KR20110027969A - Method of manufacturing polymer for delivering nucleic acids and compositions for delivering nucleic acids comprising the polymer - Google Patents

Abstract

PURPOSE: A method for preparing polymers for nucleic acid delivery is provided to transfer nucleic acid through injection or nasal cavity. CONSTITUTION: A polymer for nucleic acid delivery is prepared by binding hyaluronic acid and disulfide bond-introduced polyethyleneimine. A method for preparing the polymers comprises: a step of adding 1-ethyl-3- [3-(dimethylamino)-propyl]carbodiimide, N, N'-dicyclohexylcarbodiimide(DCC), 1-hydroxy bentriazole monohydrate(HOBt), or N-hydroxysuccinimide(NHS) compound to hyaluronic acid; and a step of binding polyethylene imine in disulfide bond is introduce. A composition for nucleic acid delivery contains polymer of chemical formula 1 or 2.

Description

Method for manufacturing polymer for delivering nucleic acids and compositions for delivering nucleic acids comprising the polymer

The present invention relates to a method for producing a nucleic acid delivery polymer and a composition for delivering a nucleic acid containing the polymer, and in particular, a method for producing a nucleic acid delivery polymer that effectively delivers nucleic acids into cells and has little toxicity and containing the polymer. A composition for nucleic acid delivery.

Small interfering ribonucleic acid (siRNA), antisense oligonucleic acids, plasmid deoxyribonucleic acid and the like have recently gained industrial importance as a medicine for nucleic acid materials. In the case of nucleic acid materials, efficient delivery to cells or tissues is the first task to be developed as a pharmaceutical.

For intracellular delivery of nucleic acid material, viral vectors or nonviral vectors using polymers or nanoparticles have been studied. Among these, viral vectors have the advantage of having higher gene transfer efficiency than nonviral vectors, but their application to humans is very limited due to safety issues when using viruses. Thus, development of non-viral vectors is underway as a safer alternative than viral vectors.

Currently, cationic polymers such as cationic liposomes, TAT peptides, or polyethylenimine (PEI) are widely used as nucleic acid materials, particularly siRNA carriers. In particular, polyimenimine (PEI), a cationic polymer, compresses nucleic acid material having a negative charge to make colloidal particles, and has a pH buffering capacity in lysosomes in cells to convert plasmid deoxyribonucleic acid into various cells. Has been reported (Boussif et al., Proc. Natl. Acad. Sci. USA 92 (1995) 7297-7301; Godbey et al., J. Controlled Release 60 (1999) 149-160).

However, the cationic polymer causes the cell aggregation in the body and electrostatically binds to the blood protein, which decreases the activity of the siRNA, and it has been found to cause fatal toxicity to body organs such as the liver and lungs. It is known to be difficult to use. In particular, in the use of polyethyleneimine, problems related to intracellular gene transfer efficiency and cytotoxicity still remain, and many studies to solve this problem have been conducted. For example, to reduce the cytotoxicity of polyethyleneimine (PEI), many modifications have been made to dextran sulfate, human serum albumin, polyethylene glycol, and the like. Commonly showed lower gene transfer efficiency than PEI itself.

Therefore, a technique for efficiently delivering oligonucleotides such as small interfering ribonucleic acid and antisense oligonucleotide into the cell while having low cytotoxicity is a necessary technique in the art. On the other hand, hyaluronic acid is a linear polymer polysaccharide obtained by alternating β-DN-acetylglucosamine and β-D-glucuronic acid, which does not have species and organ specificity, and has excellent biocompatibility even when implanted or injected into a living body. It is known to represent.

The inventors of the present invention, while studying the technology for delivering nucleic acids efficiently into the cell with little cytotoxicity, confirming that the nucleic acid delivery polymer prepared by the present invention has little cytotoxicity and excellent in intracellular delivery efficiency. The present invention has been completed.

Therefore, an object of the present invention is to provide a method for preparing a nucleic acid delivery polymer comprising the step of binding a disulfide bond introduced polyethyleneimine (polyethyleneimine) and hyaluronic acid (hyaluronic acid).

Another object of the present invention is to provide a nucleic acid delivery composition comprising a nucleic acid delivery polymer represented by the following formula (1) or (2).

The present invention is also directed to a mixture of (a) hyaluronic acid and 3- (2-pyridyldithio) propionyl hydrazide (3- (2-pyridyldithio) propionyl hydrazide, PDPH) in 1-ethyl-3- [3- ( Dimethylamino) propyl] carbodiimide (1-ethyl-3- [3- (dimethylamino) propyl] carbodiimide (EDC) and N, N'-dicyclohexylcarbodiimide (DCC) Adding at least one compound to introduce a pyridyldisulfide group into hyaluronic acid;

(b) combining hyaluronic acid to which a pyridyldisulfide group is introduced in step (a) and a nucleic acid to which a thiol group is introduced;

(c) It is an object of the present invention to provide a method for producing a nucleic acid delivery complex comprising the step of forming a complex by adding a polyethyleneimine introduced disulfide bond (polyethyleneimine).

Another object of the present invention is to provide a nucleic acid delivery composition containing a compound represented by the following formula (3).

The present invention provides a method for preparing a nucleic acid delivery polymer comprising the step of binding a disulfide bond introduced polyethyleneimine (polyethyleneimine) and hyaluronic acid (hyaluronic acid).

In another aspect, the present invention provides a nucleic acid delivery composition comprising a polymer for nucleic acid delivery represented by the following formula (1) or (2).

<Formula 1>

In Chemical Formula 1

n and m are each independently an integer selected from 8 to 20;

p and q are each independently integers selected from 16 to 2500;

<Formula 2>

In Chemical Formula 2

n and m are each independently an integer selected from 8 to 20;

r is an integer selected from 26 to 2500.

The present invention is also directed to a mixture of (a) hyaluronic acid and 3- (2-pyridyldithio) propionyl hydrazide (3- (2-pyridyldithio) propionyl hydrazide, PDPH) in 1-ethyl-3- [3- ( Dimethylamino) propyl] carbodiimide (1-ethyl-3- [3- (dimethylamino) propyl] carbodiimide (EDC) and N, N'-dicyclohexylcarbodiimide (DCC) Adding at least one compound to introduce a pyridyldisulfide group into hyaluronic acid;

(b) combining hyaluronic acid to which a pyridyldisulfide group is introduced in step (a) and a nucleic acid to which a thiol group is introduced;

(C) provides a method for producing a nucleic acid delivery complex comprising the step of forming a complex by adding a polyethyleneimine introduced disulfide bond (polyethyleneimine).

The present invention also provides a nucleic acid delivery composition containing a compound represented by the following formula (3).

<Formula 3>

In Chemical Formula 3

p is an integer selected from 16 to 2500;

R is a pyridyl group.

Hereinafter, the present invention will be described in more detail.

In the present invention, polyethyleneimine (PEI) is a polymer obtained by polymerizing ethyleneimine, but the molecular weight of the polyethyleneimine may be preferably 100 Da to 20 kDa, and more preferably 800 Da to 10 kDa.

Polyethyleneimine (PEI-SS) in which disulfide bonds are introduced in the present invention is not limited thereto, and may be prepared by reacting the polyethyleneimine with cystamine bisacrylamide represented by the following Chemical Formula 4.

<Formula 4>

Specifically, the polyethyleneimine may be prepared by adding and reacting cystamine bisacrylamide (CBA) in methanol (see <Example 1-1>).

In the present invention, hyaluronic acid (HA) may be natural or synthetic hyaluronic acid, or a modification thereof, and refers to a linear polymer polysaccharide in which β-DN-acetylglucosamine and β-D-glucuronic acid are alternately bonded. . Although there is no restriction on the molecular weight of the hyaluronic acid, preferably 0.5 to 1,000 kDa may be used, but more preferably, 5 to 200 kD in molecular weight.

Nucleic acids in the present invention include, but are not limited to, nucleic acids intended for delivery into cells, but include deoxyribonucleotides or ribonucleotides and polymers thereof in single or double stranded form, preferably the nucleic acids have a small interference It may be ribonucleic acid (siRNA), antisense nucleic acid, nucleic acid aptamer, more preferably small interfering ribonucleic acid (siRNA), antisense nucleic acid or nucleic acid aptamer consisting of 5 to 200 base pairs. ), Most preferably siRNA.

Gene silencing in the present invention refers to a decrease in the degree of transcription of a gene targeted by a target nucleic acid sequence, ie RNAi, or a decrease in the amount or activity of a target sequence or protein.

In one aspect of the present invention, the method for producing a nucleic acid delivery polymer of the present invention is characterized in that it comprises a step of coupling a disulfide-linked polyethyleneimine (polyethyleneimine) and hyaluronic acid (hyaluronic acid).

The combination of polyethyleneimine (PEI-SS) and hyaluronic acid (HA) in which the disulfide bond is introduced may be performed through two kinds of steps as follows.

First, (a) 1-ethyl-3- [3- (dimethylamino) -propyl] carbodiimine (1-ethyl-3- [3- (dimethylamino) -propyl] carbodiimide, EDC) and N, in hyaluronic acid; 1-hydroxy bentriazole monohydrate (HOBt), N-hydroxysulfosuccinimide and at least one compound selected from the group consisting of N'-dicyclohexylcarbodiimide (DCC) Adding at least one compound selected from the group consisting of (N-hydroxysulfosuccinimide, sulfo-NHS) and N-hydroxysuccinimide (NHS), and

(b) preparing a polymer for nucleic acid delivery, including binding to polyethyleneimine into which disulfide bonds are introduced. That is, through the above step, it is possible to produce a nucleic acid delivery polymer by forming an amide bond between the amine group of PEI-SS and the carboxyl group of HA (see <Example 1-2>). Through the above process, the nucleic acid delivery polymer represented by Chemical Formula 1 may be prepared.

In addition, a polymer for nucleic acid delivery can be prepared by adding NaBH ₃ CN to polyethyleneimine and hyaluronic acid into which disulfide bonds are introduced.

More preferably, it may be prepared by dissolving NaBH ₃ CN and NaCl in a borate buffer and reacting with HA and PEI-SS. That is, through the above step, the HA terminal and the amine group of PEI-SS are covalently bonded through reductive amination (see <Example 1-3>). Through the above process, the nucleic acid delivery polymer represented by Formula 2 may be prepared.

On the other hand, the nucleic acid delivery composition of the present invention is characterized in that it comprises a nucleic acid delivery polymer represented by the formula (1) or (2).

The nucleic acid delivery composition may further include a nucleic acid to form a complex in which the nucleic acid delivery polymer and the nucleic acid are electrostatically coupled, thereby delivering the nucleic acid into a cell. In addition, since the disulfide bonds contained in the nucleic acid delivery polymer represented by Formula 1 or Formula 2 are easily degraded in cells, PEI can be broken down into PEI of low molecular weight, thereby reducing intracellular toxicity and containing HA. Biocompatibility is very good.

In one experimental example of the present invention, the cytotoxicity of the nucleic acid delivery polymer represented by Chemical Formula 1 or Chemical Formula 2 was tested, and it can be seen that there is little cytotoxicity compared to PEI (25kDa) having a high molecular weight (<experimental) See Example 2).

In an experimental example of the present invention, the gene silencing activity of the nucleic acid delivery polymer represented by Chemical Formula 1 or Chemical Formula 2 was evaluated by co-transfection. As a result, luciferase activity was effectively determined in a concentration-dependent manner. It can be seen that it can be suppressed (see <Experimental Example 3>).

In one aspect of the invention, the method for producing a nucleic acid delivery polymer of the present invention (a) hyaluronic acid and 3- (2-pyridyldithio) propionyl hydrazide (3- (2-pyridyldithio) propionyl hydrazide, PDPH 1-ethyl-3- [3- (dimethylamino) propyl] carbodiimide (1-DC-3- [3- (dimethylamino) propyl] carbodiimide, EDC) and N, N'-dicyclohex in a mixture of Introducing a pyridyldisulfide group to hyaluronic acid by adding at least one compound selected from the group consisting of dicyclohexylcarbodiimide (DCC);

(b) combining hyaluronic acid to which a pyridyldisulfide group is introduced in step (a) and a nucleic acid to which a thiol group is introduced;

(c) adding a polyethyleneimine introduced with disulfide bonds to form a complex.

The pyridyl disulfide group is introduced into the HA through step (a), and the HA into which the pyridyl disulfide group is introduced through step (b) is a nucleic acid into which a thiol group is introduced. Disulfide bonds are possible. Finally, the complex in which disulfide-conjugated HA and nucleic acid is disulfide-bonded through step (c) forms an electrostatic complex with PEI-SS. Through this step, a complex in which the compound represented by Chemical Formula 3 and the nucleic acid is disulfide-bonded is formed.

On the other hand, the nucleic acid delivery composition of the present invention is characterized in that it contains a compound represented by the formula (3) and a nucleic acid introduced with a thiol group (thiol group). The compound represented by Chemical Formula 3 is not limited thereto, but preferably 1- to a mixture of hyaluronic acid and 3- (2-pyridyldithio) propionyl hydrazide (3- (2-pyridyldithio) propionyl hydrazide (PDPH)). Ethyl-3- [3- (dimethylamino) propyl] carbodiimide (1-ethyl-3- [3- (dimethylamino) propyl] carbodiimide, EDC) and N, N'-dicyclohexylcarbodiimide (dicyclohexylcarbodiimide; It can be prepared by adding a pyridyl disulfide group to hyaluronic acid by adding at least one compound selected from the group consisting of DCC).

In addition, the composition contains a nucleic acid introduced with a thiol group prepared according to a method known to those skilled in the art, the disulfide bond with the compound represented by the formula (3) to form a nucleic acid delivery complex containing a nucleic acid Can be.

The composition may further contain PEI-SS, and may electrostatically bind to the complex in which the compound represented by Formula 3 and the nucleic acid into which the thiol group group is introduced are bound.

The complex represented by the compound represented by Formula 3 and the nucleic acid to which the thiol group group is introduced may include disulfide bonds to effectively transfer nucleic acids into cells, and disulfide bonds may be included in PEI-SS and thus PEI can be broken down to low molecular weight PEI because of its easy degradation. In addition, by containing HA, the biocompatibility is very excellent.

In one experimental example of the present invention, the compound represented by the formula (3) and a thiol group (thiol group) introduced nucleic acid is combined with the result of the co-transfection of the gene silencing activity of the complex of PEI-SS evaluation result It can be seen that the luciferase activity can be effectively inhibited in a concentration-dependent manner (see <Experimental Example 4>).

The method for preparing a nucleic acid delivery polymer of the present invention and the nucleic acid delivery composition containing the polymer can effectively deliver nucleic acids into cells and have little toxicity, and thus can be utilized as an effective delivery system for nucleic acids. Polyethyleneimine and hyaluronic acid, which are excellent in biocompatibility, may be contained to effectively deliver nucleic acids through injection formulations and nasal passages.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

< Example  1>

Disulfide  Polyethylenimine incorporating a bond ( PEI - SS ) And hyaluronic acid ( hyaluronic  acid, HA )this Combined  Preparation of Polymer for Nucleic Acid Delivery

<1-1> Disulfide  Polyethylenimine incorporating a bond ( PEI - SS ) Synthesis

The method for synthesizing polyethyleneimine in which disulfide bonds are introduced in the present invention is briefly described in FIG. 1.

As shown in FIG. 1, polyethyleneimine having a low molecular weight of 2000 Da was dissolved in 90% methanol with cystamine bisacrylamide (CBA) and reacted at 60 ° C. for 24 hours to induce disulfide bond polyethylene. Imine (polyethyleneimine) was synthesized. At this time, the molar ratio of polyethyleneimine and cystamine bisacrylamide was varied to 10: 1, 4: 1, 2: 1 to obtain a composite having different substitution rates. The obtained composite was filtered and purified using a filtration tube (cutoff 3000 Da) to prepare polyethyleneimine (PEI-SS) in which disulfide bonds were introduced.

Whether PEI-SS was synthesized as described above was confirmed by ¹ H NMR and gel permeation chromatography (GPC), and the ¹ H NMR results are shown in FIG. 2.

As shown in FIG. 2, when comparing the data before and after synthesis in the ¹ H NMR results, it can be seen that polyethyleneimine is synthesized, and the peak of cystamine bisacrylamide (3.4 ppm) and the peak of polyethyleneimine (2.5 ~ 2.9 ppm) The substitution rate can be known using the integral value of.

<1-2> PEI - SS of An amine group HA Carboxyl groups Combined HA -g- ( PEI - SS ) Synthesis

Through the process as described in FIG. 3, HA-g- (PEI-SS) was synthesized by combining disulfide-induced polyethyleneimine (PEI-SS) and hyaluronic acid (HA). That is, HA-g- (PEI-SS) was synthesized by forming an amide bond between the amine group of PEI-SS and the carboxyl group of HA.

Specifically, the carboxyl group of HA having a molecular weight of 130 kDa was converted to 1-ethyl-3- [3- (dimethylamino) -propyl] carbodiimine (1-ethyl-3- [3- () in a pH 6.5, 1M NaCl solution. dimethylamino) -propyl] carbodiimide (EDC) and 1-hydroxy bentriazole monohydrate (HOBt) and then activated with PEI-SS prepared in Example 1-1. At this time, the weight ratio of HA and PEI-SS was varied to 1: 2, 1: 5, and 1:20, respectively, to obtain a composite having different substitution rates. The resulting composite was filtered and purified using a filtration tube (cutoff 10 kDa) to prepare HA-g- (PEI-SS).

Whether HA-g- (PEI-SS) was synthesized as described above was confirmed using ¹ H NMR and the results are shown in FIG. 4.

As shown in FIG. 4, it can be observed that the PEI characteristic peak shifts to the left while HA-g- (PEI-SS) is synthesized in the ¹ H NMR result, and the substitution rate is in the HA characteristic peak region (1.9 ppm). It can be obtained by comparing the PEI-SS characteristic peak region (2.5 ~ 3.4ppm).

<1-3> PEI - SS of An amine group HA The end of Combined HA -b- ( PEI - SS ) Synthesis

As shown in FIG. 5, the HA terminal and the amine group of PEI-SS were covalently bonded through reductive amination.

Specifically, it was synthesized by dissolving NaBH ₃ CN and NaCl at a concentration of 0.3M and 1M in 0.1M borate buffer of pH 8.5, and reacting HA and PEI-SS for 3 days. After the synthesis, impurities were removed using a filtration tube (cutoff 10kDa) to synthesize HA-b- (PEI-SS).

Whether HA-b- (PEI-SS) was synthesized as described above was confirmed using ¹ H NMR and the results are shown in FIG. 6.

As shown in FIG. 6, the HA-b- (PEI-SS) was synthesized in the 1H NMR result, and the PEI characteristic peak was shifted to the left side, and the substitution rate was measured in the HA characteristic peak region (1.9 ppm) and It can obtain by comparing PEI-SS characteristic peak area (2.5-3.4 ppm).

< Experimental Example  1>

PEI - SS Wow HA end Combined  Polymer and siRNA The formation of complexes

SiRNA (Bionia, SEQ ID NO: 1 UUGUUUUGGAGCACGGAAA (dTdT)) and HA-g- (PEI-SS) prepared in <Example 1-2> in 150 mM aqueous sodium chloride solution or <Example 1 The complex was formed by reacting the HA-b- (PEI-SS) conjugate prepared in -3> at room temperature for 15 minutes. In this case, the concentration of siRNA and HA-g- (PEI-SS) or HA-b- (PEI-SS) was changed to examine the most effective reaction ratio, and the results are described in FIG. 7.

As shown in FIG. 7, it can be seen that siRNA and HA-g- (PEI-SS) or HA-b- (PEI-SS) can effectively form a complex. In particular, in the case of siRNA and HA-g- (PEI-SS), the complex is formed from a mass ratio of 1: 4 or more, and particularly, when the mass ratio is 1:15, it can be seen that the complex is most effectively formed. In addition, in the case of siRNA and HA-b- (PEI-SS), it was found that the complex was formed from 1: 2 or 1: 4 or more.

< Experimental Example  2>

PEI - SS Wow HA end Combined  Toxicity Evaluation of Polymers

In order to determine the toxicity of HA-g- (PEI-SS) and HA-b- (PEI-SS) prepared in <Example 1>, high molecular weight PEI (25kDa), low molecular weight PEI (2kDa) MTT assay was performed in comparison with PEI-SS.

Specifically, B16F1 cells cultured in DMEM medium (GIBCO-BRL, NY, USA) to which 10% fetal bovine serum and 100 U / mL penicillin-streptomycin were added were dispensed into 96-well plates. Incubate at 37˚C and 5% CO ₂ until cells grow to about 70%, incubate for 24 hours by injecting various concentrations of HA-g- (PEI-SS) into each well, followed by MTT assay. Cytotoxicity was evaluated and the results are shown in FIG. 8.

As shown in FIG. 8, it can be seen that HA-g- (PEI-SS) and HA-b- (PEI-SS) have almost no cytotoxicity compared to high molecular weight PEI (25kDa).

< Experimental Example  3>

PEI - SS Wow HA end Combined  Polymer and siRNA Gene silencing of the complex of gene silencing ) activation

Gene silencing activity of HA-g- (PEI-SS) and siRNA complexes or HA-b- (PEI-SS) and siRNA complexes prepared in Example 1 was evaluated by co-transfection.

Specifically, B16f1 cells (Korea Cell Line Bank) in 24-well plates were firstly Lipofectamine / PGL3-Luc complex (Jiang, G .; Park, KT; Kim, JS; Kim, KS; Oh, EJ; Kang, HG; Han , SE; Oh, YK; Park, TG; Hahn, SK Hyaluronic acid-polyethyleneimine conjugate for target specific intracellular delivery of siRNA.Biopolymers 2008, 89, 635-642. Thereafter, HA-g- (PEI-SS) and siRNA complexes and HA-b- (PEI-SS) and siRNA complexes were treated with the cells and further incubated for 24 hours, followed by fluorescence luminometer (Luminometer). Luciferase activity was measured for 30 seconds, and the results are shown in FIGS. 9 and 10.

9 and 10, the HA-g- (PEI-SS) and siRNA complexes or the HA-b- (PEI-SS) and siRNA complexes effectively effect luciferase activity in a concentration-dependent manner. It can be seen that.

< Example  2>

HA Wow siRNA of Disulfide  By combination HA - ss - siRNA Synthesis of

<2-1> HA Wow siRNA of Disulfide  By combination HA - ss - siRNA Synthesis of

Through the process as described in FIG. 11, HA-ss-siRNA was synthesized through disulfide bonds of hyaluronic acid (HA) and siRNA. Carboxyl group of hyaluronic acid and dihydrazide group of 3- (2-Pyridyldithio) propionyl hydrazide (PDPH) 1-ethyl-3- [3- Pyridyldisulfide groups were introduced into hyaluronic acid by binding using (dimethylamino) propyl] carbodiimide (1-ethyl-3- [3- (dimethylamino) propyl] carbodiimide (EDC)). Then, HA-ss-siRNA was synthesized by combining a siRNA having a thiol group introduced at 3 ′ of the sense strand with hyaluronic acid having the pyridyldisulfide group introduced therein. Whether the HA-ss-siRNA was synthesized was analyzed using GPC and the results are shown in FIG. 12.

< Experimental Example  4>

HA - ss - siRNA Wow PEI - SS  Gene silencing of the complex ( gene silencing ) activation

Gene silencing activity of the complex of HA-ss-siRNA prepared in <Example 2> and polyethyleneimine (PEI-SS) into which disulfide bonds were introduced was measured in the same manner as in <Experimental Example 3>, and the results are shown in FIG. 13 described.

As shown in FIG. 13, it can be seen that the complex of HA-ss-siRNA and polyethyleneimine (PEI-SS) in which disulfide bonds were introduced effectively inhibits luciferase activity in a concentration-dependent manner.

Figure 1 shows the synthesis of polyethyleneimine (PEI-SS) in which disulfide bonds are introduced by combining low molecular weight polyethyleneimine with cystamine bisacrylamide.

FIG. 2 shows the results of analysis using ¹ H-NMR whether polyethyleneimine (PEI-SS) into which disulfide bonds are introduced is formed. (CBA: cystamine bisacrylamide, PEI: polyethyleneimine, PssP: polyethyleneimine with disulfide bonds (PEI-SS))

Figure 3 shows the synthesis process of HA-g- (PEI-SS) in which the amine group of polyethyleneimine (PEI-SS) in which disulfide bonds are introduced is bonded to the carboxyl group of hyaluronic acid (HA).

4 shows the results of analyzing whether HA-g- (PEI-SS) was synthesized using ¹ H NMR.

Figure 5 shows the synthesis process of HA-b- (PEI-SS) is bonded to the amine group of the PEI-SS and the end of HA.

6 is a result of analyzing whether HA-b- (PEI-SS) was synthesized using ¹ H NMR.

Figure 7 is a photograph confirming the formation of a complex of the polymer and siRNA conjugated with PEI-SS and HA by electrophoresis.

Figure 8 is the result of measuring the cytotoxicity of PEI-SS and HA coupled polymer using MTT-assay. (PEI 25k: high molecular weight PEI (25kDa), PEI 2k: low molecular weight PEI (2kDa), PssP: PEI-SS)

9 is a graph showing that HA-g- (PEI-SS) and siRNA complexes effectively inhibit luciferase activity in a concentration-dependent manner.

10 is a graph showing that HA-b- (PEI-SS) and siRNA complexes effectively inhibit luciferase activity in a concentration dependent manner.

11 shows the synthesis of HA-ss-siRNA in which HA and siRNA are disulfide-linked.

12 is a graph showing the results of GPC analysis before and after synthesis of HA-ss-siRNA.

FIG. 13 is a graph showing that the complex of HA-ss-siRNA and PEI-SS effectively inhibits luciferase activity in a concentration-dependent manner.

<110> POSTECH ACADEMY-INDUSTRY FOUNDATION <120> Method of manufacturing polymer for delivering nucleic acids and          compositions for delivering nucleic acids comprising the polymer <130> DPP20093468KR <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 1 uuguuuugga gcacggaaa 19  

Claims (12)

Method for producing a nucleic acid delivery polymer comprising the step of coupling a disulfide bond introduced polyethyleneimine (polyethyleneimine) and hyaluronic acid (hyaluronic acid). The method according to claim 1, wherein (a) 1-ethyl-3- [3- (dimethylamino) -propyl] carbodiimine is dissolved in hyaluronic acid (1-DC-3- [3- (dimethylamino) -propyl] carbodiimide, EDC ) And one or more compounds selected from the group consisting of N, N'-dicyclohexylcarbodiimide (DCC) and 1-hydroxy bentriazole monohydrate (HOBt), N-hydroxy Adding at least one compound selected from the group consisting of sorbosuccinimide (N-hydroxysulfosuccinimide, sulfo-NHS) and N-hydroxysuccinimide (NHS); and (B) a method for producing a nucleic acid delivery polymer comprising the step of binding with a polyethyleneimine introduced disulfide bond. The method of claim 1, wherein NaBH ₃ CN is added to polyethyleneimine and hyaluronic acid to which disulfide bonds are introduced. A nucleic acid delivery composition comprising a nucleic acid delivery polymer represented by Formula 1 or Formula 2 below; <Formula 1>

In Chemical Formula 1 n and m are each independently an integer selected from 8 to 20; p and q are each independently integers selected from 16 to 2500; <Formula 2>

In Chemical Formula 2 n and m are each independently an integer selected from 8 to 20; r is an integer selected from 26 to 2500. The composition of claim 4, further comprising a nucleic acid. The composition of claim 5, wherein the nucleic acid is small interfering RNA (siRNA). (a) 1-ethyl-3- [3- (dimethylamino) propyl in a mixture of hyaluronic acid and 3- (2-pyridyldithio) propionyl hydrazide (3- (2-pyridyldithio) propionyl hydrazide, PDPH) ] Carbodiimide (1-ethyl-3- [3- (dimethylamino) propyl] carbodiimide (EDC) and at least one compound selected from the group consisting of N, N'-dicyclohexylcarbodiimide (DCC) Adding pyridyl disulfide groups to the hyaluronic acid by addition; (b) combining hyaluronic acid to which a pyridyldisulfide group is introduced in step (a) and a nucleic acid to which a thiol group is introduced; (C) a method for producing a nucleic acid delivery complex comprising the step of forming a complex by adding a polyethyleneimine introduced disulfide bond (polyethyleneimine). The method of claim 7, wherein the nucleic acid is small interfering RNA (siRNA). A nucleic acid delivery composition containing a compound represented by the following formula (3) and a nucleic acid into which a thiol group is introduced. <Formula 3>

In Chemical Formula 3 p is an integer selected from 16 to 2500; R is a pyridyl group. 10. The nucleic acid delivery composition according to claim 9, further comprising polyethyleneimine having a disulfide bond introduced therein. 10. The composition of claim 9, wherein the nucleic acid is small interfering RNA (siRNA). The method according to claim 1 or 7, wherein the polyethyleneimine into which disulfide bonds are introduced is prepared by reacting polyethyleneimine with cystamine bisacrylamide.

Images