CN110755611A - 一种纳米簇载药热敏脂质体制剂及其制法和应用 - Google Patents
一种纳米簇载药热敏脂质体制剂及其制法和应用 Download PDFInfo
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Abstract
本发明公开了一种纳米簇载药热敏脂质体制剂及其制法和应用,该制剂是纳米簇载药热敏脂质体除杂后所得,纳米簇载药热敏脂质体包含热敏性磷脂成分DPPC、促进释放的磷脂成分MPPC、促进制剂长循环的磷脂成分DSPE‑PEG2000及肿瘤治疗药物,通过薄膜分散法制备脂质体,在脂质体表面包裹一层金纳米簇或在脂质体内部包封磁性Fe3O4纳米粒。该制剂能够作为抗肿瘤药物载体,连接抗体,特异性靶向至肿瘤处,通过NIR照射使药物在肿瘤部位高效释放,并联合热疗和放疗增敏作用,提高治疗效果。本制剂还能结合金和铁在电子计算机断层扫描成像(CT)/磁共振成像(MRI)等成像方面的优势,进行医学诊断,达到多模式肿瘤精准诊疗的目的。
Description
技术领域
本发明涉及一种脂质体制剂及其制法和应用,具体为纳米簇载药热敏脂质体制剂及其制法和应用。
背景技术
热敏脂质体(thermosensitive liposomes)的质膜在正常室温下呈致密的胶晶态,包裹在其中的药物难以扩散出来,起到药物储存库的作用;当经过预先加热,温度达到脂质体相变温度时,脂质体膜由胶晶态转变到液晶态,膜的通透性增加,包埋于其中的药物迅速扩散到靶器官中,在靶部位形成较高的药物浓度,从而产生热靶向作用。DPPC因为其相变温度约为41.5℃,更接近人体正常体温,所以成为制备热敏脂质体的最佳选择。热敏脂质体作为一种新型脂质体,增加了脂质体的靶向性,减少了网状内皮系统对脂质体的摄取,从而达到降低毒性和提高药效的目的。
光热疗法(hyperthermia,HT)是通过人为方法提高人体组织温度来治疗肿瘤的一种方法。目前使用金纳米壳,金纳米棒和金纳米笼等光热方法用于实现癌症治疗引起广泛关注。这些金纳米粒子吸收近红外(NIR)区域(700-900nm)的光,因此可以呈现NIR辐射诱导的光热效应。通过设计金纳米粒的形态,可以将它们的LSPR调谐到近红外(NIR)波长。近红外辐射可以穿透身体组织和血液到几毫米的深度,使这些粒子可用于成像和光热治疗。使用脂质体作为模板,表面原位还原生成金纳米簇,形成生物可降解的等离子体共振结构。在NIR照射后,温度敏感型脂质膜被破坏,金壳降解成5-8nm纳米颗粒。亲水性或疏水性药物可以包封在这些金包覆的脂质体内,并且药物可以随着NIR触发而释放,NIR照射后产生的金纳米颗粒可以通过肾清除。
放疗(Radiation Therapy)是利用不同能量的射线治疗,一方面放射线使细胞中的水裂解,产生氧自由基,破坏细胞DNA的化学键,导致细胞凋亡,另一方面能氧化细胞膜脂质双分子层,损伤线粒体,产生附加的氧化应激效应。但高能量的射线在杀死肿瘤细胞的同时会损伤肿瘤周围的正常组织,所以要控制放射剂量。而放射增敏剂的使用有助于获得相对低量高效的放射剂量,有效增加肿瘤局部控制率,提高放疗疗效和治疗增益比。金纳米颗粒在经X射线或γ射线照射后,产生强的光电吸收效应和二次电子,加速DNA链断裂,因此可以作为放疗领域中一类新型的放射增敏剂。
抗体介导的分子靶向疗法(antibody-mediated molecular-targeting therapy)是随着对肿瘤的发生机制和生物特性的深入了解,针对于肿瘤进展的特异性生物途径的新型药物治疗方式称。其中抗体治疗通过提高疗效和降低毒性,在治疗血液系统恶性肿瘤和实体瘤方面取得了临床成功。抗体介导的分子靶向治疗通过将抗体与生物活性分子连接形成抗体-药物偶联物而得到迅速发展。通过在纳米颗粒表面上缀合抗体以有效递送至靶位点(肿瘤)以改善治疗指数并最小化正常组织中的脱靶副作用,因此抗体靶向也已被广泛利用。其中曲妥珠单抗(Trastuzumab),商品名称赫赛汀(Herceptin),于1998年在美国FDA获准上市,是一种针对乳腺癌HER2靶点的靶向治疗药物,在早期和晚期(转移性)乳腺癌的治疗中均显示出疗效,其优势在于保留了HER2受体的高亲和力,同时解决了鼠源单克隆抗体对人体的免疫源性问题。
医学影像技术(Medical imaging technology)在疾病治疗过程中扮演者重要角色,如疾病的早期诊断,手术过程中的实时成像以及治疗后的效果跟踪等,这些技术都可以大大提高疾病的治愈率,但由于每种成像方式自身的局限性,单一的成像模式不能完全提供肿瘤的形态和功能信息。所以发展多模态成像技术可以充分发挥各个成像模式的长处,大大提高肿瘤诊断的精度。其中CT成像技术用途广泛,是目前临床常用的诊断手段之一,常用于解剖学成像、骨成像和肿瘤成像。但它最大的缺点是软组织对比度较低。MRI也是目前广泛应用于临床的无创成像手段,它的软组织对比度高,具有较大的穿透深度,但价格昂贵,成像时间较长。光声成像(PAI)虽然穿透能力有限,但灵敏度高,可以对局部进行微细结构的成像,并且与对人体有害的X射线相比,其安全性更好。综上所述,CT、MRI及PAI三者各有优劣,功能互补,将这两种成像结合起来,将具有较高的医学诊断应用价值。
发明内容
发明目的:本发明的目的是提供一种纳米簇载药热敏脂质体制剂。本发明另一目的是提供该制剂的制备方法。本发明还有一目的是指出该制剂可以作为良好的抗肿瘤药物载体,并且可以应用于多模式热敏免疫精准诊疗肿瘤。
技术方案:本发明所述纳米簇载药热敏脂质体制剂,该制剂是纳米簇载药热敏脂质体除杂后所得,纳米簇载药热敏脂质体是在载药热敏脂质体表面包裹一层金纳米簇或在载药热敏脂质体内部包封Fe3O4纳米粒,前者为金纳米簇载药热敏脂质体,后者为磁性纳米簇载药热敏脂质体;其中,载药热敏脂质体是由二棕榈酰磷脂酰胆碱DPPC、1-肉豆蔻酰基-2-棕榈酰基卵磷脂MPPC、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000DSPE-PEG2000或二棕榈酰磷脂酰乙醇胺-聚乙二醇-羧基DSPE-PEG2000-COOH或二棕榈酰磷脂酰乙醇胺-聚乙二醇-巯基DSPE-PEG2000-SH以及肿瘤治疗药物,通过薄膜分散法制备得到。
所述的纳米簇载药热敏脂质体制剂,所述载药热敏脂质体的粒径为80~150nm,电位为-20~-40mv;纳米簇载药热敏脂质体粒径为80~150nm,电位0~±10mv,相转变温度均为40℃~50℃,金纳米簇载药热敏脂质体紫外最大吸收波长在700-900nm。
所述的纳米簇载药热敏脂质体制剂,所述肿瘤治疗药物为选自小分子化学治疗剂、无机金属类治疗药物、蛋白治疗药物或基因治疗药物的肿瘤治疗剂。
所述的纳米簇载药热敏脂质体制剂,所述的小分子化学治疗剂为环巴胺、盐酸阿霉素,所述的无机金属类治疗药物为四氧化三铁纳米粒,蛋白治疗药物为曲妥珠单抗HER2。
所述的纳米簇载药热敏脂质体制剂的制备方法,金纳米簇载药热敏脂质体的制备包括以下步骤:
(1)分别称取DPPC 78.5-80.5mg、DSPE-PEG2000 13.0-15.0mg、MPPC 6.0-7.0mg、肿瘤治疗药物4.0-5.0mg,溶于5-10ml氯仿中,于35℃-45℃旋转蒸发除去有机溶剂制备磷脂膜,成膜后真空过夜,于45-55℃水化,将粗体脂质体超声3-10min,超声1s间隔2s制备载药热敏脂质体;
(2)分别取步骤(1)中制备的空白脂质体1-1.5ml,然后加入浓度为90-110nM氯金酸20-30μl轻轻旋转至混合均匀,并按照氯金酸体积的1.2-1.8倍量加入浓度为400-600nM的还原剂抗坏血酸30-45μl直至颜色由透明白色发生变化,呈现深蓝色或者墨绿色,得到金纳米簇载药热敏脂质体;
(3)取步骤(2)中制备的金纳米簇脂质体,使用透析法或者使用截留分子量3KD的超滤离心管,以离心转速5000-6000rpm,离心30-40min,除去未被包载的肿瘤治疗药物及游离的小颗粒金纳米粒,得到金纳米簇载药热敏脂质体制剂。
上述所述的纳米簇载药热敏脂质体制剂的制备方法,制备纳米簇载药热敏免疫脂质体,用DSPE-PEG2000-COOH或DSPE-PEG2000-SH替换DSPE-PEG2000制备载药热敏脂质体,然后取具有活性基团的载药热敏脂质体加入活化溶液,在室温条件下反应20-30min活化,分别以DSPE-PEG2000-COOH或DSPE-PEG2000-SH与HER2摩尔比为1:100–1:200进行反应,通过化学键或酶敏感键连接得到。
所述的纳米簇载药热敏脂质体制剂的制备方法,磁性纳米簇载药热敏脂质体的制备包括以下步骤:
(1)称取FeCl3·6H2O 21-22g,FeCl2·4H2O 8-9g溶于40-50ml去离子水中,并量取5-6ml油酸于其中,利用电动搅拌器将其混合均匀,并将混合溶液加热至75-85℃,当混合体系充分混匀后,加入浓度为22-28%的氨水溶液45-55mL,反应15-20min之后将整个体系加热至95-100℃,并持续搅拌反应一段时间后将混合溶液取出,用去离子水和无水乙醇分别清洗,即得Fe3O4纳米粒;
(2)按重量比称取磷脂DPPC:DSPE-PEG2000:MPPC=79.5:14:6.5,将其分散在氯仿中,并使用旋转蒸发器在真空下干燥到圆底烧瓶上,于50-55℃下用硫酸铵溶液水化薄膜,得到空白脂质体粗体,然后根据硫酸铵梯度法将盐酸阿霉素溶液DOX按照质量比DOX:脂质=1:20加入到空白脂质体溶液中,50-60℃下搅拌孵育25-30min,然后加入1-1.5ml的Fe3O4纳米粒,在35-45℃下继续搅拌半个小时;然后利用脂质体挤出器,将粗体脂质体用孔径100nm-200nm的聚碳酸酯膜挤出20-25次,使其形成均匀的磁性纳米簇载药热敏脂质体;
(3)取步骤(2)中制备的磁性纳米簇载药热敏脂质体,使用G-25葡聚糖凝胶柱,用磷酸盐缓冲液作为洗脱液,以流速0.8-1.2ml/min对磁性纳米簇载药热敏脂质体进行洗脱,除去未被包载的肿瘤治疗药物及游离的小颗粒铁纳米粒,得到磁性纳米簇载药热敏脂质体制剂。
所述的纳米簇载药热敏脂质体制剂在制备治疗乳腺癌恶性肿瘤药物中的应用。
所述的纳米簇载药热敏脂质体制剂在联合放疗、光疗、热疗诊断试剂中的应用。
所述的纳米簇载药热敏脂质体制剂在电子计算机断层扫描成像CT/磁共振成像MRI成像诊断试剂中的应用。
构建的金纳米簇载药热敏免疫脂质体在NIR照射下,热敏脂质体温度升高至相转变温度后脂质体破裂,导致脂质体表面的金纳米簇裂解成小尺寸的金纳米粒子,选择性地在肿瘤微环境实现NPs的大小和形态转变,能够结合大颗粒的药物动力学、肿瘤富集能力强和小颗粒的穿透能力强的特点,通过制剂手段提高纳米颗粒自身的组织渗透性,深入肿瘤内部治疗。
本发明所设计的具有良好生物相容性和降解能力的等离子体共振纳米结构即金纳米簇载药热敏脂质体制剂,在光照条件下,不仅能促使脂质体控制释放药物,且光照后降解产生的小粒径金纳米粒易被肾清除,具有较小的金属蓄积毒性,这种将化疗,光疗和放疗相结合的多模式协同治疗的策略,能进一步提高肿瘤治疗效果。
本发明采取不同的化学键或对组织蛋白酶敏感的肽键将抗体HER2和金纳米簇载药热敏脂质体进行连接。由于HER2抗体的有效连接,能选择性靶向乳腺癌组织。本发明设计的通过酶敏感键将抗体与脂质体相连的金纳米簇免疫脂质体制剂,能够在肿瘤细胞内组织蛋白酶B的作用下释放抗体和脂质体,能到靶向控制药物释放的优势,既能增强在肿瘤内部的渗透又可协同抗体药物和载体药物联合治疗的协同治疗作用,有利于纳米载体在体内的代谢以提高载体在体内应用的安全性。
本发明构建的金/磁纳米簇载体可通过四氧化三铁纳米粒的磁性用于MRI成像;利用胶体金较强的X射线吸收作用、良好的表面等离子体共振(SPR)效应以及较高的光学吸收系数,用于CT成像及PAI成像,从而通过金磁纳米簇的MRI/CT/PAI多模态成像作用实现对乳腺癌等浅表性恶性肿瘤的精密诊断。
有益效果:本发明构建的纳米簇载药热敏免疫脂质体,集放疗、放疗、热疗效果于一体且在体内能实现降解代谢的可视化靶向肿瘤金/磁纳米载体诊疗系统。本制剂不仅能促进渗透,提高对肿瘤靶向性治疗效果并能降低单一手段治疗的毒副作用,具有较高的医学诊疗应用价值。该制剂能够作为良好的抗肿瘤药物载体,稳定的抗体的连接能特异性靶向至肿瘤处,通过NIR照射使药物在肿瘤部位高效释放,并联合热疗和放疗增敏作用,提高治疗效果。
附图说明
图1为本发明中热敏脂质体粒径及电位图(左a,左c)及金纳米簇热敏脂质体的粒径电位图(右b,右d);
图2为热敏脂质体(a)及金纳米簇热敏脂质体(b)的透射电镜图;
图3为金纳米簇热敏脂质体的紫外全波长扫面图;
图4为热敏脂质体(a)及金纳米簇热敏脂质体(b)的相转变温度图;
图5为Fe3O4磁性纳米粒及磁性纳米簇热敏脂质体的透射电镜图;
图6为金纳米簇热敏脂质体光热转化升温曲线(TSL为空白热敏脂质体;GTSL为金纳米热敏脂质体);
图7为磁性纳米簇热敏脂质体光热转化升温曲线(TSL为空白热敏脂质体;Fe3O4–TSL为磁性纳米簇热敏脂质体);
图8为热敏脂质体及金纳米簇热敏脂质体在有/无NIR照射下的体外释放曲线(CYC为游离环巴胺药物;CYC-TSL为为载环巴胺的热敏脂质体;CYC-TSL@NIR为载环巴胺的热敏脂质体经近红外光(NIR)照射组;CYC-GTSL为载环巴胺的金纳米簇热敏脂质体;CYC-GTSL@NIR为载环巴胺的金纳米簇热敏脂质体经近红外(NIR)光照射组);
图9为热敏脂质体及磁性热敏脂质体在有/无NIR照射下的体外释放曲线(DOX为盐酸阿霉素;DOX–TSL为载阿霉素的热敏脂质体;DOX@Fe3O4-TSL为载阿霉素的磁性纳米簇热敏脂质体;DOX@Fe3O4-TSL+NIR(1)为载阿霉素的磁性纳米簇热敏脂质体经近红外光(NIR)照射组(1);DOX@Fe3O4-TSL+NIR(2)为载阿霉素的磁性纳米簇热敏脂质体经近红外光(NIR)照射组(2));
图10为肿瘤治疗剂游离药物环巴胺在MCF-7细胞的存活率;
图11为热敏脂质体及金纳米簇热敏脂质体在MCF-7细胞的存活率(TSL为空白热敏脂质体溶液;CYC-TSL为载环巴胺热敏脂质体溶液;GTSL为金纳米簇热敏脂质体溶液;CYC-GTSL为载环巴胺的金纳米簇热敏脂质体溶液);
图12为Fe3O4磁性纳米粒、热敏脂质体及磁性热敏脂质体在MCF-7细胞的存活率(Fe3O4为四氧化三铁;Fe3O4–TSL为磁性纳米簇热敏脂质体;Fe3O4–TSL+NIR为经NIR照射的磁性纳米簇热敏脂质体;DOX@Fe3O4–TSL+NIR为经NIR照射的载阿霉素磁性纳米簇热敏脂质体);
图13为放疗后金纳米簇脂质体对DNA的损伤测定;
图14为金纳米簇脂质体的体外CT成像;
图15为金纳米簇脂质体的体内CT成像;
图16为磁性纳米簇热敏脂质体的体外MRI成像。
具体实施方式
实施例1:空白热敏脂质体TSL及金纳米簇热敏脂质体GTSL的制备与表征
按处方量准确称量DPPC:DSPE-PEG2000:MPPC=79.5:14:6.5于500mL茄形瓶中,加入5mL氯仿,振摇溶解磷脂;35℃旋转蒸发,除去有机溶剂制膜,成膜后继续抽真空2h,使有机溶剂完全除净。然后于55℃下用生理盐水水化,得到热敏脂质体粗体,再通过探头超声,超声5min,功率30%、超声1s间隔2s,直至脂质体透明澄清,然后将上述超声后的脂质体过0.22um的有机滤膜约5次,得到空白热敏脂质体。
脂质体表面上的金涂层通过等离子体共振涂覆方法合成。分别取制备的空白脂质体1ml(10mg/ml),然后各加入24μL的氯金酸(100nM)轻轻旋转至混合均匀,并按照氯金酸体积的1.5倍量加入还原剂抗坏血酸(500nM)36μL直至颜色由透明白色发生变化,呈现深蓝色或者墨绿色,得到金纳米簇热敏脂质体。
取热敏脂质体经稀释为浓度1mg/mL时,使用ZetaPlus激光粒度分析仪测定其粒径及电位。
取制备好的热敏脂质体溶液适量,将覆盖碳膜的铜网完全润湿30s,然后用滤纸吸去多余的液体,在灯光下干燥约3min,用透射电镜观察的形态及粒径大小。
取热敏脂质体经稀释为浓度1mg/mL时,使用紫外-可见光分光光度计在400-1000nm范围内扫描其紫外光谱。结果如图1、图2、图3所示。
实施例2:热敏脂质体TSL及金纳米簇热敏脂质体GTSL相转变温度表征
分别用取热敏脂质体,金纳米簇热敏脂质体置于热分析坩埚中,分别用差示扫描仪分析测定,温度扫描范围:25℃-100℃,温度上升速率为10℃/min,分析其相转变温度。结果如图4所示。
实施例3:磁性Fe3O4纳米粒及磁性纳米簇热敏脂质体Fe3O4-TSL的制备和表征
称取FeCl3·6H2O 21.6165g、FeCl2·4H2O 8.7490g溶于50ml去离子水中,并量取5.5ml油酸于其中,利用电动搅拌器将其混合均匀,并将混合溶液加热至80℃,当混合体系充分混匀后,加入浓度为25%的氨水溶液50mL,反应15min之后将整个体系加热至100℃,并持续搅拌反应1.5h后将混合溶液取出,用去离子水清洗3次,无水乙醇清洗2次即得Fe3O4纳米粒。
将磷脂DPPC:DSPE-PEG2000:MPPC=79.5:14:6.5(按重量比)分散在氯仿中,并使用旋转蒸发器在真空下干燥到圆底烧瓶上。然后加入pH 4.0硫酸铵(250mM)溶液水化薄膜,得到热敏脂质体粗体,再通过探头超声,超声5min,功率30%、超声1s间隔2s,直至脂质体透明澄清,得到空白热敏脂质体。将1ml(5mg/ml)的Fe3O4纳米溶液加入载药热敏脂质体中,在40℃下继续搅拌半个小时,然后利用脂质体挤出器,趁热将载药热敏脂质体用孔径100nm-200nm的聚碳酸酯膜挤出20次,使其形成均匀的磁性纳米簇热敏脂质体Fe3O4-TSL。
取制备好的磁性Fe3O4纳米粒及磁性纳米簇热敏脂质体溶液适量,将覆盖碳膜的铜网完全润湿30s,然后用滤纸吸去多余的液体,在灯光下干燥约3min,用透射电镜观察的形态及粒径大小。如图5所示。
实施例4:金纳米簇热敏脂质体GTSL光热转化升温曲线
取空白热敏脂质体(10mg/ml)及脂质浓度分别为1mg/ml和10mg/ml的金纳米簇热敏脂质体各1ml于比色皿中,将红外激光仪固定,使其光纤离样品溶液约2cm处进行照射,并以生理盐水作为对照。808nm激光以照射功率定为3w/cm2,照射时间为10min,红外测温枪每隔1min检测一次温度,实时监测溶液的温度变化。结果如图6所示。
实施例5:磁性纳米簇热敏脂质体Fe3O4-TSL光热转化升温曲线
将Fe3O4磁性纳米簇热敏脂质体配制成50μg/mL(浓度以Fe3O4计)的水溶液,取1mL样品加入到2mL的EP管中。之后,采用808nm NIR激光对其进行照射(3W/cm2,10min),每隔1min用红外热像仪对其进行光热成像拍照。另外,为了研究Fe3O4浓度与光热升温效果之间的关系,将Fe3O4磁性纳米簇热敏脂质体配制成一系列不同浓度(25、50、100、200、500及1000μg/mL,浓度以Fe3O4计)的水溶液,保持实验条件不变,采用电热偶每隔1min测定样品溶液的温度并记录,绘制升温曲线。同样的光照条件下,采用生理盐水作为空白对照。结果如图7所示。
实施例6:热敏脂质体TSL及金纳米簇热敏脂质体GTSL在有/无近红外NIR照射下的体外释放曲线
按H2O:PEG2000摩尔比为10:1配制释放介质。称取1mg环巴胺溶解于上述释放介质中,作为游离药物。实验方法:分别取环巴胺溶液CYC、载环巴胺的热敏脂质体CYC-TSL、在近红外照射下的载环巴胺的热敏脂质体CYC-TSL@NIR、载环巴胺的金纳米簇热敏脂质体CYC-GTSL、在近红外照射下的载环巴胺的金纳米簇热敏脂质体CYC-GTSL@NIR于透析袋中与等体积的释放介质混合,在37℃,转速100rpm进行释放。其中CYC-TSL@NIR和CYC-GTSL@NIR分别在特定的点(0、2、4、6h)从离心管中取出,进行NIR照射,设置功率3W持续照射5min,然后继续考察体外释放行为。上述所有样品均在在系列时间点0.5、1、2、4、6、8、12、24、36、48h取1ml小时取出1ml,按上述方法检测环巴胺浓度,记为Cn,以时间点为横坐标,释放率为纵坐标,绘制累计释放量。结果如图8所示。
实施例7:磁性纳米簇热敏脂质体Fe3O4-TSL光热转化升温曲线
取载阿霉素的磁性纳米簇热敏脂质体DOX@Fe3O4-TSL溶液1ml,设置功率3W持续照射5min,置于透析袋中,透析液10ml,然后将容器放置于37℃水浴,转速100rpm进行释放。在不同时间点(0.5、1、2、4、6、12、24和48h)取1ml透析液测定其480nm处吸光度值,每次取样结束后各补液1ml,继续释放,作为DOX@Fe3O4-TSL+NIR(1)溶液。另取DOX@Fe3O4-TSL溶液1ml在释放6h后,从透析袋中取出样品溶液,再次进行NIR光照5min后放回透析袋中继续释放。分别在不同的时间点取样分析,作为DOX@Fe3O4-TSL+NIR(2)溶液。
对照组:DOX(盐酸阿霉素)、DOX-TSL(载阿霉素的热敏脂质体)及DOX&Fe3O4-TSL(载阿霉素的磁性纳米簇热敏脂质体)溶液各1ml,在同样条件下释放48小时,以时间点为横坐标,释放率为纵坐标,绘制累计释放量。结果如图9所示。
实施例8:金纳米簇载药热敏脂质体的制备
按处方量准确称量DPPC:DSPE-PEG2000:MPPC:环巴胺=79.5:14:6.5:4于500mL茄形瓶中,加入5mL氯仿,振摇溶解磷脂;35℃旋转蒸发,除去有机溶剂制膜,成膜后继续抽真空2h,使有机溶剂完全除净。然后于55℃下用生理盐水水化,得到热敏脂质体粗体,再通过探头超声,超声5min,功率30%、超声1s间隔2s,直至脂质体透明澄清,然后将上述超声后的脂质体过0.22μm的有机滤膜约5次,得到载药热敏脂质体CYC-TSL。
脂质体表面上的金涂层通过等离子体共振涂覆方法合成。分别取制备的空白脂质体1ml(10mg/ml),然后各加入24μL的氯金酸(100nM)轻轻旋转至混合均匀,并按照氯金酸体积的1.5倍量加入还原剂抗坏血酸(500nM)36μL直至颜色由透明白色发生变化,呈现深蓝色或者墨绿色,得到金纳米簇载药热敏脂质体CYC-GTSL。
取该脂质体经稀释为浓度1mg/mL时,使用ZetaPlus激光粒度分析仪测定其粒径及电位。
取制备好的该脂质体溶液适量,将覆盖碳膜的铜网完全润湿30s,然后用滤纸吸去多余的液体,在灯光下干燥约3min,用透射电镜观察的形态及粒径大小。
取该脂质体经稀释为浓度1mg/mL时,使用紫外-可见光分光光度计在400-1000nm范围内扫描其紫外光谱。粒径、电位和紫外结果与实施例1相同。
实施例9:磁性纳米簇载药热敏脂质体的制备
按处方量准确称量DPPC:DSPE-PEG2000:MPPC=79.5:14:6.5于500mL茄形瓶中,加入5mL氯仿,振摇溶解磷脂;35℃旋转蒸发,除去有机溶剂制膜,成膜后继续抽真空2h,使有机溶剂完全除净。然后于55℃下用pH 4.0硫酸铵(250mM)溶液水化薄膜,得到热敏脂质体粗体,再通过探头超声,超声5min,功率30%、超声1s间隔2s,直至脂质体透明澄清,得到空白热敏脂质体。通过透析法,除去游离的硫酸铵,然后根据硫酸铵梯度法将盐酸阿霉素溶液(DOX,2mg/ml),按照质量比1:20(DOX:脂质)加入到空白脂质体溶液中,在50℃下搅拌孵育30min,然后通过G-25葡聚糖凝胶柱,用PBS洗脱,分离载药热敏脂质体和游离药物。
将1ml(5mg/ml)的Fe3O4纳米溶液加入载药热敏脂质体中,在40℃下继续搅拌半个小时,然后利用脂质体挤出器,趁热将载药热敏脂质体用孔径100nm-200nm的聚碳酸酯膜挤出20次,使其形成均匀的磁性纳米簇载药热敏脂质体。
取该脂质体经稀释为浓度1mg/mL时,使用ZetaPlus激光粒度分析仪测定其粒径及电位。粒径、电位结果与实施例1相同。
取制备好的该脂质体溶液适量,将覆盖碳膜的铜网完全润湿30s,然后用滤纸吸去多余的液体,在灯光下干燥约3min,用透射电镜观察的形态及粒径大小,结果与实施例5相同。
实施例10:酰胺键连接抗体和脂质体金纳米簇载药热敏免疫脂质体的制备
首先按处方量制备载药热敏脂质体,以DSPE-PEG2000-COOH和EDC/NHS摩尔比为1:1.1投料来活化磷脂上的羧基基团。经计算,准确称取38mg EDC和63.25mg NHS(按摩尔比EDC:NHS=1:1)于5ml容量瓶中,加Mes缓冲盐(pH 5.5)稀释定容至刻度,配制EDC/NHS活化溶液。取1ml上述具有活性基团的热敏脂质体,加入EDC/NHS活化溶液10μl,在室温条件下反应30min,活化脂质体。然后分别以DSPE-PEG2000-COOH与HER2摩尔比为1:200进行反应,制备酰胺键连接抗体。然后各加入24μL的氯金酸(100nM)轻轻旋转至混合均匀,并按照氯金酸体积的1.5倍量加入还原剂抗坏血酸(500nM)36μL直至颜色由透明白色发生变化,呈现深蓝色或者墨绿色,得到金纳米簇载药热敏免疫脂质体。金纳米簇热敏免疫脂质体的制备,除了首先按照处方量制备热敏脂质体,其他步骤相同。
实施例11:二硫键连接抗体和脂质体的金纳米簇载药热敏免疫脂质体的制备
首先按处方量制备载药热敏脂质体,然后将HER2溶于HEPES缓冲盐水(HBS,25mMHEPES,140mM NaCl,pH 8.0)中(10mg/mL),用Traut试剂:赫赛汀=20:1(mol/mol)的比例对抗体进行硫醇化,将巯基化的抗体移植到PEG化脂质体的表面:将活化的抗体与DSPE-PEG-SH中的巯基反应,在4℃下温育16-18h生成二硫键进行连接,得到二硫键连接抗体。同实施例10,进一步制备金纳米簇载药热敏免疫脂质体。金纳米簇热敏免疫脂质体的制备,除了首先按照处方量制备热敏脂质体,其他步骤相同。
实施例12:酶敏感键连接抗体和脂质体的金纳米簇载药热敏免疫脂质体的制备
首先按处方量制备载药热敏脂质体,将HER2溶于PBS中,以摩尔比1:1加入MC-Val-Cit-PABC-PNP(VC)二肽,室温搅拌24小时后,加入适量脂质体,以DSPE-PEG2000-SH:HER2=1:100(摩尔比)室温搅拌24小时后,得到酶敏感键连接抗体。同实施例10,进一步制备金纳米簇载药热敏免疫脂质体。金纳米簇热敏免疫脂质体的制备,除了首先按照处方量制备热敏脂质体,其他步骤相同。
实施例13:肿瘤治疗剂游离药物环巴胺在MCF-7细胞的存活率
取环巴胺甲醇储备液(1mg/ml)1ml于15ml的离心管,加入基础培养基9ml,配制含100μg/ml药物的培养基。取7个4ml的离心管,分别吸取1.6、1.2、0.8、0.4、0.2、0.1、0.02ml100μg/ml药物的培养基于离心管中,再加入0.4、0.8、1.2、1.6、1.8、1.9、及1.98ml的基础培养基,配制药物浓度分别为80、60、40、20、10、5、1μg/ml药物的培养基各2ml。
制备MCF-7乳腺癌肿瘤细胞悬液(含有细胞5-10×104/ml),每孔加入100μl,边缘孔用无菌PBS填充,设置调零孔(培养基、MTT、二甲基亚砜)和对照孔(细胞、培养液、MTT、二甲基亚砜)。5%CO2,37℃孵育24h后,将96孔板中的培养上清去掉,再加入100ul含不同浓度药物的培养基。5%CO2,37℃孵育分别孵育24h,每孔加入10μl MTT溶液(5mg/ml,即0.5%MTT),继续培养4h。终止培养,准备溶解结晶。每孔加入150μl二甲基亚砜,置摇床上低速振荡10min,使结晶物充分溶解。在酶联免疫检测仪OD 570nm处测量各孔的吸光值。实验结果如图10所示。
实施例14:热敏脂质体及金纳米簇热敏脂质体等在MCF-7细胞的存活率
分别将空白热敏脂质体TSL溶液、载药热敏脂质体CYC-TSL溶液、金纳米簇热敏脂质体GTSL溶液、金纳米簇载药热敏脂质体CYC-GTSL溶液用基础培养基稀释成浓度分别为5、2、1、0.5mg/ml药物培养基溶液。
制备MCF-7乳腺癌肿瘤细胞悬液(含有细胞5-10×104/ml),每孔加入100μl,边缘孔用无菌PBS填充,设置调零孔(培养基、MTT、二甲基亚砜)和对照孔(细胞、培养液、MTT、二甲基亚砜)。5%CO2,37℃孵育24h后,将96孔板中的培养上清去掉,再加入100μl含不同浓度药物的培养基。5%CO2,37℃孵育分别孵育24h,每孔加入10μl MTT溶液(5mg/ml,即0.5%MTT),继续培养4h。终止培养,准备溶解结晶。每孔加入150μl二甲基亚砜,置摇床上低速振荡10min,使结晶物充分溶解。在酶联免疫检测仪OD 570nm处测量各孔的吸光值。实验结果如图11所示。
实施例15:Fe3O4纳米粒、磁性纳米簇热敏脂质体及磁性纳米簇载药热敏脂质体在MCF-7细胞的存活率
将Fe3O4溶液、磁性纳米簇热敏脂质体Fe3O4-TSL及磁性纳米簇载药热敏脂质体DOX@Fe3O4-TSL分别用基础培养基稀释成浓度分别为10、20、50、100、200、500及1000μg/ml(以Fe3O4的浓度计)的药物培养基溶液。制备MCF-7乳腺癌肿瘤细胞悬液(含有细胞5-10×104/ml),每孔加入100μl,边缘孔用无菌PBS填充,37℃孵育24h后,将96孔板中的培养上清去掉,再加入100μl不同浓度的Fe3O4溶液、磁性纳米簇热敏脂质体Fe3O4-TSL药物培养基溶液培养。另取不同浓度的磁性纳米簇热敏脂质体Fe3O4-TSL和磁性纳米簇载药热敏脂质体DOX@Fe3O4-TSL溶液,加入到96孔板后立即用NIR进行照射,设置功率3W持续照射5min,分别作为Fe3O4-TSL+NIR和DOX@Fe3O4-TSL+NIR组。设置调零孔(培养基、MTT、二甲基亚砜)和对照孔(细胞、培养液、MTT、二甲基亚砜)。5%CO2,37℃孵育分别孵育24h,每孔加入10μl MTT溶液(5mg/ml,即0.5%MTT),继续培养4h。终止培养,准备溶解结晶。每孔加入150μl二甲基亚砜,置摇床上低速振荡10min,使结晶物充分溶解。在酶联免疫检测仪OD 570nm处测量各孔的吸光值。实验结果如图12所示。
实施例16:放疗后金纳米簇热敏脂质体等对DNA的损伤测定
取SK-BR-3细胞进行消化并离心收集。在6孔板中加入2mL的含细胞培养基,细胞浓度为5*105个/孔。将6孔板置于37℃的细胞培养箱内培养24h,待细胞贴壁。移除旧培养基,加入制备好的经强还原剂还原HAuCl4得到的金颗粒悬浮液即Au种子液、金纳米簇热敏脂质体GTSL和金纳米簇热敏免疫脂质体GTSL@HER2溶液并培养12小时。采用20-30Gy照射,照射完成以后,再继续培养12h。用4%多聚甲醛固定细胞。PBS冲洗2次,在20℃下用甲醇作用15min。然后,用PBS冲洗,使山羊血清工作液(PBS溶液中1%BSA)室温下封闭1h。加入按1:500稀释的鼠抗γ-H2AX抗体,在湿盒内4℃过夜;使用PBS冲洗10min x 4次,加入按照1:500稀释的山羊抗鼠IgG二抗再室温下放置1h,再次使用PBS冲洗10min x 4次;使用4,6-二脒基-2-苯基吲哚(DAPI)复染10min,再使用PBS冲洗10min x 2次,使用90%甘油封片,在荧光显微镜下观察。实验结果如图13所示。以上结果说明在X ray的照射下,GTSL和GTSL@HER2能够加速细胞DNA的损伤。金纳米簇载药热敏脂质体和金纳米簇载药热敏免疫脂质体也能够加速细胞DNA的损伤。
实施例17:金纳米簇热敏脂质体等的体外CT成像
取金纳米簇热敏脂质体GTSL及金纳米簇热敏免疫脂质体GTSL+HER2溶液适量,分别稀释得到金浓度为0.004M、0.008M、0.013M、0.021M、0.031M、0.042M和0.064M三种纳米溶液,并将其装入到200μL的微量离心管中。以相同摩尔浓度梯度的碘海醇溶液作为对比进行CT扫描,测量各溶液的CT信号值并作图。扫描参数:X-射线管电压为60k V,电流为133μA,单次曝光时间为50ms。实验结果如图14所示。金纳米簇载药热敏脂质体的体外CT成像结果与上述一样。
实施例18:金纳米簇热敏脂质体等的体内CT成像
取对数生长期的SK-BR-3细胞,在无菌条件下制备成1×107/mL细胞悬液,以0.1mL,分别接种于Balb/c裸鼠的前肢腋下及第四对乳腺脂肪垫皮下分别得到异位及原位乳腺癌鼠模型,待肿瘤体积长到100-200mm3,分为5组,每组2只,分别尾静脉注射0.2mL碘海醇,Au种子液、金纳米簇热敏脂质体GTSL以及金纳米簇热敏免疫脂质体GTSL-HER2,分别于注射前及注射后2h、4h及12h置于小动物PET/CT上进行扫描,仪器参数设置为:管电压80kV,电流强度500μA,单次曝光时间250ms。实验结果如图15所示。金纳米簇载药热敏脂质体的体内CT成像结果与上述一样。
实施例19:磁性纳米簇热敏脂质体Fe3O4-TSL的体外MRI成像
用0.55T MesoMR23-060H-I系统进行磁共振成像(MRI)。将样品在水中稀释,铁(Fe)浓度为0、0.04、0.08、0.02、0.4、0.8mM。T2加权MRI的仪器参数如下:点分辨率为256mm×192mm,切片厚度为5.0mm,重复时间(TR)为2800ms,回波时间(TE)为60ms,数量为通过作为Fe浓度(mM)的函数的反向弛豫时间(1/T2)的线性拟合来计算T2弛豫率的激发。通过电感耦合等离子体原子发射光谱法(ICP-AES)(Leeman Prodigy,NH)测定溶液中Fe的浓度。实验结果如图16所示。磁性纳米簇载药热敏脂质体的体外MRI成像结果与上述一样。
Claims (10)
1.一种纳米簇载药热敏脂质体制剂,其特征在于该制剂是纳米簇载药热敏脂质体除杂后所得,纳米簇载药热敏脂质体是在载药热敏脂质体表面包裹一层金纳米簇或在载药热敏脂质体内部包封Fe3O4纳米粒,前者为金纳米簇载药热敏脂质体,后者为磁性纳米簇载药热敏脂质体;其中,载药热敏脂质体是由二棕榈酰磷脂酰胆碱DPPC、1-肉豆蔻酰基-2-棕榈酰基卵磷脂MPPC、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000DSPE-PEG2000或二棕榈酰磷脂酰乙醇胺-聚乙二醇-羧基DSPE-PEG2000-COOH或二棕榈酰磷脂酰乙醇胺-聚乙二醇-巯基DSPE-PEG2000-SH以及肿瘤治疗药物,通过薄膜分散法制备得到。
2.根据权利要求1所述的纳米簇载药热敏脂质体制剂,其特征在于所述载药热敏脂质体的粒径为80~150nm,电位为-20~-40mv;纳米簇载药热敏脂质体粒径为80~150nm,电位0~±10mv,相转变温度均为40℃~50℃,金纳米簇载药热敏脂质体紫外最大吸收波长在700-900nm。
3.根据权利要求1所述的纳米簇载药热敏脂质体制剂,其特征在于所述肿瘤治疗药物为选自小分子化学治疗剂、无机金属类治疗药物、蛋白治疗药物或基因治疗药物的肿瘤治疗剂。
4.根据权利要求3所述的纳米簇载药热敏脂质体制剂,其特征在于所述的小分子化学治疗剂为环巴胺、盐酸阿霉素,所述的无机金属类治疗药物为四氧化三铁纳米粒,蛋白治疗药物为曲妥珠单抗HER2。
5.一种如权利要求1所述的纳米簇载药热敏脂质体制剂的制备方法,其特征在于金纳米簇载药热敏脂质体的制备包括以下步骤:
(1)分别称取DPPC 78.5-80.5mg、DSPE-PEG2000 13.0-15.0mg、MPPC 6.0-7.0mg、肿瘤治疗药物4.0-5.0mg,溶于5-10ml氯仿中,于35℃-45℃旋转蒸发除去有机溶剂制备磷脂膜,成膜后真空过夜,于45-55℃水化,将粗体脂质体超声3-10min,超声1s间隔2s制备载药热敏脂质体;
(2)分别取步骤(1)中制备的空白脂质体1-1.5ml,然后加入浓度为90-110nM氯金酸20-30μl轻轻旋转至混合均匀,并按照氯金酸体积的1.2-1.8倍量加入浓度为400-600nM的还原剂抗坏血酸30-45μl直至颜色由透明白色发生变化,呈现深蓝色或者墨绿色,得到金纳米簇载药热敏脂质体;
(3)取步骤(2)中制备的金纳米簇脂质体,使用透析法或者使用截留分子量3KD的超滤离心管,以离心转速5000-6000rpm,离心30-40min,除去未被包载的肿瘤治疗药物及游离的小颗粒金纳米粒,得到金纳米簇载药热敏脂质体制剂。
6.根据权利要求5所述的纳米簇载药热敏脂质体制剂的制备方法,其特征在于制备纳米簇载药热敏免疫脂质体,用DSPE-PEG2000-COOH或DSPE-PEG2000-SH替换DSPE-PEG2000制备载药热敏脂质体,然后取具有活性基团的载药热敏脂质体加入活化溶液,在室温条件下反应20-30min活化,分别以DSPE-PEG2000-COOH或DSPE-PEG2000-SH与HER2摩尔比为1:100–1:200进行反应,通过化学键或酶敏感键连接得到。
7.一种如权利要求1所述的纳米簇载药热敏脂质体制剂的制备方法,其特征在于磁性纳米簇载药热敏脂质体的制备包括以下步骤:
(1)称取FeCl3·6H2O 21-22g,FeCl2·4H2O 8-9g溶于40-50ml去离子水中,并量取5-6ml油酸于其中,利用电动搅拌器将其混合均匀,并将混合溶液加热至75-85℃,当混合体系充分混匀后,加入浓度为22-28%的氨水溶液45-55mL,反应15-20min之后将整个体系加热至95-100℃,并持续搅拌反应一段时间后将混合溶液取出,用去离子水和无水乙醇分别清洗,即得Fe3O4纳米粒;
(2)按重量比称取磷脂DPPC:DSPE-PEG2000:MPPC=79.5:14:6.5,将其分散在氯仿中,并使用旋转蒸发器在真空下干燥到圆底烧瓶上,于50-55℃下用硫酸铵溶液水化薄膜,得到空白脂质体粗体,然后根据硫酸铵梯度法将盐酸阿霉素溶液DOX按照质量比DOX:脂质=1:20加入到空白脂质体溶液中,50-60℃下搅拌孵育25-30min,然后加入1-1.5ml的Fe3O4纳米粒,在35-45℃下继续搅拌半个小时;然后利用脂质体挤出器,将粗体脂质体用孔径100nm-200nm的聚碳酸酯膜挤出20-25次,使其形成均匀的磁性纳米簇载药热敏脂质体;
(3)取步骤(2)中制备的磁性纳米簇载药热敏脂质体,使用G-25葡聚糖凝胶柱,用磷酸盐缓冲液作为洗脱液,以流速0.8-1.2ml/min对磁性纳米簇载药热敏脂质体进行洗脱,除去未被包载的肿瘤治疗药物及游离的小颗粒铁纳米粒,得到磁性纳米簇载药热敏脂质体制剂。
8.根据权利要求1所述的纳米簇载药热敏脂质体制剂在制备治疗乳腺癌恶性肿瘤药物中的应用。
9.根据权利要求1所述的纳米簇载药热敏脂质体制剂在联合放疗、光疗、热疗诊断试剂中的应用。
10.根据权利要求1所述的纳米簇载药热敏脂质体制剂在电子计算机断层扫描成像CT/磁共振成像MRI成像诊断试剂中的应用。
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