CN106074451B - 含碳纳米笼的还原刺激响应药物载体及制备方法和应用 - Google Patents
含碳纳米笼的还原刺激响应药物载体及制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种含碳纳米笼的还原刺激响应药物载体及制备方法和应用,以碳纳米笼CNC为核心,由树枝状高分子包覆在CNC表面,在CNC之间、高分子之间、以及CNC与高分子之间均有二硫键连接,所述的树枝状高分子为低代数G0、G1、G2和/或G3的聚酰胺‑胺树枝状高分子PAMAM,末端基团为氨基的PAMAM‑NH2或末端为羧基的PAMAM‑COOH。本发明还提供了制备方法和作为药物载体的应用。本发明所提供的纳米载体有很高的装载能力,在还原剂如谷胱甘肽的刺激下能灵敏地释药,激光照射也能促进药物释放。因肿瘤组织含高浓度的谷胱甘肽,本发明提供的载药粒子在肿瘤靶向治疗中有重要的药用前景。
Description
技术领域
本发明涉及一种含碳纳米笼的还原刺激响应药物载体及制备方法和应用,尤其是一种以树枝状高分子包覆的碳纳米笼(简称CNC)及制备方法和应用,由二硫键将树枝状高分子和CNC相互交联而成的纳米复合粒子,及其制备方法,该纳米复合粒子在载药后具有刺激响应性释药特别是还原刺激响应性释药的特点,属于生物材料领域。
背景技术
碳基纳米材料如石墨烯、碳纳米管、纳米钻石、富勒烯等在药物输送领域有重要的应用前景,它们的共同特点是生物相容性好,载药量高。为使药物缓释,往往希望药物载体为三维立体多孔结构或空心结构。石墨烯和纳米钻石作为药物载体,药物只能吸附在载体表面;碳纳米管是由石墨烯卷曲而成的中空管,但内径很小,一般不超过5 nm,药物也主要结合在碳纳米管外表面;富勒烯是笼状的碳纳米粒子,最常见的是由60个碳原子构筑而成的空心足球状粒子(即C60),直径仅为0.71 nm,药物可吸附在粒子表面,而内部空腔几乎不能装载药物。
近年来,内部空腔远比富勒烯大得多的CNC备受关注,这种CNC的壳层主要为石墨结构,石墨壳层中富含孔隙,孔径主要分布在2-50nm之间,内部空腔直径主要分布在10-100nm之间。这种CNC,不仅内部空腔能装载药物,壳层的内外表面亦能吸附药物,而且由于壳层为多孔结构,因此,CNC的比表面积比碳纳米管大得多,是非常好的药物载体。
当前,CNC作为载体,主要用于对重金属离子的吸附 [David M. Burke, et al.Carbon nanocages as heavy metal ion adsorbents. Desalination 280 (2011) 87–94];对儿茶素、茶单酸、组氨酸、维生素E、内分泌干扰物壬基酚的吸附[Katsuhiko Ariga,et al. One-Pot Separation of Tea Components through Selective Adsorption onPore-Engineered Nanocarbon, Carbon Nanocage. J. AM. CHEM. SOC. 2007, 129,11022-11023; Ajayan Vinu, et al. Carbon nanocage: a large-pore cage-typemesoporous carbon material as an adsorbent for biomolecules. J Porous Mater(2006) 13: 379–383.]、对溶菌酶的吸附[Ajayan Vinu, et al. Large pore cage typemesoporous carbon, carbon nanocage: a superior adsorbent for biomaterials. J.Mater. Chem., 2005, 15, 5122–5127];以及对香烟烟雾中的酚类化合物(如苯二酚)和总颗粒物(如焦油和尼古丁)的吸附 [Guangda Li, et al. General synthesis of carbonnanocages and their adsorption of toxic compounds from cigarette smoke.Nanoscale, 2011, 3, 3251-3257.]。未见CNC作为载体对肿瘤化疗药物、光敏剂等药物的吸附的报道。
作为输送药物的载体,希望其能将药物靶向释放到病灶(如肿瘤),以减少药物对正常组织的伤害。研究表明,刺激响应性药物载体可以选择性地将药物释放到肿瘤部位,是近年来肿瘤靶向治疗研究领域的热点。根据刺激方式的不同,这类载体主要有还原刺激响应性载体、热敏感性载体、pH敏感性载体、超声波刺激响应性载体、电刺激响应性载体等。由于肿瘤组织中具有较强还原性的谷胱甘肽浓度是正常组织的约500倍以上,细胞内谷胱甘肽浓度是细胞外约1000倍以上,基于人体(或动物体)内这种极显著差异的生物环境,还原刺激响应性载体能非常好地将其所携载的药物选择性地释放到肿瘤组织,其基本原理是:还原刺激响应性载体一般由二硫键交联而成,该载体在体内输送药物的过程中,在正常组织中保持载体结构的完整性,药物很少释放,一旦到达肿瘤组织尤其进入肿瘤细胞时,高浓度的谷胱甘肽将使载体中的二硫键断裂,载体破坏,从而促使载体释放药物。这类谷胱甘肽还原刺激响应性的药物载体,由于其在肿瘤组织中的释药灵敏性高,因而具有重要临床应用前景。
还原刺激响应性载体基本由高分子构成,尤其是两亲性高分子,它们在水相中能够自组装成纳米粒子,可包埋脂溶性药物,也可包埋水溶性药物。然而,载体的载药量较低。
例如:水溶性化疗药物阿霉素在由二硫键连接的聚乙二醇-聚己内酯共聚物[poly(ethylene glycol)-b-poly(ε-caprolactone) (PEG-S-S-PCL)]形成的纳米粒子内的载药量(药物质量与载体质量的比)仅6.0% [Huanli Sun, et al. Biodegradable micelleswith sheddable poly(ethylene glycol) shells for triggered intracellularrelease of doxorubicin. Biomaterials 30 (2009) 6358–6366];由二硫键连接的聚乙二醇-聚乳酸共聚物[poly(ethylene glycol)-b-poly(lactic acid) (MPEG-S-S-PLA)]自组装形成的纳米粒子对脂溶性化疗药物紫杉醇的载药量最高为9.07% [Na Song, et al.Preparation and in vitro properties of redox-responsive polymericnanoparticles for paclitaxel delivery. Colloids and Surfaces B: Biointerfaces87 (2011) 454–463];再如:由二硫键连接的聚乙二醇-聚赖氨酸-聚己内酯共聚物[ poly(ethylene glycol)-b-poly(lysine)-b-poly(caprolactone) bearing a disulfidebond (PEG-b-PLys-S-S-PCL)]自组装形成的纳米粒子,可同时包埋水溶性和脂溶性化疗药物,对水溶性阿霉素和脂溶性喜树碱的载药量分别是7.2%和4.4% [Thavasyappan Thambi,et al. Bioreducible polymersomes for intracellular dual-drug delivery. J.Mater. Chem., 2012, 22, 22028–22036]。这些载药粒子中的药物与载体的质量比均比较小。
此外,还原刺激响应性的载体,在动物体内刺激药物释放的方式主要是肿瘤组织及细胞内的还原剂谷胱甘肽使载体中的二硫键断裂,单一的刺激方式难以使药物充分释放。
而且,目前的还原刺激响应性载体的作用主要是运载药物,自身没有治疗肿瘤的功能。而肿瘤治疗需要功能强大的载药粒子。
发明内容
本发明目的在于:提供一种含碳纳米笼的还原刺激响应药物载体,由二硫键相互交联的树枝状高分子包覆的碳纳米笼。
本发明的再一目的在于:提供所述树枝状高分子包覆的碳纳米笼的制备方法。
本发明的又一目的在于:提供所述树枝状高分子包覆的碳纳米笼的用途。
本发明所示的一种含碳纳米笼的还原刺激响应药物载体,以碳纳米笼(简称CNC)为核心,由树枝状高分子包覆在CNC笼表面,在CNC之间、高分子之间、以及CNC与高分子之间均有二硫键连接,所述的树枝状高分子为指低代数G0、G1、G2和/或G3的聚酰胺-胺树枝状高分子PAMAM,末端基团为氨基的PAMAM-NH2或末端为羧基的PAMAM-COOH。
所述的CNC是指壳层具有石墨结构特点的空心多孔碳纳米粒子,表面含有羧基,粒径在20~100nm之间。
这种树枝状高分子包覆的CNC,制备过程在油包水型微乳中进行,包括下述步骤:
步骤一,配制如下各微乳:
(1)配制CNC油包水型微乳,微乳的水相是去离子水分散的CNC,水相中CNC浓度在0.05~2 mg/mL之间;
(2)配制EDC油包水型微乳,或“EDC+NHS”的油包水型微乳,所述的EDC,全称为1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride,也就是1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐;所述的NHS,全称为N-Hydroxysuccinimide,也就是N-羟基琥珀酰亚胺。EDC微乳的水相是去离子水溶解的EDC,“EDC+NHS”微乳的水相是去离子水溶解的EDC与NHS的混合物,EDC与NHS的质量比为1:(1~5) 之间;
(3)配制PAMAM-NH2油包水型微乳,微乳的水相是去离子水溶解的PAMAM-NH2,水相中PAMAM-NH2浓度在0.25-100 mg/mL之间;
(4)配制PAMAM-COOH油包水型微乳,微乳的水相是去离子水溶解的PAMAM-COOH,水相中PAMAM-COOH浓度在0.25-100 mg/mL之间;
(5)配制胱胺油包水型微乳,微乳的水相是去离子水溶解的胱胺,水相中胱胺浓度在2~500 μg/mL之间;
(6)配制二硫代二丙酸油包水型微乳,微乳的水相是去离子水溶解的二硫代二丙酸,水相中二硫代二丙酸浓度在0.01-10 mg/mL之间;
其中,所述的油包水型微乳,是由环己烷、Triton X-100、正己醇、水相溶液混合而成,在微乳体系中,Triton X-100 与正己醇的体积比在(1:1)~(3:1)之间,环己烷与“Triton X-100 + 正己醇”混合溶液的体积比在(3:1)~(3:2)之间。向环己烷、Triton X-100与正己醇三者的混合溶液所加入的水相体积的量保证混合体系不发生浑浊。
步骤二,进行如下合成反应:
(1)当采用PAMAM-NH2时,纳米载体制备方法为:将CNC微乳与EDC的微乳或与“EDC+NHS”的微乳混合0.5-3h,然后加入胱胺微乳,混合0.5-5h之后,加入PAMAM-NH2微乳,混合0.5-3 h,获得含CNC和PAMAM-NH2的微乳;同时,将二硫代二丙酸微乳与EDC/NHS微乳混合0.5-3h,然后,将该二硫代二丙酸微乳与含CNC和PAMAM-NH2的微乳混合0.5-5h,最后离心,用乙醇沉淀和去离子水洗涤,即得PAMAM包覆CNC的纳米复合粒子,该复合粒子中,CNC之间以二硫键相连、PAMAM之间以二硫键相连、CNC与PAMAM之间以酰胺键相连;反应体系中,CNC与PAMAM的质量比为1:(5-50),
EDC的量是使CNC上的部分或全部羧基活化的量,EDC与CNC的质量比最小为1:10000,胱胺与EDC质量比为1:(2-10),二硫代二丙酸与PAMAM-NH2的质量比为1:(10~25)。
(2)当采用PAMAM-COOH时,纳米载体制备方法为:将CNC微乳与PAMAM-COOH微乳混合,然后加入EDC/NHS微乳,混合0.5-3h;再加入胱胺微乳,混合0.5-5h;最后离心,用乙醇沉淀和去离子水洗涤,即得PAMAM包覆CNC的纳米复合粒子,该复合粒子中,CNC之间、PAMAM之间以及CNC与PAMAM之间均以二硫键相连; 反应体系中,CNC与PAMAM的质量比为1:(5-50),EDC的量是使CNC上的部分或全部羧基活化的量,EDC与CNC的质量比最小为1:10000,胱胺与EDC质量比为1:(2-10)。
本发明提供一种用途,在所述的树枝状高分子包覆的CNC纳米复合粒子中用物理吸附法装载肿瘤化疗药物和/或光敏剂,制成载药纳米粒子,这种载药纳米粒子在还原剂和激光照射双重刺激下响应释药,可作为肿瘤靶向治疗的材料。
将本发明构建的载药纳米粒子可通过肿瘤组织中谷胱甘肽的还原作用和外加近红外激光照射共同刺激而释放药物,近红外激光照射还诱导载药纳米粒子中的CNC产生光热转化,通过温热效应协同药物杀伤肿瘤细胞,抑制肿瘤生长,在肿瘤治疗领域有重要应用前景。
本发明提供一种全新的刺激响应性药物载体及其应用方法。本发明的载体由CNC与树枝状高分子经二硫键交联而成,不仅具有高的载药能力,而且可通过三种刺激方式释药,即由还原剂谷胱甘肽刺激载体释药;由激光照射使载体产生还原性电子,进攻二硫键而使载体结构破坏,进而释药;激光照射诱导CNC载体产生光热转换,热效应促进药物释放。此外,本发明的药物载体,其自身在激光照射下可对肿瘤进行光热治疗,兼具载体和治疗双重功能。
本发明与单纯的树枝状高分子形成的纳米粒子和单纯的CNC相比,优越性在于:本发明制备的由二硫键相互交联的CNC与树枝状高分子构建而成的纳米复合粒子在载药能力、药物刺激响应释放方式、对肿瘤的治疗能力这三方面具有显著优势和很大进步;本发明制得的所述纳米复合粒子有更高的载药能力,不仅能在还原剂(如谷胱甘肽)的刺激下响应释药,还可以通过近红外激光照射促进药物释放,而且,近红外激光还诱导纳米复合粒子中的CNC产生光热转换,使纳米复合粒子通过药物治疗和热疗的协同作用治疗肿瘤。
附图说明
附图1:(CNC-S-S-CNC)-CO-NH-(PAMAM-S-S-PAMAM)纳米复合粒子结构示意图;
附图2:CNC透射电子显微镜图;
附图3:纳米复合粒子(CNC-S-S-CNC)-CO-NH-(PAMAM-S-S-PAMAM)透射电子显微镜图;
附图4:(CNC-S-S-CNC)-S-S-(PAMAM-S-S-PAMAM) 纳米复合粒子结构示意图;
附图5:装载药物的(CNC-S-S-CNC)-S-S-(PAMAM-S-S-PAMAM)在谷胱甘肽作用下释放药物示意图;
附图6:装载药物的(CNC-S-S-CNC)-S-S-(PAMAM-S-S-PAMAM)在谷胱甘肽作用之后经过近红外激光照射所导致的药物释放和载体的光热转化示意图。
具体实施方式
实施例 1
含碳纳米笼的还原刺激响应药物载体及其制备方法:
微乳中合成“聚酰胺-胺树枝状高分子包覆的碳纳米笼”(高分子为末端基团为氨基的G0型PAMAM-NH2):
(1)具体合成方法
A.高温热分解合成碳纳米笼(Carbon nanocage, CNC)
称取1g草酸亚铁溶于10 mL无水乙醇中,溶液密封于不锈钢反应釜。将此反应釜置于马弗炉中于550°C加热反应,12h后停止加热,在炉中自然冷却至室温。取出样品,置于50mL圆底烧瓶中,加入30 mL浓度为10 %的盐酸,于70°C恒温回流12 h。将反应溶液离心,收集沉淀,用去离子水洗涤3次。向洗涤后的沉淀中加入30 mL硝酸,于70°C恒温回流12 h。然后将该反应液进行离心,沉淀经去离子水洗涤5次,洗涤后的沉淀进行冷冻干燥,所得CNC富含羧基。
B.制备微乳
将环己烷、正己醇、Triton X-100按如下体积比混合:正己醇: Triton X-100=3:2,环己烷:( 正己醇+Triton X-100)=3:1.67,也就是,将环己烷(72 mL)、Triton X-100(24 mL)、正己醇(16 mL)混合,得微乳前液,然后制备如下微乳:
◆ 制备表面富含羧基的CNC的微乳:称取CNC 10 mg,分散于10 mL去离子水中,超声波处理10 min,取该CNC溶液1 mL,并取微乳前液10 mL,二者混合,室温振荡2-3 min,即得CNC微乳;
◆ 制备PAMAM-NH2微乳:称取PAMAM-NH2 10 mg,溶于2 mL 去离子水中,然后与20mL微乳前液混合,室温振荡2-3 min,即得PAMAM-NH2微乳;
◆ 制备EDC/NHS微乳:称取10 mg的EDC和50 mg的NHS,共同溶于1 mL去离子水中,然后与10 mL微乳前液混合,室温振荡2-3 min,即得EDC/NHS微乳;
◆ 制备胱胺微乳:称取胱胺10mg,溶于10mL去离子水中。称取该混合液1mL,溶于5mL去离子水中,取0.1mL,与1mL“微乳母液”混合,室温振荡2-3min,即得胱胺微乳。
◆ 制备二硫代二丙酸微乳:
称取二硫代二丙酸10mg,溶于10 mL水溶液中搅拌2h。称取该混合液1mL,然后于10mL“微乳母液”混合,室温振荡2-3min,即得活化二硫代二丙酸微乳。
C. 微乳中合成二硫键相互交联的聚酰胺-胺树枝状高分子/CNC纳米复合粒子,结构式为(CNC-S-S-CNC)-CO-NH-(PAMAM-S-S-PAMAM):
在10 mL的CNC微乳中加入1.1μL的EDC/NHS微乳,摇床中室温振荡2 h;然后与1 mL胱胺微乳混合,摇床中室温振荡反应2 h,使CNC在微乳的纳米水核中以二硫键相互交联,即CNC-S-S-CNC;然后,向其中加入20 mL PAMAM-NH2微乳,室温振荡30 min,使PAMAM-NH2与CNC以酰胺键连接,即得(CNC-S-S-CNC)-CO-NH-PAMAM;
同时,向10 mL二硫代二丙酸微乳中加入5 mL的EDC/NHS微乳,摇床中室温振荡1h;将该微乳加入到上述(CNC-S-S-CNC)-CO-NH-PAMAM微乳中,摇床中室温振荡2 h,使PAMAM之间以二硫键交联的微乳反应体系,获得:
(CNC-S-S-CNC)-CO-NH-(PAMAM-S-S-PAMAM);
将该微乳反应体系进行在5000转/分钟离心15 min,用乙醇沉淀,洗涤3次;然后再用去离子水洗涤3次,即得二硫键相互交联的聚酰胺-胺树枝状高分子/CNC纳米复合粒子,也就是:(CNC-S-S-CNC)-CO-NH-(PAMAM-S-S-PAMAM),其结构示意图如附图1。
(2)透射电子显微镜(TEM)下分析微观形貌
取少许(CNC-S-S-CNC)-CO-NH-(PAMAM-S-S-PAMAM) 沉淀分散于去离子水中,将其滴于铜网上,于TEM(JSM-6360LV, JEOL, Japan)下观察分析。
(3)载药
精确称取5-氟尿嘧啶(5-Fu,一种肿瘤化疗药物)5 mg,溶于1 mL去离子水中,然后加入上述制备的(CNC-S-S-CNC)-CO-NH-(PAMAM-S-S-PAMAM) 约 5 mg,于摇床中室温振荡2h,然后离心,沉淀用去离子水谁洗涤1次,收集上清液和洗液,并保存沉淀。沉淀即为载药粒子(CNC-S-S-CNC)-CO-NH-(PAMAM-S-S-PAMAM)-(5-Fu)。
同时,测定266nm波长处不同浓度的5-Fu的紫外吸收光谱,制备5-Fu的标准曲线,得Y = 110.8 X – 0.001(R2=0.999)。Y为5-Fu的浓度(mg/mL),X是266nm波长处的吸光度。
(4)药物释放实验
将上述制备的(CNC-S-S-CNC)-CO-NH-(PAMAM-S-S-PAMAM)-(5-Fu)沉淀分散于去离子水中,然后等分2份,分别装入透析袋中,其中一个透析袋置于20 mL 含20 mM 的谷胱甘肽水溶液中,另一个透析袋置于20 mL含0.2 mM 的谷胱甘肽水溶液中,均于摇床中振摇,于0.5 h 取透析外液,于266nm波长检测所取样品的吸光度,根据5-Fu的标准曲线计算5-Fu浓度。
实验结果:
透射电子显微镜观察发现,所获得的CNC为空心纳米球,空心球的粒径为20-50 nm左右,如附图2所示。
制备的分散于去离子水中的“聚酰胺-胺树枝状高分子包覆的碳纳米笼 (CNC-S-S-CNC)-CO-NH-(PAMAM-S-S-PAMAM)为直径在150-350 nm左右的复合粒子,形貌与单纯的CNC显著不同,复合粒子中可见大量空心粒子,这些空心粒子即为CNC,如附图3所示。
(CNC-S-S-CNC)-CO-NH-(PAMAM-S-S-PAMAM)对肿瘤化疗药物5-Fu的装载能力很高,每100 mg的载体能负载72 mg的5-Fu。
在谷胱甘肽还原刺激下,(CNC-S-S-CNC)-CO-NH-(PAMAM-S-S-PAMAM)-(5-Fu)响应释药非常敏感,载药粒子透析30min时,含20 mM 谷胱甘肽的透析外液使载药粒子释放71.0%的药物,而含0.2 mM 谷胱甘肽的透析外液仅使载药粒子释放12.0%的药物,前者是后者的近6倍,显示出还原刺激响应释药的特点。
实施例 2
微乳中合成“聚酰胺-胺树枝状高分子包覆的碳纳米笼”(高分子为末端基团为羧基的G0型PAMAM-COOH):
(1)具体合成方法
A.制备微乳
将环己烷(45 mL)、Triton X-100 (15 mL)、正己醇(15 mL)混合,它们的体积比约为:正己醇: Triton X-100=1:1,环己烷:( 正己醇+Triton X-100)=3:2,该混合溶液称为微乳前液,然后制备如下微乳:
◆ 制备表面富含羧基的CNC的微乳:配制1 mL浓度为2 mg/mL的CNC水分散液,超声波处理10 min,将其与10 mL的微乳前液混合,室温振荡2-3 min,即得CNC微乳;
◆ 制备PAMAM-COOH微乳:配制2 mL 浓度为100 mg/mL的PAMAM-COOH水中,然后与20 mL微乳前液混合,室温振荡2-3 min,即得PAMAM-COOH微乳;
◆ 制备EDC/NHS微乳:称取10 mg的EDC和50 mg的NHS,共同溶于10 mL去离子水中,取 2 mL,将其与10 mL微乳前液混合,室温振荡2-3 min,即得EDC/NHS微乳;
◆ 制备胱胺微乳:配制500 μg/mL的胱胺水溶液2 mL,与10mL微乳前液混合,室温振荡2-3min,即得胱胺微乳。
B.微乳中合成“二硫键相互交联的聚酰胺-胺树枝状高分子/碳纳米笼”纳米复合粒子(CNC-S-S-CNC)-S-S-(PAMAM-S-S-PAMAM)
将CNC-COOH微乳与PAMAM-COOH微乳混合,室温振荡2-3min后,向该微乳中加入EDC/NHS微乳,摇床中室温振荡1 h,然后向该混合微乳中加入胱胺微乳,摇床中室温振荡反应5 h。然后通过离心(5000转/分钟,15 min),使微乳中反应物沉淀,乙醇洗涤该沉淀3次,去离子水洗涤3次,获得的沉淀即为 (CNC-S-S-CNC)-S-S-(PAMAM-S-S-PAMAM),其结构示意图如附图4所示。
(2)TEM分析、装载药物5-Fu及谷胱甘肽刺激释药实验
实验方法与实施例1相同。
实验结果:
所获得的纳米复合粒子(CNC-S-S-CNC)-S-S-(PAMAM-S-S-PAMAM)为150-500nm的粒子。粒子中隐约可见空心粒子,这些空心粒子即为CNC,其表面包覆PAMAM。
载药纳米复合粒子结构对还原剂的刺激很敏感,在谷胱甘肽溶液中进行透析发现,透析0.5h时,20 mM 的谷胱甘肽使载药粒子释放5-Fu的量是0.2 mM 谷胱甘肽释药量的约6.5倍。同样表现出良好的还原刺激响应释药特性。
实施例3
(1) 具体合成方法
A. 制备微乳
将环己烷(60 mL)、Triton X-100 (15 mL)、正己醇(5 mL)混合,它们的体积比约为:正己醇: Triton X-100=1:3,环己烷:( 正己醇+Triton X-100)=3:1,然后用该微乳前液制备以下微乳:
◆ 制备表面富含羧基的CNC的微乳:配制1 mL浓度为0.05 mg/mL的CNC水分散液,超声波处理10 min,将其与10 mL的微乳前液者混合,室温振荡2-3 min,即得CNC微乳;
◆ 制备PAMAM-COOH微乳:配制2 mL 浓度为0.25 mg/mL的PAMAM-COOH水中,然后与20 mL微乳前液混合,室温振荡2-3 min,即得PAMAM-COOH微乳;
◆ 制备EDC/NHS微乳:称取10 mg的EDC和10 mg的NHS,共同溶于10 mL去离子水中,取该溶液20 μL与10 mL微乳前液混合,室温振荡2-3 min,即得EDC/NHS微乳;
◆ 制备胱胺微乳:配制2 μg/mL的胱胺水溶液,取其1 mL,与10mL微乳前液混合,室温振荡2-3min,即得胱胺微乳。
B. 微乳中合成“二硫键相互交联的聚酰胺-胺树枝状高分子/碳纳米笼”纳米复合粒子(CNC-S-S-CNC)-S-S-(PAMAM-S-S-PAMAM)
按实施例2相同的方法将上述配制的微乳进行混合,然后离心,应用乙醇和去离子水洗涤,获得的沉淀即为 (CNC-S-S-CNC)-S-S-(PAMAM-S-S-PAMAM)。
(3)TEM分析、装载药物5-Fu及谷胱甘肽刺激释药实验
实验方法与实施例1相同。
实验结果:
所获得的纳米复合粒子(CNC-S-S-CNC)-S-S-(PAMAM-S-S-PAMAM)为100-250nm的接粒子,粒子较实施例1和实施例2的要小。被PAMAM包覆的CNC在电镜下仍然可以观察到。装载药物的能力和在谷胱甘肽刺激下释药的敏感性与实施例1相似,如透析0.5 h时,外液是20 mM 的谷胱甘肽时,载药粒子的释药量约70%,而外液是0.2 mM 谷胱甘肽时,透析0.5 h时的释药量约20%。
实施例4
(1)同时装载化疗药物和光敏剂药物:
按实施例2相同的方法制备(CNC-S-S-CNC)-S-S-(PAMAM-S-S-PAMAM),将其分散于1 mL去离子水中,其中CNC的浓度为4 mg/mL。同时,将顺铂溶解于含1%二甲基亚砜的水溶液(1 mL),顺铂浓度为4mg/mL,另配制1 mL浓度为4 mg/mL的叶绿素铜钠水溶液。将上述三种溶液混合,在摇床中室温下避光振荡24 h。离心,沉淀用去离子水洗涤2次,然后再分散于2mL去离子水中,避光保存。
(2)谷胱甘肽还原刺激响应释药:
取上述载药纳米粒子混悬液0.2 mL,加入0.2 mL的谷胱甘肽水溶液(1 mg/mL),摇床中室温避光振荡 20 min,离心,收集上清液。作为对照,向另一份0.2 mL的载药纳米粒子混悬液中加入0.2 mL去离子水,同样进行避光振荡20 min,然后离心,收集上清液。
(3)低功率密度的671-nm激光照射下的药物释放:
将0.2 mL的载药粒子置于无色透明小玻璃瓶中,玻璃瓶内径约6 mm,以671-nm激光(功率密度:0.2W/cm2)从玻璃瓶侧面对样品进行照射20 min。然后对样品进行离心,收集上清液。对照实验中,将0.2 mL的载药粒子避光静置20 min,然后离心,收集上清液。
(4)谷胱甘肽和671-nm激光共同刺激药物释放:
将0.2 mL的载药粒子与0.2 mL的谷胱甘肽水溶液(1 mg/mL)混合振荡20 min,然后将样品移入与上述相同的小玻璃瓶中,以671-nm激光照射20 min,然后离心,收集上清液。对照实验中,将0.2 mL的载药粒子与0.2 mL去离子水混合40 min,然后离心,收集上清液。
因为一个顺铂分子含有1金属铂(Pt)原子,一个叶绿素铜钠分子含有1个铜(Cu)原子,因此,采用电感耦合等离子发射光谱仪(ICP-AES)检测各上清液中Pt和Cu的浓度,即可获得药物释放的量。
实验结果:
药物释放方式与效果示意图如附图5和附图6所示。附图5是装载药物的纳米复合粒子(CNC-S-S-CNC)-S-S-(PAMAM-S-S-PAMAM)在谷胱甘肽作用下释放药物示意图,附图6是装载药物的纳米复合粒子(CNC-S-S-CNC)-S-S-(PAMAM-S-S-PAMAM)在谷胱甘肽作用之后经过近红外激光照射所导致的药物释放和载体的光热转化示意图。
谷胱甘肽能显著提升载药纳米粒子的药物释放速率,在与谷胱甘肽混合20 min后,通过离心,发现载药粒子的上清溶液为绿色,而对照组中(即与去离子水混合20 min),样品的上清液接近无色,表明包埋在(CNC-S-S-CNC)-CO-NH-(PAMAM-S-S-PAMAM)纳米粒子中的叶绿素铜钠在谷胱甘肽作用下快速释放。
ICP-AES检测结果显示,加入谷胱甘肽的载药粒子的上清液中,Pt和Cu的含量分别是对照组的约5.0倍和4.5倍。
671-nm激光照射亦能明显促进药物释放,经671-nm激光照射20 min的载药纳米粒子在离心后,其上清液为浅绿色,而未照射样品的上清液接近无色。
谷胱甘肽作用20min,再经过671-nm激光照射20min的载药纳米粒子样品,在经过离心后,其上清液中Pt和Cu的含量比单独以谷胱甘肽还原刺激或激光照射刺激的样品上清液中的含量要高,分别是对照组(无谷胱甘肽、无激光照射)的6.0和5.4倍。
Claims (5)
1.一种含碳纳米笼的还原刺激响应药物载体,以碳纳米笼CNC为核心,其特征在于,由聚酰胺-胺树枝状高分子PAMAM包覆在表面含羧基的碳纳米笼CNC-COOH表面,所述的树枝状高分子为低代数G0、G1、G2和/或G3的聚酰胺-胺树枝状高分子PAMAM,在CNC之间、PAMAM之间均有二硫键连接,当末端基团为氨基的PAMAM-NH2,CNC与PAMAM之间以酰胺键连接;或当末端为羧基的PAMAM-COOH,CNC与PAMAM之间以二硫键连接。
2.如权利要求1所述的含碳纳米笼的还原刺激响应药物载体,其特征在于,所述的CNC是指壳层具有石墨结构特点的空心多孔碳纳米粒子,表面含有羧基,粒径在20~100nm之间。
3.如权利要求1或2所述的含碳纳米笼的还原刺激响应药物载体的制备方法,其特征在于:制备过程在油包水型微乳中进行,包括下述步骤:
步骤一,配制如下各微乳:
(1)配制CNC油包水型微乳,微乳的水相是去离子水分散的CNC,水相中CNC浓度在0.05~2 mg/mL之间;
(2)配制EDC油包水型微乳,或“EDC+NHS”的油包水型微乳,所述的EDC的全称为1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride,也就是1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐,EDC微乳的水相是去离子水溶解的EDC;所述的NHS的全称为N-Hydroxysuccinimide,也就是N-羟基琥珀酰亚胺; EDC+NHS微乳的水相是去离子水溶解的EDC与NHS的混合物,EDC与NHS的质量比为1:(1~5) 之间;
(3)配制PAMAM-NH2油包水型微乳,微乳的水相是去离子水溶解的PAMAM-NH2,水相中PAMAM-NH2浓度在0.25-100 mg/mL之间;
(4)配制PAMAM-COOH油包水型微乳,微乳的水相是去离子水溶解的PAMAM-COOH,水相中PAMAM-COOH浓度在0.25-100 mg/mL之间;
(5)配制胱胺油包水型微乳,微乳的水相是去离子水溶解的胱胺,水相中胱胺浓度在2~500 μg/mL之间;
(6)配制二硫代二丙酸油包水型微乳,微乳的水相是去离子水溶解的二硫代二丙酸,水相中二硫代二丙酸浓度在0.01-10 mg/mL之间;
其中,所述的油包水型微乳,是由环己烷、Triton X-100、正己醇、水相溶液混合而成,在微乳体系中,正己醇与Triton X-100的体积比在1:(1-3) 之间,环己烷与“Triton X-100+ 正己醇”混合溶液的体积比在(3:1)~(3:2)之间,向环己烷、Triton X-100与正己醇三者的混合溶液所加入的水相体积的量保证混合体系不发生浑浊;
步骤二,进行如下合成反应:
(1)当采用PAMAM-NH2时,纳米载体制备方法为:将CNC微乳与EDC的微乳或与“EDC+NHS”的微乳混合0.5-3h,然后加入胱胺微乳,混合0.5-5h之后,加入PAMAM-NH2微乳,混合0.5-3h,获得含CNC和PAMAM-NH2的微乳;同时,将二硫代二丙酸微乳与EDC/NHS微乳混合0.5-3h,然后,将该二硫代二丙酸微乳与含CNC和PAMAM-NH2的微乳混合0.5-5h,最后离心,用乙醇沉淀和去离子水洗涤,即得PAMAM包覆CNC的纳米复合粒子,该复合粒子中,CNC之间以二硫键相连、PAMAM之间以二硫键相连、CNC与PAMAM之间以酰胺键相连;反应体系中,CNC与PAMAM的质量比为1:(5-50),
EDC的量是使CNC上的部分或全部羧基活化的量,EDC与CNC的质量比最小为1:10000,胱胺与EDC质量比为1:(2-10),二硫代二丙酸与PAMAM-NH2的质量比为1:(10~25);
(2)当采用PAMAM-COOH时,纳米载体制备方法为:将CNC微乳与PAMAM-COOH微乳混合,然后加入EDC/NHS微乳,混合0.5-3h;再加入胱胺微乳,混合0.5-5h;最后离心,用乙醇沉淀和去离子水洗涤,即得PAMAM包覆CNC的纳米复合粒子,该复合粒子中,CNC之间、PAMAM之间以及CNC与PAMAM之间均以二硫键相连;反应体系中,CNC与PAMAM的质量比为1:(5-50),EDC的量是使CNC上的部分或全部羧基活化的量,EDC与CNC的质量比最小为1:10000,胱胺与EDC质量比为1:(2-10)。
4.如权利要求1或2所述的含碳纳米笼的还原刺激响应药物载体在制备肿瘤靶向治疗药物中的应用,其特征在于:在所述的树枝状高分子包覆的CNC纳米复合粒子中,用物理吸附法装载肿瘤化疗药物和/或光敏剂,制成载药纳米粒子,在还原剂和激光刺激下响应释药。
5.如权利要求4 所述的含碳纳米笼的还原刺激响应药物载体在制备肿瘤靶向热疗药物中的应用,其特征在于:所述的载药纳米粒子通过肿瘤组织中谷胱甘肽的还原作用和外加近红外激光照射共同刺激而释放药物,近红外激光照射还诱导载药纳米粒子中的CNC产生光热转化。
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