CN114099698B - 一种pH敏感脂质体及其制备方法与应用 - Google Patents
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- 210000001835 viscera Anatomy 0.000 description 1
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Abstract
本发明涉及生物医药技术领域,具体涉及一种pH敏感脂质体及其制备方法与应用。所述pH敏感脂质体由以下物质的量的药物和脂质体载体组成:摩尔比为(2~5):(0.5~1.5):(0.5~1.5):(0.1~0.3)的(2,3‑二油酰基‑丙基)‑三甲基铵‑氯盐:二油酰磷脂酰胆碱:胆固醇琥珀酸单酯:DSPE‑PEGn‑pep;ICD诱导剂与(2,3‑二油酰基‑丙基)‑三甲基铵‑氯盐、二油酰磷脂酰胆碱和胆固醇琥珀酸单酯的总质量比为(5~12):100;腺苷通路阻断剂与ICD诱导剂的质量比为(0.5~2):(0.5~3)。ICD诱导剂和腺苷通路抑制剂两药连用克服了仅使用ICD诱导剂治疗的抗肿瘤疗效差、肿瘤免疫逃避的问题,利用脂质体载体实现ICD诱导剂和腺苷通路抑制剂的体内共同递送;POL的pH敏感特性有效保证POL在血液循环过程中保持稳定。
Description
技术领域
本发明涉及生物医药技术领域,具体涉及一种pH敏感脂质体及其制备方法与应用。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解,而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
化学疗法是最经典的癌症治疗方法之一,例如使用蒽环类药物等细胞毒性药物,如奥沙利铂、米托蒽醌、阿霉素、环磷酰胺等,可以通过诱导肿瘤细胞的免疫原性细胞死亡(ICD)来增强抗肿瘤免疫效应。在此过程中产生的三磷酸腺苷ATP可以招募DC,从而激活T细胞和记忆T细胞的成熟和向肿瘤的浸润,引发抗癌免疫反应。然而不幸的是,仅依赖于ICD诱导剂的治疗在患者中的应答率仍然很低,临床效果不尽人意。主要原因为ICD诱导后产生的免疫刺激的ATP会被肿瘤微环境中高表达的CD39和CD73迅速降解为免疫抑制性腺苷,腺苷与免疫细胞或者肿瘤细胞上表达的G蛋白偶联腺苷受体A2a(A2aR)结合,导致免疫逃逸和恶性肿瘤进展。仅使用ICD诱导剂治疗存在抗肿瘤疗效差、肿瘤免疫逃避的这一问题有待改善和解决。
发明内容
针对现有技术中存在的问题,本发明的目的是提供一种pH敏感脂质体及其制备方法与应用,本发明利用纳米脂质体实现腺苷通路阻断剂与ICD诱导剂的联合递送,提供一种更安全、更可控的癌症化学免疫联合疗法。
在使用ICD诱导剂治疗肿瘤的过程中,对于ATP-腺苷通路中关键酶的抑制(抑制CD39,抑制CD73和抑制腺苷与免疫细胞上表达的A2aR的结合)具有改善实体肿瘤免疫治疗的潜力;并且CD39抑制剂的使用除限制腺苷的产生而减轻诱导的免疫抑制外,与A2AR或CD73抑制剂相反,阻断CD39的一个不同作用是保护免疫刺激性ATP库,因为三磷酸腺苷的有效释放和积累似乎对化疗后的免疫原性反应至关重要。除此之外,CD39在调节性T细胞中高表达,而CD73在这类细胞中基本上不表达。抑制CD39对抑制髓系细胞的功能也有很强的作用,从而也会增强效应T细胞的功能。
然而,除了肿瘤部位外,CD39在全身免疫系统内、血管内皮上和不同的细胞类型和器官中也均有广泛表达,如在肾脏中,CD39表现在血管内皮和皮层和间质的收集管道内,在肝脏内、肺部和肠道等也有表达。因此,CD39强抑制剂的直接使用极有可能导致“免疫相关不良事件”。同时无靶向或者不精准的的化疗也会破坏免疫细胞的活性,免疫疗法和化学疗法两者无序的非精准组合性治疗产生的毒性也更高。本发明提供的pH敏感脂质体可以更安全、更可控地实现CD39抑制剂与ICD诱导剂的联合递送,增加病变组织内药物的积累,更有效地靶向期望的肿瘤和/或免疫细胞,并减少脱靶的副作用。
为了实现上述目的,本发明的技术方案如下所述:
在本发明的第一方面,提供一种pH敏感脂质体,所述pH敏感脂质体由以下药物和脂质体载体组成:
(2,3-二油酰基-丙基)-三甲基铵-氯盐(DOTAP),二油酰磷脂酰胆碱(DOPC),胆固醇琥珀酸单酯(CHEMS),DSPE-PEGn-pep,ICD诱导剂和腺苷通路阻断剂。
在一种或多种实施方式中,(2,3-二油酰基-丙基)-三甲基铵-氯盐(DOTAP):二油酰磷脂酰胆碱(DOPC):胆固醇琥珀酸单酯(CHEMS):DSPE-PEGn-pep的摩尔比为(2~5):(0.5~1.5):(0.5~1.5):(0.1~0.3);
ICD诱导剂与(2,3-二油酰基-丙基)-三甲基铵-氯盐(DOTAP)、二油酰磷脂酰胆碱(DOPC)和胆固醇琥珀酸单酯(CHEMS)的总质量比为(5~12):100;
腺苷通路阻断剂与ICD诱导剂的质量比为(0.5~2):(0.5~3),优选的为1:1。
在本发明的第二方面,提供一种第一方面所述pH敏感脂质体的制备方法,所述制备方法包括如下步骤:
(1)合成脂溶性ICD诱导剂前药;
(2)制备多肽-PEG-N-二硬脂酰磷脂酰乙酰胺DSPE-PEGn-pep:在PLGLAG(pep,响应于肿瘤微环境中高表达MMP-2)水溶液中加入EDC和NHS,室温活化羧基,将氨基-PEG-N-二硬脂酰磷脂酰乙酰胺DSPE-PEGn-NH2溶解于DMF中,缓慢加入其中,搅拌反应后透析纯化,冷冻干燥得到最终产物(DSPE-PEGn-pep);
(3)制备ICD诱导剂脂质体:将(2,3-二油酰基-丙基)-三甲基铵-氯盐(DOTAP),二油酰磷脂酰胆碱(DOPC),胆固醇琥珀酸单酯(CHEMS),ICD诱导剂前药溶解于适量氯仿,真空旋蒸后形成脂质薄膜,与葡萄糖溶液水合,然后强制挤出得到ICD诱导剂脂质体混悬液;
(4)制备腺苷通路阻断剂/ICD诱导剂脂质体:将步骤(3)得到的ICD诱导剂脂质体混悬液缓慢滴加到腺苷通路阻断剂溶液中,室温搅拌,即得腺苷通路阻断剂/ICD诱导剂脂质体混悬液;
(5)制备POL:
在腺苷通路阻断剂/ICD诱导剂脂质体混悬液中加入DSPE-PEGn-pep,搅拌、超滤、离心即得。
在本发明的第三方面,提供一种第一方面所述的pH敏感脂质体或者第二方面所述的制备方法制备得到的pH敏感脂质体在制备靶向抗肿瘤药物中的应用。
在一种或多种实施方式中,所述肿瘤选自黑色素瘤、肠癌、肺癌、胃癌、卵巢癌、前列腺癌、胰腺癌、乳腺癌、卵巢癌、肝癌、头颈部癌、淋巴瘤、肉瘤、慢性淋巴细胞白血病、甲状腺癌和睾丸癌和肛门癌。
本发明的具体实施方式具有以下有益效果:
(1)ICD诱导剂和腺苷通路抑制剂两药连用克服了仅使用ICD诱导剂治疗的抗肿瘤疗效差、肿瘤免疫逃避的问题,激活系统的抗肿瘤免疫反应,同时解除肿瘤微环境免疫抑制;
(2)本发明利用脂质体载体实现ICD诱导剂和腺苷通路抑制剂的体内共同递送;POL的pH敏感特性可以有效保证POL在血液循环过程中保持稳定;
(3)DSPE-PEGn-pep的使用可以在体循环中将脂质体外吸附的腺苷通路抑制剂(如:CD39抑制剂)覆盖遮蔽,克服了腺苷通路抑制剂(如:CD39抑制剂)的直接使用会导致的全身系统毒性问题;到达肿瘤微环境时,pep响应于肿瘤微环境中高表达MMP-2而发生断裂。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。
图1为本发明实施例1的OXA-C前药的合成过程和1H NMR分析;
图2为由POM-1质量增加介导的静电吸附增强引起的OXA-C脂质体的表面电荷转换;
图3为在MMP-2存在或不存在的情况下,OXA-C尺寸与不同长度的DSPE-PEG-pep的关系图;
图4为本发明实施例1的OXA-C粒径分布图;
图5为本发明实施例1的POL粒径分布图;
图6为本发明实施例1的POL和OXA-C脂质体透射电镜图。
图7为本发明实施例3的体外释放曲线;
图8为本发明实施例3中B16F10细胞在用OXA-C脂质体、POL、Combo、OXA、POM-1处理24小时后的细胞活力;
图9为本发明实施例3中B16F10细胞在用OXA-C脂质体(1)、POL(2)、Combo(3)、OXA(4)、POM-1(5)、PBS(6)处理4小时后的ATP诱导;
图10为本发明实施例3中POL、POM-1/DIR脂质体、DIR脂质体和游离DIR在荷瘤小鼠体内1小时、4小时、8小时、12小时和24小时的生物分布的荧光图像;
图11为本发明实施例3中POL、POM-1/DIR脂质体、DIR脂质体和游离DIR在荷瘤小鼠体内1小时、4小时、8小时、12小时和24小时的静脉注射后24小时肿瘤和主要器官的荧光统计分析;
图12为本发明实施例3中用POL(1),Combo(2),OXA-C脂质体(3),POM-1/脂质体(4),OXA(5),POM-1(6)和5%葡萄糖(7)不同治疗后小鼠肿瘤体积变化;
图13为本发明实施例3中不同治疗后小鼠肿瘤生存周期。
具体实施方式
应该指出,以下详细说明都是例示性的,旨在对本申请提供进一步的说明。除非另有指明,本申请使用的所有技术和科学术语具有与本申请所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是,这里所使用的术语仅是为了描述具体实施方式,而非意图限制根据本申请的示例性实施方式。如在这里所使用的,除非上下文另外明确指出,否则单数形式也意图包括复数形式,此外,还应当理解的是,当在本说明书中使用术语“包含”和/或“包括”时,其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
本发明的一种实施方式中,提供了一种pH敏感脂质体,所述pH敏感脂质体由以下药物和脂质体载体组成:
(2,3-二油酰基-丙基)-三甲基铵-氯盐(DOTAP),二油酰磷脂酰胆碱(DOPC),胆固醇琥珀酸单酯(CHEMS),DSPE-PEGn-pep,ICD诱导剂和腺苷通路阻断剂。
在一种或多种实施方式中,(2,3-二油酰基-丙基)-三甲基铵-氯盐(DOTAP):二油酰磷脂酰胆碱(DOPC):胆固醇琥珀酸单酯(CHEMS):DSPE-PEGn-pepICD诱导剂的摩尔比为(2~5):(0.5~1.5):(0.5~1.5):(0.1~0.3);
ICD诱导剂与(2,3-二油酰基-丙基)-三甲基铵-氯盐(DOTAP)、二油酰磷脂酰胆碱(DOPC)和胆固醇琥珀酸单酯(CHEMS)的三者总质量的比为(5~12):100;
腺苷通路阻断剂与ICD诱导剂的质量比为(0.5~2):(0.5~3),优选的为1:1。
在一种或多种实施方式中,所述ICD诱导剂包括但不限于强心苷、奥沙利铂、蒽环类药物、米托蒽醌、阿霉素、环磷酰胺、光热剂;
在一种或多种实施方式中,所述腺苷通路阻断剂包括CD39抑制剂、CD73抑制剂、A2aR受体抑制剂在内的小分子抑制剂和抗体药物;
优选地,所述CD39抑制剂选择POM-1;
小分子抑制剂主要可以采用静电吸附方法连接,抗体类药物通过本身羧基与构成脂质体的DSPE-PEG-NH2通过酰胺缩合反应连接。
在一种或多种实施方式中,DSPE-PEGn-pep中的n=2000~5000;优选地,n=2000,3500,5000,进一步优选地,n=2000;
ICD诱导剂可以立即杀死肿瘤细胞并诱导ICD释放ATP等损伤相关分子模式,从而刺激树突状细胞的成熟;CD39抑制剂可以通过阻断CD39来维持免疫刺激性ATP水平,抑制免疫抑制性腺苷的产生,从而增强化学免疫治疗效果。
在本发明的第二方面,提供一种第一方面所述pH敏感脂质体的制备方法,所述制备方法包括如下步骤:
(1)合成ICD诱导剂前药;
(2)制备多肽-PEG-N-二硬脂酰磷脂酰乙酰胺DSPE-PEGn-pep:在PLGLAG(pep,响应于肿瘤微环境中高表达MMP-2)水溶液中加入EDC和NHS,室温活化羧基,将氨基-PEG-N-二硬脂酰磷脂酰乙酰胺DSPE-PEGn-NH2溶解于DMF中,缓慢加入其中,搅拌反应后透析纯化,冷冻干燥得到最终产物(DSPE-PEGn-pep);
(3)制备ICD诱导剂脂质体:将(2,3-二油酰基-丙基)-三甲基铵-氯盐(DOTAP),二油酰磷脂酰胆碱(DOPC),胆固醇琥珀酸单酯(CHEMS),ICD诱导剂前药溶解于适量氯仿,真空旋蒸后形成脂质薄膜,与葡萄糖溶液水合搅拌,然后强制挤出得到ICD诱导剂脂质体混悬液;
(4)制备腺苷通路阻断剂/ICD诱导剂脂质体:将步骤(3)得到的ICD诱导剂脂质体混悬液缓慢滴加到腺苷通路阻断剂溶液中,室温搅拌,即得腺苷通路阻断剂/ICD诱导剂脂质体混悬液;
(5)制备POL:
在腺苷通路阻断剂/ICD诱导剂脂质体混悬液中加入DSPE-PEGn-pep,搅拌、超滤、离心即得。
在一种或多种实施方式中,步骤(3)中与葡萄糖溶液水合的温度为30-50℃,优选40℃;
优选地,步骤(3)中所述葡萄糖溶液的浓度为4-6%;优选地,搅拌时间为30-120min,优选60min;优选地,强制挤出采用220nm的聚碳酸酯膜过滤器进行;
在一种或多种实施方式中,步骤(4)中搅拌为室温下800-200rpm搅拌10-12min;
在一种或多种实施方式中,步骤(5)中腺苷通路阻断剂/ICD诱导剂脂质体混悬液与DSPE-PEG2000-pep的摩尔比为100:1;40-50℃搅拌反应4-5h;
在一种或多种实施方式中,当ICD诱导剂选择奥沙利铂时,奥沙利铂前药OXA-C的制备方法为:
将奥沙利铂(OXA)溶解在DMF中,加入适量H2O2溶液,并在室温下反应得到OXA-OH;
将OXA-OH重新溶解于DMF中,加入十六烷基异氰酸酯,黑暗条件下反应24-72h;加入饱和食盐水出现白色沉淀混合物,使用乙酸乙酯萃取后,真空浓缩混合物,真空干燥;通过凝胶柱层层析的方法获得最终白色粉末OXA-C;反应过程如以下化学反应式所示:
在本发明的第三方面,提供一种第一方面所述的pH敏感脂质体或者第二方面所述的制备方法制备得到的pH敏感脂质体在制备靶向抗肿瘤药物中的应用。
在一种或多种实施方式中,所述肿瘤选自黑色素瘤、肠癌、肺癌、胃癌、卵巢癌、前列腺癌、胰腺癌、乳腺癌、卵巢癌、肝癌、头颈部癌、淋巴瘤、肉瘤、慢性淋巴细胞白血病、甲状腺癌和睾丸癌和肛门癌。
下面结合具体的实施例对本发明作进一步的解释和说明。
实施例1
OXA-C的合成:将0.5g奥沙利铂(OXA)溶解在8mL DMF的30%H2O2中,并在室温下保持搅拌24小时。然后加入无水乙醇,沉淀粗产物,抽滤,干燥,得到OXA-OH;将所得固体重新溶解于5-10mL DMF中,加入1g十六烷基异氰酸酯,黑暗条件下反应48h;加入饱和食盐水出现白色沉淀混合物,使用乙酸乙酯萃取后,真空浓缩混合物,真空干燥;通过凝胶柱层层析的方法获得最终白色粉末OXA-C;最终产物的核磁共振波谱如图1所示;
DSPE-PEG2000-pep的制备:在溶于10mL超纯水的40mg PLGLAG(pep,响应于肿瘤微环境中高表达MMP-2)中加入66mg EDC和20mg NHS室温活化羧基2小时,将DSPE-PEG2000-NH2分别溶解于10mL DMF中,缓慢加入其中,搅拌反应24h后透析纯化48h,冷冻干燥得到最终产物(DSPE-PEG2000-pep);
制备OXA-C脂质体:将含有摩尔比为3:1:1:0.23的DOTAP,DOPC,CHEMS,OXA-C溶解于适量氯仿,真空旋蒸后形成脂质薄膜,40℃下与适量5%葡萄糖溶液水合,搅拌60min;然后用220nm的聚碳酸酯膜过滤器强制挤出得到OXA-C脂质体混悬液;
制备POM-1/OXA-C脂质体:将1mL上述得到的OXA-C脂质体(2mg/mL)缓慢滴加到不同浓度的1mL POM-1溶液中,室温800-1200rpm搅拌10min,即得POM-1/OXA-C脂质体;
POL的制备过程为:在POM-1/OXA-C脂质体混悬液中以100:1的摩尔比例加入DSPE-PEG2000-pep,40-50℃搅拌反应4h,用超滤管超滤离心即得。
实施例2
OXA-C的合成:将0.5g奥沙利铂(OXA)溶解在8mL DMF的30%H2O2中,并在室温下保持搅拌24小时。然后加入无水乙醇,沉淀粗产物,抽滤,干燥,得到OXA-OH;将所得固体重新溶解于5-10mL DMF中,加入1g十六烷基异氰酸酯,黑暗条件下反应48h;加入饱和食盐水出现白色沉淀混合物,使用乙酸乙酯萃取后,真空浓缩混合物,真空干燥;通过凝胶柱层层析的方法获得最终白色粉末OXA-C;最终产物分别用核磁共振波谱和质谱进行表征;
DSPE-PEG3400-pep的制备:在溶于10mL超纯水的40mg PLGLAG(pep,响应于肿瘤微环境中高表达MMP-2)中加入66mg EDC和20mg NHS室温活化羧基2小时,将不同PEG长度的DSPE-PEG3400-NH2分别溶解于10mL DMF中,缓慢加入其中,搅拌反应24h后透析纯化48h,冷冻干燥得到最终产物(DSPE-PEG3400-pep),通过核磁共振波谱检测;
制备OXA-C脂质体:将含有摩尔比为3:1:1:0.23的DOTAP,DOPC,CHEMS,OXA-C溶解于适量氯仿,真空旋蒸后形成脂质薄膜,40℃下与适量5%葡萄糖溶液水合,搅拌60min;然后用220nm的聚碳酸酯膜过滤器强制挤出得到OXA-C脂质体混悬液;
制备POM-1/OXA-C脂质体:将1mL上述得到的OXA-C脂质体(2mg/mL)缓慢滴加到不同浓度的1mL POM-1溶液中,室温800—1200rpm搅拌10min,即得POM-1/OXA-C脂质体;
POL的制备过程为:在POM-1/OXA-C脂质体混悬液中以100:1的摩尔比例加入DSPE-PEG3500-pep,40-50℃搅拌反应4h,用超滤管超滤离心即得,然后使用马尔文粒度仪测量DSPE-PEG3400-pep修饰的POL粒径,同时将POL在体外与适量MMP-2孵育后测量粒径(如图3所示)。
实施例3
将实施例1制备的材料进行表征:
纳米制剂的流体动力学直径、粒径分布和电位用动态光散射法在马尔文激光粒度仪上测量。为了观察脂质体,将合理稀释的脂质体溶液滴在铜网上,并用2%(w/v)磷钨酸溶液染色后烘干30min以上,使用透射电镜观察脂质体外观形态。
OXA-C的载药量和包封率通过电感耦合等离子仪(ICP-MS)来测得Pt元素的含量。未超滤纯化的POL溶液3000rpm低速离心20min除去游离的水不溶性OXA-C。取适量上清液加入4mL超纯硝酸在聚四氟乙烯管中密闭消解过夜后,稀释定容200mL,最终酸浓度为2%;使用Helium KED检测模式,反应池气体流量为3.5L/min,Rpq=0.25。
使用薄膜分散法制备的pH敏感脂质体粒径控制在131.97±1.42nm,粒径分布窄,如图4所示,PDI=0.26±0.01,zeta电位为64.3±1.15mV,如图5所示;TEM图像显示OXA-C脂质体分散良好并显示出具有干净背景的球形形态如图6所示,;OXA-C脂质体显示出优异的载药率(5.26±0.02%,w/w)和包封率(94.77±0.39%)。
在POL制备过程中,越来越多的POM-1的吸附可以逐渐掩盖OXA-C脂质体的阳离子表面电荷并稳定粒径。POL证体从正电到负电的电荷转换过程中OXA与POM-1的质量比约为1:3.13,如图2所示。根据不同PEG链修饰的POM-1/OXA-C脂质体的大小比较,DSPE-PEG2000-NH2被优选的应用于连接pep和脂质体,确保POM-1可以被完全遮盖,避免系统循环中其他表达CD39的组织的无序靶向。
最终POL的大小为157.67±1.04nm,PDI为0.27±0.01,zeta电位为-33.57±1.50mV。TEM揭示了脂质体的特征球形形态。POL还具有良好的淡蓝色乳光和丁达尔效应。
研究Pt的释放:鉴于脂质体的pH敏感特性和奥沙利铂前药的谷胱甘肽响应特性,使用动态透析法研究了72h内不同条件下(pH=7.4,without 10mM GSH;pH=5.0,with10mM GSH)脂质体对于Pt的释放行为。透析介质均含有0.5%吐温,透析袋截留分子量为3000Da,摇床温度设置为37℃,摆动速度为100rpm,Pt的含量使用ICP-MS测定。为了测试pH对体外药物释放的影响,模拟了两种体内环境。在模拟血液环境(pH=7.4,无谷胱甘肽)中,72小时内仅释放35.70±1.70%的铂;当pH从7.4变为5.0,谷胱甘肽浓度增加时,Pt的累积释放量显着增加,如图7所示。因此,POL的pH敏感特性可以有效保证POL在血液循环过程中保持稳定,当纳米药物到达肿瘤组织并被内吞时,内体-溶酶体的酸性环境触发磷脂双层结构的破坏,从而释放OXA-C。内体-溶酶体中高浓度的谷胱甘肽也促进了前药向其活性成分OXA的转化。
检测体外细胞毒性实验,采用CCK8法检测细胞活力。B16F10以1*105细胞数每孔的密度接种在96孔板中,培养过夜。第二天弃旧培养基,然后以设定的浓度(药物浓度为100,75,50,25,10,1uM)加入不同处理过的(POL,OXA-C脂质体,OXA,POM-1,combo,blank脂质体)完全培养基,其中,POL为最终制剂,OXA-C脂质体是只包载OXA-C的脂质体,OXA是奥沙利铂,combo是OXA和POM-1的物理混合组,blank是注射用5%葡萄糖溶液。24h或者48小时。对照组不做任何处理,空白组不接种细胞。治疗后,每孔加入10uL的CCK8,并在37℃孵育2小时,使用酶标仪测定每孔在450nm处的光密度(OD)。相对细胞活力的计算方法如下:相对细胞活力(RCV)%=(ODtest-ODblank)/(ODcontrol-ODblank),所有实验独立重复3次。使用CCK8测定制备的纳米脂质体与B16F10黑色素瘤细胞孵育24小时后的细胞毒性实验结果表示:尽管有高浓度的空白载体(100μM),纳米脂质体没有表现出任何显着的细胞毒性,表明脂质体具有出色的生物相容性。OXA-C脂质体的半数抑制浓度(IC50)为18.21μM,略高于POL组的14.07μM。两者之间没有显著差异。OXA和combo的IC50值均大于100μM,分别是两种脂质体的5倍和7倍以上。在50μM的浓度下,90.14%的细胞在OXA-C脂质体的作用下被杀死,但在游离药物处理下只有37.05%的细胞被杀死,这证明了阳离子纳米药物引起的快速细胞杀伤结果如图8所示。
已知OXA的治疗原理是立即杀死肿瘤细胞并诱导ICD释放ATP等损伤相关分子模式,从而刺激DCs的成熟。POM-1可以通过阻断CD39来维持ATP水平,从而增强化学免疫治疗效果。因此,本发明试图在体外模拟和评估这个过程。用ATP检测试剂盒检测细胞外的ATP分泌。简而言之,B16F10细胞以1×105细胞/孔的密度接种到24孔板中。12小时后加入不同处理过的培养基(POL,OXA-C脂质体,OXA,POM-1,combo,5%glutin)共培养细胞4小时。之后,收集培养上清液,之后所有的操作在冰上进行。并根据制造商的标准说明用ATP测定试剂盒测量细胞外ATP含量。与物理混合组相比,OXA-C脂质体在4小时时显著促进细胞外ATP分泌(p<0.05),如图9所示。结果表明单独OXA诱导ICD效应的能力较弱,脂质体可以显着增强其诱导肿瘤细胞免疫原性死亡的能力,从而刺激DCs的成熟。
观察纳米制剂体内分布:DiR是一种常用的用于体内成像的亲脂性近红外荧光染料,可用于标记脂质体的脂溶性生物结构。B16F10细胞(1×106)皮下注射到每只C57BL/6小鼠的右侧。当肿瘤体积达到200mm3时,静脉注射游离的DiR、DiR脂质体、POM-1/DiR脂质体和PDL,DiR的剂量为100μg/kg。在给药后1小时、4小时、8小时、12小时和24小时,通过小动物活体成像系统(IVIS光谱)获得实时光学图像,如图10所示。24小时后,处死动物(每组n=3)。收集心脏、肝脏、脾脏、肺、肾脏和肿瘤,称重并成像。活体图像软件(IVIS成像系统)用于计算体内肿瘤区域和体外所有组织中DiR的总荧光强度。首先评估了静脉注射后游离药物在荷瘤小鼠中的生物分布。Dir在注射后4小时显示出全身分布和扩散的迹象,但游离药物的快速代谢和广泛的全身分布导致荧光信号太弱,无法在此处显示超过阈值范围。DiR Lips(脂质体)在注射后1小时位于肝脏中。然后,在肿瘤区域富集的DiR脂质体被缓慢递送到肿瘤内部。不难发现,PDL在肿瘤区域的穿透行为与DiR脂质体相似。这表明富含TME的PDL对MMP-2有反应,促进了POM-1的脱落;因此,PDL被转化为带正电荷和更小粒径的DiR脂质体。然而,这种现象发生在POL注射后8小时,这归因于PEG的修饰及其优异的长期循环能力。与DiR脂质体的中等穿透能力相比,PDL的肿瘤滞留能力更优。POM-1/DiR-脂质体没有DSPE-PEG-pep修饰的纳米颗粒和POM-1暴露在外面,主要发现在肝脏和肾脏附近,而不是在肿瘤中。正如之前的研究表明,CD39是POM-1的一种高亲和力受体,它也广泛表达于整个身体的不同细胞类型和器官,如肝脏、肺、肾脏和肠道。与POL相比,POM-1/DiR脂质体在肿瘤部位的积累和保留较少。解剖的肿瘤和主要组织荧光强度的定量分析如图11所示。PDL组的瘤内荧光强度比相应的POM-1/DiR脂质体组高1.4倍。大量POM-1/DiR Lips位于网状内皮器官,包括肝、肺和脾,并引起全身免疫毒性。这进一步证明隐藏POM-1显着改善了肿瘤的药物积累并减少了非特异性组织分布。基于上述体内成像结果,POL的肿瘤分布增加可归因于(1)纳米颗粒在血液循环过程中的长循环和稳定性,(2)POM-1的合理隐藏,减少了全身免疫副作用(3)MMP-2的灵敏设计,促进了脂质体的电荷转换和尺寸减小,从而增强了对肿瘤的深入渗透。
建立小鼠黑色素瘤原位肿瘤模型: 每只C57BL/6小鼠右侧皮下注射1*106株B16-F10细胞;荷瘤7天后,将小鼠随机分为7组(n = 6)。静脉注射100 μL POL,OXA-C脂质体,POM-1/脂质体,OXA,POM-1,Combo(药物浓度均为5mg/kg)。对照组尾静脉注射相同体积的5%葡萄糖溶液。之后给药频率为三天一次,每两天记录肿瘤大小和小鼠体重。肿瘤体积的计算公式如下:V=L*W2/2,其中,L为肿瘤的最长直径,W为肿瘤的最短直径。给药14天后取血、脱颈处死,取出肿瘤和重要脏器。与POM-1组和5%glutin(对照组)组相比,两者的组合(组合)导致肿瘤显着减少(p<0.01,p<0.001)。与游离药物相比,两者联合治疗效果提高,无显着差异,而与联合用药组相比,POL进一步提高了荷瘤小鼠的存活率。POL治疗下的所有6只小鼠都经历了长期存活。POL在所有组中发挥了最高的抗肿瘤效率。体内抗肿瘤实验结果(图12和13所示)证实了POM-1和OXA的协同抗黑色素瘤作用,并表明纳米技术介导的治疗对于癌症免疫治疗至关重要。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (13)
1.一种pH敏感脂质体在制备靶向抗肿瘤药物中的应用,其特征在于,所述pH敏感脂质体由以下药物和脂质体载体组成:(2,3-二油酰基-丙基)-三甲基铵-氯盐,二油酰磷脂酰胆碱,胆固醇琥珀酸单酯,DSPE-PEGn-pep,ICD诱导剂和腺苷通路阻断剂;
所述ICD诱导剂为奥沙利铂;
所述腺苷通路阻断剂为CD39抑制剂;
所述CD39抑制剂选择POM-1;
(2,3-二油酰基-丙基)-三甲基铵-氯盐:二油酰磷脂酰胆碱:胆固醇琥珀酸单酯:DSPE-PEGn-pep的摩尔比为(2~5):(0.5~1.5):(0.5~1.5):(0.1~0.3);
ICD诱导剂与(2,3-二油酰基-丙基)-三甲基铵-氯盐、二油酰磷脂酰胆碱和胆固醇琥珀酸单酯的三者总质量的比为(5~12):100;
腺苷通路阻断剂与ICD诱导剂的质量比为(0.5~2):(0.5~3);
DSPE-PEGn-pep中的n=2000~5000;
所述肿瘤选自黑色素瘤。
2.如权利要求1所述的应用,其特征在于,腺苷通路阻断剂与ICD诱导剂的质量比为1:1。
3.如权利要求1所述的应用,其特征在于,DSPE-PEGn-pep中的n=2000,3500,5000。
4.如权利要求1所述的应用,其特征在于,DSPE-PEGn-pep中的n=2000。
5.如权利要求1所述的应用,其特征在于,所述pH敏感脂质体的制备方法包括如下步骤:
(1)合成ICD诱导剂前药;
(2)制备多肽-PEG-N-二硬脂酰磷脂酰乙酰胺DSPE-PEGn-pep:在PLGLAG水溶液中加入EDC和NHS,室温活化羧基,将氨基-PEG-N-二硬脂酰磷脂酰乙酰胺DSPE-PEGn-NH2溶解于DMF中,缓慢加入其中,搅拌反应后透析纯化,冷冻干燥得到最终产物DSPE-PEGn-pep;
(3)制备ICD诱导剂脂质体:将(2,3-二油酰基-丙基)-三甲基铵-氯盐、二油酰磷脂酰胆碱、胆固醇琥珀酸单酯、ICD诱导剂前药溶解于适量氯仿,真空旋蒸后形成脂质薄膜,与葡萄糖溶液水合搅拌,然后强制挤出得到ICD诱导剂脂质体混悬液;
(4)制备腺苷通路阻断剂/ICD诱导剂脂质体:将步骤(3)得到的ICD诱导剂脂质体混悬液缓慢滴加到腺苷通路阻断剂溶液中,室温搅拌,即得腺苷通路阻断剂/ICD诱导剂脂质体混悬液;
(5)制备POL:
在腺苷通路阻断剂/ICD诱导剂脂质体混悬液中加入DSPE-PEGn-pep,搅拌、超滤、离心即得。
6.如权利要求5所述的应用,其特征在于,步骤(3)中与葡萄糖溶液水合的温度为30-50℃。
7.如权利要求6所述的应用,其特征在于,步骤(3)中与葡萄糖溶液水合的温度为40℃。
8.如权利要求5所述的应用,其特征在于,步骤(3)中所述葡萄糖溶液的浓度为4-6%。
9.如权利要求5所述的应用,其特征在于,搅拌时间为30-120min。
10.如权利要求9所述的应用,其特征在于,搅拌时间为60min。
11.如权利要求5所述的应用,其特征在于,强制挤出采用220nm的聚碳酸酯膜过滤器进行。
12.如权利要求5所述的应用,其特征在于,步骤(4)中搅拌为室温下800-200rpm搅拌10-12min;
或者,步骤(5)中腺苷通路阻断剂/ICD诱导剂脂质体混悬液与DSPE-PEG2000-pep的摩尔比为100:1;
或者,步骤(5)中40-50℃搅拌反应4-5h。
13.如权利要求5所述的应用,其特征在于,当ICD诱导剂选择奥沙利铂时,奥沙利铂前药OXA-C的制备方法为:
将奥沙利铂溶解在DMF中,加入H2O2溶液,并在室温下反应得到OXA-OH;
将OXA-OH重新溶解于DMF中,加入十六烷基异氰酸酯,黑暗条件下反应24-72h;加入饱和食盐水出现白色沉淀混合物,使用乙酸乙酯萃取后,真空浓缩混合物,真空干燥;通过凝胶柱层层析的方法获得最终白色粉末OXA-C。
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