CN115444941B - pH响应释药的双药纳米钻石药物及其制备方法和应用 - Google Patents
pH响应释药的双药纳米钻石药物及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供了一种pH响应释药的双药纳米钻石药物及其制备方法和应用。所述药物的制备:首先,将全反式视黄醛(ATR),即全反式视黄酸的前体,与胱胺二盐酸盐通过亚胺键形成胱胺化的全反式视黄醛(ASS),然后进一步与PEG修饰的纳米钻石(ND)生成ND‑PEG‑ASS(NPA)纳米药物,该纳米药物经物理吸附阿霉素(DOX),形成双药纳米药物ND‑PEG‑ASS@DOX(NPA@D)。体外药物释放实验表明,这是一种对pH敏感的药物递送系统。细胞毒性结果表明,NPA@D纳米药物能够抑制DOX敏感的MCF‑7和具有DOX耐药性的MCF‑7/ADR细胞生长。更重要的是,与NP@D相比,NPA@D纳米药物以协同方式更有效地抑制细胞活力和迁移。此外,NPA@D纳米药物能够阻止耐药肿瘤细胞膜上过表达的P‑gp糖蛋白泵出化疗药物,从而逆转耐药MCF‑7/ADR细胞的耐药性。
Description
技术领域
本发明涉及纳米钻石药物,特别涉及pH响应释药的双药纳米药物,具体属于一种先共价连接胱胺化的全反式视黄醛,然后物理吸附阿霉素的pH响应型双药纳米钻石药物及其制备方法,以及该药物在抗肿瘤中的应用。
背景技术
阿霉素(DOX)是目前临床上广泛应用治疗乳腺癌的化疗方法,然而阿霉素对癌细胞的特异性低,尤其是对心脏的毒性限制了它的大剂量使用。此外,治疗预后效果差,易造成癌症复发、转移和耐药。联合化疗可通过调节癌症的不同信号通路来克服耐药性,最大限度地发挥治疗作用,已经引起了广泛的关注。因此,如何将其它化疗药物与DOX结合,提高化疗效果,减少副作用引起研究人员的广泛兴趣。
针对这个问题,将两种药物同时集于一种给药系统是一个不错的选择。
全反式视黄醛(ATR)是全反式视黄酸的前驱体,能在溶酶体中被氧化为全反式视黄酸(ATRA),但ATRA水溶性很差,限制了它的广泛使用。ATRA是一种维生素A衍生物,常用于临床治疗急性早幼粒细胞白血病(APL)。ATRA还是一种强大的分化剂,抑制普通癌细胞增殖,诱导恶性细胞分化,通过阻碍参与干细胞维持的多种信号通路来发挥作用。还可以作为一种细胞衰老剂来预防和抑制许多癌恶性肿瘤的生长。研究表明,ATRA可以增强化疗药物的细胞毒性,例如已被证明可以增加鳞状头颈部癌和卵巢癌细胞对顺铂的体外敏感性,类视黄酸和细胞毒性药物联合使用也增强了疗效。
共传递纳米药物输送体系是将不同的药剂装载于同一纳米颗粒中,达到协同治疗的效果。前期研究证明,纳米钻石具有良好的生物相容性,表面易修饰,可通过物理吸附和共价连接的方式负载各种化疗药物,减少化疗药物的泄露。此外,纳米钻石能对化疗药物进行缓释,逆转肿瘤细胞的耐药性。受之前实验的启发,为了改善全反式视黄醛(ATR)的水溶性,我们通过可与肿瘤微酸环境响应的亚胺键将全反式视黄醛进行胱胺化,然后与聚乙二醇修饰的纳米钻石进行共价连接,之后在Na3Cit介质中将DOX负载,最终形成pH敏感的双药纳米药物体系,并研究了该药物在肿瘤细胞内的释药及抗肿瘤效应。
发明内容
本发明的目的在于提供一种胱胺化的全反式视黄醛与PEG化的纳米钻石进行共价连接,并物理吸附阿霉素,获得pH响应释药的双药纳米钻石靶向药物及其制备方法,以及该药物在抗肿瘤中的应用。
本发明提供的一种pH响应释药的双药纳米钻石药物的制备方法,包括如下步骤:
(1)称取5-10mg真空干燥的羧基化纳米钻石(ND-COOH),加入适量的MES缓冲液(0.1M,pH 5.8),超声分散20-30min,然后称取1.0-2.0mg 1-乙基-(3-二甲氨基丙基)碳二亚胺盐酸盐(EDC)和1.2-2.5mg N-羟基丁二酰亚胺(NHS),依次加入上述分散体系中,室温下匀速搅拌反应5-8h,待反应结束后,离心去除上清液,并用BBS缓冲液(0.1M,pH 8.4)进行洗涤,之后迅速加入适量BBS缓冲液(0.1M,pH 8.4),超声分散20-30min,然后加入8mg-10mg氨基-聚乙二醇-羧基(2HN-PEG-COOH),室温搅拌反应过夜;待反应结束,离心并用蒸馏水洗涤,得到产物ND-PEG-COOH(NP),冷冻干燥备用;
(2)称取25-35mg的全反式视黄醛(ATR),加入少量无水甲醇溶解,然后称取其3倍摩尔量的胱胺二盐酸盐(Cystamine dihydrochloride,分子式为H2N-CH2-CH2-S-S-CH2-CH2-NH2)溶于3-5mL无水甲醇,待完全溶解后,置于磁力搅拌器上进行搅拌,将ATR溶液慢速滴入,氮气氛围保护,在40℃油浴条件下反应20-30h;反应结束后,旋蒸除去溶剂,得到产物ATR-H2N-CH2-CH2-S-S-CH2-CH2-NH2(ASS),加入DMSO,将产物溶解,之后置于冰箱,在-20℃保存。
(3)称取5-10mg上述制备好的ND-PEG-COOH(NP),加入适量的MES缓冲液(0.1M,pH5.8),进行超声分散20-30min,然后称取1.0-2.0mg EDC和1.2-2.5mg NHS,依次加入上述分散体系中,室温下匀速搅拌反应5-8h,待反应结束后,离心并用BBS缓冲液(0.1M,pH 8.4)进行洗涤;之后加入一定体积的BBS缓冲液(0.1M,pH 8.4)进行超声分散20-30min,置于磁力搅拌器上,在快速搅拌的状态下,逐滴加入上述制备的ASS,室温条件下反应20-30h;反应结束后,离心收集上清液,然后固体用无水乙醇洗涤至上清液无色,测定ASS的偶联量,真空干燥固体样品得到NP-H2N-CH2-CH2-S-S-CH2-CH2-NH2-ATR(NPA),待干燥完成后称取5-8mg,加入5-8mL Na3Cit缓冲液(1.0M),超声分散20-30min,然后加入1.0-2.0mg DOX,室温避光搅拌5-8h,反应结束后,离心收集上清液,并用蒸馏水洗涤至上清液无色,收集洗涤液,计算DOX的负载量,将得到的NP-H2N-CH2-CH2-S-S-CH2-CH2-NH2-ATR@DOX(NPA@D)冷冻干燥,避光保存。
将以上方法制备得到的双药纳米钻石药物NPA@D,用CCK-8法,活死细胞染色法和实时动态监测法检测NPA@D对人乳腺癌细胞(MCF-7)活性的影响,表明该药物对比只负载阿霉素的纳米钻石药物(NP@D)对MCF-7细胞具有较大的杀伤力,并具有药物缓释作用;另一方面,该纳米药物能提高乳腺癌耐阿霉素的细胞(MCF-7/ADR)对阿霉素的敏感性,而且能逆转其耐药性,其可在制备抗肿瘤药物中应用。
与现有技术相比本发明的有益效果:
本发明以胱胺化的全反式视黄醛与经过聚乙二醇改性的纳米钻石共价偶联,接着以物理吸附的方式进行阿霉素负载,制备成双药纳米药物(纳米钻石-聚乙二醇-全反式视黄醛@阿霉素(NPA@D)),利用肿瘤组织微环境偏酸性,在肿瘤细胞内pH调控释药获得荧光信号;用多种不同的细胞毒性测试方法测试该药物与MCF-7肿瘤细胞作用,表明该纳米药物具有药物的协同作用,其与单独负载阿霉素的纳米药物的细胞杀伤力相比,对肿瘤细胞具有较大的杀伤力;NPA@D具有缓释药物特性;重要的是,NPA@D纳米药物能逆转其对阿霉素的耐药性,能对耐阿霉素的癌细胞(MCF-7/ADR)有较明显的杀伤力。因此,NPA@D既能提高化疗药物阿霉素对肿瘤细胞的杀伤力,又能逆转耐药细胞对阿霉素的耐受性。抗肿瘤治疗中具有广阔的应用前景,进而可在制备抗肿瘤药物中应用。
附图说明
图1胱胺化的全反式视黄醛的高分辨质谱表征
图2各药物在日光灯照射下的照片,其中(A)ATR,ASS和DOX在二甲基亚砜中;(B)ND、NP、NPA和NPA@D在磷酸缓冲液中;(C)ND、NPA和NPA@D经离心后在离心管壁上
图3各种药物的红外光谱图,其中(A)ND,DOX和ATR;(B)NP、NPA NP@D和NPA@D
图4各种纳米药物的拉曼光谱图,其中(A)ND,NP和NPA;(B)NP@D和NPA@D
图5纳米药物NPA酸性条件下的释药并用高分辨质谱检测,其中(A)释药缓冲液中的ATR;(B)释药缓冲液中的ATRA;(C)ATR的预测值;(D)ATRA的预测值
图6纳米药物NPA@D在pH 5.0、6.5、7.4的体外药物释放曲线
图7体外细胞毒性试验,其中(A)Calcein AM/PI染色以可视化通过各种处理48h处理的MCF-7细胞活力;绿色是活细胞的Calcein AM(CA)染色,红色是死细胞的碘化丙啶(PI)染色;(B)MCF-7/ADR细胞在72h的各种处理后的活细胞和死细胞染色图像
图8细胞毒性检测,其中(A)CCK-8法检测不同时间下不同药物对MCF-7细胞活性的影响;(B)不同纳米药物对MCF-7细胞毒性的动态变化;(C)ATR和DOX对MCF-7细胞毒性的动态变化
图9药物与MCF-7共孵育后细胞迁移情况,其中(A)细胞划痕变化图;(B)细胞迁移抑制率
图10Transwell检测不同药物对体外MCF-7细胞迁移影响,其中(A)显微镜下细胞迁移图;(B)迁移变化率柱状图
图11分别用DOX与NPA@D共孵育处理的MCF-7细胞成像,其中(A)DOX处理细胞成像;(B)NPA@D处理细胞成像;(C)共定位成像
图12分别用DOX与纳米药物NPA@D共孵育处理的MCF-7细胞和MCF-7/ADR成像,其中(A)游离DOX与MCF-7细胞和MCF-7/ADR共孵育4h;(B)纳米药物NPA@D与MCF-7细胞和MCF-7/ADR共孵育4h
具体实施方式
以下结合附图并通过具体实施例来进一步说明本发明的技术方案,但本发明并非局限在实施例范围内。
实施例中所用的试剂和试剂的生产厂商:
载体为纳米钻石(ND,直径大约140nm),购买于元素六公司。
氨基-聚乙二醇-羧基:(2HN-PEG-COOH,分子量为2000)购买于北京索莱宝科技有限公司。
全反式视黄醛:(ATR,分子量为284),购买于北京索莱宝科技有限公司。
胱胺二盐酸盐:(C4H12N2S2,分子量为225)购买于上海阿拉丁试剂有限公司。
阿霉素(DOX):又名盐酸多柔比星(C27H29NO11·HCl,分子量为579.99),购买于上海阿拉丁试剂有限公司。
EDC:1-乙基-(3-二甲氨基丙基)碳二亚胺盐酸盐(C8H17N3 HCl,分子量191.70),购买于北京索莱宝科技有限公司。
NHS:N-羟基丁二酰亚胺(C4H5NO3,分子量为115.09),购买于北京索莱宝科技有限公司。
MES:2-(N-吗啡啉)乙磺酸(C6H13O4NS H2O,分子量为213.2),购买于北京索莱宝科技有限公司。
BBS:硼酸(H3BO3,分子量61.83),购买于北京索莱宝科技有限公司。
其它所用试剂或仪器未注明生产厂商者,均为可通过正规渠道商购买获得的常规产品。
实施例1
(1)纳米钻石-聚乙二醇(ND-PEG-COOH)纳米粒子的制备的制备
称取10mg的羧基化纳米钻石(ND),加入10mL MES缓冲液(0.1M,pH 5.8),超声分散30min,然后称取2.0mg EDC和2.5mg NHS,依次加入上述分散体系中,室温下匀速搅拌反应6h,待反应结束后,离心去除上清液,并用BBS(0.1M,pH 8.4)缓冲液进行洗涤,之后迅速加入10mL BBS(0.1M,pH 8.4)缓冲液,超声分散30min,然后加入8mg H2N-PEG-COOH,室温搅拌反应12h。待反应结束,离心并用蒸馏水洗涤,冷冻干燥备用。
(2)通过亚胺键将胱胺与全反式视黄醛偶联(ASS)
称取28.4mg ATR,加入2.0mL无水甲醇溶解,然后称取67.5mg胱胺二盐酸盐(Cystamine dihydrochloride,结构式为H2N-CH2-CH2-S-S-CH2-CH2-NH2)溶于3mL无水甲醇,待完全溶解后,置于磁力搅拌器上进行搅拌,将ATR溶液慢速滴入,并通氮气30min,在40℃油浴条件下反应24h。反应结束后,40℃旋蒸除去溶剂,得到产物ATR-H2N-CH2-CH2-S-S-CH2-CH2-NH2(ASS),加入2mL DMSO,将产物溶解,之后置于冰箱,在-20℃保存。
(3)NP-H2N-CH2-CH2-S-S-CH2-CH2-NH2-ATR@DOX(NPA@D)的制备及ATR和DOX负载量的测定
称取10mg上述制备好的ND-PEG-COOH(NP),加入10mL MES(0.1M,pH 5.8)缓冲液,进行超声分散30min,然后称取2.0mg EDC和2.5mg NHS,依次加入上述分散体系中,室温下匀速搅拌反应6h,待反应结束后,离心并用BBS(0.1M,pH 8.4)缓冲液进行洗涤。之后加入10mL BBS(0.1M,pH 8.4)缓冲液进行超声分散30min,置于磁力搅拌器上,在快速搅拌的状态下,逐滴加入上述制备的ASS,室温条件下反应24h,反应结束后,离心收集上清液,然后固体用无水乙醇洗涤至上清液无色,测定ASS的偶联量,真空干燥固体样品得到NP-H2N-CH2-CH2-S-S-CH2-CH2-NH2-ATR(NPA),待干燥完成后称取5mg上述制备的样品,加入5mL Na3Cit(1.0M)缓冲液,超声分散30min,然后加入1.0mg DOX,室温避光搅拌6h,反应结束后,离心收集上清液,并用蒸馏水洗涤至上清液无色,收集洗涤液,计算DOX的负载量,将得到的NP-H2N-CH2-CH2-S-S-CH2-CH2-NH2-ATR@DOX(NPA@D)冷冻干燥,避光保存。经计算可得到NP对ATR负载量为35μg/mg,对DOX的负载量为140μg/mg。
实施例2
ASS的质谱表征
取部分上述制备的ASS,冷冻干燥去除溶剂,然后加入色谱级甲醇,制备成浓度为1×10-6g/mL的溶液,用0.22μm的薄膜过滤,然后加入到质谱测样的玻璃瓶中,上机测样。如图1所示,ATR的分子HR/AM值为是299.23719(图1A),当形成ASS后,图1B所示,HR/AM值变为419.25441,与图1C理论计算值419.25492基本一致。所以可以确定ASS的成功合成。
实施例3
纳米药物在日光灯下颜色表征
分别取ND、NP、NPA和NPA@D纳米药物各3mL(浓度均为1mg/mL)超声分散30min后,加入到玻璃瓶中,在日光灯下静置拍照,以不同的药物在溶液中的颜色如图2A从左到右依次为ATR(黄色)、ASS(棕红色)、DOX(亮红色)为对照。将各种纳米药物的溶液置于10mL的离心管中,以10000rpm离心5min,去除上清液,将附着在离心管壁上的纳米药物进行拍照,观察其在日光灯下的颜色。当药物被负载到ND表面上时,会改变ND表面的颜色,图2B由左到右依次为依次是灰白色(ND)、灰白色(NP)、黄色(NPA)和淡粉色(NPA@D)。将纳米药物离心之后,根据他们在离心管壁上的颜色,如图2C所示,可以明显看到纳米药物颜色的差异,从上到下,依次为灰白色(ND)、黄色(NPA)和淡粉色(NPA@D)。根据纳米药物表面颜色的不同,我们可以确定最终纳米颗粒上成功负载了ATR和DOX。
实施例4
纳米药物的红外光谱表征
为了确认NPA@D的成功合成,将制备好的干燥的纳米药物分别与KBr按1:100的质量比混合研磨,然后压片,进行测定,根据其红外光谱图确定产物是否成功制备。
图3B为所制备纳米药物的红外光谱图,与图3A中的ND、DOX和ATR相对比,可以发现,NP上出现在1618cm-1的酰胺Ⅱ带特征峰,还有1390cm-1处酰胺键中的C-N伸缩振动峰,表明PEG成功偶联在ND表面。将NPA与ATR的红外谱图对比,1710cm-1处的C=O吸收振动峰消失,说明ATR的醛基消失,出现了550cm-1处的二硫键特征峰,证明ASS成功偶联在NP上。在NPA@D的谱图中,可以看到,既有NPA上所有的特征峰,又出现了DOX在1409cm-1苯环上质子伸缩振动峰,1580cm-1苯环的C=C伸缩振动峰。证实了NPA@D的成功制备
实施例5
纳米药物的拉曼散射光谱表征
取少量制备好的纳米药物置于载玻片上,进行压片,然后进行测定,根据其拉曼散射光谱图,确定产物是否合成。
拉曼光谱可以作为一种对纳米药物表征的有效方法,由图4A可知,ND在1332cm-1出现了尖锐的特征吸收峰,还有在1580cm-1处代表石墨的G波段,偶联PEG之后,由于PEG的覆盖,纳米钻石的特征峰和石墨层的G波段都明显减弱。在进一步偶联ASS之后,NPA的谱图中在555cm-1处出现了特征吸收峰,代表了二硫键的伸缩振动,和ASS中的1680cm-1处的C=N峰,证实了NPA的成功制备。纳米颗粒进一步负载DOX后,由图4B可以发现,ND上的特征峰都被掩蔽,不再显现出来。这可能是因为外层的DOX影响了拉曼信号,同样也证实了NPA@D的成功合成。
实施例6
纳米药物的粒径和Zeta电位表征
取少量不同的纳米药物分别超声分散在蒸馏水中,取1.5mL制备的样品加入样品池中,在25℃的条件下测定其粒径和多分散性指数(PDI)。用注射器将制备的样品加入到可折叠毛细管样品池测定各种纳米药物的Zeta电位。如表1所示,ND在层层连接不同的药物之后,它的粒径逐步变大,NPA@D的粒径可达272.6±2.7nm,满足纳米药物的EPR效应尺寸。根据药物的电负性,表面电位也会发生相应的改变,由于DOX显正电性,所以NPA被DOX覆盖之后,会逆转一部分负电性,导致电位从-23.2mV变为-19.9mV。
表1不同纳米药物的粒径大小,电位和多分散指数
实施例7
纳米药物的NPA体外释药表征
取5mg的NPA于2mL醋酸钠缓冲溶液(pH 4.5,含Tween 80,w:v=10%)超声分散30min,然后置于37℃的磁力搅拌器上搅拌3h。之后,离心取出上清液,冷冻干燥。干燥物用色谱级无水甲醇溶解,用注射器过微孔滤膜,通过高分辨质谱仪进行检测。
结果如图5A所示,发现在释药后的溶液中能找到ATR的分子离子峰,峰位置与ATR的预测值一致(图5C),除此之外,由图5B所示,还能找到ATR被氧化后的全反式视黄酸(ATRA)的分子离子峰,峰位置与ATRA的预测值一致(图5D),说明NPA可以在溶酶体中发生断裂,将ATR释放出来,且有部分ATR能够转化为ATRA,与胞质蛋白结合后被转运到细胞核中,进一步发挥药效。
实施例8
纳米药物的NPA@D体外释药
采用搅拌法研究了NPA@D中ATR和DOX的释放率。将超声分散的3mg NPA@D(1mg/mL)装入装有10%(w/v)Tween 80的PBS缓冲溶液(pH 7.4、6.0和4.5)的离心管中。每个样品在37℃。然后,在不同的间隔时间,取出2mL样品并补充等体积的新鲜缓冲液。通过紫外分光光度计量化ATR和DOX释放以确定累积释放率。
如图6A所示,在pH 7.4下,ATR的释放速率在30小时内没有明显增加,然而,ATR的释放率在pH 6.5时略有增加,在pH 5.0时显着增加,在30h时约70%的ATR内释放。图6B中DOX在pH 6.5时释放约35%,在pH 5.0时分别释放80%。ATR在不同pH下的累积释放量略低于DOX,这可能是由于ATR在纳米颗粒上的共价结合,所以它需要先从结合物上断裂,然后连续扩散,这比DOX要复杂一些。因此,这些结果证明NPA@D具有pH响应性,从而促进肿瘤靶向药物递送并保护正常细胞。
实施例9
NPA@D纳米药物的细胞毒性测试
(1)Calcein AM/PI活死细胞染色
为了观察不同药物对MCF-7细胞生长的影响,将处于对数生长期的MCF-7细胞按1.5×105的密度接种于25mm2的细胞培养瓶中,待细胞贴壁之后,处于对数生长期时,向各组中分别加入含ATR:0.75μg/mL、DOX:3μg/mL、NPA:0.75μg/mL、NP@D:3μg/mL和NPA@D:3μg/mL的培养液进行孵育(以DOX的负载量进行计算),以只加培养液的细胞为对照组。孵育培养48h后,吸去培养液,根据Calcein AM/PI试剂盒的说明书进行染色,置于荧光显微镜下观察并拍照记录。同样将MCF-7/ADR细胞生长按1.5×105的密度接种于25mm2的细胞培养瓶中,待细胞贴壁之后,处于对数生长期时,向各组中分别加入含ATR:20μg/mL、DOX:80μg/mL、NPA:20μg/mL、NP@D:80μg/mL和NPA@D:80μg/mL的培养液进行孵育(以DOX的负载量进行计算),以只加培养液的细胞为对照组。孵育培养72h后,吸去培养液,根据Calcein AM/PI试剂盒的说明书进行染色,置于荧光显微镜下观察并拍照记录。
如图7A所示,对照组、NP、ATR和NPA组的细胞几乎都被钙黄绿素Calcein AM染成绿色,表明凋亡的细胞很少。在NP@D或NPA@D组中,几乎所有细胞都被碘化丙啶PI(红色)染色。值得注意的是,NPA@D比NP@D杀死更多的细胞,呈现出明亮的红色信号。此外,在图7B中,活细胞和死细胞双重染色也更好地显示了MCF-7/ADR细胞效应,可以看出游离DOX组的细胞几乎没有产生红色荧光,表明由于MCF-7/ADR细胞的耐药性,细胞没有被杀死。然而,由于游离DOX的急性毒性,受影响的MCF-7细胞在成像前已经完全死亡并漂浮,因此没有呈现结果。但是NP@D和NPA@D组均能有效杀伤耐药细胞,且后一组的协同作用优于前组,说明基于ND的递送系统可逆转细胞的多药耐药性。而且ATR可以增强DOX对乳腺癌细胞的毒性。
(2)CCK-8法检测纳米药物细胞毒性
为了定量检测纳米药物对MCF-7细胞的毒性,用CCK-8法检测在不同时间下各药物组对肿瘤细胞的影响。首先,将MCF-7细胞以5.0×103接种于3个96孔板中,孵育20h后,移去旧的培养液,然后加入200μL培养液配制的各药物组ATR:0.75μg/mL、DOX:3μg/mL、NPA:0.75μg/mL、NP@D:3μg/mL和NPA@D:3μg/mL(以DOX的负载量进行计算),以加培养液的细胞为空白对照,每组设6个复孔,分别培养24h,48h,72h后,移去培养液,用PBS缓冲液洗涤3次,然后加入配制的新的培养液200μL(含20μLCCK-8),继续孵育1h之后用酶标仪检测在450nm处的吸光度。
从图8A中可知,在任何时间ATR对细胞均不显现毒性,在24h时,NP@D与NPA@D对细胞抑制没有明显差异,但随着时间的延长,在48h和72h时,NPA@D对细胞的毒性均强于NP@D,推测是由于NPA@D内吞进细胞之后,对酸环境敏感的亚胺键断裂释放的ATR被转化为全反式视黄酸(ATRA)需要一定的时间,所以在该双药纳米药物进入细胞之后,24h时并没有显现对细胞的协同毒性,但当时间延长至48h以及72h时,双药纳米药物的协同效果强于单负载的纳米药物,表明药物具有良好的协同效果。
(3)实时动态检测NPA@D纳米药物细胞毒性
为了进一步检测纳米药物对细胞的实时毒性,以MCF-7为细胞模型,用实时无标记细胞功能分析仪(xCELLigence RTCA S16,ACEA Biosciences)实时检测纳米药物对细胞增殖的影响。首先,在E-Plate 16的孔中加入50μL培养液,将E-Plate 16放到RTCA S16系统上,系统自动扫描,确定接触良好,检测基线,然后移出E-Plate 16,在孔中加入100μL混合均匀的细胞悬液,使每孔中的细胞数目为5×103个,将E-Plate 16置于超净台中室温放置30min,然后将E-Plate 16放到培养箱中的RTCA S16上,开始检测细胞增殖曲线。待细胞贴壁后,处于对数生长期时,暂停RTCA S16,取出E-Plate 16,移去旧培养液,加入新配制得不同药物的培养液,ATR:0.75μg/mL、DOX:3μg/mL、NPA:0.75μg/mL、NP@D:3μg/mL和NPA@D:3μg/mL(以DOX的负载量进行计算),每个实验组设置3个复孔,对照组加入150μL培养液,继续开始检测。
如图8B所示,在21h时向处于对数生长期的MCF-7细胞中加入不同纳米药物,不做药物处理的细胞做对照组,可以发现,NP@D在加药之后17小时显现出细胞毒性,而NPA@D在加药之后的21小时才显现出细胞毒性,虽然NPA@D显现毒性的时间较晚,但是在50h时,它的曲线下降速度变快,说明毒性超过了NP@D,这是因为ASS中亚胺键发生断裂释放ATR并转化成为ATRA,与DOX显现了协同效果造成的,在70h后两者表现为全部杀死细胞。而NP与NPA处理组与对照组的细胞生长趋势一致,表明这两种纳米材料都没有细胞毒性,之后细胞在生长到一定阶段,达到平台期,细胞指数保持不变。说明NP是一种生物安全的纳米药物载体,而且NPA没有细胞毒性,从而证明单一的ATR对MCF-7细胞没有毒性,但ATR和DOX联合使用时,会增强DOX的化疗效果。图8C是用ATR和DOX游离药物对MCF-7细胞做处理,与B图对比,可以发现,DOX在加药后的5h左右显现细胞毒性,加药26小时之后,细胞指数降为0,细胞全部死亡。上述结果表明纳米药物相对DOX具有缓释药物特性。而ATR不显现细胞毒性,与对照组细胞生长趋势一致。
实施例10
NPA@D纳米药物的抑制细胞迁移测试
(1)细胞划痕法检测NPA@D纳米药物的细胞迁移
为了进一步研究纳米药物NPA@D对MCF-7细胞迁移抑制的影响,通过细胞划痕法进行实验。首先,将MCF-7细胞以2×105/孔的密度接种于6孔板中,待细胞密度达到90%,移去旧培养液,用10μL的移液枪枪头在皿底划十字划痕,之后用PBS(pH 7.4)缓冲液清洗3次,去除漂浮的细胞,然后加入配制的含有不同药物的培养液,继续孵育,然后从培养箱取出6孔板,置于光学显微镜下观察划痕愈合情况,拍照记录划痕宽度,采用公式分别计算划痕愈合率和细胞迁移抑制率。
公式中,WC为细胞划痕愈合率,SWt代表作用36h或48h时的划痕宽度,SW0代表初始划痕宽度。
公式中,MIR为细胞迁移抑制率,WCtreament代表实验组细胞划痕愈合率,WCcontrol代表空白对照组的划痕愈合率。
从图9中可以看到,不加药物的对照组,随着时间的延长,划痕在逐渐变窄,说明细胞具有正常的迁移能力,加入ATR或不同的纳米药物之后,细胞之间的划痕愈合速率减慢。这是因为ATR和纳米药物都抑制了肿瘤细胞的迁移能力。由于DOX具有较强的细胞毒性,所以随着时间的延长,细胞全部死亡,划痕也消失。对比NP@D与NPA@D组,可以发现,纳米药物NPA@D对MCF-7细胞迁移的抑制能力更强,这是由于两种药物的协同作用效果导致的。从图9B可知,纳米药物与肿瘤细胞共培养作用48h时,纳米药物NPA@D抑制肿瘤细胞迁移率要高于NP@D 12%,达到80%的抑制率,表明两种药物具有良好的协同抑制效果。
(2)Transwell法检测NPA@D纳米药物的细胞迁移
为了研究NPA@D纳米药物对细胞迁移能力的影响,以MCF-7细胞为模型,采用Transwell法进行检测。首先,将细胞用不含FBS的培养液悬浮,加入Transwell上室,细胞密度为1.5×104/孔,再往上室加入用不含FBS的培养液配制的不同药物,在Transwell下室加入600μL含10%FBS的培养液,每组设置3个复孔,以不加任何药物小室的细胞作为空白对照,分别孵育24h和48h。孵育结束后,将上室培养液用移液枪吸出,然后用PBS(pH 7.4)缓冲液轻轻洗涤小室,然后用4%的多聚甲醛对聚碳酸酯膜上的细胞进行固定30min,之后用结晶紫染液(0.2%)对小室染色20min,染色结束后用PBS(pH 7.4)缓冲液轻轻洗涤小室几次,再用棉签将膜上部的细胞轻轻刮去,最后在光学倒置显微镜下观察拍照。
结果如图10A所示,与对照组相比,各加药组对MCF-7细胞的迁移有明显的抑制。NPA@D组对比其他纳米药物组,能够迁移到Transwell下室的细胞很少,经过统计可以得到,如图10B所示,在36h时,NPA@D对肿瘤细胞迁移的抑制率较NP@D高9%;在48h,NPA@D组对细胞迁移的抑制率达85%左右,比NP@D高15%,与划痕实验结果相吻合。证明了双药纳米药物对肿瘤细胞有着协同抑制迁移的效果。
实施例11
NPA@D纳米药物的内吞与定位
为了研究NPA@D纳米药物被细胞的内吞过程,以MCF-7细胞为细胞模型,采用激光共聚焦显微镜进行观察。首先,将MCF-7细胞以1.5×104/皿的密度接种于激光共聚焦皿中,待细胞完全贴壁后,移去旧的培养液,加入配制好的含有药物的培养液,分别继续孵育2h、6h、10h,以加DOX的实验组孵育1h为对照组,作用结束后,移去旧的培养液,用PBS(pH 7.4)缓冲液清洗3次,接下来用4%的多聚甲醛固定细胞8min,固定结束后,加入Hoechst33258染色液避光染核15min,最后再用PBS(pH 7.4)缓冲液清洗。置于激光共聚焦显微镜下拍照观察,设置Hoechst33258激发波长为405mn,DOX的激发波长为488nm。
为了进一步探究NPA@D纳米药物的内吞过程是否与溶酶体有关,采用绿色溶酶体探针进行共定位研究。待细胞完全贴壁后,移去旧的培养液,加入配制好的含有NPA@D纳米药物的培养液5μg/mL,继续孵育2h,然后加入绿色溶酶体探针继续孵育30min,待孵育结束后,用PBS(pH 7.4)清洗,4%的多聚甲醛固定,加入Hoechst33258染色液避光染核15min,最后再用PBS(pH 7.4)缓冲液清洗。置于激光共聚焦显微镜下拍照观察,设置Hoechst33258激发波长为405mn,DOX的激发波长为488nm。
从图11A中可知,用游离DOX共孵育细胞1h时,游离DOX已进入细胞核。但用纳米药物NPA@D与细胞进行共孵育,从图11B可以观察到,在2h时,DOX的红色荧光只出现在细胞质,随着时间延长,DOX的红色荧光开始向细胞核转移,当时间到达10h时,红色荧光全部集中在细胞核,说明纳米药物NPA@D具有缓释药物作用,延迟了其进入细胞核的时间。
如图11C所示,用绿色溶酶体探针对细胞中的溶酶体进行标记,NPA@D进入细胞之后,DOX的红色荧光用来标记纳米药物位置,两者图像重叠后发现绿色探针很好的与红色探针重叠,其共定位指数为R=0.90。众所周知,共定位指数大于0.5时,认为共定位效果良好。因此可以表明纳米药物进入细胞之后,首先被溶酶体摄取,这样在溶酶体微酸性环境中导致纳米药物NPA@D中酸响应的共价键断裂释放ATR,其在溶酶体中进一步被转化为ATRA,与DOX发挥协同抗肿瘤效果。
实施例12
NPA@D纳米药物逆转耐药细胞耐药性
为了研究纳米钻石药物体系是否能够逆转耐药细胞的耐药性,选用MCF-7细胞和耐药MCF-7/ADR细胞为模型,以3.0×104/皿的密度接种于激光共聚焦皿中,待细胞完全贴壁后,移去旧的培养液,每种细胞都分别加入游离DOX(8μg/mL)和NPA@D(8μg/mL)纳米药物进行孵育,2h后,洗去培养液,4%的多聚甲醛固定,加入Hoechst33258染色液避光染核15min,最后再用PBS(pH 7.4)缓冲液清洗。置于激光共聚焦显微镜下拍照观察。
如图12所示,图12A是游离DOX与MCF-7细胞和MCF-7/ADR细胞共孵育4h后的成像图,从图中可以看到,MCF-7细胞显现出的大部分DOX红色荧光且集中在细胞核,而MCF-7/ADR与游离DOX孵育后,细胞内几乎没有DOX红色荧光出现,这是因为耐药细胞的p-糖蛋白可以将游离DOX泵出胞外导致化疗失败,而肿瘤细胞中游离DOX通过被动扩散进入细胞且1小时可进入到细胞核。将纳米药物与MCF-7细胞和MCF-7/ADR细胞共孵育4h后,由图12B可知,不论MCF-7细胞还是MCF-7/ADR细胞中均出现了明显的红色荧光信号,这是由于纳米药物能够内吞进肿瘤细胞,与游离DOX相比,相同时间主要位于细胞质,主要是由于药物DOX从ND上解离需要时间。有趣的是,纳米药物也能内吞进耐药细胞,源于纳米药物能逃逸耐药细胞的p-糖蛋白的识别,导致其能在耐药细胞累积,因此出现红色荧光信号,表明纳米药物能逆转耐药细胞的耐药性。
将化疗药物ATR用可与肿瘤微环境响应的化学键进行修饰改造,形成ASS,随后与PEG功能化的纳米钻石上偶联,最终将DOX吸附在NPA的表面,形成纳米钻石双药体系(NPA@D)。利用两种药物的协同治疗作用,提高化疗药物DOX对肿瘤细胞的杀伤力。由于ASS是通过与肿瘤微环境特异性响应的亚胺键偶联的,所以能够实现药物的可控释放。通过激光共聚焦实验探究了MCF-7细胞和MCF-7/ADR细胞对DOX和NPA@D的摄取,结果发现MCF-7细胞对游离DOX和NPA@D都有摄取,而MCF-7/ADR细胞只对NPA@D有大量摄取,表明纳米药物NPA@D能够逆转肿瘤细胞的耐药性。该研究为多功能纳米药物体系的联合治疗提供了新思路,并为临床对肿瘤的化疗用药提供理论支持。
Claims (3)
1.一种pH响应释药的双药纳米钻石药物的制备方法,其特征在于,包括如下步骤:
(1)称取5-10mg真空干燥的羧基化纳米钻石(ND-COOH),加入适量浓度0.1M、pH5.8的MES缓冲液,超声分散20-30min,然后称取1.0-2.0mg 1-乙基-(3-二甲氨基丙基)碳二亚胺盐酸盐(EDC)和1.2-2.5mg N-羟基丁二酰亚胺(NHS),依次加入上述分散体系中,室温下匀速搅拌反应5-8h,待反应结束后,离心去除上清液,并用浓度0.1M、pH 8.4的BBS缓冲液进行洗涤,之后迅速加入适量相同的BBS缓冲液,超声分散20-30min,然后加入8mg-10mg氨基-聚乙二醇-羧基,室温搅拌反应过夜;待反应结束,离心并用蒸馏水洗涤,得到产物ND-PEG-COOH(NP),冷冻干燥备用;
(2)称取25-35mg的全反式视黄醛(ATR),加入少量无水甲醇溶解,然后称取其3倍摩尔量的胱胺二盐酸盐溶于3-5mL无水甲醇,待完全溶解后,置于磁力搅拌器上进行搅拌,将ATR溶液慢速滴入,氮气氛围保护,在40℃油浴条件下反应20-30h;反应结束后,旋蒸除去溶剂,得到产物ATR-H2N-CH2-CH2-S-S-CH2-CH2-NH2(ASS),加入DMSO,将产物溶解,之后置于冰箱,在-20℃保存;
(3)称取5-10mg上述制备好的ND-PEG-COOH(NP),加入适量浓度0.1M、pH 5.8的MES缓冲液,进行超声分散20-30min,然后称取1.0-2.0mg EDC和1.2-2.5mg NHS,依次加入上述分散体系中,室温下匀速搅拌反应5-8h,待反应结束后,离心并用浓度0.1M、pH 8.4的BBS缓冲液进行洗涤;之后加入适量相同的BBS缓冲液进行超声分散20-30min,置于磁力搅拌器上,在快速搅拌的状态下,逐滴加入上述制备的ASS,室温条件下反应20-30h;反应结束后,离心收集上清液,然后固体用无水乙醇洗涤至上清液无色,真空干燥固体样品得到NP-H2N-CH2-CH2-S-S-CH2-CH2-NH2-ATR(NPA),待干燥完成后称取5-8mg,加入5-8mL浓度1.0M的Na3Cit缓冲液,超声分散20-30min,然后加入1.0-2.0mg DOX,室温避光搅拌5-8h,反应结束后,离心收集上清液,并用蒸馏水洗涤至上清液无色,收集洗涤液,将得到的NP-H2N-CH2-CH2-S-S-CH2-CH2-NH2-ATR@DOX(NPA@D)冷冻干燥,避光保存。
2.如权利要求1所述方法制备得到的pH响应释药的双药纳米钻石药物。
3.如权利要求2所述的pH响应释药的双药纳米钻石药物在制备抗肿瘤药物中的应用,所述的肿瘤为乳腺癌。
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