KR101484441B1 - Glycol Chitosan-Methyl Acrylate-Polyethyleneimine nanoparticle for Gene Delivery - Google Patents

Abstract

The present invention relates to a non-viral gene delivery system and uses thereof and, more specifically, to a non-viral gene delivery system using a cationic polymer glycol chitosan-methyl acrylate-polyethyleneimine (GMP) based on polyethylenimine (PEI) which has high transfection efficiency and low cytotoxicity. According to the present invention, a polymer for transporting non-viral hexane and a complex based on the same including nanoparticles having a GMP structure, can provide significantly low cytotoxicity and high transfection efficiency and is effective in gene delivery in cells as used as an effective hexane delivery system, thereby being very useful for medical industries.

Description

Glycoprotein nanoparticles for gene transfer {Glycol Chitosan-Methyl Acrylate-Polyethyleneimine nanoparticle for Gene Delivery}

The present invention relates to a non-viral gene carrier and a use thereof, and more particularly, the present invention relates to a non-viral gene carrier and a method for producing the same. The present invention relates to a polyethylenimine (PEI) -based cationic polymer GMP (Glycol Chitosan-Methyl Acrylate -Polyethyleneimine. ≪ / RTI >

Gene therapy refers to the treatment of inherited or acquired gene abnormalities that are difficult to treat by conventional methods by genetic engineering methods. More specifically, the gene therapy includes the steps of expressing a therapeutic protein by administering a genetic material such as DNA and RNA into the body for treatment and prevention of chronic diseases such as inherited or acquired genetic defects, viral diseases, cancer or cardiovascular diseases Or inhibiting the expression of a specific protein. It is a method expected as an alternative means of existing medical treatment as well as overcoming incurable disease, because it can fundamentally treat the cause of disease by analyzing at the gene level.

This gene therapy differs from conventional drug therapy in that a gene carrier, which is a means of delivering genes, is essential. DNA or RNA is a molecule with a large molecular weight which is negatively charged and easily hydrolyzed in vivo and is not easy to be injected into cells or tissues alone. Therefore, it is preferable that DNA or RNA, which can smoothly inject a genetic material into cells or tissues This is because a substance is necessary. Therefore, the most important substance in gene therapy is a gene transporter that transmits a therapeutic gene in vivo.

Such gene delivery systems can be broadly classified into viral vector-mediated systems and non-viral vector-mediated systems.

A viral vector made using retrovirus or adenovirus has an advantage of high transfection efficiency into a cell, but in vivo immunogenicity problems and inherent problems such as genetic recombination. In order to overcome the problem of stability of these viral vectors, various polymeric gene delivery systems have been developed as an alternative to traditional viral vector-based gene delivery methods. However, polymeric vectors have a problem of having intracellular trafficking barriers such as endosomal escape and nuclear localization.

The cationic polymer forms a polymer-gene-gene complex with the gene to induce transfection of eukaryotic cells. The specific mechanism is considered to be the release of the gene from the endosome into the cytoplasm after the complex binds to the cell surface due to the electrical interaction between the positive charge on the polymer-gene complex surface and the negative charge on the cell surface. Specific examples of such cationic polymers include cationic homopolymers such as polyethyleneimine (PEI), poly-L-lysine, polyamino ester, polyamidoamine, polypropyleneimine and polyethyleneglycol (PEG) -block-poly - < / RTI > cationic block copolymers or graft copolymers based on homopolymers such as lysine and PEG-PEI.

The polymers are useful for condensing plasmid DNA (pDNA) into nanoscale particles, protecting pDNA from degradation, protecting pDNA particles from undesirable interactions, and enhancing intracellular delivery to the cytoplasm and nucleus There is an advantage that it can be. Among them, polyethyleneimine (PEI) is the most widely used non-viral vector in recent gene therapy research.

 However, polymeric vectors have intracellular trafficking barriers such as endosomal escape and nuclear localization, and are problematic in that they are highly cytotoxic and bio-toxic and have low nucleic acid delivery efficiency .

Thus, the present inventors have found that a novel non-viral gene carrier, which is based on polyethyleneimine (PEI), has the advantage of efficiently compressing plasmid DNA (pDNA) and rapidly releasing a PEI / DNA complex in endosomes, The present inventors completed the present invention by confirming that a new structural complex of MA-PEI (Glycol Chitosan-Methyl Acrylate-Polyethylenimine) was first identified and that this novel complex can increase gene transfer efficiency without cytotoxicity.

Korean Patent No. 10-1035364

The main object of the present invention is to provide a polymer for excellent nonviral nucleic acid delivery, which has high gene transfer efficiency and low cytotoxicity, and its use.

Another object of the present invention is to provide a method for producing the nonviral nucleic acid delivery polymer.

It is still another object of the present invention to provide a nonviral nucleic acid delivery complex wherein the nonviral nucleic acid delivery polymer is bound to a nucleic acid.

In order to accomplish the above object, the present invention provides a non-viral nucleic acid-transferring polymer comprising GMP having a GC-MA-PEI (Glycol Chitosan-Methyl Acrylate-Polyethylenimine) structure.

In one embodiment of the present invention, the PEI may be PEI 800D.

In order to achieve the above object, the present invention also provides a process for preparing a glycol chitosan-methyl acrylate (GC-MA), which comprises the steps of: (a) reacting methyl acrylate with glycol chitosan; And (b) a step of synthesizing glycol chitosan-methyl acrylate-polyethyleneimine (GC-MA-PEI) by reacting PEI (polyethyleneimine) with glycol chitosan-methyl acrylate synthesized in step (a) Lt; / RTI >

In one embodiment of the present invention, the PEI may be PEI 800D.

In one embodiment of the present invention, the methyl acrylate of step (a) may be reacted with methanol at room temperature for 10 minutes, and then the glycol chitosan dissolved in distilled water may be added.

In step (a), the glycol chitosan is added to the reaction vessel, the gas inside the reaction vessel is replaced with nitrogen gas, and the reaction is carried out at 35 to 39 ° C and 150 to 200 rpm It may be a reaction for 3 to 5 days.

In one embodiment of the present invention, the step (a) may be a step of removing methanol with an evaporator after the reaction in an agitating incubator, dialyzing, and then lyophilization.

In one embodiment of the present invention, the step (b) comprises dissolving the glycol chitosan-methyl acrylate (GC-MA) synthesized in the step (a) in a 9: 1 (v: v) After that, 30 to 70 ml of methanol may be further added and the reaction may be carried out for 5 to 20 minutes.

In one embodiment of the present invention, the step (b) is performed by adding PEIpolyethylenimine to GC-MAglycol chitosan-methyl acrylate, which is further reacted with the methanol, and the gas inside the reaction vessel is replaced with nitrogen gas. At 35 to 39 DEG C and 150 to 200 rpm for 4 to 6 days.

In one embodiment of the present invention, the step (b) may be performed by removing methanol with an evaporator after the reaction in an agitating incubator, dialyzing, and then lyophilization.

In order to achieve the above object, the present invention also provides a non-viral nucleic acid delivery complex wherein a target gene is bound to a GMP constituting a GC-MA-PEI (Glycol Chitosan-Methyl Acrylate-Polyethylenimine) structure.

In one embodiment of the present invention, the target gene may be composed of DNA.

In one embodiment of the present invention, the weight ratio of GMP: DNA may be 0.5 to 20: 1.

In one embodiment of the present invention, the complex can be compressed into nanoparticles of 100 to 800 nm.

In one embodiment of the present invention, the PEI may be PEI 800D.

Non-viral nucleic acid delivery polymers and complexes based thereon comprising nanoparticles of the Glycyl Chitosan-Methyl Acrylate-Polyethylenimine (GMP) structure according to the present invention provide significantly lower cytotoxicity and higher transfection efficiency Since it can be used as an effective nucleic acid transporter, it is efficient for intracellular gene transfer and is very useful in the medical industry.

1 is a schematic diagram showing a synthesis reaction of GMP nanoparticles.
Figure 2 shows ¹ H NMR spectra of GC, GC-MA and GMP nanoparticles.
Figure 3 shows the size of the complex according to various weight ratios of GMP nanoparticles and plasmid DNA.
Figure 4 shows the zeta potential of the complex according to various weight ratios of GMP nanoparticles and plasmid DNA.
5 shows agarose gel electrophoresis results according to various weight ratios of GMP nanoparticles and plasmid DNA.
FIG. 6 shows the results of the degree of fluorescence of picogreen according to various weight ratios of GMP nanoparticles and plasmid DNA.
Figure 7 shows the cytotoxicity of GMP nanoparticles in HeLa cell line (a), HEK293 cell line (b), and HepG2 cell line (c).
Fig. 8 shows the transfection efficiency of GMP nanoparticles in HeLa cell line (a), HEK293 cell line (b), and HepG2 cell line (c).
FIG. 9 shows the intracellular locations of glycol chitosan (control 1), PEI 25 kD (control 2) and GMP nanoparticles in a HeLa cell line using a confocal microscope.

The terms used in the present invention are defined as follows.

"Gene" means any nucleic acid sequence or portion thereof that has a functional role at the time of protein coding or transcription, or in the control of other gene expression. The gene may consist of only a portion of the nucleic acid encoding or expressing any nucleic acid or protein that encodes the functional protein. The nucleic acid sequence may comprise an exon, an intron, an initiation or termination region, a promoter sequence, another regulatory sequence, or a gene abnormality within a particular sequence adjacent to the gene.

The term "polynucleotide" or "nucleic acid" refers to nucleotide polymers of any length, including ribonucleotides as well as deoxyribonucleotides. This term refers only to the primary structure of the molecule, and thus to DNA or RNA of a double or single chain. This also applies to known types of modifications, for example, labeling, methylation, "caps", nucleotide substitutions of one or more naturally occurring nucleotides, debonding (eg, methyl phosphonate, phosphotriester, phosphoamidate, carbarnate (Eg, phosphorothioate, phosphorodithioate, etc.), proteins (eg, nucleases, toxins, antibodies, signal peptides, poly-Llysine, etc.) Having an alkyl compound, a modified bond (e.g., alpha (alpha), beta (alpha) anomeric nucleic acid, etc.), as well as inter-nucleotide modification including non-modification of the polynucleotide. Generally, the nucleic acid moieties provided by the present invention may comprise fragments of a genome and a short oligonucleotide linker or series of oligonucleotides, microorganisms or viral operons, or regulatory elements derived from eukaryotic genes, Lt; RTI ID = 0.0 > nucleotides < / RTI > that can be expressed as recombinant transcription units

The term "vector" means a nucleic acid molecule capable of transporting another nucleic acid to where it is associated. The term " expression vector "includes a plasmid, a cosmid, or a phage capable of synthesizing a protein encoded by each recombinant gene carried by the vector. Preferred vectors are those capable of self-replication and expression of the associated nucleic acid.

"Transfection" refers to a method of directly expressing a genetic trait in a cell by directly introducing nucleic acid (DNA, PNA, etc.) into a cultured animal cell. In the present invention, the term "transfection" is used in combination with terms such as "transfection" and "intracellular delivery". The introduced nucleic acid is generally introduced by introducing the desired gene into a medium such as a plasmid. When the introduced gene was stabilized in the cells, it was often interrupted by the chromosome. Cells into which the nucleic acid has been introduced are called transgenic plants.

"Zeta potential" means an electrodynamic potential difference resulting from the difference in the positive charge density in the diffusion double layer of mobile water that can easily be separated from the particles and grains attached to the charged particle surface. It may also be expressed as an electric potential difference or zeta potential between the cell surface and the surrounding culture liquid.

"Luciferase" (luciferase) is an enzyme that accelerates the oxidation of luciferin to convert chemical energy into light energy and emits light. It measures the expression continuously and in real time in vivo, It functions as a reporter gene that allows the effect to be verified. Can be obtained directly from an insect such as a firefly or a glow-worm or by expression from a microorganism comprising a recombinant DNA fragment encoding this enzyme.

"Carrier" is a generic term for a polymeric material that is responsible for the transport of a carrier, in which an active material in an organism is associated with another material or is a transport of a material through the cell membrane. Examples of carriers include, but are not limited to, buffers such as phosphate, citrate and other organic acids, antioxidants such as ascorbic acid, proteins such as low molecular weight polypeptides (less than about 10 residues), serum albumin, gelatin or immunoglobulins, polyvinylpyrrolidone And other carbohydrates including glucose, mannose, or dextrin, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, and hydrophilic polymers such as sodium, Salt-forming counterion, and / or a non-ionic surfactant such as TWEEN, polyethylene glycol (PEG) and PLURONICS.

"Treatment" is an approach to obtaining beneficial or desired results, including clinical results. "Treatment" or "alleviation" of a disease, disorder or condition refers to a decrease or decrease in the severity of a condition, disorder, or disease state and / or undesirable clinical symptoms, Is slow or long. For example, in the treatment of obesity, weight loss, for example, weight loss of at least 5% is an example of desirable treatment outcome. For the purposes of the present invention, a beneficial or desired clinical result may include alleviation or amelioration of one or more symptoms, reduction of disease severity, stabilized (i.e., not worsening) condition of the disease, , Delay or slowing of disease progression, amelioration or palliation of the disease state, and sedation (partial or total). "Therapy" can also mean prolonging survival compared to expected survival in the absence of treatment. In addition, treatment does not need to occur by the administration of a single dose, but occurs occasionally when administering a series of doses. Thus, a therapeutically effective amount, an amount sufficient to alleviate, or an amount sufficient to treat a disease, disorder or condition, may be administered by one or more administrations.

The term " disorder "is any condition that can benefit from treatment with a molecule identified using the transgenic animal model of the invention. This includes chronic and acute diseases or diseases, including pathological conditions that make mammals susceptible to suspicious diseases.

A "therapeutically effective amount" refers to the amount of active compound in a composition, system, subject, or composition that will elicit a biological or medical response in a human, system, subject, or human being sought by a researcher, veterinarian, .

A "prophylactically effective amount" refers to an amount of tissue or tissue that is sought by a researcher, veterinarian, physician, or other clinician to prevent obesity or an obesity-related disorder, condition or disease in a subject at risk for obesity or obesity- , The amount of active compound in the composition, system, subject, or composition that will elicit the biological or medical response in humans.

"Gene therapy" refers to treating a disease by correcting a mutated gene, treating a genetic disease, or regulating protein expression using a gene or RNAi. In other words, it is a method of treating a disease by transplanting a normal gene from the outside into a patient's cell and changing the phenotype of the cell.

"About" is used herein to refer to a reference quantity, a level, a value, a number, a frequency, a percentage, a dimension, a quantity, Level, value, number, frequency, percentage, dimension, size, quantity, weight or length of a variable, such as 5, 4, 3, 2 or 1%.

Throughout this specification, the words " comprising "and" comprising ", unless the context requires otherwise, include the stated step or element, or group of steps or elements, but not to any other step or element, And that they are not excluded.

Hereinafter, the present invention will be described in detail.

The present invention relates to the transfection of nucleic acids. In particular, as a novel non-viral gene carrier, the use of GMP as a gene delivery carrier, which has a high transfection efficiency and low cytotoxicity, constitutes a GC-MA-PEI (Glycol Chitosan-Methyl Acrylate-Polyethyleneimine) .

GMP  polymer

In one aspect, the present invention relates to GMP (Glycol Chitosan-Methyl Acrylate-Polyethyleneimine) -based gene transporter or gene transfer vector.

A non-viral gene transfer vector is a generic term for a carrier that carries a gene into a cell without using a virus. The nucleic acid constituting the gene has a negative charge, so that a cation site on the cation phase and an anion site on the nucleic acid side are electrically A typical example is a vector in which a nucleic acid is coated using an interaction.

The cationic polymer of the present invention has the advantage that GMP (Glycol Chitosan-Methyl Acrylate-Polyethyleneimine) efficiently compresses plasmid DNA (pDNA) based on polyethyleneimine (PEI) and rapidly emits a PEI / DNA complex in endosomes But it also has the characteristics of significantly increasing transfection efficiency and cytotoxicity by forming a polymer of a novel structure of GC-MA-PEI.

GMP (Glycol Chitosan-Methyl Acrylate-Polyethyleneimine) of the present invention can use MA to connect GC and PEI. A more specific GMP synthesis process is shown in Fig.

At this time, PEI of various molecular weights and forms can be used.

When branched, the molecular weight of the backbone PEI repeat units is preferably in the range of about 400 to 50,000 daltons, more particularly preferably in the range of about 600 to 2000 daltons. When linear, the molecular weight of the backbone PEI repeat units is preferably in the range of about 200 to 50,000 Daltons.

In addition, the weight average molecular weight of the GMP of the present invention may vary depending on the PEI used. In certain embodiments, the weight average molecular weight of the GMP may range from about 500 Daltons to about 100,000 Daltons. In one embodiment, the weight average molecular weight can range from about 800 daltons to about 20,000 daltons. The molecular weight can be measured by methods known to those skilled in the art, for example, by size exclusion chromatography or agarose gel electrophoresis. Preferably, a PMP having a weight average molecular weight of about 800 daltons is used.

The GMP of the present invention effectively promotes the condensation of the nucleic acid to be delivered, in particular DNA, to form a stable complex and the internalization of the DNA into the cell, and after internalization, the complex helps to escape from the endosome to the cytoplasmic space by reduction of the disulfide bond .

GMP - nucleic acid complex

In another aspect, the present invention relates to a cationic polymer GMP of the present invention and a gene to be transferred, that is, a nucleic acid-bound complex and its use.

There is no limitation on the type of the nucleic acid, which is a dielectric substance to be delivered, but preferably, the nucleic acid is DNA and most preferably plasmid DNA (pDNA). The DNA may also comprise mixed RNA / DNA molecules or mixed protein / DNA molecules.

At this time, the weight ratio of GMP: DNA was in the range of 0.1 to 10: 1, and in one embodiment of the present invention, the zeta potential value of the stabilizer was about 20 to 30 mV in weight ratio.

In particular, the gene carriers and complexes of the present invention for delivering intracellular nucleic acids have the following advantages.

(1) "GMP" of the present invention efficiently condenses DNA.

The particle size of the GMP / nucleic acid (e. G., DNA) complex of the present invention affects the internalization of the delivery complex by the cell, thus affecting the transfection efficiency.

The PMP of the present invention allows DNA to be condensed into nanoparticles of up to 500 nm in diameter to have a nano-size suitable for gene delivery.

That is, when bound to DNA, it has the advantage of more efficiently enriching DNA by pre-periodic interactions at a relatively low N / P ratio (polymer nitrogen (+) / pDNA phosphate (-)). In other words, it exhibits a positive charge under neutral conditions, effectively concentrating the DNA to nanoscale size (about 500 nm or less), and can have a positive zeta potential with a charge ratio of 1 or more.

(2) The GMP of the present invention contributes to the transfection efficiency with the excellent proton-buffering capacity of the cationic gene carrier. As the core promotes the buffering ability of the PMP as the secondary and tertiary amines increase due to bonding with the PEI graft as the outer layer to PEI, it can have excellent transfection efficiency.

(3) The GMP / DNA complex of the present invention has low cytotoxicity.

Compared with conventional polymeric cationic carriers PEI and the like, the PMP / DNA complexes of the present invention exhibit remarkably low toxicity.

As described above, the GMP / DNA complex of the present invention has excellent transfection efficiency and low cytotoxicity, and thus results in improving the expression rate of genes in cells.

Transfection

Therefore, the present invention also includes a method for introducing said GMP / nucleic acid complex into a cell and its application.

Cells suitable for use in accordance with the methods described herein include prokaryotes, yeast, or higher eukaryotic cells including plant and animal cells, particularly mammalian cells.

In certain embodiments, the cell is a cancer cell. In certain preferred embodiments, cell lines, such as breast cancer (MCF-7, MDA-MB-438 cell line), U87 glioblastoma cell line, B16F0 cell (melanoma), HeLa cell (cervical cancer), A549 Cells (lung cancer) and HepG2 (liver cancer) cells, but are not limited thereto. In one embodiment of the present invention, 293 cells, HeLa cells and HepG2 were used.

Culturing of the cells for transfection (transfection) can be suitably carried out by those skilled in the art using conventional knowledge known in the art. For example, growth is carried out in DMEM medium containing 10% heat inactivated fetal bovine serum (FBS) with L-glutamine and penicillin / streptomycin (pen / strep). Those skilled in the art will understand that certain cells must be cultured in a particular medium because they require specific nutrients such as growth factors and amino acids. The optimal density of cells depends on the cell type and the purpose of the experiment. It is also cultured under appropriate conditions (eg, 37 ° C, 5-10% CO ₂ ) depending on the cell type. The incubation time depends on the purpose of the experiment. Generally, in a gene transfection experiment, cells are cultured for 24 to 48 hours to express the target gene.

On the other hand, the results of nucleic acid delivery, i.e., transfection, according to the GMP of the present invention can be analyzed in different ways.

In the case of gene transfection and antisense nucleic acid delivery, the target gene expression level can be detected by a reporter gene, such as a green fluorescent protein (GFP) gene, a luciferase gene, a beta -galactosidase gene expression, and the like. Those skilled in the art will be familiar with how these reporters work and how they can be introduced into a gene delivery system. Nucleic acids and their products, proteins, peptides or other biomolecules are delivered by the methods described herein, and the targets regulated by these biomolecules are delivered by a variety of methods, such as immunofluorescence, enzyme immunoassay, Can be measured by radio-graph or in-situ hybridization. When immunofluorescence is used to detect the expression of the encoded protein, an antibody labeled with a fluorescent substance that binds to the target protein is used (e. G., Added to the slide under conditions suitable for binding the antibody to the protein). Subsequently, cells containing the protein are identified by detecting the fluorescent signal. If the delivered molecule is capable of regulating gene expression, the level of target gene expression may also be measured by methods such as autoradiography, in situ hybridization, and in situ PCR. However, the identification method may vary depending on the nature of the delivered biomolecule, its expression product, the target regulated thereby and / or the final product produced by delivery of the biomolecule.

In addition, the present invention relates to a composition for treating a target disease comprising a complex containing GMP and a target gene, and a use thereof, from another aspect.

That is, the complex of the present invention can be administered as a therapeutic agent for diseases. DNA corresponding to all or part of a coding region of a gene whose expression is suppressed or abnormally expressed in a disease state is administered to a patient in need of treatment.

The exact formulation, route of administration and dosage of the compositions and pharmaceutical compositions described herein may be chosen by a physician, depending on the condition of the patient

In general, the dosage range of the composition to be administered to a patient is from 0.5 to 1000 mg, or from 1 to 500 mg, or from 10 to 500 mg, or from 50 to 100 mg, per kg body weight of the patient. The dose may be a single dose or a continuous dose provided twice or more over a period of one or more days as required by the patient. If human doses are not established, suitable human doses may be estimated from the ED ₅₀ or ID ₅₀ values, as determined by toxicity studies and efficacy studies in animals, or may be estimated from in vitro or in vivo studies Can be estimated from other suitable values

Dosage amounts and dosing intervals may be adjusted individually to provide a plasma concentration of the active moiety in an amount sufficient to maintain a modulating effect or minimum effective concentration (MEC). The MEC depends on each compound and can be measured from in vitro data. The dosage required to achieve the MEC will vary depending on the individual characteristics and the route of administration. However, HPLC testing or biopsy can be used to measure plasma concentrations. Further, the administration interval can be determined using the MEC value. The composition should be administered using a dosing regime such that the plasma concentration exceeds the MEC for a time period of 10 to 90%, preferably 30 to 90%, or most preferably 50 to 90%.

The dosage of the composition may vary depending upon the subject to be treated, the body weight of the subject, the severity of the disease, the manner of administration, and the judgment of the prescribing physician.

In addition, the complex of the present invention may further comprise a nucleic acid delivery-improving agent.

Various methods for preventing release of biomolecules from cell membranes, endosomal membranes, and carriers, which are problematic in nucleic acid delivery into cells, can be applied together.

A nucleic acid-carrier complex such as DNA must first pass through the cell membrane. When this is carried out with endocytosis, the nucleic acid-carrier complex is then internalized. The carrier with nucleic acid-cargo is encapsulated by the cell membrane by pocket formation, and this pocket is subsequently pinch off. This result is a cellular membrane-bound macromolecule-bound structure that contains nucleic acid cargo and a carrier. The nucleic acid-carrier complex should then escape into the cytoplasm from the endosomal membrane and avoid enzymatic degradation in the cytoplasm. The nucleic acid cargo must be separated from the carrier. In general, anything designed to overcome one or more of the barriers described above may be considered as a delivery enhancer.

The non-viral delivery enhancer can be either a polymeric system or a lipid system. This transfer modifier is generally a polycation that balances the negative charge of the nucleic acid. A branched version of a polycation such as PEI and Starburst dendrimer can mediate endosomal release (Boussif, et al. (1995) Proc. Natl. Acad. Sci USA vol.92: 7297-7301]. PEI is a highly branched polymer with terminal amines that can be ionized at pH 6.9 and internal amines that can be ionized at pH 3.9. This structure changes the pH of the vesicle, resulting in vesicle swelling and eventually endosome capture Lt; / RTI >

Another means of improving delivery is to devise a ligand on the carrier. The ligand should have a cellular receptor that is targeted in cargo delivery. Biomolecule delivery into the cell is then initiated by receptor recognition. When the ligand binds to its specific cell receptor, entoshiosis is stimulated. Examples of ligands that have been used in various cell types to facilitate biomolecular transfer include, but are not limited to, galactose, transferrin, glycoprotein asialourocomycode, adenovirus fiber, malaria secretory protein, epithelial growth factor, , Fibroblast growth factor and folate

Release of biomolecules such as DNA into the cytoplasm of a cell can be facilitated by agents that mediate endo-collapse, reduce degradation, or bypass all of these processes together. Chloroquine, which increases endosomal pH, can be used to reduce the degradation of endocytosed materials by inhibiting lysosomal hydrolases.

As described above, the present invention relates to a non-viable gene carrier, and more particularly, to a non-viable gene transporter which is based on GMP, which has a structure of GC-MA-PEI (Glycol Chitosan-Methyl Acrylate-Polyethyleneimine) And to a polymer / DNA complex which is a non-viral gene transfer prepared by using the polymer as a carrier.

The present invention will be very useful as an effective non-viral gene delivery system since it has high transfection efficiency, low cytotoxicity and high gene expression efficiency.

< Example >

Hereinafter, the present invention will be described in more detail with reference to Examples. It should be apparent to those skilled in the art that these embodiments are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples

Statistical analysis was performed using unpaired Students t-test (GraphPad Prism 5). All results were expressed as mean ± standard deviation (SD). Group differences were statistically significant at P <0.05 (*), P <0.01 (**), and P <0.001 (***).

Preparation for experiment

Polyethyleneimine, low molecular weight, water free) and PEI 25kD (polyethylenimine, low molecular weight, water free), glycolic acid, glycerol chitosan (≥60%, titration, crystalline), methyl acrylate (99%), methanol (polyethylenimine, high molecular weight, water free) was purchased from Sigma-Aldrich (Seoul, South Korea). FBS (Fetal bovine serum), DMEM (Dulbecco's modified Eagle's medium), and 100 × (100 μl) were purchased from DaeilLab Service Co., Ltd. (Seoul, South Korea) Antibiotic-antimycotic agents were purchased from Gibco (Gaithersburg, MD, USA). Luciferase assay kit was purchased from Promega (Madison, Wis., USA) and Micro BCA Protein Assay Kit was purchased from Pierce (Rockford, IL, USA). Aquacide II was purchased from Merck KGaA (Frankfurter, Germany).

< Example  1>

GMP ( Glycol Chitosan - Methyl Acrylate - Polyethyleneimine ) Synthesis of

In order to obtain GMP, a total of two steps of synthesis are required. Detailed synthetic reactions are shown in Fig.

One -One. GC - MA ( Glycol Chitosan - Methyl Acrylate ) synthesis

First, use MA to connect GC and PEI. MA is a linker mainly used in Michael addition reaction with primary amine, which is a material used in dendrimer synthesis.

To obtain GC-MA, methyl acrylate (0.658 ml) was first placed in a round bottom flask and reacted with methanol (63 ml) at room temperature for 10 minutes. Then, add a drop of glycol chitosan (15 mg) dissolved in distilled water to a round bottom flask. Then, the gas inside the flask was exchanged with nitrogen gas, and the mixture was reacted at 180 rpm for 4 days while maintaining the temperature in a shaking incubator at 37 ° C. After 4 days, the methanol was removed using an evaporator, and the final product was obtained by dialysis (MWCO: 100-500) for 1 day and then lyophilized.

1-2. GMP ( Glycol Chitosan - Methyl Acrylate - 폴리에틸렌imen ) synthesis

The GC-MA obtained in Example 1-1 was dissolved in a mixed solution of methanol and distilled water (methanol: distilled water = 9: 1, volume ratio), and transferred to a round bottom flask. After addition of methanol (50 ml), the reaction was allowed to proceed for 10 minutes. PEI (PEI 800D, polyethylenimine, low molecular weight, water free) was added to the flask, and the gas in the flask was replaced with nitrogen. Maintained at 37 ° C and reacted at 180 rpm for 5 days. After 5 days, the methanol was removed with an evaporator, and the final product was obtained by dialysis (MWCO: 1000) for 1 day and then lyophilized.

1-3. Confirmation of GMP synthesis

In order to identify the synthesized material by the above synthesis method, ¹ H NMR spectrum was measured using a 300 MHz NMR spectrometer (Bruker, AVANCE 300). As a result, GC-MA-PEI exhibited a 2.5-2.8 ppm Signal (see Fig. 2). As a result, a large amount of amine group was synthesized, and the expected nanoparticles were formed.

< Example  2>

GCMP / Plasmid DNA  Measurement of complex size and zeta Potential  Measure

The particle size and surface charge of the GMP / plasmid complexes affect the internalization of the delivery complex by the cell, thus affecting the transfection efficiency.

In order to determine the particle size of the GMP / plasmid complex, particle size was measured according to the manufacturer's method using an ELS-Z2 instrument (Photal, Otsuka Electroninc, Otsuka, Japan). The zeta potential measurements were also confirmed by the manufacturer's method via ZetasizerNano-Zs (Malvern Instruments Ltd., Worcestershire, U.K.).

As a control, plasmid DNA was used. As a result of measuring the size of the control group, the size of the complex was determined to be 500 nm or less after the formation of the complex according to the mass ratio of the plasmid DNA to the nanoparticle, (See FIG. 3). From these results, it can be confirmed that the nanoparticles can be introduced into the cells after forming the complex. We also measured the zeta potential of the complex according to the mass ratio of the plasmid DNA and nanoparticles to predict the binding force between the surface - negatively charged plasmid DNA and the positively charged banded nanoparticles. As a result, it was confirmed that the plasmid DNA and nanoparticle complex had a positive charge between 20 and 30 mV (see FIG. 4). This confirms that GMP nanoparticles can bind to plasmid DNA.

< Example  3>

Confirmation of complex formation by electrophoresis

To confirm the complex formation of GMP nanoparticles with plasmid DNA, electrophoresis using agarose gel was performed. The pCN-Luci DNA and GMP nanoparticles at a concentration of 0.25 μg / μL were mixed at a ratio of 1: 1 to 1:10 by mass for 30 minutes at room temperature. Ethidium bromide (0.5 μg / mL of the gel electrophoresis on a 0.7% agarose gel. Was performed at 100 V for 30 minutes and a UV-illumination photograph of the resulting gel was obtained.

As a result, as shown in FIG. 5, the gel phase migration was delayed at all mass ratios including the 1: 1 mass ratio as compared with the control group, and it was confirmed that the pDNA and the GMP nanoparticles were complexed with each other. Especially, when the plasmid DNA and GMP nanoparticles were in a ratio of 1: 8 mass ratio, they were found to have optimum mixing conditions.

< Example  4>

Pico Green  Confirmation of complex formation by quantification

The complex formation on agarose gel was confirmed through Example 3 above, but fluorescence was observed using PicoGreen reagent for confirmation once again.

GMP / pDNA complexes were mixed in HEPES buffer (20 mM Hepes, 150 mM NaCl, pH 7.4) in the range of 0.4 to 4 at different weight ratios (polymer / pDNA) and reacted at room temperature for 30 min. The reacted complexes were mixed with TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) and reacted with picogreen reagent for 2 minutes and observed with a spectrophotometer (Quantech digital filter fluorometer, Thermo scientific). At this time, fluorescence was measured by fixing the excitation wavelength at 480 nm and the emission wavelength at 520 nm.

As a result, as can be seen from FIG. 6, it was found that fluorescence was significantly reduced in the range of 0.5 to 4, and the complex was effectively formed.

< Example  5>

Intracellular  Check gene transfer efficiency

5-1. Cell culture

HeLa cells (human ovarian cancer cell line), 293 cells (human kidney cell line) and HepG2 cells (human hepatocellular carcinoma cell line) were cultured at 37 ° C in an incubator (5% CO ₂ , 95% humidity) environment. All of the cell lines were cultured in DMEM (Dubelco's modified Eagles's medium) containing 10% FBS and 1% antibiotic, and the cells were washed with DPBS (Dulbecco's Phosphate Buffered Saline) solution.

5-2. Identification of intracellular toxicity

In order to use GMP according to the present invention for gene therapy or the like, there is no cytotoxicity. Therefore, WST assay was performed to confirm intracellular toxicity.

For this, HeLa cells, HEK293 cells, and HepG2 cells were used to confirm the gene transfer efficiency. GMP nanoparticles synthesized after culturing for 15 minutes at 15,000 cells / well in a 96-well plate were cultured for 24 hours, and PEI 25 kD and PEI800D were diluted per concentration and treated with cells. After incubation for 24 hours, WST-1 reagent (Daeil Lab Service, Seoul, South Korea) was added to each well. After 2 hours, the absorbance was measured at 450 nm with ELISA (VERSAmax, Molecular Devices, Sunnyvale, CA).

As a result, it was confirmed that the control PEI25kD used for conventional gene transfer was highly cytotoxic. In the case of GMP nanoparticles according to the present invention, PEI 800D Lt; / RTI &gt; (see FIG. 7).

5-3. GMP  Confirmation of transfer efficiency of nanoparticles in gene cell

In order to confirm the intracellular gene transfer efficiency of GMP nanoparticles, HeLa cells, HepG2 cells and HEK293 cells were plated at a concentration of 25,000 / well in a 48-well plate for 24 hours, and pDNA and GMP nanoparticles The complex was formed at a mass ratio of 1: 1 to 1:20 for 30 minutes, and the cells were treated. After the treatment, the cells were cultured for 24 hours and then dissolved in repoterlysis buffer (promega), and the intracellular gene expression efficiency was measured by luciferase assay and protein quantification. As a control, PEI 25kD, which is known to have high gene transfer efficiency, and PEI800D, which was used in the synthesis of GMP nanoparticles, were used at respective mass ratios. LB 9507 photometer (Berthold, Germany) was used for enzyme activity analysis. Protein quantitation was performed by measuring absorbance at 570 nm using ELISA (VERSAmax, Molecular Devices, Sunnyvale, Calif.

As a result, the GMP complex according to the present invention exhibited a similar or higher level of transfer efficiency than PEI25kD, which is known to have high gene transfer efficiency but also high cytotoxicity, and showed much higher efficiency than PEI800D used in the synthesis 8). In addition, as the amount of GMP nanoparticles increased, the transfer efficiency tended to increase in all three cell lines. Experiments were conducted until the ratio of the maximum GMP nanoparticles to the plasmid DNA was 20: 1 , And the gene transfer efficiency was found to be very high. As the positive charge increases, complex formation with DNA is increased and it is considered that the introduction into the cell through the amine group of the surface is facilitated.

5-4. Confocal  Fluorescence microscopy experiment

HeLa cells were plated on a 35 mm soverebrass bottom dish (SPL) at a concentration of 5000 cells / well and cultured at 37 ° C for 24 hours. An ^{Alexa Fluor ® 488 5-SDP Ester} (Alexa Fluor ® 488 Sulfodichlorophenol Ester) was prepared according to the manufacturer's system to refer to fluorescence in the GMP nanoparticles. Fluorescence-bound GMP nanoparticles were treated with HeLa cells and observed for 2 hours using a confocal microscope (LSM5 live configuration variotwo VRGB).

The nuclear staining for the cells was performed with bisBenzimide H 33342 trihydrochloride, reacted at room temperature for 5 minutes, washed with DPBS (Dulbecco's Phosphate Buffered Saline), and observed with a confocal microscope (LSM5 live configuration variotwo VRGB).

As a result, most of the GMP nanoparticles were found to be introduced into the cell and located in the cytoplasm, and some of them were transferred to the nucleus. GMP nanoparticles according to the present invention exhibited much better cell-to-cell transfer efficiency than glycol chitosan, compared to glycol chitosan and PEI25 kD, which are similar to or higher than that of PETI25KD, (See FIG. 9).

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that the present invention may be embodied in various other forms without departing from the spirit or essential characteristics thereof. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (15)

A non-viral nucleic acid delivery polymer comprising a GMP constituting a GC-MA-PEI (Glycol Chitosan-Methyl Acrylate-Polyethylenimine) structure. The method according to claim 1,
Wherein the PEI is PEI800D. (a) a step of synthesizing glycol chitosan-methyl acrylate (GC-MA) to react methyl acrylate with glycol chitosan; And
(b) synthesizing GC-MA-PEI (glycol chitosan-methyl acrylate-polyethyleneimine) by adding PEI (polyethyleneimine) to the glycol chitosan-methyl acrylate synthesized in step (a) GMP synthesis method. The method of claim 3,
Wherein the PEI is PEI800D. The method of claim 3,
Wherein the methyl acrylate of step (a) is reacted with methanol at room temperature for 10 minutes, and then the glycol chitosan dissolved in distilled water is added. 6. The method of claim 5,
In the step (a), after the glycol chitosan is added, the gas inside the reaction vessel is exchanged with a nitrogen gas, and the reaction is carried out at 35 to 39 ° C. and 150 to 200 rpm for 3 to 5 days in a stirring incubator Lt; / RTI &gt; The method according to claim 6,
Wherein the step (a) is carried out in an agitating incubator, after removing the methanol with an evaporator, dialyzing, and then lyophilized. The method of claim 3,
In step (b), the glycol chitosan-methyl acrylate (GC-MA) synthesized in the step (a) is dissolved in a 9: 1 (v: v) mixture of methanol: distilled water and 30 to 70 ml of methanol And the mixture is allowed to react for 5 to 20 minutes. 9. The method of claim 8,
In step (b), PEIpolyethylenimine is added to GC-MAglycol chitosan-methyl acrylate, which is further reacted with methanol. The gas inside the reaction vessel is exchanged with nitrogen gas, and the reaction mixture is stirred at 35 to 39 ° C, 150 to 200 rpm To react for 4 to 6 days. 10. The method of claim 9,
Wherein the step (b) is performed by removing methanol with an evaporator after the reaction in a stirred incubator, dialyzing, and lyophilized. A non-viral nucleic acid delivery complex having a target gene bound to a GMP constituting GC-MA-PEI (Glycol Chitosan-Methyl Acrylate-Polyethylenimine) structure. 12. The method of claim 11,
Wherein the target gene is composed of DNA. 13. The method of claim 12,
Wherein the GMP: DNA weight ratio is 0.5 to 20: 1. 12. The method of claim 11,
Wherein said complex is compressed into nanoparticles of 100 to 800 nm. 12. The method of claim 11,
Wherein the PEI is PEI800D.

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