CN108309938B - 主动定制白蛋白冕的药物传递载体及其在药学中的应用 - Google Patents
主动定制白蛋白冕的药物传递载体及其在药学中的应用 Download PDFInfo
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Abstract
本发明属于药物制剂新辅料和新剂型领域,涉及以内源性的白蛋白为靶点的药物传递系统的设计和应用。所使用的主动定制白蛋白冕的载体材料,以马来酰亚胺作为靶头,聚乙二醇作为连接臂增加靶头的柔韧性,疏水性材料(例如PLGA、硬脂酸等)作为锚定部位。该载体材料制备的纳米递送制剂可包载多种抗肿瘤药物,并能通过其表面马来酰亚胺主动结合的白蛋白冕,与肿瘤血管和肿瘤细胞高表达的白蛋白受体(SPARC、gp60)相互作用,克服多种生物学传递屏障,有效提高纳米粒在肿瘤部位的蓄积、肿瘤细胞的摄取和抗肿瘤活性。该纳米递送制剂稳定性好,安全性高,靶向性佳,可用于静脉注射,有较大的市场应用前景。
Description
技术领域
本发明属于药物制剂新辅料和新剂型领域,涉及以内源性的白蛋白为靶点的药物传递载体的设计和应用,包括马来酰亚胺通过聚乙二醇(PEG)连接臂对疏水性材料进行修饰的载体结构设计、合成及应用。
背景技术
癌症又称恶性肿瘤,目前最有效的治疗方式是化疗,但是化疗在杀死肿瘤细胞的同时,也杀死自身健康细胞。为了降低毒副作用,肿瘤靶向纳米药物传递系统应运而生。靶向纳米药物传递系统的设计原则是对纳米递送形式(例如纳米粒、脂质体、胶束、纳米乳、纳米凝胶和纳米囊泡等)表面进行小分子、配体或抗体修饰,利用肿瘤细胞膜上过度表达的受体或者转运体特异性识别、高亲和力地结合纳米药物,进而来提高肿瘤细胞的药物摄取量,增加抑瘤效果、降低毒副作用。然而,靶向递送形式一旦进入到血液后,由于被血中的调理素、补体、巨球蛋白、免疫球蛋白等物质吸附, 递送形式表面形成蛋白水化冕,进而被网状内皮系统识别、吞噬,从体循环中清除,不能充分发挥设计所赋予的靶向功能。纳米递送形式表面蛋白冕的形成是一个动态的过程,最初表面会吸附大量低亲和力、高能动性的白蛋白,随后这些白蛋白会被高亲和力、低能动性的免疫球蛋白、调理素因子等替换,此时纳米递送形式表面的白蛋白含量会大大降低。白蛋白是人血中含量最多的蛋白,其34位半胱氨酸位点含有高反应活性的游离巯基,被广泛应用于马来酰亚胺前药的设计。另有文献报道,过度增殖的肿瘤组织摄取大量白蛋白作为营养供应,主要通过被动EPR效应和主动白蛋白受体(SPARC和gp 60)转运作用。在此,我们旨在开发能够主动定制白蛋白冕的纳米递送形式,研究其在体内循环时间、肿瘤组织的靶向和渗透作用、肿瘤细胞的摄取以及体内抗肿瘤活性。
发明内容
本发明旨在提供一种新型的主动定制白蛋白冕的药物传递载体,该载体为马来酰亚胺通过聚乙二醇(PEG)连接臂对疏水性材料进行修饰,该载体可以广泛应用于主动定制白蛋白冕。
本发明通过以下技术方案实现上述目的:
其中:
聚合度X可为0-45,即PEG的分子量介于0-2000;聚合度优选11-45,此时PEG的分子量介于500-2000,聚乙二醇链作为连接臂能增加马来酰亚胺的柔韧性。
R为疏水性材料,该疏水性材料可为聚合物、磷脂、脂肪酸、环糊精或疏水性药物中的一种或几种。
其中,所述的聚合物可为聚乳酸羟基乙酸共聚物(PLGA)、聚己内酯(PCL)、聚乳酸(PLA)、聚-3-羟基丁酸-3-羟基己酸酯(PHBHHx)及其衍生物中的一种或几种;
磷脂可为二硬脂酰基磷脂酰乙醇胺(DSPE)、二硬脂酰基磷脂酰丝氨酸(DSPS)、二硬脂酰基磷脂酰肌醇(DSPI)、二棕榈酰基磷脂酰乙醇胺(DPPE)、二油酰基磷脂酰乙醇胺(DOPE)、二豆蔻酰基磷脂酰乙醇胺(DMPE)、二月桂酰基磷脂酰乙醇胺(DLPE)、二花生四烯酰基磷脂酰乙醇胺(DAPE)及其衍生物中的一种或几种;
脂肪酸可为硬脂酸、棕榈酸、豆蔻酸、月桂酸、癸酸、辛酸、花生四烯酸、油酸、亚油酸、亚麻酸、二十二碳六烯酸、二十碳五烯酸等及其衍生物中的一种或几种;
环糊精可为α、β、γ-环糊精及其衍生物中的一种或几种;
疏水性药物可为紫杉烷类、喜树碱类、蒽醌类、二氢吡啶类、非甾体抗炎药类、维生素类等及其衍生物中的一种或几种。
本发明所述的马来酰亚胺修饰的载体通过如下方法制备:首先,合成6-氨基己酸,酰氯化得到高反应活性的6-马来酰亚胺己酰氯,然后再和疏水性R上的-OH或者PEG修饰的疏水性R上的PEG末端的-OH成酯即可。
进一步地,本发明提供了以聚乳酸羟基乙酸、硬脂酸为疏水性材料,马来酰亚胺分别修饰的载体PLGA-Mal和SA-PEG-Mal。
本发明还提供了两种载体材料的制备过程:
PLGA-Mal制备过程,如下步骤所述:分别将6-氨基己酸,马来酸酐置于三颈瓶,加入冰醋酸溶解,135 ℃油浴条件下回流6 h,量取少许醋酸酐,以1滴/秒的速度滴加到反应液中,回流2 h,利用硅胶柱分离得到6-马来酰亚胺己酸。称取6-马来酰亚胺己酸加入到100mL的茄型瓶,加草酰氯搅拌溶解样品。在70℃油浴、搅拌条件下,回流2 h,得到6-马来酰亚胺己酰氯。反应液用旋转蒸发器旋干,除尽草酰氯。将得到6-马来酰亚胺己酰氯溶解到重蒸的二氯甲烷中,在40 ℃油浴条件下磁力搅拌15 min。同时将PLGA溶解在重蒸的二氯甲烷,用恒压滴液漏斗以1滴/秒的速度滴加液体完成后,反应24 h,进一步分离得到PLGA-Mal。
该聚合物为淡黄色半固体,易溶于二氯甲烷、二甲基亚砜等有机溶剂。
SA-PEG-Mal的制备方法,如下步骤所述:分别将6-氨基己酸,马来酸酐置于三颈瓶,加入冰醋酸溶解,135 ℃油浴条件下回流6 h,量取少许醋酸酐,以1滴/秒的速度滴加到反应液中,回流2 h,利用硅胶柱分离得到6-马来酰亚胺己酸。称取6-马来酰亚胺己酸加入到100 mL的茄型瓶,加草酰氯搅拌溶解样品。在70℃油浴、搅拌条件下,回流2 h,得到6-马来酰亚胺己酰氯。反应液用旋转蒸发器旋干,除尽草酰氯。将得到6-马来酰亚胺己酰氯溶解到重蒸的二氯甲烷中,在40 ℃油浴条件下磁力搅拌15 min。同时将聚乙二醇硬脂酸溶解在重蒸的二氯甲烷,用恒压滴液漏斗以1滴/秒的速度滴加液体完成后,反应24 h,进一步分离纯化得到SA-PEG-Mal。
SA-PEG-Mal是一种黄色油状物,易溶于二氯甲烷、二甲基亚砜等有机溶剂。
本发明所述的马来酰亚胺通过聚乙二醇连接臂对疏水性材料进行修饰的载体材料,可以作为药物载体或者修饰剂在纳米粒、脂质体、胶束、囊泡、纳米凝胶、纳米乳等药物传递类型的应用。
马来酰亚胺修饰的载体,作为药物载体或者修饰剂,所制备的纳米粒、脂质体、胶束、囊泡、纳米凝胶、纳米乳等药物传递类型,是一种稳定性好、能主动结合白蛋白,具有较好的体内循环效果、肿瘤靶向和肿瘤细胞摄取的材料。
马来酰亚胺修饰的载体,作为药物载体或者修饰剂所制备的药物传递类型,可以用于修饰包载紫杉烷类、喜树碱类、蒽醌类、二氢吡啶类、非甾体抗炎药中的任一物质或其衍生物;基因类药物为DNA或siRNA的纳米药物传递系统。
马来酰亚胺修饰的载体制备制剂中,药物和马来酰亚胺修饰的载体的比例可在1/10-1/30之间,优选1/15-1/25, 高于1/15时药物不能充分包载在纳米载体中,低于1/25时,药物能包载充分,会降低制剂的载药量。
马来酰亚胺修饰的载体,在作为药物载体或者修饰剂时,其修饰方法可采用乳化溶剂挥发法和薄膜分散法。
乳化溶剂挥发法采用下述步骤:将上述的药和载体完全溶解于适量的二氯甲烷中,在适当温度和乳化超声时间下,得到具有主动定制白蛋白的纳米粒。
薄膜分散法采用下述步骤:将上述载体、药、胆固醇、磷脂(重量比为1:1:1:30)完全溶解于适量的二氯甲烷中,在适当温度下,旋干成膜,加入水化剂水化,并探头超声后,得到具有主动定制白蛋白冕的脂质体。
本发明将马来酰亚胺通过聚乙二醇(PEG)连接臂对疏水性材料进行修饰,并运用于主动定制白蛋白冕纳米递送形式的设计和制备,该纳米递送形式具有包封率高、稳定性好、肿瘤靶向等优势,能显著提高小鼠乳腺癌细胞的摄取,大大增强抗肿瘤效率,克服了肿瘤生物学传递屏障。运用该系列聚合物材料制成的纳米递送制剂,在药学中具有广阔的应用前景。
本发明具有以下有益效果:制备了新型的靶向血浆白蛋白的载体材料PLGA-Mal和SA-PEG-Mal,载体制备过程温和,易操作。运用该马来酰亚胺修饰的载体制备出能主动定制白蛋白冕的纳米药物递送形式,并具有粒径较小且均一,包封率高,稳定性好,肿瘤靶向性好等优点。体外细胞实验和体内活体成像证明PEG的链长影响药物递送效率,PEG2000长度的马来酰亚胺修饰的载体所制备的纳米药物传递类型具有最优的抗肿瘤效果。
附图说明
图1为本发明实施例1的PLGA-Mal的 1HNMR谱图
图2为本发明实施例2的SA-PEG-Mal的IR和 1HNMR谱图
图3为本发明实施例3的PLGA NPs, Mal NPs (PLGA-Mal NPs, PLGA-PEG500-MalNPs, PLGA-PEG2000-Mal NPs) 和PLGA-PEG2000 NPs的动态光散射测定胶束粒径图
图4为本发明实施例3的PLGA NPs, Mal NPs 和PLGA-PEG2000 NPs的透射电镜图
图5为本发明实施例4的SA-PEG-Mal修饰的PLGA纳米粒的透射电镜和动态光散射粒径图
图6为本发明实施例5的SA-PEG-Mal修饰的脂质体的动态光散射粒径图
图7为本发明实施例5的SA-PEG-Mal修饰的脂质体的透射电镜图
图8为本发明实施例6的PLGA NPs, Mal NPs 和PLGA-PEG2000 NPs的(A)DSC和(B)XRD图
图9为本发明实施例7的PLGA NPs, Mal NPs 和PLGA-PEG2000 NPs的血浆稳定性图
图10为本发明实施例7的PLGA NPs, Mal NPs 和PLGA-PEG2000 NPs的长期稳定性图
图11为本发明实施例8的PLGA NPs, Mal NPs 和PLGA-PEG2000 NPs的体外释放图
图12为本发明实施例9的PLGA NPs, PLGA-PEG2000-Mal NPs 和PLGA-PEG2000NPs吸附的前20种含量蛋白图
图13为本发明实施例9的PLGA-PEG2000-Mal NPs表面共价结合前20种含量蛋白图
图14为本发明实施例10的PLGA NPs, Mal NPs 和PLGA-PEG2000 NPs的48h细胞毒性图
图15为本发明实施例11的PLGA NPs, Mal NPs 和PLGA-PEG2000 NPs的2 h细胞摄取图
图16为本发明实施例12的PLGA NPs, PLGA-PEG2000-Mal NPs 和PLGA-PEG2000NPs的24 h细胞凋亡图
图17为本发明实施例13的PLGA NPs, Mal NPs 和PLGA-PEG2000 NPs的药时曲线图
图18为本发明实施例13的第一、二次注射PLGA NPs的药时曲线图
图19为本发明实施例13的第一、二次注射PLGA-PEG2000-Mal NPs的药时曲线图
图20为本发明实施例13的第一、二次注射PLGA-PEG2000 NPs的药时曲线图
图21为本发明实施例14的Cy7标记的PLGA NPs, PLGA-PEG2000-Mal NPs 和PLGA-PEG2000 NPs的离体器官荧光分布图
图22为本发明实施例14的Cy7标记的PLGA NPs, PLGA-PEG2000-Mal NPs 和PLGA-PEG2000 NPs的在肿瘤细胞的分布图
图23为本发明实施例15的PLGA NPs, Mal NPs 和PLGA-PEG2000 NPs的在体肿瘤生长曲线图。
具体实施方式
下面通过实施例的方式进一步说明本发明,但并不将发明限制在所述的实施例范围之中。
实施例1
马来酰亚胺修饰的载体PLGA-Mal的制备:
分别将6-氨基己酸,马来酸酐,置于三颈瓶,加入冰醋酸溶解,135 ℃油浴条件下回流6 h,量取少许醋酸酐,以1滴/秒的速度滴加到反应液中,回流2 h,利用硅胶柱分离得到6-马来酰亚胺己酸。称取6-马来酰亚胺己酸加至100 mL的茄型瓶,加草酰氯搅拌溶解样品。在70℃油浴、搅拌条件下,回流2 h,得到6-马来酰亚胺己酰氯。反应液用旋转蒸发器旋干,除尽草酰氯。将得到6-马来酰亚胺己酰氯溶解到重蒸的二氯甲烷中,在40 ℃油浴条件下磁力搅拌15 min。同时将PLGA溶解在重蒸的二氯甲烷,用恒压滴液漏斗以1滴/秒的速度滴加液体完成后,反应24 h,进一步分离得到PLGA-Mal。其反应式如下:
步骤中PLGA的分子量为8000,但是并不限于此,本发明的PLGA亦可是一端为羧基修饰的PLGA,但是并不局限于以上物质。PLGA的分子量可以为8000-38000范围内。
采用核磁共振测定1H-NMR氢谱来确定实施例1中PLGA-Mal的结构,选用的溶剂为CDCl3,结果如图1。6.7 ppm为马来酰亚胺上-CH=CH-上的典型质子H,4.5-5.5 ppm间的质子峰为PLGA中的H。
实施例2
马来酰亚胺修饰的载体SA-PEG-Mal的制备方法,如下步骤所述:分别将6-氨基己酸,马来酸酐置于三颈瓶,加入冰醋酸溶解,135 ℃油浴条件下回流6 h,量取少许醋酸酐,以1滴/秒的速度滴加到反应液中,回流2 h,利用硅胶柱分离得到6-马来酰亚胺己酸。称取6-马来酰亚胺己酸加入100 mL的茄型瓶,加草酰氯搅拌溶解样品。在70℃油浴、搅拌条件下,回流2 h,得到6-马来酰亚胺己酰氯。反应液用旋转蒸发器旋干,除尽草酰氯。将得到6-马来酰亚胺己酰氯溶解到重蒸的二氯甲烷中,在40 ℃油浴条件下磁力搅拌15 min。同时将聚乙二醇硬脂酸溶解在重蒸的二氯甲烷,用恒压滴液漏斗以1滴/秒的速度滴加液体完成后,反应24 h,进一步分离纯化得到SA-PEG-Mal。
SA-PEG-Mal是一种黄色油状物,易溶于二氯甲烷、二甲基亚砜等有机溶剂。步骤中硬脂酸并不限于此,可为棕榈酸、豆蔻酸、月桂酸、癸酸、辛酸、花生四烯酸、油酸、亚油酸、亚麻酸、二十二碳六烯酸、二十碳五烯酸等及其衍生物;PEG的聚合度可为0-45之间。采用核磁共振测定1H-NMR氢谱来确定实施例1中SA-PEG--Mal的结构,选用的溶剂为CDCl3,结果如图2。6.7 ppm为马来酰亚胺上-CH=CH-上的典型质子H。
实施例3
乳化溶剂挥发法制备包载多西他赛的纳米粒
称取1 mg 多西他赛或香豆素6,溶于适量的二氯甲烷,加入20 mg实施例1制备的PLGA-Mal或PLGA,PLGA-PEG500-Mal,PLGA-PEG2000-Mal,PLGA-PEG2000,加入5 mL含有0.5%PVA去离子水, 探头超声300 W 5 min,采用离心法除去未包裹的药物。
将实施例3中制备的纳米粒通过动态光散射和透视电镜测定其粒径大小和形态。结果如图3,纳米粒的粒径约为150 nm,粒径分布窄;透视电镜图表明载药纳米粒为粒径均匀的球形。
实施例4
乳化溶剂挥发法制备SA-PEG-Mal修饰包载多西他赛的纳米粒
称取1 mg 多西他赛或香豆素6,加入20 mg PLGA (La:Ga=75:25,Mw=38000) ,再分别加入实施例2所制备的SA-PEG-Mal载体2 mg,完全溶于适量二氯甲烷中,加入5 mL的含有0.5%PVA去离子水, 探头超声300 W 5 min,采用离心法除去未包裹的药物。
将实施例4中制备的纳米粒通过动态光散射和透视电镜测定其粒径大小和形态。结果如图4,纳米粒的粒径约为160 nm,粒径分布窄;透视电镜图表明载药纳米粒为粒径均匀的球形。
实施例5
薄膜分散法制备载多西他赛或香豆素6的SA-PEG-Mal修饰的脂质体
称取1 mg 多西他赛或香豆素6,溶于适量二氯甲烷中,加入2 mg实施例2制备的SA-PEG-Mal,以及30 mg的大豆磷脂和1 mg的胆固醇。旋干成膜,加入2 mL的去离子水水化30 min,探头超声300 W,5 min,采用微柱离心法除去未包裹的药物。
将实施例5中制备的脂质体通过动态光散射和透视电镜测定脂质体的粒径大小和形态。结果如图6、7,脂质体的粒径约为100 nm,粒径分布窄;透视电镜图表明载药脂质体为粒径均一的球形。
实施例6
PLGA NPs, Mal NPs 和PLGA-PEG2000 NPs的多西他赛纳米粒的DSC和XRD实验
按照实例3制备的纳米粒,冻干后通过DSC和XRD分析DTX在纳米粒中的存在状态,分析样品包括:DTX原料药,空白PLGA NPs,DTX原料药和空白PLGA NPs物理混合物,冻干的PLGA NPs,Mal NPs 和PLGA-PEG2000 NPs。
图8结果表明DTX以无定型或分子态包裹在纳米粒中。
实施例7
PLGA NPs, Mal NPs 和PLGA-PEG2000 NPs的多西他赛纳米粒的稳定性试验
按照实例2制备纳米粒,置于37℃ 10% FBS pH 7.4溶液中,通过动态光散射测定纳米粒在0.5 h, 12 h, 24 h, 48 h, 72 h的粒径。同时,纳米粒在4℃ pH 7.4溶液中的长期稳定性也进行了考察。
图9、图10结果表明PLGA-PEG500-Mal NPs,PLGA-PEG2000-Mal NPs和PLGA-PEG2000 NPs具有较好的稳定性。
实施例8
PLGA NPs, Mal NPs 和PLGA-PEG2000 NPs的多西他赛纳米粒的体外释放试验
采用透析法考察实例2制备的纳米粒体外释药特征。移取含300 μg的载药纳米粒溶液于透析袋中,透析袋两端夹紧,分别置于含有30 mL pH 7.4 PBS (含1% Tween 80) 的释放介质的锥形瓶中,在37 ℃恒温振荡器中以100 r/min进行体外释放度考察。分别在2、4、6、8、10、12、24 、48 和72h取样2 mL,同时补充2 mL新鲜释放介质,样品经0.45 μm微孔滤膜过滤,取20 μL进行HPLC测定。
图11结果表明PLGA NPs, Mal NPs 和PLGA-PEG2000 NPs的多西他赛纳米粒释药缓慢,有利于更多的药物达到肿瘤部位。
实施例9
PLGA NPs, PLGA-PEG2000-Mal NPs 和PLGA-PEG2000 NPs的多西他赛纳米粒的蛋白冕含量测定
采用LC-MS测定实例2制备的纳米粒表面蛋白冕含量。将实施例2制备的纳米粒和10% FBS37 ℃ 孵化1h,16000g离心10 min,收集含有蛋白冕的纳米粒,测定纳米粒表面的总蛋白量。这些纳米粒经pH7.4水洗至完全去除软冕,然后再用6% SDS洗完全除去硬冕,合并软冕硬冕进行蛋白组学测定,共价结合的蛋白冕是两者的差值。
图12结果表明PLGA-PEG2000-Mal NPs的多西他赛纳米粒能够富集白蛋白。图13结果表明PLGA-PEG2000-Mal NPs能够共价结合白蛋白。
实施例10
细胞毒性实验
将处于对数生长期的4T1和3T3细胞以3×103/孔/0.2mL的培养液埋于96孔板中,24 h后将实施例2制备的PLGA NPs, Mal NPs 和PLGA-PEG2000 NPs的多西他赛纳米粒按不同浓度分别加入各孔,每孔加入200 μL含纳米粒溶液,每个浓度5个平行孔,置培养箱中孵育。孵育48 h后,取出96孔板,每孔加入20 μL的5 mg/mL MTT,再放入培养箱中孵育4 h,甩板,将96孔板倒扣于滤纸中充分吸干残留液体,每孔加入200 μL DMSO于振荡器中振荡10min,酶标仪测定各孔在490 nm处的吸光度。计算抑制率:
细胞存活率(%)= A加药孔/A对照孔×100%
MTT法测定纳米粒细胞毒性结果如图14 ,细胞抑制率随药物浓度增加而增大,并且Mal NPs对细胞的抑制作用有较好的选择性。
实施例11
细胞摄取实验
将SPARC高表达的4T1和SPARC低表达的3T3细胞以8×104/孔/1mL的培养液埋于12孔板中,24 h后加入实施例2制备的PLGA NPs, Mal NPs 和PLGA-PEG2000 NPs的罗丹明B修饰的纳米粒(20 μg/mL Rhod-PCL),平行3孔,置培养箱中孵育2 h。弃上清,PBS洗三遍,DAPI染色,使用共聚焦显微镜进行观察。同时,PBS洗三遍后,使用流式细胞仪对定量细胞摄取纳米粒进行了考察。
细胞摄取的结果见图15,相比于成纤维细胞3T3,Mal NPs 能被SPARC高表达的4T1细胞选择摄取。
实施例12
细胞凋亡实验
将处于对数生长期的4T1和3T3细胞以2.5×105/孔/1mL的培养液埋于6孔板中,24h后将实施例2制备的PLGA NPs, PLGA-PEG2000-Mal NPs 和PLGA-PEG2000 NPs的多西他赛纳米粒, 以1 μg/mL浓度加入孔中,每个浓度平行3次,置培养箱中孵育24 h。PBS洗三遍,分别用Annexin V-FITC 和PI染色,使用流式细胞仪定量测定。
细胞凋亡结果如图16, 相比于成纤维细胞3T3,PLGA-PEG2000-Mal NPs对4T1细胞凋亡有较好的选择性。
实施例13
药动学实验
将实施例2制备的PLGA NPs, Mal NPs 和PLGA-PEG2000 NPs的多西他赛纳米粒(5mg DTX /kg)静脉注射给禁食12 h 的SD大鼠。给药后在设计的时间点眼眶取血0.3 ml,血液立即移入经肝素处理的试管中,离心10 min (13000 g),分离血浆,于-80℃冰箱中冷冻直至分析。
7天后,再将实施例2制备的PLGA NPs, PLGA-PEG2000-Mal NPs 和PLGA-PEG2000NPs(5 mg DTX /kg)二次注射到上述该组大鼠中,考察加速血液清除(ABC)现象。
药动学结果如图17-图20,PLGA-PEG2000-Mal NPs具有较好的长循环效果,并且二次注射不会引起ABC现象。
实施例14
Mal纳米粒的肿瘤靶向性考察
采用活体成像法考察实例2制备Cy7标记的PLGA NPs, PLGA-PEG2000-Mal NPs 和PLGA-PEG2000 NPs的肿瘤蓄积情况。以2 mg/kg Cy7尾静脉给予Balb/c小鼠体内,分别在4h和24 h后,将小鼠的心,肝,脾,肺,肾和肿瘤取出,通过活体成像仪观察纳米粒的分布情况。之后将肿瘤进行了冰冻切片染色,DAPI染细胞核,FITC标记4T1肿瘤细胞,激光共聚焦下观察纳米粒的分布。
肿瘤靶向性结果实验结果见图21、图22,结果表明PLGA-PEG2000-Mal NPs具有更好的肿瘤靶向性,并且纳米粒能被肿瘤细胞摄取。
实施例15
Mal纳米粒的抗肿瘤活性研究
荷瘤裸鼠随机分为7组,每组5只,分别为生理盐水组(空白对照组),DTX-Sol和实例2制备Cy7标记的PLGA NPs, PLGA-PEG2000-Mal NPs 和PLGA-PEG2000 NPs纳米制剂组(5mg DTX /kg),采取尾静脉注射的给药方式,隔天给药一次,共给药4次。给药后,隔天测量肿瘤大小,并绘制肿瘤生长曲线。
药效学结果实验结果见图23,结果表明PLGA-PEG2000-Mal NPs具有最好的肿瘤抑制效果。
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