WO2021197275A1 - 一种胍基衍生物及其基因递释系统 - Google Patents

一种胍基衍生物及其基因递释系统 Download PDF

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WO2021197275A1
WO2021197275A1 PCT/CN2021/083643 CN2021083643W WO2021197275A1 WO 2021197275 A1 WO2021197275 A1 WO 2021197275A1 CN 2021083643 W CN2021083643 W CN 2021083643W WO 2021197275 A1 WO2021197275 A1 WO 2021197275A1
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gene
delivery system
hydrogel
viral vector
gene delivery
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刘敏
王董理
宋杰
王婧
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复旦大学
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Definitions

  • the invention belongs to the field of pharmacy and biology, and relates to a guanidine derivative with high transfection efficiency and good biocompatibility and a gene delivery system thereof, in particular to a cationic material modified with 1,3-isophthaloylguanidine , And a gene complex formed by enveloping genes, and a hydrogel gene delivery system formed by dispersing the gene complex in a polymer solution.
  • Gene therapy refers to the use of non-biological or biological methods to introduce genes into the patient’s organs, tissues and cells, by expressing proteins, interfering with protein expression, or affecting cell pathways to correct or improve intracellular pathological conditions, thereby achieving the purpose of treating diseases .
  • Non-biological methods for introducing genes into cells include microinjection, gene gun, electroporation, and ultrasound introduction. These methods are suitable for in vitro gene introduction, but are difficult to apply to in vivo gene introduction.
  • the biological method is to carry the gene through the gene carrier and enter the target cell to achieve the purpose of gene transfection.
  • the vectors that carry genes are mainly divided into two categories: viral vectors and non-viral vectors.
  • Viral vectors take advantage of the characteristics of viruses that can infect host cells and have high transfection efficiency. At present, most of the gene vectors that enter clinical research are viral vectors. However, viral vectors also have many problems, such as complex preparation processes, low gene loading, strong immunogenicity, and potential pathogenicity and tumorigenicity, which limit their wide application.
  • the non-viral vector is obtained by chemical synthesis, has the advantages of controllable quality and batch-to-batch difference, low immunogenicity and high gene carrying capacity, and has good application prospects. Compared with viral vectors, non-viral vectors have the disadvantage of low transfection efficiency, which limits the application of non-viral vectors. Therefore, improving the transfection efficiency of non-viral vectors is the focus of gene therapy research.
  • Non-viral vectors need to overcome the following physiological barriers in the process of gene delivery: 1
  • the blood barrier protection gene effectively avoids the phagocytosis of the reticuloendothelial system and the degradation of nucleases in the blood; 2
  • the cell barrier carries genes across the amphiphilic cell membrane into the cell; 3
  • the intracellular transport barrier assists the escape of gene endosomes/lysosomes and crosses the nuclear membrane to achieve gene transfection.
  • Each of these physiological barriers directly affects the efficiency of gene transfection.
  • An ideal gene carrier needs to overcome the above-mentioned physiological barriers in order to deliver the gene to a specific part of the cell to achieve the purpose of gene therapy.
  • Functional group modification is a common strategy for overcoming the above-mentioned physiological barriers.
  • functional groups such as hydrophobic groups, penetrating peptides, homing molecules, imidazole groups, guanidine groups, fusion proteins, and nuclear localization signal peptides on existing non-viral vectors.
  • phenyl modification can improve the stability of gene complexes and help non-viral vectors carry genes across cell membranes.
  • guanidine modification helps non-viral vectors compress genes and span cell and nuclear membranes.
  • the present invention designs a functional group that can be used for gene delivery—1,3-isophthaloyl guanidine. Connect it with a cationic material through a chemical bond to obtain a new type of non-viral vector, which contains the gene complex formed by the gene, and the gene complex is dispersed in the hydrogel gene delivery system formed by the polymer solution to improve The purpose of gene transfection efficiency.
  • the purpose of the present invention is to provide a guanidine derivative with high transfection efficiency and good biocompatibility and a gene delivery system thereof, specifically related to 1,3-isophthaloylguanidine modified cationic materials, and A gene complex formed by enveloping genes, and a hydrogel gene delivery system formed by dispersing the gene complex in a high-molecular polymer solution.
  • the first aspect of the present invention provides a non-viral vector with high transfection efficiency and good biocompatibility.
  • the structure of the non-viral vector is shown in formula I:
  • R represents a cationic material, which can be polyamide-amine, polyethyleneimine, polylysine, polyarginine, chitosan, polypropyleneimine, polyurethane, polyphosphazene, polymethacrylic acid-N,N -Dimethylaminoethyl, protamine, spermine or cationic lipids;
  • n is any number between 1-100.
  • non-viral vector according to the first aspect of the present invention, wherein the structure of the non-viral vector is as shown in formula II:
  • PAMAM is a polyamide-amine dendrimer with ethylenediamine as the core.
  • the branching generation number can be any number between 1-8; n is any number between 1-100.
  • non-viral vector according to the first aspect of the present invention, wherein the non-viral vector has good gene compression, cellular uptake, endosome/lysosome escape, and nuclear membrane traversal capabilities.
  • the second aspect of the present invention provides a gene complex, the gene complex comprising the non-viral vector of the first aspect and the gene encapsulated by the vector;
  • the gene is selected from one or more of a reporter gene, a therapeutic gene or a genetic vaccine.
  • the gene complex according to the second aspect of the present invention wherein the gene is selected from one or more of the following: DNA, siRNA, mRNA, shRNA, lncRNA, antisense nucleotides.
  • the third aspect of the present invention provides a hydrogel gene delivery system, the hydrogel gene delivery system is to disperse the gene complex described in the second aspect in a biocompatible polymer solution form.
  • hydrogel gene delivery system according to the third aspect of the present invention, wherein the hydrogel gene delivery system is a slow release gene delivery system.
  • the biocompatible high molecular polymer includes polyvinyl alcohol, polylactic acid, polyethylene glycol, polylactic acid (glycolic acid)-poly Ethylene glycol-polylactic acid (glycolic acid) block copolymer, polyphosphate, polycarbonate, chitin, chitosan, alginate, hyaluronic acid, heparin, chondroitin sulfate, cellulose, agar, starch Derivatives, dextran derivatives, collagen, gelatin, fibrin, silk protein, polyphosphazene, polyglycolic acid, polycaprolactone, polypropylene fumarate, polyhydroxyethyl methacrylate, Any one or more of poly(N-isopropylacrylamide), poly(methacrylic acid oligoethylene glycol), carbomer or poloxamer.
  • the biocompatible high molecular polymer includes polyvinyl alcohol, polylactic acid, polyethylene glycol, polylactic acid (glycolic acid
  • the fourth aspect of the present invention provides the non-viral vector of the first aspect, the gene complex of the second aspect, or the hydrogel gene delivery system of the third aspect.
  • the drug is a gene vaccine drug for inducing mucosal immunity of the respiratory tract to protect against viral infectious diseases.
  • the medicine is used for the treatment of cancer, genetic disease, infectious disease, cardiovascular disease or autoimmune disease.
  • the fifth aspect of the present invention provides a medicine for the treatment of cancer, genetic disease, infectious disease, cardiovascular disease or autoimmune disease.
  • the medicine includes: the non-viral vector of the first aspect and the gene of the second aspect The compound or the third aspect of the hydrogel gene delivery system.
  • the sixth aspect of the present invention provides a method for treating cancer, genetic disease, infectious disease, cardiovascular disease or autoimmune disease, the method comprising: administering the first aspect to a subject in need The drug loaded by the non-viral vector, the gene complex of the second aspect or the hydrogel gene delivery system of the third aspect.
  • the seventh aspect of the present invention provides a method for immune protection against viral infectious diseases, the method comprising: administering to a subject in need the drug carried by the non-viral vector of the first aspect, and the gene complex of the second aspect Or the third aspect of the hydrogel gene delivery system.
  • the present invention has designed a functional molecule that can be used for gene delivery—1,3-isophthaloyl guanidine.
  • a new type of non-viral vector is obtained through chemical bond and cationic material connection, named R-BGG, the structure is as follows:
  • BGG stands for 1,3-isophthaloyl guanidine
  • R stands for cationic material, which can be polyamide-amine, polyethyleneimine, polylysine, polyarginine, chitosan, polypropyleneimine, Polyurethane, polyphosphazene, polymethacrylic acid-N,N-dimethylaminoethyl, protamine, spermine and cationic lipids
  • X represents a linking group, which can be -NH-, carbon-nitrogen bond (-NH- CH 2 -, -NH-CH 2 -CH 2 -, -NH-CH 2 -CH 2 -CH 2 -, -NH-CH 2 -CH 2 -CH 2 -CH 2 -, -NH-CH 2 -CH 2 -CH 2 -CH 2 -or -NH-CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -), amide bond (-NH
  • the present invention synthesizes 5-bromomethyl-N,N'-di-tert-butoxycarbonyl-1,3-isophthaloylguanidine according to the synthesis route shown in the following figure, and adopts polyamide-amine 5th generation (G5)
  • the amino group of the compound reacts with the bromomethyl group of the above compound to obtain G5-BGG after deprotection; based on the above reaction principle, BGG can be used for the chemical modification of a variety of cationic materials to obtain a new type of non-viral vector.
  • the novel non-viral vector designed by the present invention can compress gene drugs such as DNA, siRNA, mRNA, shRNA, lncRNA or antisense nucleotides to form gene complexes, and has high gene compression, cell uptake and abilities to cross the nuclear membrane. High gene transfection efficiency and good biocompatibility.
  • the administration mode of the BGG gene complex designed in the present invention can be injection administration, nasal mucosal administration, pulmonary administration, skin administration, ocular administration or oral mucosal administration, and is used for cancer and infectious diseases. , Treatment of genetic diseases, cardiovascular diseases or autoimmune diseases.
  • the BGG gene complex constructed by the present invention can be dispersed in a biocompatible polymer solution to form a hydrogel gene delivery system.
  • the administration method in the organism can be injection or nasal mucosal administration. , Pulmonary administration, skin administration, ocular administration or oral mucosal administration, for the treatment of cancer, genetic diseases, infectious diseases, cardiovascular diseases or autoimmune diseases.
  • the present invention provides the preparation method of BGG and G5-BGG, the cytological evaluation of the gene complex formed by G5-BGG encapsulated gene, and the material basis of the hydrogel gene delivery system constructed by the gene complex for tumor treatment.
  • the test results of the present invention show that the transfection efficiency of G5-BGG is significantly higher than that of the positive control PEI 25K; G5-BGG containing the therapeutic gene pORF-hTRAIL significantly inhibits the growth of tumor cells in vitro, and the G5-BGG/pORF-hTRAIL hydrogel gene is loaded
  • the delivery system significantly inhibited the growth of tumors in nude mice models of subcutaneous tumors.
  • the cell uptake test examines the ability of the G5-BGG/pDNA complex to enter the cell.
  • Green fluorescent protein transfection test and luciferase quantitative transfection test were used to investigate the transfection efficiency of G5-BGG/pDNA complex cells.
  • the MTT method was used to investigate G5-BGG/pORF-hTRAIL complex cells Learn the anti-tumor effect.
  • Figure 1 shows the particle size potential characterization of the gene complex
  • the particle size of the composite formed by G5-BGG and pGL3 is significantly smaller than the particle size of the composite formed by the other three materials and pGL3.
  • G2-AM can compress pGL3 to form nanoparticles with a particle size of about 150nm. Zeta potential Around 20mV.
  • Figure 2 shows the agarose gel electrophoresis image
  • the gene When the mass ratio of G5-BGG, G5 and G5-GUA to pGL3 is 1:1, the gene can be completely compressed; when the mass ratio of G5-BEN to pGL3 is 2:1, the gene can be completely compressed.
  • Figure 3 shows the cytotoxicity of G5, G5-GUA, G5-BEN and G5-BGG materials
  • Figures A, B, and C are HEK 293T, HeLa and SGC7901 cells, respectively.
  • concentration of G5 G5-GUA, G5-BEN and G5-BGG is 100 ⁇ g/mL and below, the survival rates of HEK293T, HeLa and SGC7901 cells are all above 85%.
  • Figure 4 shows the cellular uptake of G5-BGG/pGL 3 complex
  • Figures A, C, and E show semi-quantitative data uptake by flow cytometry
  • Figures B, D, and F show the fluorescence curves of uptake by flow cytometry
  • Figures A and B are HEK293T cells
  • Figures C and D are HeLa cells
  • Figures E and F are SGC 7901 cells.
  • the uptake of G5-BGG/pGL3 is the highest among the three types of cells, and the uptake is significantly different from the uptake of G5/pGL3, G5-GUA/pGL3 and G5-BEN/pGL3 (***p ⁇ 0.001); G5-BGG/ The uptake of pGL3 was 25K higher than the positive control PEI.
  • Figure 5 shows a picture of the qualitative transfection of HEK 293T cells with G5-BGG/pEGFP-N2
  • G5-BGG Different modification ratios of G5-BGG have higher transfection efficiency, and the transfection efficiency is higher than that of G5, G5-GUA, G5-BEN and the positive control PEI 25K.
  • Figure 6 shows a picture of the qualitative transfection of HEK 293T cells by PEI 1800-BGG/pEGFP-N2
  • the transfection efficiency of PEI 1800-BGG is significant PEI 1800, which is equivalent to the positive control PEI 25K.
  • FIG. 7 shows G5-BGG/pGL 3 quantitative transfection and serum stability
  • Figures A, C, and D are the quantitative transfection results of HEK 293T, HeLa and SGC 7901 cells, respectively.
  • the transfection efficiency is G5-BGG>G5-BEN>G5-GUA>G5 under the same mass ratio conditions. ; At an appropriate mass ratio, the transfection efficiency of G5-BGG can be better than the positive control PEI 25K.
  • Figure B shows the quantitative transfection results of G5-BGG/pGL 3 on HEK 293T cells under different percentage serum concentrations.
  • the serum concentration is in the range of 0%-40% (v/v), G5-BGG/pGL 3 There was no significant change in transfection efficiency; the transfection efficiency of G5-BGG/pGL 3 decreased slightly when the serum concentration was in the range of 60% to 100% (v/v).
  • Figure 8 shows the cytological efficacy of the G5-BGG/pTRAIL complex
  • Figure A shows the survival rate of HeLa cells after administration.
  • the inhibitory rate of G5-BGG/pTRAIL on HeLa cells is 77.8%, which is higher than that of G5/pTRAIL (10.1%), G5-GUA/pTRAIL (17.9%) and G5-BEN/ pTRAIL (21.7%), there is a significant difference (***p ⁇ 0.001); the inhibition rate of G5-BGG/pTRAIL is higher than the positive control PEI25K/pTRAIL.
  • Figure B shows the survival rate of SGC 7901 cells after administration.
  • the inhibition rate is G5-BGG/pTRAIL (71.1%), which is higher than G5/pTRAIL (2.2%), G5-GUA/pTRAIL (19.4%) and G5-BEN/pTRAIL (21.7%), there is a significant difference (***p ⁇ 0.001); the inhibition rate of G5-BGG/pTRAIL is higher than the positive control PEI 25K/pTRAIL.
  • Figure 9 shows the characterization of the PVA hydrogel and the release of the gene complex
  • Figure A is a photo of PVA solution and PVA hydrogel.
  • Figures B and C are graphs of elastic modulus (G') and viscous modulus (G”) with strain and frequency, respectively. Both figures show that G'is on the order of 103 after PVA crosslinking, which is greater than G” ( 10 2 orders of magnitude), a PVA hydrogel is formed; and when the strain is less than 0.1 and the frequency is less than 1rad/s, the G'and G" values of the PVA solution are both less than 1, and G">G' is a solution state.
  • Figure D shows the cumulative release of the gene complex from the PVA hydrogel. It was 40% released in the first 2 hours and then slowly released. The cumulative release at 72 hours was 81%, indicating that the PVA hydrogel can slowly release the gene complex.
  • Figure 10 shows the efficacy and in vivo safety evaluation of the G5-BGG/pTRAIL hydrogel gene delivery system against HeLa subcutaneous xenografts
  • Figure A shows the tumor volume change curve after administration.
  • Figure B shows the tumor weight data.
  • the G5-BGG group has the smallest tumor weight, which is significantly different from other groups.
  • Figures C, D, E, and F are the alanine aminotransferase (ALT), aspartate aminotransferase (AST), urinary creatinine (CR), and uric acid (UA) index data in mice 2 days after the administration, and there is no between each group Significant difference.
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • CR urinary creatinine
  • U uric acid
  • Methyl 5-bromomethyl isophthalate (6g, 20.98mmol) was dissolved in 100mL of glacial acetic acid, and then 100mL of 40% hydrobromic acid was slowly added. After reacting at 120°C for 12h, it was cooled to room temperature, and the reaction system was gradually changed. It was added dropwise to 1000 mL of ice water, filtered under reduced pressure to obtain a solid crude product. The crude product was recrystallized from acetonitrile. After vacuum drying, the intermediate 5-bromomethyl isophthalic acid (3.2 g, 12.41 mmol) was obtained. Yield It is 59.13%.
  • the dialysate is firstly used with dimethyl sulfoxide, then changed to water, and the product is obtained by freeze-drying.
  • the modification ratio is determined to be 1, which is abbreviated as G5-BGG by hydrogen nuclear magnetic resonance spectroscopy.
  • G5 reacts with 5-bromomethyl-N,N'-di-tert-butoxycarbonyl-1,3-isophthaloylguanidine in different molar ratios to obtain materials with different modification ratios, with a modification ratio of 18 , 38 and 95 are abbreviated as G5-BGG(18), G5-BGG(38) and G5-BGG(95), respectively.
  • G5-GUA 1H-pyrazole-1-carboxamidine hydrochloride and DIEA were dissolved in water, reacted at room temperature for 24 hours, and then deeply dialyzed with water. After lyophilization, G5-GUA was obtained. The modification ratio of G5-GUA was determined by the free amino group quantitative method, and the modification ratio was 3, which was used as the reference substance in the follow-up experiment.
  • G5-BEN was obtained by freeze-drying in water, and its modification ratio was determined to be 2 by proton nuclear magnetic resonance spectroscopy, which was used as a reference substance for subsequent experiments.
  • Gene complex is prepared by mixing a solution of the volume of the support material pGL 3 solution and the like, of pGL3 final concentration of 40 ⁇ g / mL, immediately vortexed 30s, room temperature for 30min, to obtain a freshly prepared complex solution, the concentration ratio adjusting materials according to quality.
  • the ratio of carrier to pGL3 in the composite is expressed by mass ratio, and the particle size and Zeta potential of each sample are measured with Malvern NanoZS particle size analyzer. The results are shown in Figure 1.
  • the agarose gel electrophoresis blocking experiment investigates the compression ability of the carrier material on pGL3.
  • the specific experimental plan is as follows: Weigh 0.3g agarose into an Erlenmeyer flask, add 30ml of 1 ⁇ TAE buffer, and place it in a microwave oven (medium-high heat) Heat 2-3 times non-continuously until the Agarose is completely dissolved. Cool at room temperature to 50°C and add 3 ⁇ L gel red, mix well, pour the gel, and insert the comb. Leave it at room temperature for 40 minutes or a little longer, and pull out the comb after the glue has solidified. Pour 1 ⁇ TAE into the electrophoresis tank in advance, put the glue in, and the liquid can cover the glue surface.
  • Pave a 96-well cell culture plate add 200 ⁇ l of 10% serum-containing DMEM culture medium to each well, containing 3 ⁇ 10 3 cells/well, and incubate overnight at 37°C and 5% CO 2. Add the material to the 96-well plate. Holes 20 ⁇ l, each sample is set with 3 multiple holes, placed in a cell incubator and incubated for 4h, replaced with 10% serum-containing DMEM medium and continued to incubate for 48h, prepare 5mg/mL MTT solution, filter with 0.22 ⁇ m filter membrane, Add to a 96-well plate with 20 ⁇ l per well. After culturing for 4 hours, discard the cell culture medium. Add 200 ⁇ l DMSO to each well. After standing at room temperature for 10 minutes, shake to completely dissolve the purple crystals. The microplate reader measures the absorbance at 490nm wavelength. The results are shown in Figure 3.
  • TOTO-3 labeled pGL3 plasmid Take 10 ⁇ L of TOTO stock solution (1mM) and dissolve it in 500 ⁇ L of PBS to obtain TOTO working solution. Take another 500 ⁇ L 0.8 mg/mL DNA solution, add it to the TOTO working solution, vortex to mix, and place at room temperature for 30 minutes to obtain a TOTO-3 labeled DNA solution with a concentration of 0.4 mg/mL and store at 4°C. When used, it is diluted to the required concentration, the excitation wavelength is 642nm, and the emission wavelength is 660nm.
  • Example 4 different materials and TOTO-3 labeled pGL3 plasmids were used to prepare gene complexes of different materials (the mass ratio was 12:1).
  • the cells were trypsinized, resuspended, and counted.
  • the cell density was diluted to 1 ⁇ 10 5 cells/mL with complete culture medium, and 1ml of cell suspension was added to each well of a 12-well plate, and cultured overnight to make the cells adhere to the wall; Fresh gene complex, trypsinize the cells after 4h incubation, resuspend in PBS, and detect by flow cytometry.
  • the results are shown in Figure 4.
  • G5-BGG/pGL3 uptake is the highest.
  • Quantitative cell transfection experiment inoculate the cells into a 48-well plate at 2 ⁇ 10 4 cells/well, each well containing 0.5mL DMEM medium (containing 10% serum), and culture overnight until the cell confluence reaches 70%-80% , Replace the original cell culture medium with 450 ⁇ L of fresh cell culture medium per well and add the freshly prepared carrier material/pGL 3 complex (the mass ratio is 4:1, 8:1, 12:1 and 16:1), per well 50 ⁇ l, containing 2 ⁇ g pGL 3 , cultured at 37°C for 4h, change to fresh cell culture medium, continue to incubate at 37°C for 48h, then discard the culture medium, add 100 ⁇ l of cell lysate to each well, leave it for 1-2min, pipette Pipette to assist cell lysis, then transfer the cell lysate to a 1.5mL centrifuge tube, centrifuge at 15000 ⁇ g, 4°C for 2min, and transfer the supernatant to an EP tube for determination.
  • DMEM medium
  • Serum stability investigation Cells were seeded on a 48-well plate at 2 ⁇ 10 4 cells/well, each well contained 0.5 mL DMEM medium (containing 10% serum), and cultured overnight until the cell confluence reached 70%-80%. Prepare DMEM culture medium with different percentage serum concentration, replace the original cell culture medium with 450 ⁇ L cell culture medium with different serum concentration, and add freshly prepared carrier material/pGL 3 complex (mass ratio 12), 50 ⁇ l per hole, Contain 2 ⁇ g pGL 3 , cultured at 37°C for 4h, change to fresh cell culture medium, continue to culture at 37°C for 48h, then discard the culture medium, add 100 ⁇ l cell lysate to each well, place for 1-2min, pipette To assist cell lysis, then transfer the cell lysate to a 1.5mL centrifuge tube, centrifuge at 15000 ⁇ g, 4°C for 2min, and transfer the supernatant to an EP tube for determination.
  • DMEM medium containing 10% serum
  • the Promega kit operation guide prepare the luciferase substrate into a working solution, then take 40 ⁇ l of the supernatant and place it in the test tube, add 90 ⁇ l of the luciferase substrate, and put it into the ultra-weak luminescence analyzer to measure the luminescence Intensity, the collection time is 10s, the time interval is 0.1s, and then the total protein content of the cells is measured by the MicroBCA method.
  • the transfection result is expressed by the fluorescence intensity per unit mass of total protein (RLU/mg protein).
  • RLU/mg protein fluorescence intensity per unit mass of total protein
  • HeLa cells and SGC 7901 cells were seeded on a 96-well plate at a density of 3 ⁇ 10 3 cells/well, each well contained 200 ⁇ L DMEM medium (10% serum), and cultured overnight until the cell confluence reached 70%-80%.
  • TRITC tetramethylrhodamine
  • Sample preparation for in vivo administration Use PBS to configure each material to 20 mg/mL, and the plasmid concentration to 1.6 mg/mL. Add 220 ⁇ L of plasmid to a 1.5mL EP tube, then add 220 ⁇ L of material solution dropwise, vortex for 30s and then let it stand for 30min. Mix 440 ⁇ L of gene complex with an equal volume of 10% PVA (Mw 89000-98000) solution. Prepare terephthalic acid solution with a concentration of 10mg/mL.
  • each nude mouse is injected with 100 ⁇ L of PVA/gene complex mixed solution next to the tumor, and 50 ⁇ L of terephthalic acid is injected at the same position.
  • PVA and terephthalic acid can be cross-linked to form a gel, which can make the gene complex Slowly release next to the tumor to achieve the purpose of continuous treatment. It was administered only once, and the tumor diameter and the body weight of the nude mice were measured before the administration, and the tumor diameter was measured every two days thereafter. The mice were sacrificed on the 14th day after the administration, and the tumors were weighed. The results are shown in Figure 10.
  • the in vivo safety was investigated using normal non-tumor-bearing ICR mice, and the preparation and administration of the gene complex were the same as above. Two days after the administration, blood was taken from each mouse, and the serum was taken after centrifugation at 3000 rpm for 5 min. In vivo safety select liver function indicators alanine aminotransferase (ALT), aspartate aminotransferase (AST) and renal function indicators urine creatinine (CR), uric acid (UA) reaction. The concentrations of the four substances in the serum were determined using an automatic biochemical analyzer. The results are shown in Figure 10.

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Abstract

本发明属于药学与生物领域，涉及一种具有高转染效率和良好生物相容性的胍基衍生物及其基因递释系统，具体涉及1,3-间苯二甲酰胍修饰的阳离子材料(其结构如式I所示)，及其包载基因形成的基因复合物，以及上述基因复合物分散于高分子聚合物溶液形成的水凝胶基因递释系统。式I中：R代表阳离子材料，可以是聚酰胺-胺、聚乙烯亚胺、聚赖氨酸、聚精氨酸、壳聚糖、聚丙烯亚胺、聚氨酯、聚磷腈、聚甲基丙烯酸-N,N-二甲氨基乙酯、精蛋白、精胺和阳离子脂质；X代表连接基团，可以是-NH-、碳氮键(-NH-CH2-、-NH-CH2-CH2-、-NH-CH2-CH2-CH2-、-NH-CH2-CH2-CH2-CH2-、-NH-CH2-CH2-CH2-CH2-CH2-或-NH-CH2-CH2-CH 2-CH2-CH2-CH2-)、酰胺键(-NH-CO-)、亚胺键(-N＝C-)、脲(-NH-CO-NH-)、硫脲(-NH-CS-NH-)或脒(-N＝CH-NH-)；n可以是1～100之间的任意数字。

Description

一种胍基衍生物及其基因递释系统
相关申请的交叉引用
本申请要求2020年03月30日提交的第CN202010239778.3号中国发明专利申请的优先权，所述申请以引用的方式整体并入本文。
技术领域
本发明属于药学与生物领域，涉及一种具有高转染效率和良好生物相容性的胍基衍生物及其基因递释系统，具体涉及1,3-间苯二甲酰胍修饰的阳离子材料，及其包载基因形成的基因复合物，以及上述基因复合物分散于高分子聚合物溶液形成的水凝胶基因递释系统。
背景技术
基因治疗是指利用非生物或生物学方法将基因导入到患者器官、组织和细胞中，通过表达蛋白、干扰蛋白表达或影响细胞通路，以纠正或改善细胞内病理状态，从而达到治疗疾病的目的。将基因导入细胞的非生物学方法包括显微注射、基因枪、电穿孔和超声导入等，这些方法适用于体外基因导入，难以应用于体内基因导入。生物学方法是通过基因载体包载携带基因，进入目的细胞达到基因转染的目的。目前包载基因的载体主要分为两类：病毒载体和非病毒载体。病毒载体利用病毒可以感染宿主细胞的特点，具有较高的转染效率，目前大部分进入临床研究的基因载体为病毒载体。但病毒载体也存在着诸多问题，如制备工艺复杂、基因携载量低、免疫原性强以及潜在的致病性和致瘤性等，使其广泛应用收到了限制。非病毒载体由化学合成得到，具有质量及批次间差异可控、免疫原性低以及基因携载量高等优点，具有较好的应用前景。与病毒载体相比，非病毒载体存在转染效率低的缺点，限制了非病毒载体的应用。因此提高非病毒载体的转染效率是基因治疗研究中的重点。
非病毒载体在递送基因的过程中需要克服以下生理屏障：①血液屏障保护基因有效规避血液中网状内皮系统的吞噬和核酸酶的降解；②细胞屏障携带基因跨越两亲性细胞膜进入胞内；③胞内转运屏障协助基因内涵体/溶酶体逃逸，跨越核膜，实现基因转染。其中每一道生理屏障都直接影响到基因的转染效率。理想的基因载体需要克服上述生理性屏障，才能将基因递送至细胞内特定部位，发挥基因治疗的目的。
功能性基团修饰是克服上述生理屏障的常用策略，通过对现有非病毒载体进行疏水基、穿膜肽、寻靶分子、咪唑基、胍基、融合蛋白和核定位信号肽等功能性基团修饰， 以增强基因压缩、基因入胞、内涵体/溶酶体逃逸和核膜跨越等能力。作为疏水基团，苯基修饰可提高基因复合物的稳定性，并有助于非病毒载体携带基因跨越细胞膜。通过与基因和生物膜的磷酸基形成氢键，胍基修饰有助于非病毒载体压缩基因、跨越细胞膜和核膜。
基于苯基的疏水作用、以及平面结构的间苯双胍基与磷酸基的分子识别作用，本发明设计了一种可用于基因递送的功能性基团—1,3-间苯二甲酰胍，将其通过化学键与阳离子材料连接得到一种新型非病毒载体，包载基因形成的基因复合物，并将基因复合物分散于高分子聚合物溶液形成的水凝胶基因递释系统，以达到提高基因转染效率的目的。
发明内容
本发明的目的在于提供一种具有高转染效率和良好生物相容性的胍基衍生物及其基因递释系统，具体涉及1,3-间苯二甲酰胍修饰的阳离子材料，及其包载基因形成的基因复合物，以及上述基因复合物分散于高分子聚合物溶液形成的水凝胶基因递释系统。
本发明的第一方面提供了一种具有高转染效率和良好生物相容性的非病毒载体，所述的非病毒载体的结构如式I所示：
Figure PCTCN2021083643-appb-000001
式I中：
R代表阳离子材料，可以是聚酰胺-胺、聚乙烯亚胺、聚赖氨酸、聚精氨酸、壳聚糖、聚丙烯亚胺、聚氨酯、聚磷腈、聚甲基丙烯酸-N,N-二甲氨基乙酯、精蛋白、精胺或阳离子脂质；
X代表连接基团，可以是-NH-、碳氮键(-NH-CH 2-、-NH-CH 2-CH 2-、-NH-CH 2-CH 2-CH 2-、-NH-CH 2-CH 2-CH 2-CH 2-、-NH-CH 2-CH 2-CH 2-CH 2-CH 2-或-NH-CH 2-CH 2-CH 2-CH 2-CH 2-CH 2-)、酰胺键(-NH-CO-)、亚胺键(-N＝C-)、脲(-NH-CO-NH-)、硫脲(-NH-CS-NH-)或脒(-N＝CH-NH-)；
n是1～100之间的任意数字。
根据本发明第一方面的非病毒载体，其中，所述的非病毒载体的结构如式II所示：
Figure PCTCN2021083643-appb-000002
式II中：
PAMAM为乙二胺为核心的聚酰胺-胺型树枝状聚合物，枝化代数可以是1～8之间的任意代数；n是1～100之间的任意数字。
根据本发明第一方面的非病毒载体，其中，所述非病毒载体具有良好的压缩基因、细胞摄取、内涵体/溶酶体逃逸和跨越核膜的能力。
本发明的第二方面提供了一种基因复合物，所述基因复合物包括第一方面的非病毒载体及由所述载体包载的基因；
优选地，所述基因选自报告基因、治疗基因或基因疫苗中的一种或多种。
根据本发明第二方面的基因复合物，其中，所述基因选自以下一种或多种：DNA、siRNA、mRNA、shRNA、lncRNA、反义核苷酸。
本发明的第三方面提供了一种水凝胶基因递送系统，所述水凝胶基因递送系统为将第二方面所述的基因复合物分散于具有生物相容性的高分子聚合物溶液中形成。
根据本发明第三方面的水凝胶基因递送系统，其中，所述水凝胶基因递送系统为缓慢释放基因递送系统。
根据本发明第三方面的水凝胶基因递送系统，其中，所述的具有生物相容性的高分子聚合物包括聚乙烯醇、聚乳酸、聚乙二醇、聚乳酸(羟基乙酸)-聚乙二醇-聚乳酸(羟基乙酸)嵌段共聚物、聚磷酸酯、聚碳酸酯、甲壳质、壳聚糖、海藻酸盐、透明质酸、肝素、硫酸软骨素、纤维素、琼脂、淀粉衍生物、葡聚糖衍生物、胶原、明胶、血纤蛋白、蚕丝蛋白、聚磷腈、聚乙醇酸、聚己内酯、聚反丁烯二酸丙二醇酯、聚甲基丙烯酸羟乙酯、聚(N-异丙基丙烯酰胺)、聚(甲基丙烯酸低聚乙二醇)、卡波姆或泊洛沙姆的任意一种或者几种。
本发明的第四方面提供了第一方面的非病毒载体、第二方面的基因复合物或第三方面的水凝胶基因递释系统在制备给药方式为注射给药、鼻粘膜给药、肺部给药、皮肤给药、眼部给药或口腔粘膜给药的药物中的应用；
优选地，所述药物为用于诱导呼吸道粘膜免疫从而对病毒感染性疾病免疫保护的基因疫苗药物。
根据本发明第四方面的应用，所述药物为用于癌症、遗传性疾病、感染性疾病、心 血管疾病或自身免疫性疾病的治疗的药物。
本发明的第五方面提供了一种治疗癌症、遗传性疾病、感染性疾病、心血管疾病或自身免疫性疾病的药物，所述药物包括：第一方面的非病毒载体、第二方面的基因复合物或第三方面的水凝胶基因递释系统。
本发明的第六方面提供了一种用于治疗癌症、遗传性疾病、感染性疾病、心血管疾病或自身免疫性疾病的方法，所述方法包括：对有需要的受试者给予第一方面的非病毒载体所负载的药物、第二方面的基因复合物或第三方面的水凝胶基因递释系统。
本发明的第七方面提供了一种病毒感染性疾病的免疫保护方法，所述方法包括：对有需要的受试者给予第一方面的非病毒载体所负载的药物、第二方面的基因复合物或第三方面的水凝胶基因递释系统。
基于苯基的疏水作用、以及平面结构间苯双胍基与磷酸基的分子识别作用，本发明设计了一种可用于基因递送的功能性分子—1,3-间苯二甲酰胍，将其通过化学键与阳离子材料连接得到一类新型非病毒载体，命名为R-BGG，结构如下图：
Figure PCTCN2021083643-appb-000003
R-BGG的结构式
其中BGG代表1,3-间苯二甲酰胍；R代表阳离子材料，可以是聚酰胺-胺、聚乙烯亚胺、聚赖氨酸、聚精氨酸、壳聚糖、聚丙烯亚胺、聚氨酯、聚磷腈、聚甲基丙烯酸-N,N-二甲氨基乙酯、精蛋白、精胺和阳离子脂质；X代表连接基团，可以是-NH-、碳氮键(-NH-CH 2-、-NH-CH 2-CH 2-、-NH-CH 2-CH 2-CH 2-、-NH-CH 2-CH 2-CH 2-CH 2-、-NH-CH 2-CH 2-CH 2-CH 2-CH 2-或-NH-CH 2-CH 2-CH 2-CH 2-CH 2-CH 2-)、酰胺键(-NH-CO-)、亚胺键(-N＝C-)、脲(-NH-CO-NH-)、硫脲(-NH-CS-NH-)或脒(-N＝CH-NH-)；n可以是1～100之间的任意数字。
具体的，本发明按照下图合成路线合成了5-溴甲基-N,N’-二叔丁氧羰基-1,3-间苯二甲酰胍，通过聚酰胺-胺5代(G5)的氨基与上述化合物的溴甲基反应，脱保护基后得到G5-BGG；基于上述反应原理，BGG可用于多种阳离子材料的化学修饰，得到一类新型非病毒载体。
本发明所设计的新型非病毒载体可压缩DNA、siRNA、mRNA、shRNA、lncRNA 或反义核苷酸等基因药物形成基因复合物，具有较高基因压缩、细胞摄取和跨越核膜的能力，呈现高的基因转染效率和良好的生物相容性。
本发明所设计的BGG基因复合物的给药方式可以是注射给药、鼻粘膜给药、肺部给药、皮肤给药、眼部给药或口腔粘膜给药，用于癌症、感染性疾病、遗传性疾病、心血管疾病或自身免疫性疾病的治疗。
本发明构建的BGG基因复合物，可分散于具有生物相容性的高分子聚合物溶液，形成水凝胶基因递释系统，在生物体内的给药方式可以是注射给药、鼻粘膜给药、肺部给药、皮肤给药、眼部给药或口腔粘膜给药，用于癌症、遗传性疾病、感染性疾病、心血管疾病或自身免疫性疾病的治疗。
本发明提供了BGG和G5-BGG的制备方法、G5-BGG包载基因形成基因复合物的细胞学评价，以及上述基因复合物构建的水凝胶基因递释系统用于肿瘤治疗的物质基础。本发明试验结果表明：G5-BGG转染效率显著高于阳性对照PEI 25K；G5-BGG包载治疗基因pORF-hTRAIL显著抑制体外肿瘤细胞的生长，载G5-BGG/pORF-hTRAIL水凝胶基因递释系统显著抑制皮下瘤裸鼠模型肿瘤的生长。
1.G5-BGG的合成
按下图所示的合成路线，得到5-溴甲基-N,N’-二叔丁氧羰基-1,3-间苯二甲酰胍，通过乙二胺为核心的聚酰胺-胺型树枝状聚合物5代(G5)的氨基与上述化合物的溴甲基反应，脱保护基后得到G5-BGG；作为对照组，同时合成了G5-BEN和G5-GUA。 1H NMR表征各化合物的分子结构。
Figure PCTCN2021083643-appb-000004
G5-BGG、G5-BEN和G5-GUA的合成路线图
2.G5-BGG/pDNA细胞学评价
细胞摄取试验考察G5-BGG/pDNA复合物入胞能力。绿色荧光蛋白转染试验和虫荧光素酶定量转染试验考察G5-BGG/pDNA复合物细胞转染效率，包载治疗基因pORF-hTRAIL后，MTT法考察G5-BGG/pORF-hTRAIL复合物细胞学抗肿瘤效果。
3.载G5-BGG/pDNA水凝胶递释系统体内抗肿瘤作用和安全性评价
构建PVA与对苯二硼酸交联的PVA水凝胶，包载G5-BGG/pORF-hTRAIL复合物，瘤旁注射考察载G5-BGG/pORF-hTRAIL水凝胶基因递释系统体内抗肿瘤作用和安全性评价。
附图的简要说明
以下，结合附图来详细说明本发明的实施方案，其中：
附图1示出了基因复合物粒径电位表征
G5-BGG与pGL3形成的复合物粒径显著小于其他三种材料与pGL3形成成的复合物的粒径，质量比为12时G2-AM能压缩pGL3形成粒径150nm左右的纳米粒，Zeta电位在20mV左右。
附图2示出了琼脂糖凝胶电泳图
G5-BGG、G5和G5-GUA与pGL3的质量比为1:1时，可完全压缩基因；G5-BEN与pGL3的质量比为2:1时，可完全压缩基因。
附图3示出了G5、G5-GUA、G5-BEN和G5-BGG材料细胞毒性
图A、B、C分别为HEK 293T、HeLa和SGC7901细胞。G5、G5-GUA、G5-BEN和G5-BGG浓度为100μg/mL及以下时，HEK 293T、HeLa和SGC 7901细胞的存活率均在85％以上。
附图4示出了G5-BGG/pGL 3复合物细胞摄取
图A、C、E为流式细胞摄取半定量数据，图B、D、F流式细胞摄取荧光曲线图。图A、B为HEK293T细胞，图C、D为HeLa细胞，图E、F为SGC 7901细胞。三种细胞中G5-BGG/pGL3均摄取最高，摄取量与G5/pGL3、G5-GUA/pGL3和G5-BEN/pGL3摄取量有显著性差异(***p<0.001)；G5-BGG/pGL3摄取量高于阳性对照PEI 25K。
附图5示出了G5-BGG/pEGFP-N2对HEK 293T细胞定性转染图片
不同修饰比G5-BGG均具有较高转染效率，转染效率高于G5、G5-GUA、G5-BEN和阳性对照PEI 25K。
附图6示出了PEI 1800-BGG/pEGFP-N2对HEK 293T细胞定性转染图片
PEI 1800-BGG的转染效率显著PEI 1800，与阳性对照PEI 25K相当。
附图7示出了G5-BGG/pGL 3定量转染和血清稳定性
图A、C、D分别为HEK 293T、HeLa和SGC 7901细胞定量转染结果图，在三种细胞中，同一质量比条件下，转染效率G5-BGG>G5-BEN>G5-GUA>G5；在适宜质量比时，G5-BGG的转染效率可优于阳性对照PEI 25K。图B为G5-BGG/pGL 3在不同百分浓度血清条件下对HEK 293T细胞的定量转染结果图，血清浓度在0％～40％(v/v)范围内，G5-BGG/pGL 3转染效率无明显变化；血清浓度在60％～100％(v/v)范围内，G5-BGG/pGL 3转染效率略下降。
附图8示出了G5-BGG/pTRAIL复合物细胞学药效
图A为给药后HeLa细胞存活率，G5-BGG/pTRAIL对HeLa细胞的抑制率为77.8％，高于G5/pTRAIL(10.1％)、G5-GUA/pTRAIL(17.9％)和G5-BEN/pTRAIL(21.7％)，存在的显著性差异(***p<0.001)；G5-BGG/pTRAIL抑制率高于阳性对照PEI25K/pTRAIL。图B为给药后SGC 7901细胞存活率，抑制率为G5-BGG/pTRAIL(71.1％)，高于G5/pTRAIL(2.2％)、G5-GUA/pTRAIL(19.4％)和G5-BEN/pTRAIL(21.7％)，存在的显著性差异(***p<0.001)；G5-BGG/pTRAIL抑制率高于阳性对照PEI 25K/pTRAIL。
附图9示出了PVA水凝胶的表征与基因复合物的释放
图A为PVA溶液和PVA水凝胶照片。图B、C分别为弹性模量(G’)和粘性模量(G”)随应变strain、频率frequency变化图，两图中均显示PVA交联后G’在10 3数量级，大于G”(10 2数量级)，形成了PVA水凝胶；而strain小于0.1、frequency小于1rad/s 时，PVA溶液的G’和G”值均小于1，且G”>G’，为溶液状态。图D为基因复合物自PVA水凝胶中累积释放量，在前2h释放40％，后缓慢释放，72h时累积释放量为81％，说明PVA水凝胶可缓慢释放基因复合物。
附图10示出了载G5-BGG/pTRAIL水凝胶基因递释系统对抗HeLa皮下移植瘤药效与体内安全性评价
图A为给药后肿瘤体积变化曲线，G5-BGG组抑瘤效果显著优于其他组(n＝6，***p<0.001)。图B为肿肿瘤重量数据，G5-BGG组瘤重最小，与其他组相比存在显著性差异。图C、D、E、F分别是给药后2天小鼠体内谷丙转氨酶(ALT)、谷草转氨酶(AST)、尿肌酐(CR)、尿酸(UA)指标数据，各组之间不存在显著性差异。
实施发明的最佳方式
下面通过具体的实施例进一步说明本发明，但是，应当理解为，这些实施例仅仅是用于更详细具体地说明之用，而不应理解为用于以任何形式限制本发明。
本部分对本发明试验中所使用到的材料以及试验方法进行一般性的描述。虽然为实现本发明目的所使用的许多材料和操作方法是本领域公知的，但是本发明仍然在此作尽可能详细描述。本领域技术人员清楚，在上下文中，如果未特别说明，本发明所用材料和操作方法是本领域公知的。
通过下述实施例将有助于进一步理解本发明，但并不限制本发明的内容。
实施例1
5-溴甲基-N,N’-二叔丁氧羰基-1,3-间苯二甲酰胍的合成
将5-甲基间苯二甲酸(10g，55.54mmol)溶于150mL甲醇中，后逐滴缓慢加入4mL浓硫酸，80℃条件下回流6h后冷却至室温，缓慢加入饱和NaHCO 3溶液至pH 8.0左右。二氯甲烷萃取，分液漏斗分离收集二氯甲烷，有机相用饱和氯化钠溶液洗涤三次，无水硫酸镁干燥除去有机相中水分，减压干燥除去有机溶剂，得到中间体5-甲基间苯二甲酸甲酯(10.6g，50.94mmol)，产率为91.72％。 1H-NMR(400MHz,CDCl 3):δ＝8.40(H,s,Ph-H),7.96(2H,s,Ph-H),3.88(6H,s,-OCH 3),2.38(3H,s,-CH 3)ppm。
将5-甲基间苯二甲酸甲酯(8g，38.45mmol)溶于200mL四氯化碳中，加入7.2g溴代琥珀酰亚胺(NBS)和0.16g过氧化二苯甲酰(BPO)作为引发剂，氮气保护条件下90℃条件下回流反应8h，冷却至室温。减压蒸馏出去有机溶剂，所得固体用冰乙醇洗涤三次，真空干燥的到中间体5-溴甲基间苯二甲酸甲酯(7.2g，25.18mmol)，产率为65.48％。 1H-NMR(400MHz,CDCl 3):δ＝8.61(1H,s,Ph-H),8.26(2H,s,Ph-H),4.55(2H,s,-CH 2Br),3.97(6H,s,-OCH 3)ppm。
将5-溴甲基间苯二甲酸甲酯(6g，20.98mmol)溶于100mL冰醋酸中，再缓慢加入100mL 40％氢溴酸，120℃条件下反应12h后冷却至室温，将反应体系逐滴滴加至1000mL冰水中，减压过滤的到固体粗产物，粗产物用乙腈重结晶，真空干燥后的到中间体5-溴甲基间苯二甲酸(3.2g，12.41mmol)，产率为59.13％。 1H-NMR(400MHz,DMSO-d6): δ＝13.20(2H,s,-COOH),8.41(1H,s,Ph-H),8.26(2H,s,Ph-H),4.88(2H,s,-CH 2Br)ppm。
将5-溴甲基间苯二甲酸(1g，3.88mmol)溶于5mL氯仿中，冰盐水浴使体系温度降到0℃以下，无水和氮气保护条件下逐滴缓慢1.2mL草酰氯并在滴加过程中始终保持反应体系温度在0℃以下，反应过夜。将反应后体系减压蒸发至体系剩余2mL左右，得到中间体5-溴甲基间苯二甲酰氯，不做纯化直接用于后续反应。
将Boc-胍(2g，12.56mol)溶于5mL N,N二甲基甲酰胺中，加入1.5mL N,N-二异丙基乙胺(DIEA)，冰水浴和氮气保护条件下逐滴滴加上述5-溴甲基间苯二甲酰氯，并在滴加过程中保持反应体系温度不高于4℃，滴加结束后室温条件下反应12h。反应结束后将反应体系减压蒸发出去可挥发性溶剂，剩余液体加入10倍量的水，用乙酸乙酯萃取，分液漏斗分离收集有机相，有机相使用无水硫酸钠干燥。粗产物通过200目硅胶柱色谱分离纯化(洗脱液：石油醚/乙酸乙酯＝8/1)，得到白色固体5-溴甲基-N,N’-二叔丁氧羰基-1,3-间苯二甲酰胍(276mg，0.51mmol)，产率为13.18％。 1H-NMR(400MHz,DMSO-d6):δ＝11.03(2H,s,-NH-),9.68(2H,s,＝NH),8.76(1H,s,Ph-H),8.63(2H,m,Ph-H),8.30(2H,s,-NH-),4.84(2H,s,-CH2Br),1.48(18H,s,-Boc)ppm。
实施例2
G5-BGG、G5-GUA和G5-BEN的合成
将五代乙二胺为核心的聚酰胺-胺型树枝状聚合物(G5)和5-溴甲基-N,N’-二叔丁氧羰基-1,3-间苯二甲酰胍分别溶解于甲醇和N,N-二甲基甲酰胺中，混匀后加入少量DIEA，氮气保护60℃搅拌12h，减压蒸发有机溶剂，得到黄色粘稠状固体，然后将固体溶解在含有50％三氟乙酸的二氯甲烷中，并将溶液在室温下搅拌6小时后减压蒸发有机溶剂，后加入适量水溶解，10％NaOH溶液将体系pH调节至8左右后透析(截流分子量500～1000Da)，透析液先用二甲基亚砜，后换为水，冻干得到产物，通过核磁共振氢谱确定其修饰比例为1，简写为G5-BGG。核磁图谱为 1H-NMR(400MHz,D 2O):δ＝8.04(3H,s,Ph-H),3.92(10H,s,-NH-CH 2-)3.47-3.20(520H,m,-NH-CH 2-),3.13-2.49(1014H,m,-CH 2-CH 2-),2.38(504H,s,-CH 2CO-)ppm。
通过相同的方法按不同摩尔比的G5和5-溴甲基-N,N’-二叔丁氧羰基-1,3-间苯二甲酰胍反应，得到不同修饰比的材料，修饰比18、38和95，分别简写为G5-BGG(18)、G5-BGG(38)和G5-BGG(95)，核磁共振氢谱结果如下：G5-BGG(18) 1H-NMR(400MHz,D 2O):δ＝8.44-7.66(52H,m,Ph-H),3.76(24H,s,-NH-CH 2-)3.42-3.04(520H,m,-NH-CH 2-),3.02-2.43(825H,m,-CH 2-CH 2-),2.31(504H,s,-CH 2CO-)ppm；G5-BGG(38) 1H-NMR(400MHz,D 2O):δ＝8.53-7.55(114H,m,Ph-H),3.82(54H,s,-NH-CH 2-)3.60-3.08(514H,m,-NH-CH 2-),3.08-2.44(803H,m,-CH 2-CH 2-),2.32(504H,s,-CH 2CO-)ppm；G5-BGG(95) 1H-NMR(400MHz,D 2O):δ＝8.48-7.81(286H,m,Ph-H),3.76(104H,s,-NH-CH 2-)3.49-3.03(520H,m,-NH-CH 2-),3.03-2.40(742H,m,-CH 2-CH 2-),2.29(504H,s,-CH 2CO-)ppm。
将G5、1H-吡唑-1-甲脒盐酸盐和DIEA溶于水中，室温条件下反应24h后用水深度透析，冻干后得到G5-GUA。G5-GUA的修饰比例采用游离氨基定量方法确定，修饰比为3，作后续实验对照品。
将G5溶解在甲醇中，后加入适量苄溴和DIEA，氮气保护60℃搅拌6h，减压蒸发有机溶剂后透析(截流分子量500～1000Da)，透析液先用二甲基亚砜，后换为水，冻干得到G5-BEN，通过核磁共振氢谱确定其修饰比例为2，作后续实验对照品。核磁谱图为 1H-NMR(400MHz,D 2O):δ＝8.03(8H,s,Ph-H),3.90(35H,s,-NH-CH 2-),3.44-3.18(520H,m,-NH-CH 2-),3.13-2.48(1014H,s,-CH 2-CH 2-),2.37(504H,s,-CH 2CO-)ppm。
实施例3
PEI 1800-BGG的合成
将PEI 1800和5-溴-N,N’-二叔丁氧羰基-1,3-间苯二甲酰胍溶解于N,N-二甲基甲酰胺中，搅拌条件下加入醋酸钯(Pd(OAc) 2)、1,1'-联萘-2,2'-双二苯膦(BINAP)和碳酸铯(CsCO 3)，氮气保护，120℃反应过夜。所得混合物经8000rpm离心5min后，吸取上清液，弃沉淀。在冰浴条件下在上清液中加入含有50％三氟乙酸的二氯甲烷中，室温搅拌过夜。减压蒸发有机溶剂，后加入适量蒸馏水溶解，用10％的NaOH溶液将体系pH调节至8左右后透析(截留分子量500-1000Da)，透析液先用二甲基亚砜，后用蒸馏水，收集透析袋中液体，冻干得PEI 1800-BGG，通过核磁共振氢谱确定其修饰比例为1。 1H NMR(400MHz,D 2O)δ7.83(3H,s,Ph-H),3.19(s,34H,-NH-CH 2-),2.93–2.10(m,140H,-CH 2CH 2-)ppm。
实施例4
G5-BGG/pDNA复合物的制备和表征
基因复合物制备方法为将pGL 3溶液与载体材料溶液等体积混合，pGL3终浓度为40μg/mL，立刻涡旋30s，室温放置30min，得新鲜制备的复合物溶液，材料浓度根据质量比调整。复合物中载体与pGL3的比例用质量比表示，用马尔文的NanoZS粒径仪分别测量各样品的粒径和Zeta电位。结果见附图1。
实施例5
琼脂糖凝胶电泳阻滞实验
琼脂糖凝胶电泳阻滞实验考察载体材料对pGL3的压缩能力，具体实验方案如下：称取0.3g琼脂糖于锥形瓶中，加入30ml的1×TAE缓冲液，在微波炉(中高火)中非连续加热2-3次，至Agarose全部溶解。室温冷却至50℃加入3μL gel red，混合均匀后灌胶，插入梳子。室温放置40min或稍长时间，待胶凝固后拔出梳子。将电泳槽中预先倒入1×TAE，放入胶，液体没过胶面即可。随后，将样品(制备方法同上)分别加至样品槽中进行电泳[电泳条件：电压120V，1×TAE，40min，每孔10μl]。DNA条带在紫外下观察并拍照。结果见附图2。
实施例6
材料细胞毒性实验
铺96孔细胞培养板，每孔加入200μl含10％血清的DMEM培液，含有3×10 3cells/ 孔，37℃，5％CO 2条件下培养过夜，将材料加入96孔板中，每孔20μl，每个样品设置3个复孔，放入细胞培养箱中孵育4h，换含10％血清的DMEM培液继续培养至48h，配制5mg/mL的MTT溶液，0.22μm的滤膜过滤，加入到96孔板中，每孔20μl，培养4h后，弃去细胞培养液，每孔加入200μl DMSO，室温放置10min后，摇晃，使紫色结晶完全溶解，酶标仪测定490nm波长下的吸光度。结果见附图3。
实施例7
细胞摄取实验
TOTO-3标记pGL3质粒制备：取10μL TOTO储备液(1mM)，溶于500μL的PBS中，得TOTO工作液。另取500μL 0.8mg/mL的DNA溶液，加入上述TOTO工作液中，涡旋混匀，室温放置30min，得TOTO-3标记的DNA溶液，浓度为0.4mg/mL，4℃保存。使用时稀释至所需浓度，激发波长为642nm，发射波长为660nm。
按照实施例4，使用不同材料和TOTO-3标记的pGL3质粒制备不同材料的基因复合物(质量比为12:1)。
将细胞用胰酶消化、重悬、计数，使用完全培养液将细胞密度稀释为1×10 5个/mL，于12孔板中每孔加1ml细胞悬液，培养过夜使细胞贴壁；加入新鲜基因复合物，孵育4h后胰酶消化细胞，PBS重悬，流式细胞仪检测。结果见附图4，在HEK 293T、HeLa和SGC 7901三种细胞中，G5-BGG/pGL3均摄取最高。
实施例8
绿色荧光蛋白质粒的细胞转染实验
将细胞以2×10 4个/孔接种到48孔板上，每孔含0.5mL DMEM培液(含10％血清)，培养过夜至细胞汇合度达到70％-80％，每孔将原细胞培液替换为450μL新鲜细胞培液后加入新鲜制备的G5-BGG/pEGFP-N2、G5-BGG(18)/pEGFP-N2、G5-BGG(38)/pEGFP-N2、G5-BGG(95)/pEGFP-N2、G5/pEGFP-N2、G5-GUA/pEGFP-N2、G5-BEN/pEGFP-N2、PEI1800/pEGFP-N2、PEI 1800-BGG/pEGFP-N2和PEI 25K/pEGFP-N2复合物(质量比为12)，每孔50μl，含2μg pEGFP-N2质粒，37℃培养4h后，换成新鲜的细胞培液，37℃继续培养至48h，置荧光显微镜下观察。结果见图5和附图6。
实施例9
虫荧光素酶定量转染实验及血清稳定性考察
细胞定量转染实验：将细胞以2×10 4个/孔接种到48孔板上，每孔含0.5mL DMEM培液(含10％血清)，培养过夜至细胞汇合度达到70％-80％，每孔将原细胞培液替换为450μL新鲜细胞培液后加入新鲜制备的载体材料/pGL 3复合物(质量比为4:1、8:1、12：1和16:1)，每孔50μl，含2μg pGL 3，37℃培养4h后，换成新鲜细胞培液，37℃继续培养至48h，然后弃去培养液，每孔加入100μl的细胞裂解液，放置1-2min，用移液器吹打辅助细胞裂解，然后将细胞裂解液转移至1.5mL的离心管中，15000×g，4℃，离心2min，取上清液转移至EP管中待测定。
血清稳定性考察：将细胞以2×10 4个/孔接种到48孔板上，每孔含0.5mL DMEM培液(含10％血清)，培养过夜至细胞汇合度达到70％-80％。配置不同百分浓度血清的DMEM培养液，每孔将原细胞培液替换为450μL不同血清浓度细胞培液后加入新鲜制备的载体材料/pGL 3复合物(质量比为12)，每孔50μl，含2μg pGL 3，37℃培养4h后，换成新鲜细胞培液，37℃继续培养至48h，然后弃去培养液，每孔加入100μl的细胞裂解液，放置1-2min，用移液器吹打辅助细胞裂解，然后将细胞裂解液转移至1.5mL的离心管中，15000×g，4℃，离心2min，取上清液转移至EP管中待测定。
根据Promega试剂盒操作指南，将虫荧光素酶底物配制成工作溶液，然后取40μl上清液置于测试管中，加入90μl虫荧光素酶底物，放入超弱发光分析仪中测定发光强度，采集时间为10s，时间间隔为0.1s，然后用MicroBCA法测定细胞的总蛋白含量，转染结果用单位质量总蛋白所发出的荧光强度表示(RLU/mg protein)。结果见附图7，三种细胞中G5-BGG转染效率最高，在40％血清浓度及以下，G5-BGG/pDNA较稳定，转染效率无明显变化。
实施例10
细胞药效实验
用pORF-hTRAIL作为治疗基因进行体外细胞凋亡实验研究以评价BGG修饰的载体材料包载治疗基因后杀伤肿瘤细胞的能力。将HeLa细胞和SGC 7901细胞以3×10 3个/孔的密度接种到96孔板上，每孔含200μL DMEM培液(10％血清)，培养过夜至细胞汇合度达到70％-80％，每孔换成180μL新鲜的细胞培液，然后加入新鲜制备的载体材料/pORF-hTRAIL基因复合物(HeLa细胞质量比为12，SGC 7901细胞的质量比为4)，每孔20μL，含0.8μg pORF-hTRAIL，37℃培养6h或12h后换成新鲜培养液，继续培养至48h，配制5mg/mL的MTT溶液，0.22μm的滤膜过滤，加入到96孔板中，每孔20μl，培养4h后，弃去细胞培养液，每孔加入200μl DMSO，室温放置10min后，摇晃，使紫色结晶完全溶解，酶标仪测定490nm波长下的吸光度，根据吸光度计算细胞存活率。结果见附图8，G5-BGG/pTRAIL组细胞存活率最低。
实施例11
水凝胶基因递释系统的构建及表征
配置聚乙烯醇(PVA，Mw 89000-98000)溶液，浓度为10％；配置对苯二硼酸溶液，浓度为10mg/mL。制备基因复合物，将基因复合物与10％PVA溶液等体积混合，混匀后加入1/2体积的对苯二硼酸溶液，加入后对苯二硼酸与PVA即交联形成水凝胶，流变仪测定PVA溶液及PVA水凝胶模量值，结果见附图9。
四甲基罗丹明(TRITC)标记小牛胸腺的制备：取2mg小牛胸腺溶解到1mL 0.2M的Na 2CO 3缓冲液(pH9.7)中，加入TRITC 1mg，4℃反应8h。反应液用G15凝胶柱分离，收集前段红色部分，冻干，的TRITC标记的小牛胸腺。激发波长为541nm，发射波长为567nm。
使用PBS将G5-BGG配置为20mg/mL，质粒浓度为1.6mg/mL。1.5mL的EP管中加 入150μL TRITC标记的小牛胸腺，再逐滴滴加150μLG5-BGG溶液，涡旋30s后静置30min。将300μL基因复合物与等体积10％PVA(Mw 89000-98000)溶液混合均匀，混匀后加入300μL 10mg/mL的对苯二硼酸溶液，静置30min使充分交联形成凝胶。在凝胶中加入3mL PBS，在不同的时间点每次取出1mL释放液，在补充1mL PBS。将取出的溶液用十二烷基硫酸钠(SDS)溶液破坏其中基因复合物，酶标仪测定荧光强度，计算释放量，结果见附图9。
实施例12
体内药效学实验及体内安全性考察
HeLa细胞皮下瘤模型构建：将HeLa细胞用胰酶消化，PBS洗涤后定容至每毫升4×10 7个细胞，每只裸鼠右侧近腋窝皮下注射100μL，至肿瘤体积大小为100mm 3(瘤体积＝肿瘤长径×肿瘤短径 2÷2)左右时开始给药。
体内给药样品制备：使用PBS将各材料配置为20mg/mL，质粒浓度为1.6mg/mL。1.5mL的EP管中加入220μL质粒，再逐滴滴加220μL材料溶液，涡旋30s后静置30min。将440μL基因复合物与等体积10％PVA(Mw 89000-98000)溶液混合均匀。配置对苯二硼酸溶液，浓度为10mg/mL。
给药方式：每只裸鼠瘤旁注射100μL的PVA/基因复合物的混合溶液，同一位置再注射50μL对苯二硼酸，PVA与对苯二硼酸可交联形成凝胶，可使基因复合物在瘤旁缓慢释放，达到持续治疗的目的。仅给药1次，给药前测量肿瘤瘤径和裸鼠体重，之后每两天测量瘤径。给药后第14天将老鼠处死，将肿瘤称重。结果见附图10。
体内安全性使用正常未荷瘤ICR小鼠考察，基因复合物制备和给药方式同上。给药后两天后对每只小鼠取血，3000rpm离心5min后取血清。体内安全性选择肝功能指标谷丙转氨酶(ALT)、谷草转氨酶(AST)和肾功能指标尿肌酐(CR)、尿酸(UA)反应。血清中四种物质浓度使用全自动生化分析仪测定，结果见附图10。
尽管本发明已进行了一定程度的描述，明显地，在不脱离本发明的精神和范围的条件下，可进行各个条件的适当变化。可以理解，本发明不限于所述实施方案，而归于权利要求的范围，其包括所述每个因素的等同替换。

Claims (13)

  1. 一种具有高转染效率和良好生物相容性的非病毒载体，其特征在于，所述的非病毒载体的结构如式I所示：
    Figure PCTCN2021083643-appb-100001
    式I中：
    R代表阳离子材料，可以是聚酰胺-胺、聚乙烯亚胺、聚赖氨酸、聚精氨酸、壳聚糖、聚丙烯亚胺、聚氨酯、聚磷腈、聚甲基丙烯酸-N,N-二甲氨基乙酯、精蛋白、精胺或阳离子脂质；
    X代表连接基团，可以是-NH-、碳氮键(-NH-CH 2-、-NH-CH 2-CH 2-、-NH-CH 2-CH 2-CH 2-、-NH-CH 2-CH 2-CH 2-CH 2-、-NH-CH 2-CH 2-CH 2-CH 2-CH 2-或-NH-CH 2-CH 2-CH 2-CH 2-CH 2-CH 2-)、酰胺键(-NH-CO-)、亚胺键(-N＝C-)、脲(-NH-CO-NH-)、硫脲(-NH-CS-NH-)或脒(-N＝CH-NH-)；
    n是1～100之间的任意数字。
  2. 根据权利要求1所述的非病毒载体，其特征在于，所述的非病毒载体的结构如式II所示：
    Figure PCTCN2021083643-appb-100002
    式II中：
    PAMAM为乙二胺为核心的聚酰胺-胺型树枝状聚合物，枝化代数可以是1～8之间的任意代数；n是1～100之间的任意数字。
  3. 根据权利要求1或2所述的非病毒载体，其特征在于，所述非病毒载体具有良好的压缩基因、细胞摄取、内涵体/溶酶体逃逸和跨越核膜的能力。
  4. 一种基因复合物，其特征在于，所述基因复合物如权利要求1至3中任一项所述的非病毒载体及由所述载体包载的基因；
    优选地，所述基因选自报告基因、治疗基因或基因疫苗中的一种或多种。
  5. 按权利要求4所述的基因复合物，其特征在于，所述基因选自以下一种或多种：DNA、siRNA、mRNA、shRNA、lncRNA、反义核苷酸。
  6. 一种水凝胶基因递送系统，其特征在于，所述水凝胶基因递送系统为将权利要求4或5所述的基因复合物分散于具有生物相容性的高分子聚合物溶液中形成。
  7. 根据权利要求6所述的水凝胶基因递送系统，其特征在于，所述水凝胶基因递送系统为缓慢释放基因递送系统。
  8. 根据权利要求6或7所述的水凝胶基因递释系统，其特征在于，所述的具有生物相容性的高分子聚合物包括聚乙烯醇、聚乳酸、聚乙二醇、聚乳酸(羟基乙酸)-聚乙二醇-聚乳酸(羟基乙酸)嵌段共聚物、聚磷酸酯、聚碳酸酯、甲壳质、壳聚糖、海藻酸盐、透明质酸、肝素、硫酸软骨素、纤维素、琼脂、淀粉衍生物、葡聚糖衍生物、胶原、明胶、血纤蛋白、蚕丝蛋白、聚磷腈、聚乙醇酸、聚己内酯、聚反丁烯二酸丙二醇酯、聚甲基丙烯酸羟乙酯、聚(N-异丙基丙烯酰胺)、聚(甲基丙烯酸低聚乙二醇)、卡波姆或泊洛沙姆的任意一种或者几种。
  9. 权利要求1至3中任一项所述的非病毒载体、权利要求4至5中任一项所述的基因复合物或权利要求6至8中任一项所述的水凝胶基因递释系统在制备给药方式为注射给药、鼻粘膜给药、肺部给药、皮肤给药、眼部给药或口腔粘膜给药的药物中的应用；
    优选地，所述药物为用于诱导呼吸道粘膜免疫从而对病毒感染性疾病免疫保护的基因疫苗药物。
  10. 根据权利要求9所述的应用，其特征在于，所述药物为用于癌症、遗传性疾病、感染性疾病、心血管疾病或自身免疫性疾病的治疗的药物。
  11. 一种治疗癌症、遗传性疾病、感染性疾病、心血管疾病或自身免疫性疾病的药物，其特征在于，所述药物包括：权利要求1至3中任一项所述的非病毒载体、权利要求4至5中任一项所述的基因复合物或权利要求6至8中任一项所述的水凝胶基因递释系统。
  12. 一种用于治疗癌症、遗传性疾病、感染性疾病、心血管疾病或自身免疫性疾病的方法，其特征在于，所述方法包括：对有需要的受试者给予权利要求1至3中任一项所述的非病毒载体所负载的药物、权利要求4至5中任一项所述的基因复合物或权利要求6至8中任一项所述的水凝胶基因递释系统。
  13. 一种病毒感染性疾病的免疫保护方法，其特征在于，所述方法包括：对有需要的受试者权利要求1至3中任一项所述的非病毒载体所负载的药物、权利要求4至5中任一项所述的基因复合物或权利要求6至8中任一项所述的水凝胶基因递释系统。
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