with small pores and contains phagocytes such as Kupffer cells that possess 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 delivery vehicle components is 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 polyesters or polycarbonates can better control the degradation of the delivery vehicle.

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 through an apolipoprotein E-dependent manner. In the case of APE, it has been shown that the core structure of the polymer's tertiary amino alcohol may play an important role in targeting specific organs. These targeting strategies have limitations and, in general, rationally designed targeting approaches, such as incorporation of tissue-specific receptors with ligands obtained from high-throughput screening to find successful targeting solutions for specific tissues, are alternative strategies.

recently reported the in vitro active targeting of sialic acid-terminal glycoproteins on the surface of dendritic cells. 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 targeting ligands. Therefore, new strategies targeting specific tissues need to be explored to address the clinical challenge.

II. Polyelectrolyte complex (PEC)

As a general model, 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 polyelectrolytes is the main driving force for PEC particle self-assembly.

PEC particles are polydisperse systems, often between 10 nm and 1 μm in diameter. Particle size is usually measured by indirect techniques that maintain the integrity of nanoparticles in solution, primarily 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. These dynamic properties of PEC particles can inherently compromise their integrity upon dilution and addition of other charged compounds, resulting in electrostatic shielding and multiple ion exchanges from these nanomaterials. As a result, the stability of PEC particles may be severely compromised in biological environments where high concentrations of small electrolytes and charged proteins are found. However, PEC particles can be easily stabilized through cross-linking of polyelectrolyte components, and in this way exchange with free polymers in solution can be minimized. On the other hand, structural damage due to physiological electrolytes to PEC particles can be beneficial, for example by increasing the permeability of these nanomaterials, leading to release of preloaded cargo.

Finally, only certain polyelectrolytes mixed under appropriate conditions form dispersed and stable PEC particles. While making these nanomaterials, several factors need to be optimized, in particular the molecular weight, concentration and charge density of the polyelectrolytes, the pH and ionic strength of the (aqueous) medium or the relative proportions and mixing order of the two polyelectrolytes. These parameters are usually interdependent and must be optimized in parallel. Often stable colloidal PEC particles are prepared with high dilution and low ionic strength, and can be prepared with a significant mismatch in their molecular weight with weak ionic groups in more than one of the poly ions, ideally in one of the polymers. Under different conditions, coacervates separate from solution and coalesce into a dense material known as a compact polyelectrolyte complex, which has also shown great potential for some biomedical applications, such as synthetic cartilage and enzyme-active biocomposites.

1. Cationic compounds

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 with (-) charge and the cationic compound with (+) charge are mixed with each other, they can be condensed. More specifically, a molecule with a strong positive charge can condense or compress while surrounding the negatively charged nucleic acid, thereby reducing the particle size.

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

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

On the other hand, in terms of forming multiparticulates with anionic compounds, there is no specific upper limit to the degree of cationization of cationic compounds. As a criterion, a cationic compound having a cationization degree of 2 or less can be used.

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

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

The cationic compound used in the present invention may be a natural product or a synthetic product 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 to which bile acids or bile acid derivatives are covalently bonded.

Preferred cationic polypeptides are protamine, polylysine and polyarginine;

cationic polysaccharides such as chitosan, polyben, glycol chitosan;

cationic polymers such as polyethyleneimine (PEI);

Cationic lipids such as 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, etc., dendrimers such as polypropylamine dendrimers or dendrimers based on pAMAM, PEI:poly(ethylenimine), poly polyimine(s) such as (propyleneimine), polyallylamine, cyclodextrin-based polymers, dextran-based polymers, glycobackbone-based polymers such as chitosan, silane backbone-based polymers such as PMOXA-PDMS copolymers, a combination of one or more cationic blocks (eg selected from the aforementioned cationic compounds) and one or more hydrophilic or hydrophobic blocks (eg polyethylene glycol); and the like.

Multiparticulates containing cationic compounds have the added advantage of improving transfection efficiency by formulating smaller uniform particle sizes. Cationic compounds tend to condense and package negatively charged nucleic acids. Poly-L-lysine (PLL) is the first cationic polymer investigated for DNA transfection. In addition, PEI (poly-ethylenimine), a new 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 ability of several amino groups on PEI to quench 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, eventually leading to osmotic expansion and destruction of endosomes. However, PEI-based formulations are cytotoxic because they bind to serum proteins and erythrocytes due to their high positive charge, causing plasma membrane disruption. Moreover, cell lines treated with PEI polymer exhibit autophagy, necrosis and cell death.

To address the aforementioned problems, next-generation cation-based polymers, poly[(2-dimethylamino)ethylmethacrylate] (pDMAEMA), polyamidoamine (PAMAM) dendrimers, and biodegradable poly(β-amino ester) (PBAE) have been developed. polymers have been developed. Because of their tertiary amine end groups, pDMAEMA and PBAE aid in endosome escape and have excellent transfection efficiency. Although PBAE is less toxic compared to PEI, care 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 the PACE polymer is based on the presence of a branched amino group with a cleavable ester moiety that aids in endosome disruption and is readily hydrolyzable under biological conditions. PACE-based cationic polymers exhibit 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 total amino acid residues is preferably 70% or more, more preferably 80% or more, still more preferably 90% or more. , more preferably 99% or more basic RNA is preferably used.

In particular, basic cationic polypeptides composed only of basic amino acid residues may be mentioned preferably.

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

Specifically, polylysine or polyarginine is preferably used.

Also, 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 may 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 selected from the group consisting of, for example, 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) and polyamidoamine (PAMAM), , preferably PS.

A candidate for the cationic polypeptide is protamine, a polycationic peptide with a variety of applications. In particular, it is used for drug delivery and has been reported to delay the absorption of injected insulin suspensions. 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, when complexed with DNA, protamine neutralizes the negative charge of DNA phosphate groups. Commonly called poly-electrolyte complexes (PECs), these materials consist of macromolecules of biological macromolecules that contain groups that can dissociate in water and interact to create charged components. Among animal testes, PS is present in abundance in sperm nuclei of fish, including salmon, and is known as a protein involved in the expression of genetic information through association with or dissociation with DNA, such as histones. Usually, the molecular weight of PS extracted from sperm nuclei of fish is about 4,000 to 10,000, and more than 70% of 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 pharmaceutically acceptable salts thereof. Specifically, the salt of PS of the present invention encompasses salt forms by acidic substances including hydrochloride or sulfate. PS is soluble in water without forming a salt, and can be used in any form because the salt form is also soluble in water.

In order to obtain a more compact 'particle' structure, a modified form by treating PS with acrylamide (denoted as mPS) and a modified form by treating HA with an acrylate group (denoted as mHA) can also be used. In this case, the final gel is It can also be constructed by photo-crosslinking through UV irradiation.

Poly-L-lysine (PLL: poly-L-lysine) is linked by a biodegradable peptide bond, so it can be delivered into cells by forming complexes and ionic complexes, but it is somewhat toxic, so it is difficult to show high delivery efficiency alone Due to limitations, it can be used by attaching endosome-destroying substances or endosome fusion peptides, including CPP and pH-dependent CPP.

Currently, the most commonly used nucleic acid stabilizing material is polyethylenimine (PEI), which is positively charged, but has a disadvantage in that its use is limited due to toxicity in the body. On the other hand, PS is not cytotoxic and has the advantage of allowing nucleic acids to be safely released into the cytoplasm by promoting penetration into negatively charged cell membranes (endosome membranes) and endosomal escape of nucleic acids.

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

In the cell penetrating peptides (CPP), peptides that activate cell permeability by pH (a) have little membrane permeability at high peptide concentrations above the effective concentration (EC) and physiological pH range (pH 7.0), (b) In the acidic pH range of endosomes (pH5.5) and at low peptide concentrations below the effective concentration (EC), macromolecular-sized defects can be efficiently formed in the membrane, and the domain and pH-dependent membrane that increase cell membrane permeability It is a peptide composed of an active domain.

Cell Penetrating Peptides (CPP) that activate cell permeability by pH type amino acid sequence note Melittin GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 1) Membrane activation over a wide pH range LP GWWLALALALALALALASWIKRKRQQ (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 is Unmodified mRNA; Modified mRNA; self-amplifying mRNA; And Trans-amplifying mRNA; It provides a nucleic acid / PS core particle containing.

According to the present invention, the nucleic acid/PS core particle has a ratio of nucleic acid and PS of 1:0.4

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

The weight ratio of the nucleic acid and PS may be 1:0.2 to 5, preferably 1:0.4 to 3.0. In an environment where the w/w ratio is 1:2.5 or less, it is further condensed, but when PS is added at a ratio of 1:5 or more, the particles may be micro or larger in size.

Although the particles of the present invention are stably maintained in high-purity water due to their physical and chemical stability, the particles can rapidly aggregate when salt is added to the colloidal system. Another disadvantage of these first generation nucleic acid/PS particles, so-called 'particles', may be the low release of nucleic acids from the particles.

To overcome these 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 amphipathic 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), conductive metal elements such as aluminum (Al), or metal oxides thereof, may be nanoparticles selected from the group consisting of one or more combinations. there is.

Human serum albumin (HSA) may be a promising candidate to address these problems. HSA, with a molecular weight of about 65,000 Da and a slightly negative surface charge under physiological conditions, is a non-toxic macromolecule widely used in particulate formulations and other pharmaceuticals. HSA is a protective colloid. This colloid is only entrapped 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 high HSA concentrations (1000 μg/ml), the particles are completely coated with HSA, and 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 the polysaccharides used in nucleic acid-based therapy as a cationic compound, the present invention specifically selected chitosan to highlight applicable properties and characteristic features that make it highly suitable for nucleic acid delivery. The positively charged amino group in the glucosamine monomer of chitosan or the 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 apparently 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 invention may be included in 1 to 50% by weight, more specifically 2 to 40% by weight, and 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 nucleic acid, and if it exceeds 50% by weight, the size of the nanoparticles becomes too large and the size of the nanoparticles There is a possibility that the stability is lowered and the loss rate increases during filter sterilization.

The cationic compound and the nucleic acid are bonded through electrostatic interaction to form a complex. As a specific example, the ratio of the charge amount of the nucleic acid (P) and the cationic compound (N) (N / P; the ratio of the positive charge of the cationic compound to the negative 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. Since it is difficult to form a complex containing a sufficient amount of nucleic acid when the ratio (N/P) is less than 0.5, it is advantageous to form a complex containing a sufficient amount of an anionic drug when the ratio is greater than 0.5. On the other hand, when the ratio (N/P) exceeds 100, there is a risk of toxicity, so it is better to set it to 100 or less.

2. Anionic compounds

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

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

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 a nucleic acid. That is, the negatively charged nucleic acid forms an ionic complex encapsulated inside the cationic compound due to an 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 step of coating the anionic compound layer is a step of coating the outer surface of the particle with an anionic compound by electrostatic attraction to the anionic compound layer.

The composite particle 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 proteins including serum albumin, anionic polysaccharides including polyglutamic acid and polyaspartic acid. 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 anionic polysaccharides such as polyacrylic acid and its salts. In particular, hyaluronan, polyglutamic acid and salts thereof may be suitably mentioned.

As an anionic compound, a commercially available product can be used.

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

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

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

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, such as high water solubility, long half-life in blood, similar molecular weight (65-70 kDa), and similar number. Number of amino acid residues (585 amino acids for HSA and 583 amino acids for BSA). There is no appreciable property difference in constructing the nanomedicine. Therefore, both types of albumin have been widely used to design multifunctional nanoparticles. Technologies developed as alternative albumins can be easily replaced.

Serum albumin, one of high molecular materials in vivo, is a very stable protein with a half-life of 19 days in blood as well as excellent biocompatibility, and shows effective cancer accumulation ability due to its excellent affinity with cancer tissues. Therefore, serum albumin can be chemically bound to various low-molecular-weight chemical drugs, proteins, antibodies, etc. to improve the stability of various drugs in vivo and can be used to effectively deliver the drugs to target cancer sites. For example, a study has been reported that chemically binds representative hydrophobic anticancer drugs such as doxorubicin (Dox) or methotrexate (MTX) to albumin to improve cancer targeting and improve drug stability in the blood. . In addition, with respect to the use of albumin as a drug delivery system, based on the high targetability of albumin to cancer tissue, when a fluorescent substance is attached to albumin or labeled with a radioactive isotope and injected into a small animal transplanted with cancer, it is imaged. It has been reported that a positive amount of albumin is selectively accumulated in cancer tissue, and it can be seen that this is caused by an enhanced permeability and retention (EPR) effect occurring in loose blood vessels around cancer tissue. As such, the excellence of albumin as an anticancer drug delivery system has been known, but albumin itself lacks binding ability to be used as a strong negatively charged siRNA delivery system. Therefore, while maintaining the unique characteristics of albumin, it is necessary to modify it 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 may be 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 multiparticles with the cationic polypeptide.

On the other hand, in terms of forming multiparticulates with cationic polypeptides, there is no particular upper limit on the degree of anionization of the anionic polysaccharide. As a reference, an anionic polysaccharide 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 the polymer) - (Number of cationic functional groups contained in the 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.

As the anionic polysaccharide, any molecule having a negative charge and having biocompatibility may be used without limitation.

Heparin has a structure containing a hydroxy group, a carboxyl group and an amino group, and unfractionated heparin, molecular weight heparin, low molecular weight heparin, heparin fragments, recombinant heparin, heparin analogues, heparan sulfate, sulfonated polysaccharides having heparin activity, etc. can be used. there is.

The hyaluronic acid (HA) is a large complex oligosaccharide composed of up to 50,000 pairs of disaccharides glucuronic acid-β(1-3) and 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 of the human body either systemically or topically because of its ability to provide targeted active substances to diseased or diseased areas. It is known that HA is formed in injured carotid artery (for uninjured contralateral artery) and colorectal carcinoma in laboratory animals and is retained in the skin of such laboratory 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 particle and the hyaluronic acid may be in the range of 1:0.5 to 2 (core particle:HA).

Hyaluronic acid of the present invention is one of complex polysaccharides composed of amino acids and uronic acids, 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 a therapeutic agent that selectively targets cancer.

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

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

Since the present invention further includes an outer surface layer and a hyaluronic acid layer of the core particle composed of the nucleic acid and the cationic polypeptide, the nucleic acid can be protected from enzymes that degrade nucleic acid in vivo.

In addition, the present invention reduces the particle size to nano size by condensing nucleic acid as a pharmaceutically active ingredient having a micro size or nano size size similar thereto, and also coats the outermost layer with (-) charged hyaluronic acid. Accordingly, it is possible to provide particles that easily pass through the skin barrier.

3. Natural polysaccharides

Natural polysaccharides can be obtained from animals (eg chitosan, hyaluronic acid), plants (eg starch, cellulose, cyclodextrin, pectin), algae (eg alginates) or bacteria (eg dextran). It has a wide range of applications, from agriculture and food industry to cosmetics, pharmaceuticals and biomedical fields. Polysaccharides are formed by monosaccharides forming carbohydrate structures that are neutral or charged, linear or branched and have varying degrees of hydrophilicity.

The widespread use of natural polysaccharides in pharmaceuticals for drug delivery is biocompatibility, biodegradability and low toxicity. It has also been used in combination with synthetic materials as a strategy to mitigate the toxicity of the latter. Their structural diversity makes it possible to select the most suitable polysaccharides for each case, depending on 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 bioadhesive properties due to the presence of specific functional groups, making them useful starting materials for nanocarrier design with improved retention times, especially for intestinal and pulmonary applications. The presence of these groups also allows certain modifications of the polymer structure to enhance its delivery or targeting capabilities. In addition, natural polysaccharides can be neutral (eg cellulose, pullulan, starch, dextran), negatively (eg hyaluronic acid, alginates, chondroitin sulfate), or positively charged (eg chitosan). ) can be

Types of Polysaccharides Animal Polysaccharides Chitin
Chitosan
Glycosamminoglycans Vegetal Polysaccharides Land Vegetal Marine Vegetal Cellulose
PEMtins
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 the polysaccharides used in nucleic acid-based therapy, we specifically selected hyaluronic acid to highlight applicable properties and characteristic features that make it highly suitable 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 comes from the chemical structure, which gives the 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. In fact, negatively charged species are "unfilterable" due to the repulsive force established by the negative charge of the glomerular membrane.

modified ionic molecule

<Modified cation molecule>

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

A particle comprising at least one cationic polypeptide and a complex to which a bile acid or a bile acid derivative is covalently bonded, wherein the covalently bonded bile acid or bile acid derivative has an ionic group.

The particles of the present invention are formed by electrostatic interactions between cationic polypeptides and complexes to which bile acids or derivatives thereof are covalently bonded, or electrostatic interactions between anionic polysaccharides to which bile acids or derivatives thereof are covalently bonded, cationic polypeptides, and nucleic acids. Formed by the action, it is possible to induce cellular internalization of nucleic acids more safely.

That is, the core particle of the present invention is a particle 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 which bile acid is surface-modified.

In the case of using the surface-located particles of the bile acid of the present invention to deliver the nucleic acid to target cells, the complex was well delivered through the intestinal mucosa and was 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 complex delivery particles 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 molecules include deoxycholic acid (DCA), taurodeoxycholic acid, taurocholic acid (TCA), glycocholic acid and glycochenodeoxycholine. It may be selected from the group consisting of acid (glycochenodeoxycholic acid).

The bile acids are absorbed with a high efficiency of 95% or more through the ileum of the small intestine and absorbed into the liver. Since it is specifically recognized by the apical sodium-dependent bile acid transporter (ASBT) present in the distal intestine, it can act as a targeting ligand for oral delivery.

That is, the bile acids absorbed through the ileum are located on the surface of the nucleic acid delivery particles, thereby increasing the absorption of the nucleic acid delivery particles into target cells through the intestinal wall, which is the biggest problem of the existing oral administration method through ASBT. play a role in As a result, the particles for nucleic acid delivery of the present invention are easily absorbed through the ileum where bile acids are absorbed, so they can be used for oral use, thereby increasing the bioavailability of the drug.

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

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

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

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

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

In the present invention, the ionic molecule in 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 complex in which the bile acid or bile acid derivative is ionically bonded 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 cationic polypeptides have limitations in binding and delivery is possible only in the range where they can have optimal strong binding ability, the formation of ionic complexes according to the electrostatic interaction between cationic polypeptides and nucleic acids within the above range is possible. It's done well.

<Modified anion molecule>

In addition, the present invention includes a nucleic acid/cationic polypeptide particle comprising at least one anionic polysaccharide or nucleic acid to which a bile acid or a bile acid derivative is covalently bonded, wherein the covalently bonded bile acid or bile acid derivative is a nucleic acid having an ionic group. A pharmaceutical composition containing the particles as an active ingredient is provided.

Taurocholic acid of the present invention is a kind of bile acid, which is secreted in the body and is known as a substance that helps absorption of lipids or vitamins during digestion, and is characterized in that more than 90% of taurocholic acid is reabsorbed through enterohepatic 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 the particles are prepared by combining taurocholic acid with hyaluronic acid, the oral absorption rate in the small intestine can be improved and the delivery through the enterohepatic 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, the hyaluronic acid-taurocholic acid conjugate of the present invention has a feed mole ratio (hyaluronic acid: taurocholic acid) in the range of 1:1 to 1:100. acid) and has a negative charge.

If the hyaluronic acid-taurocholic acid conjugate has a positive charge, 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 contains 1:1 hyaluronic acid and taurocholic acid.

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

When prepared by mixing the hyaluronic acid and taurocholic acid at an addition molar ratio of less than 1:1, it is not effective in coating the composite/PS, and the hyaluronic acid and taurocholic acid are added at a molar ratio exceeding the range of 1:100. When prepared by mixing, it is not economically efficient because only hyaluronic acid-taurocholic acid conjugates having similar binding ratios are produced.

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.

Specifically, the complex particle for oral administration of the present invention has the nucleic acid/PS located therein, has a form in which the hyaluronic acid-taurocholic acid conjugate is coated on the surface of the nucleic acid/PS, has negative charge characteristics, and has a diameter of the particle 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 coating the following HA-TCA conjugate may exceed 250 nm, and the advantage as a particle may disappear. /PS It is preferable to manufacture such that the diameter of the core particle is 100 nm or less. In addition, since the charge characteristic of the nucleic acid PS core particle is negatively charged, it is difficult to bond (coating) with the negatively charged HA-TCA conjugate, so it is preferable to prepare the nucleic acid PS core particle to be positively charged.

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 multiparticles.

III. Polyelectrolyte Multilayer (PEM)

Both synthetic and natural polyelectrolytes have been used as building blocks for layer-by-layer (LbL)-based nano- and micro-objects. The main advantage of using synthetic polymers is that several parameters can be tuned over a wider range (e.g. ionic strength and assembly pH) and resistance to more physical and chemical stresses can be tuned. 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 recreating 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 biomedical field considering their structural similarity with natural extracellular matrix and biodegradable properties. Polysaccharides are of particular interest because they are highly hydrated, highly biocompatible and often biodegradable. It can also be easily processed into PEM film.

LbL PEMs are used in biomedical applications including biosensors, drug delivery, biomaterial coatings and tissue engineering. In addition, various types of LbL-based objects including individual coated cells, encapsulated cell aggregates, hollow capsules or free-standing membranes can be prepared using colloidal templates or supporting materials. 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.

A method for preparing multilayer polyelectrolytes (PEMs) by successive deposition of oppositely charged polyelectrolytes provides user-friendly preparation under ambient and physiological conditions, the possibility to integrate various biomolecules, fine control over and production of PEM structures. It is receiving high attention due to its robustness. This nanoscale control can be achieved simply by altering 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 varying polyelectrolyte concentrations can produce very thick assemblies with minimal deposition steps.

Complexation of transfection reagents with nucleic acids for better stability lends itself to substrate-mediated transfection approaches. These complexes can be produced in large quantities by coating and serve as the transport of active ingredient nucleic acids. The use of PEMs is a promising approach in the field of matrix-mediated active substance delivery. The ability to incorporate drugs into PEMs makes this system related to nucleic acid release and desired functions. A simple technique for PEM deposition is the layer by layer (LbL) technique, in which a polymer electrolyte is deposited alternately on a substrate. It consists of alternating positively and negatively charged materials and electrostatic interactions that stabilize the structure.

Cationic polyelectrolytes such as poly(L-lysine), chitosan, and poly(ethylenimine) (PEI) and anionic polyelectrolytes such as alginic acid, hyaluronic acid, and polyacrylic acid are good for cell-biomaterial interactions in many PEM operations. Among cationic polymers, PEI is widely used for complexation of negatively charged nucleic acids because it forms non-covalent bonds with strong cationic charge density. By complexing the nucleic acids, the 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 on every third atom, allowing it to release protons from endosomes. Because of their buffering capacity, chloride anions flow into the endosome, causing water influx and consequent 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.

The choice of molecular weight, chemical structure and phosphoric acid/nitrogen (N/P) ratio are 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 particle size for transfection.

Hyaluronic acid (HA) is a negatively charged glycosaminoglycan that occurs naturally in the extracellular matrix, synovial fluid, umbilical cord, and blood. Biopolymer properties such as biocompatibility, biodegradability and non-immunogenicity make hyaluronic acid a suitable material for wound healing, knee osteoarthritis treatment or tissue engineering scaffolds. What is 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, whereas low molecular weight hyaluronic acid activates pro-inflammatory chemokines and cytokines. HA is not a typical transfection reagent due to its negative charge, but is frequently used for nucleic acid delivery in combination 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.

Multilayer Polyelectrolyte Nanoparticles

The present invention homogenizes a polyelectrolyte complex (PEC) and a polyelectrolyte multilayer (PEM) formed by electrostatic interaction between active ingredients, cationic compounds and anionic compounds, and It relates to multilayer electrolyte nanoparticles (Polyelectrolyte multilayer nanoparticles: PEMN) encapsulated.

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

Over the past decade, research on nanoparticles (NPs) applied to medical applications has increased significantly, from inorganic to organic systems. In the case of inorganic nanoparticles, 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-assembling systems (micelles and liposomes), and polymer nanoparticles being the most commonly used and reported systems.

Regarding polymeric NPs, they are commonly used in the biomedical field due to their characteristics of compatibility, biodegradability and low cytotoxicity, and natural polymers such as chitosan, modified starch and natural gum are very common. However, the development of polymer NPs is not limited to biopolymer systems because they can also be obtained as biocompatible synthetic materials. Polylactide (PLA), polycaprolactone (PCL), polyglycolic acid (PGA) and acrylate-based polymers are also used. Similarly, polymeric NPs are classified according to their structures, the most common being nanospheres, nanocapsules, polymer nanoconjugates, and polyelectrolyte complexes (PECs).

Regarding PEC, they can be obtained in several ways, one of the most used being the traditional polyelectrolytic complexation process (bottom-up method). This technology is based on spontaneous interpolymeric aggregation given by electrostatic attraction when cationic and anionic polyelectrolytes are mixed in an aqueous solution. The preparation of this polymer electrolyte composite is in principle an easy methodology to perform. However, it can also be the other way round as it depends on several internal and external variables. The internal variables are the polymer's chemical properties, molecular weight and fractional charge. Extrinsic variables depend on many process conditions, such as polyelectrolyte ratio, mixture volume, rate and order of addition, and agitation rate during polyelectrolyte complexation, as well as several dissolution medium conditions (temperature, pH, or ionic strength). These factors lead to the fact that it is difficult to obtain polyelectrolyte composites, especially in the nanometer scale. Therefore, standardization and extension of PECs 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 of homogenizing PEC and PEM by applying high energy by High Pressure Homogenization (UHPH) and Ultra High Pressure Homogenization (UHPH).

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

Sonication and/or extrusion through a screen membrane at high pressure, high pressure and super pressure can be used to produce a homogeneous PEMN suspension from the PEC and PEM prepared in the present invention. did Sonication in PEMN formation breaks down large PEMs, entangles and/or disperses PEMs, activates or functionalizes surfaces, and emulsifies PEMs.

In these processing steps, high-powered ultrasound gives an intense effect. When sonicating suspensions at high intensities, the sound waves propagating in the liquid medium frequently alternate between high pressure (compressive) and low pressure (lean) cycles depending on the frequency. During the low-pressure cycle, high-intensity ultrasound creates tiny vacuum bubbles or voids in the liquid. When the bubble reaches a volume at which it can no longer absorb energy, it collapses violently during the high-pressure cycle. Large particles can be disrupted by surface erosion (through cavitational collapse of the surrounding liquid) or particle size reduction (due to interparticle collisions 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 crystallite size and structure changes.

Polyelectrolyte multilayer nanoparticels (PEMN) containing an active ingredient, a cationic compound and an anionic compound according to the present invention, preferably, a complex layer formed by electrostatic interaction between a nucleic acid having a negative charge and a cationic compound, and an anionic polymer It is a composite particle containing an outer layer.

In the present invention, PEMN includes (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 PEMN encapsulated with the nucleic acid.

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

Particles prepared by self-assembly by mixing the selected nucleic acid, cationic compound and anionic compound were referred to as PEM. One or more of the nucleic acids, cationic compounds, and anionic compounds may be selected, and PEM may be prepared by mixing the selected compounds.

A second method was used in which the anionic nucleic acid, the cationic compound, and the anionic compound were continuously vortexed or/and sonicated for 30 seconds in the reaction solution to stabilize them at room temperature for 1 hour after the reaction was completed.

A third method was used to prepare a membrane prepared and eluted by a layer-by-layer layering technique in which anionic nucleic acids, cationic compounds, and anionic compounds are alternately layered on a selected substrate.

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

In the present invention, (a) a suspension solution containing core particles prepared by electrostatic interaction between anionic nucleic acid and a cationic compound as active ingredients in a suspension solution, and mixing the anionic compound with the suspension solution to obtain the core 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) PEM prepared and eluted by a layer-by-layer method in which active ingredients anionic nucleic acids, cationic compounds, and anionic compounds are alternately deposited on a substrate and eluted.

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

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

Here, the nucleic acid, cationic compound or anionic compound may be one or more selected from each. Each ratio can be varied according to the purpose.

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

In the present invention, the cationic compound is a polypeptide, peptide. an allyl-based amine polymer, or a cationic polysaccharide. By using these cationic compounds, the stability of the core particle can be improved.

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

The present invention is characterized in that an aqueous solution of nucleic acid (A) and an aqueous solution of cationic compound (B) are prepared separately, and these aqueous solutions are mixed with each other. Thus, the aggregation of the core particles can be prevented 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 all cationic compounds 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) within the ranges described above, core particles that are difficult to agglomerate can be produced.

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, coats the nucleic acid with a positively charged molecule to prepare a core particle, and additionally coats the anionic polysaccharide for a second time. A method for manufacturing the PEM is provided.

Preferably, the nucleic acid PEM of the present invention may be a multi-particle obtained by mixing nucleic acid, cationic compound and anionic compound.

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

The nucleic acid consists 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, especially arginine (70-80%), so the cation per mole is 1.26 ± 10 ²⁵ . Thus, when complexed with the complex, PS neutralizes the negative charge of the phosphate group of the complex. Commonly called core particles, these materials are composed of macromolecules of biological macromolecules containing groups that can dissociate in water and interact to form charged components.

A method may be provided in which the charge ratio between the nucleic acid and the cationic compound of the present invention is 1:0.4 or more, that is, nucleic acid is 1 and PS is 0.4 or more.

The binding between the core particles that aggregate by the electrostatic interaction of the nucleic acid and PS is determined by the charge of each of the nucleic acid and PS, and in the particles 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.

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

In the core particle presented in the present invention, the nucleic acid necessarily further includes a cationic compound to form an ionic complex. That is, an ionic complex formed of a negatively charged nucleic acid encapsulated inside the cationic compound due to an electrostatic interaction with the cationic compound is located in the core portion of the particle. The layer of the core particle formed by the contact between the nucleic acid and the cationic compound has a strong positive charge, which increases adhesion to cells and makes it difficult to pass through tissue barriers.

In the present invention, in order to solve this problem, a PEM may be prepared by secondarily coating the outer surface of the cationic compound layer using an anionic compound having good biocompatibility and negative charge to form an anionic compound layer.

The step of coating the anionic compound layer is a step of coating the anionic compound layer on the outer surface of the cationic molecule layer by electrostatic attraction by putting an anionic compound therein.

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 analogues, derivatives, and metabolites of cholesterol, tocopherol and fatty acids, respectively.

In the present invention, the nucleic acid may be included in 0.05 to 10% by weight, more specifically 0.1 to 5% by weight, and 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, the amount of the carrier used is too large compared to the drug, and side effects caused by the carrier may occur, and if it exceeds 10% by weight, the size of the nanoparticles is too large. There is a concern that the stability of the nanoparticles may decrease and the loss rate during filter sterilization may increase.

The cationic compound is bonded to the nucleic acid by electrostatic interaction to form a complex, and the complex is encapsulated inside the nanoparticle structure of the anionic compound. Accordingly, the cationic compound includes all types of compounds capable of forming complexes with nucleic acids through electrostatic interaction, 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 by an ultrasonicator. may include doing.

It is preferable to use a bath type ultrasonicator as the ultrasonicator. Bath-type ultrasonicators are known to provide weak ultrasonication of 20 to 40 W/L (Watt/L) and provide a very uneven distribution, whereas probe-type ultrasonicators provide approximately 20,000 W/L. can be added. This means that the probe-type sonicator is about 1,000 times superior to the bath-type sonicator due to the concentrated and uniform ultrasonic intensity input.

In general, it has been known that nucleic acids are impossible or difficult to be pulverized by ultrasonication due to their instability, but by encapsulating nucleic acids in polymer nanoparticles as in the present invention, it is possible to use a sonicator for more convenient and efficient manufacturing of delivery systems. possible.

The present invention also relates to a method for preparing a PEM-containing composition containing an active ingredient prepared by the above-described method. That is, the method for producing a 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 produced by the above-described manufacturing method does not disappear even when the pH is changed thereafter. Accordingly, according to the present invention, a PEM-containing composition can be easily prepared.

In addition, a targeting molecule may be covalently linked to the anion molecule. The composite particle can be used as an active carrier. The targeting molecule may be a compound that binds to the cell surface, an antibody or peptide, an 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 with one or two or more nucleic acids selected from the group consisting of non-amplified RNA and self-amplifying mRNA. You may.

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

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

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

The lyophilization aid used in the present invention is added to help the lyophilized composition maintain a cake shape or to help the anionic compound composition dissolve uniformly in a short time in the process of reconstitution after lyophilization. Specifically, it may be at least one selected from the group consisting of lactose, mannitol, sorbitol and sucrose. The content of the lyophilization aid may be 1 to 90% by weight, more specifically 10 to 60% by weight, based on the total dry weight of the lyophilized 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 manufacturing method according to the present invention, are stable in blood, and the size may be specifically 10 to 200 nm And, more specifically, it may be 10 to 150 nm.

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

As a first important element, a biodegradable pharmaceutical composition includes a polymer carrier-transport complex made by a nucleic acid carrier and a biodegradable polymer carrier molecule.

As a second important element, the biodegradable pharmaceutical composition may contain at least one additional pharmaceutically active ingredient. In this regard, the pharmaceutically active component is a compound that is therapeutically effective in healing, ameliorating, or preventing a specific symptom, and is preferably effective against cancer, 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 polymeric carrier-transmitter complex by itself causes a non-specific innate immune response that allows the biodegradable polymeric carrier-transmitter complex to be used as an immunostimulatory drug (without the addition of other pharmacologically active components). causes If treated together with other pharmacologically active components, preferably particularly immunogenic components, preferably antigens, the nucleic acids of the present invention assist in the specific adaptive immune response elicited by the other pharmacologically active components. serves as an adjuvant.

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

If mRNA is used as the nucleic acid molecule of the nucleic acid carrier and the nucleic acid molecule of the complex of the biodegradable polymeric carrier carrier, the biodegradable vaccine is generally constituted as a biodegradable pharmaceutical composition and is preferably an immune response of the immune system of the patient to be treated, for example, Helps or elicits the innate immune response. Additionally or alternatively, preferably, if the nucleic acid of the complex of the nucleic acid transporter and the biodegradable polymeric carrier transporter encodes any of the aforementioned antigens or proteins eliciting an adaptive immune response, the biodegradable vaccine may elicit 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; May contain preservatives.

In addition, the present invention provides several applications and uses of biodegradable polymeric carrier molecules, and a biodegradable carrier-transportation complex made by a nucleic acid carrier and a biodegradable polymeric carrier molecule, a pharmaceutical composition containing them, or a kit containing them ) is provided.

According to one embodiment, the present invention is preferably a biodegradable polymeric carrier molecule, a nucleic acid delivery material and a biodegradable polymeric carrier molecule made by a biodegradable polymeric carrier molecule, as a medicament for gene therapy or treatment of a disease as defined herein. A first medical use of a polymeric carrier-transport composite 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 a pharmaceutical composition. A pharmaceutical composition in the context of the present invention generally comprises a biodegradable polymeric carrier molecule or nucleic acid deliverable and a biodegradable polymeric carrier-carryout complex made by the biodegradable polymeric carrier molecule, optionally including other ingredients, for example For example, preferably a biodegradable nucleic acid as defined herein and optionally a pharmaceutically acceptable carrier and/or vehicle.

Each of the anionic nucleic acid group, cationic compound group, and anionic compound group may be of 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 multi-particle obtained by mixing nucleic acid, cationic compound and anionic compound.

The ratio of nucleic acid to cationic compound, i.e. the net charge of the particle, is important. Therefore, in the present invention, a delivery platform based on nucleic acid multiparticles using cationic 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 is intended to overcome concerns about rapid extracellular nucleic acid degradation and aggregation with serum proteins, thereby providing the possibility for systemic administration of nucleic acids.

Another important issue is the biodistribution of nucleic acid carriers after systemic delivery. Certain cationic fatty nanoparticles were delivered via intravenous injection, mainly 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 as targets 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 a neutral molecule, cholesterol, may be attached to the nucleic acid. The multiparticles can simultaneously possess the coding of proteins, the pharmacological function by the contained pharmaceutical active ingredient, the imaging function by the imaging material, and the protection function of the nucleic acid drug by cholesterol.

Since the nucleic acid itself is defenseless against enzymes that degrade nucleic acids composed of nucleotides in the human body, it is difficult to completely deliver its efficacy to the inside of the target tissue and has a problem in that it is excreted by the kidney filtration function. Therefore, the present invention provides a method for overcoming the above problems in order to develop a complex into an excellent drug, as well as a polyelectrolyte.

IV. 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, at least one selected from the group consisting of 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 ultra-high pressure may be up to 350 to 400 MPa.

1) Extrusion homogenization

The present invention was carried out using high extrusion pressures. An extrusion done under a higher pressure will have a higher flow rate than an otherwise identical extrusion done under a lower pressure, will be less clogged and fouled, will allow the membrane to withstand a high degree of fouling or clogging during the production process, and will have a smaller size. PEM will be produced. The pressure that can be used is limited only by the extrusion equipment used and the tolerance of the membrane. Pressures greater than about 400 psi are used.

The methods and devices in the present invention can be used to make PEMs of any desired average diameter. Generally, as described above, membranes are selected that have an average pore diameter close to the desired average PEM diameter. The average PEM size is reduced by, for example, 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 make it The size of the PEM can be determined using any technique known in the art.

PEMs produced using the methods or devices of the present invention may be further processed using any processing technology. To reduce the degree of contamination, the membrane is passed alternately in the forward and backward directions. The average diameter of the extruded PEM can be further reduced by sonication. The number of shredding cycles using intermittent high-frequency waves can be screened by Dynamic Light Scattering (DLS) evaluation for conditions that lead to efficient PEM synthesis.

The methods and devices of the present invention can be practiced with any number of stacked membranes. One of the techniques in the art to which this invention pertains is that extrusion through a larger number of stacked membranes produces a PEM with a smaller average diameter and exhibits a lower flow rate than other similar extrusions through a smaller number of stacked membranes. know that The number of membranes used for stacking is limited only by the tolerance of the extrusion equipment. In a preferred embodiment, the stacked stack consists of between 2 and 10 membranes. 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 film in the stacked pile is different from at least one of the other films in the pile. The difference can be said to be in certain properties that affect extrusion. As described herein, differences may exist, for example, in membrane composition, coating, pore size, pore density, pore angle, pore shape, or membrane size.

Extrusion can be performed by multiple passes through a single membrane or stacked membranes. If an embodiment using stacked membranes is used for extrusion, multiple passes may be unnecessary to obtain a PEM of the desired diameter. In particular, a stepwise decreasing method is used. In the step-down method, multiple passes of the suspension are made through a membrane of decreasing pore diameter. In a particularly preferred embodiment of the step-down process, first through a membrane with a pore diameter of about 0.4 μm, secondly through a membrane with a pore diameter of about 0.2 μm, and, if necessary, through a third, fourth, five Fourth and sixth, it is performed 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 flushing agent prior to extrusion. The membrane may be treated with a flushing agent after at least one pass has been made and prior to at least one pass made through the membrane. Flush chemicals can be any type of material or composition that removes material from clogged or fouled membrane pores or prevents clogging, fouling or creating a 'sieving effect'. The flushing agent consists of organic alcohol. The flushing agent consists of ethanol.

Any extrusion apparatus that accepts suitable membranes and can withstand high extrusion pressures can be used to practice the methods and apparatus of the claimed invention. The extrusion apparatus of the present invention and the apparatuses useful for practicing the method of the present invention may consist of a hydrophilic, curved aperture or a hydrophilic, curved aperture screen membrane. The film is a polyester (PETE) film.

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

Commercially available devices that are suitable for suitable membranes and can be used in the present invention are THE MINI-EXTRUDER ^TM , 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).

Extrusion equipment must be capable of withstanding high extrusion pressures. As a general rule, higher pressures result in improved performance, eg increased flow rate, less membrane fouling and clogging, and a faster decrease in the size of the extruded PEM. At a minimum, the extrusion equipment must be capable of withstanding extrusion pressures greater than about 400 psi. Also, membrane pedestal holders or covers that optimize the available surface area can be used if they can withstand the extrusion pressures they are subjected to. The cartridge holder device provides the advantage of minimizing membrane manipulation and greater efficiency in membrane stacking. In addition, the cartridge holder device provides an overall improved efficiency in production, for example, while exchanging a clogged or contaminated cartridge holder device, a clogged or contaminated membrane can be exchanged while the product flow is diverted to a new cartridge holder device. .

2) Pressure homogenization

The first application of homogenization for stabilization of food and dairy emulsions was presented at the Paris World's Fair in 1900 by Auguste Gaulin, an invention for "mixing milk" using pressures of up to 30 MPa. Since then, conventional homogenization has extended the pressure range to 50 MPa. HPH, also referred to as dynamic HPH, has been frequently highlighted for its potential for food pasteurization. The latest ultra-high pressure homogenizer enables pressures 10-15 times higher than conventional pressures and realizes 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 the pressure drop caused by the application of dynamic high pressure, shear stress, turbulent flow, cavitation, shock wave, temperature increase can be associated with emerging physical technologies as a result of several mechanisms such as

Homogenization processes may be used, particularly ultra-high pressure homogenization (UHPH), also referred to as dynamic high pressure. UHPH is a new technology recently studied in the fields of food, cosmetics and pharmaceuticals, 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. Indeed, 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 technology 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, standard homogenizers operate with upstream pressures of 20 to 60 MPa, as in the dairy industry. UHPH has benefited from the latest developments in high pressure (HP) technology, namely the development of HP hardeners, and the concept of high pressure resistant materials (stainless steel, ceramics, seals). Sophisticated homogenizing valves with seats and needles made of ceramics or coated with artificial diamonds specially developed for UHPH equipment capable of withstanding pressures of up to 350 to 400 MPa are currently being studied.

The PEMN fabricated in the present invention is quasi-electric light scattering (QELS), also known as Dynamic Light Scattering (DLS), according to 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 of the PEMN prepared in the present invention may be 0.01 to 0.5. For example, the polydispersity index of 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, or 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.

In the PEMN prepared in the present invention, the active ingredient nucleic acid can be released at a constant rate.

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

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

The PEMN prepared in the present invention may additionally contain at least one selected from the group consisting of ionic salts, small molecule drugs, high molecular drugs, high molecular drugs including drugs, and polymers including fluorescent substances.

The high-molecular drug may be any one or more selected from the group including proteins, antibodies, and nucleic acids.

A therapeutically effective amount of the PEMN can be administered to a subject, thereby treating a disease in a subject in need thereof, including the process of treating the disease.

Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited only to these examples.

V. Applications of Complexes Containing MLKL mRNA

In the present invention, necroptosis-like cell death may be induced in a tumor by the complex containing the MLKL mRNA. The RIPK3 is known as a key regulator of cell death by necroptosis, and recruits MLKL, an executor, to signal necroptosis, which is terminated by rupture of the cytoplasmic membrane and leakage of cell contents from dead cells. phosphorylate Experimental data to date have indicated that RIPK3 and MLKL are essential mechanisms for all necroptotic cell death responses.

In the present invention, the nanoparticles containing the MLKL mRNA may induce efflux of DAMP (Damage-associated molecular pattern) from tumor cells by ultrasound irradiation.

In addition, in the present invention, the nanoparticle containing the MLKL mRNA may be targeted to a tumor.

In addition, in one embodiment of the present invention, when cancer cells are treated with nanoparticles containing MLKL mRNA, cell death occurs in a mechanism similar to necroptosis rather than apoptosis, resulting in damage-associated molecular pattern (DAMP) with preserved immunogenicity. ) was confirmed to be leaked, and as a result, maturation of dendritic cells was induced, suggesting the possibility of combination therapy with anticancer immunotherapy. In particular, nanoparticles containing MLKL mRNA can induce an anticancer immune response more effectively by inducing activation of NK cells and eosinophils, unlike conventional apoptosis that can only involve activation of dendritic cells.

In addition, in another embodiment of the present invention, it was confirmed that the nanoparticles containing MLKL mRNA according to the present invention were selectively accumulated in tumors compared to other tissues.

Therefore, according to the above embodiments, the present invention can provide a pharmaceutical composition for the prevention or treatment of cancer, comprising the nanoparticles containing MLKL mRNA according to the present invention as an active ingredient in another aspect.

The pharmaceutical composition includes a composition for ultrasound immunotherapy.

As another aspect of the present invention, the present invention may provide a method for preventing or treating cancer, comprising administering to a subject a nanoparticle containing MLKL mRNA according to the present invention.

As another aspect of the present invention, the present invention may provide a use of nanoparticles containing MLKL mRNA according to the present invention for preventing, improving or treating cancer.

As another aspect of the present invention, the present invention can provide the use of nanoparticles containing MLKL mRNA according to the present invention to produce a drug used for cancer.

In addition, the composition may include cytokines, chemokines, suicide gene products, immunogenic proteins or peptides, apoptosis inducers, angiogenesis inhibitors, heat shock proteins, tumor antigens, β-catenin inhibitors, STING pathway activators, immune checkpoint blockers (Immune-checkpoint blockers, ICB), innate immune activators, antibodies, dominant negative receptors and decoy receptors, inhibitors of myeloid derived suppressor cells (MDSC), inhibitors of the IDO pathway, and inhibitors of apoptosis; or It may further include any one selected from the group consisting of peptides, antibacterial agents, antiviral agents, drugs, adjuvants, chemotherapeutic agents, and kinase inhibitors.

The immune checkpoint blockers (ICB) are PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, LAG3 inhibitors, TIM3 inhibitors, OX-40 stimulators, 4-1BB stimulators , CD40L stimulators, CD28 stimulators, and GITR stimulators.

The PD-1 inhibitor is an antibody antagonistic to PD-1, and the PD-L1 inhibitor may be an antibody antagonistic to PD-L1.

Any one selected above may be any one selected from the group including protein and mRNA.

The immune checkpoint blockers (ICB) may be any one selected from the group including antibodies, proteins and mRNAs.

In addition, the composition may further contain RIPK3 (receptor-interacting protein kinase 3) mRNA as an active ingredient.

Moreover, in the present invention, the composition may further contain a DPP4 inhibitor (dipeptidyl peptidase 4 inhibitor, DPP4i) as an active ingredient.

In the present invention, the composition may be administered simultaneously, separately or sequentially with the immune checkpoint inhibitor or DPP4 inhibitor, and may be administered singly or multiple times.

In one embodiment of the present invention, in addition to the excellent anticancer effect of nanoparticles containing MLKL mRNA in a cancer animal model, complete remission was observed in 40% of subjects when used in combination with an immune checkpoint inhibitor, confirming that the anticancer effect was maximized. In addition, nanoparticles (cNB) containing MLKL mRNA also showed excellent anticancer effects in cancer animal models, and it was confirmed that the anticancer effect was further maximized when treated in combination with the DPP4 inhibitor sitagliptin.

As used herein, the term "prevention" refers to any activity that suppresses or delays cancer by administration of the pharmaceutical composition according to the present invention.

As used herein, the term "treatment" refers to any activity in which cancer is improved or beneficially altered by administration of the pharmaceutical composition according to the present invention, and attempts are made to obtain useful or desirable results, including clinical results. means A useful or desirable clinical outcome, whether detectable or not, is alleviation or amelioration of one or more symptoms or conditions, reduction of disease extent, stabilization of disease state, inhibition of disease occurrence, inhibition of disease spread, delay or slowing of disease progression. , delay or slowing of disease onset, improvement or alleviation of disease state, and reduction (partial or total), but are not necessarily limited thereto. Also, "treatment" can mean prolonging the survival of a patient beyond what would be predicted in the absence of treatment. In addition, "treatment" may refer to inhibiting disease progression, temporarily slowing disease progression, or may relate to permanently halting disease progression.

As used herein, the term “immune checkpoint inhibitor” refers to an immune checkpoint protein involved in T cell suppression when some cancer cells evade immunity while utilizing the immune checkpoint of T cells, which are immune cells in the body. ), a type of immunocancer drug that activates T cells to attack cancer cells by blocking the activation of CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) inhibitor, PD-1 (Programmed cell death protein 1) inhibitor , PD-L1 (Programmed death-ligand 1) inhibitor, LAG-3 (Lymphocyte-activation gene 3) inhibitor, TIM-3 (T-cell immunoglobulin and mucin-domain containing-3) inhibitor, TIGIT (T cell immunoreceptor with Ig) and ITIM domains) inhibitors and VISTA (V-domain Ig suppressor of T cell activation) inhibitors.

In the present invention, the immune checkpoint inhibitor may be an antibody that specifically binds to one or more proteins selected from the group consisting of CTLA-4, PD-1 and PD-L1, but is not limited thereto.

As used herein, the term "dipeptidyl peptidase 4 inhibitor (DPP4i)" refers to a relatively small molecular weight chemical compound that selectively inhibits the enzyme activity through structural analysis of the active pocket that is very important for the enzyme activity of DPP4. It is mainly used for the treatment of type 2 diabetes. In the present invention, the DPP4 inhibitor may be an activity inhibitor of the DPP4 protein, and more specifically, an antibody against DPP-4, sitagliptin, vildagliptin, saxagliptin, lina linagliptin, dutogliptin, gemigliptin, alogliptin, anagliptin, evogliptin, berberine, diprotin ) and at least one selected from the group consisting of lupiol (Lupeol), but is not limited thereto.

In the present invention, the cancer may be one in which RIPK3 or MLKL protein is deficient or the expression of the protein is down-regulated, but is not limited thereto.

In the present invention, the cancer is lung cancer, gastric cancer, glioma, liver cancer, melanoma, kidney cancer, urothelial cancer, head and neck cancer, Merkel-cell carcinoma, prostate cancer, hematological cancer, breast cancer, colon cancer, colon cancer , rectal cancer, pancreatic cancer, brain cancer, ovarian cancer, bladder cancer, bronchial cancer, skin cancer, cervical cancer, endometrial cancer, esophageal cancer, thyroid cancer, bone cancer, and combinations thereof. Since the containing nanoparticles are based on the principle of exerting an anticancer effect by inducing necroptosis in cancer cells, the pharmaceutical composition according to the present invention can exert an effect on all types of cancer regardless of the type of cancer. .

Meanwhile, the pharmaceutical composition according to the present invention may further include suitable carriers, excipients and/or diluents commonly used to prepare pharmaceutical compositions in addition to active ingredients. In addition, it can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions according to conventional methods.

Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose , microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oil. When formulating the composition, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is based on the type, severity, and activity of the drug in the patient. , sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field.

Considering all of the above factors, it is important to administer the amount that can obtain the maximum effect with the minimum amount without side effects, which can be determined by those skilled in the art. Specifically, the effective amount of the pharmaceutical composition according to the present invention may vary depending on the patient's age, sex, condition, body weight, absorption rate, inactivity rate and excretion rate of the active ingredient in the body, disease type, and concomitant drugs.

The pharmaceutical composition of the present invention can be administered to a subject by various routes. For example, it may be administered by oral administration, intranasal administration, transbronchial administration, arterial injection, intravenous injection, subcutaneous injection, intramuscular injection, or intraperitoneal injection. The daily dose may be divided and administered once or several times a day.

In the present invention, the composition may be in the form of an intravenous or intraperitoneal injection, but is not limited thereto.

As another aspect of the present invention, the present invention may provide a pharmaceutical composition for inhibiting metastasis of cancer, comprising nanoparticles containing MLKL mRNA according to the present invention as an active ingredient.

As another aspect of the present invention, the present invention may provide a method for inhibiting cancer metastasis, comprising administering nanoparticles containing MLKL mRNA according to the present invention to a subject.

As another aspect of the present invention, the present invention may provide a use of the nanoparticles containing MLKL mRNA according to the present invention for preventing or inhibiting cancer metastasis.

In one embodiment of the present invention, it was confirmed that the nanoparticles containing MLKL mRNA had excellent ability to inhibit cancer metastasis in an animal model of lung metastasis, and it was confirmed that side effects such as lymphoproliferation did not appear.

As used herein, the term “metastasis” refers to a state in which a malignant tumor has spread to other tissues away from an organ where it has developed. As a malignant tumor that started in one organ progresses, it spreads from an organ, which is the primary site where it first occurred, to other tissues, and spreading from the primary site to other tissues can be referred to as metastasis. Metastasis can be said to be a phenomenon accompanying the progression of malignant tumors, and metastasis may occur while acquiring new genetic traits as malignant tumor cells proliferate and cancer progresses. Metastasis can occur when tumor cells that have acquired new genetic traits invade blood vessels and lymph glands, circulate along the blood and lymph, and settle and proliferate in other tissues. Depending on the tissue in which metastasis occurs, various cancer diseases such as liver cancer, kidney cancer, lung cancer, stomach cancer, colon cancer, rectal cancer, and pancreatic cancer may be induced. The composition according to the present invention can prevent and treat the spread of cancer by inhibiting metastasis.

As used herein, the term “inhibition” refers to any activity that suppresses the cancer metastasis by administration of the composition according to the present invention.

The terms or words used in this specification and claims should not be construed as being limited to ordinary or dictionary meanings, and the inventors may appropriately define the concept of terms in order to explain their invention in the best way. It should be interpreted as a meaning and concept consistent with the technical idea of the present invention based on the principle that there is.

Hereinafter, a preferred embodiment is presented to aid understanding of the present invention. However, the following examples are provided to more easily understand the present invention, and the content of the present invention is not limited by the following examples.

Example 1. Design and manufacture of mRNA nucleic acid templates of interest

The nucleic acids of the present invention suggest an mRNA of interest having a structure capable of protein synthesis in cells. mRNAs of interest for use in the present invention include, but are not limited to, MLKL, an ORF encoding soluble PD-L1.

1-1. Design of nucleic acid templates

The nucleic acid may be a non-amplifying mRNA (non-amplifying mRNA) construct; and self-amplifying 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 includes (a) an mRNA construct for expressing RNA dependent RNA polymerase (RdRp); and (b) an mRNA replicon comprising an ORF capable of being replicated by the RdRp and encoding the polypeptide of interest, wherein the two types of components are in the same single RNA. cis-replicon RNA constructs or trans-replicon RNA constructs in two different RNAs.

In order to prepare the nucleic acid of the present invention, target protein DNA having a T7 promoter capable of producing a target protein ORF is designed and constructed and recombined into 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 is a DNA fragment capable of efficiently synthesizing the target protein, and was prepared in the order of <T7 promoter - 5'UTR - target protein DNA - muag - A64 - C30 - histone-SL>.

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, and 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, and contains muag (mutated alpha-globin-3'UTR), A64 poly(A) sequence, poly(C) sequence (C30) and It was placed upstream of the histone stem-loop sequence (histone_SL) sequence.

When the mRNA product of interest is secreted into blood, a signal peptide (Sec, SEQ ID NO: 8) and a nucleotide sequence (Albumin binding domain, SEQ ID NO: 13) for life-span extension in blood may be added.

The unit elements constituting the target protein DNA fragment are as follows.

Nucleotide sequence constituting target protein nucleic acid 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_SL (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 ATTC TGCCTA ATAAAAAACA TTTACATTGC TGCGTC (SEQ ID NO: 11) A30LA70 (SEQ ID NO: 12) Albumin binding domain CAGCATGATG AAGCGGTGGA TGCGAACAGC CTGGCGGAAG CGAAAGTGCT GGCGAACCGC GAACTGGATA AATATGGCGT GAGCGATTTT TATAAACGCC TGATTAACAA AGCGAAAACC GTGGAAGGCG TGGAAGCGCT GAAACTGCAT ATTCTGGCGG CGCTGCCGTA A (SEQ ID NO: 13, 171 nt)
(QHDEAVDANS LAEAKVLANR ELDKYGVSDF YKRLINKAKT VEGVEALKLH ILAALP: SEQ ID NO: 14) (G4S)3-linker GGAGGCGGTG GTAGTGGAGG TGGCGGGTCC GGTGGAGGTG GAAGC (SEQ ID NO: 15)
(GGGGSGGGGSGGGGS; SEQ ID NO: 16)

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

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

The 3'UTR may be any one selected from structures including muag-A64-C30-histone-SL and 2 BgUTR-A30LA70.

If the target protein is a fusion protein, the linker is a (G4S)3-linker and. Sec can be used for the purpose of secreting a target protein to the outside of the cell.

where the 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 the wild-type sequence) mutated UTR of (alpha-)globin, poly (A) RNA sequence consisting of 64 adenine nucleotides for the tail, 30 cytosine nucleotides for the poly(C) sequence, and (5'-CAAAGGCUCU UUUCAGAGCC ACCA-3' SEQ ID NO: 20) histone stem-loop.

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

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

<mRNA of interest>

MLKL

Necroptosis is a caspase-independent lytic form of programmed cell death, which is involved in host defense. Necroptosis may also participate in several human pathologies including ischemia-reperfusion injury, degenerative and inflammatory diseases. Cells tend to receive death signals when receptor-interacting serine/threonine-protein kinase (RIPK)-1 cannot be localized by E3 ligases such as cellular inhibitor of apoptosis proteins (cIAP). Within the necrosome, RIPK3 is in turn activated by autophosphorylation followed by cytoplasmic pseudokinase, mixed lineage kinase domain-like (MLKL) phosphorylation. Although the exact mechanisms of MLKL activation may vary by species, RIPK3-mediated phosphorylation of MLKL in necrosomes occurs before translocation of MLKL to the plasma membrane, where MLKL compromises membrane integrity and triggers apoptosis.

Although our understanding of the necroptotic pathway is still in its infancy, it is of great importance for the upstream events governing MLKL activation and the final events by which MLKL disrupts membrane integrity. In comparison, the molecular mechanisms of the intervening steps, such as translocation of MLKL to the plasma membrane and triggering necrosis, are poorly understood. To date, proteins of the ESCRT-III complex, Flotillins and Alix:Syntenin-1 have been reported to regulate MLKL localization, primarily in the context of endosomal compartmentalization or exosomal shedding of activated MLKL to negate apoptosis. It is unclear which of these pathways regulate MLKL trafficking to the plasma membrane, where membrane integrity is disrupted leading to cell death.

MLKL (NM_152649.2) cDNA ORF clone, Homo sapiens (human) -> NP_689862.1 Homo sapiens mixed lineage kinase domain-like (MLKL), transcript variant 1, mRNA.

ATGGAAAATT TGAAGCATAT TATCACCCTT GGCCAGGTCA TCCACAAACG GTGTGAAGAG

ATGAAATACT GCAAGAAACA GTGCCGGCGC CTGGGCCACC GCGTCCTCGG CCTGATCAAG

CCTCTGGAGA TGCTCCAGGA CCAAGGAAAG AGGAGCGTGC CCTCTGAGAA GTTAACCACA

GCCATGAACC GCTTCAAGGC TGCCCTGGAG GAGGCTAATG GGGAGATAGA AAAGTTCAGC

AATAGATCCA ATATCTGCAG GTTTCTAACA GCAAGCCAGG ACAAAATACT CTTCAAGGAC

GTGAACAGGA AGCTGAGTGA TGTCTGGAAG GAGCTCTCGC TGTTACTTCA GGTTGAGCAA

CGCATGCCTG TTTCACCCAT AAGCCAAGGA GCGTCCTGGG CACAGGAAGA TCAGCAGGAT

GCAGACGAAG ACAGGCGAGC TTTCCAGATG CTAAGAAGAG ATAATGAAAA AATAGAAGCT

TCACTGAGAC GATTAGAAAT CAACATGAAA GAAATCAAGG AAACTTTGAG GCAGTATTTA

CCACCAAAAT GCATGCAGGA GATCCCGCAA GAGCAAATCA AGGAGATCAA GAAGGAGCAG

CTTTCAGGAT CCCCGTGGAT TCTGCTAAGG GAAAATGAAG TCAGCACACT TTATAAAGGA

GAATACCACA GAGCTCCAGT GGCCATAAAA GTATTCAAAA AACTCCAGGC TGGCAGCATT

GCAATAGTGA GGCAGACTTT CAATAAGGAG ATCAAAACCA TGAAGAAATT CGAATCTCCC

AACATCCTGC GTATATTTGG GATTTGCATT GATGAAACAG TGACTCCGCC TCAATTCTCC

ATTGTCATGG AGTACTGTGA ACTCGGGACC CTGAGGGAGC TGTTGGATAG GGAAAAAGAC

CTCACACTTG GCAAGCGCAT GGTCCTAGTC CTGGGGGCAG CCCGAGGCCT ATACCGGCTA

CACCATTCAG AAGCACCTGA ACTCCACGGA AAAATCAGAA GCTCAAACTT CCTGGTAACT

CAAGGCTACC AAGTGAAGCT TGCAGGATTT GAGTTGAGGA AAACACAGAC TTCCATGAGT

TTGGGAACTA CGAGAGAAAAA GACAGACAGA GTCAAATCTA CAGCATATCT CTCACCTCAG

GAACTGGAAG ATGTATTTTA TCAATATGAT GTAAAGTCTG AAATATACAG CTTTGGAATC

GTCCTCTGGG AAATCGCCAC TGGAGATATC CCGTTTCAAG GCTGTAATTC TGAGAAGATC

CGCAAGCTGG TGCTGTGAA GCGGCAGCAG GAGCCACTGG GTGAAGACTG CCCTTCAGAG

CTGCGGGAGA TCATTGATGA GTGCCGGGCC CATGATCCCT CTGTGCGGCC CTCTGTGGAT

GAAATCTTAA AGAAACTCTC CACCTTTTCT AAGTAG

-------------------------------------------------- --------------------

Mlkl (NM_029005.3) cDNA ORF clone, Mus musculus (house mouse) -> NP_083281.1 Mus musculus mixed lineage kinase domain-like (Mlkl), transcript variant 2, mRNA.

ATGGATAAAT TGGGACAGAT CATCAAGTTA GGCCAGCTCA TCTATGAACA GTGTGAAAAG

ATGAAATACT GCCGGAAACA ATGCCAGCGT CTAGGAAACC GTGTGCACGG CCTGCTACAG

CCTCTCCAGA GACTCCAGGC CCAAGGAAAG AAGAACCTGC CCGATGACAT TACTGCTGCC

CTGGGCCGTT TTGATGAAGT CCTGAAGGAG GCTAACCAGC AGATAGAAAA GTTCAGCAAG

AAGTCCCATA TTTGGAAGTT TGTGAGTGTG GGCAATGATA AGATCCTCTT CCATGAAGTG

AATGAGAAGC TGAGAGACGT CTGGGAGGAG CTGTTGCTGC TGCTTCAGGT TTATCATTGG

AATACCGTTT CAGATGTCAG CCAGCCAGCA TCCTGGCAGC AGGAAGATCG ACAGGATGCA

GAGGAAGACG GAAATGAAAA TATGAAAGTT ATCCTGATGC AGTTGCAAAT TAGCGTGGAA

GAAATCAACA AAACCCTGAA GCAATGCTCA CTAAAACCCA CACAGGAGAT CCCACAAGAT

CTCCAAATCA AGGAGATTCC AAAGGAACAT CTTGGACCTC CGTGGACCAA ACTGAAGACA

AGTAAAATGA GCACCATTTA TAGAGGAGAG TATCACAGAT CTCCAGTTAC CATCAAAGTA

TTCAACAACC CCCAGGCCGA AAGTGTTGGA ATAGTGAGGT TCACTTTCAA TGACGAGATC

AAAACCATGA AGAAATTCGA TTCTCCCAAC ATCTTGCGTA TATTTGGGAT TTGCATTGAT

CAAACAGTGA AGCCCCCTGA GTTCTCCATT GTCATGGAGT ACTGTGAACT TGGAACCCTG

AGGGAACTGC TGGATAGAGA AAAAGACCTC ACAATGAGTG TGCGCAGCCT CCTAGTCCTG

AGGGCAGCCA GAGGCTTATA CAGGCTACAC CATTCGGAAA CACTCCACAG AAACATCAGC

AGCTCCAGTT TCCTCGTAGC CGGAGGCTAC CAAGTAAAGC TTGCAGGATT TGAGTTAAGC

AAAACACAGA ATTCCATCAG CCGGACAGCA AAGAGCACTA AAGCAGAGAG ATCCAGTTCA

ACGATATATG TCTCCCCTGA GAGACTGAAA AATCCATTTT GCCTTTATGA CATAAAAGCT

GAAATATATA GCTTTGGAAT TGTACTCTGG GAAATTGCCA CTGGAAAGAT CCCATTTGAA

GGCTGTGATT CTAAGAAGAT CCGGGAGCTG GTGGCTGAGG ACAAGAAGCA GGAACCAGTG

GGTCAGGATT GCCCTGAGTT GTTGCGGGAA ATCATTAATG AGTGTCGTGC CCATGAGCCC

TCCCAACGGC CCTCTGTGGA CGGAATCTTG GAGAGACTGT CTGCGGTTGA AGAATCCACG

GACAAGAAGG TGTAA

Water-soluble PD-L1 (sPD-L1)

Drugs for cancer treatment are largely divided into small-molecule drugs and high-molecular drugs, and polymer drugs with specificity are in the limelight as therapeutic agents compared to small-molecule drugs that have relatively large side effects due to lack of specificity. Recently, as a method for treating cancer, blocking of PD-1/PD-L1 binding among immune checkpoint inhibitory proteins has a great effect on cancer treatment, and has fewer side effects than other immune checkpoint inhibitory proteins.

Large pharmaceutical companies such as Bristol-Myers Squibb are making efforts to develop therapeutic drugs through PD-1/PD-L1 immune checkpoint inhibition, and anti-cancer drugs such as YERVOY (ipilimumab) and OPDIVO (nivolumab) use antibody formats. are being developed using

Since PD-1 and PD-L1 are expressed not only in cancer cells but also in human immune cells, antibody drugs can cause autoimmune diseases by killing normal immune cells.

In addition, in order to eliminate the binding of TILs (Tumor-infiltrating lymphocytes) that are bound to PD-1/PD-L1 with tumors, a therapeutic agent with excellent cell penetrating ability is required. exist.

In addition, although PD-L1 variants were discovered through screening in previous studies, this is a PD-L1 with few mutations because it has relatively low binding ability and can have immunogenicity as a therapeutic drug due to the introduction of many mutations. Many variants that bind to PD-1 with high binding affinity have been proposed (Republic of Korea, Patent Publication 10-2020-0002654).

(Korean Patent Publication No. 10-2020-0002654), which has a high affinity with PD-1, can effectively inhibit the binding between wild-type PD-L1 and PD-1, and at the same time minimize the possibility of immunogenicity. An L1 variant was discovered. By substituting some amino acid sequences of wild-type PD-L1 with other amino acid sequences (E169D, R195A, P198S) and optimizing, the binding force with PD-1 is greatly improved, and the possibility of immunogenicity is reduced by minimizing the mutation site. The base sequence was proposed as follows.

DNA sequence #24: PD-L1 Variant JY-DAS

TTTACTGTCA CGGTTCCCAA GGACCTATAT GTGGTAGAGT ATGGTAGCAA TATGACAATT 60

GAATGCAAAT TCCCAGTAGA AAAACAATTA GACCTGGCTG CACTAATTGT CTATTGGGAA 120

ATGGAGGATA AGAACATTAT TCAATTTGTG CATGGAGAGG AAGACCTGAA GGTTCAGCAT 180

AGTAGCTACA GACAGAGGGC CCGGCTGTTG AAGGACCAGC TCTCCCTGGG AAATGCTGCA 240

CTTCAGATCA CAGATGTGAA ATTGCAGGAT GCAGGGGTGT ACCGCTGCAT GATCAGCTAT 300

GGTGGTGCCG ACTACAAGCG AATTACTGTG AAAGTCAATG CCCCATACAA CAAAATCAAC 360

CAAAGAATTT TGGTTGTGGA TCCAGTCACC TCTGAACATG AACTGACATG TCAGGCTGAG 420

GGCTACCCCA AGGCCGAAGT CATCTGGACA AGCAGTGACC ATCAAGTCCT GAGTGGTAAG 480

ACCACCACCA CCAATTCCAA GAGAGATGAG AAGCTTTTCA ATGTGACCAG CACACTGAGA 540

ATCAACACAA CAACTAATGA GATTTTCTAC TGCACTTTTA GGGCTTTAGA TTCTGAGGAA 600

AACCATACAG CTGAATTGGT CATCCCAGAA CTACCTCTGG CACATCCTCC AAATGAAAGG 660

660

1.2. Preparation of target protein nucleic acid

mRNAs of interest for use in the present invention include, but are not limited to, MLKL, an ORF encoding soluble PD-L1.

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), using this as a template for forward primer 5 '- 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 contain ECoRI/BamHI recognition sequences.

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

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

To synthesize the capped target protein mRNA, first, pUC19 vector into which DNA having <T7 promoter - 5'UTR - target protein ORF DNA - muag - A64 - C30 - histone-SL> was inserted was used as a template, and forward primers SEQ ID NO: 20 and Reverse primer SEQ ID NO: 21 was used to amplify. 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, the HighYield T7 ARCA mRNA Synthesis Kit (m5CTP/Ψ-UTP) (Jena BioSCienCe, USA) synthesizes an expression cassette containing the capped target protein modified mRNA in vitro did

Specifically, the reaction solution (6 mM 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 an ethanol precipitation method or a spin column membrane.

As a control, the synthesis of the capped target protein mRNA 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).

Addition of video material

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

Example 2. Manufacturing design of multilayer polyelectrolyte nanoparticles (nanoparticles)

The target proteins of nucleic acids used in the present invention include therapeutic proteins (sTNFR2, EPO, G-CSF), extracellular matrix degrading enzymes (HYAL 1, Hyaluronidase PH20, MMP1, MMP 8, MMP 13), and extracellular matrix degrading enzymes. ORFs encoding inhibitors (TIMP 1, TIMP 2, TIMP 3 and TIMP 4) and target proteins for vaccination (RAV-G and RSV-F proteins), but are not limited thereto.

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

Preparation of the core particles or nanoparticles of the present invention is by adding an anionic compound to a suspension solution in which an anionic nucleic acid and a cationic compound are continuously vortexed for 30 seconds in a reaction solution, and continuously vortexed for 30 seconds or / And a first method of stabilizing at room temperature for 1 hour after the reaction was completed by ultrasonic treatment was used.

Particles prepared by self-assembly by mixing the selected nucleic acid, cationic compound and anionic compound were referred to as *?**?*nanoparticles*?**?*. One or more of the nucleic acids, cationic compounds, and anionic compounds may be selected, and nanoparticles may be prepared by mixing the selected compounds.

A second method was used in which the anionic nucleic acid, the cationic compound, and the anionic compound were continuously vortexed or/and sonicated for 30 seconds in the reaction solution to stabilize them at room temperature for 1 hour after the reaction was completed.

A third method was used to prepare a membrane prepared and eluted by a free standing layer by layer layering technique in which an anionic nucleic acid, a cationic compound, and an anionic compound are alternately layered on a selected substrate. Preferably, the anionic compound is first 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 is a cationic compound or anionic compound Selected and prepared. Subsequently, nanoparticles prepared by various methods were eluted. After preparing each layer, the substrate with the prepared layer was immersed in the cleaning solution and washed.

Example 3. Preparation of core particles

3.1. Preparation of core particle 1.0

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

The selected nucleic acid contains n nucleotides and thus has 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, especially arginine (70-80%), so that the number of cations per mole is 1.26 × 10 ²⁵ . PS neutralizes the negative charge of nucleic acid phosphate groups. Therefore, theoretically, a nucleic acid core particle can be prepared with a charge ratio of 1:0.4 or more between the selected nucleic acid and PS, that is, 1 for nucleic acid and 0.4 or more for PS.

When cationic PS is added to an anionic nucleic acid solution, an interaction between PS and nucleic acid occurs, and a mixture of PS and nucleic acid can spontaneously form particles.

The nucleic acid-based core particle of the present invention has a weight ratio of the nucleic acid and PS (Protamine) (μg / mL) in sterile distilled water ( DW) or isotonic solution (CM) to prepare core particles composed of nucleic acid/PS particles.

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

Then, the reactants were stabilized at room temperature for 1 hour and lyophilized to obtain prepared nucleic acid/PS core particles 1.1.

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'. In detail, the core particle using PS is The core particles were 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, refer to the inventor's application patent (Korean Patent Application: 10-2019-0007873).

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

Particles may also be used with a Phoenix RS-VA10 Vortexer (Phoenix Instrument, Germany) (10 seconds, 25 W) and a SpeedMixer™ DAC 150.1 CM 41 (HausChild & Co KG, Germany) (5 minutes, 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 particle 1.1 prepared in Example 2.1 was determined by Photon Correlation S nanoparticle troscopy (PCS) using a Malvern Zetasizer 3000 HSA (Herrenberg, Germany). 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). Software data analysis for size distribution calculation of core particle 1.0 was based on the fitting method of non-negative constrained least-squares (NNLS). The 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 by Malvern Zetasizer 3000 HSA (Malvern Panalytical technologies, Germany), initial complex particles of nucleic acid and PS were formed in the first step, and when strongly positively charged PS was additionally added, nucleic acid /PS particles were formed by self-assembly.

Referring to FIG. 4, the core particle 1.1-DW sample was a very large particle at 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 or higher. The most suitable size stable multiparticulates with a small size distribution can be prepared at PS concentrations where the nucleic acid:PS (μg/ml) ratio is higher than the 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, which increased as the PS concentration increased and then stabilized. At PS concentrations where the nucleic acid:PS ratio is higher than the mass ratio of about 30:60, the core particle 1.0-coated 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, and 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 particles 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 results of this study are similar to the theoretical expectations. The physical and chemical particle characteristics and biological activity of Core Particle 1.0 are determined by the ratio between the positively charged nitrogen (N) of PS, which is a cationic compound, 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 charge.

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

These results were not limited to selected mRNAs but were generally observed with other nucleic acids.

seal and release

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

Confirmation of binding between nucleic acid and 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 under 80 Volt conditions using 1x TBE buffer.

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

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

The stability of core particle 1.0 and the release of nucleic acids were investigated at 37°C in PBS. After 24 hours, nucleic acid release did not occur under the conditions of 30:90 and 30:150 nucleic acid:PS ratios. The 30:75 condition showed a small release of nucleic acid, and 8.0% of unbound nucleic acid was found. The nucleic acid:PS (μg/ml) ratio of the core particle was 90.0% at a mass ratio of 30:15, and 63.0% of the nucleic acid was found in the solution at a mass ratio of 30:30.

As the PS increased at a nucleic acid:PS (μg/ml) ratio of 1.0 or more in the core particle 1.0, the mRNA was strongly bound to PS and the degree of release 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 with other nucleic acids.

Example 4. Core particle 2.0

4.1. Manufacture of core particle 2.0

When the core particle 2.0 is prepared using the cationic compound PS and DOTAP together, the concentration of the positive charge generated from the PS varies from 15% (low), 45%/55% (medium), and 85% (high). made it

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

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

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

4.2. Evaluation of core particle 2.0

When the core particle 2.0 is prepared using the cationic compound PS and DOTAP together, the concentration of the positive charge generated from the PS varies from 15% (low), 45%/55% (medium), and 85% (high). made it

The physical and chemical particle characteristics and biological activity of core particle 2 are determined by the ratio between the positively charged nitrogen (N) of PS and DOTAP, which are cationic compounds, 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 charge.

A nucleic acid consisting of 2,000 bases has a negative charge of about 340.5 g/mol. Thus, 1 g nucleic acid equals 2.94 mmol negative charge. At physiological pH, phosphate groups in the RNA backbone are anionic based on the associated pKa of the phosphate group. The amount of positive charge on PS is determined by the sulfate concentration provided by the manufacturer. PS has a positive charge of 3.6 mmol per gram of material. Potentiometric titration (supplement) binds 0.35 mmol at pH 7.2 (preparation conditions). DOTAP has a molecular weight of 698.5 g/mol and 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 results 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, it was confirmed that a minimum mass ratio of 30:60 was ideal for preparing nucleic acid/PS core particles 1.0 having a stable small monomodal size distribution in isotonic solution.

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 must be considered, which can lead to more complex Core Particle 2.0 configurations.

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

In order to evaluate the RNA sealed in the core particle 2.0, gel retardation assay was performed to evaluate the amount. All core particles 2.0 confirmed RNA loading. In the core particle 2.0 synthesized with MP, the amount of RNA loaded twice as 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 for interaction sites and releases RNA from cationic compounds. The added heparin concentration increased from 10-fold to 100-fold excess compared to the RNA concentration (% w/w).

Particles with an N/P ratio of 2 consisting only of DOTAP/RNA or PS/RNA were evaluated in comparison with core particles having similar DOTAP/PS/RNA absolute ratios but obtained from other assembly routes. 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% of RNA bound in Core Particle 2.0 clearly varied depending on the manufacturing route in Core Particle 2.0. PC (medium) core particle 2 had almost no mRNA release, whereas DC (low) had moderate RNA release, and MP (medium) core particle 2.0 had 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 complex particles' including nucleic acid/PS/HSA complex particle 1.1, nucleic acid/PS-TCA/HSA complex particle 1.2, nucleic acid/CS/HSA complex particle 1.3 or nucleic acid/CS-TCA/HSA complex particle 1.4 was collectively called.

5.1. Preparation of HSA multiparticulates

The nucleic acids of the present invention suggest an mRNA of interest having a structure capable of protein synthesis in cells. mRNAs of interest for use in the present invention include, but are not limited to, MLKL, an ORF encoding soluble PD-L1.

The HSA multiparticles 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 137mM, KCl 2.7mM, Na2HPO4 10mM, KH2PO4 1.8mM; pH 7.4) or It was prepared at a concentration of 2.5 mg/ml using RPMI 1640 cell medium without phenol red.

2000㎍의 HSA를 15

150㎍의 PS에 첨가한 다음 5초 동안 혼합하였다. 그 후, 상기 제조한 30㎍ 핵산을 1ml의 최종 용적에 도달시켜 첨가하고, 5초 동안 와동하여 입자를 제조하였다. 0

2000 μg of HSA for 15

It was added to 150 μg of PS and mixed for 5 seconds. Then, 30 μg nucleic acid prepared above was added to reach a final volume of 1 ml, and vortexed for 5 seconds to prepare particles.

Alternatively, the order of addition of the three compounds may be arbitrarily determined.

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

Then, the reactants were left at room temperature for 1 hour and lyophilized for 2 days to obtain multiparticles prepared by *??**?*Nucleic acid/PS/HSA composite particles 1.1 or HSA composite particles 1.1*?**? *.

Here, the nucleic acid may be any one type selected from the above several 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, refer to the inventor's application patent (Korean Patent Application: 10-2019-0007873).

Instead of PS, it can be prepared by selecting PS and DOTAP, and their concentrations can be used by varying the ratio of positive charge generated from PS to 15% (low), 45%/55% (medium) and 85% (high). may be

5.2. Evaluation of HSA multiparticulates

Effect of HSA on particle size

500㎍의 HSA를 첨가하고 혼합하면 dh는 250

280nm의 범위이고 PDI가 ≤ 0.1이었다. 핵산/PS/HSA 복합입자 1.1-DW는 1000

2000㎍의 HSA를 증가시키면 dh가 커지고 PDI가 0.2 이상이 되었다(빨간색 막대 그래프). Referring to FIG. 7, the HSA multiparticle 1.1-DW was 100 μg in 1 ml of distilled water containing 30 μg nucleic acid and 90 μg PS.

When 500 μg of HSA is added and mixed, the dh is 250

range of 280 nm and a PDI of ≤ 0.1. Nucleic acid/PS/HSA complex particle 1.1-DW is 1000

Increasing 2000 μg of HSA increased dh and resulted in a PDI greater than 0.2 (red bar graph).

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

2000㎍의 HSA를 첨가하고 혼합하면 안정성은 개선되었다. 이 입자의 dh는 사용된 등장액에 따라 1

2mm에서 250

400nm로 감소하였다. 페놀 레드가 없는 세포 배지 RPMI 1640 등장액에서 dh = 245nm 및 PDI ≤ 0.15를 갖는 입자를 형성하였다. 이를 핵산/PS/HSA 복합입자 1.1-RPMI이라고 지칭하였다. Referring to FIG. 7, HSA multiparticles 1.1-isotonic solution was added to 1 ml isotonic solution containing 30 μg nucleic acid and 90 μg PS.

Stability was improved when 2000 μg of HSA was added and mixed. The dh of this particle is 1 depending on the isotonic solution used.

250 at 2mm

decreased to 400 nm. Particles with dh = 245 nm and PDI ≤ 0.15 were formed in cell medium RPMI 1640 isotonic solution without phenol red. This was referred to as nucleic acid/PS/HSA multiparticulate 1.1-RPMI.

150㎍ 범위로 평가되었다. Summarizing the results of FIG. 7, the optimal concentration of HSA is about 100 μg/ml in the HSA composite particle 1.1-DW, about 1000 μg/ml in the nucleic acid/PS/HSA composite particle 1.1-isotonic solution, and the optimal concentration of PS is 15 μg/ml.

A range of 150 μg was evaluated.

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

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

Example 6. HA nanoparticles

6.1. Preparation of HA nanoparticles

The nucleic acids of the present invention suggest an mRNA of interest having a structure capable of protein synthesis in cells. mRNAs of interest for use in the present invention include, but are not limited to, MLKL, an ORF encoding soluble PD-L1.

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

Particles formed by self-assembly by mixing the core particle and anionic polysaccharide or particles formed by ultrasonic spraying while mixing mRNA, cationic polypeptide, and anionic polysaccharide were referred to as nanoparticles. In the present invention, the following nucleic acid/PS/HA nanoparticle 1.1 and nucleic acid/PS/HA-TCA nanoparticle 1.2 were referred to as 'HA nanoparticles'.

The preparation of the nucleic acid / PS / HA nanoparticles is performed by dissolving 1 mg / mL of hyaluronic acid (HA, 83 kDa), an anionic polysaccharide, in sterile distilled water or isotonic solution for surface modification of the previously prepared core particle 1.0. Prepared as a 0.1% stock solution.

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

After mixing the 0.1% core particle 1.0 solution and the 0.1% HA solution prepared above at a feed mole ratio (nmol) of 1:0.5, 1:1, 1:1.5 or 1:2, they were treated for 5 minutes and freeze-dried. Thus, HA nanoparticles-DW were obtained.

Subsequently, a sonicator or an extruder may be appropriately used to adjust the size of the particles. Lyophilization gave HA nanoparticles 1.1-DW.

Alternatively, in another preparation of the HA nanoparticle 1.1, the feed mole ratio (nmol) of the nucleic acid/PS]:HA or [nucleic acid/PS]:HA is 1:0.5, 1:0.5, 1: After mixing nucleic acid, PS and HA for about 5 minutes according to 1, 1: 1.5 or 1: 2, a sonicator or extruder can be used appropriately to adjust the size of the particles, and freeze-drying Thus, HA nanoparticles 1.1 were obtained.

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

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

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

Homogenization using an extruder

The control is a nanoparticle prepared from a suspension solution in which an active ingredient nucleic acid, a cationic compound, and an anionic compound are mixed with an aqueous solution obtained by a conventional method (bottom-up method, bottom-up method).

Preliminary experiments were conducted on the nanoparticle production under various conditions using a LIPEX ^TM extruder (Northern Lipids, Canada) for the suspension solution containing the bottom-up nanoparticles.

PORETICS ^TM PETE membrane (Cat.No.T01CP02500) with an average pore size of 0.1 μm stacked in 5 layers at 200 psi using the LIPEX ^TM extruder (Northern Lipids, Canada) set for the above-prepared nanoparticle suspension solution After stabilizing the solution obtained by passing back and forth five times at room temperature for 1 hour, the formed HA nanoparticles were harvested and analyzed.

6.2. Evaluation of HA nanoparticles

Morphological analysis and confirmation of encapsulation

Particle size, polydispersity index (PDI) and zeta potential analysis were determined using a Zetasizer nano ZSP (Malvern Instrument, UK) equipped with a red He/Ne laser (633 nm). Particle size and PDI were measured using Dynamic Light Scattering (DLS) with a scattering angle of 173° at 20 °C and a quartz flow cell (ZEN0023), while 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 caused by Brownian motion and using a correlation function in the instrument corresponding to the Stokes-Einstein relation. These correlation functions are obtained by cumulative analysis and fit simple exponentials from which mean size (z-mean diameter) and distribution width estimate (polydispersity index) are obtained. Similarly, the correlation function is also suitable for multiple exponentials where 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-average diameter and the PDI ranges from 0 to 1, corresponding to monodisperse and very broad distributions, respectively. All nanoparticles were dispersed in ultrapure water using a dilution factor of ~1:100 v/v. All measurements were performed in triplicate and reported as mean ± standard deviation.

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

DLS analysis result of prepared HA nanoparticles 1.1 molar ratio of addition
(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 materials selected from the above example, nucleic acid, PS and HA, were mixed while sonication for 1 hour on an ice pack without heating in the reaction solution, and the remaining materials not treated in the previous step were added to the complex solution formed by mixing on ice without heating. After the reaction was completed by sonication on the pack for 1 hour, maintained at room temperature for 1 hour, HA nanoparticles 1.1 formed using a LIPEX ^TM extruder (Northern Lipids, Canada) were analyzed using Zetasizer, and the results were As shown in Table 5.

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

Results of DLS analysis of HA nanoparticles 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 nanoparticles of 1.0 were prepared at a molar ratio of 1:1.5 of nucleic acid/PS]; HA, centrifuged, lyophilized, rehydrated with 5% w/w Trehalose solution, and particle sizes before and after rehydration were investigated. did

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

HA nanoparticles 1.1 were lyophilized and then rehydrated, resulting in DLS analysis 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 with other nucleic acids.

HA nanoparticle drug release profile

The stability of HA multiparticle 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 multiparticle 1.1 was obtained by performing electrophoresis for 20 minutes at 80 Volt condition using 1x TBE buffer in a 2% agarose gel for the supernatant obtained under various conditions, and then the nucleic acid was 0.5 μg / ml of EtBr ( Ethidium bromide) was used for staining.

[Nucleic acid/PS]:HA hardly released mRNA under the addition molar ratio of 1:1.5 and 1:2. [Nucleic acid/PS]:HA 90.0% at molar ratios of 1:0.5 and 63.0% mRNA at 1:1 were found in solution. The 1:1.5 condition showed a small release of nucleic acid and 8.0% of unbound nucleic acid was found. It can be judged that, due to the influence of positively charged HA, as the amount of HA added increases, it reacts strongly with nucleic acid to form more stable particle forms.

[Nucleic acid/PS]:HA addition molar ratio of 1:1.5 mass ratio of the multiparticles, the drug release profile over time was investigated (FIG. 9).

The results of reading the band intensity in FIG. 9 are shown in Table 7. HA multiparticle 1.1 released 100% of the level of the sealed nucleic acid accumulated in the PBS solution for 120 hours and 50% of the nucleic acid between 72 and 96 hours. Emission was observed.

Release profiles of nucleic acids over time from HA nanoparticles 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 nanoparticles using FS_nanoparticles

7.1. Production of FS_Nanoparticles (Free standing LbL-based nanoparticles)

FS_nanoparticles (Free standing LbL-based nanoparticles) are silicon wafers (Si), polystyrene (PS), polypropylene (PP) and Teflon (PTFE) silicon wafers (Si), polystyrene (PS), polypropylene (PP) and It was built on four substrates of Teflon (PTFE). It was washed with ethanol before film deposition and thoroughly rinsed with water before drying with a nitrogen flow.

2.9이므로 산성 pH에서 음전하이다. PS는 pKa가

9.2-13.3이므로 양전하를 띠고 수용액에 용해되기 위해서는 산성 환경이 필요하다. pH 조정을 위해, pH 5.5 아세트산 나트륨 완충액 (0.1m)을 추가 염 (0.15m NaCl)의 존재하에서 제조하였다.Multi-electrolyte solutions were freshly prepared at various concentrations (1, 3 or 5 mg/mL) in 0.15 M NaCl pH 7.4 unless otherwise noted. HA has a pKa of

2.9, so it is negatively charged at acidic pH. PS is the pKa

9.2-13.3, so it has a positive charge and requires an acidic environment to dissolve in aqueous solutions. 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 up 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. Deionized water was used in all experiments unless otherwise specified.

Anionic HA, cationic PS, active ingredient nucleic acid, PS, and HA were deposited on the washed slide, and nanoparticles were prepared by repeatedly depositing them as one unit. The washed slides were continuously immersed in PS and HA (10 mL) solutions for 15 min each and then rinsed twice in 0.15 M NaCl pH 4.5 between solutions.

Alternatively, FS_nanoparticles were first produced using a polypropylene support washed with ethanol and water. A polyelectrolyte solution was alternately deposited on a template to form a PS and HA bilayer using a dipping robot specially designed for automated production of multilayer polyelectrolyte or inkjet printing.

Between each polyelectrolyte solution deposition step, the slide is immersed in an acetate buffer solution. After the deposition was completed, it was investigated whether or not the film was separated from the substrate without a cleaning treatment step. For this, four types of substrates were selected in terms of hydrophobicity: Si, PP, PS, and PTFE. A weak van der Waals interaction was established between the polyelectrolyte and the substrate, enabling the production of defect-free FS nanoparticles that peel off easily from the substrate.

The (PS/HA) 100 multilayer film made of Si was not robust and the film made of PTFE substrate was inseparable. Conversely, the nanoparticles built on the PP and PS substrates could be separated just by allowing them to dry. It is easy to handle with tweezers and can be cut into any shape with scissors.

Another LbL technique involves depositing a cationic compound, poly (4-vinylpyridiniomethanecarboxylate) (PVPMC) on selected substrates, then using a specially designed dipping robot or inkjet printing to create anionic HA, cationic PS, Active ingredients nucleic acid, PS, and HA were deposited, and nanoparticles were prepared by repeatedly depositing them as one unit. The layer-by-layer (LbL) nanoparticles grew linearly as the number of layers increased. The degradation rate of PVPMC-based multilayers can be well controlled by changing the salt concentration in the aqueous solution. The PVPMC-based multilayer film was completely degraded within 15 minutes in 0.9% physiological saline.

7.2. Ultra-high pressure homogenization (UHPH)

The control is a nanoparticle prepared from a suspension solution in which an active ingredient nucleic acid, a cationic compound, and an anionic compound are mixed with an aqueous solution obtained by a conventional method (bottom-up method, bottom-up method).

The development of nanoparticles was carried out in several steps according to the anionic nucleic acid, hybridization order and ratio of cationic and anionic compounds, UHPH applied pressure and recirculation cycle. Initially, three polymer solutions of anionic nucleic acid, cationic compound and anionic compound were independently prepared in ultrapure water at a concentration of 1 mg/mL.

In Case I, a suspension solution A was prepared by mixing an anionic nucleic acid solution and a cationic compound solution while shaking (~ 300 rpm, 25° C.) to form core particles by electrostatic interaction. Subsequently, an anionic compound was added while shaking the suspension solution A, and ultrasonic treatment was performed to prepare a suspension solution B containing nanoparticles formed by electrostatic interaction between the core particles and the anionic compound.

Case 2 is a suspension solution containing nanoparticles formed by electrostatic interaction between the anionic nucleic acid solution, cationic compound solution and anionic compound solution at a concentration of 1 mg/mL, each added while shaking, and sonicated. C was prepared.

In Case 3, solution D containing nanoparticles prepared and eluted in a layer-by-layer manner was prepared on a substrate.

Nanoparticles were prepared by homogenizing the prepared suspension solutions B, C, and solution D with UHPH using a Nano DeBEE 2000 Series Homogenizer (BEE International, USA).

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 were pressure (68.9, 137.9, 172.4 and 2000 MPa) and number of recirculation cycles (1 and 4 times).

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

In the present invention, the purification of the particles is performed by adding 100% EtOH to the buffer containing the particles to make them 70% EtOH, centrifuging at 4 ° C for 5 minutes, washing twice with 70% EtOH, and centrifugation at 4 ° C for 5 minutes. A fourth method of adding sterile distilled water or isotonic solution after separation;

A fifth method of adding 20 μL isopropanol followed by tapping, treating at -20 ° C for 15 minutes, centrifuging at 4 ° C for 5 minutes, washing twice with 70% EtOH, drying the precipitate and adding sterile distilled water or isotonic solution; and

After adding 2.5X amount of 100% EtOH and 3M sodium acetate, tapping, treating at -20℃ for 15 minutes, centrifuging at 4℃ for 5 minutes, washing twice with 70% EtOH, drying the precipitate, and using sterile distilled water or isotonic solution. A sixth method of adding; was used as an ideal method selected from methods including.

In the drug release analysis method in the present invention, FBS was added to the particles to prepare a reaction solution with a final 50% FBS concentration, followed by reaction over time in a 37˚C, CO ₂ incubator, centrifugation, and the supernatant was analyzed by Gel retardation analysis.

In the present invention, lyophilization 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 lyophilize. proceeded. Freeze-drying time was determined according to the amount and was 1 hour/100 μL. The freeze-dried sample was stored at room temperature using a container separated from the lyophilizer while maintaining a vacuum state.

Example 8. Biological evaluation of multiparticulates

8.1. Experimental Materials and Methods

8.1.1. Cell culture and cellular uptake imaging

Mouse colorectal cancer cell line (CT26) and human colorectal cancer cell line (HT29) were purchased from ATCC (MD, USA), RPMI1640 medium (Gibco, USA) containing 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic. were cultured in a CO2 incubator at 37 °C. 1 × 10 ⁵ CT26 cells were inoculated in a 35-pi cell culture dish with a glass bottom, and cultured for 36 hours.

Cells were washed with PBS and incubated with nanoparticles containing the mRNA of interest in serum-free RPMI medium in the dark for 6 hours. Then, the cells were washed twice with PBS, fixed with 2% paraformaldehyde solution, and then stained with DAPI (4,6-diamidino-2-phenylindole) for 10 minutes. Cells were then washed twice with PBS, and DAPI (blue; λEx = 405 nm, λEm = 450–500 nm) and Ce6 (red; λEx = 633 nm, λEm = 650–730 nm) were examined with a confocal laser scanning microscope. (Leica TCS SP8; Leica Microsystems, Germany).

Meanwhile, for experiments using nanoparticles containing the mRNA of interest, 1 × 10 ⁵ CT26 cells were seeded in a 35-pi cell culture dish with a glass bottom and cultured for 36 hours. Cells were washed with PBS and incubated with nanoparticles containing 0.1 mg/mL mRNA of interest in serum-free RPMI medium for 6 hours. Then, the cells were washed twice with PBS, fixed with 2% paraformaldehyde solution, and then stained with DAPI for 10 minutes. Cells were then washed twice with PBS, DAPI (blue; λEx = 405 nm, λEm = 450–500 nm) and Cy5.5 (red; λEx = 633 nm, λEm = 650–730 nm) confocal laser Analysis was performed using a scanning microscope (Leica TCS SP8; Leica Microsystems).

8.1.2. Cell viability evaluation

The cytotoxicity of the nanoparticles containing the mRNA of interest was evaluated using a colorimetric assay using a cell counting kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., USA). 1 × 10 ⁴ CT26 cells were seeded in each well of a 96-well plate and cultured for 24 hours, followed by incubation with nanoparticles containing 1 to 100 μg/ml of the mRNA of interest for 24 hours, followed by 2 in PBS. washed twice. Subsequently, the cells were incubated for 30 minutes with a medium containing 10% CCK-8 solution, and absorbance was measured at 450 nm using a microplate reader (VERSAmax™; Molecular Devices Corp., USA).

8.1.3. Cell death assay using Annexin V/PI staining

2 × 10 ⁶ CT26 cells were cultured for 36 hours in a 100 mm culture dish. Then, the cells were incubated with the nanoparticles containing the mRNA of interest for 6 hours and washed twice with PBS. After trypsinizing the cells and resuspending them in 500 μl of 1% FBS containing Annexin V binding buffer, the cells were further incubated for 1 hour and then treated with Alexa Fluor™ 488 Annexin V and propidium iodide (PI) for 10 minutes. ) was stained. Subsequently, 200 μl of the cell suspension was placed in a glass bottom 35-pi cell culture dish, washed with PBS after 5 minutes, and immediately analyzed using a confocal laser scanning microscope (Leica TCS SP8; Leica Microsystems, Germany). did Each fluorophore was observed under the following conditions: Alexa Fluor™ 488-Annexin V, green, λEx = 488 nm, λEm = 500-550 nm; Propidium iodide, red, λEx = 514 nm, λEm = 590-650 nm.

8.1.4. Western blot

In a 150 mm culture dish, 4 × 10 ⁶ CT26 cells were cultured for 36 hours, and further incubated with the nanoparticles containing the mRNA of interest for 6 hours. Cells were washed twice with PBS and trypsinized. Trypsinized cells were resuspended in 500 μl of RPMI1640 medium containing 10% FBS. 200 μl of cell suspension. Cells treated with nanoparticles containing the mRNA of interest were cultured for an additional hour and then the supernatant was harvested. The harvested supernatant was concentrated twice using a desalting column (Amicon Ultra-0.5 Centrifugal Filter Unit-3K, Merck KGaA, Germany) according to the manufacturer's instructions. Proteins were separated by electrophoresis using SDS-PAGE gels (12% for GAPDH and HMGB1 in conditioned media, 16% for caspase-3, and HMGB1 in cell pellets). As primary antibodies, anti-caspase-3 antibody (1 : 1000 and 1 : 500, 9662S and 9661, Cell Signaling, USA), anti-HMGB1 antibody (1 : 660, ab79823, Abcam, UK) and anti-GAPDH antibody (1:1000, sc-32233, Santa Cruz Biotechnology, USA). As a secondary antibody, HRP-conjugated anti-rabbit IgG (1:1000, Invitrogen, USA) was used. The blotted membrane was analyzed using a real-time optical imaging system (LAS-3000; Fuji Photo Film, Japan).

On the other hand, experiments using nanoparticles containing the mRNA of interest were conducted in the same manner as above, and anti-HSP70 antibody (1:650), anti-HMGN1 antibody (1:500), and anti-IL-33 were used as primary antibodies. Antibody (1:500) and anti-GSDME (DFNA5) antibody (1:100) were treated. As a secondary antibody, HRP-conjugated anti-rat IgG (1 : 1000, Invitrogen, USA) was treated.

8.1.5. Evaluation of maturity of bone marrow-derived dendritic cells

Bone marrow was obtained from 8-week-old BALB/c mice. Bone marrow-derived dendritic cells (BMDC) were cultured in RPMI containing GM-CSF (20 ng/mL), IL-4 (20 ng/mL), 2-mercaptoethanol (50 μM) and 10% (v/v) FBS. It was prepared by culturing cells in bone marrow for one week with 1640 medium. 1 × 10 ⁷ CT26 cells were treated with 10 μM) of nanoparticles containing the mRNA of interest. After 6 hours, 3 mL of the CT26 cell culture supernatant was transferred to a 6-well plate and cultured with 4 × 10 ⁶ BMDCs per well for 24 hours. Finally, BMDCs were stained with FITC-conjugated anti-CD86 antibody for 1 hour and analyzed using a flow cytometer (Guava EasyCyte, Merck Millipore, USA).

8.1.6. In vivo biodistribution analysis

The body distribution of nanoparticles containing the mRNA of interest was evaluated using a 4-week-old BALB/c nude mouse model bearing CT26 tumors. Specifically, a CT26 cell suspension (100 μL) containing 1×10 ⁶ cells was subcutaneously injected into the flank of the mouse to inoculate the tumor, and the tumor grew to approximately 150 mm ³ for 14 days. Nanoparticles containing the mRNA of interest were injected intravenously into the tail of mice and monitored for 24 hours using a real-time optical imaging system (IVIS Lumina K; PerkinElmer Inc., USA; λEx = 660 nm, λEm = 710 nm). Then, major organs including liver, lung, kidney, spleen, heart and tumor were harvested and analyzed using a real-time optical imaging system.

8.1.7. Anti-tumor growth test

A CT26 tumor-bearing model using 4-week-old BALB/c mice was prepared by subcutaneous injection of a CT26 cell suspension (100 μL) containing 1×10 ⁶ cells into the flank of the mouse. Tumors were grown to approximately 60-70 mm ³ volume for 8 days and classified into 7 groups according to experimental purpose: i) saline, ii) [MLKL mRNA:PS]/HSA complexes, iii) [sPD -L1:PS]/HSA conjugate iv) [MLKL mRNA +sPD-L1 mRNA:PS]/HSA conjugate, v) MLKL mRNA:PS]/HA conjugate, vi) [sPD-L1:PS]/HA multiparticulates and vii) [MLKL mRNA + sPD-L1 mRNA:PS]/HA multiparticulates at a dose of 2 ug/kg on days 8, 11 and 14, respectively. Tumor volume was calculated as maximum diameter × minimum diameter ² × 0.5. Complete inhibition of tumor growth (CIG) was defined as tumor burdens smaller than the initial point.

On the other hand, in the case of experiments using nanoparticles containing the mRNA of interest, the following 7 groups were classified according to the purpose of the experiment: i) saline, ii) [MLKL mRNA:PS]/HSA complex particles, iii) [sPD- L1:PS]/HSA complex iv) [MLKL mRNA +sPD-L1 mRNA:PS]/HSA complex, v) MLKL mRNA:PS]/HA complex, vi) [sPD-L1:PS]/HA complex particles and vii) [MLKL mRNA + sPD-L1 mRNA:PS]/HA multiparticulates at a dose of 2 ug/kg were administered on days 8, 11, 14 and 17, respectively. DPP4i was administered daily at a dose of 25 mg/kg from day 8 to day 17.

8.1.8. Anti-tumor efficacy test for metastatic tumors

A CT26 tumor bearing model using 4-week-old BALB/c mice was prepared by subcutaneous injection of 1 × 10 ⁶ CT26 cells. On day 7, 4 × 10 ⁵ cells were intravenously injected to induce metastatic lung cancer. On day 8, mice were divided into 7 groups and treated as follows: i) saline, ii) [MLKL mRNA:PS]/HSA multiparticulates, iii) [sPD-L1:PS]/HSA multiparticulates iv) [ MLKL mRNA +sPD-L1 mRNA:PS]/HSA conjugate, v) MLKL mRNA:PS]/HA conjugate, vi) [sPD-L1:PS]/HA conjugate, and vii) [MLKL mRNA + sPD-L1 mRNA:PS]/HA multiparticles were intravenously administered to tumor-bearing mice on days 8, 11 and 14 at a dose of 2 ug/kg.

Tumor volume was calculated as maximum diameter × minimum diameter ² × 0.5.

On day 22, the lungs were removed from the mice, stained with 15% India ink solution, and then treated with Fekete's solution (70 mL of EtOH, 10 mL of formalin, 5 mL of acetic acid, and 15 mL of deionized water) for 48 h. Fixed.

8.1.9. ex vivo histology

On the 22nd day, the lungs, spleens and tumors of the mice were removed and fixed with 4% paraformaldehyde solution. Tissues were embedded in paraffin and cut into slices with a thickness of 4 μm on slide glass. To observe CD8+T lymphocytes, tissues were stained with anti-mouse CD8 alpha-Alexa Fluor™ 488 conjugated antibody (1:200) and mounted with DAPI Fluoromount G (Southern Biotech, USA). Slides were observed using a confocal laser scanning microscope (Leica TCS SP8). In addition, tissue slides stained with H&E (hematoxylin and eosin) were observed using a microscope slide scanner (Aperio CS2; Leica Biosystems, Germany). In the case of experiments using nanoparticles containing the mRNA of interest, to observe CD80 + dendritic cells and CD49b + NK cells (natural killer cells), tissues were conjugated with FITC anti-mouse CD80 antibody (1: 100) and FITC anti-mouse It was stained with CD49b conjugated antibody (1:100).

8.2. Evaluation of cytotoxicity and apoptosis inducing ability of nanoparticles containing each MLKL mRNA

The cytotoxicity of nanoparticles containing MLKL mRNA was evaluated in colon cancer cell line CT26 (colon carcinoma). First, seeding was performed in a 96-well plate at a density of 1×10 ⁴ cells/well, followed by culturing for 36 hours. Thereafter, the prepared nanoparticles containing MLKL mRNA were treated at various concentrations (1-100 μg/ml), and 24 hours later, cell viability was measured using the CCK-8 assay (see Experimental Materials and Methods above for specific methods). ).

As a result, as shown in FIG. 3d, next, in order to evaluate the nanoparticle-sensitized apoptosis inducing ability containing MLKL mRNA, 2×10 ⁶ CT26 cells were cultured in a 100-pi dish for 36 hours, and a certain concentration Nanoparticles containing MLKL mRNA of were treated for 6 hours. The cells were redispersed in a 48-well plate, further cultured for 24 hours, and cell viability was measured using the CCK-8 assay.

As a result, as shown in FIGS. 3e and 3f, it was found that the cytotoxicity was increased by the nanoparticles containing MLKL mRNA (FIG. 3f).

8.3. Confirmation of necroptosis-like apoptosis inducing ability of nanoparticles containing each MLKL mRNA

8.3.1. Ability of nanoparticles containing MLKL mRNA to induce necroptosis-like apoptosis

In order to verify the apoptosis mechanism of nanoparticles containing MLKL mRNA, cell morphology was observed through confocal microscopy after Annexin V/PI staining. Specifically, 2×10 ⁶ RIPK-3 deficient CT26 colorectal cancer cell line was cultured in a 100-pi dish for 36 hours, and then treated with nanoparticles containing MLKL mRNA for 6 hours. Thereafter, the mixture was redispersed in 500 μl of Annexin V binding buffer and incubated for an additional 1 hour. Cells were stained with Alexa FluorTM 488 Annexin V and PI (propidium iodide) and observed under a confocal microscope.

As a result, as shown in FIG. 4a , damage to cell morphology due to cell membrane rupture was observed in cells treated with nanoparticles containing MLKL mRNA. That is, when nanoparticles containing MLKL mRNA were treated, leakage of chromatin, cytoplasmic components, and membrane fragments due to cell membrane collapse was observed, which is a behavior very similar to apoptosis by necroptosis. Interestingly, regardless of RIPK3 and MLKL expression, cells exhibited morphological characteristics of necroptosis when treated with nanoparticles containing MLKL mRNA (Fig. This suggests that it occurred in a /MLKL-independent manner.

8.3.2. Ability of nanoparticles containing MLKL mRNA to induce necroptosis-like apoptosis

Next, the apoptosis inducing ability of nanoparticles containing MLKL mRNA was investigated.

First, as shown in FIG. 4f , characteristics of apoptosis (condensation of chromatin and externalization of phosphatidylserine) were observed in cells treated with doxorubicin (DOX). In comparison, in cells treated with nanoparticles containing MLKL mRNA, damage to cell morphology due to cell membrane rupture was observed. That is, it supports the occurrence of cell death by necroptosis that appears in nanoparticles containing MLKL mRNA.

In addition, as shown in FIG. 4B , it was confirmed that cell necroptosis could occur in a RIPK3/MLKL-independent manner regardless of RIPK3 and MLKL expression in the nanoparticles containing MLKL mRNA of Example 4.1.

8.4. Evaluation of DAMP efflux behavior and evaluation of dendritic cell maturation

8.4.1. Assessment of damage-associated molecular pattern (DAMP) efflux behavior

Unlike apoptosis, necroptosis causes the release of intact DAMPs from cells, thus effectively mediating dendritic cell maturation and activation. Accordingly, in order to verify the efflux of immunogenic DAMP through nanoparticle-mediated necroptosis-like cell death including each MLKL mRNA, the present inventors confirmed the efflux of DAMP protein using Western blot. .

Specifically, CT26 cells were cultured for 36 hours in a 150-pi dish at a density of 4×10 ⁶ cells/dish, and then replaced with a culture medium containing nanoparticles containing MLKL mRNA and further cultured for 6 hours. Afterwards, the cells and the culture medium were extracted separately and SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) was performed to separate proteins by size, and in the case of nanoparticles containing MLKL mRNA, anti-GAPDH, anti-HMGB1, anti- Chemiluminescence imaging (LAS-3000; Fuji Photo Film) was performed by staining each protein with caspase-3 antibody and, in the case of cNB, with anti-HSP70, anti-HMGN1, anti-IL-33, and anti-GSDME (DFNA5) antibodies. , Japan). Among the released DAMPs, HMGB1 (High mobility group box 1) is known to have different immunological functions depending on the oxidation-reduction state, and therefore HMGB1 is considered a representative marker for necroptosis.

First, as shown in FIGS. 4c and 4d, the nanoparticles containing MLKL mRNA have an increased expression of cleaved caspase-3, a characteristic of apoptosis, and an oxidized form of HMGB1 protein. (oxHMGB1) was confirmed to exist. The above results support the ability to induce necroptosis-like apoptosis in response to nanoparticles containing MLKL mRNA.

Additionally, the expression type and amount of DAMP protein expressed in existing apoptosis and necroptosis caused by nanoparticles containing MLKL mRNA were confirmed. As a result, as shown in FIG. 4h, in the case of CT26 cells inducing apoptosis through doxorubicin, the release of HMGB1 and HSP70, which induce maturation of dendritic cells, was observed. On the other hand, in the case of HMGN1, a DAMP that induces chemotaxis of leukocytes, GSDME, a DAMP that induces the activation of natural killer cells, and IL-33, a DAMP that induces the activation of eosinophils, in CT26 cells that induce apoptosis through doxorubicin, there is no efflux. However, it was confirmed that the nanoparticles containing MLKL mRNA were specifically effluxed only in CT26 cells. These results show that in the case of necroptosis induced through nanoparticles containing MLKL mRNA, unlike apoptosis in which only activation of dendritic cells can be involved, activation of NK cells and eosinophils is induced, resulting in a more effective anti-cancer immune response. This means that it can be induced.

8.4.2. Evaluation of maturity of dendritic cells

The CT26 culture solution after ultrasonic irradiation extracted from the experiment of the above example was treated with bone marrow-derived dendritic cells for 24 hours, and then FITC-anti-CD86 was labeled and flow cytometry was performed. CD86 is a representative costimulatory molecule expressed in dendritic cells, and the degree of maturation of dendritic cells can be determined through its expression level.

As a result, as shown in FIG. 4e, the cell culture solution extracted from the group treated with nanoparticles containing MLKL mRNA showed 1.52-fold and 1.16-fold higher amounts of CD86 compared to the control group or the DOX-treated group, respectively. It was confirmed that expression was induced. This is thought to be due to the high efficiency induction of dendritic cell maturation without oxidation of DAMPs and tumor-related antigens by necroptosis-like cell death following cell membrane rupture.

Taken together, the above results suggest that RIPK3/MLKL-independent necroptosis-like cell death can be induced using nanoparticles containing MLKL mRNA. It has been proven that it effectively induces maturation. Therefore, necroptosis-like cell death as described above has high immunogenicity, suggesting the possibility of combination therapy with anti-cancer immunotherapy.

8.5. Confirmation of tumor accumulation behavior of nanoparticles containing MLKL mRNA

After demonstrating the ability of nanoparticles containing MLKL mRNA to induce RIPK3-independent necroptosis and maturation of dendritic cells through Example 5, the present inventors injected nanoparticles containing MLKL mRNA into tumor-bearing mice. After systemic administration (systemic administration), the tumor accumulation behavior of nanoparticles containing MLKL mRNA was evaluated using an optical imaging technique.

As a result, as shown in FIG. 5a , strong fluorescence signals were observed in the tumors of live mice during the entire test period. In addition, as shown in FIG. 5b, the ex vivo image of the tissue excised 24 hours after administration showed the strongest fluorescence signal in the tumor tissue, indicating the high tumor targeting efficiency of the nanoparticles containing MLKL mRNA (FIG. 5b). Top). Quantitatively, fluorescence intensities in tumor tissue were 2.74-fold and 5.66-fold higher than those in liver and lung, respectively (Fig. 5b bottom). Taken together, these results reveal that nanoparticles containing MLKL mRNA can effectively reach tumors by a passive targeting mechanism.

8.6. Evaluation of cancer treatment efficacy and metastasis inhibitory ability of nanoparticles containing each mRNA of interest using animal models

8.6.1. Confirmation of anticancer effect and combination treatment effect of nanoparticles containing the mRNA of interest in cancer animal models

In order to evaluate the therapeutic efficacy of the nanoparticles containing the mRNA of interest in a cancer animal model, a cancer animal model was prepared by subcutaneously transplanting the CT26 cell line (1 × 10 ⁶ ) used in the in vitro validation experiment to a mouse. . Nanoparticles containing the mRNA of interest according to the present invention were intravenously injected into the animal model at a dose of 2 μg/kg, and physiological saline and DOX) were administered as a control group. Then, treatment efficacy was evaluated according to the presence or absence of administration of nanoparticles containing the mRNA of interest. The dosing schedule is shown in Figure 6a.

As a result, as shown in FIGS. 6B to 6D , in the case of the nanoparticles containing the mRNA of interest, the volume of the tumor was reduced by 88.3% compared to the physiological saline treatment group.

In addition, in order to confirm the possibility of combination treatment, nanoparticles containing sPD-L1 mRNA, a representative immune checkpoint inhibitor, were administered together to compare treatment efficacy. As shown in FIGS. 6B to 6D, nanoparticles containing MLKL mRNA When the particles and nanoparticles containing sPD-L1 mRNA were co-administered and ultrasound was irradiated, 40% complete remission was observed, and the weight of the excised cancer tissue was compared to the nanoparticles containing sPD-L1 mRNA alone administration group. It was found to be reduced by 97.03% compared to , and it was confirmed that the anticancer effect was further maximized when administered in combination.

8.6.2. Confirmation of cancer metastasis inhibitory effect of nanoparticles containing the mRNA of interest in metastatic cancer animal models

In order to confirm the cancer metastasis inhibitory effect of the nanoparticles containing MLKL mRNA according to the present invention, the CT26 cell line was subcutaneously transplanted into mice, and then the CT26 cell line was additionally injected intravenously to prepare an animal model of lung metastasis cancer, and India Ink Cancer metastasis inhibitory ability was evaluated through india ink staining. The administration schedule and the time of ultrasound irradiation are shown in FIG. 7a.

As a result, it was confirmed that cancer growth was significantly inhibited in the group administered with the nanoparticles containing MLKL mRNA and the nanoparticles containing sPD-L1 mRNA (see FIGS. 7B to 7D ). When CD8+ cells in the cancer tissue were confirmed by immunofluorescence staining, it was confirmed that a significantly larger number of CD8+ T cells were distributed in the NB+US+aPDL1 group (see FIG. 7e).

In addition, as shown in FIGS. 7f and 7g , as a result of H&E staining and India ink staining, it was confirmed that cancer metastasis to the lung was significantly suppressed in the NB+US+aPDL1 group compared to the control group.

In addition, as a result of examining whether side effects such as lymphoid hyperplasia appear through H&E staining by removing the spleen, as shown in FIG. 7h, the NB + US + aPDL1 group has no side effects compared to the control group was able to confirm

Through the above results, necroptosis-like cell death was selectively induced in the group administered with nanoparticles containing MLKL mRNA and nanoparticles containing sPD-L1 mRNA, thereby efficiently suppressing tumors and inducing an anti-cancer immune response. It has been proven that it can be done, and it is expected that various combination treatment technologies will be developed accordingly.

8.6.3. Confirmation of anticancer effect and combination treatment effect of nanoparticles containing MLKL mRNA in cancer animal models

In order to evaluate the therapeutic efficacy of the nanoparticles containing MLKL mRNA in cancer animal models, the nanoparticles containing MLKL mRNA were administered and then the tumor growth inhibitory effect was evaluated. A cancer animal model was prepared by subcutaneously transplanting the CT26 cell line (1 × 10 ⁶ cells) used in the in vitro validation experiment to mice. Nanoparticles containing MLKL mRNA were intravenously injected into the animal model at a dose of 2ug/kg. After that, the treatment efficacy was evaluated. In order to evaluate the efficacy of anti-cancer immunotherapy by eosinophils and NK cells, sitagliptin, one of DPP4 (Dipeptidyl peptidase 4) inhibitors (DPP4i) that inhibits eosinophil activity, was added to cNB. was administered concomitantly with The dosing schedule is shown in FIG. 8A.

As a result, as shown in FIGS. 8b and 8c, in the case of the group administered with nanoparticles containing MLKL mRNA, the tumor volume was reduced by 24.3 ± 11.1% compared to the control group. In addition, in order to confirm the anticancer immune effect through eosinophils and NK cells, the treatment efficacy was compared by administering sitagliptin, DPP4i, within the tumor. As a result, the tumor volume was reduced by 70.4 ± 26.9%, and the extracted cancer tissue It was found that the weight of was reduced by 76.3 ± 25.7%, confirming that the anticancer effect was further maximized during combined treatment (left graphs in FIGS. 8B and 8C). On the other hand, when treated with an antibody that inhibits eosinophils (aCD170), it was confirmed that the anticancer effect that appeared when combined with a DPP4 inhibitor disappeared (left graph of FIG. 8c). Therefore, in the case of necroptosis-like apoptosis induced by nanoparticles containing MLKL mRNA, it is thought that the activation of eosinophils in tumors is induced and the resulting anti-cancer immune response is accompanied.

In addition, for the observation of splenomegaly caused by tumor-derived cytokines and spilled drifting cancer cells, the spleen was removed and weighed. As a result, compared to the control group, nanoparticles containing MLKL mRNA were investigated and DPP4 inhibitors were used in combination. When administered, it was confirmed that the decrease was about 33.8 ± 21.7% (right graph of FIG. 8c). In comparison, there was no significant difference in spleen weight in the other groups. The above result is thought to be due to the suppression of cancer cells and tumor-derived cytokines leaked into the bloodstream by the anti-cancer immune response of eosinophils when nanoparticle irradiation containing MLKL mRNA and DPP4 inhibitor are administered in combination.

Additionally, when CD80+ dendritic cells in cancer tissues were identified by immunofluorescence staining, significantly high fluorescence of CD80+ dendritic cells was observed in all groups in which nanoparticles containing MLKL mRNA capable of effectively releasing HMGB1 were examined ( Fig. 8d). On the other hand, in the case of CD49b+NK cells induced by eosinophils, the group irradiated with nanoparticles containing MLKL mRNA (cNB+US) and the group irradiated with nanoparticles containing MLKL mRNA and administered with a DPP4 inhibitor in combination (cNB+US+ DPP4i) showed high fluorescence, but when treated with an antibody that inhibits eosinophils (cNB+US+DPP4i+aCD170), it was confirmed that the intensity of fluorescence significantly decreased (FIG. 8d). This supports that necroptosis-like cell death through nanoparticles containing MLKL mRNA can activate NK cells through eosinophil activation as well as effective dendritic cell activation.

Through the above results, it was demonstrated that necroptosis-like cell death can be selectively induced under the nanoparticles containing the mRNA of interest, and thereby efficient tumor suppression and anticancer immune response can be induced. This is thought to be due to the induction of a more effective anti-cancer immune response through the activation of eosinophils and NK cells along with the activation of dendritic cells shown in the existing apoptosis. Accordingly, it is expected that the development of various combination treatment technologies will be possible.

The above description of the present invention is for illustrative purposes, and those skilled in the art 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, the embodiments described above should be understood as illustrative in all respects and not limiting.

Claims (16)

(a) MLKL (mixed lineage kinase domain like protein) mRNA as an active ingredient;
(b) cationic compounds; and
(c) an anionic compound;
(d) formed by electrostatic interactions between them;
(e) targeted delivery to the tumor site; a pharmaceutical composition characterized by the complex The method of claim 1, wherein the composition is a cytokine, chemokine, suicide gene product, immunogenic protein or peptide, apoptosis inducer, angiogenesis inhibitor, heat shock protein, tumor antigen, β-catenin inhibitor, activator of the STING pathway , Immune-checkpoint blockers (ICB), innate immune activators, antibodies, dominant negative receptors and decoy receptors, inhibitors of myeloid derived suppressor cells (MDSC), inhibitors of the IDO pathway, and inhibitors of apoptosis. A pharmaceutical composition characterized by further adding any one selected from the group consisting of binding proteins or peptides, antibacterial agents, antiviral agents, drugs, adjuvants, chemotherapeutic agents and kinase inhibitors as an active ingredient The method of claim 2, wherein the immune-checkpoint blockers (ICB) are PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, LAG3 inhibitors, TIM3 inhibitors, OX-40 stimulators , 4-1BB stimulator, CD40L stimulator, CD28 stimulator, GITR stimulator characterized in that at least one selected from the group consisting of a pharmaceutical composition characterized by The pharmaceutical composition of claim 3, wherein the PD-1 inhibitor is an antagonistic antibody to PD-1, and the PD-L1 inhibitor is an antagonistic antibody to PD-L1. The pharmaceutical composition according to claims 2 to 4, wherein the selected one is selected from the group consisting of protein and mRNA. The pharmaceutical composition according to claim 2, wherein the immune checkpoint blockers (ICB) is any one selected from the group consisting of antibodies, proteins and mRNAs. The method of claim 6, wherein the ICB mRNA
(a) a nucleotide sequence encoding a secretion signal peptide (Sec); and
(b) a nucleotide sequence encoding a peptide domain for extending the life-span of the mRNA in blood; a pharmaceutical composition comprising The method of claim 1 to 7, wherein the composition
A pharmaceutical composition further comprising receptor-interacting protein kinase 3 (RIPK3) mRNA as an active ingredient According to claims 1 to 8,
The composition further comprises a DPP4 inhibitor (dipeptidyl peptidase 4 inhibitor, DPP4i) as an active ingredient, a pharmaceutical composition. According to claim 9,
The DPP4 inhibitor is a pharmaceutical composition characterized by an activity inhibitor of DPP4 protein According to claim 10,
The activity inhibitor of the DPP4 protein is an antibody against DPP-4, sitagliptin, vildagliptin, saxagliptin, linagliptin, dutogliptin, zemi At least one selected from the group consisting of gemigliptin, alogliptin, anagliptin, evogliptin, berberine, diprotin and lupeol A pharmaceutical composition characterized by The pharmaceutical composition according to claim 1, wherein the cationic compound is a cationic polyelectrolyte. The pharmaceutical composition according to claim 12, wherein the cationic polyelectrolyte is any one selected from the group consisting of protamine and chitosan. According to claim 1,
The anionic compound is a pharmaceutical composition characterized by an anionic polyelectrolyte 15. The pharmaceutical composition according to claim 14, wherein the anionic polyelectrolyte is selected from the group consisting of hyaluronic acid and human serum albumin. The method of claim 1, wherein the tumor site targeted delivery
(a) EPR (Enhanced Permeability and Retention) effect by the size of the complex: and
(b) a specific binding ability to a specific molecule of an anionic polyelectrolyte; a pharmaceutical composition characterized by at least one selected from the group comprising.

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