技术领域technical field
本发明属于生物医学材料技术领域，具体涉及一种具有肿瘤细胞生物还原性微环境敏感的载药纳米胶囊及其制备方法。The invention belongs to the technical field of biomedical materials, and in particular relates to a drug-loaded nanocapsule sensitive to the bioreductive microenvironment of tumor cells and a preparation method thereof.
背景技术Background technique
化学治疗(Chemotherapy，简称化疗)为临床上恶性肿瘤的治疗提供了可行的手段，也在一定程度上提高了患者的生存率，但是由于此策略中使用的抗肿瘤化学药物自身的一些缺点，例如低水溶性、高毒性、缺乏对肿瘤部位特异性识别等，导致这一方法在临床使用中受限。纳米药物载体为解决这一问题提供了一条思路。通过纳米药物载体递送抗肿瘤药物分子，可以在避免上述药物自身缺点的同时，保证药物在血液中安全运输，提高其在肿瘤部位的蓄积，并且能够通过肿瘤微环境的信号刺激实现药物在肿瘤细胞内部的可控释放，从而最大限度的提高药物分子的生物利用度，达到抑制肿瘤生长或消退肿瘤的目的。Chemotherapy (Chemotherapy, referred to as chemotherapy) provides a feasible means for the clinical treatment of malignant tumors, and also improves the survival rate of patients to a certain extent, but due to some shortcomings of the anti-tumor chemical drugs used in this strategy, such as Low water solubility, high toxicity, and lack of specific recognition of tumor sites have limited the clinical use of this method. Nano-drug carriers provide a way to solve this problem. The delivery of anti-tumor drug molecules through nano-drug carriers can avoid the above-mentioned shortcomings of the drugs themselves, while ensuring the safe transportation of drugs in the blood, improving their accumulation in tumor sites, and realizing the drug in tumor cells through signal stimulation of the tumor microenvironment. The internal controllable release can maximize the bioavailability of drug molecules and achieve the purpose of inhibiting tumor growth or regressing tumors.
纳米胶囊是一种区别于传统纳米载体诸如脂质体、纳米粒子、胶束、组装大分子等的新型纳米载体。在结构上，纳米胶囊具有稳定的壳层结构和由壳层结构构筑的大腔体组成。纳米胶囊的壳层厚度通常在数纳米至几十纳米，而大腔体结构是荷载化学药物的优良腔室。与所有纳米载体构建面临的问题一样，如何构建具有纳米尺寸并且分散性良好的纳米胶囊是制约其发展的主要原因之一。利用三维网络状的水凝胶(hydrogel)构建纳米胶囊壳层(shell)结构是解决其纳米胶囊团聚(aggregation)的可行策略。通过此方法构建的水凝胶纳米胶囊因其壳层可以吸附大量的水分子，使得其在水溶液中具有非常好的分散性。Nanocapsules are a new type of nanocarriers that are different from traditional nanocarriers such as liposomes, nanoparticles, micelles, and assembled macromolecules. Structurally, nanocapsules have a stable shell structure and a large cavity constructed by the shell structure. The shell thickness of nanocapsules is usually several nanometers to tens of nanometers, and the large cavity structure is an excellent chamber for loading chemicals. Like all the problems faced by the construction of nanocarriers, how to construct nanocapsules with nanometer size and good dispersion is one of the main reasons restricting its development. Using three-dimensional network hydrogel (hydrogel) to construct nanocapsule shell (shell) structure is a feasible strategy to solve its nanocapsule aggregation (aggregation). The hydrogel nanocapsules constructed by this method have very good dispersibility in aqueous solution because their shell can absorb a large amount of water molecules.
在构筑纳米胶囊壳层结构的同时引入具有肿瘤微环境响应的功能基团可以实现基于肿瘤微环境刺激的纳米胶囊解体，达到在特定肿瘤或肿瘤细胞部位药物释放的目的。例如，引入巯基(-SH)基团可以通过其氧化形成二硫键(-S-S-)实现胶囊壳层的交联，达到稳定胶囊结构的目的；而在肿瘤细胞内部高还原性谷胱甘肽(3-10mM)的作用下，二硫键的断裂可以触发胶囊的迅速解体，实现药物快速释放。另一方面，除了基于肿瘤部位增强的渗透和滞留作用(EPR)下的被动靶向(passive targeting)效应，引入主动靶向(activetargeting)配体(targeting ligand)是促进主动的引导纳米载药系统面向肿瘤部位的有效富集。叶酸是小分子靶向配体的典型代表之一。通过叶酸与肿瘤(如，乳腺癌，肺癌，卵巢癌等)细胞上高表达(与正常细胞相比，表达量可达到100-300倍)的叶酸受体的特异性结合，可以引导纳米载药胶囊更高效的富集于肿瘤部位。但现有的抗肿瘤药物纳米胶囊递送体系有以下缺点：生物相容性差，对正常组织有毒副作用，安全性低等。The introduction of functional groups that respond to the tumor microenvironment while constructing the shell structure of the nanocapsule can realize the disintegration of the nanocapsule based on the stimulation of the tumor microenvironment, and achieve the purpose of drug release at specific tumor or tumor cell sites. For example, the introduction of a sulfhydryl (-SH) group can form a disulfide bond (-S-S-) through its oxidation to realize the cross-linking of the capsule shell and achieve the purpose of stabilizing the capsule structure; Under the action of (3-10mM), the breaking of the disulfide bond can trigger the rapid disintegration of the capsule and realize the rapid release of the drug. On the other hand, in addition to the passive targeting effect based on the enhanced penetration and retention (EPR) at the tumor site, the introduction of active targeting ligands (targeting ligand) is to promote the active guided nano drug delivery system. Efficient enrichment for tumor sites. Folic acid is one of the typical representatives of small molecule targeting ligands. Through the specific binding of folic acid to folic acid receptors that are highly expressed (compared with normal cells, the expression level can reach 100-300 times) on tumor (such as breast cancer, lung cancer, ovarian cancer, etc.) cells, nano-drug loading can be guided Capsules are more efficiently enriched in tumor sites. However, the existing nanocapsule delivery systems for anti-tumor drugs have the following disadvantages: poor biocompatibility, toxic side effects on normal tissues, and low safety.
发明内容Contents of the invention
针对现有技术中的上述不足，本发明提供了一种具有肿瘤细胞生物还原性微环境敏感的载药纳米胶囊及其制备方法，可有效解决现有的载物纳米胶囊生物相容性差，对正常组织有毒副作用，安全性低等问题。Aiming at the above-mentioned deficiencies in the prior art, the present invention provides a drug-loaded nanocapsule sensitive to the bioreductive microenvironment of tumor cells and a preparation method thereof, which can effectively solve the problem of poor biocompatibility of the existing drug-loaded nanocapsules. Normal tissue has toxic side effects, low safety and other issues.
为实现上述目的，本发明解决其技术问题所采用的技术方案是：In order to achieve the above object, the technical solution adopted by the present invention to solve the technical problems is:
一种具有肿瘤细胞生物还原性微环境敏感的载药纳米胶囊的制备方法，包括以下步骤：A method for preparing drug-loaded nanocapsules sensitive to the bioreductive microenvironment of tumor cells, comprising the following steps:
(1)胱胺结合的透明质酸(HA-Cys)的制备(1) Preparation of cystamine-bound hyaluronic acid (HA-Cys)
在含有透明质酸的PBS(0.01mol/L，pH＝7.4)中加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺和1-羟基苯并三唑反应4-6h，其目的是用于活化羧基，然后加入胱胺二盐酸盐，室温条件下搅拌反应22-26h，利用去离子水透析除去未反应的分子和缩合剂等；其中透明质酸、1-(3-二甲氨基丙基)-3-乙基碳二亚胺、1-羟基苯并三唑和胱胺二盐酸盐的摩尔比为1:3-5:3-5:3-5；Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 1-hydroxybenzotriazole to PBS (0.01mol/L, pH=7.4) containing hyaluronic acid to react 4- 6h, the purpose is to activate the carboxyl group, then add cystamine dihydrochloride, stir and react at room temperature for 22-26h, and use deionized water to dialyze to remove unreacted molecules and condensation agents; among them, hyaluronic acid, 1- The molar ratio of (3-dimethylaminopropyl)-3-ethylcarbodiimide, 1-hydroxybenzotriazole and cystamine dihydrochloride is 1:3-5:3-5:3-5 ;
(2)酯化胱胺结合的透明质酸(GM-HA-SH)的制备(2) Preparation of esterified cystamine-bound hyaluronic acid (GM-HA-SH)
将胱胺结合的透明质酸溶解于PBS/二甲基甲酰胺混合液中(PBS和二甲基甲酰胺的体积比为4:1，PBS浓度为0.01mol/L，pH＝7.4)，加入甲基丙烯酸缩水甘油酯和三乙胺，室温条件下搅拌反应14-15天，反应结束后，利用去离子水透析72h(透析袋截留分子量3500道尔顿)，然后加入二硫苏糖醇，室温条件下搅拌10-12h，接着用去离子水透析72h(透析袋截留分子量3500道尔顿)，最后冷冻干燥，制得侧链含烯键的酯化胱胺结合的透明质酸；其中胱胺结合的透明质酸、甲基丙烯酸缩水甘油酯、三乙胺和二硫苏糖醇的摩尔比为1:20-23:20-23:5-8；Dissolve cystamine-bound hyaluronic acid in PBS/dimethylformamide mixture (the volume ratio of PBS and dimethylformamide is 4:1, the concentration of PBS is 0.01mol/L, pH=7.4), add Glycidyl methacrylate and triethylamine were stirred and reacted at room temperature for 14-15 days. After the reaction, dialyzed with deionized water for 72 hours (dialysis bag molecular weight cut-off 3500 Daltons), and then added dithiothreitol, Stir at room temperature for 10-12 hours, then dialyze with deionized water for 72 hours (the molecular weight cut-off of the dialysis bag is 3500 Daltons), and finally freeze-dry to obtain hyaluronic acid bound to esterified cystamine containing ethylenic bonds in the side chain; The molar ratio of amine-bound hyaluronic acid, glycidyl methacrylate, triethylamine and dithiothreitol is 1:20-23:20-23:5-8;
(3)多功能化透明质酸(Z-HA-SH)的制备(3) Preparation of multifunctional hyaluronic acid (Z-HA-SH)
利用2-(二甲氨基)甲基丙烯酸乙酯和β-丙内酯开环反应制备羧酸甜菜碱甲基丙烯酸酯单体，以羧酸甜菜碱甲基丙烯酸酯单体为原料，溴化亚铜为催化剂，1,1,4,7,10,10-六甲基三亚乙基四胺为配体，2-(2-溴异丁氧基)甲基丙烯酸乙酯为引发剂，再加入步骤(2)中所制备的侧链含烯键的酯化胱胺结合的透明质酸(GM-HA-SH)，油浴下反应20-24h，反应温度为60℃，制备得到侧链为羧酸甜菜碱甲基丙烯酸酯聚合物的巯基化透明质酸；其中酯化的胱胺结合的透明质酸上的烯键、羧酸甜菜碱甲基丙烯酸酯、溴化亚铜、1,1,4,7,10,10-六甲基三亚乙基四胺，2-(2-溴异丁氧基)甲基丙烯酸乙酯的摩尔比为0.8-1.2:8-12:1.5-2.5:3-5:0.8-1.2；Utilize 2-(dimethylamino)ethyl methacrylate and β-propiolactone ring-opening reaction to prepare carboxybetaine methacrylate monomer, take carboxybetaine methacrylate monomer as raw material, bromide Cuprous as catalyst, 1,1,4,7,10,10-hexamethyltriethylenetetramine as ligand, 2-(2-bromoisobutoxy) ethyl methacrylate as initiator, and then Add the hyaluronic acid (GM-HA-SH) combined with the esterified cystamine prepared in step (2) containing ethylenic bonds in the side chain, react in an oil bath for 20-24h, and the reaction temperature is 60°C to prepare the side chain A mercaptolated hyaluronic acid which is a carboxybetaine methacrylate polymer; where esterified cystamine binds olefinic bonds on the hyaluronic acid, carboxybetaine methacrylate, cuprous bromide, 1, The molar ratio of 1,4,7,10,10-hexamethyltriethylenetetramine to 2-(2-bromoisobutoxy)ethyl methacrylate is 0.8-1.2:8-12:1.5-2.5 :3-5:0.8-1.2;
(4)氨基化叶酸的制备(4) Preparation of aminofolate
将叶酸溶解于二甲基亚砜中，然后添加二环己基碳二亚胺和N-羟基丁二酰亚胺，于50℃搅拌反应10-12h，向反应混合物中加入乙二胺和少量的吡啶，室温搅拌反应10-14h；反应结束后，利用0.45微米孔径的滤器过滤除去不溶物，再用去离子水透析72h(透析袋截留分子量3500道尔顿)，最后冷冻干燥，制得；其中叶酸、二环己基碳二亚胺、N-羟基丁二酰亚胺、乙二胺和吡啶的摩尔比为0.8-1.2:1.2-1.5:1-3:8-12:0.001-0.002；Dissolve folic acid in dimethyl sulfoxide, then add dicyclohexylcarbodiimide and N-hydroxysuccinimide, stir and react at 50°C for 10-12h, add ethylenediamine and a small amount of Pyridine, stirring at room temperature for 10-14h; after the reaction, use a filter with a pore size of 0.45 microns to remove insoluble matter, then dialyze with deionized water for 72h (molecular weight cut-off of the dialysis bag is 3500 Daltons), and finally freeze-dry to obtain; wherein The molar ratio of folic acid, dicyclohexylcarbodiimide, N-hydroxysuccinimide, ethylenediamine and pyridine is 0.8-1.2:1.2-1.5:1-3:8-12:0.001-0.002;
(5)载药纳米胶囊前体的制备(5) Preparation of drug-loaded nanocapsule precursor
将溶解有活性物质的有机溶剂与溶解有多功能化透明质酸的PBS溶液(0.01mol/L，pH＝7.4)按体积比为1:20混合，在超声探头作用下，利用超声乳化法制备纳米胶囊，将得到的乳液置于黑暗环境下挥发除去有机溶剂，再利用去离子水透析，除去未被包裹的活性物质，最后冷冻干燥，制得载药纳米胶囊前体；其中活性物质和氨基化叶酸的质量比为1-2:2.5-4；Mix the organic solvent in which the active substance is dissolved with the PBS solution (0.01mol/L, pH=7.4) in which the multifunctional hyaluronic acid is dissolved in a volume ratio of 1:20, and prepare it by ultrasonic emulsification under the action of an ultrasonic probe Nanocapsules, the obtained emulsion is placed in a dark environment to volatilize to remove organic solvents, and then dialyzed with deionized water to remove uncoated active substances, and finally freeze-dried to obtain drug-loaded nanocapsule precursors; wherein the active substances and amino groups The mass ratio of folic acid is 1-2:2.5-4;
(6)具有肿瘤细胞生物还原性微环境敏感的载药纳米胶囊的制备(6) Preparation of drug-loaded nanocapsules sensitive to tumor cell bioreductive microenvironment
将步骤(5)所得的载药纳米胶囊前体分散于PBS溶液(0.01mol/L，pH＝7.4)中，加入二环己基碳二亚胺和N-羟基丁二酰亚胺，室温搅拌反应4-6h，然后加入步骤(4)所制备的氨基化叶酸，室温搅拌反应12-14h，再利用去离子水透析透析纯化，最后冷冻干燥，制得；其中载药纳米胶囊前体外围羧基、二环己基碳二亚胺、N-羟基丁二酰亚胺、氨基化叶酸的摩尔比为1:1-2:1.5-3:8-12:0.1-0.3。Disperse the drug-loaded nanocapsule precursor obtained in step (5) in a PBS solution (0.01mol/L, pH=7.4), add dicyclohexylcarbodiimide and N-hydroxysuccinimide, and stir the reaction at room temperature 4-6h, then add the aminated folic acid prepared in step (4), stir and react at room temperature for 12-14h, then use deionized water to dialysis and purify, and finally freeze-dry to obtain; wherein the drug-loaded nanocapsule precursor peripheral carboxyl, The molar ratio of dicyclohexylcarbodiimide, N-hydroxysuccinimide and aminated folic acid is 1:1-2:1.5-3:8-12:0.1-0.3.
进一步地，步骤(1)中透明质酸、1-(3-二甲氨基丙基)-3-乙基碳二亚胺、1-羟基苯并三唑和胱胺二盐酸盐的摩尔比分别为1:3:3:3。Further, the molar ratio of hyaluronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 1-hydroxybenzotriazole and cystamine dihydrochloride in step (1) 1:3:3:3 respectively.
进一步地，步骤(2)中胱胺结合的透明质酸、甲基丙烯酸缩水甘油酯、三乙胺和二硫苏糖醇的摩尔比分别为：1:20:20:5。Further, the molar ratios of cystamine-bound hyaluronic acid, glycidyl methacrylate, triethylamine and dithiothreitol in step (2) are respectively: 1:20:20:5.
进一步地，羧酸甜菜碱甲基丙烯酸酯单体的制备方法为：Further, the preparation method of carboxybetaine methacrylate monomer is:
①将2-(二甲氨基)甲基丙烯酸乙酯溶于无水丙酮中，使2-(二甲氨基)甲基丙烯酸乙酯的浓度为0.2mol/L；①Dissolve 2-(dimethylamino)ethyl methacrylate in anhydrous acetone so that the concentration of 2-(dimethylamino)ethyl methacrylate is 0.2mol/L;
②氮气保护下，将β-丙内酯溶于无水丙酮中，使β-丙内酯的浓度为1.2mol/L；②Under nitrogen protection, dissolve β-propiolactone in anhydrous acetone so that the concentration of β-propiolactone is 1.2mol/L;
③将步骤②所得溶液滴加到步骤①所得溶液中反应4-6h，反应温度为10℃；其中2-(二甲氨基)甲基丙烯酸乙酯和β-丙内酯的摩尔比为1:1.2；③The solution obtained in step ② is added dropwise to the solution obtained in step ① to react for 4-6h, and the reaction temperature is 10°C; wherein the molar ratio of 2-(dimethylamino)ethyl methacrylate and β-propiolactone is 1: 1.2;
④反应结束后，用无水丙酮沉淀产物，然后再用无水丙酮和无水乙醚洗涤，真空干燥，制得。④ After the reaction, the product is precipitated with anhydrous acetone, then washed with anhydrous acetone and anhydrous ether, and vacuum-dried to obtain the product.
进一步地，步骤③中滴加速度为1滴/秒，滴加时间控制在15min之内。Further, in step ③, the dropping speed is 1 drop/second, and the dropping time is controlled within 15 minutes.
进一步地，步骤(3)中酯化的胱胺结合的透明质酸(GM-HA-SH)上的烯键、羧酸甜菜碱甲基丙烯酸酯聚合物、溴化亚铜、1,1,4,7,10,10-六甲基三亚乙基四胺，2-(2-溴异丁氧基)甲基丙烯酸乙酯的摩尔比分别为1:10:2:4:1。Further, the ethylenic bond on the cystamine-bound hyaluronic acid (GM-HA-SH) esterified in step (3), carboxybetaine methacrylate polymer, cuprous bromide, 1,1, The molar ratios of 4,7,10,10-hexamethyltriethylenetetramine and 2-(2-bromoisobutoxy)ethyl methacrylate are 1:10:2:4:1, respectively.
进一步地，步骤(4)中叶酸、二环己基碳二亚胺、N-羟基丁二酰亚胺、乙二胺和吡啶的摩尔比分别为1:1.2:2.0:10:0.001。Further, the molar ratios of folic acid, dicyclohexylcarbodiimide, N-hydroxysuccinimide, ethylenediamine and pyridine in step (4) are 1:1.2:2.0:10:0.001, respectively.
进一步地，步骤(5)中活性物质和氨基化叶酸的质量比为1:2.5。Further, in step (5), the mass ratio of the active substance to the aminoated folic acid is 1:2.5.
进一步地，步骤(5)中活性物质为抗肿瘤药物、荧光分子、光敏剂、四氧化三铁磁粒子或金纳米粒子，优选为抗肿瘤药物，具体为阿霉素、紫杉醇或吉西他滨。Further, the active substance in step (5) is an antitumor drug, a fluorescent molecule, a photosensitizer, ferromagnetic particles or gold nanoparticles, preferably an antitumor drug, specifically adriamycin, paclitaxel or gemcitabine.
进一步地，步骤(5)中有机溶剂为苯、甲苯、氯仿、环己烷、二氯甲烷或三氯甲烷，优选为三氯甲烷或二氯甲烷。Further, the organic solvent in step (5) is benzene, toluene, chloroform, cyclohexane, dichloromethane or chloroform, preferably chloroform or dichloromethane.
进一步地，步骤(6)中载药纳米胶囊前体外围羧基、二环己基碳二亚胺、N-羟基丁二酰亚胺、氨基化叶酸的摩尔比分别为1:1.2:2.0:10:0.2。Further, the molar ratios of drug-loaded nanocapsule precursor peripheral carboxyl, dicyclohexylcarbodiimide, N-hydroxysuccinimide, and aminated folic acid in step (6) are respectively 1:1.2:2.0:10: 0.2.
制备过程中所用的PBS均是浓度为0.01mol/L，pH＝7.4的PBS。The PBS used in the preparation process is PBS with a concentration of 0.01 mol/L and a pH of 7.4.
通过上述方法制备的具有肿瘤细胞生物还原性微环境敏感的载药纳米胶囊。A drug-loaded nanocapsule sensitive to the bioreductive microenvironment of tumor cells prepared by the above method.
本发明提供的一种具有肿瘤细胞生物还原性微环境敏感的载药纳米胶囊及其制备方法，具有以下有益效果：The invention provides a drug-loaded nanocapsule sensitive to the bioreductive microenvironment of tumor cells and its preparation method, which has the following beneficial effects:
骨架材料透明质酸(HA)是构成细胞外基质和胞间质的主要成分，具有很好的生物安全性；骨架材料透明质酸(HA)可以与肿瘤部位过表达的受体结合，增强对肿瘤细胞的亲和性；利用具有高生物相容性的透明质酸(HA)三维网状结构构建胶囊壳层结构，通过吸附大量水分子，赋予其类似纳米水凝胶的性质；该纳米胶囊外围为亲水的两性离子链段(羧酸甜菜碱甲基丙烯酸酯单体聚合物)，具有较好的抗非特异性蛋白吸附特性，可以帮助纳米胶囊载体实现血液中的长循环；粒径在EPR效应(实体瘤的高通透性和滞留效应，enhancedpermeability and retention effect)范围内，可实现被动靶向通过血液循环富集于肿瘤部位；具有叶酸靶向配体，可以通过主动靶向富集于具有叶酸受体高表达的肿瘤(例如，乳腺癌，肺癌，卵巢癌等)部位；HA壳层结构中具有还原性敏感的二硫键(disulfide bond)，可实现在肿瘤细胞内部高谷胱甘肽水平(glutathione，GSH)条件下断裂，实现胶囊壳层结构溶胀和解体，可以帮助药物载体从溶酶体有效逃逸，避免药物分解，同时实现在肿瘤细胞内部药物快速释放；抗肿瘤效率高，对正常组织毒副作用小。The skeleton material hyaluronic acid (HA) is the main component of the extracellular matrix and intercellular substance, and has good biological safety; The affinity of tumor cells; the capsule shell structure is constructed by using the three-dimensional network structure of hyaluronic acid (HA) with high biocompatibility, and the nano-hydrogel-like properties are endowed by absorbing a large number of water molecules; the nanocapsules The periphery is a hydrophilic zwitterionic segment (carboxybetaine methacrylate monomer polymer), which has good anti-non-specific protein adsorption properties and can help nanocapsule carriers achieve long circulation in the blood; the particle size is between Within the scope of EPR effect (high permeability and retention effect of solid tumors, enhanced permeability and retention effect), passive targeting can be achieved and enriched in the tumor site through blood circulation; with folic acid targeting ligand, it can be enriched through active targeting In tumors with high expression of folate receptors (such as breast cancer, lung cancer, ovarian cancer, etc.); HA shell structure has a reduction-sensitive disulfide bond (disulfide bond), which can achieve high glutathione in tumor cells Peptide level (glutathione, GSH) breaks under conditions to realize swelling and disintegration of the capsule shell structure, which can help drug carriers escape from lysosomes effectively, avoid drug decomposition, and achieve rapid release of drugs inside tumor cells; high anti-tumor efficiency, Toxic and side effects on normal tissues are small.
附图说明Description of drawings
图1为本发明具有肿瘤细胞生物还原性微环境敏感的载药纳米胶囊的示意图；Figure 1 is a schematic diagram of the drug-loaded nanocapsules sensitive to the bioreductive microenvironment of tumor cells of the present invention;
图2为利用透明质酸(HA)制备酯化胱胺结合的透明质酸(GM-HA-SH)化学反应过程图；Fig. 2 is the chemical reaction process diagram of hyaluronic acid (GM-HA-SH) that utilizes hyaluronic acid (HA) to prepare esterified cystamine binding;
图3为Z-HA-SH、GM-HA-SH和HA-Cys的1H-NMR表征图以及Z-HA-SH结构示意图；Figure 3 is a1 H-NMR characterization diagram of Z-HA-SH, GM-HA-SH and HA-Cys and a schematic diagram of the structure of Z-HA-SH;
图4为制备末端氨基化的叶酸(folic acid，FA)的化学反应过程图；Fig. 4 is the chemical reaction process chart of the folic acid (folic acid, FA) that prepares terminal amination;
图5为具有肿瘤细胞生物还原性微环境敏感的载药纳米胶囊透射电子显微镜图(TEM)；Figure 5 is a transmission electron microscope image (TEM) of a drug-loaded nanocapsule sensitive to the bioreductive microenvironment of tumor cells;
图6为具有肿瘤细胞生物还原性微环境敏感的载药纳米胶囊扫描电子显微镜图(SEM)；Figure 6 is a scanning electron micrograph (SEM) of drug-loaded nanocapsules sensitive to the bioreductive microenvironment of tumor cells;
图7为具有肿瘤细胞生物还原性微环境敏感的载药纳米胶囊在水溶液中的粒径分布图；Figure 7 is a particle size distribution diagram of drug-loaded nanocapsules sensitive to tumor cell bioreduction microenvironment in aqueous solution;
图8为具有肿瘤细胞生物还原性微环境敏感的载药纳米胶囊在不同谷胱甘肽(GSH)浓度条件下释放曲线图；Figure 8 is a graph showing the release curves of drug-loaded nanocapsules sensitive to the bioreductive microenvironment of tumor cells under different glutathione (GSH) concentrations;
图9为具有肿瘤细胞生物还原性微环境敏感的载药纳米胶囊对乳腺癌细胞(4T1)的毒性作用图(以自由药盐酸阿霉素DOX·HCl为对照组)；Figure 9 is a diagram of the toxic effect of drug-loaded nanocapsules sensitive to the bioreductive microenvironment of tumor cells on breast cancer cells (4T1) (with free drug doxorubicin hydrochloride DOX·HCl as the control group);
图10为具有肿瘤细胞生物还原性微环境敏感的载药纳米胶囊对荷瘤小鼠的体内抑制肿瘤生长数据(以自由药盐酸阿霉素DOX·HCl和生理盐水Saline为对照组)；Figure 10 is the in vivo tumor growth inhibition data of drug-loaded nanocapsules sensitive to tumor cell bioreduction microenvironment on tumor-bearing mice (with free drug doxorubicin hydrochloride DOX·HCl and saline Saline as the control group);
图11为具有肿瘤细胞生物还原性微环境敏感的载药纳米胶囊在荷瘤小鼠主要脏器以及肿瘤部位的蓄积随时间变化情况(左列为定性分析，右列为定量分析；以自由药盐酸阿霉素DOX·HCl为对照组)；Figure 11 shows the accumulation of drug-loaded nanocapsules sensitive to the bioreductive microenvironment of tumor cells in the main organs and tumor sites of tumor-bearing mice over time (the left column is qualitative analysis, and the right column is quantitative analysis; Doxorubicin hydrochloride DOX·HCl is the control group);
图12为主要脏器的HE切片图(以自由药盐酸阿霉素DOX·HCl和生理盐水Saline为对照组；线圈或箭头所示为脏器损伤)。Fig. 12 is a HE section view of major organs (free drug doxorubicin hydrochloride DOX·HCl and saline were used as control group; coils or arrows indicate organ damage).
具体实施方式detailed description
具有肿瘤细胞生物还原性微环境敏感的载药纳米胶囊的示意图，如图1所示，其制备过程如下：A schematic diagram of a drug-loaded nanocapsule sensitive to the bioreductive microenvironment of tumor cells is shown in Figure 1, and its preparation process is as follows:
实施例1胱胺结合的透明质酸(HA-Cys)的制备The preparation of the hyaluronic acid (HA-Cys) that embodiment 1 cystamine binds
将5g透明质酸溶解于250ml PBS(0.01mol/L，pH＝7.4)中，加入3倍当量的1-(3-二甲氨基丙基)-3-乙基碳二亚胺和3倍当量的1-羟基苯并三唑，室温搅拌，活化羧基4h；然后加入3倍当量的胱胺二盐酸盐，室温条件下搅拌反应24h，再利用去离子水透析72h(透析袋截留分子量3500道尔顿)，除去未反应的分子和缩合剂等；其中透明质酸、1-(3-二甲氨基丙基)-3-乙基碳二亚胺、1-羟基苯并三唑和胱胺二盐酸盐的摩尔比为1:3:3:3。Dissolve 5g of hyaluronic acid in 250ml of PBS (0.01mol/L, pH=7.4), add 3 equivalents of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 3 equivalents 1-Hydroxybenzotriazole, stirred at room temperature, activated carboxyl group for 4 hours; then added 3 times equivalent of cystamine dihydrochloride, stirred and reacted at room temperature for 24 hours, and then dialyzed with deionized water for 72 hours (the molecular weight cut-off of the dialysis bag was 3500 channels Dayton), to remove unreacted molecules and condensing agents, etc.; among them, hyaluronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 1-hydroxybenzotriazole and cystamine The molar ratio of dihydrochloride is 1:3:3:3.
实施例2酯化胱胺结合的透明质酸(GM-HA-SH)的制备Example 2 Preparation of hyaluronic acid (GM-HA-SH) combined with esterified cystamine
将1g胱胺结合的透明质酸(HA-Cys)溶解于25ml PBS/二甲基甲酰胺混合液中(PBS和二甲基甲酰胺的体积比为4:1)，加入过量的甲基丙烯酸缩水甘油酯和三乙胺，于室温条件下搅拌反应14天，反应结束后，利用去离子水透析72h(透析袋截留分子量3500道尔顿)，接着加入过量的二硫苏糖醇，室温条件下搅拌10h，再利用去离子水透析纯化72h(透析袋截留分子量3500道尔顿)，最后冷冻干燥，制得侧链含烯键的酯化胱胺结合的透明质酸(GM-HA-SH)；其中胱胺结合的透明质酸、甲基丙烯酸缩水甘油酯、三乙胺和二硫苏糖醇的摩尔比为1:20:20:5。反应过程图如图2所示。Dissolve 1 g of cystamine-conjugated hyaluronic acid (HA-Cys) in 25 ml of PBS/dimethylformamide mixture (the volume ratio of PBS and dimethylformamide is 4:1), and add excess methacrylic acid Glycidyl ester and triethylamine were stirred and reacted at room temperature for 14 days. After the reaction was over, dialyzed with deionized water for 72 hours (molecular weight cut-off of the dialysis bag was 3500 Daltons), and then excessive dithiothreitol was added, and the reaction was carried out at room temperature. Stir for 10 hours, then dialysis and purify with deionized water for 72 hours (the molecular weight cut-off of the dialysis bag is 3500 Daltons), and finally freeze-dry to obtain the hyaluronic acid (GM-HA-SH ); wherein the molar ratio of cystamine-bound hyaluronic acid, glycidyl methacrylate, triethylamine and dithiothreitol is 1:20:20:5. The reaction process diagram is shown in Figure 2.
实施例3多功能化透明质酸(Z-HA-SH)的制备Example 3 Preparation of Multifunctional Hyaluronic Acid (Z-HA-SH)
利用2-(二甲氨基)甲基丙烯酸乙酯和β-丙内酯开环反应制备羧酸甜菜碱甲基丙烯酸酯单体。具体方法为：Preparation of carboxybetaine methacrylate monomer by ring-opening reaction of 2-(dimethylamino)ethyl methacrylate and β-propiolactone. The specific method is:
①将30mmol的2-(二甲氨基)甲基丙烯酸乙酯溶解到150ml无水丙酮中；①Dissolve 30mmol of 2-(dimethylamino)ethyl methacrylate in 150ml of anhydrous acetone;
②氮气保护条件下，将36mmol的β-丙内酯溶解于30ml无水丙酮中；②Under nitrogen protection conditions, dissolve 36mmol of β-propiolactone in 30ml of anhydrous acetone;
③将步骤②所得溶液滴加到步骤①所得溶液中，保持滴加速率为1滴/秒，滴加时间控制在15min之内，然后搅拌反应5h，整个反应过程中利用冰浴将反应体系温度保持在10℃；③ Add the solution obtained in step ② dropwise to the solution obtained in step ①, keep the dropping rate at 1 drop/second, control the dropping time within 15 minutes, then stir and react for 5 hours, and use an ice bath to reduce the temperature of the reaction system during the entire reaction process. Keep at 10°C;
④反应结束后，利用无水丙酮沉淀产物，并利用无水丙酮和无水乙醚交替洗涤多次，然后在真空干燥箱中干燥，得到白色粉末状产物即羧酸甜菜碱甲基丙烯酸酯单体。④ After the reaction, use anhydrous acetone to precipitate the product, and use anhydrous acetone and anhydrous ether to wash alternately for several times, and then dry it in a vacuum oven to obtain a white powder product that is carboxybetaine methacrylate monomer .
将所得羧酸甜菜碱甲基丙烯酸酯单体作为原料，溴化亚铜为催化剂，1,1,4,7,10,10-六甲基三亚乙基四胺为配体，2-(2-溴异丁氧基)甲基丙烯酸乙酯为引发剂，再加入步骤(2)中所制备的侧链含烯键的酯化胱胺结合的透明质酸(GM-HA-SH)，在无氧条件下，通过原子转移自由基聚合反应，制备得到侧链为羧酸甜菜碱甲基丙烯酸酯聚合物的巯基化透明质酸；整个反应过程中利用油浴将反应体系温度保持在60℃，搅拌反应24h；其中酯化的胱胺结合的透明质酸上的烯键、羧酸甜菜碱甲基丙烯酸酯、溴化亚铜、1,1,4,7,10,10-六甲基三亚乙基四胺的摩尔比为1:10:2:4:1。The obtained carboxylic acid betaine methacrylate monomer is used as a raw material, cuprous bromide is a catalyst, 1,1,4,7,10,10-hexamethyltriethylenetetramine is a ligand, 2-(2 -Bromoisobutoxy) ethyl methacrylate is an initiator, then add the hyaluronic acid (GM-HA-SH) combined with the esterified cystamine in the side chain containing ethylenic bond prepared in step (2), in Under anaerobic conditions, thiolated hyaluronic acid whose side chain is carboxybetaine methacrylate polymer was prepared by atom transfer radical polymerization; the temperature of the reaction system was kept at 60°C by using an oil bath during the entire reaction process , stirred for 24 hours; among them, the ethylenic bond on the hyaluronic acid bound by the esterified cystamine, carboxybetaine methacrylate, cuprous bromide, 1,1,4,7,10,10-hexamethyl The molar ratio of triethylenetetramine is 1:10:2:4:1.
将所得产物冷冻干燥，溶解于重水中(D2O)，做600MHz 1H NMR扫描，其结果如图3所示。The obtained product was freeze-dried, dissolved in heavy water (D2O), and scanned by 600 MHz 1H NMR. The results are shown in FIG. 3 .
实施例4氨基化叶酸的制备The preparation of embodiment 4 aminofolic acid
将450mg叶酸溶解于50ml二甲基亚砜中，向其中添加1.2倍当量二环己基碳二亚胺和2.0倍当量的N-羟基丁二酰亚胺，50℃搅拌反应10h，向反应混合物中加入10倍当量的乙二胺和0.001倍当量的吡啶，室温搅拌反应12h，反应结束后，利用0.45微米孔径的滤器过滤除去不溶物，再用去离子水透析72h(透析袋截留分子量3500道尔顿)，最后冷冻干燥；其中叶酸、二环己基碳二亚胺、N-羟基丁二酰亚胺、乙二胺和吡啶的摩尔比为1:1.2:2.0:10:0.001。反应过程图如图4所示。Dissolve 450 mg of folic acid in 50 ml of dimethyl sulfoxide, add 1.2 equivalents of dicyclohexylcarbodiimide and 2.0 equivalents of N-hydroxysuccinimide, stir and react at 50°C for 10 h, and add to the reaction mixture Add 10 times the equivalent of ethylenediamine and 0.001 times the equivalent of pyridine, and stir at room temperature for 12 hours. After the reaction, remove the insoluble matter by filtering through a filter with a pore size of 0.45 microns, and then dialyze with deionized water for 72 hours (the molecular weight cut-off of the dialysis bag is 3500 dal Dayton), and finally freeze-dried; wherein the molar ratio of folic acid, dicyclohexylcarbodiimide, N-hydroxysuccinimide, ethylenediamine and pyridine is 1:1.2:2.0:10:0.001. The reaction process diagram is shown in Figure 4.
实施例5载药纳米胶囊前体的制备与表征Preparation and Characterization of Example 5 Drug-loaded Nanocapsule Precursor
将100mg阿霉素盐酸盐分散于5ml氯仿溶剂中，加入3倍当量的三乙胺(24ul)，超声水浴处理1min；将250mg多功能化透明质酸(Z-HA-SH)溶解于100ml PBS(0.01mol/L，pH＝7.4)中，与溶解有阿霉素的氯仿混合，在超声探头作用下(600W)处理5min，利用超声乳化法制备纳米胶囊；将得到的红色乳液置于黑暗环境下室温搅拌12h，以挥发除去溶剂中的氯仿，再利用去离子水透析72h(透析袋截留分子量3500道尔顿)，除去未被包裹的阿霉素后，冷冻干燥，形成载药纳米胶囊前体。Disperse 100mg of doxorubicin hydrochloride in 5ml of chloroform solvent, add 3 times the equivalent of triethylamine (24ul), and treat in an ultrasonic water bath for 1min; dissolve 250mg of multifunctional hyaluronic acid (Z-HA-SH) in 100ml In PBS (0.01mol/L, pH=7.4), mix with the chloroform that dissolves doxorubicin, process 5min under the effect of ultrasonic probe (600W), utilize ultrasonic emulsification method to prepare nanocapsule; The red emulsion that obtains is placed in dark Stir at room temperature for 12 hours to remove chloroform in the solvent by volatilization, and then dialyze with deionized water for 72 hours (dialysis bag molecular weight cut-off 3500 Daltons), remove unwrapped doxorubicin, freeze-dry to form drug-loaded nanocapsules precursor.
实施例6具有肿瘤细胞生物还原性微环境敏感的载药纳米胶囊的制备Example 6 Preparation of drug-loaded nanocapsules sensitive to tumor cell bioreduction microenvironment
避光条件下，将100mg载药纳米胶囊前体分散在50ml PBS(0.01mol/L，pH＝7.4)中，按照纳米胶囊外围pCBMA末端羧基摩尔数(约0.046mmol)，加入1.2倍当量的二环己基碳二亚胺(10.58mg)和2倍当量的N-羟基丁二酰亚胺(10.59mg)，室温搅拌反应4h，活化羧基，再加入10倍当量的氨基化叶酸(222.41mg)，室温搅拌反应12h，利用去离子水透析72h(透析袋截留分子量3500道尔顿)，最后冷冻干燥，得到具有肿瘤细胞生物还原性微环境敏感的载药纳米胶囊。Under light-shielding conditions, disperse 100 mg drug-loaded nanocapsule precursor in 50 ml PBS (0.01mol/L, pH=7.4), and add 1.2 times the equivalent of di Cyclohexylcarbodiimide (10.58mg) and 2 times the equivalent of N-hydroxysuccinimide (10.59mg), stirred at room temperature for 4h to activate the carboxyl group, and then added 10 times the equivalent of aminated folic acid (222.41mg), The reaction was stirred at room temperature for 12 hours, dialyzed with deionized water for 72 hours (the molecular weight cut-off of the dialysis bag was 3500 Daltons), and finally freeze-dried to obtain drug-loaded nanocapsules sensitive to the bioreductive microenvironment of tumor cells.
将所得的载药纳米胶囊分散到纯净水中，取20ul滴加到铜网上，自然干燥，做透射电子显微镜表征，如图5所示，纳米胶囊体系呈分散良好的球形结构，可见明显空腔结构，水分挥发后其平均粒径明显变小(46.00±6.09nm)。Disperse the obtained drug-loaded nanocapsules into pure water, drop 20 ul onto the copper grid, let it dry naturally, and perform transmission electron microscope characterization, as shown in Figure 5, the nanocapsule system has a well-dispersed spherical structure, and an obvious cavity structure can be seen , the average particle size becomes significantly smaller (46.00±6.09nm) after the water evaporates.
取20ul滴加到硅片上，自然干燥，喷金处理，做扫描电子显微镜表征，结果如图6所示，纳米胶囊呈分散的球体结构，水分挥发后其粒径与透射电子显微镜基本一致(55.52±11.88nm)。Take 20ul and add it dropwise on the silicon wafer, dry naturally, spray gold, and perform scanning electron microscope characterization. The results are shown in Figure 6. The nanocapsules are in a dispersed spherical structure. After the water evaporates, its particle size is basically consistent with that of the transmission electron microscope ( 55.52±11.88nm).
取100ul，添加900ul水稀释，利用马尔文激光粒度分析仪表征，得到该纳米胶囊体系在液相中的水动力学尺寸分布，如图7所示，分散在水中后，纳米胶囊吸收大量水分，使得粒径增大(189.2nm，多分散指数PDI＝0.167)。Take 100ul, add 900ul of water to dilute, use the Malvern laser particle size analyzer to characterize, obtain the hydrodynamic size distribution of the nanocapsule system in the liquid phase, as shown in Figure 7, after being dispersed in water, the nanocapsule absorbs a large amount of water, This resulted in an increase in particle size (189.2 nm, polydispersity index PDI = 0.167).
相关表征结果表明，所制备的纳米胶囊体系分散性良好，尺寸适宜，有利于基于肿瘤部位增强的渗透和滞留作用(EPR)效应实现被动靶向。Relevant characterization results show that the prepared nanocapsule system has good dispersion and appropriate size, which is conducive to passive targeting based on the enhanced penetration and retention (EPR) effect at the tumor site.
实施例7不同谷胱甘肽(GSH)浓度条件下载药纳米胶囊体系的药物释放行为Example 7 Drug release behavior of drug nanocapsule system under different glutathione (GSH) concentration conditions
称取适量实例6中的冻干载药纳米胶囊，分散于PBS(0.01mol/L，pH＝7.4)中，配置成阿霉素浓度为300ug/ml的分散液，然后分装于相同体积(约1.5ml)透析袋中(透析袋截留分子量1000道尔顿)，每袋装入1ml，将其分别浸没于以下缓冲液体系中，每个条件设置三个平行：Weigh an appropriate amount of freeze-dried drug-loaded nanocapsules in Example 6, disperse them in PBS (0.01mol/L, pH=7.4), and configure a dispersion solution with a doxorubicin concentration of 300ug/ml, and then distribute them in the same volume ( About 1.5ml) in dialysis bags (molecular weight cut-off of dialysis bag is 1000 Daltons), each bag is filled with 1ml, which are respectively immersed in the following buffer system, and three parallels are set for each condition:
1)20ml的PBS(0.01mol/L，pH＝7.4)，不含GSH；1) 20ml of PBS (0.01mol/L, pH=7.4), without GSH;
2)20ml GSH浓度为10mM的PBS(0.01mol/L，pH＝7.4)；2) 20ml of PBS with a GSH concentration of 10mM (0.01mol/L, pH=7.4);
3)20ml GSH浓度为2.8μM的PBS(0.01mol/L，pH＝7.4)。3) 20 ml of PBS (0.01 mol/L, pH=7.4) with a GSH concentration of 2.8 μM.
将以上体系置于37℃恒温水浴摇床震荡，在设计的时间点(0-72h)分别取1ml透析袋外部的缓冲液用于测定所含药物浓度，并添加1ml缓冲液以保持其体积始终为20ml。以已知浓度的阿霉素药物作为参比，根据测试结果绘制阿霉素释放曲线，如图8所示。Place the above system in a constant temperature water bath shaker at 37°C, take 1ml of the buffer solution outside the dialysis bag at the designed time point (0-72h) to determine the concentration of the drug contained, and add 1ml buffer solution to keep its volume constant for 20ml. Taking doxorubicin with known concentration as a reference, the release curve of doxorubicin was drawn according to the test results, as shown in FIG. 8 .
结果表明，阿霉素的释放行为与GSH浓度水平相关，高浓度(GSH＝10mM)条件下，阿霉素达到了最大的释放行为，而低浓度或无GSH缓冲液中，阿霉素释放很少。具体表现为，GSH＝10mM条件下，阿霉素在前12h有突释现象，累计释放达到69.49％，延长试验时间，释放量进一步增加，在24h后达到81.91％，并一直保持此水平直到整个测试周期(72h)。作为对照，当GSH＝2.8μM和GSH＝0的缓冲液体系在12h只检测到5％甚至更低的药物释放，当测试时间到达72h，分别探测到～17％和～12％药物释放。这些结果表明，本发明的载药纳米胶囊体系其药物释放是依赖于GSH浓度水平，并且肿瘤细胞微环境的高GSH浓度可以有利于这一载药体系实现快速的胞内药物释放，进一步有益于肿瘤治疗。The results show that the release behavior of doxorubicin is related to the concentration level of GSH. Under the condition of high concentration (GSH=10mM), doxorubicin has reached the maximum release behavior, while in low concentration or no GSH buffer, the release of doxorubicin is very slow. few. Specifically, under the condition of GSH=10mM, doxorubicin had a burst release phenomenon in the first 12 hours, and the cumulative release reached 69.49%. After prolonging the test time, the release amount further increased, reaching 81.91% after 24 hours, and maintained this level until the whole Test cycle (72h). As a control, when the GSH=2.8μM and GSH=0 buffer systems only detected 5% or even lower drug release at 12h, when the test time reached 72h, ~17% and ~12% drug release were detected respectively. These results show that the drug release of the drug-loaded nanocapsule system of the present invention is dependent on the GSH concentration level, and the high GSH concentration of the tumor cell microenvironment can help this drug-loaded system to achieve rapid intracellular drug release, which is further beneficial tumor treatment.
实施例8肿瘤细胞生长抑制作用Embodiment 8 Tumor cell growth inhibitory effect
采用商用试剂盒通过CCK8方法对本发明中的载药纳米胶囊细胞生长抑制作用进行验证。具体方法为：将小鼠乳腺癌4T1细胞(5000个)接种于96孔细胞培养板上，当细胞达到90％融合时，分别加入不同浓度(0.001，0.01，0.10，1.0，10.0和100.0μg/ml)的自由药(盐酸阿霉素，DOX·HCl)和载药纳米胶囊(DOX/FA-Z-NC)，继续培养36h，再加入CCK8试剂(每孔10μl)，在37℃条件下继续避光孵育2h，用酶标仪测定各个孔在450nm处的光吸收值。每个实验组设置5个平行样，以不加任何药物或纳米胶囊的细胞组作为空白对照并记作100％细胞存活，计算并将实验组数据表示成细胞存活率百分比。实验结果如图9所示。The cell growth inhibitory effect of the drug-loaded nanocapsules in the present invention was verified by the CCK8 method using a commercial kit. The specific method is: inoculate mouse breast cancer 4T1 cells (5000 cells) on a 96-well cell culture plate, and when the cells reach 90% confluence, add different concentrations (0.001, 0.01, 0.10, 1.0, 10.0 and 100.0 μg/ ml) of free drug (doxorubicin hydrochloride, DOX·HCl) and drug-loaded nanocapsules (DOX/FA-Z-NC), continue to culture for 36h, then add CCK8 reagent (10μl per well), continue at 37°C Incubate for 2 h in the dark, and measure the absorbance of each well at 450 nm with a microplate reader. Five parallel samples were set up for each experimental group, and the cell group without any drug or nanocapsule was used as a blank control and recorded as 100% cell survival, and the data of the experimental group were calculated and expressed as the percentage of cell survival. The experimental results are shown in Figure 9.
研究结果表明，含有药物的实验组均可对肿瘤细胞的生长产生抑制作用，并且随着阿霉素浓度的增高其肿瘤细胞生长抑制作用更加明显。与自由药(盐酸阿霉素，DOX·HCl)比较，载药纳米胶囊(DOX/FA-Z-NC)具有较相近的肿瘤细胞生长毒性。The research results showed that the experimental group containing the drug could inhibit the growth of tumor cells, and the inhibitory effect on tumor cell growth was more obvious as the concentration of doxorubicin increased. Compared with free drug (doxorubicin hydrochloride, DOX·HCl), the drug-loaded nanocapsules (DOX/FA-Z-NC) had similar tumor cell growth toxicity.
实施例9肿瘤模型动物体内抗肿瘤能力评价Example 9 Evaluation of anti-tumor ability in tumor model animals
选取BALB/c雌性裸鼠(3-4周，18-19g)建立皮下乳腺癌肿瘤模型，待肿瘤生长至180cm3时，将荷瘤裸鼠随机分组，每组5只。通过尾静脉注射方式分别将200μl生理盐水(空白对照组)，自由药(盐酸阿霉素，DOX·HCl)和载药纳米胶囊(DOX/FA-Z-NC)注射入荷瘤裸鼠体内，阿霉素总计计量为4μg/kg(裸鼠体重)。每4天注射一次，共计5次给药。每隔两天记录肿瘤体积和荷瘤裸鼠体重变化，观察期为23天。统计肿瘤体积变化和体重变化，结果如图10所示。BALB/c female nude mice (3-4 weeks, 18-19g) were selected to establish a subcutaneous breast cancer tumor model. When the tumor grew to 180 cm3 , the tumor-bearing nude mice were randomly divided into groups of 5. Inject 200 μl of normal saline (blank control group), free drug (doxorubicin hydrochloride, DOX·HCl) and drug-loaded nanocapsules (DOX/FA-Z-NC) into tumor-bearing nude mice by tail vein injection, respectively. The total dose of mycin was 4 μg/kg (body weight of nude mice). Injections were given every 4 days for a total of 5 doses. The tumor volume and body weight changes of tumor-bearing nude mice were recorded every two days, and the observation period was 23 days. The changes in tumor volume and body weight were counted, and the results are shown in FIG. 10 .
结果表明，载药纳米胶囊(DOX/FA-Z-NC)表现出比自由药(盐酸阿霉素，DOX·HCl)组更好的肿瘤生长抑制作用。在第23天，与原始肿瘤体积大小相比，DOX/FA-Z-NCs组的肿瘤体积仅为320.15±30.2％，自由药组为477.6±65.5％，而空白对照组为881.7±48.3％。这一结果说明本发明的载药纳米胶囊体系能够较好的抑制模型动物实体瘤生长。The results showed that the drug-loaded nanocapsules (DOX/FA-Z-NC) exhibited better tumor growth inhibition than the free drug (doxorubicin hydrochloride, DOX·HCl) group. On day 23, compared with the original tumor volume, the tumor volume of the DOX/FA-Z-NCs group was only 320.15±30.2%, that of the free drug group was 477.6±65.5%, and that of the blank control group was 881.7±48.3%. This result shows that the drug-loaded nanocapsule system of the present invention can better inhibit the growth of solid tumors in model animals.
实施例10载药纳米胶囊在动物体内脏器及肿瘤分布研究Example 10 Study on distribution of drug-loaded nanocapsules in internal organs and tumors of animals
选取BALB/c雌性裸鼠(3-4周，18-19g)建立皮下乳腺癌肿瘤模型，待肿瘤生长至180cm3时，将荷瘤裸鼠随机分组，每组3只。通过尾静脉注射将自由药(盐酸阿霉素，DOX·HCl)和载药纳米胶囊(DOX/FA-Z-NC)分别注射入荷瘤裸鼠体内，阿霉素总计计量为5μg/kg(裸鼠体重)。在2h，7h和24h分别处死裸鼠，并小心剥离主要脏器(心、肝、脾、肺、肾)和肿瘤，利用活体成像仪对其进行扫描，以表征载药纳米胶囊在动物体内分布以及随时间变化趋势，实验结果如图11所示。BALB/c female nude mice (3-4 weeks, 18-19g) were selected to establish a subcutaneous breast cancer tumor model. When the tumor grew to 180 cm3 , the tumor-bearing nude mice were randomly divided into groups of 3. Free drug (doxorubicin hydrochloride, DOX·HCl) and drug-loaded nanocapsules (DOX/FA-Z-NC) were injected into tumor-bearing nude mice through tail vein injection, and the total dose of doxorubicin was 5 μg/kg (nude mice). mouse body weight). Nude mice were sacrificed at 2h, 7h and 24h, and the main organs (heart, liver, spleen, lung, kidney) and tumors were carefully stripped, and scanned with an in vivo imager to characterize the distribution of drug-loaded nanocapsules in the animal body As well as the trend over time, the experimental results are shown in Figure 11.
结果表明，相对于自由药组，载药纳米胶囊(DOX/FA-Z-NC)可以随时间逐步实现在肿瘤部位的稳定累积(平均信号强度789.71±21.27；单位counts)，而在肝脏和肾脏表现出了有效地清除，从而表明本发明的载药纳米胶囊可以携带药物达到肿瘤部位，且减少对主要脏器的损伤。作为对比，自由药组在肿瘤的蓄积随着时间逐渐减少(肿瘤平均信号强度329.32±22.50；单位counts)，到达24h时其在心脏的蓄积仍然明显(183.42±5.29；单位counts)，具有较强的心脏毒性。The results showed that, compared with the free drug group, the drug-loaded nanocapsules (DOX/FA-Z-NC) could gradually achieve stable accumulation in the tumor site over time (average signal intensity 789.71±21.27; unit counts), while in the liver and kidney It shows effective clearance, thus indicating that the drug-loaded nanocapsules of the present invention can carry drugs to tumor sites and reduce damage to major organs. In contrast, the accumulation in the tumor of the free drug group gradually decreased over time (the average signal intensity of the tumor was 329.32±22.50; unit counts), and its accumulation in the heart was still obvious at 24 hours (183.42±5.29; unit counts), which had a strong of cardiotoxicity.
实施例11主要脏器的组织学研究Example 11 Histological study of major organs
在实施例9中，于第23天，处死荷瘤裸鼠，并小心剥离主要脏器(心、肝、脾、肺、肾)并对其进行石蜡包埋、切片和HE染色处理。通过观察所得切片，探讨生理盐水组，自由药(盐酸阿霉素，DOX·HCl)和载药纳米胶囊(DOX/FA-Z-NC)对主要脏器生理结构的影响，结果如图12所示。In Example 9, on the 23rd day, the tumor-bearing nude mice were sacrificed, and the main organs (heart, liver, spleen, lung, kidney) were carefully dissected and processed for paraffin embedding, sectioning and HE staining. By observing the obtained slices, the effects of normal saline group, free drug (doxorubicin hydrochloride, DOX HCl) and drug-loaded nanocapsules (DOX/FA-Z-NC) on the physiological structure of major organs were explored, and the results are shown in Figure 12 Show.
结果表明，不进行治疗(生理盐水组)，模型动物皮下肿瘤出现了在肝脏和肺部(如图中箭头所示)的转移，表明已对主要脏器造成了伤害。自由药组表现出了肝脏部位干细胞的坏死(图中黑线圈出)和心脏部位的心肌颗粒性病变，表明仅仅给自由药治疗，会对肝脏和心脏造成一定的损伤。而载药纳米胶囊均未发现上述损伤。综合以上结果，表明本发明的载药纳米胶囊体系可以有效地减少抗肿瘤药物阿霉素对主要脏器造成损伤，并且也能避免肿瘤细胞向脏器的转移。The results showed that without treatment (normal saline group), the subcutaneous tumors in the model animals metastasized to the liver and lungs (as indicated by the arrows in the figure), indicating that major organs had been damaged. The free drug group showed necrosis of stem cells in the liver (circled in black in the figure) and myocardial granular lesions in the heart, indicating that only free drug treatment would cause certain damage to the liver and heart. However, the above-mentioned damages were not found in the drug-loaded nanocapsules. Based on the above results, it is shown that the drug-loaded nanocapsule system of the present invention can effectively reduce the damage caused by the anti-tumor drug doxorubicin to major organs, and can also avoid the transfer of tumor cells to organs.
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