KR20050097189A - Gene delivery system by urocanic acid-modified chitosan - Google Patents

Abstract

The present invention relates to chitosan derivatives (UAC) modified with urocanic acid with imidazoling as gene carriers.

In the present invention, UAC has a surface charge value larger than that of chitosan itself, thereby reducing the particle size of the complex. UAC / DNA complexes are not toxic, have high transduction efficiency, and have low transfection efficiency. complementary to improve the genetic balhyeonryul and excellent biocompatibility.

Description

Gene delivery system using chitosan derivatives having imidazole ring {Gene Delivery system by Urocanic Acid-Modified Chitosan}

The present invention relates to chitosan derivatives as new gene carriers.

Gene therapy involves injecting new genes or genetic material created by DNA recombination into a patient's cell to correct genetic defects or add new functions to cells to cause cancer, infectious diseases, and It refers to a method of preventing or treating all genetic defects such as immune diseases. Recently, the concept of gene therapy has been expanded and applied to the treatment of acquired diseases such as AIDS caused by human immunodeficiency virus (HIV) infection as well as congenital genetic diseases transmitted to offspring.

These gene therapy methods include direct injection of proteins, drug therapy to help or inhibit the expression of specific genes in the body, and injection of DNA with genetic information into the body to express the injected gene. However, direct protein injection and drug therapy have the risk of causing an immune response to foreign substances, the need for continuous infusion and expensive production costs. To overcome these problems with protein drugs, the idea was to use genes. DNA has the ability to produce proteins in vivo. Therefore, the basic concept of gene therapy is to inject DNA in vivo in the method of injecting the existing protein directly, so that the injected DNA continuously produces the protein.

There are some issues that need to be addressed before this new level of treatment can be used as a common and effective treatment in many medical institutions. Once you have a gene with a genetic code that can produce some specific protein. Next, there must be a gene expression system called a plasmid that can express the gene. Finally, there is a need for a carrier that can safely transport an expression containing a specific gene to a desired location. Since DNA is susceptible to hydrolysis in vivo and has low efficiency of entering into cells, many researches on gene delivery systems have been actively conducted to solve these problems and to apply gene therapy.

Until recently, viral vectors with high transfection efficiency have been used as gene carriers in clinical trials. However, this is not only complicated in the manufacturing process, but also limited in safety issues such as immunogenicity, infectivity, inflammation, non-specific DNA insertion and the size of the acceptable DNA is limited to apply to humans. Therefore, non-viral vectors are currently in the spotlight as substitutes for viral vectors.

Nonviral vectors have the advantage of being able to be repeatedly administered with a minimum of immune response, capable of specific delivery to specific cells, being stable for storage, and easy to produce large quantities. It is mainly used as a positive liposome-based liposome-based N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride { N- [1- (2,3 -dioleyloxy) propyl] -N, N, N -trimethylammonium chloride (DOTMA)}, alkylammonium, cationic cholesterol derivatives, gramicidin, etc. Although it has been reported to enter the cell membrane, a complicated manufacturing process, low inclusion rate of DNA having a large molecular weight, decrease in transfection activity by serum components on in vivo ( in vivo) , particle size and the like has been a problem. In addition, lipofectin, which is currently used most, is also a cytotoxic problem.

Among non-viral vectors, cationic polymers have attracted a lot of attention because they can form complexes with negatively charged DNA and ionic bonds. These include poly-L-lysine (PLL), poly (4-hydroxy-L-proline ester), polyethyleneimine (PEI), poly [α- (4-aminobutyl) -L-glycolic acid], polyamidoamine Dendrimers, poly [N, N '-(dimethylamino) ethyl] methacrylate (PDMAEMA), which compress DNA to form nanoparticles, protecting DNA from enzymatic degradation, and rapidly penetrating into cells to endo Help get out of the moth. However, most non-viral gene carriers have lower immunorejection response and cytotoxicity than viral gene carriers, but the gene expression rate is relatively low. This is the most important problem.

Chitosan is also a material that offers ample potential as a nonviral gene carrier because of its biocompatibility, biodegradability, low toxicity and high cationicity. However, due to the low transfection efficiency, there are many difficulties in practical applications. For efficient gene expression, there are several steps to be taken. For effective penetration into cells, complexes formed by binding to DNA must be condensed down to a size of 150 nm, prevent DNA degradation from various enzymes, enter the cell, and the complex must exit the endosomes well, and the cell nucleus It must enter and dissociate the DNA from the carrier. (M. Koeping-Hoeggaerd et. Al. (2001) "Chitosan as a nonviral gene delivery system.Structure-property relationships and characteristics compared with polyethylenimine in vitro and after lung administration in vivo" Gene Therapy 8: 1108-1121)

Current studies at home and abroad report that the low release rate of the polymer / DNA complex from the endosome is the main reason for the low gene expression rate. To solve this problem, chloroquine (E. Wagner (1998) "Effects of membrane-active agents in gene delivery" J. Control Rel. 53: 155-158), which may lead to instability of the endosomal membrane. Or inactivated adenoviruses (E. Wagner et. Al. (1992) "Coupling of adenovirus to transferrin-polylysine / DNA complexes greatly enhances receptor-mediated gene delivery and expression of transfected genes" Proc. Natl. Acad. Sci. 88 : 6099-6103) or by adding GALA or KALA, peptides that destroy endosomes, induce endothelial membrane instability due to pH change and try to release DNA complexes quickly. It is becoming. However, due to cytotoxicity, immunogenicity and other side effects, it is not suitable for actual clinical application. In addition, PEI is known to exhibit high gene expression in cationic polymers, which is known to release PEI / DNA complexes rapidly from the endosome and enter the cell nucleus because PEI is hydrogenated to produce a proton sponge effect. (WT Godbey et. Al. (1999) "Polyethylenimine and its role in gene delivery" J. Control Rel. 60 (1999) 149-160. However, PEI is also highly cytotoxic and not biodegradable and therefore has practical application in clinical practice. Also, recently, it has been known that polymers such as histidine with imidazoling act as proton sponges such as PEI to help release them into the cytoplasm. The imidazole ring of histidine has a pKa value of about 6.15, which is a protein constituent unit and has excellent biocompatibility. (D. W. Pack et. Al. (2000) "Design of imidazole-containing endosomolytic biopolymers for gene delivery" Biotechnology and Bioengineering 67 217-223).

It is an object of the present invention to provide a novel gene delivery vehicle that can enhance the gene expression rate by inducing the rapid release from the endosomes by supplementing the shortcomings of chitosan, which is nontoxic and excellent in biocompatibility but low in transfection efficiency.

The present invention relates to chitosan derivatives modified with urocanic acid having imidazoling as gene carriers. More specifically, the present invention relates to a polymer compound comprising the following general formula (1) as a main repeating unit.

(Where x> n and x is an integer greater than or equal to 1)

Preferably, the polymer compound has a molecular weight of 5K or more, and may be used up to about 1,000K.

In the present invention, the compound is a polymer in which urocanic acid having an imidazole ring is bonded to chitosan (hereinafter referred to as the polymer compound of the present invention or UAC).

The present invention also relates to a complex of chitosan derivatives represented by Chemical Formula 1 and DNA. The complex is an ionic bond product of chitosan derivatives and DNA.

 Hereinafter, the carrier and the complex of the present invention will be described in more detail.

The gene carrier of the present invention of Formula 1 is a natural polymer, nontoxic and excellent biocompatibility of the positive polysaccharide chitosan, by binding the urokanoic acid having an imidazole ring to help release quickly from the endosome to the cytosol It is a cationic high molecular compound prepared to increase the efficiency of transfection. This reaction was bound via active ester intermediate in the presence of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC).

When the UAC of the present invention is mixed with DNA in an aqueous solution, it forms a cooperative bond, that is, a complex between the UAC of the present invention, which is a cationic polymer, and the DNA, which is anionic polymer, and an electrostatically complementary chain.

Another aspect of the present invention is a therapeutic agent for genetic diseases comprising the complex as an active ingredient. Applications of gene therapy include cancer, acquired immunodeficiency syndrome, atopy, diabetes, osteoporosis, and other immune related diseases. It is also widely used as a cytokine therapy and DNA vaccine.

Hereinafter, the present invention will be described in detail with reference to Examples, which are merely illustrative of the present invention and do not limit the scope of the present invention.

Example 1 Preparation of Chitosan Derivatives (UAC) Containing Urocanoic Acid

50 K of water-soluble chitosan was used, and urokanoic acid (UA) (mol%: 20, 50, 70) was purified using EDC [1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride]. Bound to chitosan. The carboxyl group of UA was activated by N-hydroxysuccinimide (NHS) / EDC in 10 ml of 2-Morphoholinoethanesulfonic acid (MES) buffer solution. The dissolved solution was poured into a solution of 0.5 g of chitosan and reacted at room temperature for 24 hours. Thereafter, the reaction was terminated by addition of hydroxylamine, and the pH was adjusted to 8 or more by addition of NaOH. Thus, all unreacted UA was removed and a white precipitate was obtained. The product was dialyzed through a dialysis membrane (Spectra / Por membrane; MWCO = 12,000-14,000) for 4 days, and the precipitate was lyophilized by centrifugation with washing with water several times, and the dried product was again dried in 0.1M acetic acid. Dissolved. Each UAC substitution value was calculated by ninhydrin assay, which was reconfirmed by NMR measurement. The combined urokanoic acid accounts for 16, 27.7 and 36.3 mol% of the total and is named UAC20, UAC50 and UAC70 respectively. 1 shows the ¹ H-NMR spectrum of UAC70.

Example 2 Complexation of UAC with DNA

To calculate the charge ratio, DNA was calculated as one charge per molecular weight of 330, and UAC was calculated as two charges per repeat unit of chitosan, including urokanoic acid. DNA was allowed to fluoresce using ethidium bromide. UAC and DNA [plasmin Enhanced Green Fluorescent Protein (pEGFP) (0.1 μg) prepared in Example 1 were spontaneously complexed for 30 minutes at various charge ratios in 10 mM phosphate buffer (PBS, pH 7.4). Afterwards, the formation of the complex was confirmed by electrophoresis at 100 V for 40 minutes in a 1% agarose gel using a Tris-acetate (TAE) running buffer.The results are shown in Fig. 2. As the substitution value of UA increases, It appears to form more complex well, and the charge ratio appears to form a complete complex when UAC (N) / DNA (P) is 5 or more.

Experimental Example 1 Inhibition of DNA Degradation from Deoxyribonuclease (DNase I) of UAC / DNA Complex

DNase I was added to the complex formed in Example 2, 2 units per 0.1 g of DNA was reacted at 37 ° C. for 30 minutes, and the results are shown in FIG. 3. UAC20 also confirmed that the DNA remained undecomposed from the charge ratio 3, which completely formed the complex, indicating that UAC plays a role in protecting DNA from enzymes.

Experimental Example 2: Particle Size and Particle Size Distribution of the UAC / DNA Complex>

UAC / DNA was prepared at various charge ratios as in the preparation method, and measured by an electrophoretic light scattering system (ELS 8000, Otsuka Electronics, Ltd., Osaka, Japan), and the results are shown in Table 1 and FIG. 4.

The chitosan derivative substituted with UA showed smaller particle size than the chitosan alone, and the size had a value around 100 nm. The particle size distribution is evenly distributed by showing one sharp peak. It is well suited to the requirement that the particle size be less than 150 nm for rapid entry into the cell.

In addition, a UAC / DNA complex was prepared at a charge ratio of 20, as in the above preparation method, dropped on a copper grid, and stained with 1% of uranyl acetate for 10 seconds to transmit electron microscopy. The shape was observed by. The results are shown in FIG. As shown in Fig. 5, the shape is spherical and the particle size is similar to or slightly smaller than the value shown in Table 1. It satisfies the condition that the particle size should be 150 nm or less for rapid entry into cells.

Experimental Example 3: Surface Charge of Particles of UAC / DNA Complex

Surface charge was also used the same electrophoretic light scattering system used in Experimental Example 2, the results are shown in FIG. The larger the UAC / DNA charge ratio was, the stronger the positive charge was, and the larger the substitution value of UA was, the more positive the charge was, which was due to the UA having a lower value than the pKa value of chitosan itself. Is estimated. In addition, the positive charge of the surface charge means that the negatively charged DNA is completely stacked in the complex, and the result is the same as the result shown in Experimental Example 1. In addition, the slight positive charge not only facilitates the inflow into the cell, but also has the advantage of reducing the aggregation phenomenon between the particles by causing the electrostatic repulsion between the particles.

Experimental Example 4: Cytotoxicity Test of UAC

HeLa and 293T cell lines were each dispensed in 96 well-plates at a concentration of 2 × 10 ⁴ per well and grown for 24 hours using Dulbecco's Modified Eagle's Minimum essential medium (DMEM) medium. The chitosan and the UAC prepared in Example 1 were treated for 48 hours at different concentrations ^. Aqueous daily solution cell division assay [CellTiter 96 ^? Aqueous One Solution Cell Proliferation Assay (Promega, Madison, USA)] was used to determine the cytotoxicity. Cytotoxicity results for 293T are shown in FIG. 7. UAC was better for cell activity than chitosan itself, and the cell activity was better with increasing UA substitution value. This proves that UA is a biocompatible material.

Experimental Example 5: Transfection Efficiency of UAC / DNA Complexes

293 T cells were dispensed in 24 well-plates at 1 × 10 ⁵ cells per well and grown in DMEM medium containing 10% fetal bovine serum (FBS) for 24 hours at 37 ° C. and 5% carbon dioxide. Then, in the same manner as in Example 2 to form a complex with luciferase (luciferase) marker gene (pGL3-Control) and put in each well with a medium without FBS. After incubation for 6 hours at 37 ° C. and 5% carbon dioxide, the medium is removed and placed in DMEM medium containing fresh 10% FBS and incubated at 37 ° C. and 5% carbon dioxide for 48 hours. After the medium was removed, 100 μl of cell lysis buffer was added thereto and left at 20 ° C. for 24 hours. After one day, it is dissolved at room temperature and then transferred to an e-tube in each well and centrifuged for 15 minutes at 10000 rpm to obtain only the supernatant. The obtained supernatant was measured for activity using a luciferase assay kit. (In the figure, RLU stands for 'relative light unit') The results are shown in FIG. As can be seen, as the substitution value of the UA increases, it can be seen that the gene expression rate is greatly increased, and compared with chitosan itself, the efficiency was increased more than 1000 times.

Experimental Example 6: Observation of Confocal Laser Scanning Microscope of UAC / DNA Complex>

A 1 cm diameter film is placed on a 24 well plate and 293 T cells are dispensed at 3 × 10 ⁵ cells per well, and the sample is treated in the same manner as in Experiment 4. Here, pEGFP N1, a green fluorescent protein marker gene, was used to form a complex with UAC50. Six hours after the sample treatment, the cells were replaced with DMEM medium containing 10% FBS, and after 24 hours, the cells were fixed with 3.7% formalin, stained with nuclei with propidium iodide (PI), and mounted on slice glass. Confocal laser scanning microscope was used. The results are shown in FIG. 9, and it can be seen that the green protein is expressed around the nucleus. This corresponds to the result shown in Experimental Example 4.

In the present invention, UAC has a surface charge value larger than that of chitosan itself, thereby reducing the particle size of the complex. UAC / DNA complexes are not toxic, have high transduction efficiency, and have low transfection efficiency. complementary to improve the genetic balhyeonryul and excellent biocompatibility.

1 is a ¹ H-NMR spectrum of a chitosan derivative (UAC) with an imidazole ring.

2 is an electrophoretic image of UAC / DNA (pEGFP N1) complexes according to DNA and various charge ratios.

Figure 3 is a Salping electrophoresis picture of the defense ability from DNase I of the UAC / DNA complex.

Figure 4 is a graph of the particle size distribution of the UAC70 / DNA complex at the charge ratio 10.

5 is a photograph of a UAC50 / DNA complex at a charge ratio of 20 observed with a transmission electron microscope.

Figure 6 is a graph of the surface charge (zeta-potential) of chitosan and UAC / DNA complex according to various charge ratios.

7 is a graph of cytotoxicity of 293 T cells of chitosan, UAC and polyethyleneimine (PEI).

8 is a graph showing the transfection efficiency of 293 T cells of UAC / DNA (pGL3-Control) complexes according to various charge ratios.

9 is a confocal laser scanning micrograph of 293 T cells of the UAC50 / DNA (pEGFP N1) (charge ratio 20) complex.

Claims (4)

A polymeric compound comprising the following formula as a major repeating unit: Wherein x> n and x is an integer of 1 or more. Gene delivery system comprising the polymer compound of claim 1. A complex of the polymer compound of claim 1 with DNA A therapeutic agent for genetic diseases comprising the complex of claim 3 as an active ingredient.