CN107596366B - 一种具有多重刺激响应型药物控释功能的诊断治疗制剂及其制备方法和应用 - Google Patents
一种具有多重刺激响应型药物控释功能的诊断治疗制剂及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种具有多重刺激响应型药物控释功能的诊断治疗制剂及其制备方法和应用,所制备的制剂以聚吡咯纳米粒子为核心,外部修饰环糊精‑化疗药物复合物和靶向基团,具有多重刺激响应型药物控释、光声成像、化疗和光热联合治疗的效果。该制剂同时结合了pH值、明胶酶、光热响应三种药物控释机制,提高了化疗药物对肿瘤的选择性;将成像和治疗相融合,可以实时监测制剂的体内分布,引导光热治疗时激光照射的位置、功率和时间;将化疗和光热治疗相结合,有效降低了肿瘤的复发率。该复合制剂有效增加了诊疗效率,改善药效,降低了毒副作用,具有很好的临床应用前景。
Description
技术领域
本发明属于生物医用材料领域,具体涉及一种具有多重刺激响应型药物控释功能的聚吡咯-环糊精诊断治疗纳米体系及其制备方法和应用。
背景技术
化疗是癌症治疗的重要手段。然而,单纯的小分子化疗药物进入人体后,经肝脏、肾脏等器官快速代谢、排泄,体内半衰期和平均滞留时间较短。先进的药物递送系统可以很好地解决这一问题。将化疗药物装载于纳米药物载体,可以大大延长化疗药物的体内半衰期,从而增加药效。但是,单纯的小分子化疗药物和普通的药物递送系统仍然存在肿瘤靶向性不足、毒副作用较大的问题。开发具有药物控释能力的智能化的药物递送系统正是为了解决这一问题。优良的纳米控释药物递送系统在血液循环系统正常的生理条件下,释放药物量很少,但是在肿瘤部位特定的刺激条件下,实现药物的快速和大量释放,由此增加了肿瘤部位对化疗药物的摄取量,减少了正常组织器官对药物的摄取,在进一步提高药效的同时,降低了药物的毒副作用。常用的药物控释的刺激条件包括pH值、温度、肿瘤高表达的酶、外加磁场等。但是,单一的刺激条件对药物释放的促进能力是有限的,比如,肿瘤的酸性微环境在不同类型的肿瘤间有很大的差异。同时采用多重刺激条件将大大增加药物的快速和彻底释放。
传统的化疗、放疗等治疗方法存在选择性差、毒副作用大等问题。光热治疗是通过体内注射光敏剂,体外给予激光照射,对肿瘤部位进行定点消融的治疗手段。光热转换剂将吸收的光能转化为热能,使肿瘤部位的温度升高,从而杀伤肿瘤细胞。由于采用激光定点照射,并且激光波长多位于近红外区,对正常组织没有损伤,相对于化疗和放疗等传统治疗方法,光热治疗的选择性高,副作用小。
光热转换剂的研发是光热治疗的研究热点。作为光热转换剂的小分子近红外染料,光稳定性差,易漂白;石墨烯、碳纳米管等碳纳米材料,光热转换效率低;金银纳米材料、硫化铜等金属纳米材料,毒性较大。
发明内容
本发明的目的之一是提供一种以明胶作为稳定剂的聚吡咯纳米粒子,以提高聚吡咯的生物安全性,解决聚吡咯表面修饰操作复杂的问题,同时赋予纳米粒子明胶酶敏感性药物控释的能力。
本发明的目的之二是提供一种同时具有多重刺激响应型药物控释功能和光声成像、光热-化疗联合治疗效果的诊断治疗一体化的纳米药物制剂,解决临床上化疗药物半衰期短、药物肿瘤靶向性差和副作用大、单一治疗模态肿瘤复发率高、诊断和治疗试剂需要分别给药等问题,用一种智能化的药物递送系统,同时也是一种纳米诊疗制剂,来实现:(1)增加化疗药物在肿瘤部位的靶向释放、减少药物副作用;(2)通过联合治疗提高治疗效果、减少肿瘤复发;(3)实现诊断和治疗的融合,减少分别给药给病人带来的不便。
为达到上述目的,本发明构建一种基于聚吡咯和环糊精复合物的药物控释递送系统兼诊断治疗制剂,该制剂以聚吡咯纳米粒子为内核,聚吡咯外层是作为稳定剂的明胶,明胶上偶联环糊精,并以环糊精的疏水空腔装载抗癌化疗药物,明胶上同时修饰靶向分子,以提高纳米粒子的靶向性。
进一步的,所述诊断治疗制剂的实体直径为10-200纳米。
进一步的,所述抗癌药物包括但不限于阿霉素、丝裂霉素、紫杉醇、喜树碱、氟尿嘧啶、甲氨蝶呤、苯丁酸氮芥、奥沙利铂、虫草素、伊立替康、索拉非尼、吉非替尼、莫达非尼、多西他赛、姜黄素、蓝萼甲素、鬼臼毒素。
进一步的,所述明胶来源于牛、猪或鱼,通过碱解法、酸解法或酶解法制备,分子量在10000-100000之间,胶强度在10-1000之间。
进一步的,所述靶向分子包括但不限于抗体及其片段、多肽、叶酸、透明质酸、适配体等各种类型的对肿瘤细胞、肿瘤微环境、肿瘤新生血管有靶向作用的分子。
诊断治疗制剂具有pH值响应性、明胶酶响应性、光热响应性的多重刺激响应的药物控释功能。
所述的具有多重刺激响应型药物控释功能的诊断治疗制剂的制备方法,包括如下步骤:
1)通过水基分散聚合法(aqueous dispersion polymerization)或称微乳化法(microemulsion)制备以明胶为稳定剂的聚吡咯纳米粒子:将单体和氧化剂分散于明胶水溶液中搅拌反应,单体发生氧化聚合生成聚吡咯纳米粒子,通过超滤、透析或离心纯化生成的聚吡咯纳米粒子;单体和氧化剂加入明胶水溶液的顺序不分先后;
2)环糊精与明胶的共价偶联:通过化学偶联反应将环糊精的功能基团与步骤1)制备的聚吡咯纳米粒子中的明胶的功能基团相偶联,从而将环糊精修饰到纳米粒子的表面,得到环糊精修饰的纳米粒子;
3)将抗癌药物包载到环糊精中:将步骤2)制备的环糊精修饰的纳米粒子与疏水抗癌药物分散于有机溶剂中,装入透析袋后,在水相中透析,得到包载抗癌药物后的的纳米粒子;并通过离心或过滤除去没有包载到环糊精中而析出的抗癌药物;
4)将靶向分子与明胶共价偶联:通过化学偶联反应将靶向分子的功能基团与步骤3)制备的包载抗癌药物后的纳米粒子中的明胶的功能基团相偶联。
进一步的,步骤1)中所述单体包括但不限于吡咯或吡咯的羧基化衍生物;所述氧化剂包括但不限于氯化铁、硫酸铁、过硫酸铵、过硫酸钾、过氧化氢、氯化铜;步骤2)中所述环糊精包括但不限于α-、β-、γ-环糊精及其羟丙基化、磺丁基化、氨基化、羧甲基化、羧基化衍生物,环糊精的功能基团包括但不限于α-、β-、γ-环糊精及其衍生物的羟基、氨基、羧基、巯基、磺基;明胶的功能基团包括但不限于明胶本身具有的氨基、羧基、巯基、羟基或经修饰引入的功能基团;步骤3)中所述有机溶剂包括但不限于二甲基亚砜、甲醇、乙醇、丙醇、异丙醇、丙二醇。
本发明的有益效果:
本发明的优点之一是明胶作为聚吡咯的稳定剂,相对于传统的人工合成的高分子稳定剂,提高了聚吡咯的生物安全性,同时引入了丰富的可修饰基团,解决了聚吡咯表面修饰流程复杂的问题,并赋予纳米粒子明胶酶响应的性能。
本发明的优点之二是所述制剂同时具有多重刺激响应的药物控释,可以提高药物在肿瘤组织的富集,减少药物的毒副作用。
本发明的优点之三是所述制剂将光热治疗和化疗融合于一体,即改善了治疗效果,减少了肿瘤复发,又实现了光热治疗和化疗药物的光热控释的巧妙结合。
本发明的优点之四是所述制剂将光声成像和光热-化疗联合治疗结合在一起,实现诊断和治疗的融合,减少病人的给药次数,实现影像引导下的治疗和监测药物的分布及评价治疗效果。
聚吡咯作为一种优良的光热转换剂,具有不易漂白、光热转换系数大、生物安全性高等优势。传统的聚吡咯纳米粒子通常以聚乙烯吡咯烷酮、聚乙烯醇等人工合成的高分子材料作为稳定剂。明胶是胶原蛋白的水解产物,其本质是分子量较大的多肽,由于缺少苯基氨基酸,免疫原性低,具有极佳的生物安全性。以明胶作为稳定剂,将进一步提高聚吡咯的生物安全性。同时,传统的聚吡咯表面修饰,如果采用层层堆叠(layer by layer)的方法,操作复杂;如果通过掺杂羧基吡咯的方法,引入的官能团单一。但是,如果将明胶作为聚吡咯的稳定剂,明胶中丰富的氨基、羧基就可以为聚吡咯的表面修饰提供丰富的官能团。最后,明胶可以被肿瘤细胞高表达的明胶酶(基质金属蛋白酶)特异性地降解,使得负载于明胶上的药物实现酶敏感性药物控释。因而,明胶作为聚吡咯的稳定剂,具有传统稳定剂不可比拟的优势。
除了光热治疗,聚吡咯同时具有光声成像的作用。光声成像同时结合了光学成像多光谱成像的特异性和超声成像组织分辨率高的优势。诊治一体化是当前纳米药物研究的热点之一,且具有很大的现实意义。传统意义上的诊断试剂和治疗试剂是相互独立的两个概念,诊断和治疗需要分别给药,流程繁琐,且增加了病人的痛苦。将诊断功能和治疗功能同时融合于同一载体,可以减少给药次数,对于光热治疗来说,将成像和治疗统一于同一载体,可以通过影像学手段确定肿瘤位置,实时监测光敏剂在肿瘤部位的富集情况,确定激光照射的部位、最佳的激光照射时间和照射功率,已达到最理想的治疗效果。
将智能化的药物递送系统与诊治一体化的纳米诊疗平台融合在一起,就可以通过影像学的手段实时监测化疗药物在体内的分布、评价治疗效果。在本发明中,我们将聚吡咯的光热效应与化疗药物光热控释巧妙结合,在提高药物靶向性的同时,实现了光热治疗和化疗联合治疗的目的。单纯的化疗治疗效果有限,光热治疗对肿瘤的消融虽然立竿见影,但是复发率高,而联合治疗可以融合两者的优势,达到协同治疗的效果,减少肿瘤的复发。
附图说明
图1:实施例1所述的该制剂的制备流程示意图。
图2:实施例1所得到的该制剂的透射电镜图(该制剂为球形纳米粒子,实体直径约70纳米,粒径均匀,可见明显的核壳样结构)。
图3:尾静脉注射实施例1制备的该制剂后,荷瘤鼠肿瘤部位的光声成像图(具体实验流程如实施例3所述。注射该制剂后的24小时内,肿瘤部位的光声信号逐渐增强,并最终达到了良好的增强肿瘤光声成像的效果)。
图4:实施例1制备的该制剂的多重刺激响应型药物控释的体外效果评价(具体实验流程如实施例4所述,第I,II,III阶段分别反映了该制剂优良的明胶酶响应性、pH值响应性和光热响应性的药物控释效果)。
图5:实施例1制备的该制剂对荷瘤鼠的皮下移植瘤的治疗效果评价,纵坐标为测量当日肿瘤体积与第0天肿瘤大小的比值(具体实验流程如实施例5所述,该制剂的光热和化疗联合治疗使肿瘤完全消融,且没有产生肿瘤的复发)。
具体实施方式
本发明具体实施方式所列举的实施例只用于说明本发明,并不限制本发明的内容。
实施例1
本发明中所述一种具有多重刺激响应型药物控释功能的诊断治疗制剂的一种制备方案如附图1所示,包括以下步骤:
(1) 以明胶为稳定剂的聚吡咯纳米粒子的合成:将500 mg深水鱼明胶加入到40mL水中,50℃加热搅拌20分钟使明胶完全溶解。冷却到室温后,向溶液中加入0.82 g六水三氯化铁,继续室温搅拌1 h。将溶液置于冰水浴中,向溶液中缓慢滴加139 μL的吡咯,持续搅拌24 h。17000 g离心40 min得到聚吡咯纳米粒子,并用去离子水重新分散后离心,反复洗涤3次,得到纯净的聚吡咯纳米粒子,将母液保存在4℃冰箱中储存。
(2) 将羧甲基环糊精偶联到聚吡咯纳米粒子:称取20 mg单取代羧甲基环糊精,溶解于1 mL去离子水中,加入6.4 mg 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和18 mgN-羟基硫代琥珀酰亚胺,置于振荡器中振荡15分钟,加入2.36 μL β-巯基乙醇终止反应,再加入10 mg溶解于1 mL磷酸盐缓冲液(0.2 M, pH=8.0)的聚吡咯纳米粒子,继续振荡2 h。将反应液加入到分子截留量为1000 kDa的透析袋中,在纯水中透析48 h,并不断更换透析液,最终得到纯净的羧甲基环糊精修饰的聚吡咯纳米粒子。
(3) 将抗癌药物阿霉素装载到羧甲基环糊精修饰的聚吡咯纳米粒子中:将10 mg羧甲基环糊精修饰的聚吡咯纳米粒子、5 mg盐酸阿霉素、3.8 μL三乙胺加入到10 mL二甲基亚砜中,水浴超声混匀,装入分子截留量为1000 kDa的透析袋中,在磷酸盐缓冲液(0.01 M,pH=7.2-7.4)中透析48 h,并不断更换透析液,最终得到纯净的载药纳米粒子。
(4) 将靶向抗体与载药纳米粒子偶联:以靶向甲状腺癌细胞的抗半乳糖凝集素3抗体为例,将10 mg载药纳米粒子、0.4 mg 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和1.1 mg N-羟基硫代琥珀酰亚胺溶解于1 mL去离子水中,振荡15分钟后,加入1.4 μL β-巯基乙醇终止反应,最后加入1 mg溶解于1 mL磷酸盐缓冲液(0.06 M, pH=7.5)的抗体,振荡2h后,将反应液装入分子截留量1000 KDa的透析袋中,在磷酸盐缓冲液(0.01 M, pH=7.2-7.4)中透析48 h,并不断更换透析液,最终得到纯净的最终形式的本发明中所述的制剂。
实施例2
本发明中所述一种具有多重刺激响应型药物控释功能的诊断治疗制剂的一种制备方案如下所述:
(1) 制备羧甲基环糊精修饰的明胶:称取单取代羧甲基环糊精,溶解于去离子水中,加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N-羟基硫代琥珀酰亚胺活化羧基,置于振荡器中振荡15分钟,加入β-巯基乙醇终止反应,再加入溶解于磷酸盐缓冲液(0.2 M,pH=8.0)的深水鱼明胶,继续振荡2 h。将反应液加入到分子截留量为2000 Da的透析袋中,在纯水中透析48 h,并不断更换透析液,最终得到纯净的羧甲基环糊精修饰的明胶。
(2) 以环糊精修饰的明胶为稳定剂的聚吡咯纳米粒子的合成:将500 mg羧甲基环糊精修饰的深水鱼明胶加入到40 mL水中,50℃加热搅拌20分钟使明胶完全溶解。冷却到室温后,向溶液中加入0.82 g六水三氯化铁,继续室温搅拌1 h。将溶液置于冰水浴中,向溶液中缓慢滴加139 μL的吡咯,持续搅拌24 h。17000 g离心40 min得到聚吡咯纳米粒子,并用去离子水重新分散后离心,反复洗涤3次,得到纯净的聚吡咯纳米粒子,将母液保存在4℃冰箱中储存。
(3) 将抗癌药物阿霉素装载到聚吡咯纳米粒子中:将10 mg聚吡咯纳米粒子、5 mg盐酸阿霉素、3.8 μL三乙胺加入到10 mL二甲基亚砜中,水浴超声混匀,装入分子截留量为1000 kDa的透析袋中,在磷酸盐缓冲液(0.01 M, pH=7.2-7.4)中透析48 h,并不断更换透析液,最终得到纯净的载药纳米粒子。
(4) 将靶向抗体与载药纳米粒子偶联:以靶向甲状腺癌细胞的抗半乳糖凝集素3抗体为例,将10 mg载药纳米粒子、0.4 mg 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和1.1 mg N-羟基硫代琥珀酰亚胺溶解于1 mL去离子水中,振荡15分钟后,加入1.4 μL β-巯基乙醇终止反应,最后加入1 mg溶解于1 mL磷酸盐缓冲液(0.06 M, pH=7.5)的抗体,振荡2h后,将反应液装入分子截留量1000 KDa的透析袋中,在磷酸盐缓冲液(0.01 M, pH=7.2-7.4)中透析48 h,并不断更换透析液,最终得到纯净的最终形式的本发明中所述的制剂。
实施例3
本发明一种具有多重刺激响应型药物控释功能的诊断治疗制剂的小动物活体光声成像效果评价,主要包括如下步骤:
(1) 裸鼠皮下瘤模型的建立:将TPC-1细胞以每只裸鼠 5×106个细胞的量接种于BALB/c nude鼠的大腿外侧皮下,裸鼠于SPF级环境下饲养;
(2) 肿瘤大小长到约200 mm3时,经尾静脉注射200 μL 5 mg/mL的实施例1制备的该制剂,分别在给药前和给药后的不同时间后,用小动物光声成像系统检测肿瘤部位的光声信号。
成像结果如附图3所示,注射该制剂后,肿瘤部位的光声信号逐渐增强,在24小时,光声信号达到最强,且弥散于整个肿瘤部位,相对于给药前,肿瘤可以清晰成像,说明该制剂具有很好的增强光声成像的效果。
实施例4
本发明一种具有多重刺激响应型药物控释功能的诊断治疗制剂的多重响应型药物控释效果的体外评价,包括pH值敏感性、明胶酶敏感性和光热敏感性药物控释评价,步骤如下所述:
(1) 首先,在实验的第I阶段研究明胶酶敏感性药物控释效果。将10 mg具体实施方式一制备的该制剂和25 μg活化的明胶酶溶解于5 mL明胶酶激活液中,装入分子截留量2500 Da的透析袋后,在50 mL明胶酶激活液中透析,每隔一定时间,取200 μL透析液测定抗癌药物阿霉素的含量,并计算累积药物释放量。两组对照组分别不加入明胶酶或者在加入明胶酶的同时,加入过量的明胶酶的抑制剂。
(2) 接下来,在该实验的第II阶段,研究该制剂的pH值敏感性药物控释效果。将在第I阶段中透析8 h的装有纳米粒子的透析袋放入50 mL不同pH值的、含有0.1% 吐温-80(Tween-80)的磷酸盐缓冲液中,继续透析16 h,每隔一定时间,取200 μL透析液测定抗癌药物阿霉素的含量,并计算包括第I阶段在内的累积药物释放量。
(3) 最后,在该实验的第III阶段,把在第I阶段中透析8 h并在第II阶段中透析8h的纳米粒子分散液用波长为808 nm的激光器照射10 min,功率设定为2 W/cm2,随后停止照射,冷却110分钟,重复照射和冷却四次,并且在每次照射前后,取分散液在30000 g转速下离心20分钟,取上清液测定释放出来的阿霉素的含量,计算从第I阶段开始的总的累积药物释放量。
实验结果如附图4所示,在第I阶段中,有明胶酶作用的该制剂药物释放量最大;在第II阶段中,在酸性条件下,该制剂的药物释放量会迅速增加;在第III阶段中,每次近红外激光照射都会引起药物释放量的迅速增加。总的来看,三种刺激综合作用下,药物的累积释放量最大,达到了100%的药物释放。该实验说明了该制剂达到了很好的三重刺激响应型药物控释效果。
实施例5
本发明一种具有多重刺激响应型药物控释功能的诊断治疗制剂的肿瘤治疗效果评价,主要步骤如下所述:
(1) 裸鼠皮下瘤模型的建立:将TPC-1细胞以每只裸鼠 5×106个细胞的量接种于BALB/c nude鼠的大腿外侧皮下,裸鼠于SPF级环境下饲养;
(2) 当肿瘤大小达到约110 mm3时,将小鼠平均分为6组,每组5只,每组分别经尾静脉分别注射200 μL 5 mg/mL的该制剂、200 μL 5 mg/mL没有装载阿霉素的该制剂或者200 μL 0.51 mg/mL单纯的盐酸阿霉素(给药量相当于该制剂的载药量),对照组注射200μL磷酸盐缓冲液。
(3) 对于需要光热治疗的实验组,在给药后24 h,用波长为800 nm的激光照射肿瘤部位10 min,激光功率设定为2 W/cm2。
(4) 每隔一天用游标卡尺测定肿瘤的长和宽,肿瘤体积近似等于长乘以宽的平方除以二。
实验如附图5所示,只注射磷酸盐缓冲液的对照组,肿瘤生长迅速,并且单纯的激光照射对肿瘤生长没有影响;单纯注射没有装载阿霉素的该制剂或者单纯注射盐酸阿霉素,肿瘤生长受到的抑制很小;注射装载有阿霉素的该制剂时,肿瘤生长受到明显的抑制,说明该制剂可以提高阿霉素的药效;注射没有装载阿霉素的该制剂后给予激光照射,肿瘤被消融掉,但是肿瘤出现复发;当注射装载有阿霉素的该制剂后给予激光照射后,肿瘤被彻底消融掉,而且没有出现复发,这说明该制剂具有很好的光热治疗的效果,而且该制剂装载的阿霉素可以起到防止肿瘤复发的协同治疗的效果。
Claims (9)
1.一种具有多重刺激响应型药物控释功能的诊断治疗制剂,其特征在于:所述诊断治疗制剂以聚吡咯纳米粒子为内核,所述聚吡咯纳米粒子包括内层的聚吡咯和外层的明胶;明胶上偶联环糊精和靶向分子,并以环糊精的疏水空腔装载抗癌药物。
2.根据权利要求1所述的一种具有多重刺激响应型药物控释功能的诊断治疗制剂,其特征在于:所述明胶为聚吡咯纳米粒子的稳定剂。
3.根据权利要求1所述的一种具有多重刺激响应型药物控释功能的诊断治疗制剂,其特征在于:所述诊断治疗制剂具有pH值响应性、明胶酶响应性、光热响应性的多重刺激响应的药物控释功能。
4.根据权利要求1所述的一种具有多重刺激响应型药物控释功能的诊断治疗制剂,其特征在于:所述诊断治疗制剂的实体直径为10-200纳米。
5.根据权利要求1所述的一种具有多重刺激响应型药物控释功能的诊断治疗制剂,其特征在于:所述抗癌药物包括但不限于阿霉素、丝裂霉素、紫杉醇、喜树碱、氟尿嘧啶、甲氨蝶呤、苯丁酸氮芥、奥沙利铂、虫草素、伊立替康、索拉非尼、吉非替尼、莫达非尼、多西他赛、姜黄素、蓝萼甲素、鬼臼毒素。
6.根据权利要求1所述的一种具有多重刺激响应型药物控释功能的诊断治疗制剂,其特征在于:所述明胶来源于牛、猪或鱼,通过碱解法、酸解法或酶解法制备,分子量在10000-100000之间,胶强度在10-1000之间。
7.根据权利要求1所述的一种具有多重刺激响应型药物控释功能的诊断治疗制剂,其特征在于:所述靶向分子包括但不限于抗体及其片段、多肽、叶酸、透明质酸、适配体。
8.权利要求1~7中任意一项所述的具有多重刺激响应型药物控释功能的诊断治疗制剂的制备方法,包括如下步骤:
1)通过水基分散聚合法制备以明胶为稳定剂的聚吡咯纳米粒子:将单体和氧化剂分散于明胶水溶液中搅拌反应,单体发生氧化聚合生成聚吡咯纳米粒子,通过超滤、透析或离心纯化生成的聚吡咯纳米粒子;单体和氧化剂加入明胶水溶液的顺序不分先后;
2)环糊精与明胶的共价偶联;通过化学偶联反应将环糊精的功能基团与步骤1)制备的聚吡咯纳米粒子中的明胶的功能基团相偶联,从而将环糊精修饰到纳米粒子的表面,得到环糊精修饰的纳米粒子;
3)将抗癌药物包载到环糊精中:将步骤2)制备的环糊精修饰的纳米粒子与疏水抗癌药物分散于有机溶剂中,装入透析袋后,在水相中透析,得到包载抗癌药物后的纳米粒子;并通过离心或过滤除去没有包载到环糊精中而析出的抗癌药物;
4)将靶向分子与明胶共价偶联:通过化学偶联反应将靶向分子的功能基团与步骤3)制备的包载抗癌药物后的纳米粒子中的明胶的功能基团相偶联。
9.根据权利要求8所述的具有多重刺激响应型药物控释功能的诊断治疗制剂的制备方法,其特征在于:步骤1)中所述单体包括但不限于吡咯或吡咯的羧基化衍生物;所述氧化剂包括但不限于氯化铁、硫酸铁、过硫酸铵、过硫酸钾、过氧化氢、氯化铜;步骤2)中所述环糊精包括但不限于α-、β-、γ-环糊精及其羟丙基化、磺丁基化、氨基化、羧甲基化、羧基化衍生物,环糊精的功能基团包括但不限于α-、β-、γ-环糊精及其衍生物的羟基、氨基、羧基、巯基、磺基;明胶的功能基团包括但不限于明胶本身具有的氨基、羧基、巯基、羟基或经修饰引入的功能基团;步骤3)中所述有机溶剂包括但不限于二甲基亚砜、甲醇、乙醇、丙醇、异丙醇、丙二醇。
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