CN115025242A - 一种他汀前药和他汀靶向递送系统及其制备方法和应用 - Google Patents
一种他汀前药和他汀靶向递送系统及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及一种他汀前药和他汀靶向递送系统及其制备方法和应用。将他汀通过聚乙二醇及酮缩硫醇键与α‑生育酚共价连接,得到具有活性氧响应能力的他汀前药。该他汀前药与两亲性纤连蛋白靶向多肽聚合物(二硬脂酰基磷脂酰乙醇胺‑聚乙二醇‑纤连蛋白靶向肽)和替卡格雷通过溶剂蒸发法进行组装构建,获得具活性氧响应性及纤连蛋白靶向能力的他汀靶向递送系统。本发明提供的他汀前药和他汀靶向递送系统具有活性氧响应释放他汀的特性。他汀靶向递送系统可主动靶向并同时递送辛伐他汀、α‑生育酚及替卡格雷至动脉斑块部位,发挥协同抗炎抗氧化功效,可用于动脉粥样硬化的治疗,在生物医学领域有良好的应用前景。
Description
技术领域
本发明涉及一种他汀靶向递送系统及其制备方法和应用,具体为他汀前药和他汀靶向递送系统及其制备方法和应用。
背景技术
动脉粥样硬化(atherosclerosis,AS)是大多数心血管疾病的主要原因,可导致急性心血管综合征,如心肌梗死、中风、冠心病和外周动脉疾病等。近年来,由于人口寿命延长及老龄化的发展,以冠状动脉和脑血管粥样硬化为主的心血管疾病的发病率和死亡率仍不断上涨,高居全球死亡率首位。研究发现,炎症是动脉粥样硬化发生发展的潜在机制,在斑块的前期形成、发展阶段、不稳定破裂以及血栓生成等过程中都起着重要的促进作用。大量的临床试验数据也表明,与传统的降脂药相比,抗炎治疗可能是治疗动脉粥样硬化的一个新方向。
他汀类药物可通过竞争性抑制内源性胆固醇合成限速酶3-羟基-3-甲基-谷氨酰-辅酶A(3-hydroxy-3-methylglutarylcoenzymeAreductase,HMG-CoA)还原酶,阻断羟甲戊酸代谢途径,使细胞内胆固醇合成减少,降低机体胆固醇水平,是治疗动脉粥样硬化的常用药物。近年来,研究发现他汀类药物除作用于HMG-CoA还原酶这一靶点之外,也表现出抗炎、抗氧化、抗血栓、免疫调节和稳定斑块等效用,这一现象被称为他汀类药物的多效性效应。
然而,他汀类诸如辛伐他汀等小分子药物有其固有的劣势,如水溶性差、血液循环中代谢失活迅速、缺乏靶向性等。此外,他汀类药物往往具有一定的肝脏靶向功能,所以很难在动脉硬化斑块部位达到有效药物浓度富集,但在临床应用中,使用高浓度的他汀类药物治疗时,往往又会出现多种副作用,如肝脏损伤、出血性中风、糖尿病和横纹肌溶解症等,从而限制了他汀类药物的治疗效果。因此,如何提高药物生物利用度,减少血液循环损失,赋予药物特异靶向性使更多药物富集于病灶部位,减轻毒副作用,这些问题成为拓宽包括他汀类药物在内的小分子药物临床使用前景的重要挑战。
纳米技术的蓬勃发展,无疑为上述问题提供了良好的解决方法。纳米药物递送系统具有增强药物水溶性以提高有效浓度、缓释控释、靶向递送、协同给药等突出优点,因其粒径特点也可通过高渗透长滞留(enhancedpermeabilityandretentioneffect,EPR)效应自发的在疾病部位富集以提高药物疗效、防止毒副作用,近年来展现出了突出的应用前景。纳米药物的控释能力是指其可在微环境中特定刺激下实现药物的可控释放,例如特定温度、pH、氧化还原环境、声光刺激等。这种可控释放的实现依赖于纳米药物自身的特定结构。例如,酮缩硫醇键可响应高浓度活性氧断裂,二硫键可在高谷胱甘肽的还原环境中自发断开。在纳米药物中引入酮缩硫醇键,药物即可利用炎症部位如动脉粥样硬化病灶处与机体正常组织的ROS浓度差实现药物的定点释放,避免对正常细胞产生损伤。纳米药物的靶向性也是重要的研究内容,可利用不同疾病位点的特征表达蛋白构建特异靶向递送系统,从而达到“精准给药”的目的。
综上所述,目前的问题在于如何进一步优化载体材料,改善他汀类小分子药物的递送能力,使其更多的富集于动脉粥样硬化病灶部位,以解决现有药物缺乏靶向、易引发毒副作用这一难题。
发明内容
本发明的目的在于提供一种他汀前药和他汀靶向递送系统及其制备方法和应用,可用于动脉粥样硬化的治疗,解决现有技术中存在的问题。本发明首先合成一种具有活性氧响应能力的他汀前药。同时,将该前药与两亲性纤连蛋白靶向多肽聚合物(DSPE-PEG-CREKA)和替卡格雷通过溶剂蒸发法进行组装构建,获得他汀靶向递送系统。
本发明的他汀靶向递送系统具有多功能性,即活性氧响应的药物释放性能、主动靶向能力及多药递送能力,可改善他汀类药物的水溶性差、体内代谢循环快速等劣势,同时也赋予了药物主动靶向及控制释放能力,避免他汀类药物临床大剂量使用时常出现的肝毒性现象。此外,多药协同治疗往往能够弥补单药调控路径单一的不足,获得更好的治疗效果。本发明提供的他汀靶向递送系统为动脉粥样硬化药物治疗提供了一种有前景的递送模式。
本发明提供的他汀靶向递送系统,该他汀靶向递送系统由他汀前药与纤连蛋白靶向多肽聚合物链构建而成,并包封疏水性小分子协同抗炎药物;所述的他汀前药为α-生育酚琥珀酸酯-聚乙二醇-氧化还原敏感键-他汀;所述的纤连蛋白靶向多肽聚合物链为二硬脂酰基磷脂酰乙醇胺-聚乙二醇-纤连蛋白靶向肽(CREKA)。
所述的氧化还原敏感键包括酮缩硫醇键、二硫键。
所述的他汀包括辛伐他汀、洛伐他汀、普伐他汀、氟伐他汀、阿托伐他汀、瑞舒伐他汀和匹伐他汀中的至少一种。优选地,所述的他汀为辛伐他汀。
所述的包封的疏水性小分子协同抗炎药物为替卡格雷。
本发明提供的辛伐他汀前药的制备方法经过以下步骤:
1)将3-巯基丙酸和无水丙酮用无水盐酸气体饱和通气反应0.5-1小时后转移至-20℃冷冻3-4小时,随后依次用冷水和正己烷洗涤三次,得到白色晶体,再将所得晶体真空干燥,得到酮缩硫醇(thioketal,TK)产物;其中3-巯基丙酸和无水丙酮的体积比为1:1.7;
2)将氨基-聚乙二醇-亚氨基-叔丁氧羰基(NH2-PEG-NH-BOC)、α-生育酚琥珀酸酯(α-TocopherolSuccinate,TOS)、1-乙基-(3-二甲基氨基丙基)碳化二亚胺盐酸盐(EDC-HCl)和N-羟基琥珀酰亚胺(NHS)溶于5mL无水N,N-二甲基甲酰胺(DMF)中,氮气保护室温下反应22-24小时后进行透析纯化处理,随后冻干得到α-生育酚琥珀酸酯-聚乙二醇-亚氨基-叔丁氧羰基(TOS-PEG-NH-BOC)产物;其中NH2-PEG-NH-BOC、α-生育酚琥珀酸酯、EDC-HCl和NHS的摩尔比为1:1.25:2:2;所述的聚乙二醇分子量大小为2000。
3)将步骤2)制备的TOS-PEG-NH-BOC溶于三乙醇胺(Triethanolamine,TEA)和二氯甲烷(Dichloromethane,DCM)中,室温反应20-30分钟后,旋蒸除去过量的DCM并进行三次乙醚沉淀处理,随后冻干得到α-生育酚琥珀酸酯-聚乙二醇-氨基(TOS-PEG-NH2)产物;其中TEA和DCM的体积比为1:6;
4)将步骤3)制备的TOS-PEG-NH2、步骤1)制备的TK、EDC-HCl和NHS溶于5mL无水N,N-二甲基甲酰胺中,氮气保护室温反应22-24小时后进行透析纯化处理,随后冻干得到α-生育酚琥珀酸酯-聚乙二醇-酮缩硫醇(TOS-PEG-TK)产物;其中TOS-PEG-NH2、TK、EDC-HCl和NHS的摩尔比为1:10:2:2;
5)将步骤4)制备的TOS-PEG-TK、二环己基碳二亚胺(Dicyclohexylcarbodiimide,DCC)、辛伐他汀(simvastatin,SIM)和4-二甲氨基吡啶(4-Dimethylaminopyridine,DMAP)溶于5mL二氯甲烷,室温反应12小时后进行透析纯化处理,随后冻干得到辛伐他汀前药链α-生育酚琥珀酸酯-聚乙二醇-酮缩硫醇-辛伐他汀(TOS-PEG-TK-SIM);其中TOS-PEG-TK、DCC、辛伐他汀和DMAP的摩尔比为1:1.25:2:2。
上述的制备方法中,步骤2)及步骤4)所述的透析条件为:2000Da透析袋,超纯水为透析介质,透析24小时,每6小时更换透析液一次。步骤5)所述的透析条件为:2000Da透析袋,先使用DMF为透析介质,透析24小时,每6小时更换透析液一次;随后再使用超纯水为透析介质,透析48小时,每6小时更换透析液一次。
本发明提供的他汀靶向递送系统制备方法经过以下步骤:
1)将他汀前药(α-生育酚琥珀酸酯-聚乙二醇-酮缩硫醇-辛伐他汀)、纤连蛋白靶向多肽聚合物(二硬脂酰基磷脂酰乙醇胺-聚乙二醇-纤连蛋白靶向肽)和替卡格雷溶于1mL丙酮,超声使其完全溶解,得到混合溶液;
2)将混合溶液滴加入剧烈搅拌的10mL蒸馏水中,室温反应30分钟后,旋蒸除去丙酮并冻干,得到最终产物;
其中,α-生育酚琥珀酸酯-聚乙二醇-酮缩硫醇-辛伐他汀、二硬脂酰基磷脂酰乙醇胺-聚乙二醇-纤连蛋白靶向肽和替卡格雷的质量比为4:1:1;其中的辛伐他汀可用洛伐他汀、普伐他汀、氟伐他汀、阿托伐他汀、瑞舒伐他汀或匹伐他汀替代。
所述的他汀靶向递送系统的粒径为50-300 nm,优选180nm。
本发明提供了一种他汀前药和他汀靶向递送系统及其制备方法和应用。该他汀靶向递送系统用于制造治疗动脉粥样硬化的药物。
本发明最终形成具有氧化还原响应释放特性,动脉粥样硬化靶向性,并可协同递送多药的纳米递送系统。在递送系统到达疾病部位并响应裂解后,他汀、α-生育酚及替卡格雷三种药物协同发挥抗炎抗氧化功能,从而高效治疗动脉粥样硬化。
本发明的技术效果如下:
本发明将他汀通过氧化还原敏感键与聚乙二醇连接制备两亲性前药,提高药物水溶性且使其具有氧化还原敏感性,氧化还原敏感键可响应动脉粥样硬化病灶部位高浓度活性氧断裂,释放他汀。
本发明中引入两亲性纤连蛋白靶向多肽聚合物DSPE-PEG-CREKA,在前药链与靶向链在水溶液中自发聚集构建纳米胶束递送系统时,靶向肽CREKA暴露在纳米胶束外侧,赋予递送系统主动靶向动脉粥样硬化斑块能力。
本发明中他汀前药链上同时连接抗炎药物α-生育酚琥珀酸酯,且在体系中包封抗血小板药物替卡格雷,能够发挥协同给药功效。
本发明最终形成具有活性氧响应释放特性,动脉粥样硬化靶向性,并可协同递送多药的纳米递送系统。在递送系统到达疾病部位并响应裂解后,辛伐他汀、α-生育酚及替卡格雷三种药物协同发挥抗炎抗氧化功能,从而高效治疗动脉粥样硬化。
总之,本发明的他汀靶向递送系统具有多功能性,即活性氧响应的药物释放性能、主动靶向能力及多药递送能力,可改善他汀类药物的水溶性差、体内代谢循环快速等劣势,同时也赋予了药物主动靶向及控制释放能力,避免他汀类药物临床大剂量使用时常出现的肝毒性现象。此外,多药协同治疗往往能够弥补单药调控路径单一的不足,获得更好的治疗效果。总之,本发明提供的他汀靶向递送系统为动脉粥样硬化药物治疗提供了一种有前景的递送模式。
附图说明
图1:他汀前药(α-生育酚琥珀酸酯-聚乙二醇-酮缩硫醇键-辛伐他汀)合成路线。
图2:他汀前药(α-生育酚琥珀酸酯-聚乙二醇-酮缩硫醇键-辛伐他汀)核磁共振氢谱图。
图3:他汀靶向递送系统(TPTS/C/T)冷冻透射电镜图。
图4:他汀靶向递送系统(TPTS/C/T)在体外氧化还原刺激下的辛伐他汀释放图。
图5:不同药物处理后通过流式细胞仪检测细胞内活性氧水平,反映靶向递送系统抗氧化能力。
图6:不同药物处理后酶联免疫吸附测定(ELISA)细胞上清液中炎性因子分泌水平,反映靶向递送系统抗炎能力。
图7:小鼠胸主动脉大体油红染色定量结果图。
图8:小鼠主动脉根部油红染色定量结果图。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。实施例中未注明具体条件的实验方法,通常按照常规条件以及手册中所述的条件,或按照制造厂商所建议的条件;所用的通用设备、材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1:他汀前药(α-生育酚琥珀酸酯-聚乙二醇-酮缩硫醇-辛伐他汀)的制备
1)量取17.08mL 3-巯基丙酸和29.32mL无水丙酮加入圆底烧瓶中,用无水盐酸气体饱和通气反应1小时,待反应停止后转移至-20℃冷冻4小时,随后再依次用冷水和正己烷洗涤3次,得到白色晶体。将所得晶体置于真空干燥器中干燥,得到酮缩硫醇(TK)产物。
2)分别称取100mg NH2-PEG-NH-BOC、33.17mgTOS、19.17mgEDC-HCl和11.51mgNHS溶于5mL无水N,N-二甲基甲酰胺(DMF)中,在氮气保护条件下室温反应24小时。将反应溶液转入透析袋中进行纯化(透析条件为:2000Da透析袋,超纯水为透析介质,透析24小时,每6小时更换透析液一次),随后进行真空冻干处理,得到白色粉末状的TOS-PEG-NH-BOC产物。
3)称取50mg上步反应制得的TOS-PEG-NH-BOC产物溶于400µLTEA和2.6mLDCM中,在室温下搅拌反应30分钟,随后通过旋转蒸发除去过量的DCM。将冷凝后所得的样品溶液滴加至乙醚中沉淀,重复三次,随后再进行真空冻干处理,得到TOS-PEG-NH2。
4)分别称取100mg上步反应制得的TOS-PEG-NH2、101mg1)步反应制得的TK、15.3mgEDC-HCl和9.3mgNHS溶于5ml无水N,N-二甲基甲酰胺(DMF)中,在氮气保护条件下室温反应24小时。将反应溶液转入透析袋中进行纯化(透析条件为:2000Da透析袋,超纯水为透析介质,透析24小时,每6小时更换透析液一次),随后进行真空冻干处理,得到白色粉末状的TOS-PEG-TK产物。
5)分别称取183.2mg上步反应制得的TOS-PEG-TK、17.3mgDCC溶于5mLDCM,0℃条件下搅拌反应30分钟。随后称取56.2mg辛伐他汀和16.46mgDMAP加入上述混合物中,室温反应12小时后,将反应溶液转入透析袋中进行纯化(透析条件为:先使用DMF为透析介质,透析24小时,每6小时更换透析液一次;随后再使用超纯水为透析介质,透析48小时,每6小时更换透析液一次),随后进行真空冻干处理,得到白色粉末状的TOS-PEG-TK-SIM(TPTS)产物,即他汀前药。
实施例1中他汀前药的合成路线及核磁氢谱图分别如图1、图2所示,充分证明了本发明中他汀前药的成功合成。
实施例2:他汀靶向递送系统的制备
分别称取8mg TOS-PEG-TK-SIM、2mgDSPE-PEG-CREKA和2mg替卡格雷溶于1mL丙酮,超声处理15分钟使其完全溶解。随后,在剧烈搅拌下,将上述混合溶液滴加至10mL蒸馏水中,室温搅拌反应30分钟后,通过旋转蒸发除去体系中的丙酮,再将所得溶液通过滤膜(0.22µm)进一步纯化并进行真空冻干处理,得到最终他汀靶向递送系统TPTS/C/T。通过冷冻透射电镜观察所得他汀靶向递送系统形貌,如图3。
实施例3:体外模拟他汀靶向递送系统氧化还原响应释药行为研究
分别称取2mgTOS-PEG-TK-SIM(TPTS)前药及2mg不含TK键的TPS前药并分别溶于2mL缓冲介质A(PBS,pH7.4),缓冲介质B(PBS+1mMH2O2,pH7.4)及缓冲介质C(PBS+10mMH2O2,pH7.4)中。随后,将各组溶液加入截留分子量为3500的透析袋中并转移至含有相应透析缓冲液介质(20mL)的离心管中,并置于37℃恒温摇床中150rpm孵育,分别在0、8、12和24小时四个时间段取样,并用相同体积透析缓冲液补齐20mL体系。用HPLC进行不同条件下辛伐他汀释放量的检测,色谱条件为:C18反相色谱柱,90%乙腈作为流动相,流速为1mL/min,226nm处波长检测。不同组体外响应释放结果如图4,随缓冲介质中H2O2浓度的加大,TPTS前药辛伐他汀的累积释放量也随之提高,而不含TK键的TPS前药与含H2O2缓冲介质共同孵育并未表现出响应释放行为,充分证明了本发明中他汀前药由于引入酮缩硫醇键从而具有活性氧响应释药能力。
实施例4:体外细胞内抗氧化能力研究
使用二氯荧光素(DCFH)探针检测细胞内活性氧(ROS)生成水平。将Raw264.7细胞以5×105个细胞/孔的密度接种在6孔板中,每孔加入1mL的DMEM高糖培养基(10%胎牛血清+1%青霉素-链霉素)并在37℃、5%CO2条件下培养12小时使细胞生长和稳定贴壁。随后分别用游离的辛伐他汀、替卡格雷、辛伐他汀前药(TPTS)、TPTS/C/T纳米胶束预处理细胞12小时,再使用脂多糖(Lipopolysaccharide,LPS)(100ng/mL)和γ干扰素(IFN-γ)(100IU/mL)对Raw264.7细胞进行炎性诱导24小时。最后,向每孔加入2×10-5mol的DCFH-DA探针,37℃孵化20分钟后,使用流式细胞仪定量检测DCFH探针的荧光强度,如图5。本发明中他汀前药具有比辛伐他汀、替卡格雷更好的细胞内抗氧化能力,他汀靶向递送系统表现出相比他汀前药更强的抗氧化能力。
实施例5:体外细胞内抗炎能力研究
将Raw264.7细胞以5×105个细胞/孔的密度接种在6孔板中,每孔加入1mL的DMEM高糖培养基(10%胎牛血清+1%青霉素-链霉素)并在37℃、5%CO2条件下培养12小时使细胞生长和稳定贴壁。随后分别用游离的辛伐他汀、替卡格雷、辛伐他汀前药(TPTS)、TPTS/C/T纳米胶束预处理细胞12小时,再使用LPS(100ng/mL)和IFN-γ(100IU/mL)对Raw264.7细胞进行炎性诱导24小时。最后,收集细胞培养上清液,通过ELISA试剂盒分别检测各组肿瘤坏死因子-α(THF-α)、单核细胞趋化蛋白-1(MCP-1)和白介素-1β(IL-1β)三种炎症因子的分泌水平,如图6。本发明中他汀前药具有比辛伐他汀、替卡格雷更好的细胞内抗炎能力,他汀靶向递送系统表现出相比他汀前药更强的抗炎能力。
实施例6:体内动脉粥样硬化治疗效果考察
选择ApoE-/-小鼠作为研究对象,通过高脂饲料喂养构建动脉粥样硬化模型。首先,将ApoE-/-小鼠喂食高脂饲料4周后随机分为生理盐水、辛伐他汀、替卡格雷、TPTS前药和TPTS/C/T纳米胶束共五组,每组n=6,随后各组用相应药物分别给药,一周两次,共给药8周,期间一直保持高脂喂养(尾静脉注射给药,剂量10mg/kg-1,TPTS前药、TPTS/C/T纳米胶束组以辛伐他汀等量)。12周实验结束后,取小鼠胸主动脉进行后续检测(对照组小鼠在高脂喂养4周后直接取材)。通过小鼠胸主动脉大体油红染色定量结果及主动脉根部切片油红染色定量结果评估药物对ApoE-/-小鼠动脉粥样硬化的治疗效果,如图7、图8。图7中小鼠胸主动脉大体油红染色定量结果体现了各组小鼠经不同药物治疗后胸主动脉处发生粥样硬化的血管表面积,图8主动脉根部切片油红染色定量结果主要体现动脉部位的斑块厚度,两者相结合用以更全面地评估小鼠主动脉的病变程度。从结果看,本发明中他汀前药相比辛伐他汀、替卡格雷单药治疗,在体内发挥出更好的动脉粥样硬化治疗效果。而他汀靶向递送系统因其具备多功能性,如活性氧响应释药性能、主动靶向及多药递送,使治疗组小鼠动脉粥样硬化病变程度大幅下降,在动脉粥样硬化治疗领域有着良好的应用前景。
Claims (10)
1.一种他汀前药和他汀靶向递送系统,其特征在于:该他汀靶向递送系统由他汀前药与纤连蛋白靶向多肽聚合物链构建而成,并包封疏水性小分子协同抗炎药物;所述的他汀前药为α-生育酚琥珀酸酯-聚乙二醇-氧化还原敏感键-他汀;所述的纤连蛋白靶向多肽聚合物链为二硬脂酰基磷脂酰乙醇胺-聚乙二醇-纤连蛋白靶向肽(CREKA)。
2.根据权利要求1所述的他汀靶向递送系统,其特征在于:所述的他汀为辛伐他汀、洛伐他汀、普伐他汀、氟伐他汀、阿托伐他汀、瑞舒伐他汀和匹伐他汀中的至少一种;优选地,所述的他汀为辛伐他汀。
3.根据权利要求1所述的他汀靶向递送系统,其特征在于:所述的包封的疏水性小分子协同抗炎药物为替卡格雷;所述的氧化还原敏感键包括酮缩硫醇键、二硫键。
4.根据权利要求2所述的他汀靶向递送系统,其特征在于:辛伐他汀前药的制备方法经过以下步骤:
1)将3-巯基丙酸和无水丙酮用无水盐酸气体饱和通气反应0.5-1小时后转移至-20℃冷冻3-4小时,随后依次用冷水和正己烷洗涤三次,得到白色晶体,再将所得晶体真空干燥,得到酮缩硫醇(TK)产物;
2)将氨基-聚乙二醇-亚氨基-叔丁氧羰基(NH2-PEG-NH-BOC)、α-生育酚琥珀酸酯(α-TocopherolSuccinate,TOS)、1-乙基-(3-二甲基氨基丙基)碳化二亚胺盐酸盐(EDC-HCl)和N-羟基琥珀酰亚胺(NHS)溶于5mL无水N,N-二甲基甲酰胺(DMF)中,氮气保护室温下反应22-24小时后进行透析纯化处理,随后冻干得到α-生育酚琥珀酸酯-聚乙二醇-亚氨基-叔丁氧羰基(TOS-PEG-NH-BOC)产物;
3)将步骤2)制备的TOS-PEG-NH-BOC溶于三乙醇胺(Triethanolamine,TEA)和二氯甲烷(Dichloromethane,DCM)中,室温反应20-30分钟后,旋蒸除去过量的DCM并进行三次乙醚沉淀处理,随后冻干得到α-生育酚琥珀酸酯-聚乙二醇-氨基(TOS-PEG-NH2)产物;其中TEA和DCM的体积比为1:6;
4)将步骤3)制备的TOS-PEG-NH2、步骤1)制备的TK、EDC-HCl和NHS溶于5mL无水N,N-二甲基甲酰胺中,氮气保护室温反应22-24小时后进行透析纯化处理,随后冻干得到α-生育酚琥珀酸酯-聚乙二醇-酮缩硫醇(TOS-PEG-TK)产物;
5)将步骤4)制备的TOS-PEG-TK、二环己基碳二亚胺(Dicyclohexylcarbodiimide,DCC)、辛伐他汀(simvastatin,SIM)和4-二甲氨基吡啶(4-Dimethylaminopyridine,DMAP)溶于5mL二氯甲烷,室温反应12小时后进行透析纯化处理,随后冻干得到辛伐他汀前药链α-生育酚琥珀酸酯-聚乙二醇-酮缩硫醇-辛伐他汀(TOS-PEG-TK-SIM)。
5.根据权利要求4所述的他汀靶向递送系统,其特征在于:步骤1)所述的3-巯基丙酸和无水丙酮的体积比为1:1.7;步骤2)所述的NH2-PEG-NH-BOC、α-生育酚琥珀酸酯、EDC-HCl和NHS的摩尔比为1:1.25:2:2;所述的聚乙二醇分子量大小为2000;步骤4)所述的TOS-PEG-NH2、TK、EDC-HCl和NHS的摩尔比为1:10:2:2;步骤5)所述的TOS-PEG-TK、DCC、辛伐他汀和DMAP的摩尔比为1:1.25:2:2;所述的辛伐他汀用洛伐他汀、普伐他汀、氟伐他汀、阿托伐他汀、瑞舒伐他汀或匹伐他汀替代。
6.根据权利要求4所述的他汀靶向递送系统,其特征在于:步骤2)及步骤4)所述的透析条件为:2000Da透析袋,超纯水为透析介质,透析24小时,每6小时更换透析液一次;步骤5)所述的透析条件为:2000Da透析袋,先使用DMF为透析介质,透析24小时,每6小时更换透析液一次;随后再使用超纯水为透析介质,透析48小时,每6小时更换透析液一次。
7.权利要求1所述的他汀靶向递送系统制备方法,其特征在于经过以下步骤:
1)将他汀前药(α-生育酚琥珀酸酯-聚乙二醇-酮缩硫醇-辛伐他汀)、纤连蛋白靶向多肽聚合物(二硬脂酰基磷脂酰乙醇胺-聚乙二醇-纤连蛋白靶向肽)和替卡格雷溶于1mL丙酮,超声使其完全溶解,得到混合溶液;
2)将混合溶液滴加入剧烈搅拌的10mL蒸馏水中,室温反应30分钟后,旋蒸除去丙酮并冻干,得到最终产物他汀靶向递送系统。
8.根据权利要求7所述的制备方法,其特征在于所述的α-生育酚琥珀酸酯-聚乙二醇-酮缩硫醇-辛伐他汀、二硬脂酰基磷脂酰乙醇胺-聚乙二醇-纤连蛋白靶向肽和替卡格雷的质量比为4:1:1。
9.根据权利要求7所述的他汀靶向递送系统制备方法,其特征在于所述的他汀靶向递送系统的粒径为50-300 nm,优选180nm。
10.权利要求1所述的他汀靶向递送系统应用于制造治疗动脉粥样硬化的药物。
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Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA2595485A1 (en) * | 2007-08-01 | 2009-02-01 | Bc Cancer Agency | Liposomal compositions for parenteral delivery of statins |
US20090074828A1 (en) * | 2007-04-04 | 2009-03-19 | Massachusetts Institute Of Technology | Poly(amino acid) targeting moieties |
WO2012093227A1 (fr) * | 2011-01-07 | 2012-07-12 | Jacquot Epouse Guilmin Liliane | Promédicaments pour une délivrance au niveau du foie d'une statine |
US20130098357A1 (en) * | 2011-10-20 | 2013-04-25 | Saint Joseph's Translational Research Institute, Inc. | Cerivastatin to treat pulmonary disorders |
US20160022835A1 (en) * | 2013-03-15 | 2016-01-28 | The Brigham And Women's Hospital, Inc. | Targeted Polymeric Inflammation-Resolving Nanoparticles |
CN108066771A (zh) * | 2017-12-15 | 2018-05-25 | 北京思如诺科技有限公司 | 一种具有高载药量环境响应型抗肿瘤纳米药物、载体以及制备方法 |
CN112402620A (zh) * | 2020-12-07 | 2021-02-26 | 南开大学 | 肿瘤微环境还原响应性的纳米药物及其制备方法 |
-
2022
- 2022-05-19 CN CN202210542474.3A patent/CN115025242B/zh active Active
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20090074828A1 (en) * | 2007-04-04 | 2009-03-19 | Massachusetts Institute Of Technology | Poly(amino acid) targeting moieties |
CA2595485A1 (en) * | 2007-08-01 | 2009-02-01 | Bc Cancer Agency | Liposomal compositions for parenteral delivery of statins |
WO2012093227A1 (fr) * | 2011-01-07 | 2012-07-12 | Jacquot Epouse Guilmin Liliane | Promédicaments pour une délivrance au niveau du foie d'une statine |
US20130098357A1 (en) * | 2011-10-20 | 2013-04-25 | Saint Joseph's Translational Research Institute, Inc. | Cerivastatin to treat pulmonary disorders |
US20160022835A1 (en) * | 2013-03-15 | 2016-01-28 | The Brigham And Women's Hospital, Inc. | Targeted Polymeric Inflammation-Resolving Nanoparticles |
CN108066771A (zh) * | 2017-12-15 | 2018-05-25 | 北京思如诺科技有限公司 | 一种具有高载药量环境响应型抗肿瘤纳米药物、载体以及制备方法 |
CN112402620A (zh) * | 2020-12-07 | 2021-02-26 | 南开大学 | 肿瘤微环境还原响应性的纳米药物及其制备方法 |
Non-Patent Citations (1)
Title |
---|
RUNZE ZHAO ET.AL.: "A multifunctional nano-delivery system enhances the chemo-co-phototherapy of tumor multidrug resistance via mitochondrial-targeting and inhibiting P-glycoprotein-mediated efflux", 《J. MATER. CHEM. B》, no. 9 * |
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