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1、 基金项目：国家自然科学基金青年基金（51103180），天津市应用基础研究计划面上项目（11JCYBJC02400） 作者简介：张琳华，女，副研究员 研究方向：生物材料与药物缓控释 *通讯作者：孙洪范，男，研究员 研究方向：生物材料与药物缓控释Email: , Tel:(022)87892052叶酸靶向紫杉醇聚合物纳米囊泡的制备及其抗肿瘤活性研究张琳华，马桂蕾，宋存先，孙洪范*（中国医学科学院&北京协和医学院生物医学工程研究所，天津市生物医学材料重点实验室，天津 300192）摘要： 目的 制备新型叶酸靶向紫杉醇聚合物纳米囊泡，研究其理化性质、细胞毒性和体内抗肿瘤活性。方法 以两亲性三嵌段共
2、聚物聚己内酯-b-聚乙二醇-b-聚己内酯（PCL-b-PEG-b-PCL）、甲氧基聚乙二醇二硬脂酰磷脂酰乙醇胺（mPEG2000-DSPE）、偶联叶酸的磷脂DSPE-PEG(2000) Folate共混物为载体材料，通过薄膜-超声分散法制备出叶酸靶向紫杉醇聚合物纳米囊泡并对其进行形态、粒径及其分布、Zeta电位、载药量及包封率、体外释放等表征，用CCK-8法研究了聚合物纳米囊泡对H1299肺癌细胞的细胞毒性，并评价其在BALB/c小鼠EMT-6乳腺癌模型中的抗肿瘤效果。结果 叶酸靶向紫杉醇聚合物纳米囊泡呈球形，具有明显的核-壳结构，粒径均匀。随着紫杉醇投药量的增加，纳米囊泡的粒径增大，包封率降
3、低。载药30%的叶酸靶向紫杉醇聚合物纳米囊泡的平均粒径为311 nm，多分散系数为0.171，Zeta电位为-30.3 mV，包封率为91.16%。体外释放研究表明载紫杉醇聚合物纳米囊泡基本无药物突释现象，具有缓释特性。细胞毒性研究表明当紫杉醇浓度为25gmL-1时，叶酸靶向紫杉醇聚合物纳米囊泡的细胞毒性低于相应浓度的紫杉醇/聚氧乙烯蓖麻油注射剂。体内抗肿瘤活性实验研究表明，叶酸靶向紫杉醇聚合物纳米囊泡对小鼠EMT-6乳腺癌具有明显抑制作用，具有与紫杉醇/聚氧乙烯蓖麻油注射剂相似的抑制肿瘤作用。结论 叶酸靶向紫杉醇聚合物纳米囊泡具有高载药量及包封率、均匀的粒径及分布、缓释特性、低毒和较好的抗肿
4、瘤作用，是一种有潜力的紫杉醇缓控释新剂型用于提高紫杉醇的药效并且降低不良反应。关键词：紫杉醇；聚合物纳米囊泡；聚己内酯-b-聚乙二醇-b-聚己内酯；叶酸靶向；细胞毒性；抗肿瘤活性Preparation, Characterization, In Vitro and In Vivo Studies on Folate-Targeted Biodegradable Polymersomes Loaded with PaclitaxelZHANG Lin-hua, MA Gui-lei, SONG Cun-xian, SUN Hong-fan. (Tianjin Key Laboratory of
5、Biomaterial Research, Institute of Biomedical Engineering, Peking Union Medical College & Chinese Academy of Medical Sciences, Tianjin 300192, China)ABSTRACT: OBJECTIVE To prepare folate-targeted biodegradable polymersomes loaded with paclitaxel, and evaluate its physical and chemical properties, in
6、 vitro cell cytotoxicity as well as in vivo antitumor activity. METHODS Folate-targeted biodegradable paclitaxel-loaded polymersomes composed of PCL-b-PEG-b-PCL、mPEG2000-DSPE and DSPE-PEG(2000) Folate were prepared by thin-film hydration and ultrasonic method, and characterized in terms of morpholog
7、y, particle size and size distribution, drug loading content, encapsulation efficiency and in vitro release. The cell cytotoxicity of folate-targeted biodegradable paclitaxel-loaded polymersomes in lung carcinoma H1299 cells was observed with CCK-8 assay. The in vivo antitumor activity was evaluated
8、 in BALB/c mice bearing the EMT-6 breast tumor models. RESULTS Folate-targeted biodegradable paclitaxel-loaded polymersomes showed spherical core-shell morphology with narrow size distribution. With the increase of drug-fed amount, the sizes of folate-targeted biodegradable paclitaxel-loaded polymer
9、somes increased, while the drug encapsulating efficiency decreased. The folate-targeted biodegradable paclitaxel-loaded polymersomes with drug-loading content of 30% were found as spherical shape with the average particle diameter of 311 nm, polydispersity coefficient of 0.171 and Zeta potential of
10、-30.3 mV. The folate-targeted biodegradable paclitaxel-loaded polymersomes showed a continuous and steady release of PTX without initial burst release. At the paclitaxel concentration of 25gmL-1, the folate-targeted biodegradable paclitaxel-loaded polymersomes displayed much less cell cytotoxicity t
11、han paclitaxel injections with Cremophor EL. The in vivo tumor inhibiting activity of folate-targeted biodegradable paclitaxel-loaded polymersomes was similar to that of paclitaxel injections with Cremophor EL in the same paclitaxel concentration. CONCLUSION The folate-targeted biodegradable paclita
12、xel-loaded polymersomes had high drug-loading content and drug encapsulating efficiency, more uniform size and size distribution, excellent drug sustainable-release behavior, less cytotoxicity, good antitumor activity similar to paclitaxel injections with Cremophor EL. Therefore, folate-targeted bio
13、degradable paclitaxel-loaded polymersomes would be a novel paclitaxel preparation in clinic for the treatment of tumor.KEY WORDS: Paclitaxel; Nano-sized Polymersomes; PCL-b-PEG-b-PCL; Folate targeted; Cell cytotoxicity; Antitumor activity紫杉醇（paclitaxel，PTX）是从红豆杉属植物紫杉的树干和树皮中提取得到的天然抗癌药，临床上主要用于卵巢癌、乳腺癌及
14、非小细胞肺癌的治疗1,2。由于PTX在水中溶解度极低，临床以聚氧乙烯蓖麻油和乙醇溶解后给药，给药后存在严重过敏反应、肾毒性、神经毒性、心脏毒性等不良反应，使其临床应用受到限制3,4。为消除毒副作用，提高PTX在水中的分散性，设计新的更能被接受的剂型以淘汰含有Cremophor的传统剂型成为当前的研究热点，如微乳、胶束、囊泡、前体药物、包合物、脂质体、纳米粒等5-11。聚合物囊泡（Polymersome）是由合成或天然改性的双亲性聚合物自组装构成、具有类似脂质体双层结构的封闭空腔球体或类球体。相对于表面活性剂、脂质体等小分子囊泡，聚合物囊泡有着分子可设计性好、囊泡强度高、稳定性好、渗透性强等优点
15、 12,13。作为药物载体时,同脂质体一样具有组织相容性和细胞透过性,但不像脂质体容易受氧化或水解,也不易泄漏药物。药物被聚合物囊泡包封后，可以起到增效、减毒、缓释、增加药物稳定性和靶向等作用；表面修饰后，则可赋予囊泡在血液中长循环和靶向等特性14,15。近年来，随着大分子自组装的飞速发展，以聚合物囊泡作为一类新型药物载体的研究得到了广泛的关注。叶酸是一种糖基化磷脂肌醇连接的膜糖蛋白，其受体在许多肿瘤细胞中过度表达，而在绝大多数正常组织中几乎不表达，同时叶酸无毒、免疫原性弱、稳定, 与叶酸受体的结合力强,对肿瘤有高度选择性16-18。因此，将叶酸连接到聚合物囊泡表面，赋予聚合物囊泡对肿瘤细胞的
16、主动靶向性，可提高药物在肿瘤组织中的浓度，减轻不良反应，实现肿瘤的靶向治疗。本研究以两亲性三嵌段共聚物PCL-b-PEG-b-PCL、甲氧基聚乙二醇二硬脂酰磷脂酰乙醇胺mPEG2000-DSPE、偶联叶酸的磷脂DSPE-PEG(2000) Folate为载体材料，通过分子自组装制备出新型叶酸靶向的载紫杉醇聚合物纳米囊泡，并对其特性进行表征，研究其细胞毒性和体内抗肿瘤活性，并与临床应用的紫杉醇/聚氧乙烯蓖麻油注射剂进行比较。1 材料及仪器己内酯单体（-CL）（纯度99%, 美国Aldrich公司）；聚乙二醇8000（纯度99%，美国Sigma公司）；甲氧基聚乙二醇二硬脂酰磷脂酰乙醇胺mPEG2000-DSPE（美国Avanti Polar Lipids 公司）；偶联叶酸的磷脂DSPE-PEG(2000) Folate，（美国Avanti Polar Lipids 公司）；紫杉醇原料药（纯度99%，中国重庆
