A kind of hydrogel for gene therapy and preparation method thereof

Abstract

The present invention discloses a hydrogel for gene therapy, comprising a vinyl functionalized nucleic acid inclusion compound and a thiol polymer which are cross-linked to form a three-dimensional network structure; the vinyl functionalized nucleic acid inclusion compound is obtained by electrostatic complexation of a vinyl macromolecular monomer and a nucleic acid nanocomposite; and the surface of the vinyl functionalized nucleic acid inclusion compound has an active double bond. The present invention also discloses a method for preparing a hydrogel for gene therapy. The process of the present invention is mild, and the hydrogel block is cross-linked by oxidizing the free thiol groups on the thiol polymer to form disulfide bonds. The process is mild and will not damage the embedded nucleic acid component; the nucleic acid loading capacity is high, and the nucleic acid nanocomposite particles form a vinyl macromolecular monomer coating layer on the surface through electrostatic complexation, which effectively improves the dispersion stability of the nucleic acid nanocomposite particles, and can be grafted onto the hydrogel skeleton to prepare a high nucleic acid loading gene therapy hydrogel.

Description

Technical Field

[0001] The invention relates to a hydrogel and a preparation method thereof, in particular to a hydrogel for gene therapy and a preparation method thereof. Background Art

[0002] Gene therapy refers to the technology of inserting normal or therapeutic exogenous genes into target cells to replace or repair defective genes in target cells to achieve the purpose of disease treatment. The success of gene therapy is inseparable from the development of various safe and efficient gene delivery systems. Among them, synthetic non-viral vectors have attracted widespread attention due to their advantages of easy preparation and high safety. For example, cationic liposomes, polyethyleneimine (PEI) polymers, polylysine polymers, etc. have been successively developed and applied in the fields of vaccines and cancer treatment. Generally, the nucleic acid nanocomplex (Polyplex) formed by such genes and vectors directly enters the human body and acts on the target position by spray coating, intravenous injection or pulmonary inhalation. Although these administration methods have achieved good results in the treatment of various diseases, due to factors such as the small size of nucleic acid nanocomplexes, easy aggregation, and high toxicity, the local retention effect of such nucleic acid drugs is poor, and multiple applications are required to maintain the therapeutic effect, so patient compliance is poor.

[0003] Establishing a hydrogel-based sustained-release system for nucleic acid drugs is an effective strategy to solve the above problems. However, since the active ingredient nucleic acid nanocomplex is very easy to aggregate when the concentration is high, the final concentration of nucleic acid nanocomplex in the hydrogel used for gene therapy is low, only 0.02-0.1 mg / mL. -1 In addition, nucleic acid nanocomplexes also have problems such as easy aggregation during freeze-drying and high carrier toxicity, which also brings difficulties to the storage and use of drugs. In addition, since hydrogels usually have large pores (micrometer level), Polyplex drugs are prone to sudden release, which reduces the controllability of the release process. Summary of the invention

[0004] Purpose of the invention: In order to overcome the deficiencies in the prior art, the purpose of the present invention is to provide a gene therapy hydrogel that can prevent burst release, has a long-lasting release, and has good biocompatibility. Another purpose of the present invention is to provide a method for preparing a gene therapy hydrogel that effectively improves the dispersion stability of nucleic acid nanocomplex particles.

[0005] Technical solution: The hydrogel for gene therapy of the present invention comprises a vinyl functionalized nucleic acid inclusion compound and a thiol polymer cross-linked to form a three-dimensional network structure; the vinyl functionalized nucleic acid inclusion compound is obtained by electrostatic complexation of a vinyl macromolecular monomer and a nucleic acid nanocomposite; and the surface of the vinyl functionalized nucleic acid inclusion compound has active double bonds.

[0006] Furthermore, the vinyl macromonomer is any one of vinyl-modified hyaluronic acid, vinyl-modified sodium alginate, vinyl-modified gelatin, and vinyl-modified chitosan. The vinyl macromonomer is preferably vinyl-modified hyaluronic acid or vinyl-modified sodium alginate.

[0007] Further, the nucleic acid nanocomplex comprises a nucleic acid and a cationic carrier. The cationic carrier is one or more of cationic liposomes, chitosan, polylysine, polyethyleneimine, polyamide-amine dendrimer polymer with ethylenediamine as the core, and polyurethane. The cationic carrier is preferably polyethyleneimine (PEI) and polylysine (PLL). The nucleic acid is any one of DNA, therapeutic gene, RNA, catalytically active nucleic acid, and antisense oligonucleotide modified nucleic acid.

[0008] Furthermore, the thiolated polymer is one or more of thiol-modified hyaluronic acid, thiol-modified sodium alginate, thiol-modified dextran, thiol-modified pullulan, thiol-modified chitosan, thiol-modified cellulose or thiol-modified gelatin. The thiol-modified polymer is preferably thiol-modified hyaluronic acid, with an average molecular weight of 5 kDa to 3000 kDa and a thiol substitution rate of 0.1 to 2.0 mmol / g.

[0009] The method for preparing the above-mentioned hydrogel for gene therapy comprises the following steps:

[0010] Step 1, mixing a vinyl macromolecular monomer and a nucleic acid nanocomplex in a solvent to obtain a vinyl functionalized nucleic acid inclusion complex;

[0011] Step 2, dissolving the vinyl-functionalized nucleic acid inclusion complex and the thiol-functionalized polymer in a solvent;

[0012] Step 3: adjusting the pH of the product obtained in step 2 to 6-10 for reaction to obtain a hydrogel for gene therapy.

[0013] Furthermore, in step 1, the mass ratio (w / w) of the vinyl macromer to the nucleic acid nanocomplex is 1 to 50, preferably 5.

[0014] Furthermore, the solvent is deionized water, PBS buffer or physiological saline.

[0015] Furthermore, in step 2, the mass volume concentration of the vinyl functionalized nucleic acid inclusion complex is 0.5-15%, and the mass volume concentration of the thiol-functionalized polymer is 0.5-30%.

[0016] Preparation principle: Nucleic acid nanocomplex particles are prone to irreversible aggregation at high concentrations, thus limiting their loading capacity in the hydrogel carrier. Vinyl macromolecular monomers and nucleic acid nanocomplexes form vinyl functionalized nucleic acid inclusion complexes through electrostatic complexation, thereby effectively avoiding irreversible aggregation between nucleic acid nanocomplex particles under high concentration conditions or during freeze-drying, and obtaining hydrogels with high nucleic acid loading capacity. In addition, the surface of the vinyl functionalized nucleic acid inclusion complex after electrostatic complexation also has active double bonds, which can be grafted onto the hydrogel skeleton through Michael addition reaction to prevent burst release, so as to achieve the controlled release process induced by polymer degradation.

[0017] Beneficial effects: Compared with the prior art, the present invention has the following significant features:

[0018] 1. The process is mild. The hydrogel block is formed by cross-linking by oxidizing the free sulfhydryl groups on the sulfhydryl polymer to form disulfide bonds. This process is mild and will not damage the embedded nucleic acid components.

[0019] 2. The nucleic acid loading capacity is high. The nucleic acid nanocomposite particles form a vinyl macromolecular monomer coating layer on the surface through electrostatic complexation. This coating layer effectively improves the dispersion stability of the nucleic acid nanocomposite particles and can be grafted onto the hydrogel skeleton through Michael addition reaction, thereby realizing the preparation of hydrogels for gene therapy with high nucleic acid loading capacity;

[0020] 3. Long-term effect of gene therapy. Nucleic acid nanocomplexes are grafted onto the hydrogel skeleton through Michael addition reaction. Therefore, the stable release of nucleic acid nanocomplexes can be achieved through the gradual degradation of hydrogel. In addition, the cross-linking degree of the hydrogel network can be controlled by adjusting the thiol content of the thiol polymer, thereby slowing down the material degradation process and achieving long-term release.

[0021] 4. All component materials used are non-toxic and have good biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a preparation flow chart of the present invention;

[0023] Figure 2 The structural formula and hydrogen nuclear magnetic resonance spectrum of PEI of the present invention are shown below;

[0024] Figure 3 This is a dynamic light scattering diagram of the nucleic acid nanocomplex prepared by mixing PEI and DNA in the present invention;

[0025] Figure 4 The structural formula and hydrogen nuclear magnetic resonance spectrum of the vinyl-modified hyaluronic acid of the present invention are shown below;

[0026] Figure 5 The structural formula and hydrogen nuclear magnetic resonance spectrum of the thiolated sodium alginate of the present invention are shown;

[0027] Figure 6 Schematic diagram of the hydrogel of the present invention being used for transfection of HeLa cells. DETAILED DESCRIPTION

[0028] Example 1

[0029] like Figure 1 , a method for preparing a hydrogel for gene therapy, comprising the following steps:

[0030] (1) Synthesis of vinyl macromonomers: 28.75 mg of polyethylene glycol (PEG-DA) was weighed and added to 10 mL of PBS solution to prepare a PEG-DA solution. 5 mg of thiohyaluronic acid (HA-SH) was weighed and added to 2 mL of PBS solution to prepare a HA-SH solution. The PEG-DA solution and the HA-SH solution were mixed and the pH was controlled to be 7 to 8 to perform Michael addition cross-linking reaction to form a solution. The solution was dialyzed with deionized water for 3 days and then freeze-dried for storage to obtain vinyl-modified hyaluronic acid.

[0031] (2) Synthesis of nucleic acid nanocomplexes: Prepare 1 μg / mL polyethyleneimine (PEI) solution with water for injection, and then dilute 1 μg of DNA with 80 μL of serum-free culture medium and mix thoroughly to form a DNA diluent. Add 20 μL of PEI solution to 80 μL of DNA diluent, vortex for 10 seconds, and mix thoroughly. Incubate at room temperature for 15 minutes to obtain a DNA-PEI nanocomplex.

[0032] (3) Synthesis of vinyl-functionalized nucleic acid inclusion complex: 5 mg of vinyl-modified hyaluronic acid was mixed with 10 mg of DNA-PEI nanocomplex, and then cryoprotected with sucrose for concentration and storage to obtain vinyl-functionalized nucleic acid inclusion complex.

[0033] (4) Gene therapy hydrogel: 0.5% (w / v) vinyl functionalized nucleic acid inclusion complex and 4% (w / v) thiolated hyaluronic acid were dissolved in deionized water, and the pH of the solution was adjusted to 8 for reaction to obtain a gene therapy hydrogel. The thiol-modified hyaluronic acid had an average molecular weight of 5 kDa to 3000 kDa and a thiol substitution rate of 0.1 to 2.0 mmol / g.

[0034] By wrapping with vinyl macromolecular monomers, the nucleic acid nanocomplex can be well protected from aggregation during the freezing process to achieve a good redispersion state. The prepared hydrogel for gene therapy includes vinyl functionalized nucleic acid inclusion complexes and thiol polymers that are cross-linked to form a three-dimensional network structure.

[0035] The structural formula of polyethyleneimine (PEI) used in step (2) and its H NMR spectrum are as follows: Figure 2 1 mL of DNA-PEI nanocomplex solution was taken and the particle size was evaluated using dynamic light scattering technology on a Malvern Zeta Sizer ZS instrument. Figure 3 As shown in Figure 2, the average particle size of the nucleic acid nanocomplex is about 450 nm. Figure 4 As shown, the hyaluronic acid surface was successfully modified with active double bonds.

[0036] Example 2

[0037] A method for preparing a hydrogel for gene therapy comprises the following steps:

[0038] (1) Synthesis of vinyl macromonomers: 80 mg of polyethylene glycol (PEG-DA) was added to 10 mL of PBS solution to prepare a 0.8% (w / v) PEG-DA solution. 2 mg of sodium thioalginate was added to 2 mL of PBS solution to prepare a 0.1% (w / v) sodium alginate solution. The 0.8% PEG-DA solution and the 0.1% sodium alginate solution were mixed and the pH was controlled to 7-8 to perform Michael addition cross-linking reaction to form a solution. The solution was dialyzed with deionized water for 3 days and then freeze-dried for storage to obtain vinyl-modified sodium alginate.

[0039] (2) Synthesis of nucleic acid nanocomplexes: Prepare polyurethane (PU) polymer at a concentration of 1 mg / mL and dilute it according to a nitrogen-phosphorus ratio (N / P) of 5:1. Then prepare a DNA solution at a concentration of 37.5 μg / mL. The specific mixing process is to take 600 μL of PU solution and add it to 150 μL of DNA solution, vortex for 10 seconds, and let it stand at room temperature for 15 minutes to obtain a DNA-PU complex.

[0040] (3) Synthesis of vinyl-functionalized nucleic acid inclusion complex: 12 mg of vinyl-modified sodium alginate was mixed with 3 mg of DNA-PU nanocomplex, and then cryoprotected with sucrose for concentration and storage to obtain vinyl-functionalized nucleic acid inclusion complex. Figure 5 As shown, the surface of the successfully modified sodium alginate has thiol groups.

[0041] (4) Gene therapy hydrogel: A vinyl functionalized nucleic acid inclusion complex with a mass volume concentration of 5% and a thiolated sodium alginate with a mass volume concentration of 8% were dissolved in deionized water, and the pH of the solution was adjusted to 8 for reaction to obtain a gene therapy hydrogel.

[0042] Example 3

[0043] A method for preparing a hydrogel for gene therapy comprises the following steps:

[0044] (1) Synthesis of vinyl macromonomers: 20 mg of polyethylene glycol (PEG-DA) was added to 10 mL of PBS solution to prepare a PEG-DA solution with a mass volume concentration of 0.2%, 5 mg of thiolated chitosan was added to 10 mL of PBS solution to prepare a 0.5% thiolated chitosan solution, the 2% PEG-DA solution and the 0.5% thiolated chitosan solution were mixed, the pH was controlled to 8 to perform Michael addition cross-linking reaction to form a solution, which was dialyzed with deionized water for 3 days and then freeze-dried for storage to obtain vinyl-modified chitosan.

[0045] (2) Synthesis of nucleic acid nanocomplexes: Prepare 4 μg / mL polylysine (PLL) solution with water for injection, dilute 2 μg RNA with 100 μL serum-free medium, and mix thoroughly to form a therapeutic gene dilution solution. Add 5 μL of PLL solution to 100 μL of therapeutic gene dilution solution, vortex for 10 seconds, mix thoroughly, and incubate at room temperature for 15 minutes to obtain RNA-PLL nanocomplexes.

[0046] (3) Synthesis of vinyl-functionalized nucleic acid inclusion complex: 10 mg of vinyl-modified chitosan was mixed with 1 mg of RNA-PLL nanocomplex, and then cryoprotected with sucrose for concentration and storage to obtain vinyl-functionalized nucleic acid inclusion complex.

[0047] (4) Hydrogel for gene therapy: A vinyl-functionalized nucleic acid inclusion complex with a mass volume concentration of 0.5% and a thiol-modified dextran with a mass volume concentration of 2% were dissolved in deionized water, and the pH of the solution was adjusted to 7 to react and form a gel to obtain a hydrogel for gene therapy.

[0048] Example 4

[0049] A method for preparing a hydrogel for gene therapy comprises the following steps:

[0050] (1) Synthesis of vinyl macromonomers: 30 mg of polyethylene glycol (PEG-DA) was weighed and added to 10 mL of PBS solution to prepare a PEG-DA solution. 6 mg of thiodextran was weighed and added to 2 mL of PBS solution to prepare a thiodextran solution. The PEG-DA solution and the thiodextran solution were mixed, and the pH was controlled to 7.5 to perform Michael addition crosslinking reaction. The mixture was dialyzed with deionized water for 3 days and then freeze-dried for storage to obtain vinyl-modified dextran.

[0051] (2) Synthesis of nucleic acid nanocomplexes: Prepare chitosan solution with a concentration of 0.02 mg / mL. Prepare catalytically active nucleic acid solution with a concentration of 0.02 mg / mL. The specific mixing process is to take 100 μL of chitosan solution and 100 μL of catalytically active nucleic acid solution and add them to 30 μL of PBS buffer (pH = 5.7), place on ice for 10 minutes to react, and obtain catalytically active nucleic acid-chitosan nanocomplexes.

[0052] (3) Synthesis of vinyl-functionalized nucleic acid inclusion complex: 6 mg of vinyl-modified dextran was mixed with 2 mg of catalytically active nucleic acid-chitosan nanocomplex, and then cryoprotected with sucrose for concentration and storage to obtain vinyl-functionalized nucleic acid inclusion complex.

[0053] (4) Hydrogel for gene therapy: A vinyl-functionalized nucleic acid inclusion complex with a mass volume concentration of 15% and a thiol-modified pullulan with a mass volume concentration of 10% were dissolved in deionized water, and the pH of the solution was adjusted to 10 to react and form a gel to obtain a hydrogel for gene therapy.

[0054] Example 5

[0055] A method for preparing a hydrogel for gene therapy comprises the following steps:

[0056] (1) Synthesis of vinyl macromonomers: 50 mg of polyethylene glycol (PEG-DA) was weighed and added to 10 mL of PBS solution to prepare a 5% (w / v) PEG-DA solution. 5 mg of thiol gelatin was weighed and added to 1 mL of PBS solution to prepare a 0.5% (w / v) thiol gelatin solution. The PEG-DA solution and the thiol gelatin solution were mixed and the pH was controlled to 8 to form a Michael addition crosslinking reaction. The mixture was dialyzed with deionized water for 3 days and then freeze-dried for storage to obtain vinyl modified gelatin.

[0057] (2) Synthesis of nucleic acid nanocomplexes: Prepare a polyamide-amine dendrimer polymer (PAMAM) solution with ethylenediamine as the core at a concentration of 1 mg / mL. Dilute it according to the nitrogen-phosphorus ratio (N / P) = 10:1, and then prepare an antisense oligonucleotide-modified nucleic acid solution with a concentration of 0.02 mg / mL. The specific mixing process is to take 50 μL of the diluted PAMAM solution and add 100 μL of the antisense oligonucleotide-modified nucleic acid solution, and place it at room temperature for 30 minutes to react to obtain an antisense oligonucleotide-modified nucleic acid-polyamide-amine dendrimer polymer nanocomplex with ethylenediamine as the core.

[0058] (3) Synthesis of vinyl-functionalized nucleic acid inclusion complex: 8 mg of vinyl-modified gelatin and 2 mg of antisense oligonucleotide-modified nucleic acid-polyamide-amine dendrimer polymer nanocomplex with ethylenediamine as the core were mixed, and then cryoprotected with sucrose for concentration and storage to obtain vinyl-functionalized nucleic acid inclusion complex.

[0059] (4) Gene therapy hydrogel: A vinyl functionalized nucleic acid inclusion complex with a mass volume concentration of 5% and a thiol-modified chitosan with a mass volume concentration of 2% were dissolved in deionized water, and the pH of the solution was adjusted to 8 to react and form a gel to obtain a gene therapy hydrogel.

[0060] The vinyl-modified gelatin in this embodiment can be replaced by any one of vinyl-modified hyaluronic acid, vinyl-modified sodium alginate, vinyl-modified chondroitin sulfate, vinyl-modified keratan sulfate, vinyl-modified xanthan gum, vinyl-modified gelatin, vinyl-modified heparin, and vinyl-modified heparan sulfate. The thiol-modified cellulose can be replaced by one or more of thiol-modified hyaluronic acid, thiol-modified sodium alginate, thiol-modified dextran, thiol-modified pullulan, thiol-modified chitosan, thiol-modified cellulose, or thiol-modified gelatin.

[0061] Example 6

[0062] A method for preparing a hydrogel for gene therapy comprises the following steps:

[0063] (1) Synthesis of vinyl macromonomer: Vinyl-modified dextran was synthesized according to the method of step (1) in Example 1, except that the thiol hyaluronic acid was replaced by thiolated dextran.

[0064] (2) Synthesis of nucleic acid nanocomplexes: Prepare 1 μg / mL PEI solution with water for injection, dilute 2 μg of DNA with 100 μL of serum-free medium, and mix thoroughly to make a DNA dilution solution. Add 4 μL of PEI solution to 100 μL of DNA dilution solution, vortex for 10 seconds, mix thoroughly, and incubate at room temperature for 15 minutes to prepare a DNA-PEI nanocomplex.

[0065] (3) Synthesis of vinyl-functionalized nucleic acid inclusion complexes: 10 mg of vinyl-modified dextran was mixed with 2 mg of DNA-PU nanocomplexes.

[0066] (4) Hydrogel for gene therapy: synthesized according to the method of step (4) in Example 1.

[0067] (5) Transfected cells: Inoculate cells one day before transfection, so that the cell density at the time of transfection is preferably 70-80%. Remove the cell growth medium, add 2 mL of fresh preheated complete medium to each well of the 96-well plate, directly add 100 μL of the gene complex dilution to the cells, shake the culture plate, and gently mix. Culture in a 37°C, 5% CO2 incubator. The expression of the transfected gene can be detected as soon as 5 hours after transfection. The transfection results after 24 hours are as follows: Figure 6 , using green fluorescent protein expression, it can be seen from the fluorescence microscope that the gene hydrogel successfully achieved gene transfection.

[0068] Comparative Example

[0069] The remaining steps of this comparative example are the same as those of Example 1, except that step (1) is removed and the vinyl-functionalized nucleic acid inclusion complex is replaced with a DNA-PEI nanocomposite embedded in non-vinyl-modified hyaluronic acid. By comparison, it is found that the vinyl macromolecular monomer encapsulation can effectively prevent the nucleic acid nanocomposite from aggregating during the freezing process, so as to achieve a good redispersion state.

Claims

1. A method for preparing a hydrogel for gene therapy, characterized in that: The following steps are involved: Step 1, mixing a vinyl macromolecular monomer and a nucleic acid nanocomplex in a solvent to obtain a vinyl functionalized nucleic acid inclusion complex; Step 2, dissolving the vinyl-functionalized nucleic acid inclusion complex and the thiol-functionalized polymer in a solvent; Step 3, adjusting the pH of the product obtained in step 2 to 6-10 for reaction to obtain a hydrogel for gene therapy; The hydrogel for gene therapy comprises a vinyl functionalized nucleic acid inclusion compound and a thiol polymer which are cross-linked to form a three-dimensional network structure; the vinyl functionalized nucleic acid inclusion compound is obtained by electrostatic complexation of a vinyl macromolecular monomer and a nucleic acid nanocomposite; and the surface of the vinyl functionalized nucleic acid inclusion compound has active double bonds; The vinyl macromonomer is any one of vinyl-modified hyaluronic acid, vinyl-modified sodium alginate, vinyl-modified gelatin, and vinyl-modified chitosan; The nucleic acid nanocomplex comprises a nucleic acid and a cationic carrier; The cationic carrier is one or more of cationic liposomes, chitosan, polylysine, polyethyleneimine, polyamide-amine dendritic polymer with ethylenediamine as the core, and polyurethane; The nucleic acid is any one of DNA, therapeutic gene, RNA, catalytically active nucleic acid, and antisense oligonucleotide-modified nucleic acid; The thiolated polymer is one or more of thiol-modified hyaluronic acid, thiol-modified sodium alginate, thiol-modified dextran, thiol-modified pullulan, thiol-modified chitosan, thiol-modified cellulose or thiol-modified gelatin.

2. The method for preparing a hydrogel for gene therapy according to claim 1, characterized in that: In the step 1, the mass ratio of the vinyl macromonomer to the nucleic acid nanocomplex is 1-50.

3. The method for preparing a hydrogel for gene therapy according to claim 1, characterized in that: The solvent is deionized water, PBS buffer or physiological saline.

4. The hydrogel obtained by the method for preparing a hydrogel for gene therapy according to claim 1, characterized in that: In the step 2, the mass volume concentration of the vinyl functionalized nucleic acid inclusion complex is 0.5-15%, and the mass volume concentration of the thiol-functionalized polymer is 0.5-30%.

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