CN114835888A - 一种肿瘤原位组装的纳米载体***、载药体系及应用 - Google Patents
一种肿瘤原位组装的纳米载体***、载药体系及应用 Download PDFInfo
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Abstract
本发明属于抗肿瘤纳米载体技术领域,公开了一种肿瘤原位组装的纳米载体***、载药体系及应用。本发明的肿瘤原位组装的纳米载药体系中,由特定结构的聚乙二醇‑聚乳酸材料制成的纳米颗粒组成,可实现纳米颗粒在血液中长循环;当纳米颗粒到达肿瘤弱酸环境,可实现纳米颗粒在肿瘤原位组装成大尺寸颗粒聚集体,能增强纳米载药体系及所载药物在肿瘤的富集和滞留,并作为一种胞外“药物仓库”缓慢释放所载药物到达胞外靶点,发挥抗肿瘤疗效,为发展胞外/膜上靶点抗肿瘤药物的递送载体***提供新思路。
Description
技术领域
本发明涉及抗肿瘤纳米载体技术领域,具体涉及一种肿瘤原位组装的纳米载体***、载药体系及应用。
背景技术
肿瘤靶向药物是指被赋予了靶向能力的药物或其制剂。其目的是使药物或其载体能瞄准特定的病变部位,并在目标部位蓄积或释放有效成分。
目前,大部分的抗肿瘤药物的作用靶点位于胞内或核内,需要递送这些药物到达胞内的靶标位置,才能发挥药效。现有技术中绝大多数的纳米抗肿瘤药物载体基本设计原理是:通过物理相互作用包埋药物或者将药物通过化学共价键键合方式携载药物,经过静脉给药后,通过配体修饰或其尺度效应(粒径通常在50~200nm之间)实现主/被动靶向至肿瘤组织;进一步,被摄取后在肿瘤细胞胞质中释放出所包载的药物,杀伤肿瘤细胞。
然而,随着新型抗肿瘤药物或候选药物的不断开发,靶点位于细胞外或细胞膜的抗肿瘤药物不断涌现。针对细胞外靶点药物,如基质金属蛋白酶抑制剂类药物(巴马司他、马立马司他、伊洛马司他、CGS 27023A等)、贝伐珠单抗等;针对细胞膜上靶点的药物,如绝大部分单抗类药物aPD-L1、aPD-1、aCTLA-4、aHER-2以及TRAIL、蜂毒肽等。这些药物不同于胞内靶点药物,靶标在胞外或膜上的抗肿瘤药物必须递送它们到达肿瘤组织后在胞外释放不被细胞摄取才能发挥其抗肿瘤效应。
因而,以往传统的胞内靶点抗肿瘤药物纳米递送体系已无法满足这些药物的递送,可能会将这些药物递送至胞内,导致药物无法到达胞外靶标部位,从而无法发挥抗肿瘤作用。
因此,如何合理设计纳米材料及载体递送靶点位于细胞外/膜上的抗肿瘤药物在胞外释放发挥抗肿瘤疗效,是纳米药物载体领域亟待解决的重大难题之一。
发明内容
本发明的目的在于克服现有技术的不足之处而提供一种肿瘤原位组装的纳米载体***、载药体系及应用。
为实现上述目的,本发明采取的技术方案如下:
第一方面,本发明提供了一种聚乙二醇-聚乳酸材料,所述聚乙二醇-聚乳酸材料为在聚乙二醇-聚乳酸材料末端修饰半胱氨酸或2-氰基苯并噻唑的材料。
第二方面,本发明另提供了一种聚乙二醇-聚乳酸材料,所述聚乙二醇-聚乳酸材料为用2,3-二甲基马来酸酐屏蔽权利要求1所聚乙二醇-聚乳酸材料的半胱氨酸残基的材料。
第三方面,本发明提供了一种所述的聚乙二醇-聚乳酸材料的制备方法,包括以下步骤:
(1)通过利用羧基化聚乙二醇引发丙交酯(D,L-LA)开环聚合得到羧基化的聚乙二醇-聚乳酸;或通过利用叔丁氧羰基保护的氨基化聚乙二醇引发丙交酯(D,L-LA)开环聚合,脱去叔丁氧羰基得到氨基化的聚乙二醇-聚乳酸;
(2)通过酰胺化反应,在所述氨基化的聚乙二醇-聚乳酸末端共价修饰上半胱氨酸基团,得到末端半胱氨酸修饰的聚乙二醇-聚乳酸材料;或在所述羧基化聚乙二醇-聚乳酸末端共价修饰上2-氰基苯并噻唑基团,得到末端2-氰基苯并噻唑修饰的聚乙二醇-聚乳酸材料。
第四方面,本发明另提供了一种所述的聚乙二醇-聚乳酸材料的制备方法,包括以下步骤:
(1)通过叔丁氧羰基的保护与去保护反应,利用叔丁氧羰基保护的氨基化聚乙二醇引发丙交酯(D,L-LA)开环聚合,脱去叔丁氧羰基得到氨基化的聚乙二醇-聚乳酸;
(2)通过酰胺化反应,在所述氨基化的聚乙二醇-聚乳酸末端共价修饰上半胱氨酸基团,得到末端半胱氨酸修饰的聚乙二醇-聚乳酸材料;
(3)利用2,3-二甲基马来酸酐屏蔽所述末端半胱氨酸修饰的聚乙二醇-聚乳酸材料的半胱氨酸残基,即得。
优选的,上述聚乙二醇-聚乳酸材料的制备方法中,所述修饰的2-氰基苯并噻唑基团为利用6-氨基-2-氰基苯并噻唑与羧基化聚乙二醇-聚乳酸通过酰胺化反应合成得到;所述修饰的半胱氨酸为L型或D型半胱氨酸;所述反应体系中有机溶剂为四氢呋喃;所述酰胺化反应催化剂为1-羟基苯并***和1-乙基-碳酰二亚胺盐酸盐。
本发明的两种生物正交基团修饰的聚乙二醇-聚乳酸(PEG-b-PLA)材料可按照下述方法合成:利用叔丁氧羰基(Boc)保护的氨基化或羧基化聚乙二醇羟基引发丙交酯(D,L-LA)开环聚合得到官能团化的聚乙二醇-聚乳酸材料;进一步,用三氟乙酸脱去Boc保护,得到氨基化的聚乙二醇-聚乳酸。随后,通过酰胺化反应在PEG-b-PLA末端修饰上半胱氨酸/2-氰基苯并噻唑等生物正交基团;进一步,用2,3-二甲基马来酸酐屏蔽半胱氨酸残基得到肿瘤酸度响应的半胱氨酸修饰的PEG-b-PLA材料。
本发明的聚乙二醇-聚乳酸材料中的生物正交基团是半胱氨酸和2-氰基苯并噻唑,两者之间的生物正交反应高效、无需催化剂,其二级反应速率常数为9.19M-1s-1。聚乙二醇-聚乳酸材料中亲水部分是聚乙二醇,为亲水性聚酯,其相对分子量为1000~10000。聚乙二醇-聚乳酸材料中疏水部分是聚乳酸,其相对分子量为1000~10000,它的优点在于:(1)疏水性,通过疏水-疏水相互作用可包载疏水性药物自组装成纳米颗粒;(2)可生物降解,并且它的最终降解产物不会对生物体有不良影响;(3)合成简单且可控。
第五方面,本发明提供了一种纳米颗粒,包括所述在聚乙二醇-聚乳酸材料末端分别修饰半胱氨酸或2-氰基苯并噻唑的材料,和/或,用2,3-二甲基马来酸酐屏蔽所述半胱氨酸残基的材料。
优选的,所述纳米颗粒的直径50-150nm,优选的,直径为60-70nm,进一步的,直径为65nm。
本发明的纳米颗粒中,在聚乙二醇-聚乳酸材料末端分别修饰2-氰基苯并噻唑的材料制备的纳米颗粒记为C-NP颗粒;在聚乙二醇-聚乳酸材料末端分别修饰半胱氨酸,再用2,3-二甲基马来酸酐屏蔽所述半胱氨酸残基的材料制备的纳米颗粒记为D-NP颗粒。
D-NP在肿瘤弱酸环境下发生酸响应重新暴露半胱氨酸残基,与表面2-氰基苯并噻唑修饰的C-NP颗粒发生生物正交反应实现颗粒间共价交联聚集。
第六方面,本发明提供了一种上述的纳米颗粒的制备方法,所述方法为纳米沉淀法或单乳化法。
第七方面,本发明提供了一种肿瘤原位组装的纳米载体***,包括上述的纳米颗粒。
本发明的肿瘤原位组装的纳米载体***,能够实现纳米颗粒在肿瘤部位发生共价交联组装,形成大尺寸颗粒聚集体,增强纳米载体在肿瘤部位的富集和滞留。
第八方面,本发明提供了一种肿瘤原位组装的纳米载药体系,包括上述的肿瘤原位组装的纳米载体***和抗肿瘤药物。
优选的,所述抗肿瘤药物为胞外或膜上靶点的抗肿瘤药物,为疏水性抗肿瘤药物。
本发明的纳米载药体系中,D-NP在肿瘤弱酸环境下发生酸响应重新暴露半胱氨酸残基,与表面2-氰基苯并噻唑修饰的C-NP颗粒发生生物正交反应实现颗粒间共价交联聚集后,形成大尺寸颗粒聚集体,增强载体和所载药物在肿瘤部位的富集和滞留,提高抗肿瘤疗效。该体系用于递送胞外靶点抗肿瘤药物,有望提高胞外靶点药物的抗肿瘤疗效,具有巨大的临床应用潜能。纳米载药体系中负载的胞外/膜上靶点抗肿瘤药物在胞外形成的颗粒聚集体可作为胞外“药物仓库”,缓慢释放所载药物到达胞外/膜上靶点,发挥抗肿瘤疗效。
第九方面,本发明将所述的聚乙二醇-聚乳酸材料、所述聚乙二醇-聚乳酸材料的制备方法、所述的纳米颗粒、所述纳米颗粒的制备方法、所述的纳米载体***、所述的纳米载药体系在制备胞外/膜上靶点抗肿瘤药物中应用。
第十方面,本发明将所述的聚乙二醇-聚乳酸材料、所述聚乙二醇-聚乳酸材料的制备方法、所述的纳米颗粒、所述纳米颗粒的制备方法、所述的纳米载体***、所述的纳米载药体系在制备抗肿瘤转移药物中应用。
与现有技术相比,本发明的有益效果为:
本发明的肿瘤原位组装的纳米载药体系中,包括特定结构的聚乙二醇-聚乳酸材料制成的纳米颗粒,具有良好的生物相容性和可降解性;基于肿瘤酸度敏感的酰胺键屏蔽纳米颗粒之间的生物正交反应,可实现纳米颗粒在血液中长循环;而当纳米颗粒到达肿瘤弱酸环境,酸度响应的D-NP发生酸响应重新暴露半胱氨酸基团,发生生物正交反应,实现纳米颗粒在肿瘤原位组装成大尺寸颗粒聚集体;形成的颗粒聚集体能增强纳米载药体系及所载药物在肿瘤的富集和滞留,避免被肿瘤相关细胞摄取,并作为一种胞外“药物仓库”缓慢释放所载药物到达胞外靶点,发挥抗肿瘤疗效,为发展胞外/膜上靶点抗肿瘤药物的递送载体***提供新思路。
附图说明
图1为生物正交基团修饰的聚乙二醇-聚乳酸材料DACys-PEG-b-PLA和CBT-PEG-b-PLA的合成路线。
图2为酸响应的半胱氨酸修饰的聚乙二醇-聚乳酸材料DACys-PEG-b-PLA的1H NMR。
图3为非酸响应的半胱氨酸修饰的聚乙二醇-聚乳酸材料SACys-PEG-b-PLA的合成路线图及1H NMR。
图4为2-氰基苯并噻唑修饰的聚乙二醇-聚乳酸材料CBT-PEG-b-PLA的1H NMR。
图5为D-NP和C-NP纳米颗粒在pH 6.5弱酸环境下交联后的凝胶渗透色谱(GPC)图。
图6为D-NP和C-NP纳米颗粒的粒径分布及其透射电子显微镜图片。
图7为D-NP和C-NP以及D-NP/C-NP混合纳米颗粒的稳定性图。
图8为D-NP/C-NP混合纳米颗粒在不同pH环境中粒径的变化曲线及其透射电镜图片。
图9为D-NP/C-NP混合纳米颗粒在pH 6.5弱酸环境下的三维激光共聚焦图。
图10为D-NP/C-NP在pH 6.5弱酸性环境下孵育不同时间后被4T1肿瘤细胞的摄取图。
图11为酸响应D-NP/C-NP颗粒与非酸响应S-NP/C-NP颗粒在肿瘤部位富集滞留的荧光成像图。
图12为酸响应D-NPBB94/C-NPBB94和非酸响应S-NPBB94/C-NPBB94载药纳米颗粒在不同pH条件下的药物释放曲线图。
图13为D-NP、C-NP和S-NP三种纳米颗粒在体内的药代动力学曲线图。
图14为D-NP/C-NP和S-NP/C-NP递送巴马司他抗肿瘤转移的肿瘤生长曲线图。
图15为体内治疗实验中各实验组小鼠肺组织的转移结节数量统计图。
图16为体内治疗实验中各实验组小鼠体重变化曲线图。
具体实施方式
为更好地说明本发明的目的、技术方案和优点,下面将结合具体实施例对本发明作进一步说明。本领域技术人员应当理解,此处所描述的具体实施例仅用以解释本发明,并不用于限定本发明。
实施例中所用的试验方法如无特殊说明,均为常规方法;所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1:生物正交基团修饰的聚乙二醇-聚乳酸的合成与表征
1、生物正交基团修饰的聚乙二醇-聚乳酸合成
DACys-PEG-b-PLA是由叔丁氧羰基保护的半胱氨酸(Boc-Cys)和氨基化聚乙二醇-聚乳酸通过酰胺化反应,进一步脱去Boc保护并用2,3-二甲基马来酸酐屏蔽半胱氨酸残基得到。
DACys-PEG-b-PLA聚合物材料的合成路线如图1所示。
所需组分的制备和预处理包括:
(1)合成叔丁氧羰基保护的半胱氨酸:
向一100mL干净圆底烧瓶加入半胱氨酸(5g,0.029mol)和50mL超纯水,加入磁子搅拌溶解半胱氨酸。再称取NaHCO3(2.436g,0.029mol)加入上述半胱氨酸水溶液中,继续搅拌至固体溶解后,再加入50mL THF混合均匀。在搅拌条件下,在0℃低温下逐滴加入二碳酸二叔丁酯(Boc2O,6.322g,0.029mol),然后继续搅拌反应30min后,自然升温至室温反应过夜。反应结束后,用旋转蒸发仪浓缩Boc-Cys/THF/H2O粗产物溶液,除去沸点较低的THF,抽滤收集澄清滤液转移至100mL圆底烧瓶中,加入搅拌磁子,检测滤液pH值为8~9。向上述产物溶液中缓慢滴加1M的HCl调节产物溶液的pH值,当产物溶液酸碱度下降至pH 6~7左右时,有大量白色沉淀会从产物溶液中析出,随后继续静置2h。进一步,抽滤得白色粉末状产物,干燥过夜得到Boc-Cys。
(2)半胱氨酸修饰的聚乙二醇-聚乳酸材料的合成
将经甲苯共沸除水的BocNH-PEG3400-OH和冻抽过夜除水的DA-LA丙交酯转移至手套箱(H2O<0.1ppm,O2<0.1ppm)中。称取BocNH-PEG3400-OH(1g,0.294mmol)和丙交酯(1.1g,7.639mmol)加入预先烘烤除水汽的50mL圆底烧瓶中,向圆底烧瓶中加入30mL无水甲苯(溶剂无水纯化装置处理),加入磁子搅拌溶解混合固体。将圆底烧瓶置于85℃油浴锅中,在搅拌条件下滴加2滴(20mg,0.049mmol)异辛酸亚锡(Sn(Oct)2)后继续搅拌反应3h。反应完毕浓缩产物沉淀在甲醇/***混合液(1/10,v/v)中,抽滤干燥得BocNH-PEG-b-PLA。随后,将BocNH-PEG-b-PLA溶于三氟乙酸/四氢呋喃混合溶液(20mL,TFA/THF,9/1,v/v),在氮气保护下室温搅拌3h,脱去Boc。随后,浓缩产物沉淀在冷的甲醇/***混合液(1/10,v/v)中,抽滤、真空条件干燥得H2N-PEG-b-PLA,收集产物进行称重并计算产率。
进一步,通过活化Boc-Cys的羧基与H2N-PEG-b-PLA末端氨基反应生成酰胺键,得到Boc-Cys-PEG-b-PLA聚合物材料。具体反应过程为:取一50mL圆底烧瓶加入搅拌磁子,称取Boc-Cys(0.126g,0.57mmol)加入圆底烧瓶并加入10mL DCM溶解固体材料,再分别称取0.145g的1-羟基苯并***(0.684mmol,HOBT)和0.131g的1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐(0.684mmol,EDC·HCl)加入上述装有Boc-Cys溶液的圆底烧瓶中,在室温下搅拌活化6h。随后,称取H2N-PEG-b-PLA(1.1g,0.162mmol)预先溶于10mL DCM,随后快速倒入上述混合反应液中,搅拌反应24h(25℃)。反应完毕,浓缩产物溶液沉淀在甲醇/***混合溶液(1/10,v/v)中,抽滤、真空条件干燥得淡黄色Boc-Cys-PEG-b-PLA,收集产物进行称重并计算产率。随后将Boc-Cys-PEG-b-PLA溶于三氟乙酸/二氯甲烷(20mL,TFA/THF,9/1,v/v),在氮气保护下室温搅拌3h,脱去Boc,得到Cys-PEG-b-PLA。
(3)利用酸酐屏蔽Cys-PEG-b-PLA末端的半胱氨酸残基
利用2,3-二甲基马来酸酐(DA)和丁二酸酐(SA)分别屏蔽Cys-PEG-b-PLA末端半胱氨酸残基的氨基。具体实验过程如下:称取200mg的Cys-PEG-b-PLA聚合物材料,通过纳米沉淀法制备空白纳米颗粒CysNP。向CysNP纳米颗粒(2mg/mL,50mL)溶液中分批缓慢加入5倍半胱氨酸残基氨基当量的DA或SA,使用NaOH溶液(1.0M)调节颗粒溶液pH至8~9之间,而后在室温下继续搅拌反应4h。收集纳米颗粒溶液使用YM-30超滤离心管(Millipore,MWCO 3000Da)进行超滤浓缩纯化,得到的纳米颗粒分别标记为DA-CysNP和SA-CysNP。分别将两颗粒溶液冷冻冻干后得到DACys-PEG-b-PLA和SACys-PEG-b-PLA材料。
(4)2-氰基苯并噻唑基团修饰的聚乙二醇-聚乳酸材料的合成
将经甲苯共沸除水的HOOC-PEG3400-OH和冻抽过夜除水的DA-LA丙交酯转移至手套箱中。取一烘烤除水干燥的50mL圆底烧瓶,加入HOOC-PEG3400-OH(1g,0.294mmol)、丙交酯(1.1g,7.639mmol)和30mL无水甲苯(溶剂无水纯化装置处理),加入磁子搅拌溶解固体反应物。将圆底烧瓶置于85℃油浴锅中,在搅拌条件下滴加2滴(20mg,0.049mmol)Sn(Oct)2后继续搅拌反应3h。反应结束浓缩产物溶液沉淀在冷甲醇/***混合液(1/10,v/v)中,抽滤、真空条件干燥得到淡黄色HOOC-PEG-b-PLA,收集产物进行称重并计算产率。
随后,通过酰胺化反应合成了CBT-PEG-b-PLA,具体反应过程如下:称取HOOC-PEG-b-PLA(1.1g,0.162mmol)、EDC·HCl(37.2mg,0.194mmol)和HOBT(41.2mg,0.194mmol)加入50mL带磁子的圆底烧瓶中,再加入10mL DCM搅拌溶解上述固体反应物,在室温下继续搅拌活化6h。而后,称取CBT(34.2mg,0.195mmol)加入上述混合反应溶液中,在室温下继续搅拌反应24h。反应完毕浓缩产物溶液滴入冷甲醇/***的混合溶液(1/10,v/v)沉淀,抽滤、真空条件下干燥后得黄色的CBT-PEG-b-PLA固体产物。
2、生物正交基团修饰的聚乙二醇-聚乳酸材料的表征
对上述生物正交基团修饰的聚乙二醇-聚乳酸材料进行核磁共振氢谱(1H NMR)分析,测定其分子结构,DACys-PEG-b-PLA 1H NMR谱见图2,CBT-PEG-b-PLA 1H NMR谱见图3。
如图2所示,DACys-PEG77-b-PLA46的1H NMR谱图中均出现各个位置氢原子的特征峰,2.43ppm的特征峰(a峰)归属于DA的甲基氢原子(-CH 3),并且根据1H NMR计算a峰积分面积为7.85个氢原子(理论为6个),表明Cys-PEG77-b-PLA46末端半胱氨酸残基的氨基几乎完全被DA屏蔽。半胱氨酸的亚甲基氢原子(-CH 2-)特征峰在2.89ppm(b峰),半胱氨酸氨基相邻的氢原子(-CH-)特征峰在4.16ppm(f峰),聚乙二醇的氢原子(-CH 2-)特征峰在3.53ppm(c峰),聚乳酸的甲基氢原子(-CH 3)特征峰在1.46ppm(d峰),聚乳酸中与羰基相邻的氢原子(-CH-)特征峰在5.19ppm(e峰),其积分面积为46个氢原子,这些1H NMR结果表明,成功合成了DACys-PEG77-b-PLA46。
如图3所示,用丁二酸酐代替2,3-二甲基马来酸酐屏蔽半胱氨酸残基,得到非酸响应的半胱氨酸化聚乙二醇-聚乳酸对照组材料。在SACys-PEG-b-PLA的1H NMR谱图上,出现一个化学位移为2.28ppm新的特征峰(a峰),归属于丁二酸酐的两个亚甲基氢原子(-CH2-),其积分氢原子数为4.95个(理论为4个),也表明Cys-PEG77-b-PLA46末端半胱氨酸残基的氨基几乎完全被SA屏蔽。这些1H NMR结果表明,成功合成了SACys-PEG77-b-PLA46。
如图4所示,聚乙二醇中亚甲基氢原子(-CH 2-)特征峰在3.53ppm(a峰),聚乳酸中与碳基相邻的氢原子(-CH-)特征峰在5.19ppm(b峰),聚乳酸中甲基氢原子(-CH 3)特征峰在1.46ppm(c峰),而化学位移在7.20ppm(d峰)、6.98ppm(e峰)和7.36ppm(f峰)的特征峰则分别是CBT的苯环氢原子特征峰。从1H NMR谱图分析结果看出,CBT-PEG-b-PLA的各个位置氢原子均在1H NMR谱图上能找到对应的特征峰,并且PLA段的重复单元数经1H NMR计算为32,证明成功合成了CBT-PEG77-b-PLA32聚合物材料。
实施例2:生物正交基团末端修饰的聚乙二醇-聚乳酸的纳米颗粒化及应用
1、D-NP和C-NP纳米颗粒的制备
通过纳米沉淀法或单乳化法制备两种生物正交基团表面修饰的纳米颗粒及其载药纳米颗粒,具体方法如下:
称取10mg DACys-PEG-b-PLA或10mg CBT-PEG-b-PLA分别溶于1mL DMSO中,涡旋完全溶解后,将材料溶液滴入水相(10mL,1×PBS,pH 7.4)中搅拌2h制备得两种纳米颗粒。将颗粒溶液装入MWCO=14000Da透析袋放入1×PBS(pH 7.4,2L)透析过夜。再使用YM-30超滤离心管(Millipore,MWCO5000Da)进行超滤浓缩,制备得D-NP和C-NP纳米颗粒。
2、D-NP和C-NP纳米颗粒的特性
经纳米沉淀法得到两种纳米颗粒D-NP和C-NP,动态光散射仪(Dynamic lightscattering,DLS)和透射电子显微镜(Transmission Electron Microscope,TEM)检测观察两纳米颗粒粒径和形貌。进一步,通过凝胶渗透色谱检测两颗粒在pH 6.5弱酸环境下生成的交联产物。
如图5所示,D-NP/C-NP在pH 6.5弱酸环境下搅拌2h后,在31.8min处出现一个新的峰,代表D-NP和C-NP发生生物正交反应生成更大分子量的交联聚集体。
如图6所示,D-NP和C-NP的粒径均为65nm左右。并且两种纳米颗粒都呈一个完整紧凑的球型形貌,大小在60nm左右,与DLS检测结果一致。
如图7所示,两种纳米颗粒都具有一个较好的稳定性。在含10%胎牛血清的1×PBS(pH=7.4)溶液中共培养96h后,两种纳米颗粒及其混合颗粒溶液粒径均无明显变化。这可能是由于PEG能够为颗粒提供一个惰性的表面,从而提高颗粒的稳定性。
3、D-NP和C-NP纳米颗粒的酸响应触发颗粒组装聚集的功能
(1)D-NP和C-NP纳米颗粒的酸响应组装聚集
D-NP/C-NP混合纳米颗粒在pH 6.5或7.4的0.02M PBS(模拟内涵体/溶酶体酸性微环境)共同孵育培养不同时间。
如图8所示,DLS检测结果表明在pH 6.5弱酸环境下,D-NP/C-NP混合颗粒的粒径不断增加,24h内尺寸能从~60nm增加至2600nm左右;而在pH 7.4中性环境下,D-NP/C-NP的混合颗粒粒径无明显变化;并且,通过TEM可以清楚地观察到的D-NP/C-NP混合颗粒在pH 6.5环境下的颗粒组装聚集程度随着共孵育时间的延长而增加,且聚集体数量也逐渐增加;而在pH 7.4环境下,两颗粒无组装聚集现象。
分别称取10mg的DACys-PEG-b-PLA和10mg的CBT-PEG-b-PLA分别溶于1mL的DMSO中,分别向DACys-PEG-b-PLA和CBT-PEG-b-PLA材料溶液中加入100μg的DiI(2mg/mL,DMSO,50μL)后涡旋混合均匀,在搅拌条件下,将上述混合材料溶液分别滴入水相(1×PBS,pH 7.4,10mL)中搅拌2h,使用YM-30超滤离心管(Millipore,MWCO 5000Da)进行超滤洗涤浓缩纯化,分别制备得DiID-NP和DiIC-NP纳米颗粒。随后,DiI染料标记的DiID-NP/DiIC-NP混合纳米颗粒在pH 6.5环境下共孵育不同时间,用3D激光共聚焦显微镜(3D confocal laser scanningmicroscope)观察混合颗粒溶液中的组装颗粒聚集体。
如图9所示,随着DiID-NP/DiIC-NP共孵育时间的延长,混合颗粒溶液中的颗粒组装聚集体数量逐渐增加,并且聚集体的尺寸也不断增大。
(2)D-NP和C-NP纳米颗粒组装聚集后被肿瘤细胞摄取的行为
首先,合成Cy5-PEG-b-PLA聚合物材料,具体为:称取Cy5-NHS(61mg,0.081mmol)活性酯和H2N-PEG-b-PLA(0.5g,0.074mmol)加入带磁子的圆底烧瓶(25mL),再向烧瓶中加入10mL THF,室温下避光搅拌反应8h。反应结束浓缩上述反应产物溶液至2mL左右逐滴滴入冷甲醇/***混合溶液(1/10,v/v)中,在搅拌和低温(-20℃)条件下,沉淀结束后抽滤、干燥得到深红色Cy5-PEG-b-PLA产物。
制备Cy5标记的Cy5D-NP纳米颗粒:称取10mg DACys-PEG-b-PLA溶于1mL DMSO中,再掺入1mg Cy5-PEG-b-PLA(1mL,10mg/mL,DMSO),涡旋混合均匀后,将混合材料滴入水相(10mL,1×PBS,pH 7.4)中搅拌2h制备Cy5D-NP。将颗粒溶液装入MWCO=14000Da透析袋放入1×PBS(pH 7.4,2L)透析过夜。再使用YM-30超滤离心管(Millipore,MWCO 5000Da)进行超滤浓缩,制备得Cy5D-NP纳米颗粒。Cy5C-NP纳米颗粒的制备如上述过程一致,利用CBT-PEG-b-PLA代替DACys-PEG-b-PLA经相同步骤制备得到。
然后,将鼠源4T1肿瘤乳腺癌细胞以接种数量2×104个细胞/孔加到24孔板中,加入0.5mL RPMI 1640(含10%FBS),在37℃、5%CO2温湿环境下孵育12h。预先将Cy5D-NP和Cy5C-NP纳米颗粒(颗粒终浓度为1mg/mL)在pH 6.5条件下搅拌不同时间(0.5h,1h,2h和4h)后,用RPMI 1640培养基稀释上述交联不同时间的颗粒(颗粒终浓度为0.1mg/mL),加入24孔板中与4T1细胞共同孵育2h。其中,pH 7.4条件下,两颗粒混合作为对照组。孵育结束后,1×PBS洗涤细胞除去胞外颗粒,随后消化细胞得细胞悬液用FACS检测肿瘤细胞内的Cy5荧光强度。
如图10所示,随着Cy5D-NP/Cy5C-NP混合颗粒孵育时间的延长,4T1肿瘤细胞对混合颗粒的摄取能力逐渐减弱,表明随着时间的延长,D-NP和C-NP的组装聚集程度不断增加,形成大尺寸的颗粒聚集体能避免被肿瘤细胞摄取。
4、D-NP和C-NP肿瘤原位组装增强肿瘤富集和滞留的功能
分别称取10mg的DACys-PEG-b-PLA、10mg的CBT-PEG-b-PLA和10mg的SACys-PEG-b-PLA分别溶于1mL的DMSO中,分别向DACys-PEG-b-PLA、CBT-PEG-b-PLA和SACys-PEG-b-PLA材料溶液中加入100μg的DiD(2mg/mL,DMSO,50μL)后涡旋混合均匀,在搅拌条件下,将上述混合材料溶液分别滴入水相(1×PBS,pH 7.4,10mL)中搅拌2h,使用YM-30超滤离心管(Millipore,MWCO 5000Da)进行超滤洗涤浓缩纯化,分别制备得DiDD-NP、DiDC-NP和DiDS-NP纳米颗粒。
将4T1荷瘤小鼠随机分为两组,每组3只。将DiD标记的混合颗粒溶液经尾静脉注射给药,颗粒给药量为每只小鼠注射2mg。分别在给药4h,8h,12h,24h,48h,72h和96h时间点,用小动物活体成像仪观察肿瘤部位的荧光(Ex:644nm,Em:665nm)。
如图11所示,与非酸响应S-NP/C-NP颗粒组比较,D-NP/C-NP经尾静脉注射后能在肿瘤部位更多的富集以及更长时间的滞留。
5、D-NP和C-NP包载胞外靶点抗肿瘤药物巴马司他的体外释放行为
为了研究D-NP/C-NP纳米载体***递送胞外靶点抗肿瘤药物的抗肿瘤疗效,选用巴马司他(Batimastat,BB94)作为一种胞外靶点抗肿瘤药物模型。BB94是一种有效的广谱的胞外基质金属蛋白酶(matrix metalloproteinase,MMP)抑制剂,可抑制肿瘤组织胞外MMP-1,MMP-2,MMP-9,MMP-7和MMP-3等酶的活性,从而抑制肿瘤的生长和转移。
单乳化法制备包载巴马司他(BB94)的三种纳米载药颗粒,具体为:分别称取10mg的DACys-PEG-b-PLA、10mg的CBT-PEG-b-PLA和10mg的SACys-PEG-b-PLA聚合物材料溶于600μL二氯甲烷中,分别向三种材料溶液中加入1.5mg的巴马司他(BB94),涡旋混合均匀。取一干净50mL离心管加入8mL 1×PBS,用移液枪吸取材料混合溶液注射至离心管底部。将超声探头深入离心管底部伸入有机相中,启动超声(65W),超声2min,超声10s,间隔2s。超声完毕,将颗粒乳化液经旋转蒸发除去DCM,再使用YM-30超滤离心管(Millipore,MWCO 5000Da)对澄清颗粒溶液进行超滤洗涤和浓缩,得到D-NPBB94、C-NPBB94和S-NPBB94载药纳米颗粒。吸取100μL颗粒溶液冻干后重新溶于1mL DMSO中,配制BB94标准曲线溶液,检测标准曲线各颗粒样品在285nm处的吸光度,根据标准曲线计算各载药颗粒的载药量。
纳米颗粒包载BB94的载药量(drug loading content,DLC)及包封效率(encapsulation efficiency,EE)通过以下公式算得:
载药量(%)=(颗粒包载BB94的总质量/包载BB94的纳米颗粒总质量)×100%;包封率(%)=(颗粒包载BB94的总质量/BB94总的投药量)×100%。
BB94从两种载药纳米颗粒中的释放行为研究在含有0.02mol L-1的磷酸盐缓冲液(phosphate buffered saline,PBS,pH=7.4或5.5)中进行。做三组平行实验,分别取1mL的D-NPBB94/C-NPBB94或S-NPBB94/C-NPBB94混合载药纳米颗粒([BB94]=90μg mL-1)重悬于pH=7.4或6.5的0.02M PBS中,将两组载药混合纳米颗粒置于透析袋(Float-A-Lyzer,MWCO=14000)中,再将透析袋置于三组含15mL的PBS缓冲液(pH=7.4或6.5)的50mL离心管中,释放过程于37℃摇床(80rpm)下进行。在指定的时间将释放外液全取出,并补充等量的新鲜缓冲液。通过HPLC分析释放外液中BB94的浓度。
如图12所示,BB94可从D-NPBB94/C-NPBB94或S-NPBB94/C-NPBB94缓慢释放出来,96h内释放量达到60%左右。
6、D-NP和C-NP体内药代动力学实验及其体内抗肿瘤疗效
(1)药代动力学实验
选取9只ICR小鼠随机均等的分成3个小组,通过尾静脉分别注射Cy5标记的纳米颗粒溶液(Cy5D-NP、Cy5C-NP和Cy5S-NP,[NP]=2mg/只)。在预先设定的时间点从小鼠眼眶取血、离心后取上层血清。配制各颗粒的标准曲线溶液,用多功能孔板检测***(CytationTM 5,BioTek,Winooski,VT,USA;Ex:620nm,Em:650nm)检测各血清样品中的颗粒含量。
如图13所示,Cy5D-NP、Cy5C-NP和Cy5S-NP三种纳米颗粒在小鼠体内具有类似的循环过程。
(2)体内抗肿瘤治疗试验
随机将20只成瘤(肿瘤大小~150mm3)的4T1荷瘤小鼠分为四组。在第1、3、5天通过尾静脉分别给小鼠注射PBS、游离BB94(2%吐温20助溶)、S-NPBB94/C-NPBB94和D-NPBB94/C-NPBB94,其中BB94给药剂量为5mg/kg小鼠体重,每隔一天量取小鼠的体重和肿瘤块长度、宽度。在治疗27天后,处死小鼠取出完整肺组织并对转移结节计数。并检测各实验组小鼠体重变化。肿瘤体积的计算公式如下:体积(mm3)=0.5×长×宽2。
如图14所示,与PBS组对比,free BB94和S-NPBB94/C-NPBB94对原位肿瘤的抑制效果都不明显;而D-NPBB94/C-NPBB94治疗组的小鼠肿瘤生长被显著抑制,表明可原位组装的D-NP/C-NP纳米载体***能提高胞外靶点药物巴马司他的抗肿瘤疗效。
如图15所示,在治疗27天后,D-NPBB94/C-NPBB94能显著减少小鼠肺组织的转移结节数,与PBS组对比减少了94%,表明D-NP/C-NP纳米载体***递送巴马司他具有良好的抗肿瘤转移功效。
如图16所示,在治疗的末期,PBS组和Free BB94治疗组的小鼠体重有明显急剧下降的趋势,但是D-NPBB94/C-NPBB94治疗组小鼠在整个治疗周期,小鼠体重无明显变化,表明D-NP/C-NP递送BB94不会对小鼠造成严重的毒副作用造成体重急剧下降。
最后所应当说明的是,以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制,尽管参照较佳实施例对本发明作了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的实质和范围。
Claims (10)
1.一种聚乙二醇-聚乳酸材料,其特征在于,所述聚乙二醇-聚乳酸材料为在聚乙二醇-聚乳酸材料末端修饰半胱氨酸或2-氰基苯并噻唑的材料。
2.一种聚乙二醇-聚乳酸材料,其特征在于,所述聚乙二醇-聚乳酸材料为用2,3-二甲基马来酸酐屏蔽权利要求1所述聚乙二醇-聚乳酸材料的半胱氨酸残基的材料。
3.一种权利要求1所述的聚乙二醇-聚乳酸材料的制备方法,其特征在于,包括以下步骤:
(1)通过羧基化聚乙二醇引发丙交酯开环聚合,得羧基化的聚乙二醇-聚乳酸;或通过叔丁氧羰基保护的氨基化聚乙二醇引发丙交酯开环聚合,脱去叔丁氧羰基,得氨基化的聚乙二醇-聚乳酸;
(2)通过酰胺化反应,在所述氨基化的聚乙二醇-聚乳酸末端共价修饰上半胱氨酸基团,得到末端半胱氨酸修饰的聚乙二醇-聚乳酸材料;或在所述羧基化聚乙二醇-聚乳酸末端共价修饰上2-氰基苯并噻唑基团,得到末端2-氰基苯并噻唑修饰的聚乙二醇-聚乳酸材料。
4.一种权利要求2所述的聚乙二醇-聚乳酸材料的制备方法,其特征在于,包括以下步骤:
(1)通过叔丁氧羰基保护的氨基化聚乙二醇引发丙交酯开环聚合,脱去叔丁氧羰基,得氨基化的聚乙二醇-聚乳酸;
(2)通过酰胺化反应,在所述氨基化的聚乙二醇-聚乳酸末端共价修饰上半胱氨酸基团,得末端半胱氨酸修饰的聚乙二醇-聚乳酸材料;
(3)利用2,3-二甲基马来酸酐屏蔽所述末端半胱氨酸修饰的聚乙二醇-聚乳酸材料的半胱氨酸残基,即得。
5.一种纳米颗粒,其特征在于,包括权利要求1所述的聚乙二醇-聚乳酸材料和/或权利要求2所述的聚乙二醇-聚乳酸材料。
6.一种权利要求5所述的纳米颗粒的制备方法,其特征在于,所述方法为纳米沉淀法或单乳化法。
7.一种肿瘤原位组装的纳米载体***,其特征在于,包括权利要求5所述的纳米颗粒。
8.一种肿瘤原位组装的纳米载药体系,其特征在于,包括权利要求6所述的肿瘤原位组装的纳米载体***和抗肿瘤药物。
9.权利要求1或2所述的聚乙二醇-聚乳酸材料、权利要求3或4所述的制备方法、权利要求5所述的纳米颗粒、权利要求6所述的制备方法、权利要求7所述的纳米载体***、权利要求8所述的纳米载药体系在制备胞外/膜上靶点抗肿瘤药物中的应用。
10.权利要求1或2所述的聚乙二醇-聚乳酸材料、权利要求3或4所述的制备方法、权利要求5所述的纳米颗粒、权利要求6所述的制备方法、权利要求7所述的纳米载体***、权利要求8所述的纳米载药体系在制备抗肿瘤转移药物中的应用。
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