CN102585036A - Amphiphilic chitosan derivative, its preparation method and its application in medicinal preparation - Google Patents

Abstract

The invention relates to the medical auxiliary material chitosan derivative field, and more specifically relates to an amphiphilic chitosan derivative (I), its preparation method and its application in a medicinal preparation. The chitosan derivative of the present invention is a polymer capable of carrying out biodegradation in vivo possessing amphiphilic polymer molecules, which is characterized in that one end contains hydrophilic group, the other end contains hydrophobic group, and is suitable for medicines, matching with medicines or being as a medicine carrier, and especially can be the medicine carrier with small toxicity and no hemolytic reaction capable of intravenous injection administration.

Description

Translated fromChinese

两亲性壳聚糖衍生物及其制备方法与在药物制剂中的应用Amphiphilic chitosan derivative and its preparation method and application in pharmaceutical preparations

技术领域technical field

本发明涉及以天然且可生物降解的壳聚糖为原料进行两亲性衍生化产品及其制备方法，以及其作为药物载体在制备药物中的应用。The invention relates to an amphiphilic derivatized product using natural and biodegradable chitosan as a raw material and a preparation method thereof, as well as its application as a drug carrier in the preparation of medicines.

背景技术Background technique

纳米技术与可生物降解材料均为当前药物传输系统发展的热点研究领域。聚合物胶束研究融合两者的优势，即可生物降解，又可形成药物纳米载体。聚合物胶束由亲水壳和疏水核组成，既具备形成亲水胶体的药用性能，又适合疏水药物的增溶和包裹。聚合物胶束所具有的由亲水壳和疏水核组成特征使其可以达到体内长循环、减少网状内皮细胞吞噬的目的。Both nanotechnology and biodegradable materials are hot research areas in the development of current drug delivery systems. The research on polymer micelles combines the advantages of both, which can be biodegradable and form drug nanocarriers. Polymer micelles are composed of a hydrophilic shell and a hydrophobic core, which not only have the medicinal properties of forming a hydrophilic colloid, but also are suitable for the solubilization and encapsulation of hydrophobic drugs. The polymer micelle is composed of a hydrophilic shell and a hydrophobic core, so that it can achieve the purpose of long circulation in the body and reduce the phagocytosis of reticuloendothelial cells.

壳聚糖是可生物降解的天然来源的聚合物，具有良好的生物相容性，且来源广泛，价格便宜。发明人前期研究成果：N-辛基-O-磺酸基壳聚糖(NOSC)紫杉醇载药胶束(平其能；张灿.壳聚糖衍生物及其制备方法与在制药中的应用，CN03112981.1)。另外CN200610037810.X还公开了N-辛基-N’-羧甲基壳聚糖(OCC)。Chitosan is a biodegradable polymer of natural origin, has good biocompatibility, and has a wide range of sources and is cheap. Inventor's previous research results: N-octyl-O-sulfonic chitosan (NOSC) paclitaxel drug-loaded micelles (Ping Qineng; Zhang Can. Chitosan derivatives and their preparation methods and their application in pharmaceuticals , CN03112981.1). In addition CN200610037810.X also discloses N-octyl-N'-carboxymethyl chitosan (OCC).

NOSC的临界胶束浓度(Critical micelle concentration，CMC)值为0.45mg/mL(Colloids andSurfaces B：Biointerfaces 39(2004)69-75)，OCC胶束的CMC为5-20μg/mL，CMC值小，预示耐稀释性能增强，体内胶束的稳定性好，药物在纳米粒中不易泄露。The critical micelle concentration (Critical micelle concentration, CMC) value of NOSC is 0.45 mg/mL (Colloids and Surfaces B: Biointerfaces 39 (2004) 69-75), the CMC of OCC micelle is 5-20 μ g/mL, CMC value is small, It indicates that the resistance to dilution is enhanced, the stability of the micelles in the body is good, and the drug is not easy to leak in the nanoparticles.

已发明的两亲性壳聚糖衍生物只能溶解在水中，不能溶于有机溶剂。The invented amphiphilic chitosan derivatives can only be dissolved in water and cannot be dissolved in organic solvents.

发明内容Contents of the invention

本发明用可生物降解的天然来源的壳聚糖作为原料，利用无毒或毒性小的原料摸索出易进行大规模生产的制备工艺，使其形成具有两亲性的聚合物分子，即一端含亲水基、另一端含疏水基并且仍可在体内进行生物降解的聚合物，以适合用于药物、与药物组配或作为药物载体，尤其是作为毒性小、不会产生溶血反应的可静脉注射给药的药物载体。The present invention uses biodegradable chitosan of natural origin as raw material, utilizes non-toxic or less toxic raw material to explore a preparation process that is easy to carry out large-scale production, and makes it form an amphiphilic polymer molecule, that is, one end contains A polymer with a hydrophilic group and a hydrophobic group at the other end that can still be biodegradable in vivo, suitable for use in drugs, compounded with drugs, or as a drug carrier, especially as an intravenous Drug carrier for injection administration.

本发明研究的两亲性质的壳聚糖衍生物，一方面不但可用于难溶药物的增溶和包裹，控制药物释放，而且可以在水中形成纳米级胶束；另一方面使得中间体和终产物在有机溶剂，诸如DMF、DMSO、甲醇或乙醇中有优良的溶解性，使得反应充分，保证较高的产率。首先由亲水的壳和疏水的核组成的纳米胶束，由此可以延长体内循环、减少网状内皮细胞的吞噬，最终提高治疗效果。其次终产物优良的有机溶剂溶解性为更多载药方式提供了可行性操作依据。The amphiphilic chitosan derivatives studied in the present invention can not only be used for solubilization and encapsulation of poorly soluble drugs to control drug release, but also can form nano-sized micelles in water; on the other hand, intermediates and final The product has excellent solubility in organic solvents such as DMF, DMSO, methanol or ethanol, which makes the reaction sufficient and ensures a higher yield. Firstly, the nano-micelle is composed of a hydrophilic shell and a hydrophobic core, which can prolong the circulation in the body, reduce the phagocytosis of reticuloendothelial cells, and finally improve the therapeutic effect. Secondly, the excellent organic solvent solubility of the final product provides a feasible operation basis for more drug loading methods.

本发明的两亲性壳聚糖衍生物如下：Amphiphilic chitosan derivatives of the present invention are as follows:

其中R＝-(CH₂)_n，n＝6～18；Wherein R=-(CH₂ )_n , n=6～18;

x＝50％～60％；y＝8％～10％；z＝20％～30％。x=50%～60%; y=8%～10%; z=20%～30%.

其中x表示辛基取代度；y表示N乙酰化程度；z表示不同酸酐的酰化取代度。x+y+z≤100％.Where x represents the degree of octyl substitution; y represents the degree of N acetylation; z represents the degree of acylation substitution of different anhydrides. x+y+z≤100%.

x优选60％；y优选10％；z优选30％.x is preferably 60%; y is preferably 10%; z is preferably 30%.

n优选12.n is preferably 12.

本发明的壳聚糖衍生物也可称为N-长链烷基-N’-酰化-O-羟丙基壳聚糖，其制备方法依次包括下列步骤：Chitosan derivative of the present invention can also be referred to as N-long chain alkyl-N'-acylated-O-hydroxypropyl chitosan, and its preparation method comprises the following steps successively:

1)取壳聚糖加入甲醇或乙醇中，室温搅拌，加入6-18C的长链烷基醛，反应12-18小时后，加入KBH₄水溶液，继续搅拌8-12小时，生成N-长链烷基壳聚糖；1) Add chitosan to methanol or ethanol, stir at room temperature, add 6-18C long-chain alkylaldehyde, react for 12-18 hours, add KBH₄ aqueous solution, continue stirring for 8-12 hours, and generate N-long chain Alkyl chitosan;

2)取N-长链烷基壳聚糖加入N，N-二甲基甲酰胺(DMF)中搅拌8-12小时，再加入邻苯二甲酸酐(或其他酸酐)的DMF溶液，在130℃和N₂保护下反应12-18小时，制得N-长链烷基-N’-酰化壳聚糖；2) Take N-long-chain alkyl chitosan and add N,N-dimethylformamide (DMF) to stir for 8-12 hours, then add the DMF solution of phthalic anhydride (or other anhydrides), at 130 ℃ and N₂ under protection for 12-18 hours to prepare N-long chain alkyl-N'-acylated chitosan;

3)取N-长链烷基-N’-酰化壳聚糖加入多聚磷酸的DMF溶液中，50℃机械搅拌8-12小时，制得N-长链烷基-N’-酰化-O-羟丙基壳聚糖。3) Add N-long chain alkyl-N'-acylated chitosan into the DMF solution of polyphosphoric acid, and mechanically stir at 50°C for 8-12 hours to obtain N-long chain alkyl-N'-acylated chitosan -O-Hydroxypropyl Chitosan.

更优选的制备方法包括(此部分酸酐化以邻苯二甲酰化为例叙述，其他酸酐化方法类似)：More preferred preparation methods include (this partial acid anhydride is described using phthalylation as an example, and other acid anhydride methods are similar):

1)取壳聚糖加入甲醇中室温搅拌，加入6-18C的长链烷基醛，反应14-16小时后，加入KBH₄水溶液，继续搅拌10小时，生成N-长链烷基壳聚糖；1) Add chitosan to methanol and stir at room temperature, add 6-18C long-chain alkyl aldehyde, react for 14-16 hours, add KBH₄ aqueous solution, continue stirring for 10 hours, and generate N-long-chain alkyl chitosan ;

2)取N-长链烷基壳聚糖加入DMF中搅拌9-10小时，再加入邻苯二甲酸酐DMF溶液，在110-130℃和N₂保护下反应14-16小时，制得N-长链烷基-N’-邻苯二甲酰壳聚糖。2) Add N-long-chain alkyl chitosan into DMF and stir for 9-10 hours, then add phthalic anhydride DMF solution, and react at 110-130°C under_N2 protection for 14-16 hours to obtain N - Long-chain alkyl-N'-phthaloyl chitosan.

3)取N-长链烷基-N’-邻苯二甲酰壳聚糖加入50％NaOH水溶液和DMF中，50℃机械搅拌10小时，制得N-长链烷基-N’-邻苯二甲酰-O-羟丙基壳聚糖(APHPC)。3) Add N-long-chain alkyl-N'-phthaloyl chitosan into 50% NaOH aqueous solution and DMF, and mechanically stir at 50°C for 10 hours to obtain N-long-chain alkyl-N'-o Phthaloyl-O-hydroxypropyl chitosan (APHPC).

更优选地，生成的N-长链烷基壳聚糖的反应液用稀盐酸液中和，用丙酮沉淀，过滤，乙醚冲洗后干燥后再进入第二步反应。生成N-长链烷基-N’-邻苯二甲酰壳聚糖的反应液，过滤，用大量水与DMF混合溶液冲洗后干燥后再进入第三步反应。APHPC滤液优选用透析袋(MWCO 14000)透析，冷冻干燥。More preferably, the generated N-long-chain alkyl chitosan reaction solution is neutralized with dilute hydrochloric acid solution, precipitated with acetone, filtered, rinsed with ether and dried before entering the second step of reaction. Generate the reaction solution of N-long-chain alkyl-N'-phthaloyl chitosan, filter, rinse with a large amount of water and DMF mixed solution, and then dry and then enter the third step of reaction. The APHPC filtrate is preferably dialyzed against a dialysis bag (MWCO 14000) and freeze-dried.

最优选的N-长链烷基-N’-邻苯二甲酰-O-羟丙基壳聚糖(APHPC)的制备方法包括：The preparation method of most preferred N-long chain alkyl-N'-phthaloyl-O-hydroxypropyl chitosan (APHPC) comprises:

1)N-长链烷基壳聚糖的制备1) Preparation of N-long chain alkyl chitosan

取壳聚糖加入甲醇中室温搅拌24小时，加入6-18C的长链烷基醛，反应14-16小时后，加入KBH₄的水溶液，继续搅拌10小时，即生成N-长链烷基壳聚糖，反应液用稀盐酸液中和，用甲醇沉淀，过滤，滤饼即为N-长链烷基壳聚糖，干燥后，得到浅黄色粉末。Add chitosan to methanol and stir at room temperature for 24 hours, add 6-18C long-chain alkyl aldehyde, react for 14-16 hours, add KBH₄ aqueous solution, and continue stirring for 10 hours to form N-long-chain alkyl shell For polysaccharides, the reaction solution is neutralized with dilute hydrochloric acid solution, precipitated with methanol, filtered, and the filter cake is N-long chain alkyl chitosan. After drying, a light yellow powder is obtained.

2)N-长链烷基-N’-邻苯二甲酰-壳聚糖的制备2) Preparation of N-long-chain alkyl-N'-phthaloyl-chitosan

取N-长链烷基壳聚糖加入DMF中搅拌8-12小时，再加入邻苯二甲酸酐的DMF溶液，在130℃和N₂保护下反应12-18小时，反应液倾倒在去离子水中，过滤，滤饼用大量水+DMF冲洗，制得N-长链烷基-N’-邻苯二甲酰壳聚糖。Take N-long-chain alkyl chitosan and add it to DMF and stir for 8-12 hours, then add the DMF solution of phthalic anhydride, react at 130°C and_N2 protection for 12-18 hours, and pour the reaction solution into a deionized water, filter, and wash the filter cake with a large amount of water+DMF to obtain N-long-chain alkyl-N'-phthaloyl chitosan.

3)N-长链烷基-N’-邻苯二甲酰-O-羟丙基壳聚糖(APHPC)的制备3) Preparation of N-long-chain alkyl-N'-phthaloyl-O-hydroxypropyl chitosan (APHPC)

取N-长链烷基-N’-邻苯二甲酰壳聚糖加入到50％NaOH和DMF溶液中，50℃机械搅10小时，制得APHPC。反应液用稀盐酸液中和pH至7，过滤，滤液用透析袋(MWCO 14000)透析，以去除分子量小于14,000的小分子，冷冻干燥，得淡黄色APHPC。Add N-long-chain alkyl-N'-phthaloyl chitosan into 50% NaOH and DMF solution, and mechanically stir at 50°C for 10 hours to prepare APHPC. The reaction solution was neutralized to pH 7 with dilute hydrochloric acid solution, filtered, and the filtrate was dialyzed with a dialysis bag (MWCO 14000) to remove small molecules with a molecular weight less than 14,000, and then freeze-dried to obtain light yellow APHPC.

本发明的壳聚糖衍生物在水、甲醇、N，N-二甲基甲酰胺(DMF)、二甲亚砜(DMSO)中均有良好的溶解性，在化学结构修饰过程中的溶剂选择和在聚合物胶束进行包裹难溶性药物的方式上有了很大程度的选择性。Chitosan derivatives of the present invention have good solubility in water, methanol, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and the solvent selection in the chemical structure modification process And there is a great degree of selectivity in the way of encapsulating insoluble drugs in polymer micelles.

合成反应式如下：The synthetic reaction formula is as follows:

其中a、b、c、n分别表示相应重复单元的聚合度。在壳聚糖的2-NH₂接入疏水长链烷基(链长为6-18C)，剩余氨基上接入邻苯二甲酸酐，在6-OH进行羟丙基(引入亲水基)，制得APHPC。Wherein a, b, c, n represent the degree of polymerization of the corresponding repeating unit, respectively. In 2-NH₂ of chitosan, insert hydrophobic long-chain alkyl (chain length is 6-18C), insert phthalic anhydride on the remaining amino group, and carry out hydroxypropyl group in 6-OH (introduce hydrophilic group) , APHPC was obtained.

为了获得能够形成聚合物胶束以及能够包裹药物尤其是难溶性药物的聚合物胶束，除了考虑毒性、溶血反应以外，必须筛选合适的能够接入的疏水及亲水性基团。并不是所有只要形成亲水亲脂性的两亲性壳聚糖衍生物都适合作为药物配体或药物载体的，亲水、亲脂都有一个度，且亲水、亲脂基团之间必须有一平衡，这平衡既与基团亲水、亲脂程度有关，亦与其所带电荷有关，还与其体积甚至立体结构有关。本发明在壳聚糖结构上引入邻苯二甲酰基团，很好地调节了亲水亲油基团之间的平衡，保证了材料良好水溶性的同时也使得临界胶束浓度较低。In order to obtain polymer micelles that can form polymer micelles and encapsulate drugs, especially insoluble drugs, in addition to considering toxicity and hemolytic reactions, it is necessary to screen suitable hydrophobic and hydrophilic groups that can be incorporated. Not all amphiphilic chitosan derivatives that only form hydrophilic and lipophilic properties are suitable as drug ligands or drug carriers. There is a degree of hydrophilicity and lipophilicity, and there must be a gap between the hydrophilic and lipophilic groups. There is a balance, which is not only related to the degree of hydrophilicity and lipophilicity of the group, but also to its charge, and to its volume and even its three-dimensional structure. The invention introduces a phthaloyl group into the chitosan structure, well adjusts the balance between the hydrophilic and lipophilic groups, ensures good water solubility of the material and lowers the critical micelle concentration.

本发明的两亲性质的壳聚糖衍生物，不但可用于难溶药物的增溶和包裹，控制药物释放，而且可以在水中形成纳米级胶束。由于由亲水的壳和疏水的核组成的纳米胶束，可以延长体内循环、减少网状内皮细胞的吞噬，并最终提高治疗效果。The amphiphilic chitosan derivative of the invention can not only be used for solubilization and encapsulation of poorly soluble drugs, and control drug release, but also can form nano-scale micelles in water. Due to the nanomicelle composed of a hydrophilic shell and a hydrophobic core, it can prolong the circulation in vivo, reduce the phagocytosis of reticuloendothelial cells, and ultimately improve the therapeutic effect.

本发明的两亲性壳聚糖衍生物适合用于药物、与药物组配或作为药物载体，尤其是作为毒性小、不会产生溶血反应的可静脉注射给药的药物载体。同时在胃肠道中稳定，也可作为口服难溶性药物的载体。The amphiphilic chitosan derivative of the present invention is suitable for use in medicine, compounded with medicine or as a medicine carrier, especially as a medicine carrier capable of intravenous injection with little toxicity and no hemolytic reaction. At the same time, it is stable in the gastrointestinal tract and can also be used as a carrier for oral insoluble drugs.

以APHPC为例，胶束的制备方法方法如下：Taking APHPC as an example, the preparation method of micelles is as follows:

按每mL水中溶解4-20mg的APHPC的比例，可以每mL水中溶解10-20mg的APHPC，亦可以每mL水中溶解12-16mg的APHPC，将制得的APHPC溶于水中，制备成粒径为90-150nm的APHPC胶束。According to the ratio of 4-20mg of APHPC dissolved in every mL of water, 10-20mg of APHPC can be dissolved in every mL of water, or 12-16mg of APHPC can be dissolved in every mL of water, and the prepared APHPC can be dissolved in water to prepare a particle size of 90-150nm APHPC micelles.

本发明的壳聚糖衍生物可作为载体制备的药物组合物，对难溶药物有增溶作用。所述难溶药物优选：紫杉醇、环孢素A、替尼铂甙、羟基喜树碱、喜树碱、长春酰胺、依托泊甙、尼莫地平、阿霉素、多烯紫杉醇，灯盏花素、银杏内酷、水飞蓟素、柔红霉素、丝裂霉素、氨甲喋呤，尤其是对紫杉醇、多烯紫杉醇、环孢素A、替尼泊甙、羟基喜树碱、喜树碱、长春酰胺、依托泊甙或尼莫地平。The chitosan derivative of the invention can be used as a pharmaceutical composition prepared by a carrier, and has a solubilizing effect on poorly soluble drugs. The insoluble drugs are preferably: paclitaxel, cyclosporin A, teniplatin, hydroxycamptothecin, camptothecin, vincamide, etoposide, nimodipine, doxorubicin, docetaxel, scutellarin , Ginkgolide, silymarin, daunorubicin, mitomycin, methotrexate, especially paclitaxel, docetaxel, cyclosporine A, teniposide, hydroxycamptothecin, camptothecin, vinblastamide, Etoposide or nimodipine.

药物和本发明的的壳聚糖衍生物的摩尔比优选4∶10～10∶10。更优选6∶10～8∶10。The molar ratio of the drug to the chitosan derivative of the present invention is preferably 4:10˜10:10. More preferably 6:10 to 8:10.

N-长链烷基-N’-邻苯二甲酰-O-羟丙基壳聚糖胶束对难溶性药物如紫杉醇、多烯紫杉醇、环孢素A具有较好增溶作用，尤其是APHPC对紫杉醇和多烯紫杉醇有明显的增溶效果，当其水溶液浓度为0.6％时，提高紫杉醇的溶解度达2.2-3.5mg/mL。N-long-chain alkyl-N'-phthaloyl-O-hydroxypropyl chitosan micelles have a good solubilization effect on insoluble drugs such as paclitaxel, docetaxel and cyclosporine A, especially APHPC has obvious solubilizing effect on paclitaxel and docetaxel, and when its aqueous solution concentration is 0.6%, it can increase the solubility of paclitaxel to 2.2-3.5 mg/mL.

本发明的壳聚糖衍生物作为载体较原料壳聚糖毒性低，且溶血反应符合静脉注射药用辅料标准。As a carrier, the chitosan derivative of the invention has lower toxicity than the chitosan raw material, and the hemolysis reaction meets the standard of pharmaceutical auxiliary materials for intravenous injection.

实施例6和7对于小鼠静脉注射的急性毒性试验数据表明：APHPC(OPHPC和DPHPC属于APHPC大类的材料，以下以OPHPC和DPHPC两类代表APHPC说明情况)急性毒性较原料壳聚糖有明显降低。下述溶血实验表明：APHPC在用生理盐水稀释至适当浓度，进行静脉注射给药，不会引起机体的溶血反应。Embodiment 6 and 7 show for the acute toxicity test data of mouse intravenous injection: the acute toxicity of APHPC (OPHPC and DPHPC belong to the material of APHPC big class, below with OPHPC and DPHPC two kinds of representatives APHPC explanation situation) acute toxicity is more obvious than raw material chitosan reduce. The following hemolysis experiments show that: APHPC diluted to an appropriate concentration with normal saline, administered intravenously, will not cause the body's hemolytic reaction.

具体实施方式Detailed ways

实施例1Example 1

1、N-辛基壳聚糖的制备(参见CN 03112981.1)1. Preparation of N-octyl chitosan (see CN 03112981.1)

壳聚糖(1.0g)悬浮在50mL甲醇中室温搅拌，加入辛醛(1.02g)，继续反应24小时后，加入KBH₄(0.5g)的5mL水溶液，继续搅拌过夜，反应液用2N的盐酸溶液中和，用甲醇沉淀，过滤，滤渣用甲醇和水重复洗涤3次，用真空烘箱于60℃下干燥过夜，得浅黄色粉末N-辛基壳聚糖1.0g。Chitosan (1.0g) was suspended in 50mL of methanol and stirred at room temperature. Octanal (1.02g) was added. After continuing the reaction for 24 hours, a 5mL aqueous solution of KBH₄ (0.5g) was added, and stirring was continued overnight. The reaction solution was washed with 2N hydrochloric acid The solution was neutralized, precipitated with methanol, filtered, and the filter residue was repeatedly washed with methanol and water three times, and dried overnight in a vacuum oven at 60° C. to obtain 1.0 g of light yellow powder N-octyl chitosan.

相同方法可以制备N-癸基壳聚糖、N-月桂化壳聚糖、N-十八烷基、N-胆固醇化壳聚糖等N-烷基化壳聚糖。N-alkylated chitosan such as N-decyl chitosan, N-lauryl chitosan, N-octadecyl, N-cholesteryl chitosan can be prepared by the same method.

2、N-辛基-N’-邻苯二甲酰壳聚糖的制备2. Preparation of N-octyl-N'-phthaloyl chitosan

N-辛基壳聚糖(1.5g)悬浮在DMF(40mL)中机械搅拌过夜，N₂保护和搅拌下，慢慢加入邻苯二甲酸酐1.9g，缓慢升温至130℃，继续搅拌15小时。反应完毕将反应液倒入大量去离子水中，过滤，滤饼用大量水洗。红外干燥得淡黄色粉末1.9g。相同方法可以制备N-顺丁烯二甲酰-N’-辛基壳聚糖、N-(3-甲酰丙酸)-N’-辛基壳聚糖、N-(4-甲酰丁酸)-N’-辛基壳聚糖等酸酐化壳聚糖。N-octyl chitosan (1.5g) was suspended in DMF (40mL) and mechanically stirred overnight, under_N2 protection and stirring, slowly added 1.9g of phthalic anhydride, slowly heated to 130°C, and continued to stir for 15 hours . After the reaction is completed, the reaction solution is poured into a large amount of deionized water, filtered, and the filter cake is washed with a large amount of water. After infrared drying, 1.9 g of light yellow powder was obtained. The same method can prepare N-maleyl-N'-octyl chitosan, N-(3-formyl propionic acid)-N'-octyl chitosan, N-(4-formyl butyrate acid)-N'-octyl chitosan and other acid anhydride chitosan.

3、N-辛基-N’-邻苯二甲酰-O-羟丙基壳聚糖(OPHPC)的制备3. Preparation of N-octyl-N'-phthaloyl-O-hydroxypropyl chitosan (OPHPC)

N-辛基-N’-邻苯二甲酰壳聚糖(1g)加入20mL环氧丙烷和10mL 50％NaOH+DMF(15mL)溶液中，50℃机械搅10小时，制得OPHPC。反应液用5％HCl溶液调节pH至7，过滤，滤液用透析袋(MWCO 14000)透析，以去除分子量小于14,000的小分子，冷冻干燥，得金黄色絮状物1.6g。N-octyl-N'-phthaloyl chitosan (1g) was added to 20mL propylene oxide and 10mL 50% NaOH+DMF (15mL) solution, and mechanically stirred at 50°C for 10 hours to obtain OPHPPC. The pH of the reaction solution was adjusted to 7 with 5% HCl solution, filtered, and the filtrate was dialyzed with a dialysis bag (MWCO 14000) to remove small molecules with a molecular weight less than 14,000, and freeze-dried to obtain 1.6 g of golden yellow flocs.

FT IR：2860，1386，1287，908，718cm^-1(长链烷基)，1121cm^-1(羟丙基)，1718cm^-1(邻苯二甲酰).FT IR: 2860, 1386, 1287, 908, 718cm^-1 (long chain alkyl), 1121cm^-1 (hydroxypropyl), 1718cm^-1 (phthaloyl).

′H-NMR(D₂O)：δ(ppm)：0.6-1.8(m，15H)(-NH-CH₂-(CH₂)₆-CH₃)；2.5-2.8(m，2H)(-NH-CH₂-(CH₂)₆-CH₃)；3.6(m，4H)；7.3-7.7(d，4H)(Ar-H).'H-NMR (D₂ O): δ (ppm): 0.6-1.8 (m, 15H) (-NH-CH₂ -(CH₂ )₆-CH₃ ); 2.5-2.8 (m, 2H) (-NH-CH₂ -(CH₂ )₆ -CH₃ ); 3.6(m, 4H); 7.3-7.7(d, 4H)(Ar-H).

¹³C-NMR(D₂O)：δ(ppm)：13.5-13.7(-NH-CH₂-(CH₂)₆-CH3)；21.9-31.4(-NH-CH₂-(CH₂)6-CH₃)；48.3(-NH-CH₂-(CH₂)₆-CH₃)；55(C2)；61.4(C6)；66.8(C3)；75.4(C5)；76.4(C4)；97(C1).¹³ C-NMR (D₂ O): δ (ppm): 13.5-13.7 (-NH-CH₂ -(CH₂ )₆ -CH3); 21.9-31.4 (-NH-CH₂ -(CH₂ ) 6-_CH3 ); 48.3(-NH-_CH2- (CH2)₆ -_CH3 ); 55(_C2 ); 61.4(C6); 66.8(C3); 75.4(C5); 76.4(C4); ).

元素分析测得脱乙酰化度为91.5％，N-辛基取代度为的取代度为50％，邻苯二甲酰的取代度为60.7％，羟丙基化取代度为106％。OPHPC元素分析结果为：N，2.98；C，36.89；H，5.41。Elemental analysis showed that the deacetylation degree was 91.5%, the N-octyl substitution degree was 50%, the phthaloyl substitution degree was 60.7%, and the hydroxypropylation substitution degree was 106%. The results of OPHPC elemental analysis are: N, 2.98; C, 36.89; H, 5.41.

相同方法可以制备N-辛基-N’-顺丁烯二甲酰-O-羟丙基化壳聚糖、N-辛基-N’-(3-甲酰丙酸)-O-羟丙基化壳聚糖、N-辛基-N’-(4-甲酰丁酸)-O-羟丙基化壳聚糖等酸酐化壳聚糖。The same method can prepare N-octyl-N'-maleyl-O-hydroxypropylated chitosan, N-octyl-N'-(3-formylpropionic acid)-O-hydroxypropyl Anhydrous chitosan such as ylated chitosan, N-octyl-N'-(4-formylbutyric acid)-O-hydroxypropylated chitosan.

实施例2Example 2

1，N-癸基壳聚糖的制备1. Preparation of N-decyl chitosan

用癸醛和壳聚糖反应，制备方法同N-辛基壳聚糖。Using decanal and chitosan to react, the preparation method is the same as that of N-octyl chitosan.

2，N-癸基-N’-邻苯二甲酰壳聚糖的制备2, Preparation of N-decyl-N'-phthaloyl chitosan

用N-癸基壳聚糖和邻苯二甲酸酐反应制备，方法同N-辛基-N’-邻苯二甲酰壳聚糖。相同方法可以制备N-癸基-N’-顺丁烯二甲酰壳聚糖、N-癸基-N’-(3-甲酰丙酸)壳聚糖、N-癸基-N’-(4-甲酰丁酸)壳聚糖等酸酐化壳聚糖。It is prepared by reacting N-decyl chitosan with phthalic anhydride, and the method is the same as that of N-octyl-N'-phthaloyl chitosan. The same method can prepare N-decyl-N'-maleyl chitosan, N-decyl-N'-(3-formyl propionic acid) chitosan, N-decyl-N'- (4-formylbutyric acid) chitosan and other acid anhydride chitosan.

3，N-癸基-N’-邻苯二甲酰-O-羟丙基化壳聚糖的制备3, Preparation of N-decyl-N'-phthaloyl-O-hydroxypropylated chitosan

用N-癸基-N’-邻苯二甲酰壳聚糖和多聚磷酸反应，制备方法同OPHPC。React with N-decyl-N'-phthaloyl chitosan and polyphosphoric acid, and the preparation method is the same as OPHPC.

FT IR：2860，1386，1287，908，718cm^-1(长链烷基)，1121cm^-1(羟丙基)，1718cm^-1(邻苯二甲酰).FT IR: 2860, 1386, 1287, 908, 718cm^-1 (long chain alkyl), 1121cm^-1 (hydroxypropyl), 1718cm^-1 (phthaloyl).

′H-NMR(D₂O)：δ(ppm)：0.6-1.8(m，15H)(-NH-CH₂-(CH₂)₆-CH₃)；2.5-2.8(m，2H)(-NH-CH₂-(CH₂)₆-CH₃)；3.6(m，4H)；7.3-7.7(d，4H)(Ar-H).'H-NMR (D₂ O): δ (ppm): 0.6-1.8 (m, 15H) (-NH-CH₂ -(CH₂ )₆-CH₃ ); 2.5-2.8 (m, 2H) (-NH-CH₂ -(CH₂ )₆ -CH₃ ); 3.6(m, 4H); 7.3-7.7(d, 4H)(Ar-H).

¹³C-NMR(D₂O)：δ(ppm)：13.5-13.7(-NH-CH₂-(CH₂)₆-CH3)；21.9-31.4(-NH-CH₂-(CH₂)6-CH₃)；48.3(-NH-CH₂-(CH₂)₆-CH₃)；55(C2)；61.4(C6)；66.8(C3)；75.4(C5)；76.4(C4)；97(C1).123.6-166.7(Ar C)¹³ C-NMR (D₂ O): δ (ppm): 13.5-13.7 (-NH-CH₂ -(CH₂ )₆ -CH3); 21.9-31.4 (-NH-CH₂ -(CH₂ ) 6-_CH3 ); 48.3(-NH-_CH2- (CH2)₆ -_CH3 ); 55(_C2 ); 61.4(C6); 66.8(C3); 75.4(C5); 76.4(C4); ).123.6-166.7 (Ar C)

元素分析测得脱乙酰化度为91.5％，N-癸基取代度为50％，邻苯二甲酰的取代度为29.7％，羟丙基化取代度为106％。N-癸基-N’-邻苯二甲酰-O-羟丙基化壳聚糖元素分析结果为：N，2.63，C，37.29，H，6.41。The elemental analysis shows that the deacetylation degree is 91.5%, the N-decyl substitution degree is 50%, the phthaloyl substitution degree is 29.7%, and the hydroxypropylation substitution degree is 106%. The elemental analysis results of N-decyl-N'-phthaloyl-O-hydroxypropylated chitosan were: N, 2.63, C, 37.29, H, 6.41.

相同方法可以制备N-癸基-N’-顺丁烯二甲酰-O-羟丙基化壳聚糖、N-癸基-N’-(3-甲酰丙酸)-O-羟丙基化壳聚糖、N-癸基-N’-(4-甲酰丁酸)-O-羟丙基化壳聚糖等酸酐化壳聚糖。The same method can prepare N-decyl-N'-maleyl-O-hydroxypropylated chitosan, N-decyl-N'-(3-formyl propionic acid)-O-hydroxypropyl Anhydrous chitosan, N-decyl-N'-(4-formylbutyric acid)-O-hydroxypropylated chitosan, etc.

实施例3Example 3

1，N-十八烷基壳聚糖的制备1, Preparation of N-octadecyl chitosan

用十八碳醛和壳聚糖反应，制备方法同N-辛基壳聚糖。The reaction between octadecanal and chitosan is the same as that of N-octyl chitosan.

2，N-十八碳烷基-N’-邻苯二甲酰壳聚糖的制备2, Preparation of N-octadecyl-N'-phthaloyl chitosan

用N-十八碳烷基壳聚糖和邻苯二甲酸酐反应制备，方法同N-辛基-N’-邻苯二甲酰壳聚糖。相同方法可以制备N-十八烷基-N’-顺丁烯二甲酰壳聚糖、N-十八烷基-N’-(3-甲酰丙酸)壳聚糖、N-十八烷基-N’-(4-甲酰丁酸)壳聚糖等酸酐化壳聚糖。It is prepared by reacting N-octadecyl chitosan with phthalic anhydride, and the method is the same as that of N-octyl-N'-phthaloyl chitosan. The same method can prepare N-octadecyl-N'-maleyl chitosan, N-octadecyl-N'-(3-formyl propionic acid) chitosan, N-octadecyl Anhydrous chitosan such as alkyl-N'-(4-formylbutyric acid) chitosan.

3，N-十八碳烷基-N’-邻苯二甲酰-O-羟丙基化壳聚糖的制备3, Preparation of N-octadecyl-N'-phthaloyl-O-hydroxypropylated chitosan

用N-十八碳烷基-N’-邻苯二甲酰壳聚糖和多聚磷酸反应，制备方法同OPHPC。React with N-octadecyl-N'-phthaloyl chitosan and polyphosphoric acid, and the preparation method is the same as OPHPC.

FT IR：2860，1386，1287，908，718cm^-1(长链烷基)，1121cm^-1(羟丙基)，1718cm^-1(邻苯二甲酰).FT IR: 2860, 1386, 1287, 908, 718cm^-1 (long chain alkyl), 1121cm^-1 (hydroxypropyl), 1718cm^-1 (phthaloyl).

′H-NMR(D₂O)：δ(ppm)：0.6-1.8(m，15H)(-NH-CH₂-(CH₂)₆-CH₃)；2.5-2.8(m，2H)(-NH-CH₂-(CH₂)₆-CH₃)；3.6(m，4H)；7.3-7.7(d，4H)(Ar-H).'H-NMR (D₂ O): δ (ppm): 0.6-1.8 (m, 15H) (-NH-CH₂ -(CH₂ )₆-CH₃ ); 2.5-2.8 (m, 2H) (-NH-CH₂ -(CH₂ )₆ -CH₃ ); 3.6(m, 4H); 7.3-7.7(d, 4H)(Ar-H).

¹³C-NMR(D₂O)：δ(ppm)：49.3-13.4(-NH-CH₂-(CH₂)₁₆-CH₃)；55(C2)；61.4(C6)；66.8(C3)；75.4(C5)；76.4(C4)；97(C1)；124.6-166.7(Ar C and C＝O)¹³ C-NMR (D₂ O): δ (ppm): 49.3-13.4 (-NH-CH₂ -(CH₂ )₁₆ -CH₃ ); 55 (C2); 61.4 (C6); 66.8 (C3); 75.4(C5); 76.4(C4); 97(C1); 124.6-166.7(Ar C and C=O)

元素分析测得脱乙酰化度为91.5％，N-十八烷基的取代度为50％，邻苯二甲酰的取代度为29.7％，羟丙基化取代度为106％。N-十八烷基-N’-邻苯二甲酰-O-羟丙基化壳聚糖元素分析结果为：N，2.51，C，44.29，H，6.41。The degree of deacetylation measured by elemental analysis is 91.5%, the degree of substitution of N-octadecyl is 50%, the degree of substitution of phthaloyl is 29.7%, and the degree of substitution of hydroxypropylation is 106%. The elemental analysis results of N-octadecyl-N'-phthaloyl-O-hydroxypropylated chitosan are: N, 2.51, C, 44.29, H, 6.41.

相同方法可以制备相同方法可以制备N-十八烷基-N’-顺丁烯二甲酰-O-羟丙基化壳聚糖、N-十八烷基-N’-(3-甲酰丙酸)-O-羟丙基化壳聚糖、N-十八烷基-N’-(4-甲酰丁酸)-O-羟丙基化壳聚糖等酸酐化壳聚糖。The same method can prepare N-octadecyl-N'-maleyl-O-hydroxypropylated chitosan, N-octadecyl-N'-(3-formyl Anhydrous chitosan such as propionic acid)-O-hydroxypropylated chitosan, N-octadecyl-N'-(4-formylbutyric acid)-O-hydroxypropylated chitosan.

实施例4Example 4

OPHPC溶解度测定OPPHPC Solubility Determination

根据05年版中国药典凡例，称取研成细粉的OPHPC，置于25±2℃一定容量的溶剂中，每隔5分钟强力振摇30秒钟，观察30分钟内的溶解情况，如无目视可见的溶质颗粒或液滴，即视为完全溶解。测算出饱和溶解度，测出的溶解度均为实际饱和溶解度下限。结果见表1。According to the general rules of the Chinese Pharmacopoeia of the 2005 edition, weigh OPHPPC that has been ground into a fine powder, place it in a solvent with a certain capacity at 25±2°C, shake vigorously for 30 seconds every 5 minutes, and observe the dissolution within 30 minutes. The visible solute particles or droplets are considered to be completely dissolved. The saturated solubility is measured and calculated, and the measured solubility is the lower limit of the actual saturated solubility. The results are shown in Table 1.

OPHPC和NOSC在水中溶解度均在25mg/mL以上，其中OPHPC在有机溶剂中溶解性远远好于NOSC。OPHPC在DMF、DMSO中溶解容易并且溶解度大，水中最大溶解度在30mg以上，＞25mg时会出现乳光。对比NOSC，OPHPC在溶解度和溶剂选择性上均有很大幅度地改善。The solubility of OPHPC and NOSC in water is above 25mg/mL, and the solubility of OPHPC in organic solvents is much better than that of NOSC. OPHPC is easy to dissolve in DMF and DMSO and has a large solubility. The maximum solubility in water is above 30mg, and opalescence will appear when it is >25mg. Compared with NOSC, OPHPPC has greatly improved solubility and solvent selectivity.

表1、OPHPC与NOSC溶解度(mg/mL)考察Table 1. Solubility (mg/mL) investigation of OPHPC and NOSC

实施例5Example 5

OPHPC及其类似物CMC测定CMC determination of OPHPC and its analogues

精确配置1-1000μg/mL的15个不同浓度的OPHPC及其类似物和NOSC水溶液，测定表面张力，结果如表2所示。Precisely prepare 15 different concentrations of OPHPPC and its analogues and NOSC aqueous solution of 1-1000 μg/mL, and measure the surface tension. The results are shown in Table 2.

表2 表面张力法测定材料CMC值Table 2 Determination of material CMC value by surface tension method

通过OPHPC及其类似物和NOSC的CMC值对比，本发明壳聚糖衍生物明显小于NOSC，也比CN 200610037810.X中报道的材料OCC材料的CMC值要小。预示着耐稀释性能优越，体内胶束的稳定性好，不易粒子松散药物泄露。By comparing the CMC values of OPHPC and its analogs and NOSC, the chitosan derivatives of the present invention are significantly smaller than NOSC, and also smaller than the CMC value of the OCC material reported in CN 200610037810.X. It indicates that the dilution resistance is superior, the stability of the micelles in the body is good, and it is not easy for the particles to loose and leak drugs.

实施例6Example 6

OPHPC小鼠急性毒性考察Acute Toxicity Study of OPHPC Mice

1.载体静脉注射溶液配置：1. Carrier intravenous injection solution configuration:

正确称取OPHPC 500mg，先用20mL生理盐水将其溶解，得20mL浓度为25mg/mL的OPHPC注射溶液，以25mg/mL的药液作为本次实验的最高浓度，并以1∶0.8的比例依此作递减稀释，配制20，16，12.8和10.24mg/mL共5个浓度的药液，给药体积为0.2mL/10g，单次iv给药。将原料壳聚糖对照(400mg/kg)，同上体积，单次iv给药。Correctly weigh 500 mg of OPHPC, dissolve it with 20 mL of normal saline, and obtain 20 mL of OPHPC injection solution with a concentration of 25 mg/mL. This is a descending dilution, and a total of 5 concentrations of 20, 16, 12.8 and 10.24mg/mL are prepared. The administration volume is 0.2mL/10g, and a single iv administration is performed. Raw material chitosan control (400mg/kg), same volume, single iv administration.

2.受试动物选择2. Selection of test animals

昆明种小白鼠，雌雄各半，18-22g，由东大医学院动物中心提供，合格证为苏动(质)91058。动物数为：OPHPC尾静脉给药组，10只/组，5个剂量组。原料壳聚糖对照1个剂量组。Kunming white mice, half male and half male, 18-22 g, provided by the Animal Center of Dongda University School of Medicine, certificate of conformity is Su Dong (Quality) 91058. The number of animals is: OPHPC tail vein administration group, 10 animals/group, 5 dose groups. Raw material chitosan was compared with one dose group.

3.给药方案3. Dosing regimen

(1)OPHPC静脉给药对小鼠的急性毒性：小鼠50只，随机分为5组，10只/组，受试前禁食12小时。根据预试，确定本次实验最高剂量为300mg/kg，并以1∶0.8的剂距依此递减为240，192，153和122mg/kg共5个剂量组。(1) Acute toxicity of intravenous administration of OPHPC to mice: 50 mice were randomly divided into 5 groups, 10 mice/group, and fasted for 12 hours before the test. According to the preliminary test, the highest dose of this experiment was determined to be 300 mg/kg, and the dose interval of 1:0.8 was gradually reduced to 240, 192, 153 and 122 mg/kg, a total of 5 dose groups.

(2).壳聚糖原料对照300mg/kg，1个剂量组。(2). Chitosan raw material control 300mg/kg, 1 dose group.

相同方法考察N-烷基-N’-顺丁烯二甲酰-O-羟丙基化壳聚糖、N-烷基-N’-(3-甲酰丙酸)-O-羟丙基化壳聚糖、N-烷基-N’-(4-甲酰丁酸)-O-羟丙基化壳聚糖等材料的急性毒性。In the same way, N-alkyl-N'-maleyl-O-hydroxypropylated chitosan, N-alkyl-N'-(3-formylpropionic acid)-O-hydroxypropyl Acute toxicity of chitosan, N-alkyl-N'-(4-formylbutyric acid)-O-hydroxypropylated chitosan and other materials.

结果表明，OPHPC静脉给药对小鼠的急性毒性在较高剂量给药后即进入挣扎、昏迷窒息而迅速死亡，中、低剂量给药后多有短暂的呼吸抑制，呼吸困难、急促，部分小鼠最终也因肺部的病变死亡。死亡小鼠剖体检查可见肺部不规则得出血性改变，稍晚死亡小鼠除肺部出血性改变外，部分可见肝脏脂肪样变，余未见明显异常。壳聚糖原料对照300mg/kg，在给药后8小时即全部死亡。死亡原因可能系主要系肺部出血所致。本品小鼠静脉给药的死亡情况，LD50及95％可信限见表3，OPHPC的小鼠静脉注射的LD50及95％可信限分别为168.09(153.61--183.93)mg/kg，较原料壳聚糖有明显降低。The results show that the acute toxicity of OPHPC intravenous administration to mice enters into struggling, coma and suffocation and dies quickly after administration of higher doses. The mice also eventually died from lesions in the lungs. The autopsy of the dead mice showed irregular hemorrhagic changes in the lungs. In addition to the hemorrhagic changes in the lungs, part of the dead mice showed fatty changes in the liver, and no obvious abnormalities were found in the rest. Chitosan raw material control 300mg/kg, promptly all died 8 hours after administration. The cause of death may be mainly caused by pulmonary hemorrhage. The death situation of this product mouse intravenous administration, LD50 and 95% credible limit are shown in Table 3, and the LD50 and 95% credible limit of the mouse intravenous injection of OPHPC are respectively 168.09 (153.61--183.93) mg/kg, compared with Raw material chitosan is significantly reduced.

其他部分材料：N-烷基-N’-顺丁烯二甲酰-O-羟丙基化壳聚糖、N-烷基-N’-(3-甲酰丙酸)-O-羟丙基化壳聚糖、N-烷基-N’-(4-甲酰丁酸)-O-羟丙基化壳聚糖最大溶解度给药，300mg/kg给药后观察14天内，小鼠食量没有变化，活动活动和其他行为如常，毛色光亮。实验结果表明，材料iv小鼠最大耐受剂量大于300mg/kg，观察14天未见急性毒性反应。表明该材料毒性很低，基本无毒。Other materials: N-Alkyl-N'-maleyl-O-hydroxypropylated chitosan, N-Alkyl-N'-(3-formylpropionic acid)-O-hydroxypropyl The maximum solubility of chitosan and N-alkyl-N'-(4-formylbutyric acid)-O-hydroxypropylated chitosan was administered, and the food intake of mice was observed within 14 days after administration of 300 mg/kg. No change, activities and other behaviors as usual, coat color is shiny. The experimental results showed that the maximum tolerated dose of the material iv mice was greater than 300 mg/kg, and no acute toxic reaction was observed after 14 days of observation. It shows that the toxicity of the material is very low, basically non-toxic.

表3 LD50软件计算OPHPC小鼠静脉注射的LD50和95％置信限Table 3 LD50 and 95% confidence limit calculated by LD50 software for intravenous injection of OPHPC mice

实施例7Example 7

载体溶血试验Carrier hemolysis test

N-辛基-N’-顺丁烯甲酰-O-羟丙基壳聚糖作为考察对象，常规制备2％的兔红细胞悬液适量备用。进行A，B，C三次溶血试验，A，B，C三次试验内容相同，但在A试验中N-辛基-N’-顺丁烯甲酰-O-羟丙基壳聚糖浓度为10mg/mL，C试验中载体浓度为1mg/mL(稀释用0.9％生理盐水)，实验按表4数据进行。N-octyl-N'-maleyl-O-hydroxypropyl chitosan was used as the object of investigation, and an appropriate amount of 2% rabbit erythrocyte suspension was conventionally prepared for use. Carry out A, B, C three hemolysis tests, A, B, C three test contents are the same, but in A test the concentration of N-octyl-N'-maleyl-O-hydroxypropyl chitosan is 10mg /mL, the carrier concentration in the C test was 1 mg/mL (diluted with 0.9% physiological saline), and the experiment was carried out according to the data in Table 4.

表4、载体10mg/mL，3mg/mL，1mg/mL三组溶液溶血试验Table 4. Carrier 10mg/mL, 3mg/mL, 1mg/mL three groups of solution hemolysis test

由表5可知，在N-辛基-N’-顺丁烯甲酰-O-羟丙基壳聚糖试验中，1h观察现象发现，10mg/mL第一组第一至三管均发生完全溶血和部分溶血现象，其余各管以及3mg/mL与1mg/mL组均无溶血现象。As can be seen from Table 5, in the N-octyl-N'-maleyl-O-hydroxypropyl chitosan test, 1h observed the phenomenon and found that the first to third tubes of the first group of 10mg/mL all occurred completely. Hemolysis and partial hemolysis, the other tubes and the 3mg/mL and 1mg/mL groups had no hemolysis.

表5、载体10mg/mL，3mg/mL，1mg/mL三组溶液溶血试验结果Table 5. Carrier 10mg/mL, 3mg/mL, 1mg/mL three groups of solution hemolysis test results

相同方法测试N-烷基-N’-邻苯二甲酰-O-羟丙基壳聚糖、N-烷基-N’-(3-甲酰丁酸)-O-羟丙基化壳聚糖中、低浓度均未出现溶血现象，高浓度均发生部分溶血现象。N-烷基-N’-(3-甲酰丙酸)-O-羟丙基化壳聚糖高中低浓度均未出现溶血现象。所有结果与NOSC高、中浓度均出现溶血现象相比，溶血现象有较大改善。N-alkyl-N'-phthaloyl-O-hydroxypropyl chitosan and N-alkyl-N'-(3-formylbutyric acid)-O-hydroxypropyl chitosan were tested in the same way There was no hemolysis at medium and low concentrations of polysaccharides, and partial hemolysis occurred at high concentrations. N-alkyl-N'-(3-formylpropionic acid)-O-hydroxypropylated chitosan did not appear hemolysis at high, medium and low concentrations. Compared with the hemolysis phenomenon in both high and medium concentrations of NOSC, the hemolysis phenomenon has been greatly improved in all the results.

实施例8Example 8

胶束的制备及胶束粒径的测定Preparation of Micelles and Determination of Micellar Size

将OPHPC 40mg溶解在7mL水中于50℃搅拌30min，然后室温超声30min后，用Zetasizer 3000HS instrument(Malvern Instruments，Malvern，UK)在633nm，25℃，He-Ne激光测定胶束粒径为90-150nm。Dissolve 40 mg of OPHPC in 7 mL of water and stir at 50°C for 30 minutes, then sonicate at room temperature for 30 minutes, use Zetasizer 3000HS instrument (Malvern Instruments, Malvern, UK) at 633nm, 25°C, He-Ne laser to measure the particle size of micelles to be 90-150nm .

实施例9Example 9

1、包含紫杉醇的OPHPC胶束的制备1. Preparation of OPHPPC micelles containing paclitaxel

(1).透析法：(1). Dialysis method:

OPHPC 40mg溶解在7mL水中于50℃搅拌30min。紫杉醇50mg溶解在1mL乙醇中。然后二者溶液混合，在室温超声30min后，用透析袋(MWCO 10000)在蒸馏水中室温透析过夜，离心(3000rpm)5min，用0.22μm滤膜过滤，冷冻干燥。Dissolve OPHPC 40mg in 7mL water and stir at 50°C for 30min. Paclitaxel 50mg was dissolved in 1mL ethanol. Then the two solutions were mixed, ultrasonicated at room temperature for 30 min, dialyzed overnight in distilled water with a dialysis bag (MWCO 10000), centrifuged (3000 rpm) for 5 min, filtered with a 0.22 μm filter membrane, and freeze-dried.

(2).溶剂挥发法：(2). Solvent evaporation method:

OPHPC 40mg溶解在7mL水中于50℃搅拌30min，紫杉醇30mg溶解在1mL二氯甲烷中。然后在室温搅拌下将二者溶液混合，继续搅拌过夜，使二氯甲烷挥发，离心(3000rpm×5min)，用0.22μm滤膜过滤，冷冻干燥。Dissolve 40mg of OPHPC in 7mL of water and stir at 50°C for 30min, and dissolve 30mg of paclitaxel in 1mL of dichloromethane. Then, the two solutions were mixed under stirring at room temperature, and the stirring was continued overnight to volatilize the dichloromethane, centrifuged (3000rpm×5min), filtered through a 0.22 μm filter membrane, and freeze-dried.

(3).搅拌法：(3). Stirring method:

OPHPC 40rng溶解在7mL水中于50℃搅拌30min后，加入30mg紫杉醇，继续室温搅拌2小时，离心(3000rpm×5min)，用0.22μm滤膜过滤，冷冻干燥。Dissolve OPHPC 40rng in 7mL water and stir at 50°C for 30min, add 30mg paclitaxel, continue to stir at room temperature for 2 hours, centrifuge (3000rpm×5min), filter with a 0.22μm filter membrane, and freeze-dry.

2、OPHPC胶束中紫杉醇含量的测定2. Determination of paclitaxel content in OPPHPC micelles

用HPLC(LC-6A，Shimadzu，Japan)流动相为甲醇∶乙睛∶水＝30∶40∶30(v/v)。色谱柱为DiamohsilTM C18(250x4.6mm)，柱子粒径为5μm，流速为1.0mL/min，检测波长为227nm(SPD-10A，UV detector，Shimadzu，Japan)，柱温为30℃。进样体积为20μL。The mobile phase of HPLC (LC-6A, Shimadzu, Japan) was methanol:acetonitrile:water=30:40:30 (v/v). The chromatographic column is DiamohsilTM C18 (250x4.6mm), the particle size of the column is 5μm, the flow rate is 1.0mL/min, the detection wavelength is 227nm (SPD-10A, UV detector, Shimadzu, Japan), and the column temperature is 30°C. The injection volume was 20 μL.

结果见表6。The results are shown in Table 6.

实施例10Example 10

1、包含多烯紫杉醇的OPHPC胶束的制备1. Preparation of OPHPC micelles containing docetaxel

(1).透析法(1). Dialysis

OPHPC 40mg溶解在7mL水中于50℃搅拌30min。多烯紫杉醇50mg溶解在1mL乙醇中。然后二者溶液混合，在室温超声30min后，用透析袋(MWCO 10000)在蒸馏水中室温透析过夜，离心(3000rpm×5min)，用0.22μm滤膜过滤，冷冻干燥。Dissolve OPHPC 40mg in 7mL water and stir at 50°C for 30min. Docetaxel 50 mg was dissolved in 1 mL of ethanol. Then the two solutions were mixed, ultrasonicated at room temperature for 30 min, dialyzed overnight in distilled water with a dialysis bag (MWCO 10000), centrifuged (3000 rpm × 5 min), filtered with a 0.22 μm filter membrane, and freeze-dried.

(2).溶剂挥发法(2). Solvent evaporation method

OPHPC40mg溶解在7mL水中于50℃搅拌30min，多烯紫杉醇30mg溶解在2mL氯仿中。然后在室温搅拌下将二者溶液混合，继续搅拌过夜，使氯仿挥发，离心(3000rpm×5min)，用0.22μm滤膜过滤，冷冻干燥。Dissolve 40 mg of OPHPC in 7 mL of water and stir at 50 ° C for 30 min, and dissolve 30 mg of docetaxel in 2 mL of chloroform. Then, the two solutions were mixed under stirring at room temperature, and the stirring was continued overnight to volatilize the chloroform, centrifuged (3000rpm×5min), filtered with a 0.22 μm filter membrane, and freeze-dried.

(3).搅拌法(3). Stirring method

OPHPC 40mg溶解在7mL水中于50℃搅拌30min后，加入30mg多烯紫杉醇，继续室温搅拌2小时，离心(3000rpm×5min)，用0.22μm滤膜过滤，冷冻干燥。Dissolve 40 mg of OPHPC in 7 mL of water and stir at 50 ° C for 30 min, add 30 mg of docetaxel, continue stirring at room temperature for 2 hours, centrifuge (3000 rpm × 5 min), filter with a 0.22 μm filter membrane, and freeze-dry.

载体溶液浓度选用几个梯度，相同方法制备胶束。Several gradients were selected for the concentration of the carrier solution, and micelles were prepared in the same way.

以上相同方法制备紫杉醇聚合物胶束。Paclitaxel polymer micelles were prepared by the same method as above.

2、OPHPC胶束中多烯紫杉醇含量的测定2. Determination of docetaxel content in OPHPC micelles

用HPLC(LC-6A，Shimadzu，Japan)流动相为甲醇∶水∶磷酸＝350∶140∶1(v/v).色谱柱为Spherisorb C8(150×4.6μm)，柱子粒径为5μm.流速为1.0mL/min，检测波长为210nm(SPD-10A，UV detector Shimadzu，Japan)，柱温为30℃，注射样品体积为20μL。结果见表6The HPLC (LC-6A, Shimadzu, Japan) mobile phase is methanol: water: phosphoric acid=350: 140: 1 (v/v). The chromatographic column is Spherisorb C8 (150 × 4.6 μm), and the column particle size is 5 μm. The flow rate The detection wavelength is 1.0mL/min, the detection wavelength is 210nm (SPD-10A, UV detector Shimadzu, Japan), the column temperature is 30°C, and the injected sample volume is 20μL. The results are shown in Table 6

表6 不同胶束溶液中药物含量Table 6 Drug content in different micellar solutions

实施例11Example 11

1.包含蛇床子素的OPHPC胶束的制备1. Preparation of OPHPPC micelles containing osthole

(1).透析法：OPHPC 10mg溶解在2mL水中，超声30min，使其分散均匀。蛇床子素20mg溶解在1mL乙醇中。然后二者溶液混合，在室温超声30min后，用透析袋(MWCO 10000)在蒸馏水中室温透析过夜，用0.22μm滤膜过滤，冷冻干燥。(1). Dialysis method: Dissolve 10mg of OPHPC in 2mL of water, and sonicate for 30min to make it evenly dispersed. 20 mg of osthole was dissolved in 1 mL of ethanol. Then the two solutions were mixed, and after ultrasonication at room temperature for 30 min, dialyzed overnight in distilled water with a dialysis bag (MWCO 10000), filtered through a 0.22 μm filter membrane, and freeze-dried.

(2).溶剂挥发法：OPHPC 10mg溶解在2mL水中，超声30min，使其分散均匀，蛇床子素20mg溶解在1mL氯仿中。然后在室温搅拌下将二者溶液混合，继续搅拌过夜，使氯仿挥发，离心(3000rpm×5min)，用0.22μm滤膜过滤，冷冻干燥。(2). Solvent evaporation method: 10 mg of OPHPC was dissolved in 2 mL of water, and ultrasonically disperse for 30 minutes to make it evenly dispersed, and 20 mg of osthole was dissolved in 1 mL of chloroform. Then, the two solutions were mixed under stirring at room temperature, and the stirring was continued overnight to volatilize the chloroform, centrifuged (3000rpm×5min), filtered with a 0.22 μm filter membrane, and freeze-dried.

(3).搅拌法：OPHPC 10mg溶解在2mL水中，超声30min，使其分散均匀。蛇床子素20mg溶解在1mL乙醇中。磁力搅拌条件下，将蛇床子素乙醇溶液逐滴滴加到蛇床子素溶液中，继续室温搅拌2小时，离心(3000rpm×5min)，用0.22μm滤膜过滤，冷冻干燥。(3). Stirring method: Dissolve 10mg of OPHPC in 2mL of water, and ultrasonicate for 30min to make it evenly dispersed. 20 mg of osthole was dissolved in 1 mL of ethanol. Under the condition of magnetic stirring, the osthole ethanol solution was added dropwise to the osthole solution, continued to stir at room temperature for 2 hours, centrifuged (3000rpm×5min), filtered with a 0.22 μm filter membrane, and freeze-dried.

2、OPHPC胶束中蛇床子素含量的测定2. Determination of osthole content in OPPHPC micelles

用HPLC(LC-6A，Shimadzu，Japan)流动相为乙腈∶水＝90∶10(v/v).色谱柱为Spherisorb C8(150×4.6μm)，柱子粒径为5μm.流速为1.0mL/min，检测波长为237nm(SPD-10A，UVdetector Shimadzu，Japan)，柱温为30℃，注射样品体积为20μL。结果见表7The HPLC (LC-6A, Shimadzu, Japan) mobile phase is acetonitrile: water=90: 10 (v/v). The chromatographic column is Spherisorb C8 (150 × 4.6 μm), and the particle size of the column is 5 μm. The flow rate is 1.0mL/ min, the detection wavelength is 237nm (SPD-10A, UV detector Shimadzu, Japan), the column temperature is 30°C, and the injected sample volume is 20 μL. The results are shown in Table 7

表7 不同胶束溶液中蛇床子素含量Table 7 Contents of osthole in different micellar solutions

实施例12Example 12

1.包含环孢素A的OPHPC胶束(CyA-OPHPC)的制备1. Preparation of OPHPPC micelles (CyA-OPHPC) containing cyclosporin A

(1).透析法：OPHPC 50mg溶解在10mL水中，超声30min，使其分散均匀。环孢素A 30mg溶解在1mL乙醇中。然后二者溶液混合，在室温超声30min后，用透析袋(MWCO 10000)在蒸馏水中室温透析过夜，用0.22μm滤膜过滤，冷冻干燥。(1). Dialysis method: Dissolve 50 mg of OPHPC in 10 mL of water, and ultrasonicate for 30 minutes to make it evenly dispersed. Dissolve 30 mg of cyclosporine A in 1 mL of ethanol. Then the two solutions were mixed, and after ultrasonication at room temperature for 30 min, dialyzed overnight in distilled water with a dialysis bag (MWCO 10000), filtered through a 0.22 μm filter membrane, and freeze-dried.

(2)溶剂挥发法：OPHPC 50mg溶解在10mL水中，超声30min，使其分散均匀，环孢素A 30mg溶解在1mL氯仿中。然后在室温搅拌下将二者溶液混合，继续搅拌过夜，使氯仿挥发，离心(3000rpm×5min)，用0.22μm滤膜过滤，冷冻干燥。(2) Solvent evaporation method: Dissolve 50mg of OPHPC in 10mL of water, ultrasonicate for 30min to make it evenly dispersed, and dissolve 30mg of cyclosporin A in 1mL of chloroform. Then, the two solutions were mixed under stirring at room temperature, and the stirring was continued overnight to volatilize the chloroform, centrifuged (3000rpm×5min), filtered with a 0.22 μm filter membrane, and freeze-dried.

(3)搅拌法：OPHPC 50mg溶解在10mL水中，超声30min，使其分散均匀。环孢素A 30mg溶解在1mL乙醇中。磁力搅拌条件下，将环孢素A乙醇溶液逐滴滴加到OPHPC水溶液中，继续室温搅拌1小时，离心(3000rpm×5min)，用0.22μm滤膜过滤，冷冻干燥。(3) Stirring method: Dissolve 50mg of OPHPC in 10mL of water, and ultrasonicate for 30min to make it evenly dispersed. Dissolve 30 mg of cyclosporine A in 1 mL of ethanol. Under the condition of magnetic stirring, cyclosporine A ethanol solution was added dropwise to the OPHPPC aqueous solution, continued to stir at room temperature for 1 hour, centrifuged (3000rpm×5min), filtered with a 0.22 μm filter membrane, and freeze-dried.

2、OPHPC胶束中环孢素A含量的测定2. Determination of cyclosporine A content in OPHPC micelles

用HPLC(LC-6A，Shimadzu，Japan)流动相为乙腈∶水＝90∶10(v/v).色谱柱为Spherisorb C8(150×4.6μm)，柱子粒径为5μm.流速为1.0mL/min，检测波长为237nm(SPD-10A，UVdetector Shimadzu，Japan)，柱温为30℃，注射样品体积为20μL。结果见表8The HPLC (LC-6A, Shimadzu, Japan) mobile phase is acetonitrile: water=90: 10 (v/v). The chromatographic column is Spherisorb C8 (150 × 4.6 μm), and the particle size of the column is 5 μm. The flow rate is 1.0mL/ min, the detection wavelength is 237nm (SPD-10A, UV detector Shimadzu, Japan), the column temperature is 30°C, and the injected sample volume is 20 μL. The results are shown in Table 8

表8 不同胶束溶液中环孢素A含量Table 8 Cyclosporin A content in different micellar solutions

实施例13Example 13

CyA-OPHPC的胃肠道稳定性Gastrointestinal stability of CyA-OPHPC

按中国药典配制模拟胃液(pH 1.2)和模拟肠液(pH 6.8)制备CyA：OPHPC 6∶10(w/w)的载药胶束，用模拟胃肠液1∶5(v/v)和1∶10(v/v)稀释。置于透皮吸收扩散池中，37℃下120rpm孵育测试样品。HPLC考察模拟胃液2h和模拟肠液4h时胶束中CyA含量。结果见表9.According to the Chinese Pharmacopoeia, simulated gastric juice (pH 1.2) and simulated intestinal juice (pH 6.8) were prepared to prepare CyA:OPHPC 6:10 (w/w) drug-loaded micelles, and simulated gastrointestinal juice 1:5 (v/v) and 1 : 10 (v/v) dilution. Place in a transdermal absorption diffusion cell and incubate the test sample at 120rpm at 37°C. The content of CyA in micelles was investigated by HPLC at 2h simulated gastric juice and 4h simulated intestinal juice. The results are shown in Table 9.

表9 CyA-OPHPC模拟胃液和肠液稳定性考察Table 9 CyA-OPHPC simulated gastric juice and intestinal juice stability investigation

实施例14Example 14

1、包含紫杉醇的N-癸基-N’-邻苯二甲酰-O-羟丙基化壳聚糖(DPHPC)胶束的制备1. Preparation of N-decyl-N'-phthaloyl-O-hydroxypropylated chitosan (DPHPC) micelles comprising paclitaxel

(1).透析法(1). Dialysis

DPHPC 40mg溶解在10mL水中于50℃搅拌30min。紫杉醇50mg溶解在1mL乙醇中。然后二者溶液混合，在室温超声30min后，用透析袋(MWCO 12000)在蒸馏水中室温透析过夜，离心(3000rpm×5min)，用0.22μm滤膜过滤，冷冻干燥。DPHPC 40mg was dissolved in 10mL water and stirred at 50°C for 30min. Paclitaxel 50mg was dissolved in 1mL ethanol. Then the two solutions were mixed, after ultrasonication at room temperature for 30 min, dialyzed overnight in distilled water with a dialysis bag (MWCO 12000), centrifuged (3000 rpm × 5 min), filtered with a 0.22 μm filter membrane, and freeze-dried.

(2).溶剂挥发法(2). Solvent evaporation method

DPHPC 40mg溶解在10mL水中于50℃搅拌30min，紫杉醇30mg溶解在0.8mL氯仿中。然后在室温搅拌下将二者溶液混合，继续搅拌过夜，使氯仿挥发，离心(3000rpm×5min)，用0.22μm滤膜过滤，冷冻干燥。DPHPC 40mg was dissolved in 10mL water and stirred at 50°C for 30min, paclitaxel 30mg was dissolved in 0.8mL chloroform. Then, the two solutions were mixed under stirring at room temperature, and the stirring was continued overnight to volatilize the chloroform, centrifuged (3000rpm×5min), filtered with a 0.22 μm filter membrane, and freeze-dried.

(3).搅拌法(3). Stirring method

DPHPC 40mg溶解在10mL水中于50℃搅拌30min后，加入30mg紫杉醇，继续室温搅拌2小时，离心(3000rpm×5min)，用0.22μm滤膜过滤，冷冻干燥。DPHPC 40mg was dissolved in 10mL water and stirred at 50°C for 30min, then 30mg of paclitaxel was added, continued to stir at room temperature for 2 hours, centrifuged (3000rpm×5min), filtered with a 0.22μm membrane filter, and freeze-dried.

载体溶液浓度选用几个梯度，相同方法制备胶束。Several gradients were selected for the concentration of the carrier solution, and micelles were prepared in the same way.

以上相同方法制备紫杉醇聚合物胶束。Paclitaxel polymer micelles were prepared by the same method as above.

2、DPHPC胶束中紫杉醇含量的测定2. Determination of paclitaxel content in DPHPC micelles

用HPLC(LC-6A，Shimadzu，Japan)流动相为甲醇∶水∶磷酸＝350∶140∶1(v/v).色谱柱为Spherisorb C8(150×4.6μm)，柱子粒径为5μm.流速为1.0mL/min，检测波长为210nm(SPD-10A，UV detector Shimadzu，Japan)，柱温为30℃，注射样品体积为20μL。结果见表10The HPLC (LC-6A, Shimadzu, Japan) mobile phase is methanol: water: phosphoric acid=350: 140: 1 (v/v). The chromatographic column is Spherisorb C8 (150 × 4.6 μm), and the column particle size is 5 μm. The flow rate The detection wavelength is 1.0mL/min, the detection wavelength is 210nm (SPD-10A, UV detector Shimadzu, Japan), the column temperature is 30°C, and the injected sample volume is 20μL. The results are shown in Table 10

表10 不同胶束溶液中药物含量Table 10 Drug content in different micellar solutions

实施例15Example 15

1、包含紫杉醇的N-十八烷基-N’-邻苯二甲酰-O-羟丙基化壳聚糖胶束的制备1. Preparation of N-octadecyl-N'-phthaloyl-O-hydroxypropylated chitosan micelles containing paclitaxel

(1).透析法(1). Dialysis

N-十八烷基-N’-邻苯二甲酰-O-羟丙基化壳聚糖40mg溶解在10mL水中于50℃搅拌30min。紫杉醇50mg溶解在1mL乙醇中。然后二者溶液混合，在室温超声30min后，用透析袋(MWCO 12000)在蒸馏水中室温透析过夜，离心(3000rpm×5min)，用0.22μm滤膜过滤，冷冻干燥。40 mg of N-octadecyl-N'-phthaloyl-O-hydroxypropylated chitosan was dissolved in 10 mL of water and stirred at 50°C for 30 min. Paclitaxel 50mg was dissolved in 1mL ethanol. Then the two solutions were mixed, after ultrasonication at room temperature for 30 min, dialyzed overnight in distilled water with a dialysis bag (MWCO 12000), centrifuged (3000 rpm × 5 min), filtered with a 0.22 μm filter membrane, and freeze-dried.

(2).溶剂挥发法(2). Solvent evaporation method

N-十八烷基-N’-邻苯二甲酰-O-羟丙基化壳聚糖40mg溶解在10mL水中于50℃搅拌30min，紫杉醇30mg溶解在0.8mL氯仿中。然后在室温搅拌下将二者溶液混合，继续搅拌过夜，使氯仿挥发，离心(3000rpm×5min)，用0.22μm滤膜过滤，冷冻干燥。40 mg of N-octadecyl-N'-phthaloyl-O-hydroxypropylated chitosan was dissolved in 10 mL of water and stirred at 50° C. for 30 min, and 30 mg of paclitaxel was dissolved in 0.8 mL of chloroform. Then, the two solutions were mixed under stirring at room temperature, and the stirring was continued overnight to volatilize the chloroform, centrifuged (3000rpm×5min), filtered with a 0.22 μm filter membrane, and freeze-dried.

(3).搅拌法(3). Stirring method

N-十八烷基-N’-邻苯二甲酰-O-羟丙基化壳聚糖40mg溶解在10mL水中于50℃搅拌30min后，加入30mg紫杉醇，继续室温搅拌2小时，离心(3000rpm×5min)，用0.22μm滤膜过滤，冷冻干燥。Dissolve 40 mg of N-octadecyl-N'-phthaloyl-O-hydroxypropylated chitosan in 10 mL of water and stir at 50 ° C for 30 min, then add 30 mg of paclitaxel, continue to stir at room temperature for 2 hours, and centrifuge (3000 rpm ×5min), filtered through a 0.22 μm membrane filter, and freeze-dried.

载体溶液浓度选用几个梯度，相同方法制备胶束。Several gradients were selected for the concentration of the carrier solution, and micelles were prepared in the same way.

以上相同方法制备紫杉醇聚合物胶束。Paclitaxel polymer micelles were prepared by the same method as above.

2、N-十八烷基-N’-邻苯二甲酰-O-羟丙基化壳聚糖胶束中紫杉醇含量的测定2. Determination of paclitaxel content in N-octadecyl-N'-phthaloyl-O-hydroxypropylated chitosan micelles

用HPLC(LC-6A，Shimadzu，Japan)流动相为甲醇∶水∶磷酸＝350∶140∶1(v/v).色谱柱为Spherisorb C8(150×4.6μm)，柱子粒径为5μm.流速为1.0mL/min，检测波长为210nm(SPD-10A，UV detector Shimadzu，Japan)，柱温为30℃，注射样品体积为20μL。结果见表11The HPLC (LC-6A, Shimadzu, Japan) mobile phase is methanol: water: phosphoric acid=350: 140: 1 (v/v). The chromatographic column is Spherisorb C8 (150 × 4.6 μm), and the column particle size is 5 μm. The flow rate The detection wavelength is 1.0mL/min, the detection wavelength is 210nm (SPD-10A, UV detector Shimadzu, Japan), the column temperature is 30°C, and the injected sample volume is 20μL. The results are shown in Table 11

表11 不同胶束溶液中药物含量Table 11 Drug content in different micellar solutions

Claims (8)

1. chitosan derivatives:

R=-(CH wherein₂)_n, n=6～18;

x＝50％～60％；y＝8％～10％；z＝20％～30％。

2. the chitosan derivatives of claim 1, wherein x=60%; Y=10%; Z=30%.

3. the chitosan derivatives of claim 1, wherein n=12.

4. pharmaceutical composition wherein contains the chitosan derivatives of medicine and claim 1.

5. the pharmaceutical composition of claim 4; Its Chinese traditional medicine is taxol, ciclosporin A, for Buddhist nun's platinum glucoside, NSC 107124, NSC 94600, vindesine, etoposide, nimodipine, Zorubicin, Docetaxel, cruel in Breviscarpine, the ginkgo, SLB, daunorubicin, MTC or methotrexate.

6. the pharmaceutical composition of claim 5, its Chinese traditional medicine is taxol, Docetaxel, ciclosporin A, Vumon, NSC 107124, NSC 94600, vindesine, etoposide or nimodipine.

7. the pharmaceutical composition of claim 4, the mol ratio of the chitosan derivatives of its Chinese traditional medicine and claim 1 is 4: 10～10: 10.

8. the pharmaceutical composition of claim 7, the mol ratio of the chitosan derivatives of its Chinese traditional medicine and claim 1 is 6: 10～8: 10.