WO2023231133A1 - 一种阳离子超支化淀粉基基因载体及其制备方法和应用 - Google Patents

一种阳离子超支化淀粉基基因载体及其制备方法和应用 Download PDF

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WO2023231133A1
WO2023231133A1 PCT/CN2022/103778 CN2022103778W WO2023231133A1 WO 2023231133 A1 WO2023231133 A1 WO 2023231133A1 CN 2022103778 W CN2022103778 W CN 2022103778W WO 2023231133 A1 WO2023231133 A1 WO 2023231133A1
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starch
cationic
based gene
branching enzyme
gbe
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French (fr)
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程力
孙悦
顾正彪
洪雁
李兆丰
李才明
班宵逢
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江南大学
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B35/00—Preparation of derivatives of amylopectin
    • C08B35/04—Ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to a cationic hyperbranched starch-based gene carrier and its preparation method and application, and belongs to the field of medicine.
  • the composition of genetic drug carriers is complex, and it is difficult to find the cause when side effects occur. A certain proportion and varying degrees of allergic reactions after vaccination have greatly reduced the audience's trust in genetic medicines.
  • the carriers of genetic drugs are mainly nanoliposomes.
  • Polyethylene glycol (PEG) is mainly used as a moisture carrier and stabilizer to increase the stability and validity of vaccines.
  • PEG is a polyether compound that is widely used in pharmaceuticals, cosmetics and food additives. It has been reported that few people are allergic to PEG.
  • the findings suggest that during the PEGylation process, the conformation and/or chemical structure of PEG covalently attached to the lipid nanoparticle surface may change, potentially altering and increasing the structure of PEG , making it have certain allergenicity. Therefore, it is very important to find a simple, safe and effective gene carrier.
  • PAMAM polyamide-amine
  • ethylenediamine is the core and methyl acrylate. Graft with ethylenediamine in sequence, with amino or carboxyl groups as terminal functional groups. This kind of hyperbranched polymer has a large number of terminal functional groups, good solubility, low molecular entanglement, and a high degree of geometric symmetry in the structure.
  • PAMAM has been widely used in biomedicine, catalyst carriers, membrane materials and other fields with its novel structure and unique properties, especially in the delivery of drugs and genes in the biomedical field.
  • PAMAM As a synthetic cationic gene carrier, PAMAM increasingly shows good gene loading and protection effects as the number of synthesis generations increases. Studies have found that when gene fragments interact with PAMAM macromolecules, they are often distributed in the internal gaps of its tree structure. This molecular encapsulation mechanism can effectively improve the water solubility and controlled release of the drug, and the geometry of the carrier and gene fragments The shape barely changes.
  • starch is one of the most widely used drug carriers. It is often used as a disintegrant, diluent and in the form of starch paste during wet granulation. As an adhesive.
  • natural starch has poor hydrophobicity and low resistance, which makes it difficult to meet the conditions as a gene carrier. It needs to be modified by certain physical and chemical means to achieve its needs as an ideal gene carrier.
  • enzymatic hydrolysis or acid hydrolysis can significantly reduce the molecular weight, but the molecular weight distribution of the product is uneven, and the natural branched structure is destroyed to varying degrees;
  • starch branching enzymes can increase the number of non-reducing ends of starch molecules, producing Many branched short chains realize artificial control of starch structure, thereby obtaining a natural biological macromolecule with a highly branched structure;
  • the halogen-containing group or epoxy group in the etherifying agent can etherize with the hydroxyl group in the starch molecule chemical reaction to generate a positively charged starch ether derivative.
  • This positively charged starch derivative can electrostatically interact with gene fragments to form a stable complex.
  • the cationic hyperbranched starch-based carrier with branched structure obtained in the present invention has strong ability to wrap and protect gene fragments. By comparing gene carriers with different degrees of branching and different degrees of substitution, their performance is examined, and it is helpful for the development of new ones. Safe and non-toxic gene carriers are of great significance to expand the utilization of starch resources.
  • the first object of the present invention is to provide a preparation method and application of a starch-based cationic polymer gene carrier that is highly branched and has a low degree of substitution.
  • the preparation method has simple process, easy control and low cost.
  • the preparation method of the cationic hyperbranched starch-based gene carrier includes the following steps: increasing the branching degree of starch molecules through the transglycoside and hydrolysis of starch branching enzymes, reducing the molecular weight of starch molecules, and at the same time providing certain resistance to the system , and then prepare a cationic branched starch-based gene carrier through an etherification reaction, so that the carrier can be successfully grafted with cationic groups, thereby achieving the effect of effective delivery of siRNA.
  • the degree of starch branching increases and the molecular weight decreases.
  • the particle size distribution of the prepared complex can reach the nanoscale, thus also having the advantages of a nanoscale carrier.
  • the high specific surface area of the highly branched structure increases the loading capacity of siRNA through adsorption. .
  • the cationic hyperbranched starch-based gene carrier prepared by the method of the present invention has a lower degree of substitution and can completely embed siRNA, and as the degree of substitution increases, the embedding effect is better. As the degree of branching of the carrier increases, the complex formed becomes more uniform, and the minimum size can reach about 300nm, which belongs to the category of nanoscale drug delivery.
  • a method for preparing a cationic hyperbranched starch-based gene carrier is to use starch/dextrin treated with a starch branching enzyme as a substrate, chemically modify it with a cationic etherifying agent, and prepare a cationic branched starch-based gene carrier.
  • the ⁇ -1,6 bond content of the cationic hyperbranched starch-based gene carrier is 5%-11%; the substitution degree of the cationic hyperbranched starch-based gene carrier is 0.030-0.080.
  • the cationic hyperbranched starch-based gene carrier and the gene drug form a nanocomplex of 300-400 nm.
  • the specific preparation steps of the cationic hyperbranched starch-based gene vector are as follows:
  • step (1) Disperse the substrate obtained in step (1) in absolute ethanol to form a mixture; adjust the pH of the cationic etherifying agent to 9-10, add it to the mixture and heat the reaction. After the reaction is completed, cool to room temperature to generate yellow or light Yellow initial product; add glacial acetic acid to neutralize the system until the pH is neutral, then filter, wash, and dry to obtain a cationic hyperbranched starch-based gene vector.
  • the cationic etherifying agent is a trimethylammonium chloride solution containing 3-chloro-2-hydroxypropyl.
  • the starch treated with the starch branching enzyme is branched starch RG-S.
  • the dextrin treated with starch branching enzyme is branched dextrin RG-M.
  • the starch treated with the starch branching enzyme is branched starch RG-S.
  • the dextrin treated with starch branching enzyme is branched dextrin RG-M.
  • the specific preparation steps of using starch as a substrate to prepare a cationic hyperbranched starch-based gene vector are as follows:
  • the starch is prepared into a starch solution with distilled water, kept in a water bath for gelatinization, stirred and added with a starch branching enzyme, gelatinized to inactivate the enzyme, freeze-dried, ground and sieved to obtain the substrate, which is starch treated with a starch branching enzyme, recorded as RG- S;
  • step (1) Disperse the RG-S obtained in step (1) in absolute ethanol to form a mixture, adjust the pH of the etherifying agent (3-chloro-2-hydroxypropyl)trimethylammonium chloride solution to 9-10, Heating the reaction, cooling to room temperature after the reaction is completed, a yellow or light yellow cationic starch initial product is generated.
  • the concentration of the starch aqueous solution is 10%-30% (w/v) (on a dry basis).
  • the starch branching enzymes are Ro-GBE and Gt-GBE derived from Rhodothermus obamensis and Geobacillus thermoglucosidans.
  • step (1) the step of adding the starch branching enzyme is to first add the starch branching enzyme Gt-GBE and then add the starch branching enzyme Ro-GBE.
  • the reaction temperature of the starch branching enzyme Gt-GBE is 50°C to 60°C, the amount of enzyme added is 25 to 35U/g, and the reaction time is 10-15h.
  • the reaction temperature of the starch branching enzyme Ro-GBE is 55°C to 65°C
  • the amount of enzyme added is 30 to 40U/g
  • the reaction time is 8 to 12 hours.
  • step (2) the molar ratio of dehydrated glucose units of branched starch, NaOH and CTA is 1-1.2:1-1.2:1-1.5, and the water content of the mixed system does not exceed 10%.
  • Reaction temperature 50-70°C, reaction time 1-4h;
  • the specific preparation steps for preparing cationic hyperbranched starch-based gene vectors using dextrin as a substrate are as follows:
  • step (1) Disperse the RG-M obtained in step (1) in absolute ethanol to form a mixture, adjust the pH of the etherifying agent (3-chloro-2-hydroxypropyl)trimethylammonium chloride solution to 9-10, Heating the reaction, cooling to room temperature after the reaction is completed, a yellow or light yellow cationic starch initial product is generated. Add glacial acetic acid to neutralize the reaction system to a pH value of 7, and then use vacuum filtration to fully absorb the cationic starch initial product with absolute ethanol. Wash until silver nitrate is dripped into the filtrate and no silver chloride precipitates; filter with suction and dry in a 37°C oven to constant weight to obtain a cationic dextrin gene vector, designated as C-RG-M.
  • C-RG-M cationic dextrin gene vector
  • the concentration of the dextrin aqueous solution is 10%-30% (w/v) (on a dry basis).
  • the starch branching enzymes are Ro-GBE and Gt-GBE derived from Rhodothermus obamensis and Geobacillus thermoglucosidans.
  • step (1) the step of adding the starch branching enzyme is to first add the starch branching enzyme Gt-GBE and then add the starch branching enzyme Ro-GBE.
  • the reaction temperature of the starch branching enzyme Gt-GBE is 50°C to 60°C, the amount of enzyme added is 25 to 35U/g, and the reaction time is 10-15h.
  • the reaction temperature of the starch branching enzyme Ro-GBE is 55°C to 65°C
  • the amount of enzyme added is 30 to 40U/g
  • the reaction time is 8 to 12 hours.
  • step (2) the molar ratio of the anhydroglucose units of branched dextrin, NaOH and CTA is 1-1.2:1-1.2:1-1.5, and the water content of the mixed system does not exceed 10%. , reaction temperature 50-70°C, reaction time 1-4h;
  • the prepared cationic hyperbranched starch-based gene vector is put into a dialysis bag and dialyzed in ultrapure water for 48-72 hours; the product is obtained after freeze-drying.
  • the present invention uses the above method to obtain a cationic hyperbranched starch-based gene carrier.
  • the second object of the present invention is to utilize the above-mentioned cationic hyperbranched starch-based gene carrier in the preparation of gene drugs.
  • the application is to provide the use of the above-mentioned cationic hyperbranched starch-based gene vector as a non-viral gene vector in gene therapy.
  • a cationic hyperbranched dextrin gene carrier with a low degree of substitution carrying genetic drugs is obtained by loading the genetic drug on the above-mentioned cationic hyperbranched starch-based gene carrier through electrostatic attraction.
  • the genetic medicine is DNA or RNA.
  • the application is to mix the above-mentioned cationic hyperbranched starch-based gene carrier with gene fragments according to an N/P ratio of 2.0 to 3.5, and the two can self-organize to form a nanocomposite through electrostatic interaction. material to achieve good loading of gene fragments.
  • the cationic hyperbranched starch-based gene carrier prepared by the present invention can load a large amount of genetic drugs and has good hydrophobic drug carrying and genetic drug binding capabilities;
  • the cationic hyperbranched starch-based gene carrier prepared by the present invention has a smaller degree of substitution and a controllable reaction
  • the particle size distribution of the complex formed by the cationic hyperbranched starch-based gene carrier prepared by the present invention and siRNA can reach the nanometer level.
  • corn starch involved in the following examples was purchased from Shandong Shouguang Co., Ltd., and the maltodextrin was purchased from Shandong Baolingbao Biological Co., Ltd., China; two starch branching enzymes derived from Rhodothermus obamensis and Geobacillus thermoglucosidans (EC 2.4.1.18 ) are all from this laboratory.
  • step (5) is changed to 60°C for 4 hours, and cooling to room temperature produces a yellow or light yellow cationic branched starch primary product
  • step (2) was changed to place the gelatinized dextrin solution in a four-necked flask in a 55°C water bath for 15 minutes, stir and add 30 U/g Gt-GBE, react for 10 hours, gelatinize and inactivate the enzyme for 30 minutes , freeze-dried, ground and sieved to obtain modified starch, recorded as Gt-M;
  • step (2) is changed to place the gelatinized dextrin solution in a four-necked flask in a 60°C water bath for 15 minutes, stir and add 35 U/g Ro-GBE, react for 10 hours, and gelatinize to inactivate the enzyme for 30 minutes. , freeze-dried, ground and sieved to obtain modified starch, recorded as Ro-M;
  • step (2) is changed to place the gelatinized dextrin solution in a four-necked flask in a 60°C water bath for 15 minutes, stir and simultaneously add 35U/g Ro-GBE and 30U/g Gt-GBE for reaction After 10 hours, gelatinize and inactivate enzyme for 30 minutes, then freeze-dry, grind and sieve to obtain modified starch, recorded as RG-M;
  • step (2) is changed to place the gelatinized dextrin solution in a four-necked flask in a 60°C water bath for 15 minutes, stir and simultaneously add 35U/g Ro-GBE and 30U/g Gt-GBE for reaction After 10 hours, gelatinize and inactivate enzyme for 30 minutes, then freeze-dry, grind and sieve to obtain modified starch, recorded as RG-M;
  • the sample was dissolved in heavy water (D 2 O) to form a starch milk with a concentration of 40 mg/mL, and was gelatinized in boiling water for 30 minutes.
  • the gelatinized sample was lyophilized and then dissolved in D 2 O again, and measured by 1 H NMR (Proton Nuclear Magnetic Resonance Spectroscopy).
  • the relative content of ⁇ -1,6-glycosidic bonds can be obtained by calculating the peak areas of the corresponding absorption peaks at 5.37 ppm and ⁇ -1,6-glycosidic bonds at 4.96 ppm in the spectrum.
  • Table 1 shows the ⁇ -1,6 glycosidic bond content of cationic starch-based gene vectors with different degrees of branching prepared according to the above method.
  • Starch branching enzymes Gt-GBE and Ro-GBE can increase the branching degree of dextrin to a certain extent. Comparing the transglycoside effects of branching enzymes on different substrates, it was found that the proportion of ⁇ -1,6-glycosidic bonds in corn starch increased by 156%, and the proportion of ⁇ -1,6-glycosidic bonds in maltodextrin after enzymatic hydrolysis increased by 80%.
  • % that is, the transglycoside effect of Gt-GBE and Ro-GBE on starch is better than that of dextrin; comparing the transglycoside effect of different branching enzymes on dextrin, it was found that the ⁇ -1 of dextrin after the action of Gt-GBE and Ro-GBE , the proportion of 6 bonds is significantly higher than that of the product treated with a single enzyme, which shows that the synergistic effect occurs when the two branching enzymes work together and can significantly increase the branching degree of the starch-based carrier.
  • Table 2 shows the chain length distribution of cationic starch-based gene vectors with different degrees of branching prepared according to the above method.
  • Starch branching enzymes Gt-GBE and Ro-GBE can catalyze the hydrolysis of ⁇ -1,4-glycosidic bonds in starch molecules to a certain extent, breaking long chain segments with DP>13 and producing short chains with non-reducing ends.
  • Maltodextrin is the product of acidolysis or enzymatic hydrolysis of starch, and the original structure of starch has been destroyed to a certain extent.
  • the short chain content of dextrin without the action of branching enzymes is significantly higher than that of starch after the action of Gt-GBE and Ro-GBE.
  • Table 3 shows the degree of substitution of cationic starch-based gene vectors with different degrees of branching prepared according to the above method.
  • Example 1 Utilize the cationic branched starch-based gene vector prepared in Example 1 and dissolve it in DEPC water to prepare a solution with a concentration of 6.73 mg/ml;
  • siRNA fragment targeting the human gene ABCB1 encoding P-glycoprotein was synthesized in the laboratory (the target gene was purchased from Suzhou Jinweizhi Biotechnology Co., Ltd., China, and the siRNA was synthesized in the laboratory), with a concentration of 2500ng/ ⁇ l;
  • the value is 2500ng/ ⁇ l, add an appropriate amount of ultrapure water to make the final volume 10 ⁇ l, vortex for 1 minute, and let stand at room temperature 25°C for 1 hour;
  • the cationic branched starch-based gene vector prepared in Example 2 was dissolved in DEPC water to prepare a solution with a concentration of 4.80 mg/ml;
  • the cationic branched starch-based gene vector prepared in Example 3 was dissolved in DEPC water to prepare a solution with a concentration of 13.25 mg/ml;
  • the cationic branched starch-based gene vector prepared in Example 4 was dissolved in DEPC water to prepare a solution with a concentration of 12.41 mg/ml;
  • the cationic branched starch-based gene vector prepared in Example 5 was dissolved in DEPC water to prepare a solution with a concentration of 12.74 mg/ml;
  • the cationic branched starch-based gene vector prepared in Example 6 was dissolved in DEPC water to prepare a solution with a concentration of 28.53 mg/ml;
  • the cationic branched starch-based gene vector prepared in Example 7 was dissolved in DEPC water to prepare a solution with a concentration of 11.96 mg/ml;
  • the zeta potential of the nanocomplex formed by the cationic branched dextrin gene carrier and siRNA prepared in Examples 10, 11, 12, and 14 under different N/P was measured using a Malvern laser particle size distribution instrument.
  • the results show (Table 4) that at the same degree of substitution, regardless of the degree of branching of the cationic branched dextrin gene carrier, the surface potential of the complex formed with siRNA increases as N/P increases, and the surface charge shows a gradual increasing trend.
  • the potential is -4.00–17.00mV.
  • the positive charge of the nanocomplex indicates that it can complex more closely with the negatively charged siRNA, and during the transfection process, it can quickly bind to the negatively charged cell membrane on the surface to promote the endocytosis of the complex by cells, and with the degree of branching With the increase, the surface potential of the complex gradually decreases, which indicates that the highly branched structure has great potential to reduce the cytotoxicity of the carrier.
  • Table 4 shows the surface potential of the complexes formed by cationic branched dextrin gene carriers with different branching degrees prepared according to the above method and siRNA under different N/P conditions.
  • Table 4 Surface potential of complexes formed by cationic branched dextrin gene carriers with different branching degrees and siRNA under different N/P conditions.
  • the particle size of the nanocomplex formed by the cationic branched dextrin gene carrier and siRNA prepared in Examples 10, 11, 12, and 14 under different N/P was measured using a Malvern laser particle size distribution instrument. The results show (Table 5) that as the N/P molar ratio increases, the particle size changes show a trend of first increasing and then decreasing. This may be because the particle surface potential is close to 0mV at this time, and the cationic modified starch, DNA and transfer Agglomeration easily occurs between systems, resulting in a significant increase in particle size. When N/P continues to increase, the surface is positively charged, showing mutual repulsion between particles, and can exist in the solution with a stable particle size.
  • the cationic hyperbranched dextrin C-RG-M- The particle size distribution of the complex formed by 4 and siRNA becomes more and more uniform, and the size of the formed nanotransmission system is 300-400nm.
  • Table 5 shows the particle size distribution of the complexes formed by cationic branched dextrin gene vectors with different branching degrees prepared according to the above method and siRNA under different N/P conditions.
  • Table 5 Particle size distribution of complexes formed by cationic branched dextrin gene vectors with different branching degrees and siRNA under different N/P conditions.

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Abstract

本发明公开了一种纳米级阳离子超支化淀粉基基因载体的制备方法，主要步骤包括为：将糊精溶液加热糊化后，采用淀粉分支酶的水解和转苷作用，获得一种具有丰富短链的高度分支簇状糊精分子，再经醚化反应获得不同取代度的阳离子聚合物。此聚合物降解可控，其高支化的结构能在一定程度上减小基因载体对阳离子淀粉高取代度的要求，细胞毒性明显降低。此外，该聚合物载体可与基因片段形成稳定的纳米复合物，作为高效的基因载体在基因治疗中具有广泛的应用。

Description

一种阳离子超支化淀粉基基因载体及其制备方法和应用 技术领域
本发明涉及一种阳离子超支化淀粉基基因载体及其制备方法和应用，属于医药领域。
背景技术
基因类药物载体组成复杂，发生副作用时难以找到原因。疫苗注射后出现的一定比例、不同程度的过敏反应使得受众对于基因药物的信任程度大大降低。目前，基因类药物的载体主要是纳米脂质体。聚乙二醇(PEG)主要作为水分载体和稳定剂，用于增加疫苗的稳定性和有效期。PEG是一种聚醚化合物，广泛用于制药、化妆品和食品添加剂，已有报道中，对PEG的过敏的人群很少。但是，研究结果显示，在聚乙二醇化过程中，与脂质纳米颗粒表面共价连接的聚乙二醇构象和/或化学结构可能发生变化，从而有可能改变和增加聚乙二醇的结构，使其具有一定的致敏性。因此，寻找一种结构简单、安全有效的基因载体十分重要。
在众多的基因递送载体中，聚酰胺-胺(PAMAM)作为树状大分子家族中研究最广泛、最深入的聚合物之一，因其独特的结构和性质得到了研究人员的广泛关注。PAMAM是一类具有明确结构和均一大小的高度支化的聚合物大分子，主要由核、内部(枝)和外壳(末端基团)三部分结构组成，其中乙二胺作核心，丙烯酸甲酯和乙二胺依次接枝，氨基或羧基作末端功能基团。这种超支化聚合物具有大量的末端功能基团、良好的溶解性、较低分子缠结，同时在结构上具有高度的几何对称性，可以人为控制分子质量和结构，基因负载效果好，转染效率高。近十几年来，PAMAM凭借其新奇的结构和独特的性能被广泛应用于生物医学、催化剂载体、膜材料等领域，尤其是生物医学领域中的药物及基因的递送方面。
作为一种人工合成的阳离子基因载体，PAMAM随着合成代数的增加，愈发显示出良好的基因负载和保护效果。研究发现，基因片段与PAMAM大分子发生作用时，常分布于其树状结构的内部空隙，这种分子包埋机制可有效的改善药物的水溶性和控释性，且载体与基因片段的几何形状几乎不发生改变。淀粉作为自然界中少有的具有高分支结构的天然大分子，是应用最为广泛的药物载体之一，常被用作崩解剂、稀释剂以及在湿法造粒过程中以淀粉糊的形式用作粘合剂。然而，天然淀粉疏水性差、抗性低，难以满足作为基因载体的条件，需要通过一定的物理、化学手段对其进行改性，从而实现作为理想基因载体的需求。在淀粉的改性工艺中，酶解或酸解可显著降低分子量，但产物分子量分布不均匀，且天然的分支结构被不同程度的破坏；淀粉分支酶可增加淀粉分子的非还原端数量，产生许多分支短链，实现对淀粉结构的人工调控，从而获得一种具有高度分支结构的天然生物大分子；醚化剂中的含 卤基或环氧基可以与淀粉分子中的羟基基团进行醚化反应，生成一种带有正电荷的淀粉醚衍生物，这种带正电的淀粉衍生物可以与基因片段发生静电相互作用而生成稳定的复合物。本发明中获得的具有支化结构的阳离子超支化淀粉基载体，具有较强的包裹和保护基因片段的能力，通过比较不同分支程度、不同取代度的基因载体，考察其性能，对开发新的安全无毒的基因载体，扩大淀粉质资源的利用具有重大的意义。
发明内容
本发明的第一个目的在于提供了一种高度分支且具有低取代度的淀粉基阳离子聚合物基因载体的制备方法和应用。该制备方法工艺简单，易于控制，成本低廉。
本发明提供的阳离子超支化淀粉基基因载体的制备方法包括为以下步骤：通过淀粉分支酶的转苷和水解作用，增加淀粉分子的分支程度，降低淀粉分子的分子量，同时为体系提供一定抗性，再通过醚化反应制备阳离子分支淀粉基基因载体，使载体成功接枝阳离子基团，从而实现对siRNA有效递送的效果。经酶解后的淀粉分支程度增加，分子量降低，制备的复合物粒径分布可以到达纳米级，从而也具备纳米级载体的优势，高度分支结构的高比表面积通过吸附作用提高了siRNA的负载量。
利用本发明的方法制备得到的阳离子超支化淀粉基基因载体，具有较低的取代度，可以实现对siRNA完整的包埋，且随着取代度的增加，包埋效果更好，随着淀粉基载体分支程度的增加，形成的复合物更加均匀，最小可以达到300nm左右，属于纳米级递送药物范畴。
本发明的技术方案如下：
一种阳离子超支化淀粉基基因载体的制备方法，是以淀粉分支酶处理过的淀粉/糊精为底物，采用阳离子醚化剂对其进行化学改性，制备获得阳离子分支淀粉基基因载体，所述的阳离子超支化淀粉基基因载体的α-1,6键含量5％-11％；所述的阳离子超支化淀粉基基因载体的取代度在0.030-0.080。
在本发明一种实施方式中，所述的阳离子超支化淀粉基基因载体与基因药物形成300-400nm的纳米复合物。
在本发明一种实施方式中，所述的阳离子超支化淀粉基基因载体具体制备步骤如下：
(1)底物的制备
将淀粉/糊精用蒸馏水配置成水溶液，水浴保温糊化，搅拌并加入淀粉分支酶，糊化灭酶，经冷冻干燥、研磨过筛得到底物，即淀粉分支酶处理过的淀粉/糊精；
(2)底物改性制备阳离子超支化淀粉基基因载体
将步骤(1)中获得的底物分散于无水乙醇中，形成混合物；调节阳离子醚化剂的pH至 9-10，加入到混合物中加热反应，反应完成后冷却至室温即生成黄色或淡黄色的初产物；加入冰乙酸中和体系至pH呈中性，然后过滤洗涤，干燥即得阳离子超支化淀粉基基因载体。
在本发明一种实施方式中，所述阳离子醚化剂为含3-氯-2-羟丙基的三甲基氯化铵溶液。
在本发明一种实施方式中，所述淀粉分支酶处理过的淀粉为分支淀粉RG-S。
在本发明一种实施方式中，所述淀粉分支酶处理过的糊精为分支糊精RG-M。
在本发明一种实施方式中，所述淀粉分支酶处理过的淀粉为分支淀粉RG-S。
在本发明一种实施方式中，所述淀粉分支酶处理过的糊精为分支糊精RG-M。
在本发明一种实施方式中，使用淀粉为底物制备阳离子超支化淀粉基基因载体具体制备步骤如下：
(1)分支淀粉RG-S的制备
将淀粉用蒸馏水配置成淀粉水溶液，水浴保温糊化，搅拌并加入淀粉分支酶，糊化灭酶，经冷冻干燥、研磨过筛得到底物，即淀粉分支酶处理过的淀粉，记为RG-S；
(2)分支淀粉RG-S改性制备阳离子超支化淀粉基基因载体
将步骤(1)中获得的RG-S分散于无水乙醇中，形成混合物，调节醚化剂(3-氯-2-羟丙基)三甲基氯化铵溶液的pH至9-10，加热反应，反应完后冷却至室温即生成黄色或淡黄色阳离子淀粉初产物，往其中加入冰乙酸中和反应体系至pH值为7，再通过真空抽滤将阳离子淀粉初产物用无水乙醇充分洗涤，直至滤液中滴入硝酸银没有氯化银沉淀即可；抽滤并置于37℃烘箱干燥至恒重，即得到阳离子淀粉基因载体，记为C-RG-S。
在本发明一种实施方式中，步骤(1)中，淀粉水溶液浓度10％-30％(w/v)(以干基计)。
在本发明一种实施方式中，步骤(1)中，淀粉分支酶为Rhodothermus obamensis来源和Geobacillus thermoglucosidans来源的淀粉分支酶Ro-GBE和Gt-GBE。
在本发明一种实施方式中，步骤(1)中，加入淀粉分支酶的步骤为先加入淀粉分支酶Gt-GBE再加入淀粉分支酶Ro-GBE。
在本发明一种实施方式中，步骤(1)中，淀粉分支酶Gt-GBE的反应温度50℃～60℃，加酶量25～35U/g，反应时间10-15h。
在本发明一种实施方式中，步骤(1)中，淀粉分支酶Ro-GBE的反应温度55℃～65℃，加酶量30～40U/g，反应时间8-12h。
在本发明一种实施方式中，步骤(2)中，分支淀粉的脱水葡萄糖单元、NaOH和CTA，摩尔比为1-1.2：1-1.2：1-1.5，混合体系含水量不超过10％，反应温度50-70℃，反应时间1-4h；
在本发明一种实施方式中，使用糊精为底物制备阳离子超支化淀粉基基因载体具体制备步骤如下：
(1)分支糊精RG-M的制备
将糊精用蒸馏水配置成水溶液，水浴保温糊化，搅拌并加入淀粉分支酶，糊化灭酶，经冷冻干燥、研磨过筛得到底物，即淀粉分支酶处理过的淀粉，记为RG-M；
(2)分支糊精RG-M改性制备阳离子超支化淀粉基基因载体
将步骤(1)中获得的RG-M分散于无水乙醇中，形成混合物，调节醚化剂(3-氯-2-羟丙基)三甲基氯化铵溶液的pH至9-10，加热反应，反应完后冷却至室温即生成黄色或淡黄色阳离子淀粉初产物，往其中加入冰乙酸中和反应体系至pH值为7，再通过真空抽滤将阳离子淀粉初产物用无水乙醇充分洗涤，直至滤液中滴入硝酸银没有氯化银沉淀即可；抽滤并置于37℃烘箱干燥至恒重，即得到阳离子糊精基因载体，记为C-RG-M。
在本发明一种实施方式中，步骤(1)中，糊精水溶液浓度10％-30％(w/v)(以干基计)。
在本发明一种实施方式中，步骤(1)中，淀粉分支酶为Rhodothermus obamensis来源和Geobacillus thermoglucosidans来源的淀粉分支酶Ro-GBE和Gt-GBE。
在本发明一种实施方式中，步骤(1)中，加入淀粉分支酶的步骤为先加入淀粉分支酶Gt-GBE再加入淀粉分支酶Ro-GBE。
在本发明一种实施方式中，步骤(1)中，淀粉分支酶Gt-GBE的反应温度50℃～60℃，加酶量25～35U/g，反应时间10-15h。
在本发明一种实施方式中，步骤(1)中，淀粉分支酶Ro-GBE的反应温度55℃～65℃，加酶量30～40U/g，反应时间8-12h。
在本发明一种实施方式中，步骤(2)中，分支糊精的脱水葡萄糖单元、NaOH和CTA，摩尔比为1-1.2：1-1.2：1-1.5，混合体系含水量不超过10％，反应温度50-70℃，反应时间1-4h；
在本发明一种实施方式中，制备的阳离子超支化淀粉基基因载体，是将其装入透析袋中，超纯水中透析48-72h；产物冷冻干燥后即得。
本发明利用上述方法得到了一种阳离子超支化淀粉基基因载体。
本发明第二个目的是利用上述阳离子超支化淀粉基基因载体在基因药物制备上的应用。
在本发明一种实施方式中，所述应用是在于提供上述阳离子超支化淀粉基基因载体在基因治疗中作为非病毒基因载体的应用。
在本发明一种实施方式中，负载基因药物的低取代度的阳离子超支化糊精基因载体，其是通过将基因药物通过静电引力负载在上述阳离子超支化淀粉基基因载体上得到。
在本发明一种实施方式中，所述的基因药物为DNA、RNA。
在本发明一种实施方式中，所述应用是将上述阳离子超支化淀粉基基因载体，按照2.0～3.5的N/P比与基因片段混合后，两者可通过静电相互作用自组织形成纳米复合物，实现 对基因片段的良好负载。
有益效果：
(1)本发明制备的阳离子超支化淀粉基基因载体可大量负载基因药物，具有较好的疏水药物携带和基因药物结合能力；
(2)本发明制备的阳离子超支化淀粉基基因载体的取代度较小且反应可控制；
(3)本发明制备的阳离子超支化淀粉基基因载体与siRNA形成的复合物表面带正电；
(4)本发明制备的阳离子超支化淀粉基基因载体与siRNA形成的复合物粒度分布可以达到纳米级。
附图说明
图1为实施例8～14中不同取代度、不同分支程度的阳离子超支化淀粉基基因载体在不同的N/P比的条件下(泳道自左向右依别为裸siRNA、N/P＝0.5、1.0、1.5、2.0、2.5、3.0、3.5)与siRNA形成的纳米传递系统对siRNA的保护与泄露情况的电泳图。
图2为DS＝0.077的阳离子超支化淀粉基基因载体C-RG-M-4在最佳的N/P比(N/P＝2.0)时(泳道自左向右依别为裸siRNA、2.0)与siRNA形成的纳米传递系统在4h、24h、3d、7d中对siRNA保护与泄露情况的电泳图。
具体实施方式
下面结合具体实施例对本发明进行进一步的阐述，但本发明的保护范围并不限于此。
下述实施例中涉及的玉米淀粉购自山东寿光股份有限公司，麦芽糊精购自中国山东保龄宝生物股份有限公司；来源于Rhodothermus obamensis和Geobacillus thermoglucosidans的两种淀粉分支酶(EC 2.4.1.18)均来自本实验室。
实施例1
阳离子分支淀粉C-RG-S-1的制备
(1)称取10g(以干基计)普通玉米淀粉，用蒸馏水配置为10％(w/v)的淀粉水溶液，糊化30min；
(2)将糊化后的淀粉浆置于四口烧瓶中60℃水浴保温15min，搅拌并加入35U/g Ro-GBE和30U/g Gt-GBE反应10h后，糊化灭酶30min，经冷冻干燥、研磨过筛得到改性淀粉，记为RG-S；
(3)称取上述5g(以干基计)RG-S样品分散于无水乙醇中，形成5％(w/v)淀粉-乙醇混合物；
(4)用10mol/L的NaOH溶液调节醚化剂(3-氯-2-羟丙基)三甲基氯化铵溶液(CTA)的 pH至10后，加入淀粉-乙醇混合溶液中，其中淀粉的脱水葡萄糖单元、NaOH和CTA，摩尔比为1：1：1，混合体系含水量不超过10％；
(5)60℃反应1h，冷却至室温即生成黄色或淡黄色阳离子分支淀粉初产物；
(6)向初产物中加入冰乙酸中和反应体系至pH值为7，再通过真空抽滤将阳离子分支淀粉初产物用无水乙醇充分洗涤直至滤液中滴入硝酸银没有氯化银沉淀。抽滤产物置于37℃烘箱干燥至恒重，即得到阳离子淀粉C-RG-S-1。
(7)将上述产物其装入截止分子量为1000的透析袋中，超纯水中透析72h。
实施例2
阳离子分支淀粉C-RG-S-4的制备
参照实施例1，将步骤(5)改为60℃反应4h，冷却至室温即生成黄色或淡黄色阳离子分支淀粉初产物；
其余条件不变，得到产物C-RG-S-4。
实施例3
阳离子分支糊精C-Y-M-4的制备
(1)称取10g(以干基计)DE7-9的麦芽糊精，用蒸馏水配置为10％(w/v)的糊精水溶液，糊化30min；
(2)经冷冻干燥、研磨过筛得到改性淀粉，记为Y-M；
(3)称取上述5g(以干基计)Y-M样品分散于无水乙醇中，形成5％(w/v)糊精-乙醇混合物；
(4)用10mol/L的NaOH溶液调节醚化剂(3-氯-2-羟丙基)三甲基氯化铵溶液(CTA)的pH至10后，加入淀粉-乙醇混合溶液中，其中淀粉脱水葡萄糖单元、NaOH和CTA，摩尔比为1：1：1，混合体系含水量不超过10％；
(5)60℃反应4h，冷却至室温即生成黄色或淡黄色阳离子分支糊精初产物；
(6)向初产物中加入冰乙酸中和反应体系至pH值为7，再通过真空抽滤将阳离子分支淀粉初产物用无水乙醇充分洗涤直至滤液中滴入硝酸银没有氯化银沉淀。抽滤产物置于37℃烘箱干燥至恒重，即得到阳离子淀粉C-Y-M-4。
(7)将上述产物其装入截止分子量为1000的透析袋中，超纯水中透析72h。
实施例4
阳离子分支糊精C-Gt-M-4的制备
参照实施例3，将步骤(2)改为将糊化后的糊精溶液置于四口烧瓶中55℃水浴保温15min，搅拌并加入30U/g Gt-GBE反应10h后，糊化灭酶30min，经冷冻干燥、研磨过筛得到改性淀 粉，记为Gt-M；
其余条件不变，得到产物C-Gt-M-4。
实施例5
阳离子分支糊精C-Ro-M-4的制备
参照实施例3，将步骤(2)改为将糊化后的糊精溶液置于四口烧瓶中60℃水浴保温15min，搅拌并加入35U/g Ro-GBE反应10h后，糊化灭酶30min，经冷冻干燥、研磨过筛得到改性淀粉，记为Ro-M；
其余条件不变，得到产物C-Ro-M-4。
实施例6
阳离子分支糊精C-RG-M-1的制备
参照实施例3，将步骤(2)改为将糊化后的糊精溶液置于四口烧瓶中60℃水浴保温15min，搅拌并同时加入35U/g Ro-GBE和30U/g Gt-GBE反应10h后，糊化灭酶30min，经冷冻干燥、研磨过筛得到改性淀粉，记为RG-M；
将步骤(5)改为60℃反应1h，冷却至室温即生成黄色或淡黄色阳离子分支糊精初产物；
其余条件不变，得到产物C-RG-M-1。
实施例7
阳离子分支糊精C-RG-M-4的制备
参照实施例3，将步骤(2)改为将糊化后的糊精溶液置于四口烧瓶中60℃水浴保温15min，搅拌并同时加入35U/g Ro-GBE和30U/g Gt-GBE反应10h后，糊化灭酶30min，经冷冻干燥、研磨过筛得到改性淀粉，记为RG-M；
其余条件不变，得到产物C-RG-M-4。
①α-1,6-糖苷键相对含量的测定：
样品溶解于重水(D 2O)中形成浓度为40mg/mL的淀粉乳，沸水糊化30min。糊化后的样品冻干后再次溶于D 2O中，通过 1H NMR(核磁共振氢谱)进行测定。通过计算谱图中5.37ppm处α-1,4-糖苷键与4.96ppm处α-1,6-糖苷键对应吸收峰的峰面积可得到α-1,6-糖苷键的相对含量。
表1为根据上述方法制备的不同分支程度的阳离子淀粉基基因载体的α-1,6糖苷键含量。
表1不同分支程度的淀粉基载体的α-1,6糖苷键含量
样品 Y-S RG-S Y-M Gt-M Ro-M RG-M
α-1,6键比例 4.31％ 11.04％ 5.79％ 6.29％ 8.47％ 10.16％
淀粉分支酶Gt-GBE和Ro-GBE能在一定程度上增加糊精的分支程度。对比分支酶对不同底物的转苷效果，发现玉米淀粉α-1,6-糖苷键的比例增加了156％，酶解后的麦芽糊精α-1,6-糖苷键的比例增加了80％，即Gt-GBE和Ro-GBE对淀粉的转苷作用优于糊精；对比不同分支酶对糊精的转苷效果，发现Gt-GBE和Ro-GBE作用后的糊精的α-1,6键比例明显高于单酶处理的产物，这说明两种分支酶共同作用时发生协同效应，能明显的提高淀粉基载体的分支程度。
②链长分布的测定：
称取10mg样品，溶解于2mL的醋酸钠缓冲液(50mM，pH 3.5)中，37℃下预热15min，加入100μL异淀粉酶(10000U/mL)，于恒温水浴摇床(160r/min)中脱支24h，沸水浴30min灭酶，10000r/min离心10min，取上清液稀释后过0.22μm水系滤膜，采用HPAEC-PAD测定样品链长分布。
表2为根据上述方法制备的不同分支程度的阳离子淀粉基基因载体的链长分布。
表2不同分支程度的淀粉基载体的链长分布
Figure PCTCN2022103778-appb-000001
淀粉分支酶Gt-GBE和Ro-GBE能在一定程度上催化水解淀粉分子中的α-1,4-糖苷键，使DP>13的长链链段断裂，产生具有非还原性末端的短链。麦芽糊精是淀粉的酸解或酶解产物，淀粉的原有结构已经在一定程度上被破坏。未经分支酶作用的糊精的短链含量明显高于Gt-GBE和Ro-GBE共同作用后的淀粉，对比不同分支酶对糊精的水解效果，发现Gt-GBE和Ro-GBE作用后的糊精的短链含量明显高于单酶处理的产物，这说明两种分支酶共同作用时发生协同效应，能明显的提高淀粉基载体的短链含量。
③取代度的测定：
阳离子淀粉基基因载体中氮含量的测定参考GB5009.5-2016，取代度的计算公式如下：
Figure PCTCN2022103778-appb-000002
表3为根据上述方法制备的不同分支程度的阳离子淀粉基基因载体的取代度。
表3不同分支程度的淀粉基载体的取代度
样品 C-RG-S-1 C-RG-S-4 C-Y-M-4 C-GT-M-4 C-RO-M-4 C-RG-M-1 C-RG-M-4
取代度 0.145 0.215 0.069 0.074 0.072 0.031 0.077
实施例8
(1)利用实施例1中制备的阳离子分支淀粉基基因载体溶于DEPC水配成浓度6.73mg/ml的溶液；
(2)实验室合成针对编码P-糖蛋白的人类基因ABCB1的siRNA片段，(目的基因购自中国苏州金唯智生物科技有限公司，siRNA在实验室合成)，浓度为2500ng/μl；
(3)按照N/P＝0.5、1.0、1.5、2.0、2.5、3.0、3.5的比例将步骤(1)和步骤(2)中的溶液按体积混合，控制每种N/P下siRNA浓度均为2500ng/μl，加入适量的超纯水使最终体积为10μl，涡旋振荡1min，室温25℃静置1h；
(4)取不同N/P的样品5μl与1μl 6×上样缓冲液(loading buffer)混合后用微量移液器转移1％的琼脂糖加样孔中，用裸siRNA水溶液作对照，在TAE缓冲溶液中(0.5×)中80V电压下电泳30min。电泳结束后，将琼脂糖转移至干净的蒸馏水中清洗干净，用凝胶成像仪拍摄出紫外光照片(图1a)。
实施例9
参照实施例8，将步骤(1)改为
利用实施例2中制备的阳离子分支淀粉基基因载体溶于DEPC水配成浓度4.80mg/ml的溶液；
其余条件不变，用凝胶成像仪拍摄出紫外光照片(图1b)。
实施例10
参照实施例8，将步骤(1)改为
利用实施例3中制备的阳离子分支淀粉基基因载体溶于DEPC水配成浓度13.25mg/ml的溶液；
其余条件不变，用凝胶成像仪拍摄出紫外光照片(图1c)。
实施例11
参照实施例8，将步骤(1)改为
利用实施例4中制备的阳离子分支淀粉基基因载体溶于DEPC水配成浓度12.41mg/ml的溶液；
其余条件不变，用凝胶成像仪拍摄出紫外光照片(图1d)。
实施例12
参照实施例8，将步骤(1)改为
利用实施例5中制备的阳离子分支淀粉基基因载体溶于DEPC水配成浓度12.74mg/ml的溶液；
其余条件不变，用凝胶成像仪拍摄出紫外光照片(图1e)。
实施例13
参照实施例8，将步骤(1)改为
利用实施例6中制备的阳离子分支淀粉基基因载体溶于DEPC水配成浓度28.53mg/ml的溶液；
其余条件不变，用凝胶成像仪拍摄出紫外光照片(图1f)。
实施例14
参照实施例8，将步骤(1)改为
利用实施例7中制备的阳离子分支淀粉基基因载体溶于DEPC水配成浓度11.96mg/ml的溶液；
其余条件不变，用凝胶成像仪拍摄出紫外光照片(图1g)。
实施例8、9、10、11、12、13、14在不同N/P比下得到的凝胶电泳图如图1所示，在一定的N/P比时，阳离子淀粉基基因载体都表现出良好的siRNA保护效果。为了比较相同分支程度、不同取代度的阳离子基因载体的siRNA负载效果，对比图1(a)、1(b)和1(f)、1(g)，发现较低的取代度需要在较高的N/P下才能实现较好的包埋，但是随着阳离子基因载体的质量的增加，复合物的表面电位增加，潜在的生物毒性增加。另一方面，为探究淀粉结构对siRNA负载影响，需要进一步比较相似取代度，不同分支程度的阳离子淀粉基作基因载体的负载效果，同时，为了获得具有特殊官能团的载体促进纳米颗粒通过生物屏障，提高siRNA的生物利用度，制备小尺寸的纳米级阳离子淀粉基基因载体十分必要。
实施例15
利用马尔文激光粒度分布仪测定实施例10、11、12、14中制备的不同N/P下的阳离子分支糊精基因载体与siRNA形成的纳米复合物的zeta电位进行测定。结果显示(表4)，相同的取代度下，不论阳离子分支糊精基因载体的分支程度高低，与siRNA形成的复合物的表面电位随着N/P的增加，电荷呈现逐渐增加的趋势，表面电位为-4.00–17.00mV。纳米复合物带正电表明它能与带负电的siRNA络合更加紧密，且在转染过程中能与表面带负电的细胞膜快速结合促进细胞对复合物的内吞作用，且随着分支程度的增加，复合物的表面电位逐渐减小，这说明高支化的结构对降低载体的细胞毒性具有巨大潜力。
表4为根据上述方法制备的不同分支程度的阳离子分支糊精基因载体在不同N/P条件下与siRNA形成的复合物的表面电位。
表4不同分支程度的阳离子分支糊精基因载体在不同N/P条件下与siRNA形成的复合物的表面电位。
N/P C-Y-M-4 C-GT-M-4 C-RO-M-4 C-RG-M-4
0.5 -3.59±1.77 -2.47±0.46 -1.95±0.56 -2.38±0.27
1.0 -0.35±0.15 2.15±0.90 0.61±0.10 -1.59±0.35
1.5 4.50±0.13 4.38±0.25 3.67±0.47 4.06±0.38
2.0 10.22±0.92 7.92±1.56 6.48±0.45 6.11±0.03
2.5 10.89±1.69 12.50±0.42 8.70±0.31 8.50±1.99
3.0 13.40±0.46 13.55±0.35 13.85±0.49 11.00±0.28
3.5 17.23±0.06 15.30±0.57 16.10±1.13 13.25±0.21
实施例16
利用马尔文激光粒度分布仪测定实施例10、11、12、14中制备的不同N/P下的阳离子分支糊精基因载体与siRNA形成的纳米复合物的粒度进行测定。结果显示(表5)，随着N/P摩尔比的升高，粒度变化均呈现先增大后减小的趋势，可能是因为此时粒子表面电位接近0mV，阳离子改性淀粉、DNA与传递系统之间易发生团聚，从而导致粒度大幅增加，当N/P继续增大，表面带正电，表现出颗粒间互斥的现象，从而能够以稳定的粒径形态存在于溶液中。相同的取代度下，随着阳离子分支糊精基因载体分支程度的增加，复合物粒子之间的空间阻力增加，因此，在不同的N/P下，阳离子超支化糊精C-RG-M-4和siRNA形成的复合物的粒度分布越来越均匀，所形成的纳米传输系统的尺寸为300-400nm。
表5为根据上述方法制备的不同分支程度的阳离子分支糊精基因载体在不同N/P条件下与siRNA形成的复合物的粒度分布情况。
表5不同分支程度的阳离子分支糊精基因载体在不同N/P条件下与siRNA形成的复合物的粒度分布情况。
N/P C-Y-M-4 C-GT-M-4 C-RO-M-4 C-RG-M-4
0.5 659.30±28.99 607.55±2.05 620.25±14.64 312.63±14.47
1.0 635.47±19.80 1090.33±63.50 509.50±18.53 333.83±15.90
1.5 1462.33±25.58 513.97±26.97 718.25±12.94 704.40±22.48
2.0 481.03±19.10 394.97±103.74 413.75±40.52 330.60±7.33
2.5 800.43±59.57 382.27±23.95 463.80±41.44 350.10±20.45
3.0 972.05±100.34 553.60±45.16 538.03±20.08 378.15±2.47
3.5 952.65±81.10 710.95±105.00 552.15±79.97 324.03±13.30
实施例17
将实施例14中的阳离子分支糊精基因载体溶液和siRNA溶液以N/P＝2.0的比例按体积混合，控制siRNA浓度均为2500ng/μl，加入适量的超纯水使最终体积为10μl，涡旋振荡1min，室温25℃分别静置4h、24h、3d、7d。
取静置不同时间的样品5μl与1μl 6×上样缓冲液(loading buffer)混合后用微量移液器转移1％的琼脂糖加样孔中，用裸siRNA水溶液作对照，在TAE缓冲溶液中(0.5×)中80V电压下电泳30min。电泳结束后，将琼脂糖转移至干净的蒸馏水中清洗干净，用凝胶成像仪拍摄出紫外光照片(图2a、2b、2c、2d)。
比较不同时间阳离子分支糊精基因载体对siRNA的包埋效果，如图2所示，阳离子分支糊精基因载体-siRNA复合物室温静置7天后，仍表现出良好的包埋效果，并未发生siRNA的泄露。

Claims (10)

  1. 一种阳离子超支化淀粉基基因载体的制备方法，其特征在于，是以淀粉分支酶处理过的淀粉或糊精为底物，采用阳离子醚化剂对其进行化学改性，制备获得阳离子分支淀粉基基因载体，所述的阳离子超支化淀粉基基因载体的α-1,6-糖苷键含量5％-11％；所述的阳离子超支化淀粉基基因载体的取代度在0.030-0.080。
  2. 根据权利要求1所述方法，其特征在于，所述的阳离子超支化淀粉基基因载体具体制备步骤如下：
    (1)底物的制备
    将淀粉/糊精用蒸馏水配置成水溶液，加热保温糊化，搅拌并加入淀粉分支酶酶解，酶解后糊化灭酶，经冷冻干燥、研磨过筛得到底物，即淀粉分支酶处理过的淀粉/糊精；
    (2)底物改性制备阳离子超支化淀粉基基因载体
    将步骤(1)中获得的底物分散于无水乙醇中，形成混合物；调节阳离子醚化剂的pH至9-10，然后加入到混合物中进行加热反应，反应完成后冷却至室温即生成黄色或淡黄色的初产物；向初产物中加入冰乙酸中和至pH呈中性，然后过滤、洗涤，干燥即得阳离子超支化淀粉基基因载体。
  3. 根据权利要求2所述方法，其特征在于，所述阳离子醚化剂为含3-氯-2-羟丙基的三甲基氯化铵溶液。
  4. 根据权利要求2所述方法，其特征在于，步骤(1)中，淀粉分支酶为Rhodothermus obamensis来源的淀粉分支酶Ro-GBE和Geobacillus thermoglucosidans来源的淀粉分支酶Gt-GBE。
  5. 根据权利要求2所述方法，其特征在于，步骤(1)中，加入淀粉分支酶的步骤为先加入淀粉分支酶Gt-GBE再加入淀粉分支酶Ro-GBE。
  6. 根据权利要求2所述方法，其特征在于，步骤(1)中，淀粉分支酶Gt-GBE的反应温度50℃～60℃，加酶量25～35U/g，反应时间10-15h。
  7. 根据权利要求2所述方法，其特征在于，步骤(1)中，淀粉分支酶Ro-GBE的反应温度55℃～65℃，加酶量30～40U/g，反应时间8-12h。
  8. 根据权利要求1～8任一项所述方法，其特征在于，将制备完的阳离子超支化淀粉基基因载体装入透析袋中，超纯水中透析48-72h，然后冷冻干燥。
  9. 根据权利要求1～8任一项所述方法制备得到的阳离子超支化淀粉基基因载体。
  10. 权利要求9所述阳离子超支化淀粉基基因载体在基因药物制备上的应用。
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