Redox sensitive cationic polymer gene vector as well as preparation and application thereof

Abstract

The invention discloses a redox sensitive cationic polymer gene vector as well as preparation and application thereof. According to the invention, hydrophobic small molecules with oxidation / reduction responsive S-S bonds are adopted to carry out side chain modification on polyglycidyl amine, and the cationic polymer gene delivery carrier material with specific tumor microenvironment response capability is prepared. The material has good biocompatibility, and can effectively load siRNA molecules and form siRNA compound nanoparticles with good stability and excellent gene transfection capacity. The compound nanoparticles can be effectively absorbed by cells, and can effectively respond to an oxidation / reduction tumor microenvironment to realize targeted release of siRNA. The oxidation / reduction response cationic polymer gene vector provided by the invention has a relatively great clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical polymer materials, specifically relating to an oxidation / reduction sensitive cationic polymer gene carrier and its preparation and application. Background Technology

[0002] With the rapid development of biotechnology, gene therapy, as a revolutionary medical approach, has demonstrated significant application value in the treatment of various diseases, including hereditary diseases, cancer, and viral infections. Gene therapy delivers exogenous genes (such as DNA and RNA) to target cells to repair or regulate the expression of abnormal genes, thereby achieving precise treatment of diseases. Among these, RNA interference (RNAi) technology, due to its ability to directly regulate gene expression levels and its advantages of high efficiency, precision, and reversibility, is considered an important strategy for treating tumors. RNAi therapeutic agents mainly include siRNA, miRNA, and shRNA. However, numerous technical bottlenecks in the delivery of RNA molecules, such as their inherent instability, susceptibility to enzymatic degradation, poor cell membrane penetration, and potential immunogenicity, severely restrict their clinical application. Therefore, developing safe and efficient RNA delivery vectors has become a key issue that urgently needs to be addressed in the field of gene therapy.

[0003] To address the aforementioned technical challenges, the development of gene vectors has become a core research direction. As "transport vehicles" for RNA molecules, gene vectors not only need to effectively protect RNA from degradation by nucleases but also need to facilitate its efficient entry into target cells and the release of its function. Currently, gene vectors are mainly divided into two categories: viral vectors and non-viral vectors. While viral vectors have high transfection efficiency, their inherent drawbacks, such as immunogenicity, mutagenicity, and difficulty in large-scale production, limit their widespread application. In contrast, non-viral vectors, due to their high safety, good biocompatibility, ease of modification, and large-scale production, show great promise for clinical applications.

[0004] Cationic polymers, due to their unique positive charge, can form stable complexes with negatively charged RNA molecules through electrostatic interactions, effectively protecting RNA from degradation by nucleic acid-degrading enzymes and promoting cellular uptake and intracellular release. Polyethyleneimine (PEI), as a representative cationic polymer, is considered the "gold standard" due to its excellent gene transfection efficiency; however, its excessive positive charge leads to high cytotoxicity and poor stability, severely limiting its clinical application. Therefore, developing novel cationic polymer gene vectors through molecular design, functionalization modification, and complex optimization to improve their biocompatibility, targeting, and delivery efficiency has become a key research focus.

[0005] Furthermore, existing delivery vehicles lack a specific response to the tumor microenvironment, making it difficult to achieve precise drug release at the target site. This leads to premature drug release or accumulation in non-target tissues during delivery, reducing therapeutic efficacy and increasing toxic side effects on normal tissues. Simultaneously, the uncontrollable nature of drug release further limits the delivery efficiency of these vehicles, impacting their application value in cancer treatment. Summary of the Invention

[0006] To address the above problems, the present invention aims to provide a reduction-sensitive cationic polymer gene vector, its preparation method, and its application.

[0007] The specific technical solution adopted in this invention is as follows:

[0008] A redox-sensitive cationic polymer gene vector, the structural formula of which is shown in formula (Ⅰ):

[0009]

[0010] Where n represents the degree of polymerization, which is 70-3000, corresponding to a main chain molecular weight of 5000-300000 g / mol; m represents the number of modification units, with an m / n ratio of 10-100%, which corresponds to a hydrophobic modification molar ratio of 10-100%.

[0011] In the formula, the substituent R is One of them.

[0012] Furthermore, the m / n ratio is between 10-50%, and more preferably between 25-50%.

[0013] A gene therapy drug includes the cationic polymer gene vector of the present invention and a gene drug loaded on the gene vector, wherein the gene drug is one of siRNA, mRNA, and shRNA.

[0014] A method for preparing a gene therapy drug includes the following steps: preparing a cationic polymer gene carrier into a carrier solution of 1-10 mg / mL, preparing a gene drug into a drug solution of 0.1-10 μg / μL, mixing the carrier solution and the drug solution in a buffer solution and blowing evenly, and incubating at room temperature on a shaker at 50-200 rpm for 0.5-2 h, thereby preparing the gene therapy drug.

[0015] Furthermore, when the carrier solution and the gene drug solution are mixed, the mass ratio of the carrier to the gene drug is 15:1-60:1.

[0016] Furthermore, the solvent for the carrier solution is one of methanol, ethanol, THF, and DMSO.

[0017] Furthermore, the drug solution is a DEPC aqueous solution of nucleic acid, and the DEPC water is ultrapure water treated with diethyl pyrocarbonate and sterilized by high temperature and high pressure.

[0018] Furthermore, the buffer solution is a citrate-disodium hydrogen phosphate buffer solution with a pH of 3.4-6.0.

[0019] The application of the cationic polymer gene vector described in the above technical solution in the preparation of gene therapy drugs.

[0020] The application of the gene therapy drug described in the above technical solution in the cell transfection process.

[0021] The application of the gene therapy drug described in the above technical solution in RNA interference.

[0022] The beneficial effects of this invention are as follows:

[0023] 1) The reduction-sensitive cationic polymer carrier of the present invention can effectively load gene drugs, form nanoparticles with good particle size and distribution through electrostatic interaction, and the formed nanoparticles have good stability.

[0024] 2) The reduction-sensitive cationic polymer gene carrier of this invention can effectively reduce the cytotoxicity of materials. Once the gene loaded on the carrier enters the lesion cells, the gene drug begins to be released. Through controlled release, it exerts a therapeutic effect at specific lesion sites.

[0025] 3) Nucleic acid-loaded nanoparticles prepared based on reduction-sensitive cationic polymers, i.e. gene therapy drugs, are very stable under neutral physiological conditions, but degrade in the reducing environment inside cells, which is conducive to achieving effective cell transfection of loaded genes.

[0026] 4) Cellular uptake is a critical rate-limiting step in cell transfection, directly determining whether exogenous nucleic acids can enter the cell and exert their effects. The gene therapy drug prepared in this invention exhibits good cellular uptake.

[0027] 5) High loading efficiency of the vector for gene drugs is a prerequisite for achieving high delivery efficiency. The reduction-sensitive cationic polymer gene vector of the present invention has high loading efficiency. Attached Figure Description

[0028] Figure 1 Synthetic route diagram of polyglycidylamine;

[0029] Figure 2 HOOC-SS-C 12 The synthetic route;

[0030] Figure 3 HOOC-SS-C6-3 The synthetic route;

[0031] Figure 4 Synthetic route for PGAm hydrophobic modification;

[0032] Figure 5 GPC curve of polyepoxychloropropane;

[0033] Figure 6 HOOC-SS-C 12 of 1 H NMR spectrum;

[0034] Figure 7 HOOC-SS-C 6-3 of 1 H NMR spectrum;

[0035] Figure 8 P10 / C 6-3 / 30 1 H NMR spectrum;

[0036] Figure 9 Nanoparticle size and PDI;

[0037] Figure 10 The potential of nanoparticles;

[0038] Figure 11 siRNA loading efficiency of nanoparticles (n=3);

[0039] Figure 12 Storage stability of nanoparticles (n=3);

[0040] Figure 13 GSH-responsive release of nanoparticles;

[0041] Figure 14 ROS-responsive release from nanoparticles;

[0042] Figure 15 shows the gene transfection of nanoparticles with 50% and 30% modified polymers;

[0043] Figure 16 (a) Cytotoxicity of 50% modified polymer carrier; (b) Cytotoxicity of 50% modified polymer carrier;

[0044] Figure 17 Cellular uptake of nanoparticles;

[0045] Figure 18 (a) Flow cytometry quantification histograms for each group of nanoparticles; (b) Flow cytometry quantification results for each group of nanoparticles (n=3). Detailed Implementation

[0046] The present invention will be further described below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited thereto.

[0047] Special note: The naming method for each modified product is as follows: alkanoates are C16-C ... n n is the number of carbons; enols are C m1-m2 In this context, m1 represents the number of carbon atoms, and m2 represents the position of the double bond. Aromatic alcohols are indicated by the first three letters of their English name or by the abbreviation of their functional group. For example, HOOC-SS-C4 is the product of butanol coupled with a disulfide compound.

[0048] The designation of PGAm (polyglycidylamine) is as follows: P10 indicates that the starting material, polyephichloropropane (PECH), has a molecular weight of 10 kDa; P20 indicates a molecular weight of 20 kDa; and P30 indicates a molecular weight of 30 kDa. The designation is followed by the modification percentage. For example, P10 / C4 / 30 indicates that the polyglycidylamine synthesized from 10 kDa polyephichloropropane (PECH) has 30% of its side-chain amino groups modified with butanol, meaning that 30% of the amino groups in the polyglycidylamine are modified with butanol.

[0049] A reduction-sensitive cationic polymer gene vector, the structural formula of which is shown in formula (I).

[0050]

[0051] Where n represents the degree of polymerization, which is 70-3000, corresponding to a main chain molecular weight of 5000-300000 g / mol; m represents the number of modification units, with an m / n ratio of 10-50%, which corresponds to a hydrophobic modification molar ratio of 10-50%.

[0052] In the formula, the substituent R is

[0053] One of them.

[0054] Example 1: Synthesis of polyglycidylamine, see [link to example]. Figure 1 .

[0055] Taking the synthesis of polyepoxychloropropane (PECH) with a molecular weight of 10 kDa as an example: A 50 mL two-necked flask was selected. Under N2 protection, the initiator tetra-n-octylamine bromide (TOAB, 0.131 g, 0.24 mmol) was dissolved in 3 mL of anhydrous toluene. Epichlorohydrin (ECH, 2 mL, 25.940 mmol) was added, and the mixture was stirred at room temperature. The reaction system was cooled to -30 °C, and a triisobutylaluminum solution (iBu3Al, 1 M, 0.7 mL) was added dropwise to the reaction solution. After the addition was complete, the reaction was stirred for 3 h. After the reaction was completed, methanol precipitation was used, and the product was dried under vacuum to obtain a colorless, viscous liquid, which is polyepoxychloropropane (PECH) with a molecular weight of 10 kDa. Its structure is obtained through... 1 H NMR and 13 The PECH was characterized by C10 NMR, and its molecular weight was determined by GPC. The GPC curve is shown in the figure. Figure 5 (Standard curve: polystyrene: 1.15kDa-270kDa; mobile phase: tetrahydrofuran; flow rate: 1mL / min; column temperature: 35℃). 1 H NMR (400MHz, Chloroform-d) δ: 3.80–3.66 (CH2O, CH2Cl, 4H), 3.65–3.57 (CHO, 1H). 13 C NMR (101MHz, Chloroform-d) δ: 79.10 (CHO), 69.65 (CH2O), 43.68 (CH2Cl).

[0056] Poly(propylene azide) oxide (PGA): A 25 mL two-necked flask was used. PECH (10 kDa) synthesized above was added, along with sodium azide in a 1.1 molar equivalent to PECH, and 10 mL of N,N-dimethylformamide (DMF). The mixture was stirred at 110 °C for 12 h. After the reaction, the sodium chloride precipitate was removed by filtration. The filtrate was slowly added to water with stirring to precipitate the precipitate, and the supernatant was discarded. This precipitation step was repeated three times. The product was then dried overnight in a vacuum drying oven to obtain a pale yellow viscous liquid, which is poly(propylene azide) oxide (PGA). Its structure is determined by… 1 H NMR and 13 Characterized by C NMR. 1 H NMR (400MHz, Chloroform-d) δ: 3.78–3.55 (CH2CHO, 3H), 3.49-3.27 (CH2N3, J=23.5, 15.1, 10.4, 2.8Hz, 2H). 13 C NMR (101MHz, Chloroform-d): δ: 78.77 (CHO), 69.60 (CH2O), 51.77 (CH2N3).

[0057] Polyglycidylamine (PGAm): A 50 mL round-bottom flask was used. The synthesized PGA, 1.1 molar equivalents of triphenylphosphine (PPh3) compared to PGA, and 15 mL of DMF were added sequentially. The mixture was stirred at room temperature for 3 h, followed by the addition of 5 mL of ultrapure water and stirring for another 0.5 h. After the reaction, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 1000 Da and dialyzed in methanol for 3 days. The solution was then concentrated using a rotary evaporator to obtain a pale yellow viscous liquid polyglycidylamine PGAm (1.6009 g, yield 66.7%). PGAm was dissolved in methanol to prepare a 1 M stock solution, where 1 M represents the concentration of amino groups in the repeating units of PGAm in methanol. The molecular weight of the polymer PGAm was determined by GPC to determine the number of repeating units and thus the amino content of the polymer PGAm. Its structure was determined by… 1 HNMR and 13 Characterized by C NMR. 1 H NMR (400MHz, Deuterium Oxide) δ: 3.80–3.53 (CH2CHO, 3H), 2.88-2.65 (CH2NH2, J=13.5, 5.0Hz, 2H). 13 C NMR (101MHz, Methanol-d4) δ: 80.26 (CHO), 70.00 (CH2O), 42.11 (CH2NH2).

[0058] Example 2 Synthesis of small alkanool molecules

[0059] With HOOC-SS-C 12 For example, see the synthesis of [the substance]. Figure 2 A 250 mL single-necked round-bottom flask was used. 2,2'-dithiodiacetic acid (4.0000 g, 22.0 mmol) and p-toluenesulfonic acid (PTSA) (4.7250 g, 27.4 mmol) were dissolved in 50 mL of toluene with stirring. 1-Dodecanol (2.0451 g, 11.0 mmol) was added, and the mixture was stirred at 120 °C for 36 h. After the reaction was complete, the toluene was removed by rotary evaporation. The solution was dissolved in dichloromethane (DCM) and transferred to a 500 mL separatory funnel. Aqueous phase was added at a volume ratio of organic phase to water of 1:1, along with 5 mL of saturated NaHCO3 solution. The mixture was shaken and allowed to stand overnight. The solution was washed three times with water and once with saturated NaCl solution. The resulting organic layer was dried over anhydrous Na2SO4, filtered, and concentrated by rotary evaporation to remove DCM, yielding the crude product. The crude product was purified by silica gel column chromatography (ethyl acetate: n-hexane = 20:1, v / v) to obtain pure HOOC-SS-C. 12 (2.0182 g, yield 54.8%), its structure is determined by... 1 Characterized by H NMR, see Figure 6 . 1 HNMR(400MHz,Chloroform-d)δ4.15(t,J=6.7Hz,2H),3.61(d,J=13.7Hz,4H),1.66(q,J=6.9Hz,2H),1.27(d,J=9.9Hz,18H),0.87(t,J=6.8Hz,3H).

[0060] Example 3 Synthesis of small enol molecules

[0061] With HOOC-SS-C 6-3 For example, see the synthesis of [the substance]. Figure 3 A 250 mL single-necked round-bottom flask was selected. Under N2 protection, 2,2'-dithiodiacetic acid (3.6444 g, 20.0 mmol) and 30 mL of 30% acetic anhydride were added and stirred at room temperature for 2 h. Then, 50 mL of anhydrous toluene was added and rotary evaporated. Immediately afterward, 50 mL of anhydrous DCM was added. DMAP (0.2443 g, 2 mmol) and 3-hexen-1-ol (1.0016 g, 10 mmol) were dissolved in 3 mL of anhydrous DCM and added sequentially to the reaction mixture. The mixture was stirred at room temperature for 24 h. After the reaction was complete, the reaction mixture was transferred to a 500 mL separatory funnel. An aqueous phase was added at a volume ratio of organic phase to water of 1:1, along with 2 mL of 1 M hydrochloric acid solution. The mixture was shaken and allowed to stand overnight. The product was washed three times with water and once with saturated NaCl solution. The resulting organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated by rotary evaporation to remove DCM, yielding the crude product. Purification was then performed by silica gel column chromatography (ethyl acetate:n-hexane = 20:1, v / v) to obtain the product HOOC-SS-C. 6-3 (1.6745 g, yield 63.3%). Its structure is determined through... 1 Characterized by H NMR, see Figure 7 . 1 H NMR(400MHz,Chloroform-d)δ5.59–5.46(m,1H),5.31(dtt,J=10.6,7.3,1.6Hz,1H),4.15(t,J=6.9H z, 2H), 3.61 (d, J = 14.8Hz, 4H), 2.45–2.37 (m, 2H), 2.06 (pd, J = 7.5, 1.5Hz, 2H), 0.97 (t, J = 7.5Hz, 3H).

[0062] The synthesis of alkanol small molecules can be performed according to the method described in Example 2, and the synthesis of enol small molecules can be performed according to the method described in Example 3.

[0063] Example 4 Hydrophobic Modification of Polyglycidylamine

[0064] With P10 / C 6-3 For example, see the synthesis of / 30. Figure 4 HOOC-SS-C 6-3 39.65 mg (0.15 mmol) and benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (pyBOP) (312.23 mg, 0.6 mmol) were dissolved in 10 mL of LMF, and 500 μL of the PGAm methanol mother liquor (1 M) prepared in Example 1 was added. The mixture was stirred at room temperature for 3 days. After the reaction was completed, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 1000 Da and dialyzed in methanol for 3 days. The solution was then concentrated by rotary evaporation to obtain a colorless, transparent, viscous solid, which is the hydrophobic small molecule modified polyglycidylamine P10 / C. 6-3 / 30 (actual modification ratio 26.3%, the product is named according to the molar ratio of the feed in this application embodiment), its structure is obtained through 1 Characterized by H NMR, see Figure 8 It was prepared into a 10 mg / mL methanol solution and stored.

[0065] Example 5: Preparation of Nanoparticles

[0066] The reduction-sensitive cationic polymer gene vector was dissolved in methanol to prepare a 5 mg / mL gene vector solution; the gene drug siRNA was dissolved in diethyl pyrocarbonate-treated water (DECP water) to prepare a 0.5 g / L gene drug solution. The gene vector solution and gene drug solution were mixed in a citrate-disodium hydrogen phosphate buffer solution at pH 4.0 according to a mass ratio of 30:1. The mixture was stirred evenly and incubated at 100 rpm for 1 h at room temperature to obtain the gene therapy drug.

[0067] The aforementioned gene therapy drugs, at 25°C, had their nanoparticle size, polydispersity index (PDI), and surface potential (ζ) measured using dynamic light scattering (DLS). Figure 9 As shown, the modified nanoparticles all have a particle size of around 200 nm, exhibiting good dispersibility and uniform distribution. Figure 10 As shown, the nanoparticles have a weak positive charge on their surface, which is beneficial for cell uptake, and the charge change generally shows an increasing trend with the increase of side chain length.

[0068] In addition, the applicant found through experiments that when polyglycidylamine is not modified with hydrophobic small molecules, its structure is purely hydrophilic, making it difficult to form nanoparticles and resulting in poor performance.

[0069] Example 6: Determination of siRNA loading efficiency

[0070] The siRNA loading efficiency of nanoparticles was detected using the Quant-iT RiboGreen RNA Detection Kit. 0.5 g / L siRNA solution and 5 mg / mL gene vector solution were mixed in a 1:15 to 1:60 siRNA-to-gene vector mass ratio in a 10 mM citrate-disodium hydrogen phosphate buffer solution (pH 4.0). The mixture was then incubated at 100 rpm for 1 h at room temperature to obtain the nanoparticle solution. Following the same method, a nanoparticle solution with a fixed siRNA concentration of 2.5 ng / μL was prepared. This solution was then diluted 10-fold with 10 mM citrate-disodium hydrogen phosphate buffer solution (pH 4.0). The siRNA loading of the nanoparticles was detected using the Quant-iT RiboGreen RNA Detection Kit, with the Ribogreen assay reagent diluted 2000-fold with 1×TE buffer as the working solution. 100 μL of the diluted nanoparticle solution and an equal volume of Ribogreen working solution were added to each well of a 96-well black plate, and the fluorescence value (λ) was detected using a microplate reader. ex =485nm, λ em =535nm). SiRNA solutions with concentration gradients were prepared, and fluorescence values ​​were detected using the same procedure to plot a standard curve. The fluorescence values ​​detected in the nanoparticle solutions were substituted into the regression equation to calculate the corresponding free siRNA concentration, thereby calculating the siRNA loading efficiency of the corresponding nanoparticles.

[0071] like Figure 11 As shown, PGAm of various molecular weights, after modification, achieved complete siRNA loading at all mass ratios. The molecular weight of the polymer and the length of the modified side chain had virtually no effect on siRNA loading. At the 30:1 mass ratio used in cell experiments, the vector could load siRNA well, achieving approximately 100% loading.

[0072] Figure 11 In the middle bar chart, "15", "30", "45", and "60" represent the mass ratios of gene vector to gene drug siRNA as 15, 30, 45, and 60, respectively.

[0073] Example 7: Nanoparticle Stability Experiment

[0074] The prepared nanoparticles were stored at 4°C, and the particle size changes were measured by dynamic light scattering (DLS) at storage times of 0, 1, and 3 days. Figure 13 As shown, PGAm modified with short carbon chains is relatively more stable during the 3-day storage period, with little change in particle size.

[0075] Figure 12In the diagram, 0D, 1D, and 3D represent the test results after storing the data for 0 days, 1 day, and 3 days, respectively.

[0076] Example 8: Reduction-sensitive release of nanoparticles

[0077] First, following the preparation method of the nanoparticle solution in Example 6, P10 / C4 / 30 was selected as the gene vector, and a nanoparticle solution containing 50 ng / μL of siRNA was prepared at a mass ratio of siRNA to gene vector of 1:30. Subsequently, SDS solutions of different concentrations (w / v) were prepared: 0.02%, 0.04%, 0.06%, and 0.08%, and a 10 mM GSH solution was also prepared. The experiment was divided into two groups: in the first group, 10 μL of nanoparticle solution was mixed with 10 μL of SDS solution of different concentrations and 10 μL of GSH solution, respectively; in the second group, 10 μL of nanoparticle solution was mixed with 10 μL of SDS solution of different concentrations and 10 μL of sterile water, respectively, to maintain a consistent total volume. All mixed solutions were incubated at room temperature for 10 hours. The released free siRNA was determined by agarose gel electrophoresis (100V, 10min), and the siRNA bands were imaged using an imaging system (iBright FL1500, Thermo Fisher, USA). Figure 13 As shown, siRNA began to be released when the SDS concentration was 0.04%. The group with GSH released more siRNA, proving that the nanoparticles have a GSH response and can achieve targeted release at the tumor site.

[0078] Figure 13 In the study, the Free siRNA group used siRNA solution (50 ng / μL) as a positive control, comparing the release of siRNA with the presence of SDS and GSH. Without siRNA consisted only of nanoparticle solution, without SDS and GSH. The GSH group consisted of experiments with both nanoparticle solution and GSH.

[0079] Example 9: Oxidation-Sensitive Release of Nanoparticles

[0080] First, following the preparation method of the nanoparticle solution in Example 6, P10 / C4 / 30 was selected as the gene vector, and a nanoparticle solution containing 50 ng / μL of siRNA was prepared at a mass ratio of siRNA to gene vector of 1:30. Subsequently, SDS solutions of different concentrations (w / v) were prepared: 0.02%, 0.04%, 0.06%, and 0.08%, respectively, along with a 10 mM H2O2 solution. The experiment was divided into two groups: in the first group, 5 μL of nanoparticle solution was mixed with 1 μL of SDS solution of different concentrations and 1 μL of H2O2 solution, respectively; in the second group, 5 μL of nanoparticle solution was mixed with 1 μL of SDS solution of different concentrations and 1 μL of sterile water, respectively, to maintain a consistent total volume. All mixed solutions were incubated at room temperature for 10 hours. The released free siRNA was determined by agarose gel electrophoresis (100V, 10min), and the siRNA bands were imaged using an imaging system (iBright FL1500, Thermo Fisher, USA). Figure 14 As shown, when the SDS concentration was 0.04%, the group with H2O2 began to release a small amount of siRNA. As the SDS concentration increased, the group with H2O2 released more siRNA, demonstrating that the nanoparticles have an oxidative response and can achieve targeted release at the tumor site.

[0081] Figure 14 In the study, the Free siRNA group used siRNA solution (50 ng / μL) as a positive control, comparing the release of siRNA in the presence of SDS and H2O2. The Without group consisted only of nanoparticle solution, without SDS and H2O2. The H2O2 group consisted of both nanoparticle solution and H2O2.

[0082] Example 10: Cell transfection and cytotoxicity of nanoparticles

[0083] 0.5 g / L siRNA solution and 5 mg / mL gene vector solution were mixed at a siRNA:gene vector mass ratio of 1:30 in a 10 mM citrate-disodium hydrogen phosphate buffer solution (pH 4.0) and incubated for 30 min. Human cervical cancer cells (HeLa-Luc cells) stably expressing firefly luciferase were seeded into 96-well white plates (10,000 cells per well, 100 μL LMEM). After culturing at 37°C for 24 h, the original culture medium was discarded, and 200 μL of fresh culture medium and 20 μL of nanoparticle solution were added. Incubation was continued at 37°C for another 24 h, and the luminescence value was measured to assess the expression level of luciferase. Normalized analysis was performed using the untreated group as a negative control. Results are shown below. Figure 15 .

[0084] Figure 15 In the diagram, the horizontal axis P10 and the underlined 50%, and the vertical axis C4 represent the gene vector as P10 / C4 / 50, and so on.

[0085] Example 11: Cytotoxicity of Nanoparticles

[0086] HeLa-Luc cells were seeded into 96-well clear plates (10,000 cells per well, 100 μL DMEM). After culturing at 37°C for 24 h, the original medium was discarded, and 200 μL of fresh medium and 20 μL of polymer@siLuc nanoparticle solution were added. After further incubation at 37°C for 24 h, 20 μL of MTT reagent was added to each well. After culturing for another 4 h, the medium was discarded, and 150 μL of DMSO was added to dissolve the blue-purple formazan crystals produced by the live cells. The absorbance at 490 nm was measured using a microplate reader. Normalized analysis was performed using the untreated group as a negative control. The results are shown below. Figure 16 .

[0087] Combined analysis of cell transfection and toxicity, such as Figure 15 , 16 As shown, P20 / C4 / 30 exhibited relatively good transfection results with virtually no cytotoxicity; the entire fatty alcohol modified series showed low cytotoxicity.

[0088] Example 12: Cellular uptake of nanoparticles

[0089] The siRNA used in this invention for detecting cellular uptake is Cy5-siRNA (Cy5-siRNA), a red fluorescent dye. A 0.5 g / L Cy5-siRNA solution and a 5 mg / mL gene vector solution were mixed at a Cy5-siRNA to gene vector mass ratio of 1:30 in a 10 mM citrate-disodium hydrogen phosphate buffer solution (pH 4.0). The mixture was incubated for 30 min. HeLa-Luc cells were seeded in 6-well plates (100,000 cells per well) and incubated for 24 hours. The original culture medium was replaced with 1 mL of fresh medium in each well, and 200 μL of polymer@Cy5-siRNA nanoparticles were added. After incubation for 2 or 6 hours, the culture medium was removed, the cells were washed three times with PBS, and fixed with 4% paraformaldehyde (1 mL / well) at 4°C for 30 min. After removing the fixative, the cells were washed three times with PBS. 1 mL of DMSO solution (5 μg / mL) of 4',6-diamidinyl-2-phenylindole (DAPI) was added to each well, and the cells were stained in an incubator for 15 min. Cell uptake images were observed and captured using a fluorescence microscope (EVOS M5000, Thermo Fisher, USA). Results are shown below. Figure 17 .

[0090] To quantify cellular uptake of nanoparticles, HeLa-Luc cells were seeded in 6-well plates (400,000 cells per well) and incubated for 24 hours. Then, 1 mL of fresh culture medium was added to each well, followed by the addition of 80 μL of polymer@Cy5-siRNA nanoparticles. Incubation continued for 6 hours. The culture medium was removed, cells were digested with trypsin, centrifuged to remove the supernatant, washed with PBS, and resuspended in 1 mL of PBS. Cy5 fluorescence intensity was measured in each well using flow cytometry (CytoFLEX, Beckman Coulter, USA). Figure 17 As shown, red indicates siRNA and blue indicates cell nuclei. Nanoparticles began to be taken up by cells at 2 hours, and with increasing time, the overlapping pink area increased by 6 hours, indicating increased cellular uptake. Flow cytometry results are shown below. Figure 18 As shown, all of these materials have high cellular uptake, indicating good cellular uptake.

Claims

1. An oxidation-reduction sensitized cationic polymeric gene carrier, characterized by, The polymer structure is shown in general formula (I) Wherein n represents the degree of polymerization, n is 70-3000, corresponding to the main chain molecular weight of 5000-300000 g / mol; m represents the number of modification units, the ratio of m / n is 10-100%; wherein the substituent R is one of the following.

2. The redox-sensitive cationic polymer gene vector as described in claim 1, characterized in that, The ratio of m / n is 10-50%.

3. A gene therapy medicine, characterized by, The cationic polymer gene carrier of claim 1 and a gene drug loaded on the gene carrier, wherein the gene drug is one of siRNA, mRNA and shRNA.

4. The method of producing a gene therapy medicine according to claim 3, wherein The preparation method of the gene therapy drug comprises the following steps: preparing the cationic polymer gene carrier into a carrier solution with a concentration of 1-10 mg / mL, preparing a gene drug solution with a concentration of 0.1-10 μg / μL, mixing the carrier solution and the drug solution in a buffer solution, blowing and beating to be uniform, and incubating at room temperature in a shaking bed at 50-200 rpm for 0.5-2 h.

5. The production method according to claim 4, wherein When the carrier solution and the drug solution are mixed, the mass ratio of the carrier to the gene drug is 15:1-60:

1.

6. The production method according to claim 4, wherein The solvent for preparing the carrier solution is one of methanol, ethanol, THF and DMSO.

7. The production method according to claim 4, wherein The drug solution is a DEPC water solution of nucleic acid, and the DECP water is a DECP treated high-temperature and high-pressure sterilized ultrapure water.

8. The production method according to claim 4, wherein The buffer solution is a citric acid-disodium hydrogen phosphate buffer solution with a pH value of 3.4-6.

0.

9. The cationic polymer gene carrier of claim 1 in the preparation of a gene therapy drug.

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