WO2015188423A1

WO2015188423A1 - Guanidine hypoglycemic drug-polysaccharide conjugate, preparation method therefor and use thereof

Info

Publication number: WO2015188423A1
Application number: PCT/CN2014/082486
Authority: WO
Inventors: 姜虎林; 王凤珍; 邢磊
Original assignee: 中国药科大学
Priority date: 2014-06-11
Filing date: 2014-07-18
Publication date: 2015-12-17
Also published as: CN103977422A; CN103977422B

Abstract

A guanidine hypoglycemic drug-polysaccharide conjugate, a preparation method therefor and a use thereof are disclosed. The conjugate is formed by connecting primary amine on guanidine drug and aldehyde group on oxidative polysaccharide through a Schiff base bond. Compared with an original guanidine hypoglycemic drug, on the one hand, the conjugate can be used as a hypoglycemic macromolecule prodrug with the enhanced pharmacologic action, less adverse reaction and higher safety; on the other hand, the conjugate can be combined with therapeutic gene for treating diabetes mellitus, in particular obese diabetic, and the curative effects of the conjugate on cellular level and animal model are better than those of the original guanidine hypoglycemic drug and the therapeutic gene. These results prove that the guanidine hypoglycemic drug-polysaccharide conjugate has great potential in the aspect of drug combined gene therapy of diabetes mellitus due to the excellent biocompatibility and high gene transfer efficiency. The synthesizing and preparing method of the conjugate is simple, the process is mature and the yield is high.

Description

胍类降糖药-多糖共轭物及其制备方法和用途 Anthraquinone hypoglycemic agent-polysaccharide conjugate, preparation method and use thereof

技术领域 Technical field

本发明涉及一种胍类降糖药-多糖共轭物，具体涉及一种具备良好生理活性和生物可降解性的胍类降糖药-多糖共轭物作为高分子前药用作基因载体，本发明还涉及该共轭物的制备方法及该共轭物载基因用于糖尿病的治疗，属于药物与基因技术领域。 The invention relates to a steroidal hypoglycemic agent-polysaccharide conjugate, in particular to a steroidal hypoglycemic agent-polysaccharide conjugate having good physiological activity and biodegradability as a polymer prodrug for use as a gene carrier. The invention also relates to a method for preparing the conjugate and the conjugate-loaded gene for the treatment of diabetes, and belongs to the field of medicine and gene technology.

背景技术 Background technique

据《美国医学会杂志》（JAMA )最新研究表明，中国已成为糖尿病人口大国，成人糖尿病患者数量估计超过 1亿，其中 I I型糖尿病占 90%以上。随着生活水平提高，人们饮食结构和生活方式发生了变化，导致肥胖症患者也逐年增加，有研究表明， 80%以上的 I I 型糖尿病患者同时伴有肥胖。肥胖症发病率的上升是诱发 I I型糖尿病的发病率迅速上升的最重要因素之一。肥胖者常伴有内分泌代谢紊乱、高胰岛素血症及脂肪、肌肉、肝细胞的胰岛素受体数目减少或其亲和力降低，对胰岛素不敏感，导致胰岛素抵抗（IR) , 从而葡萄糖利用障碍，发生糖尿病，此多为非胰岛素依赖性糖尿病（ NIDM) , 即 I I 型糖尿病。该病是一组以高血糖为特征的代谢性疾病，其典型症状是 "三多一少"，即多饮、多食、多尿、消瘦。非典型症状：乏力、口干、视力下降、皮肤瘙痒、手脚麻木，疼，走路如踩棉花样等。 According to the latest study by the Journal of the American Medical Association (JAMA), China has become a large country with a large population of diabetes. The number of adult patients with diabetes is estimated to exceed 100 million, of which type I type I diabetes accounts for more than 90%. With the improvement of living standards, people's diet structure and lifestyle have changed, leading to an increase in obesity patients. Studies have shown that more than 80% of patients with type I diabetes are accompanied by obesity. The rise in the incidence of obesity is one of the most important factors influencing the rapid rise in the incidence of type I diabetes. Obese people are often accompanied by endocrine and metabolic disorders, hyperinsulinemia, and decreased number of insulin receptors in fat, muscle, and hepatocytes, or decreased affinity, are not sensitive to insulin, cause insulin resistance (IR), and thus glucose utilization disorders, diabetes This is mostly non-insulin dependent diabetes mellitus (NIDM), which is type 2 diabetes. The disease is a group of metabolic diseases characterized by hyperglycemia. The typical symptoms are "three more and one less", that is, polydipsia, polyphagia, polyuria, and wasting. Atypical symptoms: fatigue, dry mouth, decreased vision, itchy skin, numbness of hands and feet, pain, walking like cotton.

近年来，对糖尿病病理生理学的认识水平和在此基础上的治疗水平有了很大的提高。 I I型糖尿病早期患者可通过改善生活方式（如健康饮食、适量运动、安全减肥、戒烟及避免二手烟暴露等）得以控制。但大多数患者需要口服降糖药物来帮助控制体内血糖，有些甚至仍需要胰岛素注射。然而，绝大多数 I I型糖尿病及糖尿病相关并发症（如导致失明和肾功能衰竭等）的发病原因还不清楚，尚无根治手段。近年来，随着基因重组技术及转基因技术的迅速发展，尤其是糖尿病相关基因的克隆的研究，糖尿病基因治疗有了较大的发展。 In recent years, the level of understanding of the pathophysiology of diabetes and the level of treatment based on it have been greatly improved. Early stage II diabetes can be controlled by improving lifestyles such as healthy eating, moderate exercise, safe weight loss, smoking cessation and exposure to secondhand smoke. But most suffer People need oral hypoglycemic drugs to help control blood sugar in the body, and some even require insulin injections. However, the causes of most type 2 diabetes and diabetes-related complications (such as blindness and renal failure) are still unclear, and there is no cure. In recent years, with the rapid development of genetic recombination technology and transgenic technology, especially the cloning of diabetes-related genes, diabetes gene therapy has developed greatly.

目前应用于基因治疗的载体主要有病毒载体和非病毒载体两种。病毒载体转染效率高，但存在免疫原性高、毒性大、目的基因容量小、靶向特异性差、制备较复杂及费用较高等缺点，因此人们愈来愈重视用于递送基因的非病毒载体研究。 The vectors currently used for gene therapy mainly include viral vectors and non-viral vectors. Viral vector transfection efficiency is high, but it has the disadvantages of high immunogenicity, high toxicity, small target gene capacity, poor targeting specificity, complicated preparation and high cost. Therefore, people pay more and more attention to non-viral vectors for gene delivery. the study.

发明内容 Summary of the invention

目的：为了克服现有技术中存在的不足，本发明提供一种胍类降糖药- 多糖共轭物，该共轭物是由氧化多糖的醛基与胍类降糖药的伯胺反应通过西弗碱键生成，该共轭物荷较高的正电，可以进一步通过静电相互作用与基因结合形成复合物；从而提供一种新型有效递送基因用于 I I型糖尿病治疗的非病毒载体；该递送***是一种对人体无毒副作用，尤其对肥胖症糖尿病有更好疗效的降糖药与基因共递送***。 OBJECTIVE: To overcome the deficiencies in the prior art, the present invention provides a steroidal hypoglycemic agent-polysaccharide conjugate which is obtained by reacting an aldehyde group of an oxidized polysaccharide with a primary amine of a steroidal hypoglycemic agent. The Schiff base bond is generated, and the conjugate is charged with a high positive charge, and can further form a complex with the gene through electrostatic interaction; thereby providing a novel effective delivery gene for the non-viral vector for the treatment of type II diabetes; The delivery system is a hypoglycemic agent and gene co-delivery system that has no toxic side effects on the human body, especially for obesity diabetes.

技术方案：为解决上述技术问题，本发明釆用的技术方案为：一种胍类降糖药-多糖共轭物，所述胍类降糖药-多糖共轭物为多糖氧化产生的醛基与胍类降糖药上的伯胺反应通过西弗碱键连接。 Technical Solution: In order to solve the above technical problems, the technical solution adopted by the present invention is: a steroid type hypoglycemic agent-polysaccharide conjugate, wherein the steroid type hypoglycemic agent-polysaccharide conjugate is an aldehyde group produced by oxidation of polysaccharides The primary amine reaction with the steroidal hypoglycemic agent is linked by a Schiff base bond.

其中，胍类降糖药选自二甲双胍、丁双胍和苯乙双胍； Wherein, the steroidal hypoglycemic agent is selected from the group consisting of metformin, butyl bismuth and phenformin;

多糖选自壳聚糖、壳寡糖、葡聚糖、透明质酸、肝素、软骨素、琼脂糖、海藻酸中的一种或几种。作为优选方案，所述胍类降糖药-多糖共轭物为二甲双胍-壳聚糖。 The polysaccharide is selected from one or more of chitosan, chitooligosaccharide, dextran, hyaluronic acid, heparin, chondroitin, agarose, and alginic acid. Preferably, the steroidal hypoglycemic agent-polysaccharide conjugate is metformin-chitosan.

本发明还提供上述胍类降糖药 -多糖共轭物的制备方法，包括以下步骤: The invention also provides a preparation method of the above-mentioned steroidal hypoglycemic agent-polysaccharide conjugate, comprising the following steps:

1 )氧化多糖的合成：将多糖溶于水或弱酸缓冲液中，然后加入一定量的氧化剂，在 4-90 °C下搅拌适宜时间，过滤后加入过量还原剂继续搅拌一段时间，之后用水充分透析，冻干得粉末固体的氧化多糖； 1) Synthesis of oxidized polysaccharide: Dissolve the polysaccharide in water or weak acid buffer, then add a certain amount of oxidant, stir at 4-90 °C for a suitable time, add excess reducing agent after filtration and continue to stir for a while, then fully Dialysis, freeze-drying to obtain a powdered solid oxidized polysaccharide;

2 )称取适量氧化多糖固体溶于一定溶剂后，加入胍类降糖药，搅拌适宜时间，透析冻干即得到共轭物。 2) Weighing the proper amount of oxidized polysaccharide solid dissolved in a certain solvent, adding the steroidal hypoglycemic agent, stirring for a suitable time, and lyophilizing to obtain a conjugate.

所述弱酸缓冲液选自 NaAc_HAc、 NaH₂P0₄_Na₂HP0₄、 KH₂P0₄-K₂HP0_4;；和 / 或，所述氧化剂选自高碘酸钠、高碘酸钾；和 /或，所述还原剂选自乙二醇、亚硫酸氢钠。所述的制备方法中，其中多糖单体与氧化剂的摩尔比介于 0. 01-100。其中反应温度介于 4-90 °C。其中氧化时间介于 0. 1-72 h。其中反应溶剂选自水，或不同 pH的（pH 1-pH 14 )溶液中，该溶液包括醋酸、磷酸、氢氧化钠等溶液。 The weak acid buffer is selected from the group consisting of NaAc_HAc, NaH ₂ P0 ₄ _Na ₂ HP0 ₄ , KH ₂ P0 ₄ -K ₂ HP0 ₄ ; and/or the oxidizing agent is selected from the group consisting of sodium periodate, potassium periodate; Alternatively, the reducing agent is selected from the group consisting of ethylene glycol and sodium hydrogen sulfite. 01至100。 The molar ratio of the polysaccharide monomer to the oxidizing agent is between 0. 01-100. The reaction temperature is between 4 and 90 °C. 01至72小时。 The oxidation time is between 0. 1-72 h. Wherein the reaction solvent is selected from the group consisting of water, or a solution of different pH (pH 1-pH 14), and the solution includes a solution of acetic acid, phosphoric acid, sodium hydroxide or the like.

所述的胍类降糖药-多糖共轭物胍类降糖药 -多糖共轭物作为非病毒基因载体的应用。 The use of the steroidal hypoglycemic agent-polysaccharide conjugate steroid hypoglycemic agent-polysaccharide conjugate as a non-viral gene carrier.

所述的胍类降糖药-多糖共轭物在用于治疗 II型糖尿病药物中的应用。所述的胍类降糖药-多糖共轭物，用作治疗型聚合物前药，通过静脉注射，腹腔注射，粘膜给药或肺部吸入给药。 The use of the steroidal hypoglycemic agent-polysaccharide conjugate in the treatment of a type II diabetes drug. The steroidal hypoglycemic agent-polysaccharide conjugate is used as a therapeutic polymer prodrug by intravenous injection, intraperitoneal injection, mucosal administration or pulmonary inhalation.

胍类降糖药 -多糖共轭物 /基因复合物的制备方法，步骤如下：将上述胍类降糖药-多糖共轭物用水配置成 0. 001%-10%的共轭物溶液，得到聚合物前药溶液；将治疗有效量的基因配置成 0. 001%-10%的基因溶液，与共轭物溶液混合，经涡旋处理，胍类降糖药-多糖共轭物与基因通过静电相互作用进行复合得到 10- l OOOnm复合物溶液。 The steroidal hypoglycemic agent-polysaccharide conjugate/gene complex is prepared as follows: The steroidal hypoglycemic agent-polysaccharide conjugate is placed in a 0.001%-10% conjugate solution to obtain The polymer prodrug solution; the therapeutically effective amount of the gene is configured to be 0. 001%-10% of the gene solution, mixed with the conjugate solution, vortexed, the steroidal hypoglycemic agent-polysaccharide conjugate and the gene pass static electricity Interaction The composite was obtained to obtain a 10-l OOOnm complex solution.

所述基因选自固醇调控元件结合蛋白基因（shSREBP-la 、 shSREBP-lc 或 shSREBP- 2 )、瘦素基因（lept in cDNA )、胰岛素基因（insul in gene )、胰高糖素样肽 -1基因（GLP-1 gene ), 钙响应性激酶基因（shCaMKI I )。 The gene is selected from a sterol regulatory element binding protein gene (shSREBP-la, shSREBP-lc or shSREBP-2), a lept in cDNA, an insulin gene (insul in gene), a glucagon-like peptide- 1 gene (GLP-1 gene), a calcium-responsive kinase gene (shCaMKI I ).

具体方案如下： The specific plan is as follows:

多糖按照上述的氧化方法进行反应，进一步在氧化多糖上引入胍类降糖药，使其荷更多的正电，在水溶液中可以通过静电相互作用与基因形成复合物。因此该胍类降糖药-多糖共轭物即是一种优的聚合物前药，又是一种良好的传递基因的载体材料。该共轭物可用于静脉注射，腹腔注射，粘膜给药或肺部给药。该共轭物与基因形成的复合物，粒径在 10-1000nm, 均匀度好，再分散性好，载药量和载基因量高。 The polysaccharide is reacted according to the above oxidation method, and a steroidal hypoglycemic agent is further introduced onto the oxidized polysaccharide to make it more positively charged, and a complex can be formed with the gene by electrostatic interaction in the aqueous solution. Therefore, the steroidal hypoglycemic agent-polysaccharide conjugate is an excellent polymer prodrug and a good carrier material for transmitting genes. The conjugate can be used for intravenous, intraperitoneal, mucosal or pulmonary administration. The complex formed by the conjugate and the gene has a particle size of 10-1000 nm, good uniformity, good redispersibility, and high drug loading and gene loading.

胍类降糖药-多糖共轭物的合成、表征及其与基因形成复合物的制备方法、表征及其细胞药效考察详细说明如下： The synthesis, characterization and preparation of steroid-like hypoglycemic agents-polysaccharide conjugates and their preparation and characterization of gene-forming complexes are detailed as follows:

一、胍类降糖药 -多糖共轭物的合成 I. Synthesis of steroidal hypoglycemic agents - polysaccharide conjugates

1. 氧化多糖的合成 1. Synthesis of oxidized polysaccharides

取一定量的多糖和氧化剂分别溶解于 25 mL 弱酸缓冲液中，在 4 °(通 N₂条件下溶解，然后将氧化剂溶液慢慢滴加到多糖溶液中，在 4 °C下继续搅拌 48h后，加入过量的还原剂终止反应，将所得产物用透析袋分别在含 0. 2 M NaCl的弱酸缓冲液和去离子水中透析；最后将产品冻干，保存在 -20 °C 下备用。 A certain amount of polysaccharide and oxidant were dissolved in 25 mL of weak acid buffer, dissolved at 4 ° (dissolved under N ₂ conditions, then the oxidant solution was slowly added dropwise to the polysaccharide solution, and stirring was continued at 4 ° C for 48 h. The reaction was terminated by adding an excess of reducing agent, and the obtained product was dialyzed against a weak acid buffer containing 0.2 M NaCl and deionized water in a dialysis bag; finally, the product was lyophilized and stored at -20 ° C for use.

2. 胍类降糖药 -多糖共轭物的合成 2. Synthesis of steroidal hypoglycemic agents - polysaccharide conjugates

将胍类降糖药加到一定质量浓度的多糖溶液中， 4 °C下搅拌反应 48h 后，用截留分子量 3500的透析袋在去离子水中透析，冻干，将终产物保存在 -20 下备用。 The steroidal hypoglycemic agent is added to a certain concentration of polysaccharide solution, and the reaction is stirred at 4 ° C for 48 hours. Thereafter, it was dialyzed against deionized water using a dialysis bag having a molecular weight cut off of 3,500, lyophilized, and the final product was stored at -20 for use.

本发明与传统治疗糖尿病的基因载体相比具有以下特点： Compared with the traditional gene carrier for treating diabetes, the invention has the following characteristics:

本发明制备胍类降糖药-多糖共轭物，作为非病毒载体，合成方法简单，反应步骤少，产率高，环境污染少。 The invention prepares a steroidal hypoglycemic agent-polysaccharide conjugate, and as a non-viral carrier, the synthesis method is simple, the reaction step is small, the yield is high, and the environmental pollution is small.

二、胍类降糖药-多糖共轭物的表征及毒性考察 2. Characterization and toxicity of steroidal hypoglycemic agents-polysaccharide conjugates

1. 胍类降糖药-多糖共轭物的表征方法 1. Characterization method of steroid type hypoglycemic agent-polysaccharide conjugate

本发明制备胍类降糖药-多糖共轭物，可通过氢核磁鉴定结构及凝胶渗透色谱测定分子量进行表征。 The steroid type hypoglycemic agent-polysaccharide conjugate prepared by the invention can be characterized by hydrogen nuclear magnetic identification structure and molecular weight measurement by gel permeation chromatography.

2. 胍类降糖药-多糖共轭物的毒性考察 2. Toxicity of steroidal hypoglycemic agents-polysaccharide conjugates

胍类降糖药-多糖共轭物的细胞毒性具体测定方法是：釆用不同的细胞系评价胍类降糖药 -多糖共轭物的细胞毒性。将细胞按 ΐ χ ΐ ο⁴个细胞 /孔的量接种在 96孔平底板中，在 37 °C下， 5% C0₂培养箱，于 DMEM培养基中培养 18h后，加入用不同浓度的共轭物继续培养一定时间后，再加入 20 μ ΐ MTS , 培养 4 h后，用酶标仪在 490 讓检测吸光值。 The specific cytotoxicity of the steroidal hypoglycemic agent-polysaccharide conjugate is determined by: 釆 The cytotoxicity of the steroidal hypoglycemic agent-polysaccharide conjugate is evaluated using different cell lines. The cells were seeded in a 96-well flat bottom plate at a rate of ⁴ cells/well, and cultured in a DMEM medium for 18 hours at 37 ° C in a 5% C0 ₂ incubator. After the yoke was cultured for a certain period of time, 20 μM ΐ MTS was added and cultured for 4 h, and the absorbance was measured at 490 with a microplate reader.

三、胍类降糖药 -多糖共轭物与基因的复合物的制备方法 Third, anthraquinone hypoglycemic agent - preparation method of complex of polysaccharide conjugate and gene

所有的胍类降糖药 -多糖共轭物 /基因复合物均是新鲜制备，具体方法是，将含基因溶液加到等体积下的共轭物溶液中，轻轻地涡旋 l min, 室温下保持 30 min。其中，基因选自固醇调控元件结合蛋白基因（ shSREBP-la 、 shSREBP-lc或 shSREBP-2 ),瘦素基因（ lept in c匪 A ),胰岛素基因（ insul in gene ),胰高糖素样肽 -1基因（GLP-1 gene ),钙响应性激酶基因（ shCaMKI I ); 四、胍类降糖药 -多糖共轭物与基因的复合物的表征方法 1. 胍类降糖药 -多糖共轭物 /基因粒径和电位表征。 All steroidal hypoglycemic agents-polysaccharide conjugates/gene complexes are freshly prepared by adding the gene-containing solution to an equal volume of the conjugate solution and gently vortexing for 1 min at room temperature. Hold for 30 min. Among them, the gene is selected from the sterol regulatory element binding protein gene (shSREBP-la, shSREBP-lc or shSREBP-2), leptin gene (lept in c匪A), insulin gene (insul in gene), glucagon-like Peptide-1 gene (GLP-1 gene), calcium-responsive kinase gene (shCaMKI I ); IV. Characterization method of complex of steroidal hypoglycemic agent-polysaccharide conjugate and gene 1. Deuterated hypoglycemic agents - polysaccharide conjugates / gene size and potential characterization.

将胍类降糖药 -多糖共轭物 10 mg溶解于 10 ml水中，超声溶解， 0.45 μηι滤膜过滤，作为储备液。分别将 1 mL含 80 g的基因水溶液加入到等体积胍类降糖药-多糖共轭物水溶液中（其由储备液稀释制备，溶液中胍类降糖药-多糖共轭物与基因按不同的质量比配置），轻轻涡旋 l min, 室温下保持 30 min, 用动态光散射分别测定其粒径和电位。 The steroid type hypoglycemic agent-polysaccharide conjugate 10 mg was dissolved in 10 ml of water, sonicated, and filtered through a 0.45 μηι filter membrane as a stock solution. 1 mL of 80 g of the aqueous solution of the gene was added to an equal volume of anthraquinone hypoglycemic agent-polysaccharide conjugate aqueous solution (diluted from a stock solution, the steroidal hypoglycemic agent-polysaccharide conjugate in the solution was different from the gene) The mass ratio is configured., gently vortex for 1 min, hold at room temperature for 30 min, and measure the particle size and potential by dynamic light scattering.

2. 胍类降糖药-多糖共轭物对基因压缩和保护能力通过电泳表征。共轭物 /DNA的以不同的质量比，加入上样缓冲液，最后的体积为 12 μΐ. 为了评价聚合物对 DNA的保护能力， DNasel作为降解酶。复合物被加到 1% 琼脂糖凝胶中，用 TAE缓冲液作为电解质， 50 V下跑 40 min。 2. The ability of the steroidal hypoglycemic agent-polysaccharide conjugate to encode and protect genes is characterized by electrophoresis. The conjugate/DNA was added to the loading buffer at a different mass ratio, and the final volume was 12 μΐ. To evaluate the polymer's ability to protect DNA, DNasel was used as a degrading enzyme. The complex was applied to a 1% agarose gel using TAE buffer as the electrolyte and running at 50 V for 40 min.

3. 胍类降糖药 -多糖共轭物 /基因复合物的形貌通过透射电镜观察。取 1滴胍类降糖药 -多糖共轭物 /DNA复合物滴到铜网上，干燥 10 min, 在电镜下观察其形态。 3. The morphology of the steroidal hypoglycemic agent-polysaccharide conjugate/gene complex was observed by transmission electron microscopy. One drop of the steroidal hypoglycemic agent-polysaccharide conjugate/DNA complex was dropped onto the copper mesh, dried for 10 min, and its morphology was observed under an electron microscope.

5、胍类降糖药 -多糖共轭物 /基因复合物转染效率考察 5, steroidal hypoglycemic agents - polysaccharide conjugate / gene complex transfection efficiency

釆用特定细胞系评价胍类降糖药 -多糖共轭物 /基因复合物的体外转染效率。将细胞按 10 X 10⁴个细胞 /孔的量接种在 24孔平底板中，在 37 °C 下， 5% C0₂培养箱下，于 DMEM培养基中培养 18-24 h后，吸取培养基，用无血清的含共轭物 /pGL3复合物培养基孵育 4 h后，换用新鲜的含血清培养基，于 37°C下培养一定时间。荧光素酶活性测定方法按照生产厂家的说明进行测定。用 BCA蛋白测试试剂盒提取蛋白后，其浓度用酶标仪测定，再将每个样品荧光素酶活性进行标准化，考察转染效率。 The in vitro transfection efficiency of the steroidal hypoglycemic agent-polysaccharide conjugate/gene complex was evaluated using a specific cell line. The cells were seeded in a 24-well flat bottom plate at a rate of 10×10 ⁴ cells/well, and cultured in a DMEM medium for 18-24 h at 37 ° C in a 5% CO ₂ incubator. After incubating for 4 h in serum-free conjugate/pGL3 complex medium, switch to fresh serum-containing medium and incubate at 37 ° C for a certain period of time. The method for measuring luciferase activity was measured according to the manufacturer's instructions. After extracting the protein using the BCA protein test kit, the concentration was measured by a microplate reader, and the luciferase activity of each sample was normalized to examine the transfection efficiency.

6、胍类降糖药 -多糖共轭物 /基因复合物细胞药效考察釆用特定细胞系评价胍类降糖药 -多糖共轭物 /功能基因复合物的细胞药效。将细胞按 30 X 10⁴个细胞 /孔的量接种在 6孔平底板中，在 37 °C 下， 5% C0₂培养箱下，于 DMEM培养基中培养 18-24 h后，吸取培养基，将该细胞按 30 X 10⁴个细胞 /孔的量接种在 6孔平底板中，孵育 18-24 h 后，吸取培养基，用含胍类降糖药 -多糖共轭物 /基因复合物的无血清培养基培养一定时间后，换用新鲜的含血清培养基，于 37 °C下继续孵育一定时间。蛋白和 RNA的提取分别按照试剂盒的说明进行操作。用 wes tern b lot 检测细胞中的蛋白含量；逆转录 RNA后，用 d-PCR 检测细胞中的 mRNA含量。 6. Drug efficacy of steroidal hypoglycemic agents-polysaccharide conjugates/gene complexes 细胞 Cellular efficacy of a steroidal hypoglycemic agent-polysaccharide conjugate/functional gene complex was evaluated using a specific cell line. The cells were seeded in a 6-well flat bottom plate at a dose of 30 X 10 ⁴ cells/well, and cultured in a DMEM medium for 18-24 h at 37 ° C in a 5% CO ₂ incubator. The cells were seeded in a 6-well flat bottom plate at a dose of 30 X 10 ⁴ cells/well. After incubation for 18-24 h, the medium was aspirated, and the steroid-containing hypoglycemic agent-polysaccharide conjugate/gene complex was used. After culturing for a certain period of time in serum-free medium, switch to fresh serum-containing medium and continue incubation at 37 °C for a certain period of time. Protein and RNA extraction were performed according to the instructions in the kit. The protein content in the cells was detected by wes tern b lot; after reverse transcription of the RNA, the mRNA content in the cells was measured by d-PCR.

有益效果：本发明提供的胍类降糖药-多糖共轭物，为基于生物相容性多糖载体材料，具有很低的细胞毒性； Advantageous Effects: The steroidal hypoglycemic agent-polysaccharide conjugate provided by the invention is a biocompatible polysaccharide carrier material and has low cytotoxicity;

本发明为基因与降糖药共递送***，具有显著的转染效率； The invention relates to a co-delivery system of genes and hypoglycemic agents, which has remarkable transfection efficiency;

本发明中药物的适应症：糖尿病，尤其适用于肥胖症的 I I型糖尿病；本发明提供的胍类降糖药-多糖共轭物，既可作为单一治疗型高分子前药，又可作为基因递送的优良载体，与降糖药达到协同治疗的目的； Indications for the drug of the present invention: Diabetes, especially for Type II diabetes of obesity; The steroidal hypoglycemic agent-polysaccharide conjugate provided by the present invention can be used as a single therapeutic polymer prodrug or as a gene An excellent carrier for delivery, and the purpose of synergistic treatment with hypoglycemic agents;

本发明制备的胍类降糖药-多糖共轭物能有效地与基因形成复合物，可用于静脉注射、腹腔注射、粘膜给药或肺部给药，具有安全性高，粒径控制在 10-1000 nm, 在基因治疗领域具有广阔的应用前景。 The steroidal hypoglycemic agent-polysaccharide conjugate prepared by the invention can effectively form a complex with a gene, and can be used for intravenous injection, intraperitoneal injection, mucosal administration or pulmonary administration, and has high safety and particle size control at 10 -1000 nm, has broad application prospects in the field of gene therapy.

附图说明 DRAWINGS

图 1为本发明实施例中的二甲双胍-壳聚糖的氢核磁表征； 1 is a hydrogen nuclear magnetic characterization of metformin-chitosan in an embodiment of the present invention;

图 2为本发明实施例中的二甲双胍-壳聚糖的细胞毒性考察； 2 is a view showing the cytotoxicity of metformin-chitosan in the examples of the present invention;

图 3为二甲双胍-壳聚糖 /DNA复合物的电位表征； Figure 3 is a potential characterization of metformin-chitosan/DNA complexes;

图 4为二甲双胍-壳聚糖 /m 复合物的形态表征；图 5为二甲双胍-壳聚糖 /基因复合物的细胞转: Figure 4 is a morphological characterization of metformin-chitosan/m complex; Figure 5 shows the cell turnover of metformin-chitosan/gene complex:

图 6为二甲双胍-壳聚糖 /基因复合物的细胞药; Figure 6 is a cytopharmaceutical of metformin-chitosan/gene complex;

具体实施方式 detailed description

下面结合附图对本发明作更进一步的说明。 The present invention will be further described below in conjunction with the accompanying drawings.

实施例 1 Example 1

Metformin Metformin Metformin Metformin

二甲双胍-壳聚糖的合成: Synthesis of metformin-chitosan:

chitosait

Chitosait

Chitosan-fftf z-Metforrain Chitosan-fftf z-Metforrain

(CS-MET) (CS-MET)

将 0. 209 g壳聚糖和 0. 575 g t碘酸钾分别溶解于 25 mL pH 4. 5的醋酸缓冲液中（分别含 0. 05 mo l /L的 Dissolve 0. 209 g chitosan and 0. 575 g t potassium iodate in 25 mL of pH 4. 5 acetic acid buffer (0. 05 mo l / L respectively)

在 40°C下继续搅拌 1 h后，加入与 Stirring at 40 ° C for 1 h, then adding

所得产物用截留分子量 3500的透析酸缓冲液和去离子水中透析，冻干。称取一定量的氧化壳聚糖，溶解于 1M 的氢氧化钠溶液中，再加入一定量的二甲双胍（摩尔量为氧化壳聚糖单体摩尔量的 2倍）， 4 °C下搅拌反应 48 h后，用截留分子量 3500的透析袋在去离子水中透析，冻干，将终产物保存在 -20 °(下备用。实施例 2 二甲双胍-壳聚糖的结构鉴定和分子量表征如图 1所示，二甲双胍-壳聚糖共轭物通过氢核磁鉴定结构。与壳聚糖 2位碳上质子信号峰（用虛线标示）相比，壳聚糖-二甲双胍结构中壳聚糖 2位碳上质子信号峰基本消失（ δ _Η = 3. 2 ppm ), 说明该结构中壳聚糖上的 C-C键成功的被高碘酸钾氧化断裂，通过壳聚糖 -二甲双胍¹ H丽 R图谱，分别显示了二甲双胍（ δ _Η = 3. 0 ppm; -N (CH₃) ₂ )及壳聚糖（ δ _H =2. 0 ppm; -C0CH₃ ) 的特征信号峰，进一步表明壳聚糖 -二甲双胍的形成. 壳聚糖-二甲双胍分子量通过凝胶渗透色谱检测：柱温为 25 V , 流速 0. 5 mL/min, 流动相为 0. 5 M乙酸铵溶液。壳聚糖原料分子量为 100 kDa , 氧化后其重均分子量为 19. 01 kDa , 接技二甲双胍后，形成的壳聚糖 -二甲双胍重均分子量为 28. 82 kDa。氧化壳聚糖的二甲双胍 -壳聚 The resulting product was dialyzed with a molecular weight cutoff of 3,500. Dialysis in acid buffer and deionized water, lyophilization. Weigh a certain amount of oxidized chitosan, dissolve it in 1M sodium hydroxide solution, add a certain amount of metformin (the molar amount is twice the molar amount of oxidized chitosan monomer), and stir the reaction at 4 °C. After h, it was dialyzed against deionized water using a dialysis bag with a molecular weight cut off of 3,500, lyophilized, and the final product was stored at -20 ° (substitute. Example 2: Molecular identification and molecular weight characterization of metformin-chitosan are shown in Fig. 1. , Metformin-chitosan conjugates were identified by hydrogen nuclear magnetic resonance. Chitosan 2-mer carbon protons in chitosan-metformin structure compared to chitosan 2-position carbon upper proton signal peaks (indicated by dashed lines) The signal peak disappeared (δ _Η = 3. 2 ppm ), indicating that the CC bond on the chitosan in this structure was successfully oxidatively cleaved by potassium periodate, and the chitosan-metformin ¹ H Li map was shown. The characteristic signal peaks of metformin (δ _Η = 3. 0 ppm; -N (CH ₃ ) ₂ ) and chitosan ( δ _H = 2.0 ppm; -C0CH ₃ ) further indicate the formation of chitosan- metformin. The molecular weight of chitosan-metformin was determined by gel permeation chromatography: column temperature was 2 5克。 The flow rate is 0. 5 mL / min, the mobile phase is 0. 5 M ammonium acetate solution. The molecular weight of the chitosan raw material is 100 kDa, and the weight average molecular weight after oxidation is 19.1 kDa, after the formation of metformin, formed The weight average molecular weight of chitosan-metformin is 28.82 kDa. Metformin-shell polymerization of oxidized chitosan

壳聚糖的初始 Initial of chitosan

重均分子量糖的重均分子 Weight average molecular weight

分子量 ( kDa ) Molecular weight ( kDa )

( kDa ) 量（ kDa ) ( kDa ) quantity ( kDa )

100 19. 01 28. 82 实施例 3 二甲双胍-壳聚糖的细胞毒性考察釆用 L02和 HepG2细胞系评价根据实施例 1合成的二甲双胍-壳聚糖的细胞毒性。 L02和 HepG2细胞分别为人正常肝细胞和人肝癌细胞。将这两种细胞以 1 X 10⁴个细胞 /孔的量接种在 96孔平底板中，在 37 °C, 5 % C0₂培养箱中，用 DMEM培养基孵育 18 h后，用不同浓度的聚合物处理 24 h后，用 20 μ ΐ MTS溶液处理 4 h后，用酶标仪检测其在 490 nm下的吸光值。如图 2所示，在浓度 O-lOO g/ml范围内，二甲双胍-壳聚糖对 L02和 HepG2 均无细胞毒性。 100 19. 01 28. 82 Example 3 Cytotoxicity of metformin-chitosan The cytotoxicity of metformin-chitosan synthesized according to Example 1 was evaluated using the L02 and HepG2 cell lines. L02 and HepG2 cells are human normal liver cells and human liver cancer cells, respectively. The two cells were seeded in a 96-well flat bottom plate at a dose of 1×10 ⁴ cells/well, and incubated in DMEM medium for 18 h at 37 ° C in a 5 % C0 ₂ incubator at different concentrations. After the polymer was treated for 24 h, it was treated with 20 μM ΐ MTS solution for 4 h, and its absorbance at 490 nm was measured by a microplate reader. As shown in Figure 2, metformin-chitosan was not cytotoxic to both L02 and HepG2 in the concentration range of O-100 g/ml.

实施例 4 Example 4

二甲双胍-壳聚糖与基因复合物的制备 Preparation of metformin-chitosan and gene complex

将实施例 1二甲双胍-壳聚糖 10 mg溶解于 10 ml水中，超声溶解， 0.45 μηι滤膜过滤，作为储备液。分别将 1 mL含 80 μ_§的基因水溶液加入到等体积二甲双胍-壳聚糖共轭物水溶液中（其由储备液稀释制备，溶液中二甲双胍-壳聚糖共轭物与基因的质量比分别为 1: 1, 1: 5, 1: 10, 1: 20, 1: 30 ), 轻轻涡旋 1 min, 室温下保持 30 min, 用动态光散射测定复合物的大小和表面电荷。如图 3所示，二甲双胍 -壳聚糖基因复合物的粒径随质量比的增大而减小，电荷随质量比的增大而增大，最后二甲双胍 -壳聚糖基因复合物的粒径和电荷均趋于稳定，约 100 nm和 20 mV。 10 mg of metformin-chitosan of Example 1 was dissolved in 10 ml of water, sonicated, and filtered through a 0.45 μηι filter to obtain a stock solution. 1 mL of an aqueous solution containing 80 μ _§ of the gene was added to an equal volume of an aqueous solution of metformin-chitosan conjugate (diluted by stock solution, the mass ratio of metformin-chitosan conjugate to gene in solution was 1: 1, 1: 5, 1: 10, 1: 20, 1: 30), gently vortex for 1 min, hold at room temperature for 30 min, and measure the size and surface charge of the composite by dynamic light scattering. As shown in Fig. 3, the particle size of the metformin-chitosan gene complex decreases with the increase of the mass ratio, and the charge increases with the increase of the mass ratio. Finally, the particle size of the metformin-chitosan gene complex Both the charge and the charge tend to be stable, about 100 nm and 20 mV.

实施例 5 Example 5

二甲双胍-壳聚糖共轭物对 DNA压缩和保护能力通过电泳表征 Characterization of DNA compression and protection by metformin-chitosan conjugate by electrophoresis

将二甲双胍-壳聚糖与 DNA按不同质量比（0.5-30)复合，然后将该复合物于 50 V条件下在 1%琼脂糖凝胶上跑 40 min, 结果表明二甲双胍 -壳聚糖共轭物在质量比 5: 1条件下对 DNA就有结合能力。为了评价该共轭物对匪 A的保护能力，将质量比为 5: 1的二甲双胍-壳聚糖 /DNA复合物与 DNasel 酶复合 30 min后，用 EDTA将酶失活，然后用 SDS解离二甲双胍-壳聚糖 /DNA, 于于 50 V条件下在 1%琼脂糖凝胶上跑 40 min, 通过电泳考察二甲双胍-壳聚糖共轭物对匪 A的结合能力 50 V下跑 40 min, 结果表明二甲双胍 -壳聚糖共轭物对 DNA有很好的保护能力。 Metformin-chitosan was combined with DNA at different mass ratios (0.5-30), and then the complex was run on a 1% agarose gel for 40 min at 50 V. The results showed that metformin-chitosan was conjugated. The substance has binding ability to DNA at a mass ratio of 5:1. In order to evaluate the pair of conjugates 保护A's protective ability, after combining the metformin-chitosan/DNA complex with mass ratio of 5:1 for 30 min with DNasel enzyme, inactivate the enzyme with EDTA, then dissociate metformin-chitosan/DNA with SDS On a 1% agarose gel for 40 min at 50 V, the binding ability of metformin-chitosan conjugate to 匪A was investigated by electrophoresis for 40 min at 50 V. The results showed that metformin-chitosan Conjugates have good protection against DNA.

实施例 6 Example 6

二甲双胍-壳聚糖 /基因复合物的表征 Characterization of metformin-chitosan/gene complex

如图 4所示，通过透射电镜观察按实施例 4合成的二甲双胍-壳聚糖共轭物 /DNA复合物的形貌。取 1 滴二甲双胍-壳聚糖共轭物 /DNA复合物滴到铜网上，干燥 10 min, 在电镜下观察，二甲双胍-壳聚糖 /基因复合物呈类球形。 As shown in Fig. 4, the morphology of the metformin-chitosan conjugate/DNA complex synthesized in accordance with Example 4 was observed by transmission electron microscopy. A drop of metformin-chitosan conjugate/DNA complex was dropped onto the copper mesh and dried for 10 min. Under the electron microscope, the metformin-chitosan/gene complex was spherical.

实施例 7 Example 7

二甲双胍-壳聚糖 /基因复合物的细胞转染考察 Cell Transfection of Metformin-Chitosan/Gene Complex

釆用 L02和 HepG2细胞系评价根据实施例 1合成的二甲双胍-壳聚糖的体外转染效率。 L02 和 HepG2 细胞分别为人正常肝细胞和人肝癌细胞，在 37 °C下， 5% C0₂培养箱（Thermo Sc ient if i c )下，于 DMEM培养基中培养。将两种细胞按 10 X 10⁴个细胞 /孔的量接种在 24孔平底板中，孵育 18 h后，吸取培养基，用含共轭物 /PGL3复合物无血清培养基继续孵育 4 h, 之后换用含血清的培养基，于 37 °C下培养 24 h后测定荧光素酶活性。其测定方法按照生产厂家的说明进行。如图 5所示，用 BCA蛋白测试试剂盒提取蛋白后，其浓度用酶标仪测定。最后将每个样品荧光素酶活性进行标准化，考察转染效率。结果表明甲双胍-壳聚糖 /DNA 复合物相对于壳聚糖 /DNA复合物，在 L02和 HepG2细胞中均有很高的基因转染效率。 The in vitro transfection efficiency of metformin-chitosan synthesized according to Example 1 was evaluated using the L02 and HepG2 cell lines. L02 and HepG2 cells were human normal liver cells and human liver cancer cells, respectively, and cultured in DMEM medium at 37 ° C in a 5% CO ₂ incubator (Thermo Sc ient if ic). The two cells were seeded in a 24-well flat bottom plate at a dose of 10×10 ⁴ cells/well. After incubation for 18 h, the medium was aspirated and the incubation was continued for 4 h with the conjugate/PGL3 complex-free serum medium. Thereafter, the medium containing serum was used, and the luciferase activity was measured after incubation at 37 ° C for 24 hours. The measurement method was carried out in accordance with the manufacturer's instructions. As shown in Fig. 5, after the protein was extracted using the BCA protein test kit, the concentration was measured by a microplate reader. Finally, the luciferase activity of each sample was normalized to investigate the transfection efficiency. The results indicate that the metformin-chitosan/DNA complex is complexed with chitosan/DNA High transgenic efficiency in both L02 and HepG2 cells.

实施例 8 Example 8

二甲双胍-壳聚糖 /基因复合物的细胞药效考察 Cellular efficacy of metformin-chitosan/gene complex

釆用 L02细胞系评价根据实施例 1合成的二甲双胍 -壳聚糖载功能基因的细胞药效。 L02 为人正常肝细胞，在 37 °C下， 5 % C0₂培养箱（Thermo Sc ient if ic ) 下，于 DMEM培养基中培养。将该细胞按 30 χ 10⁴个细胞 /孔的量接种在 6 孔平底板中，孵育 18 h 后，吸取培养基，用含共轭物 /shSREBP-lc 复合物的无血清培养基继续培养 24 h。之后换用新鲜的含血清培养基，于 37 °C下培养 24 h。蛋白和 RNA的提取分别按照试剂盒的说明进行提取。用 wes tern blot 检测细胞中的 AMPK、 pAMPK及 SREBP-lc的蛋白含量；用 d-PCR检测细胞中的 SREBP-lc的 mRNA含量。如图 6所示，结果表明，二甲双胍-壳聚糖 /shSREBP-lc复合物能使 L02细胞中的 AMPK磷酸化，即提高 pAMPK蛋白含量，同时该复合物下调 SREBP-lc的蛋白含量，其相应的 SREBP-lc的 mRNA含量也降低。 The cell viability of the metformin-chitosan-loaded functional gene synthesized according to Example 1 was evaluated using the L02 cell line. L02 is a human normal hepatocyte, and cultured in DMEM medium at 37 ° C in a 5 % C0 ₂ incubator (Thermo Sc ient if ic ). The cells were seeded in a 6-well flat bottom plate at 30 χ 10 ⁴ cells/well. After incubation for 18 h, the medium was aspirated and cultured in serum-free medium containing the conjugate/shSREBP-lc complex. h. Then switch to fresh serum-containing medium and incubate at 37 °C for 24 h. Protein and RNA were extracted separately according to the instructions in the kit. The protein content of AMPK, pAMPK and SREBP-lc in cells was detected by wes tern blot; the mRNA content of SREBP-lc in cells was detected by d-PCR. As shown in Figure 6, the results indicate that the metformin-chitosan/shSREBP-lc complex phosphorylates AMPK in L02 cells, ie increases the pAMPK protein content, while the complex down-regulates the protein content of SREBP-lc. The mRNA content of SREBP-lc is also reduced.

其他胍类降糖药-多糖共轭物，因为胍类降糖药、多糖的物理化学性质相似，因此根据列举的实施例可以看出，都是可以实现和达到的。 Other anthraquinone hypoglycemic agent-polysaccharide conjugates, because the physicochemical properties of the steroidal hypoglycemic agents and polysaccharides are similar, can be seen and achieved according to the enumerated examples.

以上所述仅是本发明的优选实施方式，应当指出：对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。 The above description is only a preferred embodiment of the present invention, and it should be noted that those skilled in the art can also make several improvements and retouchings without departing from the principles of the present invention. It should be considered as the scope of protection of the present invention.

Claims

权利要求书 Claim

1. 一种胍类降糖药-多糖共轭物，其特征在于：所述胍类降糖药 -多糖共轭物为多糖氧化产生的醛基与胍类降糖药上的伯胺反应通过西弗碱键连接。 A steroidal hypoglycemic agent-polysaccharide conjugate, characterized in that: the steroidal hypoglycemic agent-polysaccharide conjugate is an aldehyde group produced by oxidation of a polysaccharide and a primary amine on a steroidal hypoglycemic agent is reacted. Sieffer base linkage.

2. 根据权利要求 1 所述的胍类降糖药-多糖共轭物，其特征在于：其中，胍类降糖药选自二甲双胍、丁双胍和苯乙双胍； 2. The steroidal hypoglycemic agent-polysaccharide conjugate according to claim 1, wherein: the steroidal hypoglycemic agent is selected from the group consisting of metformin, buformin and phenformin;

多糖选自壳聚糖、壳寡糖、葡聚糖、透明质酸、肝素、软骨素、琼脂糖、海藻酸中的一种或几种。 The polysaccharide is selected from one or more of chitosan, chitooligosaccharide, dextran, hyaluronic acid, heparin, chondroitin, agarose, and alginic acid.

3. 根据权利要求 1 所述的胍类降糖药-多糖共轭物，其特征在于：所述胍类降糖药-多糖共轭物为二甲双胍-壳聚糖。 The steroidal hypoglycemic agent-polysaccharide conjugate according to claim 1, wherein the steroidal hypoglycemic agent-polysaccharide conjugate is metformin-chitosan.

4. 一种胍类降糖药 -多糖共轭物的制备方法，包括以下步骤： 4. A method for preparing a steroidal hypoglycemic agent-polysaccharide conjugate, comprising the steps of:

1 )氧化多糖的合成：将多糖溶于水或弱酸缓冲液中，然后加入一定量的氧化剂，反应温度 4-90 °C下搅拌适宜时间，过滤后加入过量还原剂继续搅拌一段时间，之后用水充分透析，冻干得粉末固体的氧化多糖； 1) Synthesis of oxidized polysaccharide: Dissolve the polysaccharide in water or weak acid buffer, then add a certain amount of oxidant, stir at a reaction temperature of 4-90 °C for a suitable time, add excess reducing agent after filtration, continue stirring for a while, then use water Fully dialyzed, lyophilized to obtain a powdered solid oxidized polysaccharide;

5. 根据权利要求 4 所述的胍类降糖药 -多糖共轭物的制备方法，其特征在于：所述弱酸缓冲液选自 NaAc-HAc , NaH₂P0₄_Na₂HP0₄、 KH₂P0₄-K₂HP0_4;；和 /或，所述氧化剂选自高碘酸钠、高碘酸钾；和 /或，所述还原剂选自乙二醇、亚硫酸氢钠。 The method according to claim 4, wherein the weak acid buffer is selected from the group consisting of NaAc-HAc, NaH ₂ P0 ₄ _Na ₂ HP0 ₄ , KH ₂ P0. _{4 -} K ₂ HP0 _4; ; and/or, the oxidizing agent is selected from the group consisting of sodium periodate, potassium periodate; and/or the reducing agent is selected from the group consisting of ethylene glycol and sodium hydrogen sulfite.

6. 权利要求 1 -5所述的胍类降糖药-多糖共轭物胍类降糖药-多糖共轭物作为非病毒基因载体的应用。 6. Use of the steroidal hypoglycemic agent-polysaccharide conjugate steroid hypoglycemic agent-polysaccharide conjugate according to claims 1 to 5 as a non-viral gene vector.

7. 权利要求 1-5 所述的胍类降糖药-多糖共轭物在用于治疗 II型糖尿病药物中的应用。 7. Use of a steroidal hypoglycemic agent-polysaccharide conjugate according to claims 1-5 for the treatment of a type II diabetes drug.

8. 根据权利要求 7 所述的胍类降糖药-多糖共轭物，用作治疗型聚合物前药，通过静脉注射，腹腔注射，粘膜给药或肺部吸入给药。 The steroidal hypoglycemic agent-polysaccharide conjugate according to claim 7, which is used as a therapeutic polymer prodrug by intravenous injection, intraperitoneal injection, mucosal administration or pulmonary inhalation.

9. 胍类降糖药 -多糖共轭物 /基因复合物的制备方法，步骤如下：将上述胍类降糖药-多糖共轭物用水配置成 0. 001%-10%的共轭物溶液，得到聚合物前药溶液；将治疗有效量的基因配置成 0. 001%-10%的基因溶液，与共轭物溶液混合，经涡旋处理，胍类降糖药-多糖共轭物与基因通过静电相互作用进行复合得到 10- l OOOnm复合物溶液。 The conjugate solution of the steroid type hypoglycemic agent-polysaccharide conjugate/gene complex is as follows: The above steroid type hypoglycemic agent-polysaccharide conjugate is set to 0. 001%-10% conjugate solution a polymer prodrug solution is obtained; a therapeutically effective amount of the gene is configured to be 0. 001%-10% of the gene solution, mixed with the conjugate solution, vortexed, steroidal hypoglycemic agent-polysaccharide conjugate and gene The 10 - l OOOnm complex solution was obtained by recombination by electrostatic interaction.

10.根据权利要求 9所述的胍类降糖药 -多糖共轭物 /基因复合物的制备方法，其特征在于：所述基因选自固醇调控元件结合蛋白基因、瘦素基因、胰岛素基因、胰高糖素样肽 -1基因、钙响应性激酶基因。 The method for preparing a steroidal hypoglycemic agent-polysaccharide conjugate/gene complex according to claim 9, wherein the gene is selected from the group consisting of a sterol regulatory element binding protein gene, a leptin gene, and an insulin gene. , glucagon-like peptide-1 gene, calcium-responsive kinase gene.