CN115429755B - pH响应电荷翻转及缺氧响应药物释放的胶束递药系统及其制备方法 - Google Patents
pH响应电荷翻转及缺氧响应药物释放的胶束递药系统及其制备方法 Download PDFInfo
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- CN115429755B CN115429755B CN202110598058.0A CN202110598058A CN115429755B CN 115429755 B CN115429755 B CN 115429755B CN 202110598058 A CN202110598058 A CN 202110598058A CN 115429755 B CN115429755 B CN 115429755B
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Abstract
本发明属于生物技术领域,涉及一种pH响应电荷翻转及缺氧响应药物释放的胶束递药系统及其制备方法。本发明制剂由HJC0152缺氧响应前药、吉西他滨前药和合成的聚乙二醇‑聚赖氨酸‑2,3‑二甲基马来酸‑聚苯丙氨酸嵌段聚合物以及注射溶媒等制成装载HJC0152和吉西他滨的胶束递药系统。本发明的载药纳米胶束递药系统,粒径小、分布均一、包封率高,载药胶束在肿瘤微酸环境下发生电荷由负到正的翻转,能促进胶束递药系统向肿瘤深部进行穿透。同时,通过缺氧响应前药策略,能保证药物在体循环中的稳定性,以及定点精准释放,具有良好的生物安全性。
Description
技术领域
本发明属于生物技术领域,涉及具有靶向、穿透、药物释放三重功效的给药系统,具体涉及一种pH响应电荷翻转及缺氧响应药物释放的胶束递药系统及其制备方法。
背景技术
据报道,纳米药物递释系统的发展改善了传统化疗药物体内循环时间短、毒副作用大的问题,在癌症的治疗中有着非常重要的应用。基于EPR效应和主动靶向修饰策略开发的被动/主动靶向纳米制剂可进一步提高荷载药物在靶部位的蓄积[2]。然而,尽管纳米递药系统能够一定程度上提高药物的循环时间,增加肿瘤蓄积,但是对于一些低血供实体瘤的治疗(例如胰腺癌),肿瘤高间质液压(IFP)和致密的胞外基质严重限制了纳米制剂和荷载药物的肿瘤渗透,使得药物难以穿过基质屏障到达肿瘤深部发挥疗效,因而无法彻底清除肿瘤细胞,这也是导致肿瘤复发、转移,病人预后不良的重要原因。因此,针对该类肿瘤,能够实现肿瘤基质屏障突破并精准按需递送药物至肿瘤深处的纳米递送策略是亟需的。
有学者提出通过递送透明质酸酶基质纤维化抑制剂等方式来缓解基质压力,但研究结果表明,所采用策略虽然能在一定程度上缓解基质压力,但在酶降解基质后的很快一段时间内,基质屏障又重塑;此外,对于通路抑制剂的首次递送,仍是要先解决突破基质屏障,才能实现对肿瘤深部基质情况的调节。
纳米制剂的粒径和电荷是影响其肿瘤穿透性的重要因素,有研究表明,胞外基质的重要组成成分之一胶原纤维的间距为40nm左右。另外有文献公开了,粒径在30nm的胶束相对于粒径为50nm、70nm、100nm的胶束具有更好的肿瘤穿透性,可以穿透渗透性差的胰腺癌和乳腺癌等肿瘤的基质间隙,然而小粒径带来的穿透增强效果依然有限,并且在其后续的入胞过程中受到一定的限制。
荷正电性的物质不仅能够穿透肿瘤基质,增强摄取,还具有核定位的特性,然而,其在血液循环中却又容易被RES捕获并吞噬,在非靶部位蓄积造成毒副作用,与其相反,电中性或电负性的纳米制剂具有优异的长循环特性,因此,粒径小于40nm,且具有肿瘤微环境响应电荷翻转(由电中性/电负性向电正性翻转)的纳米递送系统或许可以实现有效的肿瘤穿透递送。
由于肿瘤异常的糖酵解和缺氧条件,其微环境pH值相较于生理中性pH较低,该特点比高GSH、高ROS、高代谢酶表达等为住的细胞内微环境特点更容易触发且适用范围更广,因此,针对低pH值特性开发的电荷翻转型递送系统具有普适性。
聚合物胶束是由合成的两亲性嵌段共聚物在水中自组装形成的一种热力学稳定的胶体体系,其粒径通常在10-100nm左右,具良好的生物相容性,其内部疏水性环境可以高效荷载疏水性药物,并且,其载药量相对于脂质体或聚合物纳米粒较高。同时,聚合物材料合成处方容易控制,胶束制备方法简单易行,已被广泛应用于现阶段的纳米递送系统的开发,例如韩国的Gennexol PM和俄罗斯的已获批上市,胶束递送系统的产业化前景广阔。
同时,肿瘤内部缺氧是所述类低血供肿瘤的另一大特点,针对该特性,设计缺氧响应的药物释放策略将能更符合精准按需药物递送的目标。
基于现有技术的现状,本申请的发明人拟提供一种具有靶向、穿透、药物释放三重功效的给药系统。
发明内容
本发明的目的是基于现有技术的现状,提供一种具有靶向、穿透、药物释放三重功效的给药系统,具体涉及一种pH响应电荷翻转促肿瘤穿透及缺氧响应原型药物释放的胶束递药系统及其构建方法。
本发明提供了一种pH响应电荷翻转及缺氧响应药物释放的胶束递药系统,其组成特征为缺氧响应前药分子、水解酶响应前药分子、pH响应电荷翻转嵌段共聚物;所述胶束的粒径平均粒径为32.5nm。
本发明中,所述改造为缺氧响应前药的药物为HJC0152 hydrochloride,其与缺氧响应分子硝基咪唑分子间通过氨基甲酸酯键共价相连。
本发明中,所述改造为水解酶响应前药的药物为盐酸吉西他滨,其与油酸分子间通过酰胺键共价相连。
本发明中,所述pH响应电荷翻转聚合物为聚乙二醇-聚赖氨酸-2,3-二甲基马来酸-聚苯丙氨酸嵌段聚合物,先通过聚乙二醇-氨基引发的开环聚合反应形成聚乙二醇-聚赖氨酸-聚苯丙氨酸嵌段聚合物,再通过赖氨酸上氨基触发2,3-二甲基马来酸酐开环形成最终的pH响应电荷翻转聚合物。
本发明中,所述的胶束递药系统是通过薄膜水化法制备的,其对HJC0152前药的包封率为85~90%,载药量为5~8%;对吉西他滨前药的包封率为90~95%,载药量为1~3%,平均粒径为32.5nm,zeta电势为-5.6mV。
本发明的pH响应电荷翻转及缺氧响应药物释放的胶束递药系统按图1所示合成路线制备。
本发明提供了一种缺氧响应前药分子的合成方法,包括以下步骤:
(1)称取2-羟甲基-1-甲基-5-硝基咪唑,三乙胺,溶解于二甲基亚砜(DMSO)中,之后,在氮气保护下,加入溶于DMSO的对硝基苯基氯甲酸酯,随后反应6~12小时;反应结束后,旋蒸除去溶剂,柱色谱分离(流动相:石油醚∶乙酸乙酯=90∶10),获得淡黄色产物1;在所述反应中,对硝基苯基氯甲酸酯为2-羟甲基-1-甲基-5-硝基咪唑摩尔数的1~1.5倍,三乙胺用量为催化量;
(2)称取步骤(1)中得到的淡黄色产物1,HJC0152 hydrochloride,4-二甲氨基吡啶(DMAP),在氩气保护下加入无水DMSO。反应12~24小时后,旋蒸除去溶剂,柱色谱分离(流动相:石油醚∶乙酸乙酯=80∶20),得到淡黄色晶体2。所述的化合物1用量为HJC0152hydrochloride摩尔数的1~2倍;所述的DMAP用量为HJC0152 hydrochloride摩尔数的2~3倍。
本发明提供了一种水解酶响应前药分子的合成方法,包括以下步骤:
(1)将盐酸吉西他滨、无水三乙胺、十八烷酰氯在氩气保护的冰浴条件下加无水DMSO搅拌反应24小时,待反应结束后,旋蒸除去反应溶剂,柱色谱分离(流动相:甲醇∶二氯甲烷=10∶90)到白色晶体3。所述的十八烷酰氯用量为盐酸吉西他滨摩尔数的1~1.5倍;所述的无水三乙胺用量为盐酸吉西他滨摩尔数的2~3倍。
本发明提供了一种pH响应电荷翻转嵌段共聚物的合成方法,包括以下步骤:
(1)将Nε-苄氧羰基-L-赖氨酸、三光气,溶解于30~50毫升无水四氢呋喃中,氩气保护条件下于50~70℃油浴中反应4~10个小时。随后冷却至室温,逐滴滴加到于-20℃预冷的正己烷中,沉淀析出产物,抽滤得到白色化合物4。所述的三光气的用量为Nε-苄氧羰基-L-赖氨酸摩尔数的0.25~0.6倍,正己烷用量为四氢呋喃体积的10~15倍;
(2)将苯丙氨酸、三光气溶解于30~50毫升无水四氢呋喃中,氩气保护条件下于50~70℃油浴中反应4~10个小时。随后冷却至室温,逐滴滴加到于-20℃预冷的正己烷中,沉淀析出产物,抽滤得到白色化合物5。所述的三光气的用量为苯丙氨酸摩尔数的0.3~0.6倍,正己烷用量为四氢呋喃体积的10~15倍;
(3)将聚乙二醇-氨基,化合物4溶解于无水DMSO中,氩气保护下于50~70℃油浴中反应24~48小时后,再加入溶于DMSO中的化合物5,继续同样的条件下反应24~48小时。最后,旋蒸除去反应溶剂,加无水乙醚析出产物,离心得到浅黄色化合物6。所述的赖氨酸NCA单体(化合物4)为聚乙二醇摩尔数的10~20倍,所述的苯丙氨酸NCA单体(化合物5)为聚乙二醇摩尔数的20~30倍。所述的乙醚用量为DMSO体积的3~10倍;
(4)将化合物6溶解于无水三氟醋酸中,加入溶于冰醋酸的氢溴酸溶液(30%),于室温下反应3个小时。随后旋蒸除去反应溶剂,加无水乙醚析出产物,离心得到淡黄色化合物7。所述的三氟醋酸的用量为10~30毫升,氢溴酸溶液的用量为0.5~3毫升,乙醚用量为DMSO体积的3~10倍;
(5)将化合物7、2,3-二甲基马来酸酐、三乙胺溶解于无水DMSO中,氩气保护下于30~60℃油浴中反应24~48小时后,转移至截留分子量为3500Da的透析袋中,于纯水中透析除去过量的反应物,最后冷冻干燥得到黄色嵌段共聚物8。所述的2,3-二甲基马来酸酐的用量为化合物7摩尔数的20~40倍;三乙胺的用量为化合物7摩尔数的30~60倍。
本发明提供了载药胶束递药系统的制备方法,包括以下步骤:
薄膜水化法:称取一定量的嵌段共聚物8、两种前药分子溶于DMSO中,然后将溶液转移至单口瓶中,旋蒸除去有机溶液,再置于真空干燥箱中抽干过夜,最后加纯水或生理盐水,于超声条件下,薄膜水化形成载药胶束。所述的方法中,HJC0152前药用量为嵌段共聚物8质量的5~10%;吉西他滨前药用量为嵌段共聚物8质量的1~3%;水化所用液体体积为10-100毫升。
本发明的优点与效果:
1、本发明中所述的胶束递药系统构成原料包括聚乙二醇、赖氨酸、苯丙氨酸等,在体内降解后不会对机体产生明显毒性。
2、本发明中所述的胶束递药系统含有的聚苯丙氨酸嵌段为大π体系,能够很好地与同样含有大π体系的前药分子形成π-π堆积效应,利于形成粒径小、包封率高的稳定的载药纳米胶束递药系统。
3、本发明中所述的薄膜水化法制备的胶束粒径小,分散均一,且具有pH响应电荷翻转特性,为后续实现更深程度的肿瘤穿透提供可能性。
4、本发明中所述的HJC0152前药包含缺氧敏感基团硝基咪唑,在常氧条件下,保持稳定的化学结构,降低HJC0152的细胞毒性;在缺氧条件下,被还原成氨基咪唑,通过电子回共引起HJC0152原型药物的离去,实现缺氧响应的药物释放。
5、本发明中所述的吉西他滨前药包含不稳定的酰胺键,能够在水解酶等作用下,实现酰胺键的水解,进而释放出吉西他滨游离药物。
6、本发明中所述的胶束递药系统能够响应于肿瘤微环境中低pH值的特点,实现电荷的由负往正的翻转,从而实现对肿瘤的更深程度的穿透作用。
附图说明
图1为HJC0152前药、吉西他滨前药、聚乙二醇-聚赖氨酸-2,3-二甲基马来酸-聚苯丙氨酸嵌段聚合物的合成路线图。
图2为HJC0152前药、吉西他滨前药、聚乙二醇-聚赖氨酸-2,3-二甲基马来酸-聚苯丙氨酸嵌段聚合物的核磁共振(NMR)谱图。
图3为胶束递药系统的粒径和zeta电势图。
图4为胶束递药系统pH响应zeta电势变化情况结果。
图5为胶束递药系统缺氧响应细胞毒性变化考察结果。
图6为胶束递药系统缺氧响应STAT3磷酸化抑制考察结果。
图7为胶束递药系统pH响应肿瘤球穿透增强考察结果。
图8为胶束递药系统在荷瘤小鼠中的肿瘤靶向考察结果。
图9为胶束递药系统在荷瘤小鼠中的抗肿瘤治疗效果考察结果。
具体实施方式
以下通过具体实施方式详细说明本发明的技术方案,应理解一下的具体实施方案仅为示例性,任何改动或变化只要不脱离本发明的技术方案设计,都应在本发明权利的要求保护范围之内。
实施例1:pH响应电荷翻转促肿瘤穿透及缺氧响应原型药物释放的胶束递药系统的构建,其两种前药分子以及嵌段共聚物合成方法如附图1所示,包括以下步骤:
(1)称取2-羟甲基-1-甲基-5-硝基咪唑1g,三乙胺10mg,溶解于DMSO中,之后,在氮气保护下,加入溶于DMSO的对硝基苯基氯甲酸酯1.27g,随后反应6小时。反应结束后,旋蒸除去溶剂,柱色谱分离(流动相:石油醚∶乙酸乙酯=90∶10),获得淡黄色产物1;
(2)称取500mg步骤(1)中得到的淡黄色产物1,HJC0152 hydrochloride612mg,4-二甲氨基吡啶(DMAP)244mg,在氩气保护下加入10毫升无水DMSO。反应12小时后,旋蒸除去溶剂,柱色谱分离(流动相:石油醚∶乙酸乙酯=80∶20),得到淡黄色晶体2。
(3)将1g盐酸吉西他滨、无水三乙胺673mg、十八烷酰氯1g在氩气保护的冰浴条件下加10毫升无水DMSO搅拌反应24小时,待反应结束后,旋蒸除去反应溶剂,柱色谱分离(流动相:甲醇∶二氯甲烷=10∶90)到白色晶体3。
(4)将1g Nε-苄氧羰基-L-赖氨酸、三光气264mg,溶解于30毫升无水四氢呋喃中,氩气保护条件下于70℃油浴中反应4个小时。随后冷却至室温,逐滴滴加到300毫升于-20℃预冷的正己烷中,沉淀析出产物,抽滤得到白色化合物4。
(5)将1g苯丙氨酸、三光气552mg溶解于30毫升无水四氢呋喃中,氩气保护条件下于50℃油浴中反应4个小时。随后冷却至室温,逐滴滴加到300毫升于-20℃预冷的正己烷中,沉淀析出产物,抽滤得到白色化合物5。
(6)将1g聚乙二醇-氨基,612mg化合物4溶解于30毫升DMSO中,氩气保护下于50℃油浴中反应24小时后,再加入764mg溶于10毫升DMSO中的化合物5,继续同样的条件下反应24小时。最后,旋蒸除去反应溶剂,加100毫升无水乙醚析出产物,离心得到浅黄色化合物6。
(7)将1g化合物6溶解于10毫升无水三氟醋酸中,加入0.5毫升溶于冰醋酸的氢溴酸溶液(30%),于室温下反应3个小时。随后旋蒸除去反应溶剂,加50毫升无水乙醚析出产物,离心得到淡黄色化合物7。
(8)将200mg化合物7、2,3-二甲基马来酸酐126mg、三乙胺101mg溶解于10毫升无水DMSO中,氩气保护下于40℃油浴中反应24小时后,转移至截留分子量为3500Da的透析袋中,于纯水中透析除去过量的反应物,最后冷冻干燥得到黄色嵌段共聚物8。
(9)薄膜水化法:称取10mg嵌段共聚物8、HJC0152前药0.5mg、吉西他滨前药0.1mg,溶于5毫升DMSO中,然后将溶液转移至单口瓶中,旋蒸除去有机溶液,再置于真空干燥箱中抽干过夜,最后加20毫升纯水,于超声条件下,薄膜水化形成载药胶束。
通过上述方法,成功合成了两种前药化合物以及pH响应电荷翻转的聚合物材料,核磁共振氢谱验证各化合物合成成功(附图2)。随后通过薄膜水化法制备载药胶束,其形态为规整的圆球状,粒径分布均一,平均粒径在32.5nm左右,zeta电势在-5.6mV(附图3),通过测量纳米粒在酸性和中性环境中的zeta电势变化,验证了载药胶束的pH响应电荷翻转特性(附图4)。同时,通过体外细胞水平的缺氧响应细胞毒性(附图5)以及对STAT3磷酸化通路的抑制考察实验(附图6)也证明了载药胶束递送系统具有良好的缺氧响应细胞毒性增强以及STAT3磷酸化抑制能力。
实施例2:荧光探针标记的pH响应电荷翻转促肿瘤穿透及缺氧响应原型药物释放的胶束递药系统的构建
(1)称取2-羟甲基-1-甲基-5-硝基咪唑1.5g,三乙胺10mg,溶解于DMSO中,之后,在氮气保护下,加入溶于DMSO的对硝基苯基氯甲酸酯2.85g,随后反应12小时。反应结束后,旋蒸除去溶剂,柱色谱分离(流动相:石油醚∶乙酸乙酯=90∶10),获得淡黄色产物1;
(2)称取1000mg步骤(1)中得到的淡黄色产物1,HJC0152 hydrochloride612mg,4-二甲氨基吡啶(DMAP)366mg,在氩气保护下加入20毫升无水DMSO。反应24小时后,旋蒸除去溶剂,柱色谱分离(流动相:石油醚∶乙酸乙酯=80∶20),得到淡黄色晶体2。
(3)将1g盐酸吉西他滨、无水三乙胺1g、十八烷酰氯1.5g在氩气保护的冰浴条件下加15毫升无水DMSO搅拌反应24小时,待反应结束后,旋蒸除去反应溶剂,柱色谱分离(流动相:甲醇∶二氯甲烷=10∶90)到白色晶体3。
(4)将2g Nε-苄氧羰基-L-赖氨酸、三光气1g,溶解于50毫升无水四氢呋喃中,氩气保护条件下于50℃油浴中反应10个小时。随后冷却至室温,逐滴滴加到500毫升于-20℃预冷的正己烷中,沉淀析出产物,抽滤得到白色化合物4。
(5)将2g苯丙氨酸、三光气1.1g溶解于50毫升无水四氢呋喃中,氩气保护条件下于50℃油浴中反应10个小时。随后冷却至室温,逐滴滴加到500毫升于-20℃预冷的正己烷中,沉淀析出产物,抽滤得到白色化合物5。
(6)将500mg聚乙二醇-氨基,612mg化合物4溶解于30毫升DMSO中,氩气保护下于50℃油浴中反应48小时后,再加入573mg溶于10毫升DMSO中的化合物5,继续同样的条件下反应48小时。最后,旋蒸除去反应溶剂,加150毫升无水乙醚析出产物,离心得到浅黄色化合物6。
(7)将1.5g化合物6溶解于15毫升无水三氟醋酸中,加入1毫升溶于冰醋酸的氢溴酸溶液(30%),于室温下反应5个小时。随后旋蒸除去反应溶剂,加100毫升无水乙醚析出产物,离心得到淡黄色化合物7。
(8)将400mg化合物7、2,3-二甲基马来酸酐378mg、三乙胺202mg溶解于15毫升无水DMSO中,氩气保护下于60℃油浴中反应24小时后,转移至截留分子量为3500Da的透析袋中,于纯水中透析除去过量的反应物,最后冷冻干燥得到黄色嵌段共聚物8。
(9)薄膜水化法:称取30mg嵌段共聚物8、HJC0152前药2mg、吉西他滨前药0.8mg,异硫氰酸荧光素(FITC)0.15mg,溶于10毫升DMSO中,然后将溶液转移至单口瓶中,旋蒸除去有机溶液,再置于真空干燥箱中抽干过夜,最后加60毫升纯水,于超声条件下,薄膜水化形成FITC荧光标记的载药胶束。
(10)称取20mg嵌段共聚物8、HJC0152前药1mg、吉西他滨前药0.2mg,DID荧光探针0.06mg,溶于10毫升DMSO中,然后将溶液转移至单口瓶中,旋蒸除去有机溶液,再置于真空干燥箱中抽干过夜,最后加50毫升生理盐水,于超声条件下,薄膜水化形成DID荧光标记的载药胶束。
在肿瘤球模型上考察FITC标记的载药胶束在不同pH条件下对肿瘤球的穿透效果,结果显示当pH值降低时,载药胶束具有更好的肿瘤球穿透效果,表明构建的载药胶束递送系统具有良好的pH响应穿透增强效果(附图7)。同时,也在动物水平上考察了DID标记的载药胶束的胰腺癌肿瘤靶向及穿透效果评价。结果显示,pH响应的胶束递药系统具有良好的肿瘤靶向性以及肿瘤深部穿透功能。(附图8)。
实施例3:pH响应电荷翻转促肿瘤穿透及缺氧响应原型药物释放的胶束递药系统的构建
(1)称取2-羟甲基-1-甲基-5-硝基咪唑1g,三乙胺10mg,溶解于DMSO中,之后,在氮气保护下,加入溶于DMSO的对硝基苯基氯甲酸酯1.9g,随后反应12小时。反应结束后,旋蒸除去溶剂,柱色谱分离(流动相:石油醚∶乙酸乙酯=90∶10),获得淡黄色产物1;
(2)称取500mg步骤(1)中得到的淡黄色产物1,HJC0152 hydrochloride306mg,4-二甲氨基吡啶(DMAP)183mg,在氩气保护下加入10毫升无水DMSO。反应24小时后,旋蒸除去溶剂,柱色谱分离(流动相:石油醚∶乙酸乙酯=80∶20),得到淡黄色晶体2。
(3)将1g盐酸吉西他滨、无水三乙胺1g、十八烷酰氯1.5g在氩气保护的冰浴条件下加10毫升无水DMSO搅拌反应24小时,待反应结束后,旋蒸除去反应溶剂,柱色谱分离(流动相:甲醇∶二氯甲烷=10∶90)到白色晶体3。
(4)将1g Nε-苄氧羰基-L-赖氨酸、三光气528mg,溶解于40毫升无水四氢呋喃中,氩气保护条件下于50℃油浴中反应12个小时。随后冷却至室温,逐滴滴加到400毫升于-20℃预冷的正己烷中,沉淀析出产物,抽滤得到白色化合物4。
(5)将1g苯丙氨酸、三光气828mg溶解于40毫升无水四氢呋喃中,氩气保护条件下于50℃油浴中反应8个小时。随后冷却至室温,逐滴滴加到400毫升于-20℃预冷的正己烷中,沉淀析出产物,抽滤得到白色化合物5。
(6)将1g聚乙二醇-氨基,918mg化合物4溶解于40毫升DMSO中,氩气保护下于50℃油浴中反应48小时后,再加入955mg溶于15毫升DMSO中的化合物5,继续同样的条件下反应48小时。最后,旋蒸除去反应溶剂,加100毫升无水乙醚析出产物,离心得到浅黄色化合物6。
(7)将1g化合物6溶解于15毫升无水三氟醋酸中,加入1毫升溶于冰醋酸的氢溴酸溶液(30%),于室温下反应1.5个小时。随后旋蒸除去反应溶剂,加100毫升无水乙醚析出产物,离心得到淡黄色化合物7。
(8)将500mg化合物7、2,3-二甲基马来酸酐158mg、三乙胺126mg溶解于20毫升无水DMSO中,氩气保护下于60℃油浴中反应24小时后,转移至截留分子量为3500Da的透析袋中,于纯水中透析除去过量的反应物,最后冷冻干燥得到黄色嵌段共聚物8。
(9)薄膜水化法:称取50mg嵌段共聚物8、HJC0152前药3mg、吉西他滨前药0.6mg,溶于15毫升DMSO中,然后将溶液转移至单口瓶中,旋蒸除去有机溶液,再置于真空干燥箱中抽干过夜,最后加80毫升纯水,于超声条件下,薄膜水化形成载药胶束。
通过给荷瘤小鼠尾静脉注射生理盐水、游离HJC0152与吉西他滨溶液和载药胶束溶液,结果显示pH响应电荷翻转及缺氧响应药物释放的胶束递药系统可显著降低小鼠原位胰腺癌肿瘤的大小,延长荷瘤小鼠的生存期,同时具有良好的生物安全性(图9)。
Claims (6)
1.一种pH响应电荷翻转及缺氧响应药物释放的胶束递药系统,其特征在于,该制剂由HJC0152缺氧响应前药、吉西他滨水解酶响应前药、合成的聚乙二醇-聚赖氨酸-2,3-二甲基马来酸-聚苯丙氨酸嵌段共聚物以及注射溶媒制成装载HJC0152和吉西他滨的纳米给药系统;其中所述的氨基酸嵌段共聚物浓度为5~20mg/mL,HJC0152浓度为0.25~2mg/mL,吉西他滨浓度为0.05~0.5mg/mL,HJC0152前药、吉西他滨前药和氨基酸嵌段共聚物材料的质量比为1:0.25:20~1:0.25:10;
所述HJC0152缺氧响应前药由HJC0152 hydrochloride与缺氧响应分子硝基咪唑分子间通过氨基甲酸酯键共价相连而得;
所述吉西他滨水解酶响应前药由盐酸吉西他滨与油酸分子通过酰胺键共价相连而得。
2.按权利要求1所述的pH响应电荷翻转及缺氧响应药物释放的胶束递药系统,其特征在于,所述的HJC0152前药中含有缺氧响应化学键。
3.按权利要求1所述的pH响应电荷翻转及缺氧响应药物释放的胶束递药系统,其特征在于,所述的吉西他滨前药中的酰胺键为水解酶响应化学键。
4.按权利要求1所述的pH响应电荷翻转及缺氧响应药物释放的胶束递药系统,其特征在于,所述的氨基酸嵌段共聚物为聚乙二醇-聚赖氨酸-聚苯丙氨酸嵌段共聚物的衍生物。
5.按权利要求1所述的pH响应电荷翻转及缺氧响应药物释放的胶束递药系统,其特征在于,所述的注射溶媒有注射用水或生理盐水。
6.权利要求1所述的pH响应电荷翻转及缺氧响应药物释放的胶束递药系统的制备方法,其特征在于,其包括步骤:
(1)称取2-羟甲基-1-甲基-5-硝基咪唑,三乙胺,溶解于二甲基亚砜(DMSO)中,在氮气保护下,加入溶于DMSO的对硝基苯基氯甲酸酯,反应6~12小时,反应结束后,旋蒸除去溶剂,柱色谱分离,流动相:石油醚∶乙酸乙酯=90∶10,得淡黄色产物1;
(2)称取步骤(1)得到的淡黄色产物1,HJC0152 hydrochloride,4-二甲氨基吡啶(DMAP),在氩气保护下加入毫升无水DMSO,反应24~48小时后,旋蒸除去溶剂,柱色谱分离,流动相:石油醚∶乙酸乙酯=80∶20,得淡黄色晶体2,即HJC0152前药;
(3)将盐酸吉西他滨、无水三乙胺、十八烷酰氯在氩气保护的冰浴条件下加无水DMSO搅拌反应24~48小时,反应结束后,旋蒸除去反应溶剂,柱色谱分离,流动相:甲醇∶二氯甲烷=10∶90,得白色晶体3,即吉西他滨前药;
(4)将Nε-苄氧羰基-L-赖氨酸、三光气,溶解于无水四氢呋喃中,氩气保护条件下于50~70℃油浴中反应4~10个小时,冷却至室温,滴加到-20℃预冷的正己烷中,沉淀析出产物,抽滤得白色化合物4;
(5)将苯丙氨酸、三光气溶解无水四氢呋喃中,氩气保护条件下于50~70℃油浴中反应,随后冷却至室温,逐滴滴加到于-20℃预冷的正己烷中,沉淀析出产物,抽滤得到白色化合物5;
(6)将聚乙二醇-氨基,化合物4溶解于DMSO中,氩气保护下于50~70℃油浴中反应24~48小时后,再加入溶于DMSO中的化合物5,继续同样的条件下反应24~48小时,最后,旋蒸除去反应溶剂,加无水乙醚析出产物,离心得到浅黄色化合物6;
(7)将化合物6溶解于无水三氟醋酸中,加入溴化氢溶于冰醋酸后得到的溶液,所述溴化氢的质量分数为30%,于室温下反应,随后旋蒸除去反应溶剂,加无水乙醚析出产物,离心得到淡黄色化合物7;
(8)将化合物7、2,3-二甲基马来酸酐、三乙胺溶解于毫升无水DMSO中,氩气保护下于40~60℃油浴中反应24~48小时后,转移至截留分子量为3500 Da的透析袋中,于纯水中透析除去过量的反应物,最后冷冻干燥得到黄色嵌段共聚物8;
(9)取嵌段共聚物8、HJC0152前药、吉西他滨前药,溶于DMSO中,然后将溶液转移至单口瓶中,旋蒸除去有机溶液,再置于真空干燥箱中抽干过夜,最后加纯水,于超声条件下,薄膜水化形成载药胶束。
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Dual-pH Sensitive Charge-Reversal Polypeptide Micelles for Tumor-Triggered Targeting Uptake and Nuclear Drug Delivery;Han Shisong et. al.;《Small》;20151231;第11卷(第21期);第2543-2554页 * |
Penetrating Micelle for Reversing Immunosuppression and Drug Resistance in Pancreatic Cancer Treatment;Chen Qinjun et. al.;《Small》;20220531;第18卷(第18期);2107712 (1 of 16) * |
具有潜在肿瘤靶向作用的电荷翻转型聚氨基酸前药胶束的制备;张静 等;功能材料;20170331;48(第03期);第03246-03252页 * |
肿瘤微环境响应型纳米制剂研究进展;李晓锋;《药学进展》;20191231;第43卷(第11期);第865-875页 * |
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