CN102166190B - 一种双重靶向肿瘤的紫杉醇纳米脂质体及其制备方法 - Google Patents
一种双重靶向肿瘤的紫杉醇纳米脂质体及其制备方法 Download PDFInfo
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Abstract
本发明属于生物医药领域，更具体地，本发明公开了一种双重靶向肿瘤的紫杉醇纳米脂质体,所述的双靶向紫杉醇纳米脂质体由双重靶向肿瘤的多肽、脂质连接物和紫杉醇脂质体三部分组成。本发明还公开了所述的双重靶向肿瘤的紫杉醇纳米脂质体的制备方法及其作为制备抑制肿瘤药物的用途。
Description
技术领域
本发明属于医药领域，更具体地，本发明公开了一种双重靶向肿瘤的纳米脂质体及其制备方法。
背景技术
近年来恶性肿瘤的发病率和死亡率呈明显上升趋势，已经成为威胁人类健康和生命的主要疾病。国际癌症研究机构的报道显示，1975年到2000年间，全球癌症病例数增长了一倍，每年新发病例约1200万，死亡患者超过700万。2010年癌症将跃居为全球的首要死因，2030年肿瘤患者人数将为现在的三倍，新发病例数将增至2000-2600万。
化疗是恶性肿瘤综合治疗中的主要手段之一，合理的化疗策略可显著延长肿瘤患者的生存时间，极大改善患者的生存质量。紫杉醇是一种二萜类化合物，最早是从太平洋紫杉树皮中获得的天然产物，因其能够稳定肿瘤细胞的微管，抑制有丝分裂而具有独特抗癌活性(Wani，M.C.，et al.，Plant antitumoragents.VI.The isolation and structure of taxol，a novel antileukemicand antitumor agent from Taxus brevifol ia.J Am Chem Soc，1971.93(9)：p.2325-7.)，目前被广泛应用于卵巢癌(Thomas，H.and P.Rosenberg，Roleof weekly paclitaxel in the treatment of advanced ovarian cancer.CritRev Oncol Hematol，2002.44Suppl：p.S43-51.)、乳腺癌(Saloustros，E.，D.Mavroudis，and V.Georgoulias，Paclitaxel and docetaxel in thetreatment of breast cancer.Expert Opin Pharmacother，2008.9(15)：p.2603-16.)、头颈部癌(Aisner，J.and H.Cortes-Funes，Paclitaxel in headand neck and other cancers：future prospects.Semin Oncol，1997.24(1Suppl 2)：p.S2-113-S2-115.)和非小细胞肺癌的治疗(Greco，F.A.，Paclitaxel-based combination chemotherapy in advanced non-small celllung cancer.Lung Cancer，2001.34Suppl 4：p.S53-6.)。但紫杉醇是一种脂溶性物质，在水溶液和其它许多溶剂中都难以溶解(Ceruti，M.，et al.，Preparation，characterization，cytotoxicity and pharmacokinetics ofliposomes containing water-soluble prodrugs of paclitaxel.J ControlRelease，2000.63(1-2)：p.141-53.)。临床上使用的紫杉醇制剂(Taxol，商品名为泰素)采用的溶媒为非离子表面活性剂聚氧乙烯蓖麻油(Cremophor EL，CrEL)和无水乙醇(1∶1，V/V)混合物，但该剂型伴随很多难以解决的临床问题(Hennenfent，K.L.and R.Govindan，Novel formulations of taxanes：areview.Old wine in a new bottle？Ann Oncol，2006.17(5)：p.735-49.)。传统紫杉醇制剂的临床应用可导致一系列过敏反应(Hennenfent，K.L.and R.Govindan，Novel formulations of taxanes：a review.Old wine in a new bottle？AnnOncol，2006.17(5)：p.735-49.)，轻者表现瘙痒、皮疹，重者表现为血管性水肿、低血压、呼吸衰竭等(Weiss，R.B.，et al.，Hypersensitivity reactions from taxol.J ClinOncol，1990.8(7)：p.1263-8.Wiemik，P.H.，et al.，Phase I clinical andpharmacokinetic study of taxol.Cancer Res，1987.47(9)：p.2486-93.)。95％以上的患者过敏反应发生于第一次或第二次紫杉醇制剂给药期间，80％的症状发生于药物输注的前10分钟，且往往紫杉醇仅仅输注了1mg。由于CrEL可诱导动物产生类似的反应(Wiernik，P.H.，et al.，Phase I clinical and pharmacokinetic study oftaxol.Cancer Res，1987.47(9)：p.2486-93.)，因此认为是CrEL的存在导致了上述不良反应的发生。且不良反应的发生与输注速度相关(Lorenz，W.，et al.，Histamine release and hypotensive reactions in dogs by solubilizing agents and fattyacids：analysis of various components in cremophor El and development of acompound with reduced toxicity.Agents Actions，1982.12(1-2)：p.64-80.)，但延长输注时间并不能消除过敏反应的出现。研究者尝试用其他溶剂来替代CrEL，如聚乙二醇，但动物实验发现聚乙二醇的存在可降低紫杉醇的抗肿瘤活性。因此CrEL仍是目前紫杉醇的标准溶媒(Wiernik，P.H.，et al.，Phase I clinical andpharmacokinetic study of taxol.Cancer Res，1987.47(9)：p.2486-93.)。
临床上普遍采用大剂量的糖皮质激素和组胺受体拮抗剂作为其使用前的常规预处理，但其疗效尚不确切。美国西北大学芬伯格医学院的药物警戒项目中药物不良反应事件及报告研究(Research on Adverse Drug Events and Reports，RADAR)发现，1997年到2007年间美国、欧洲和日本报道了171例使用聚氧乙烯蓖麻油紫杉醇后发生过敏反应的病例，其中58例患者发生死亡(34％)，96例报道采用了预处理方案的患者中21例发生死亡，死亡率达22％。BoehnkeMichaud等的研究(Michaud，L.B.，V.Valero，and G.Hortobagyi，Risks and benefitsof taxanes in breast and ovarian cancer.Drug Saf，2000.23(5)：p.401-28.)也表明，预处理方案采用后仍有约40％的患者会发生轻症的过敏，3％的患者过敏反应可致命。同时预处理方案的施行限制了肿瘤合并溃疡、糖尿病、高血压等的患者对该药的使用，且预处理方案中激素的使用还可能增加治疗相关的死亡率。
采用CrEL作为溶媒还可导致周围神经系统毒性(Onetto，N.，et al.，Overview of Taxol safety.J Natl Cancer Inst Monogr，1993(15)：p.131-9.)。电生理检查发现紫杉醇CrEL溶液使用后发生周围神经病变的患者存在轴突降解和脱髓鞘。环胞霉素CrEL溶液静脉给药后有约25％的患者会发生周围神经病变，口服给药则无此不良反应出现，而CrEL并不经胃肠道吸收(de Groen，P.C.，et al.，Central nervous system toxicity after liver transplantation.The role of cyclosporineand cholesterol.N Engl J Med，1987.317(14)：p.861-6.)。治疗剂量的紫杉醇或环胞霉素CrEL溶液所产生的CrEL血浆浓度可导致小鼠背根神经节神经元轴突肿胀、空泡变性和降解(Windebank，A.J.，M.D.Blexrud，and P.C.de Groen，Potentialneurotoxicity of the solvent vehicle for cyclosporine.J Pharmacol Exp Ther，1994.268(2)：p.1051-6.)。目前的研究表明，CrEL产生的环氧乙烷衍生物是导致神经损伤的最主要的因素(Brat，D.J.，A.J.Windebank，and S.Brimijoin，Emulsifier forintravenous cyclosporin inhibits neurite outgrowth，causes deficits in rapid axonaltransport and leads to structural abnormalities in differentiating N1E.115neuroblastoma.J Pharmacol Exp Ther，1992.261(2)：p.803-10.)。
紫杉醇CrEL溶液每周三次，每次输注3小时的用法使紫杉醇的血浆浓度超出了机体清除代谢的能力，因此紫杉醇CrEL溶液在体内的消除呈非线性动力学，亦称为零级消除动力学(Sparreboom，A.，et al.，Cremophor EL-mediatedalteration of paclitaxel distribution in human blood：clinical pharmacokineticimplications.Cancer Res，1999.59(7)：p.1454-7.van Tellingen，O.，et al.，CremophorEL causes(pseudo-)non-linear pharmacokinetics of paclitaxel in patients.Br JCancer，1999.81(2)：p.330-5.)。动物实验证实，紫杉醇非线性药代动力学特征与CrEL有关(Sparreboom，A.，et al.，Nonlinear pharmacokinetics of paclitaxel in miceresults from the pharmaceutical vehicle Cremophor EL.Cancer Res，1996.56(9)：p.2112-5.)，人体内的实验也获得了类似的结果(van Tellingen，O.，et al.，CremophorEL causes(pseudo-)non-linear pharmacokinetics of paclitaxel in patients.Br JCancer，1999.81(2)：p.330-5.)。紫杉醇的清除率的降低导致机体组织暴露于高浓度的药物时间延长，产生严重全身毒副反应的几率增加。CrEL还可在血液中形成微小颗粒，包裹紫杉醇分子，从而影响药物分子在肿瘤组织中的分布、代谢与排泌(Sparreboom，A.，et al.，Cremophor EL-mediated alteration of paclitaxeldistribution in human blood：clinical pharmacokinetic implications.Cancer Res，1999.59(7)：p.1454-7.)。
紫杉醇的耐药机制很多，P-糖蛋白的表达升高是重要的原因之一。P-糖蛋白是由多药耐药基因编码的一种跨膜转运蛋白，具有ATP酶活性，能将化疗药物从细胞内泵出，降低细胞内的有效药物浓度而致耐药。有研究认为，CrEL可以通过调节P-糖蛋白、抑制多药耐药基因的表达而增强紫杉醇的抗肿瘤作用，但在体内实验中未获得成功(Woodcock，D.M.，et al.，Reversal of the multidrugresistance phenotype with cremophor EL，a common vehicle for water-insolublevitamins and drugs.Cancer Res，1990.50(14)：p.4199-203.Schuurhuis，G.J.，et al.，The polyoxyethylene castor oil Cremophor EL modifies multidrug resistance.Br JCancer，1990.62(4)：p.591-4.Friche，E.，et al.，The solvents cremophor EL andTween 80 modulate daunorubicin resistance in the multidrug resistant Ehrlich ascitestumor.Cancer Commun，1990.2(9)：p.297-303.)。相反有研究发现，CrEL可通过阻滞细胞周期，降低肿瘤细胞对紫杉醇的摄取而拮抗紫杉醇的细胞毒作用(Liebmann，J.，et al.，The influence of Cremophor EL on the cell cycle effects ofpaclitaxel(Taxol)in human tumor cell lines.Cancer Chemother Pharmacol，1994.33(4)：p.331-9.)。
研究表明，紫杉醇CrEL溶液存在稳定性和相容性的问题，如药物一经稀释紫杉醇就易发生沉淀；可从临床上常规使用的PVC输液袋和输液管路中溶出增塑剂(二乙烯己基邻苯二甲酸盐)，因此整个使用过程必须采用玻璃或非PVC输液器；与塑料或玻璃容器有非特异性吸附等等(Waugh，W.N.，L.A.Trissel，andV.J.Stella，Stability，compatibility，and plasticizer extraction of taxol(NSC-125973)injection diluted in infusion solutions and stored in various containers.Am J HospPharm，1991.48(7)：p.1520-4.Song，D.，L.F.Hsu，and J.L.Au，Binding of taxol toplastic and glass containers and protein under in vitro conditions.J Pharm Sci，1996.85(1)：p.29-31.)。
综上所述，紫杉醇现有剂型中CrEL的存在导致了一系列临床毒副反应的出现，CrEL对紫杉醇的疗效及药代动力学也可产生复杂的作用，因此临床上迫切需要研制出不含CrEL的紫杉醇新剂型，以克服表面活性剂的缺陷。
众多学者致力于紫杉醇新剂型的研发，目前已经研发成功或正在研发的新剂型包括：白蛋白纳米粒(AbraxameTM)(Ibrahim，N.K.，et al.，Phase I andpharmacokinetic study of ABI-007，a Cremophor-free，protein-stabilized，nanoparticle formulation of paclitaxel.Clin Cancer Res，2002.8(5)：p.1038-44.
Yamada，K.，et al.，Phase I and Pharmacokinetic Study of ABI-007，Albumin-bound Paclitaxel，Administered Every 3Weeks in Japanese Patients withSolid Tumors.Jpn J Clin Oncol，2010.)、紫杉醇前药二十二碳六烯酸紫杉醇(Taxoprexin)(Wolff，A.C.，et al.，Phase I study of docosahexaenoic acid-paclitaxel：a taxane-fatty acid conjugate with a unique pharmacology and toxicity profile.ClinCancer Res，2003.9(10Pt 1)：p.3589-97.)；多聚谷氨酸紫杉醇(XyotaxTM)(Sabbatini，P.，et al.，Phase II study of CT-2103 in patients with recurrent epithelialovarian，fallopian tube，or primary peritoneal carcinoma.J Clin Oncol，2004.22(22)：p.4523-31.)、紫杉醇类似物(BMS-184476(Rose，W.C.，C.Fairchild，and F.Y. Lee，Preclinical antitumor activity of two novel taxanes.Cancer Chemother Pharmacol，2001.47(2)：p.97-105.)、DJ-927Ono，C.，A.Takao，and R.Atsumi，Absorption，distribution，and excretion of DJ-927，a novel orally effective taxane，in mice，dogs，and monkeys.Biol Pharm Bull，2004.27(3)：p.345-51.Baas，P.，et al.，Phase I/IIstudy of a 3 weekly oral taxane(DJ-927)in patients with recurrent，advancednon-small cell lung cancer.J Thorac Oncol，2008.3(7)：p.745-50.)、BMS-275183(Broker，L.E.，et al.，Effect of food on the pharmacokinetic behavior ofthe potent oral taxane BMS-275183.Clin Caneer Res，2008.14(13)：p.4186-91.
Broker，L.E.，et al.，Phase I trial with BMS-275183，a novel oral taxane withpromising antitumor activity.Clin Caneer Res，2006.12(6)：p.1760-7.)、OrtataxelCassinelli，G.，et al.，Cellular bases of the antitumor activity of the novel taxane IDN5109(BAY59-8862)on hormone-refractory prostate cancer.Clin Caneer Res，2002.8(8)：p.2647-54.Polizzi，D.，et al.，A novel taxane with improved tolerability andtherapeutic activity in a panel of human tumor xenografts.Cancer Res，1999.59(5)：p.1036-40.)、RPRl09881A(Gelmon，K.A.，et al.，Phase I dose-finding study of a newtaxane，RPR 109881A，administered as a one-hour intravenous infusion days 1 and 8to patients with advanced solid tumors.J Clin Oncol，2000.18(24)：p.4098-108.
Kurata，T.，et al.，Phase I and pharmacokinetic study of a new taxoid，RPR10988lA，given as a 1-hour intravenous infusion in patients with advanced solidtumors.J Clin Oncol，2000.18(17)：p.3164-71.)、紫杉醇胶束共聚物(Genexol-PM)(Kim，S.C.，et al.，In vivo evaluation of polymeric micellar paclitaxel formulation：toxicity and efficacy.J Control Release，2001.72(1-3)：p.191-202.)、紫杉醇维生素E乳剂(TOCOSOL)、紫杉醇微球(Paclimer)(Lissianskaya，A.，et al.，Paclitaxelinjectable emulsion：Phase 2a study of weekly administration in patients withplatinum-resistant ovarian cancer.Journal of Clinical Oncology，2004.22(14)：p.460s-460s.Bogdanova，N.，et al.，Paclitaxel injectable emulsion：Phase 2a study ofweekly administration in patients with non-small cell lung cancer(NSCLC).Journalof Clinical Oncology，2004.22(14)：p.649s-649s.)、紫杉醇脂质体(力朴素)等。其中部分紫杉醇新剂型已经上市(AbraxameTM于2005年被美国食品药品管理局批准上市；紫杉醇脂质体力朴素由南京思科药业研发成功，于2003年批准在国内上市)，大部分紫杉醇新剂型尚处于临床前研究阶段，或I-III期临床试验中。
上述紫杉醇新剂型的共同点在于避免了采用CrEL作为溶媒，因此具有独特的优势，如可缩短药物的输注时间、降低过敏、骨髓抑制和脱发等不良反应的发生率等。部分剂型由于结构的改变而不再是P-糖蛋白的作用底物，因此可降低紫杉醇耐药的发生率，部分剂型甚至可以口服吸收。但紫杉醇新剂型与紫杉醇传统剂型相比能否改善肿瘤患者的预后尚未可知。
发明内容
本发明旨在研发一种新型的肿瘤靶向脂质体载药纳米粒，包封紫杉醇后可改善紫杉醇的理化性状和药代动力学行为，改善溶解度，从而避免传统制剂中CrEL导致的各项毒副反应，同时通过该载体的肿瘤主动靶向性，特异性地增加紫杉醇在肿瘤局部的浓度，在增强抗肿瘤作用的同时降低药物对正常组织器官的毒性。
本发明将紫杉醇包封于双重靶向肿瘤的纳米脂质体中，两个靶分子能维持各自原有的生物学活性，在功能上产生协同效应。
本发明提供的双重靶向肿瘤的紫杉醇纳米脂质体主要由三部分组成，双重靶向肿瘤的多肽，脂质连接物和紫杉醇脂质体。
首先，本发明采用固相合成法获得双重靶向肿瘤的多肽，所述多肽的氨基酸序列如下：ARYCRGDCFDATWLPPR。其中ARYCRGDCFDG：其核心结构为RGD三肽，即精氨酸-甘氨酸-天冬氨酸(Arg-Gly-Asp)序列，针对靶点是整合素αV家族。αV整合素为细胞黏附分子家族的重要成员之一，通过参与内皮细胞的激活和迁移、介导内皮细胞增殖、抑制内皮细胞凋亡、参与碱性成纤维细胞生长因子和VEGF诱导的血管生成、诱导环加氧酶2的产生等多种途径促进新生血管化和肿瘤的发生发展。αV整合素在生理状态下静止的血管内皮细胞和正常组织器官内呈低表达，但高表达于活化的肿瘤血管内皮细胞和肿瘤细胞表面。药物载体连接含RGD序列的短肽后可显著增强其靶向肿瘤新生血管的能力。ATWLPPR序列是针对的靶点为VEGFR2的共受体神经内皮素-1(Neuropilin-1，NRP-1)。VEGFR-2(Vascular Endothelial Growth Factor Receptor-2)是VEGF/VEGFR家族成员，主要识别低分子量的VEGF，在血管内皮细胞迁移、增殖、存活及血管通透性调节中起重要作用。NRP-1是-种非酪氨酸跨膜糖蛋白，是VEGFR-2的辅助受体，和VEGFR-2的共表达可显著促进VEGF165与VEGFR-2的结合，增强VEGF165介导的生物学作用，促进血管内皮的增殖。NRP-1亦高表达于活化的肿瘤血管内皮细胞和肿瘤细胞表面，在生理状态下静止的血管内皮细胞和正常组织器官内呈低表达。
本发明采用Fmoc固相合成法合成了多肽配体-赖氨酸-甘氨酸-甘氨酸-棕榈酸(lysine-glycine-glycine，KGG-pal)联结物，连接新型多肽和紫杉醇脂质体。
本发明采用薄膜超声分散法制备紫杉醇脂质体，挤压过滤法或高压均质法控制脂质体粒径在60-200nm。小粒径的纳米粒作为药物载体有其独特优势。恶性肿瘤的侵袭性生长和转移有赖于血管的生成，虽然相对于正常血管来说，肿瘤组织中血管对药物的选择性渗透力较弱，但最大直径仍不超过400nm。大粒径的脂质体在脾脏的截留更多，从血液中清除更快，到达肿瘤组织的脂质体明显减少。因此小粒径长循环药物脂质体更易透过血管间隙发挥到达肿瘤局部的作用。
细胞学研究证实，本发明的含有RGD及ATWLPPR序列的双重靶向肿瘤紫杉醇脂质体，因新型靶向多肽分子量小，核心载药颗粒粒径控制在60-200nm，故易穿透内皮细胞屏障进入肿瘤组织。研究证实，RGD及ATWLPPR序列连接后空间构象互不影响，能维持序列各自原有的生物学活性，从而产生协同效应。相较于只含有RGD或ATWLPPR序列的单靶向紫杉醇脂质体，研发的双重靶向肿瘤的紫杉醇脂质体有显著为优的与新生血管内皮细胞及肿瘤细胞的特异性结合力。与无靶向及单靶向肿瘤的紫杉醇脂质体相比，双重靶向肿瘤的紫杉醇脂质体有更强的与新生血管内皮细胞及肿瘤细胞特异性结合能力，及抑制肿瘤生长能力，故可被进一步应用于恶性肿瘤的靶向治疗领域。
附图说明
图1：双重靶向肿瘤的紫杉醇脂质体扫描电镜图
图2：双重靶向肿瘤的紫杉醇脂质体粒径分布图
图3：Taxol及各紫杉醇脂质体细胞摄取图
图4：Taxol及各紫杉醇脂质体MTT检测图
图5：Taxol及各紫杉醇脂质体抑瘤实验D60瘤重图
具体实施方式
实验材料
1、试剂、药品和细胞
蛋黄卵磷脂(egg phosphatide，egg PC)、胆固醇(cholesterol，CHOL)购自德国东尚公司；单甲氧基聚乙二醇2000二硬脂酰磷脂酰乙醇胺mPEG2000-DSPE购自美国Avanti Polar Lipids公司；脂质多肽均由吉尔生化(上海)有限公司合成；Taxol购自施贵宝公司；紫杉醇购自上海融禾医药科技发展有限公司；十二烷基硫酸钠(Sodium dodecyl sulfonate，SDS)、聚丙烯酰胺、蔗糖、冰醋酸、硝酸银、戊二醛、抑肽酶等均购自国药集团上海分公司；HUVEC(人脐静脉内皮细胞)，ATCC No.：CRL-2873；A549细胞(肺腺癌细胞系)，ATCC No.：CCL-185；DMEM(Dulbecco’s Modified Eagle Media，高糖细胞培养基)，购自Invitrogen公司；新生牛血清购自奥地利PAA公司；青霉素、链霉素、胰蛋白酶等购自华美公司。
2、仪器设备和耗材
MillexTM FH针式滤器和Ultracel YM100超滤管(美国Millipore公司)；SK5210HP水浴超声仪(上海科导超声仪器有限公司)；R502B旋转蒸发仪(西安太康仪器设备有限公司)；JEM-1200EXII型透射电子显微镜(日本JEOL公司)；Quanta 200FEG场发射环境扫描电子显微镜(FEI公司)；MASTERSIZER 2000激光粒度仪(英国Malvern公司)；冷冻干燥机6L(美国Labconco公司)；余同前。
3、溶液配制
PBS液(0.01M，pH7.4)：NaCl 8.0g；KCl 0.2g；Na2HP04·12H20 1.44g；KH2PO4 0.24g；超纯水800ml；调节pH值至7.4，定容至1L后高压灭菌，4℃保存。
0.25％胰酶消化液(1L)：胰酶2.5g；NaCl 8g；KCl 0.4g；Na2HP04·12H20 0.06g；KH2PO4 0.06g；NaHCO3 0.35g；酚红0.02g；加压过滤除菌，分装后-20℃保存DMEM培养基(1L)：DMEM粉2袋；NaHCO37.4g；青霉素0.12g；链霉素0.2g；HCl、NaOH调节pH值至7.2-7.4，加压过滤除菌，4℃保存；细胞冻存液：DMEM培养基70％；二甲亚砜(DMSO)10％；新生牛血清20％；分装后-20℃保存；细胞裂解液：Tris 10mmol/L；Nacl 100mmol/L；EDTA 1mmol/L ；EGTA 1mmol/L；NaF 1mmol/L；Na4P2O7·10H2O 20mmol/L；钒酸钠2mmol/L；Triton X-1001％；甘油10％；十二烷基硫酸钠0.1％；脱氧胆酸盐0.5％；PMSF 100μg/μL。
通过以下实施例进一步说明本发明，但不作为本发明的限制。
实施例1：双重靶向肿瘤多肽的合成步骤
采用9-芴甲氧羰基(fluorenylmethyloxycarbonyl，FMOC)固相合成法合成双重靶向肿瘤的多肽，采用高效液相色谱(high performance liquidchromatography，HPLC)法纯化，采用质谱鉴定。具体合成步骤如下：
1)树脂溶涨：将FMOC-AA-Wang-Resin树脂放入反应管中，加二甲基甲酰胺(N，N-dimethylformamide，DMF)(15ml/g)30min；
脱保护：吸弃DMF，加20％哌啶DMF溶液(15ml/g)5min，吸弃后再加20％哌啶DMF溶液(15ml/g)15min；
2)检测：抽掉哌啶溶液，取树脂十几粒，用乙醇洗三次，加入茚三酮，氰化钾，苯酚溶液各一滴，105℃-110℃加热5min，变深蓝色为阳性反应。
3)洗涤：DMF(10ml/g)两次，甲醇(10ml/g)两次，DMF(10ml/g)两次；
4)缩合：保护氨基酸(FOMC-Asp-OH)三倍过量，1-氧-3-双二甲胺羧基苯骈三氮唑四氟化硼盐三倍过量，均用尽量少DMF溶解，加入反应管，立刻加入N-甲基吗啉十倍过量，反应30min；
5)洗涤：DMF(10ml/g)一次，甲醇(10ml/g)两次，DMF(10ml/g)两次；
6)重复操作步骤2)--6)，依次连接FMOC-Tyr-OH、FMOC-Thr-OH、FMOC-Met-OH、FMOC-Asn-OH、FMOC-Pro-OH、FMOC-Arg-OH、FMOC-Lys-OH、FMOC-Leu-OH、FMOC-Phe-OH；
7)最后一次洗涤：DMF(10ml/g)两次，甲醇(10ml/g)两次，DMF(10ml/g)两次，DCM(10ml/g)两次；
8)裂解：裂解液(10ml/g)(含TFA 94.5％；水2.5％；EDT 2.5％；TIS 1％)120min；
9)吹干洗涤：氮气尽量吹干裂解液，乙醚洗涤六次，常温挥干；
10)密封，-20度保存。
所得为白色粉末状物质，HPLC纯化，纯度＞95％；
实施例2：非靶向、单靶向及双重靶向肿瘤的紫杉醇脂质体的制备
采用薄膜超声分散法制备各紫杉醇脂质体
非主动靶向紫杉醇脂质体配方：PC∶CHOL∶mPEG-DSPE∶PTX 9∶1∶0.5∶0.33，主动靶向紫杉醇脂质体在上述配方中加入不同的脂质多肽(ARYCRGDCFDG-KGG、ARYCRGDCFDATWLPPR-KGG)(与总脂摩尔比均为1∶542)。由于在专利1实施例中，多肽ATWLPPR及ATWLPPR-荧光脂质体均未显示出优于多肽ARYCRGDCFDG、ARYCRGDCFDG-荧光脂质体及多肽ARYCRGDCFDATWLPPR、ARYCRGDCFDATWLPPR-荧光脂质体的与细胞特异性亲和力，故未合成，且在后继试验中亦未予应用。
上述配方溶于氯仿，0.2μm平板滤器过滤，置旋转蒸发仪37℃旋转成膜后继续真空抽吸1小时进一步除去氯仿；加适量过滤除菌的9％蔗糖溶液，充分溶胀至薄膜脱落，所得混悬液经Mini-extruder挤出(1μm、0.4μm、0.2μm、0.1μm各10次)后，分装至西林瓶，置真空冷冻干燥机中，-38℃×2h，-35℃×18h，-16℃×2h，20℃×2h，干燥后加塞封盖，-20℃冰箱保存待用。各项检测均采用冻干脂质体PBS复溶液。
扫描电镜见双靶向紫杉醇脂质体呈圆形或类圆形，颗粒大小较一致，粒径＜100nm(见图1)。
实施例3：紫杉醇脂质体体外释放实验
复溶的非主动靶向紫杉醇脂质体、单靶向及双重靶向肿瘤的紫杉醇脂质体及泰素(Taxol)各3ml置分子量14KD透析袋内，袋外加20ml含45g/L牛血清白蛋白的灭菌水模拟体内环境，恒温磁力搅拌器搅拌(300rpm)，于30min、1h、2h、4h、6h、8h、24h分别取出透析液80μL备用，同时透析袋外加回80μL含45g/L牛血清白蛋白的灭菌水。取出的透析液加入320μL色谱级的甲醇溶液沉淀蛋白，漩涡混合后高速离心13000g×10min，取上清液行HPLC检测紫杉醇含量。同时检测透析前复溶的各紫杉醇脂质体及Taxol溶液中紫杉醇的浓度，以(透析液中紫杉醇浓度/原液中紫杉醇浓度)×100％作为衡量缓释能力的指标。
结果发现(见图2)，紫杉醇从CrEL辅剂中释放最快，24h累积释放达15.96％。紫杉醇采用脂质体作为药物载体后，体外的释放速率明显降低，紫杉醇脂质体、单靶向紫杉醇脂质体、双重靶向肿瘤的紫杉醇脂质体24h累积释放分别为4.40％、4.55％、3.6％，缓释作用显著。同时也提示脂质多肽连接于脂质体表面后未破坏脂质体的结构完整性，增强的缓释性能可进一步改善紫杉醇在体内的药代动力学行为。
实施例4：细胞内吞实验
HUVEC、A549细胞接种于24孔板，1×106/孔，置培养箱24h使细胞贴壁，每孔分别加入Taxol、紫杉醇脂质体、单靶向紫杉醇脂质体、双靶向紫杉醇脂质体(以紫杉醇计5μg)，复孔4个，37℃孵育4h，冰PBS液洗涤2次，加细胞裂解液100μL/孔，收集裂解液，加100μL甲醇溶液沉淀蛋白，漩涡混合5min，10000g离心30min，取上清液行HPLC检测细胞内的紫杉醇含量。
Taxol及各紫杉醇脂质体与细胞孵育后，细胞摄取情况见图3。由图可见，A549细胞和HUVEC对传统紫杉醇溶液(Taxol)的摄取量极低，紫杉醇含量达HPLC检测最低限(分别为5ng/孔和25ng/孔)。紫杉醇包封于脂质体后，由于脂质体可通过接触释放、吸附、内吞、融合、脂质交换等多种途径与细胞发生作用，故增加了细胞对紫杉醇的摄取能力。A549细胞和HUVEC对非靶向紫杉醇脂质体的摄取量分别达145ng/孔、157ng/孔。连接肿瘤特异性靶向多肽的紫杉醇脂质体进一步增强了细胞对紫杉醇的摄取能力，其中A549细胞和HUVEC对单靶点紫杉醇脂质体的摄取量分别为212ng/孔和232ng/孔，对双重靶向肿瘤的紫杉醇脂质体的摄取量分别为346ng/孔和319ng/孔。同时上述数据还提示，细胞摄取双重靶向肿瘤的紫杉醇脂质体的能力更强，证实双重靶向肿瘤的新型多肽具有与肿瘤细胞、血管内皮细胞更好的特异性的结合力。
实施例5：细胞毒性试验
A549和HUVEC细胞稀释至4×104/mL，加入96孔板，每孔100μL，置培养箱24h使细胞贴壁；采用无血清的DMEM培养液将Taxol、紫杉醇脂质体、单靶向紫杉醇脂质体、双重靶向肿瘤的紫杉醇脂质体倍比稀释为终浓度0.9375mg/L、1.875mg/L、3.75mg/L、7.5mg/L、15mg/L的工作液，每孔加入工作液100μL，再置细胞培养箱孵育72h；加5mg/mL的MTT液20μl/孔，培养箱中孵育4h，1500rpm离心10min，弃去培养液，各孔分别加入DMSO200μL，脱色摇床混匀30min后上酶标仪，选择波长530nm检测吸光值，计算IC50，公式如下：
图4可见，Taxol对A549细胞、HUVEC中的IC50值分别为6.027mg/L、6.56mg/L；紫杉醇脂质体对两种细胞的IC50值分别为3.37mg/L、4.56mg/L；单靶向紫杉醇脂质体、双重靶向肿瘤的紫杉醇脂质体对两种细胞的IC50值分别为1.46mg/L、1.16mg/L和3.40mg/L、3.25mg/L。结果提示紫杉醇脂质体表面连接血管靶向多肽后增强了对肿瘤细胞和血管内皮细胞的毒性，其中双重靶向肿瘤的紫杉醇脂质体具有最强的细胞毒性。
实施例6：体内抑瘤作用评估
A549肺腺癌细胞常规培养至对数生长期，0.25％胰酶消化成细胞悬液后取1×107/0.1mL接种于4周龄(18-20g)雌性Balb/c裸鼠右腋皮下，瘤体长至50mm3-80mm3起开始给药。裸鼠按给药不同共分5组，每组5只，于d1、d4、d8天分别给予生理盐水、Taxol、紫杉醇脂质体、单靶向紫杉醇脂质体和双重靶向肿瘤的紫杉醇脂质体。其中生理盐水组给予生理盐水0.5mL，余各组给药量按紫杉醇计为7.5mg/kg。给药后监测裸鼠体重(2次/周)、瘤体大小(2次/周)，至d60处死所有裸鼠，剥离瘤体称重，计算肿瘤抑制率。
肿瘤大小计算公式：肿瘤体积V＝ab2/2，其中a为肿瘤最长径和b为肿瘤最短径，肿瘤抑制率计算公式：(1-实验组瘤重)/对照组瘤重×100％。
结果显示(见图5)，给予生理盐水的对照组裸鼠移植瘤生长迅速，d60达1925mm3，平均瘤重为(1.62±0.09g)。给予Taxol及各紫杉醇脂质体组裸鼠瘤体积增长相对缓慢，Taxol组d60移植瘤平均体积为1104mm3，抑瘤率为46.9％；紫杉醇脂质体组d60移植瘤平均体积为912mm3，平均瘤重为(0.61±0.10g)，抑瘤率为62.3％；单靶向紫杉醇脂质体组和双重靶向肿瘤的紫杉醇脂质体组肿瘤平均体积进一步缩小，分别为590mm3和455mm3，平均瘤重分别为(0.44±0.07g)和(0.30±0.04g)，抑瘤率分别为72.8％和81.5％。间接证实双重靶向肿瘤的多肽有效连接于紫杉醇脂质体表面，促进了肿瘤组织对药物载体的摄取，提高了药物在局部的有效浓度，增强了药物的抗肿瘤能力。
Claims (3)
1.一种双重靶向肿瘤的紫杉醇纳米脂质体，其特征在于其主要由双重靶向肿瘤的多肽，脂质连接物和紫杉醇脂质体三部分组成；所述多肽和脂质连接物的结构为ARYCRGDCFDATWLPPR-KGG；紫杉醇脂质体的配方为PC:CHOL:mPEG-DSPE：PTX为9:1:0.5:0.33。
2.权利要求1所述的双重靶向肿瘤的紫杉醇纳米脂质体的制备方法，其特征在于首先合成双重靶向肿瘤的ARYCRGDCFDATWLPPR多肽，然后合成多肽连接物KGG，最后制备双重靶向肿瘤的紫杉醇纳米脂质体；紫杉醇脂质体的配方为PC:CHOL:mPEG-DSPE：PTX为9:1:0.5:0.33。
3.权利要求1所述的双重靶向肿瘤的紫杉醇纳米脂质体在制备抑制肿瘤药物中的应用。
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