KR101684265B1

KR101684265B1 - 2-aminoethyl methacrylate-grafted chitosan copolymer for gene deslivery using radiation technology and method for preparing the same

Info

Publication number: KR101684265B1
Application number: KR1020150053637A
Authority: KR
Inventors: 임윤묵; 정성린; 박종석; 권희정; 박순정; 장미경
Original assignee: 한국원자력연구원
Priority date: 2015-04-16
Filing date: 2015-04-16
Publication date: 2016-12-12
Also published as: KR20160124279A

Abstract

The present invention relates to the use of radiation irradiation technology to increase the positive charge of a biomedical low molecular weight chitosan (LMWC) by introducing 2-aminoethyl methacrylate (AEMA) (AEMA-LMWC) with improved gene-binding ability and no cytotoxicity. It was confirmed that the AEMA-LMWC complex of the present invention is applicable as a gene delivery vehicle.

Description

(2-aminoethyl methacrylate-grafted chitosan copolymer for gene transfer using gene technology and method for preparing same)

More particularly, the present invention relates to a gene transporter capable of improving gene transfer efficiency by introducing 2-aminoethyl methacrylate (2-aminoethyl methacrylate) into chitosan using radiation technology. Lt; / RTI >

Drug therapy for the treatment of genetic incapacity or cancer in modern society has recently been limited to the treatment of diseases due to serious side effects such as drug resistance and severe physical and mental pain of the patient. And the research for this is increasing. In order to achieve efficient gene therapy, it is important to develop a gene delivery system (Gene Delivery System) that can safely transmit genes into the body. The role of the gene carrier is to form a complex by electrostatic interaction with an anion-bearing gene, thereby safely delivering it into the target cell by protecting the gene from the DNase present in the blood. The role of the therapeutic gene is to prevent the abnormal growth of cells by expressing the desired protein in the cells transferred to the nucleus in the cell, or to secrete a protein capable of treating the disease so that the therapeutic effect is achieved.

In order to increase the efficiency of gene therapy, the development of gene carriers is a very important factor. The known viral vectors have an advantage of high expression efficiency, but their use is very difficult due to serious problems such as immune reaction or recombination of endogenous virus Is limited. Therefore, although the gene efficiency is somewhat low as a nonviral vector, the development of a carrier using a cationic polymer having a high efficiency of complex formation with a gene has attracted much interest by researchers, and various attempts have been made for this purpose.

In order to solve low gene efficiency which is a disadvantage of cationic polymer as a gene carrier, it is very important to introduce various functional groups into the main chain of the polymer. Various methods have been introduced so far. Non-patent documents 1 and 2 and introduction of ligands that can be absorbed into cells due to receptor-mediated endocytosis. Gene release through fast escape in endosomes [Non-Patent Document 3] Polymer modification such as enhancing intracellular absorption efficiency through the introduction of a cell penetrating peptide [Non-Patent Documents 4, 5, 6] has been performed. Until now, chemical agents such as crosslinking agents have been used to introduce such functional groups [Non-Patent Documents 7, 8, and 9], and post-synthesis processing and purification processes are very complicated, Which is a disadvantage of the conventional technique [Non-Patent Documents 10, 11].

Chitosan, a natural cationic polymer, has attracted much attention as a research material for drugs and gene delivery materials because of its excellent biocompatibility, low toxicity, and excellent cell adhesion ability. In addition to these properties, the amino group of the glucosamine unit of the chitosan unit has a positive charge, which is advantageous in forming a complex by ion interaction with a gene having a negative charge. However, the cationic nature of chitosan has limitations in binding to genes.

Radiation technology is used in various surface modification technologies such as increasing cellular reactivity to polymer materials or surface modification of hydrophobic polymers with hydrophilic polymers. Surface grafting through acid or base treatment, Graf Ting technology has been used as a representative. The use of radiation technology also has the advantage of introducing bioactive factors onto the surface of the polymer without any additional chemical reaction.

In addition, acrylate monomers having a positive charge such as 2-aminoethylmethacrylate (AEMA) have a double bond, so when irradiated, the double bonds are broken and monomers are produced. When irradiated with the acrylate monomer, the monomer is grafted to the substrate. This principle is to introduce AEMA into chitosan, and when AEMA is used as a gene carrier, it can improve not only the gene binding ability of the transporter but also the ability to attach to cells. In addition, since the use of radiation technology has the advantage of introducing monomers onto the surface of the polymer without undergoing a separate chemical reaction, problems such as safety, reproducibility, mass production, and the like, which are problems of conventional gene delivery methods, can be improved.

In this study, the first attempt was made to irradiate the polymer cations as a modification of the polymer cation. This is a new attempt as an environmentally friendly polymer modification method called "green process" because no chemical reagent is used.

Accordingly, the present inventors have increased the gene-binding efficiency of the chitosan complex by introducing an acrylate monomer, 2-aminoethyl methacrylate (AEMA), in order to increase the positive charge of the chitosan transporter. The present invention has been completed.

[1] Mi-hee Jo, Bahy A. Ali, Abdulaziz A. Al-Khedhairy, Chang Hyun Lee, Bongjune Kim, Seungjoo Haamc, Yong-Min Huh, Hae Young Ko, Soonhag Kim; A reverse complementary multimodal imaging system to visualize microRNA9-involved neurogenesis using peptide targeting transferrin receptor-conjugated magnetic fluorescence nanoparticles, Biomaterials, 33 (2012) 6456-6467. [2] Michael E. Werner, Shrirang Karve, Rohit Sukumar, Natalie D. Cummings, Jonathan A. Copp, Ronald C. Chen, Tian Zhang, Andrew Z. Wang; Folate-targeted nanoparticle delivery of chemo- and radiotherapeutics for treatment of ovarian cancer peritoneal metastasis, Biomaterials, 32 (2011) 8548-8554. [3] Kyungghwan Kima, Kitae Ryu, Tae-il Kim; Cationic methylcellulose derivatives with serum-compatible and endosome buffering ability for gene delivery systems, Carbohydrate Polymers, 110 (2014) 268277. [4] Jingjing Song, Yun Zhang, Wei Zhang, Jianbo Chen, Xiaoli Yang, Panpan Ma, Bangzhi Zhang, Beijun Liu, Jingman Ni, Rui Wang; Cell penetrating peptide TAT can kill cancer cells via membranedisruption after attachment of camptothecin, Peptides, 63 (2015) 143149. [5] Ankur Gautam, Minakshi Sharma, Pooja Vir, Kumardeep Chaudhary, Pallavi Kapoor, Rahul Kumar, Samir K. Nath, Gajendra P.S. Raghava; Identification and characterization of novel protein-derived arginine-rich cell-penetrating peptides, European Journal of Pharmaceutics and Biopharmaceutics, 89 (2015) 93106. [6] Lus Vasconcelos, Fatemeh Madani, Piret Arukuusk, Ly Prnaste, Astrid Grslund, lo Langel; Effects of cargo molecules on membrane perturbation caused by transportan-based cell-penetrating peptides, Biochimica et Biophysica Acta, 1838 (2014) 31183129. [7] Xi Zhang, Yajing Duan, Dongfang Wang, Fengling Bian; Preparation of Arginine-modified PEI-conjugated chitosan copolymer for DNA delivery, Carbohydrate Polymers, 122 (2015) 53-59. [8] Hongyan Zhua, Fei Liua, Jing Guoa, Jianpeng Xuea, Zhiyu Qianc, Yueqing Gua; Carbohydrate Polymers, 86 (2011) 11181129. Folate-modified chitosan micelles with enhanced tumor targeting evaluated by near infrared imaging system. [9] Tony Shing Chau Li, Toshio Yawata, Koichi Honke; Efficient siRNA delivery and tumor accumulation mediated by ionically cross-linked folic acid (ethylene glycol) chitosan oligosaccharide lactate nanoparticles: For the potential targeted ovarian cancer gene therapy, European Journal of Pharmaceutical Sciences, 52 (2014) 4861. [10] Seong-Cheol Park, Joung-Pyo Nam, Young-Min Kim, Jun-Ho Kim, Jae-Woon Nah, Mi-Kyeong Jang; Branched polyethylenimine-grafted-carboxymethyl chitosan copolymer enhances the delivery of pDNA or siRNA in vitro and in vivo, International Journal of Nanomedicine, 8 (2013) 3663-3677. [11] Joung-Pyo Nam, Seong-Cheol Park, Tae-Hun Kim, Jae-Yeang Jang, Changyong Choi, Mi-Kyeong Jang, Jae-Woon Nah; Encapsulation of paclitaxel into lauric acid-O-carboxymethyl chitosan-transferrin micelles for hydrophobic drug delivery and site-specific targeted delivery, International Journal of Pharmaceutics, 457 (2013) 124135.

It is an object of the present invention to provide a chitosan complex for gene transfer in which 2-aminoethyl methacrylate is conjugated using radiation technology and a method for producing the chitosan complex.

Another object of the present invention is to provide a chitosan complex conjugated with 2-aminoethyl methacrylate prepared according to the present invention and its use as a gene carrier thereof.

In order to achieve the above object,

1) mixing chitosan and 2-aminoethyl methacrylate (AEMA);

2) irradiating the mixture of step 1) with radiation;

Aminoethyl methacrylate grafted with 2-aminoethyl methacrylate to form a chitosan copolymer.

The present invention also provides a chitosan complex conjugated with 2-aminoethyl methacrylate.

The present invention also provides a drug delivery system comprising the chitosan complex and a pharmaceutically effective ingredient.

In addition, the present invention provides a pharmaceutical composition comprising the drug delivery vehicle and a pharmaceutically acceptable carrier.

The present invention relates to the use of radiation irradiation technology to increase the positive charge of a biomedical low molecular weight chitosan (LMWC) by introducing 2-aminoethyl methacrylate (AEMA) (AEMA-LMWC) with improved gene-binding ability and no cytotoxicity. The AEMA-LMWC complex of the present invention was confirmed by ¹ H-NMR and analyzed by DLS (Dynamic Light Scattering) analysis The size and surface charge of the nanoparticles were found to be in the range of 203.9 ~ 507.6 nm and higher than that of chitosan. Furthermore, it was confirmed that the AEMA-LMWC complex of the present invention is applicable as a gene delivery vehicle by confirming that it has excellent gene transfer ability and cytotoxicity through electrophoresis, transfection and CCK assay.

1 is a schematic diagram showing a process for preparing a complex in which plasmid DNA (pDNA) is bound to low molecular weight chitosan (LMWC) into which 2-aminoethyl methacrylate (AEMA) has been introduced by irradiation with radiation As shown in FIG.
Figure 2 shows the ¹ H-NMR spectra of the AEMA-LMWC complexes according to the radiation dose (5, 10 and 25 kGy)
AEMA: spectrum of 2-aminoethyl methacrylate;
LMWC: spectrum of low molecular weight chitosan; And
AEMA-LMWC (5, 10 and 25 kGy): spectrum of low molecular weight chitosan with 2-aminoethyl methacrylate introduced according to dose of radiation.
3 is a graph showing the molecular weight of the AEMA-LMWC complex according to the radiation dose (5, 10 and 25 kGy) by GPC.
FIG. 4 is a graph showing the relationship between the binding ratio (1: 0.1 to 1: 1) of the complex (pEGFP-N1 / AEMA-LMWC) in which plasmid DNA is bound to low molecular weight chitosan having 2-aminoethyl methacrylate introduced thereto through irradiation with 5 kGy : 10) The gene binding ability was confirmed:
AEMA: 2-aminoethyl methacrylate;
LMWC: Low molecular weight chitosan; And
AEMA-LMWC: Low molecular weight chitosan incorporating 2-aminoethyl methacrylate.
Fig. 5 shows the particle size (a) of the complex (pEGFP-N1 / AEMA-LMWC) in which the plasmid DNA was bound to the low molecular weight chitosan into which 2-aminoethyl methacrylate was introduced through a ratio of 5 kGy, (B): < RTI ID = 0.0 >
AEMA: 2-aminoethyl methacrylate;
LMWC: Low molecular weight chitosan; And
AEMA-LMWC: Low molecular weight chitosan incorporating 2-aminoethyl methacrylate.
6 is a chart for confirming cell viability by a complex (pEGFP-N1 / AEMA-LMWC) in which plasmid DNA is bound to low molecular weight chitosan having 2-aminoethyl methacrylate introduced thereto through irradiation with 5 kGy:
AEMA: 2-aminoethyl methacrylate;
LMWC: Low molecular weight chitosan; And
AEMA-LMWC: Low molecular weight chitosan incorporating 2-aminoethyl methacrylate.
FIG. 7 is a graph showing the transfection efficiency of a complex (pEGFP-N1 / AEMA-LMWC) in which plasmid DNA is bound to low molecular weight chitosan into which 2-aminoethyl methacrylate has been introduced by irradiation with 5 kGy to be:
Control: untreated cells;
lipo (lipofectamine): a commercially available transfection reagent;
pDNA / LMWC (1: 8, 1:16): cells treated with a complex of pDNA and LMWC mixed at a ratio of 1: 8 or 1:16;
pDNA / AEMA (1: 8, 1:16): cells treated with a complex of pDNA and AEMA mixed at a ratio of 1: 8 or 1:16; And
pDNA / AEMA-LMWC (1: 2 to 1:20): pDNA and AEMA-LMWC in a ratio of 1: 2 to 1:20.
8 is a graph showing the amount of a complex (pEGFP-N1 / AEMA-LMWC) in which a plasmid DNA is bound to low-molecular-weight chitosan having 2-aminoethyl methacrylate introduced thereinto through radiation of 5 kGy into the cell by FACS Also:
Control: untreated cells;
Control (only pDNA): cells treated with plasmid DNA (pDNA) only;
pDNA / AEMA-LMWC (1: 2): cells treated with a complex of pDNA and AEMA-LMWC mixed at a ratio of 1: 2;
pDNA / AEMA-LMWC (1: 4): cells treated with a complex of pDNA and AEMA-LMWC mixed at a ratio of 1: 4;
pDNA / AEMA-LMWC (1: 8): cells treated with a complex of pDNA and AEMA-LMWC mixed at a ratio of 1: 8; And
pDNA / AEMA-LMWC (1:16): Cells treated with a complex of pDNA and AEMA-LMWC mixed at a ratio of 1:16.

Hereinafter, the present invention will be described in detail.

The present invention

1) mixing chitosan and 2-aminoethyl methacrylate (AEMA);

2) irradiating the mixture of step 1) with radiation;

Chitosan is a commonly used name for poly- [1,4] - [beta] -D-glucosamine and is a natural high molecular weight chitin present in many crustaceans such as crustaceans of insects, cell wall of fungi, N-deacetylation. The chitosan used in the present invention can be chemically derived from chitin or can be derived at low cost from widely available materials such as those derived from fungal cell walls, insect bark and especially crustaceans, and also commercially available.

The chitosan of step 1) used in the present invention may be chitosan having a molecular weight of 1,000 to 100,000, preferably a low molecular weight having a molecular weight of 5,000 to 50,000, but is not limited thereto.

Preferably, the 2-aminoethyl methacrylate of step 1) above increases the cationicity of chitosan.

In the step 1), it is preferable to dissolve chitosan in distilled water and dissolve 2-aminoethyl methacrylate in methanol.

The radiation in step 2) is preferably one selected from a gamma ray, an electron beam, an ion beam, a neutron beam and ultraviolet ray, and more preferably a gamma ray.

The irradiation dose of the step 2) is preferably 5 to 25 kGy, but is not limited thereto.

The chitosan used in the present invention can be chemically derived from chitin or can be derived at low cost from widely available materials such as those derived from fungal cell walls, insect bark and especially crustaceans, and also commercially available.

The chitosan may be chitosan having a number average molecular weight of 1,000 to 100,000, preferably a low molecular weight having a number average molecular weight of 5,000 to 50,000, but is not limited thereto.

In a specific example of the present invention, the present inventors used 100 mg of low molecular weight chitosan and 10 mg of 2-aminoethyl methacrylate (LMWC) to introduce 10% of 2-aminoethyl methacrylate (AEMA) into low molecular weight chitosan mg were respectively dissolved in 4 mL of distilled water and 1 mL of MeOH and then placed in a single vial. To prepare a complex for each irradiation dose, a gamma ray of ⁶⁰ Co source (ACEL type C-1882 , Korea Atomic Energy Institute) at 5, 10 and 25 kGy, respectively, at a dose rate of 10 kGy / hr. After the irradiation, the membrane was dialyzed for 24 hours using a membrane (molecular weight cut off, MWCO 1 KDa) to remove unreacted materials, followed by lyophilization for 3 days to prepare powdered gene transfer AEMA-LMWC complex (See FIG. 1).

In addition, the inventors of the present invention have identified the structure analysis of the AEMA-LMWC composite by the ¹ H-NMR (Bruker AVANCE 400, Germany) spectra. As a result, the LMWC 5 K shifted to 4.7 ppm in the AEMA-LMWC composite spectrum where the C-1 peak at 4.9 ppm was irradiated at 5 kGy and the 2.8 ppm (See Fig. 2A). In addition, the synthesis was confirmed through the AEMA-LMWC complex in which the peak of 3.8 ppm and the peak of AEMA of 1.3 ppm, which were absent in the LMWC spectrum, were irradiated at 5 kGy (see FIG. In addition, it was confirmed that the introduction efficiency of AEMA is lowered as irradiation dose increases (refer to FIG. 2B).

The AEMA-LMWC composite showed a low molecular weight chitosan (LMWC) having a molecular weight of 5486. As a result, the AEMA-LMWC composite showed a molecular weight of 5 kGy As a result of increasing the molecular weight by 7507, it was confirmed that AEMA having a molecular weight of 165.2 was introduced at 30% (see FIG. 3). On the other hand, when 10 kGy and 25 kGy were irradiated, they showed molecular weights of 7457 and 7170, respectively, and it was confirmed that the efficiency decreased as the dose increased (see FIG. 3).

Therefore, the AEMA-LMWC complex was found to have the highest efficiency when the irradiation dose was 5 kGy, and the AEMA-LMWC complex with 5 kGy was used as the gene carrier.

The pharmaceutical active ingredient includes a chemotherapeutic agent, a protein medicine, or a nucleic acid medicine. The pharmaceutical active ingredient preferably includes those which are negatively charged or can be induced to negative charge.

The chemotherapeutic agent means an organic compound exhibiting a pharmacological effect for any disease. Such protein drugs or nucleic acid drugs include, for example, peptides that specifically bind to specific receptors to block or inhibit signal transduction, siRNAs that inhibit the expression of specific genes, and the like.

The nucleic acid includes an oligonucleic acid. More specifically, plasmid DNA, ribonucleic acid (RNA), small interfering ribonucleic acid (siRNA), antisense oligonucleotide, microRNA, locked nucleic acid, Nucleic acid aptamers, and the like.

The form of the drug delivery system according to the present invention is not particularly limited, but may be, for example, a formulation of liposome, micelle, emulsion, or nanoparticle.

The drug delivery system may be introduced into cells for the treatment of various diseases caused by overexpression of pathogenic proteins including tumors, arthritis, cardiovascular diseases, endocrine diseases and the like.

The drug delivery system of the present invention has excellent nucleic acid delivery efficiency and low cytotoxicity, so that it can suppress the overexpression of the pathogenic protein and thus can achieve an excellent disease treatment effect.

When the desired drug is introduced into the cell in vivo or ex vivo through the pharmaceutical composition of the present invention, it selectively decreases the expression of the target protein or modifies the mutation in the target gene, thereby overexpressing the pathogenic protein A disease caused by a target gene or a disease caused by a target gene.

In the present invention, the therapeutically effective amount of the drug delivery vehicle means an amount required for administration in order to expect the therapeutic effect of the disease. Therefore, it is desirable to provide a pharmaceutical composition containing the compound of the present invention, which is useful for the treatment of the disease, such as the type of disease, the severity of the disease, the kind of the nucleic acid to be administered, the type of the formulation, the patient's age, body weight, general health status, sex and diet, And can be adjusted according to various factors including drugs. It is preferable to administer the drug delivery vehicle to an adult in a dose of 0.001 mg / kg to 100 mg / kg, for example, once a day.

The carrier used in the composition according to the present invention includes a carrier and a vehicle commonly used in the medical field and specifically includes an ion exchange resin, alumina, aluminum stearate, lecithin, serum protein (e.g., human serum albumin) Salts or electrolytes such as protamine sulfate, disodium hydrogenphosphate, potassium hydrogen phosphate, sodium chloride and zinc salts), water, a salt or an electrolyte (for example, a mixture of various phosphates, glycine, sorbic acid, potassium sorbate, a partial glyceride mixture of saturated vegetable fatty acids) But are not limited to, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose based substrates, polyethylene glycol, sodium carboxymethylcellulose, polyarylate, wax, polyethylene glycol or wool. The composition of the present invention may further comprise, in addition to the above components, a lubricant, a wetting agent, an emulsifying agent, a suspending agent, or a preservative.

The pharmaceutical composition of the present invention may be formulated into parenteral dosage forms such as oral dosage forms or injections. Examples of formulations for oral administration include tablets, troches, lozenges, aqueous or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs. In order to formulate preparations such as tablets and capsules, binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin, excipients such as dicalcium phosphate, disintegrators such as corn starch or sweet potato starch, magnesium stearate , Calcium stearate, sodium stearyl fumarate, or polyethylene glycol wax. In addition, in the case of capsule formulations, in addition to the above-mentioned materials, a liquid carrier such as fatty oil may be contained. Formulations for oral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions and freeze-drying agents. Examples of the non-aqueous solvent and suspension solvent include vegetable oils such as propylene glycol, polyethylene glycol or olive oil, injectable esters such as ethyl oleate, and the like.

In one embodiment, the composition according to the invention can be prepared as an aqueous solution for parenteral administration. Preferably, a buffer solution such as Hank's solution, Ringer's solution or physically buffered saline can be used. Aqueous injection suspensions may contain a substrate capable of increasing the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran.

Another preferred embodiment of the composition of the present invention may be in the form of a sterile injectable preparation of an aqueous or oily suspension. Such suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents (e. G., Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example a solution in 1,3-butanediol. Vehicles and solvents that may be used include mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, nonvolatile oils are conventionally used as a solvent or suspending medium. For this purpose, any non-volatile oil including synthetic mono or diglycerides and less irritant may be used.

In a specific example of the present invention, the inventors of the present invention conducted a reaction for 30 minutes at a weight ratio of 1: 0.1 to 1:10 with pEGFP-N1 to confirm the ability of the AEMA-LMWC complex prepared above to bind to plasmid DNA (pDNA) 2-aminoethyl methacrylate (AEMA) was completely complexed with pEGFP-N1 from 1: 8 (pEGFP-N1: AEMA) based on pEGFP-N1 and low molecular weight chitosan (LMWC ) 5 K was found to form a complex in 1: 2 (pEGFP-N1: LMWC) (see FIG. 4). In addition, the AEMA-LMWC complex of the present invention has a higher positive charge than the conventional low molecular weight chitosan due to the introduction of AEMA, thereby improving the gene binding ability, and thus it is possible to bind to pDNA from 1: 0.5 (pEGFP-N1: AEMA-LMWC complex) (See FIG. 4).

In addition, the present inventors measured the particle size and surface charge of the complex of pEGFP-N1 bound to AEMA-LMWC. As a result, it was found that low molecular weight chitosan (LMWC) and 2-aminoethyl methacryl The size of AEMA could not be measured and the particle size of pEGFP-N1 / AEMA-LMWC complex with plasmid DNA (pDNA) was 1: 4, 1: 8, 1:16 Respectively, of 203.9, 341.9 and 382.8 nm (see Fig. 5A). The surface charge of pEGFP-N1 / AEMA, pEGFP-N1 / LMWC5K and pEGFP-N1 / AEMA-LMWC complexes was measured. As a result, 2-aminoethyl methacrylate (AEMA) and low molecular weight chitosan Showed negative charge at all ratios as a result of failure to form nanoparticles. However, the pEGFP-N1 / AEMA-LMWC complex showed higher charge than the low molecular weight chitosan (LMWC) due to the introduction of 2-aminoethyl methacrylate (AEMA). As the amount of AEMA-LMWC complex increased, (See FIG. 5B).

The present inventors also measured the cytotoxicity of 2-aminoethyl methacrylate (AEMA), low molecular weight chitosan (LMWC) and the AEMA-LMWC complex prepared above. As a result, 2-aminoethyl methacrylate (AEMA) As the concentration increased, the toxicity increased but the low molecular weight chitosan (LMWC) 5 K and AEMA-LMWC complexes did not show toxicity (see FIG. 6).

In order to confirm the gene transfer efficiency of the complex comprising pEGFP-N1 bound to AEMA-LMWC, transfection experiments were performed in vitro. As a result, it was confirmed that the control group pEGFP-N1 / AEMA had no gene transfer and pEGFP-N1 / LMWC 5 K was almost not. On the other hand, the pEGFP-N1 / AEMA-LMWC complex showed gene transfer efficiency from 1: 2, and the gene transfer efficiency was increased as the amount of AEMA-LMWC was increased (see FIG.

In addition, the present inventors measured the amount of the conjugate (pEGFP-N1 / AEMA-LMWC) conjugated with pEGFP-N1 to AEMA-LMWC by using a FACS caliber flow cytometer. As a result, in the case of cells treated with untreated cells and pEGFP-N1 alone, there was almost no change in the graph, but in the cells treated with the gene transfer pEGFP-N1 / AEMA-LMWC complex, (See Fig. 8). In particular, as the ratio of AEMA-LMWC increased, the number of pEGFP-N1 / AEMA-LMWC complexes increased as the complex formation ratio increased to 1: 2, 1: It was confirmed that the gene transfer efficiency was increased (see FIG. 8).

Therefore, the AEMA-LMWC composite in which 2-aminoethyl methacrylate (AEMA) is introduced into a low molecular weight chitosan (LMWC), which is a biocompatible material using the irradiation technique of the present invention, The AEMA-LMWC complex of the present invention can be applied as a gene delivery vehicle by confirming that it has a higher positive charge than the existing chitosan and has excellent gene transferring ability and no cytotoxicity.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples.

However, the following examples and experimental examples are intended to illustrate the present invention without limiting the scope of the present invention.

< Example 1 > 2-aminoethyl methacrylate (2- aminoethyl 메록acrylate ; AEMA ) Was introduced Low molecular weight Chitosan ( Low molecular weight chitosan ; LMWC ) Preparation of complex

In order to introduce 10% of 2-aminoethyl methacrylate (AEMA) into 5-kDa low molecular weight chitosan (LMWC), the present inventors used 100 mg of low molecular weight chitosan and 10 mg of 2-aminoethyl methacrylate, and one of the vials were loaded in both the (vial), producing a radiation dose by complex above ⁶⁰ Co crew gamma rays in three sample vial to (ACEL type C-1882, korea Atomic Energy Institute) was dissolved in MeOH 1 mL Were measured at dose rates of 10 kGy / hr, 5, 10 and 25 kGy, respectively. After the irradiation, the membrane was dialyzed for 24 hours using a membrane (molecular weight cut off, MWCO 1 KDa) to remove unreacted materials, followed by lyophilization for 3 days to prepare powdered gene transfer AEMA-LMWC complex (Fig. 1).

< Experimental Example 1 > 2- Aminoethyl methacrylate (AEMA) Introduced Low molecular weight Chitosan ( LMWC ) Identification of the structure of the complex

The present inventors conducted the following experiment to analyze the structure of the AEMA-LMWC composite prepared in Example 1 above.

Specifically, structural analysis was carried out through ¹ H-NMR (Bruker AVANCE 400, Germany) spectra. First, for the ¹ H-NMR analysis of 2-aminoethyl methacrylate (AEMA), a low molecular weight chitosan (LMWC), and the <Example 1> A AEMA-LMWC conjugate (a review of composite 5, 10, and 25 kGy prepared from ) Were each dissolved in 0.5 mL of D ₂ O as an NMR solvent and subjected to qualitative analysis.

As a result, as shown in FIG. 2, the C-1 peak at 4.9 ppm in the LMWC 5 K spectrum shifted to 4.7 ppm in the AEMA-LMWC composite spectrum irradiated with 5 kGy, and the C-2 peak at 3.1 ppm (Fig. 2A). &Lt; tb >< TABLE > In addition, the synthesis was confirmed through the AEMA-LMWC complex in which the 3.8 ppm peak not present in the LMWC spectrum and the 1.3 ppm peak in the AEMA characteristic peak were irradiated at 5 kGy (Fig. 2a). In addition, it was confirmed that the introduction efficiency of AEMA is lowered as irradiation dose increases (FIG. 2B).

< Experimental Example 2> 2- Aminoethyl methacrylate (AEMA) Introduced Low molecular weight Chitosan ( LMWC ) Confirm the molecular weight of the complex

The present inventors conducted the following experiments to confirm the molecular weight of the AEMA-LMWC complex prepared in Example 1 above.

Specifically, 2 mg of each of the low molecular weight chitosan (LMWC) and the AEMA-LMWC composite sample for each of the irradiation amounts (5, 10, and 25 kGy) prepared in Example 1 was dissolved in 0.1 M AMMONIUM ACETATE was dissolved in 1 ml of the sample, and the sample was filtered through a 0.2 ㎛ filter and then injected at 250 ㎛. At this time, the flow rate was 0.5 ml / min and the column size was 8.0 x 900 (mm) ID x Length. Plulan was used as a reference material.

As a result, as shown in Fig. 3, it was confirmed that the low molecular weight chitosan (LMWC) had a molecular weight of 5486. The AEMA-LMWC composite had a molecular weight of 7507 when irradiated with 5 kGy of gamma rays, It was confirmed that AEMA was introduced at 30% (FIG. 3). On the other hand, when 10 kGy and 25 kGy were irradiated, they showed molecular weights of 7457 and 7170, respectively, and it was confirmed that the efficiency decreased as the dose increased (FIG. 3).

Therefore, it was confirmed through NMR and GPC data of <Experimental Example 1> and <Experimental Example 2> that the efficiency of the AEMA-LMWC complex was highest when the dose was 5 kGy, AEMA-LMWC complex was used.

< Experimental Example 3> AEMA - LMWC The plasmid of the complex DNA ( pDNA ) Confirm the ability to bond with

The present inventors conducted the following experiments to confirm the binding ability of the AEMA-LMWC complex prepared in Example 1 with the plasmid DNA (pDNA).

Specifically, 5 K of 2-aminoethyl methacrylate (AEMA) and low molecular weight chitosan (LMWC) was used as a control. In order to confirm the complex forming ability with plasmid DNA pEGFP-N1, pEGFP- : 0.1 to 1:10 for 30 minutes. Then, 6X DNA loading dye was added thereto, and the resultant was loaded on 0.7% agarose gel and electrophoresed at 100 mV for 60 minutes.

As a result, as shown in FIG. 4, 2-aminoethyl methacrylate (AEMA) as a control group was completely complexed with pEGFP-N1 from 1: 8 (pEGFP-N1: AEMA) based on pEGFP- Molecular weight chitosan (LMWC) 5 K was found to form a complex in 1: 2 (pEGFP-N1: LMWC) (Fig. 4). In addition, the AEMA-LMWC complex of the present invention has a higher positive charge than the conventional low molecular weight chitosan due to the introduction of AEMA, thereby improving the gene binding ability, and thus it is possible to bind to pDNA from 1: 0.5 (pEGFP-N1: AEMA-LMWC complex) (Fig. 4).

< Experimental Example 4> pEGFP - N1 / AEMA - LMWC The particle size of the complex ( Particle you ) And surface charge (zeta potential )Confirm

The present inventors conducted the following experiment to measure the particle size and surface charge of the complex in which pEGFP-N1 was bound to AEMA-LMWC in <Experimental Example 3>.

Specifically, using a dynamic light scattering method using an ELS-8000 electrophoretic DLS spectrophotometer (He-Ne laser beam at a wave length of 632.8 nm at 25 ° C (scattering angle of 90 °), Otsuka electronics Inc., Japan) Were measured. (AEMA), low molecular weight chitosan (LMWC), and AEMA-LMWC complexes were weighed in a weight ratio of 1: 4, 1: 8, 1:16 based on 4 ㎍ of pEGFP-N1 After forming, the total volume was adjusted to 250 ㎕ and reacted for 30 minutes and then measured in a quartz cell (HE. 013.016-ALU cell adapter).

As a result, as shown in FIG. 5, the sizes of the low molecular weight chitosan (LMWC) and 2-aminoethyl methacrylate (AEMA), which can not form nanoparticles by themselves, could not be measured, and plasmid DNA The particle sizes of the pEGFP-N1 / AEMA-LMWC complexes were measured at 1: 4, 1: 8, and 1:16 on the basis of pDNA, respectively, and found to be 203.9, 341.9, and 382.8 nm, respectively (FIG.

The surface charge of pEGFP-N1 / AEMA, pEGFP-N1 / LMWC5K and pEGFP-N1 / AEMA-LMWC complexes was measured. As a result, 2-aminoethyl methacrylate (AEMA) and low molecular weight chitosan Showed negative charge at all ratios as a result of failure to form nanoparticles. However, the pEGFP-N1 / AEMA-LMWC complex showed higher charge than the low molecular weight chitosan (LMWC) due to the introduction of 2-aminoethyl methacrylate (AEMA). As the amount of AEMA-LMWC complex increased, (Fig. 5B).

< Experimental Example 5> in vitro ( In vitro )in AEMA - LMWC To determine the cytotoxicity of the complex

The present inventors conducted CCK assays in vitro to measure the cytotoxicity of 2-aminoethyl methacrylate (AEMA), low molecular weight chitosan (LMWC) and the AEMA-LMWC complex prepared in Example 1 above.

Specifically, L929 cells used in the experiments were cultured in DMEM medium containing 10% FBS in an incubator of 5% CO ₂ and 37 ° C environment. The cultured cells were plated at a density of 1 × 10 ³ cells / well in 96 wells and cultured in an incubator for 24 hours to stably attach the cells. After the cells were adhered, 100 μg of each of 2-aminoethyl methacrylate (AEMA), low molecular weight chitosan (LMWC) and the AEMA-LMWC composite sample prepared in Example 1 was dissolved in the medium, 1, and then cultured in an incubator for 48 hours. After incubation, the CCK solution was added to each well and the absorbance was measured at 450 nm using an ELISA instrument.

As a result, as shown in FIG. 6, 2-aminoethyl methacrylate (AEMA) showed an increase in toxicity with increasing concentration, but the low molecular weight chitosan (LMWC) 5 K and AEMA-LMWC complex showed no toxicity (Fig. 6).

< Experimental Example 6> in vitro ( In vitro )in pEGFP - N1 / AEMA - LMWC Confirm gene transfer efficiency of the complex

In order to confirm the gene transfer efficiency of the pEGFP-N1-conjugated complex of AEMA-LMWC in the above Experimental Example 3, transfection experiments were performed in vitro.

Specifically, the HCT119 cells used in the experiments were cultured in an incubator of 5% CO ₂ and 37 ° C environment using RPMI-1640 medium containing 10% FBS, respectively. Cells were cultured in an incubator for 24 hours at a density of 5 × 10 ⁴ cells / well in 24 wells. After the cells were adhered, the existing medium was removed, and a solution in which pEGFP-N1 and AEMA-LMWC were complexed with pEGFP-N1 on the basis of 1: 2 to 1:20, and pEGFP-N1 and 2-aminoethyl A solution of methacrylate (AEMA) or low molecular weight chitosan (LMWC) at a ratio of 1:16 was added to each well and incubated in an incubator for 10 hours. After 10 hours, the complex solution was removed and the medium containing 10% FBS and antibiotics was re-introduced and observed for 48 hours using a fluorescence microscope.

As a result, as shown in Fig. 7, it was confirmed that pEGFP-N1 / AEMA, which is a control group, had no gene transfer and pEGFP-N1 / LMWC 5K was almost not. On the other hand, the pEGFP-N1 / AEMA-LMWC complex showed gene transfer efficiency from 1: 2, and the gene transfer efficiency was increased as the amount of AEMA-LMWC was increased (FIG.

< Experimental Example 7> pEGFP - N1 / AEMA - LMWC Composite FACS Measure

The present inventors measured the amount of the conjugate (pEGFP-N1 / AEMA-LMWC) conjugated with pEGFP-N1 to AEMA-LMWC in the <Experimental Example 3> using a FACS caliber flow cytometer .

Specifically, AEMA-LMWC was reacted with FNR675 dye for 30 minutes and then complexed with pEGFP-N1 gene and treated in a 6-well plate. After incubation for 4 hours in an incubator, the cells in each well were washed with PBS to remove the dye and medium, and the cells were detached from the wells and dispersed in FACS solvent.

As a result, as shown in Fig. 8, there was almost no change in the graph of the untreated cells and the cells treated with pEGFP-N1 only, but in the cells treated with the gene transfer pEGFP-N1 / AEMA-LMWC complex Confirming that the graph moves (FIG. 8). In particular, as the ratio of AEMA-LMWC increased, the number of pEGFP-N1 / AEMA-LMWC complexes increased as the complex formation ratio increased to 1: 2, 1: And the gene transfer efficiency was increased (FIG. 8).

Claims

1) mixing chitosan and 2-aminoethyl methacrylate (AEMA);
2) irradiating the mixture of step 1) with radiation;
Aminoethyl methacrylate is introduced and grafted with the amino group of the 2-aminoethyl methacrylate introduced into the chitosan complex.

The method of claim 1, wherein the chitosan in step 1) is a low molecular weight chitosan having a number average molecular weight of 1,000 to 50,000 Da.

The method of claim 1, wherein the chitosan and the 2-aminoethyl methacrylate are dissolved in distilled water and methanol, respectively, and then mixed in the step 1).

The method of claim 1, wherein the radiation of step 2) is one selected from the group consisting of gamma ray, electron beam, ion beam, neutron beam and ultraviolet ray.

The method according to claim 1, wherein the irradiation dose of the step 2) is 5 to 25 kGy.

Aminoethyl methacrylate is bonded and the amino group of the 2-aminoethyl methacrylate is exposed.

A chitosan complex of claim 6; And
Nucleic acids;
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The method of claim 7, wherein the nucleic acid is selected from the group consisting of plasmid DNA, ribonucleic acid (RNA), small interfering ribonucleic acid (siRNA), antisense oligonucleotide, microRNA, Wherein the nucleic acid is at least one selected from the group consisting of nucleic acid (locked nucleic acid) and nucleic acid aptamer.

8. The drug delivery vehicle of claim 7, wherein the drug delivery vehicle has a formulation of liposomes, micelles, emulsions or nanoparticles.

A drug delivery system of claim 7; And
A pharmaceutically acceptable carrier;
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