CN113616602B - 一种重组高密度脂蛋白-聚酰胺-胺纳米复合物、制法及应用 - Google Patents

一种重组高密度脂蛋白-聚酰胺-胺纳米复合物、制法及应用 Download PDF

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CN113616602B
CN113616602B CN202111010266.0A CN202111010266A CN113616602B CN 113616602 B CN113616602 B CN 113616602B CN 202111010266 A CN202111010266 A CN 202111010266A CN 113616602 B CN113616602 B CN 113616602B
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pamam
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polyamide
density lipoprotein
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CN113616602A (zh
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王伟
张子瑶
傅聪
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China Pharmaceutical University
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Abstract

本发明公开了一种重组高密度脂蛋白‑聚酰胺‑胺纳米复合物，所述纳米复合物核心是负载免疫调节剂的重组高密度脂蛋白纳米粒，表面是负载化疗药物的pH敏感修饰聚酰胺‑胺纳米粒；该纳米复合物可响应酸性肿瘤微环境而释放表面小粒径的载药聚酰胺‑胺纳米粒而渗透至肿瘤深部，且纳米复合物可以依靠巨噬细胞清道夫受体递送免疫调节剂改善肿瘤免疫环境，从而实现肿瘤组织的高效渗透，以及化疗与免疫治疗的联合治疗；本发明提供的重组高密度脂蛋白‑聚酰胺‑胺复合物具有载体生物相容性好、酸性肿瘤微环境响应性等特点，可应用于癌症的靶向治疗，制备改善肿瘤免疫抑制环境和抑制肿瘤生长的药物。

Description

一种重组高密度脂蛋白-聚酰胺-胺纳米复合物、制法及应用
技术领域
本发明涉及药物制剂领域，具体涉及一种重组高密度脂蛋白-聚酰胺-胺纳米复合物，以及其制法及应用。
背景技术
乳腺癌是女性中一种常见的恶性肿瘤，目前，常见治疗手段有化疗、放疗、手术切除等。其中阿霉素(Doxorubicin，DOX)是一种常用的乳腺癌化疗药物，但是单独的DOX治疗效果有限，存在靶向性差、毒副作用大、容易产生耐药等问题。除此之外，免疫治疗也是一种广为研究的治疗手段。免疫治疗是指激活免疫反应，启动自我防御系统，从而破坏肿瘤细胞的治疗手段。免疫治疗毒副作用小、作用范围广，所以联合化疗和免疫治疗的策略有着很大的研究前景。
肿瘤相关巨噬细胞(Tumor-associated macrophages,TAMs)是肿瘤间质的重要成分，受肿瘤微环境的影响表现出高度的可塑性，在肿瘤增殖、侵袭、转移、血管生成等方面发挥着重要的作用。TAM是一种主要的肿瘤浸润性免疫细胞类型，通常分为两种不同功能的亚型，即经典活化的M1型巨噬细胞和交替活化的M2型巨噬细胞。M1可以响应细菌产物和干扰素-γ(IFN-γ)，募集和激活适应性免疫系统的细胞；M1的一个重要特征是其可以表达一氧化氮合酶(iNOS)、活性氧(ROS)和白介素-12(IL-12)，具有典型的抗肿瘤作用，包括直接介导的细胞毒性和抗体依赖的细胞介导的细胞毒性(ADCC)来杀死肿瘤细胞。M2具有清除碎片、促进组织重建和损伤修复的功能，并且可促进肿瘤细胞的发生和转移，抑制T淋巴细胞介导的抗肿瘤免疫反应，促进肿瘤血管生成，导致肿瘤进展。M1和M2型巨噬细胞均具有高度的可塑性，可在肿瘤微环境改变或药物治疗干预下相互转化。随着TAMs与恶性肿瘤关系的日益明确，靶向TAMs成为一种新的、有前景的癌症治疗策略。
目前已经有很多TAMs相关的治疗策略。其中激活Toll样受体(TLRs)使TAM由M2型复极化为M1型而产生抗肿瘤作用的手段有着巨大的研究优势。利用药物激动TLRs关键在于选择合适的载体递送药物进入巨噬细胞内作用于受体上并产生较少副作用。目前，常见载体有脂质体、聚合物胶束、自组装纳米粒等载体。但是这些载体靶向能力有限，不能有效靶向TAMs；且这些载体跨膜转运途径对于递送药物到巨噬细胞内的能力有限，不能高效递送药物到细胞内体膜上的TLRs；此外这些载体的免疫反应性限制了其在体内安全性，也更容易被机体清除导致药效发挥有限。肿瘤组织细胞外基质和细胞的密度高，以及渗漏的血管和受损的淋巴循环导致了较高的组织间质流体压力。这些因素构成了生物屏障，并限制了纳米载体在肿瘤组织中的渗透。因此，常见的纳米载体，如脂质体、聚合物胶束等难以渗透进入肿瘤组织深处，这也限制了其对于深处肿瘤的治疗效果。所以纳米载体能否渗透进入肿瘤深部也是影响其递送药物能力的关键因素。
发明内容
发明目的：本发明针对现有技术中存在纳米粒载体对于TAMs靶向性和递药能力差、纳米粒难以渗透肿瘤组织深部的问题，以及单独治疗手段局限性问题，提供一种重组高密度脂蛋白-聚酰胺-胺纳米复合物；以及上述纳米复合物的制法及应用。
技术方案：本发明的所述的重组高密度脂蛋白-聚酰胺-胺纳米复合物主要由硬脂酰基磷脂酰乙醇胺-聚乙二醇(DPSE-PEG)、聚酰胺-胺型树枝状高分子(PAMAM)、阿霉素、磷脂、胆固醇、胆固醇酯、载脂蛋白、靶向肽靶向肽、硬脂酸和咪喹莫特组成，所述纳米复合物由内外两部分纳米粒组成，内部纳米粒为由磷脂、胆固醇、胆固醇酯、载脂蛋白和咪喹莫特组成的负载咪喹莫特的靶向重组高密度脂蛋白纳米粒、外层纳米粒为由阿霉素和DSPE-PEG-PAMAM制得的负载阿霉素的聚酰胺-胺型树状纳米粒，所述靶向肽通过硬脂酸插入磷脂层的表面，所述外层纳米粒与内部纳米粒的磷脂层通过疏水作用结合在一起。
优选的，所述DSPE-PEG-PAMAM是硬脂酰基磷脂酰乙醇胺-聚乙二醇(DPSE-PEG)修饰的聚酰胺-胺型树枝状高分子(PAMAM)，所述硬脂酰基磷脂酰乙醇胺-聚乙二醇为DPSE-PEG2000-CHO，所述聚酰胺-胺型树枝状高分子为PAMAM-NH2，所述DPSE-PEG2000-CHO和PAMAM-NH2的单体摩尔比为1:1.2-1:2。
优选的，所述DSPE-PEG2000-CHO中PEG分子量为2000Da，其末端为醛基封端；PAMAM为G4-PAMAM，其末端为氨基封端。
优选的，所述重组高密度脂蛋白-聚酰胺-胺纳米复合物的壳层是将DSPE-PEG-PAMAM通过疏水作用结合阿霉素，形成聚酰胺-胺型树状纳米粒(DPP/DOX)，所述核层与磷脂层壳层通过疏水作用结合在一起；而纳米复合物内部为负载咪喹莫特的靶向重组高密度脂蛋白纳米粒，并在其磷脂层表面插入靶向肽。
优选的，所述磷脂为大豆磷脂、大豆卵磷脂、蛋黄卵磷脂、二肉豆蔻酰卵磷脂、二棕榈酰卵磷脂或二硬脂酰卵磷脂；所述胆固醇酯为胆固醇与脂肪酸缩合成的胆固醇脂肪酸酯；所述载脂蛋白为ApoA-Ⅰ，所述靶向肽为C18-M2pep，序列为C18-YEQDPWGVKWWY。
本发明的所述的重组高密度脂蛋白-聚酰胺-胺纳米复合物的制法，包括以下步骤：
(1)制备负载阿霉素的DPP/DOX纳米粒：将DPSE-PEG2000-CHO和PAMAM-NH2(G4)添加到DMSO溶液中；调节溶液为碱性，冷冻干燥得到DSPE-PEG-PAMAM(DPP)；滴加阿霉素的甲醇溶液，搅拌离心，过滤，即得负载阿霉素的DPP/DOX纳米粒；
(2)制备负载咪喹莫特的靶向重组高密度脂蛋白纳米粒(rHDL/R837)：将磷脂、胆固醇、胆固醇酯、咪喹莫特溶解在有机溶剂中，旋蒸，加水搅拌，超声分散，加入载脂蛋白ApoA-Ⅰ孵育，过滤，即得负载咪喹莫特的靶向重组高密度脂蛋白纳米粒；
(3)制备重组高密度脂蛋白-聚酰胺-胺纳米复合物：将负载咪喹莫特的靶向重组高密度脂蛋白纳米粒和负载阿霉素的DPP/DOX纳米粒溶解在水中，调节溶液pH为碱性，搅拌孵育并加入靶向肽C18-M2pep，即得所述重组高密度脂蛋白-聚酰胺-胺纳米复合物。
优选的，步骤(1)中，DPSE-PEG2000-CHO和PAMAM-NH2的质量比为10:3-10:4。
优选的，步骤(2)中，所述阿霉素与DPP质量比为1:10-8:10。
优选的，步骤(3)中，负载咪喹莫特的靶向重组高密度脂蛋白纳米粒、负载阿霉素的DPP/DOX纳米粒和靶向肽C18-M2pep的质量比为50:15:1-50:34:3。
优选的，步骤(1)具体为：将DPSE-PEG2000-CHO和PAMAM-NH2(G4)按照单体摩尔比1:1.2-1:2投入DMSO溶液中，加热溶解配成溶液，调节溶液为碱性，水浴下反应，反应完将溶液进行冷冻干燥即得到酸性敏感的DSPE-PEG-PAMAM(DPP)材料；然后取DPP粉末溶解在水中配成溶液，取盐酸阿霉素和三乙胺加入甲醇中溶解成阿霉素溶液，将阿霉素甲醇溶液逐渐滴加入DPP溶液中，并在室温下避光敞口搅拌24h以上使纳米粒负载阿霉素，反应结束后将溶液在4℃下用12000rpm离心10min并将上清液用0.22μm的水系微孔滤膜过滤去除没负载的阿霉素，即得到负载阿霉素的DPP/DOX纳米粒。
优选的，步骤(2)具体为：将磷脂、胆固醇、胆固醇酯、咪喹莫特溶解在有机溶剂中，然后在40℃下旋蒸除去有机溶剂并形成油膜，加水并在40℃下搅拌30min使油膜分散在水中，将整个体系利用超声探头细胞破碎仪的探头超声进一步分散，加入载脂蛋白ApoA-Ⅰ孵育12h，结束后将纳米粒溶液依次用0.45μm和0.22μm水系微孔滤膜过滤，即得到负载咪喹莫特的靶向重组高密度脂蛋白纳米粒。
优选的，步骤(3)具体为：将rHDL/R837纳米粒和DPP/DOX纳米粒溶解在水中，调节pH溶液为碱性，充分混合后室温搅拌12h孵育并加入C18-M2pep于2-8℃下孵育0.5h，即得到重组高密度脂蛋白-聚酰胺-胺纳米复合物。
本发明还公开了所述纳米复合物在制备抑制改善肿瘤免疫抑制环和抑制肿瘤生长的药物的应用。
发明原理：发明人在研究过程中发现，重组高密度脂蛋白(rHDL)是一种具有良好生物相容性的合成给药纳米平台，具有高密度脂蛋白(HDL)的大部分优点且几乎无毒性，在体内可降解为无毒物质。抗肿瘤载药rHDL纳米颗粒(NPs)是一种重要的药物递送系统(DDS)，该DDS对肿瘤细胞表现出了出色的主动靶向能力，在克服上述缺点的同时提高了抗肿瘤治疗的效果。可以利用M2巨噬细胞特异性靶向肽(M2pep)增加其对于M2型TAMs的靶向能力。B类I型清道夫受体(SR-B1)是一种HDL受体，有助于从循环脂蛋白中摄取胆固醇酯；通过SR-B1受体摄取rHDL载荷的过程是非内吞且高度特异性的；即脂蛋白特异性地与细胞表面结合，并直接在细胞内部递送疏水性药物，rHDL可与TAMs表面高表达的SR-B1特异性结合，保护和递送抗癌药物至肿瘤组织。咪喹莫特(R837)通过刺激局部免疫反应以治疗感染性皮肤病，通过Toll样受体-7(TLR-7)激活引发免疫反应，达到模拟微生物抗原的效果。诱导产生的干扰素-α(IFN-α)和肿瘤坏死因子-α(TNF-α)引发的免疫应答特别针对感染性抗原，并且(部分)通过朗格汉斯细胞向区域淋巴结的迁移增强而介导。
聚酰胺-胺型(PAMAM)树状大分子具有与靶向肽相似的化学组成和结构：具有特定的三维结构；其内部空腔有大量的酰胺骨架和叔胺结构，具有良好的生物相容性、溶解性和稳定性，因此可以作为药物载体；表面有众多的伯胺结构，易与负电性的细胞膜经静电相互作用，通过形成纳米孔、膜变薄和侵蚀导致细胞膜破裂，因此具有一定的细胞毒性，故需对PAMAM树状大分子末端基团进行表面修饰。
因此，发明人采用硬脂酰基磷脂酰乙醇胺-聚乙二醇(DPSE-PEG)修饰的PAMAM通过疏水作用结合化疗药阿霉素形成DSPE-PEG-PAMAM纳米粒(DPP/DOX)，并将其插入包封了R837的rHDL纳米粒(rHDL/R837)，制备成重组高密度脂蛋白-聚酰胺-胺纳米复合物(DPP/DOX@M2pep-rHDL/R837)。整个纳米复合物内部为rHDL/R837，药物咪喹莫特负载在核心内部；纳米复合物表面外周DPP/DOX通过DSPE-PEG结合在磷脂层的表面，会在在酸性环境中断裂释放PAMAM粒子；而M2型巨噬细胞特异性靶向肽(M2pep)也是通过硬脂酸插入磷脂层的表面。磷脂疏水端在内部与胆固醇、咪喹莫特等形成rHDL内核。载脂蛋白结合在rHDL表面。
制备重组高密度脂蛋白载免疫药物纳米粒并在其表面负载含有化疗药物的pH敏感纳米粒，其在酸性肿瘤微环境响应释放表面纳米粒渗透肿瘤深处，且依靠巨噬细胞清道夫受体递送免疫药物改善肿瘤免疫环境，达到化学治疗和免疫治疗结合的目的。
该纳米复合物通过静脉注射进入体内到达肿瘤部位时，PAMAM与rHDL表面的酸性敏感键断裂使得PAMAM/DOX与rHDL/R837分离并渗透入深层肿瘤组织，通过溶酶体途径进入肿瘤细胞并发挥DOX的抗肿瘤作用；而rHDL/R837通过TAMs的SR-B1疏水通道将R837递送入巨噬细胞并激活TLR-7引发免疫反应使TAMs由M2向M1表型极化而改善肿瘤免疫抑制微环境和抑制肿瘤。该纳米复合物通过靶向TAMs发挥免疫调节剂R837和化疗药DOX的作用，其生物相容性好、可以响应酸性肿瘤环境，应用于癌症的靶向治疗，实现化疗药肿瘤渗透治疗和肿瘤免疫治疗的结合，可以持续有效地抑制肿瘤生长。
有益效果：与现有技术相比，本发明具有如下显著优点：
(1)本发明的重组高密度脂蛋白-聚酰胺-胺纳米复合物(DPP/DOX@M2pep-rHDL/R837)，利用M2巨噬细胞特异性靶向肽(M2pep)靶向肿瘤相关巨噬细胞，提高了载体的靶向性；载脂蛋白ApoA-Ⅰ可以与巨噬细胞表面高度表达的SR-BI受体特异性结合并打开疏水通道，将咪喹莫特递送入细胞内，并激动细胞内涵体上的TLR-7受体促使M2型巨噬细胞向抗肿瘤的M1型巨噬细胞极化，显著提高了药物药效和靶向性，与游离药物的药效相比提高了约1.8倍；
(2)纳米复合物表面负载的PAMAM粒子在酸性肿瘤环境下与rHDL的连接断裂释放并携带阿霉素渗透进入肿瘤深处达到杀伤深处肿瘤的目的，解决纳米粒对于肿瘤深部杀伤不足的问题。并结合纳米复合物的靶向性，提高了纳米粒携带阿霉素在肿瘤部位蓄积的能力。
(3)本发明利用rHDL结合了化疗和免疫治疗，提高了免疫治疗的效果，减少了单独化疗的副作用。而且rHDL与天然内源性HDL相似，减少在体内的免疫识别和清除延长了体内半衰期，也提高了生物利用度和体内药效，rHDL也会参与巨噬细胞的胆固醇外流进一步提高咪喹莫特对于巨噬细胞的作用，有效改善肿瘤免疫和抑制肿瘤生长。
附图说明
图1为本发明的重组高密度脂蛋白-聚酰胺-胺纳米复合物的示意图。
图2为实施例1中DSPE-PEG-PAMAM(DPP)材料1H-NMR图谱；
图3为实施例2中DSPE-PEG-PAMAM/DOX纳米粒粒径图；
图4为实施例2中DSPE-PEG-PAMAM/DOX纳米粒透射电镜图；
图5为实施例3中rHDL/R837纳米粒粒径图；
图6为实施例3中rHDL/R837纳米粒透射电镜图；
图7为实施例4中DPP/DOX@M2pep-rHDL/R837纳米复合物粒径图；
图8为实施例4中DPP/DOX@M2pep-rHDL/R837纳米复合物透射电镜图；
图9为实施例5中DPP/DOX@M2peprHDL/R837纳米复合物不同pH粒径变化图；
图10为实施例6中不同纳米粒体外对于肿瘤细胞毒性图；
图11为实施例7中DPP/DOX@M2pep-rHDL/R837纳米复合物对于巨噬细胞极化图。
具体实施方式
下面结合附图对本发明的技术方案作进一步说明。
如图1所示，本发明的重组高密度脂蛋白-聚酰胺-胺纳米复合物的示意图，其中1为磷脂组成纳米粒基本结构(phospholipid)；2为胆固醇组成纳米粒的核心(Cholester-)；3为阿霉素(DOX)；4为咪喹莫特(R837)；5为DSPE-PEG2000；6为G4-PAMAM；7为ApoA-Ⅰ蛋白；8为C18-M2pep；所述纳米复合物为核壳结构，其核层为由磷脂、胆固醇、胆固醇酯、载脂蛋白和咪喹莫特组成的负载咪喹莫特的靶向重组高密度脂蛋白纳米粒，咪喹莫特包封在纳米粒的核心，其磷脂层壳层为由阿霉素和DSPE-PEG-PAMAM制得的负载阿霉素的DPP/DOX纳米粒，所述靶向肽通过硬脂酸插入磷脂层的表面，纳米粒表面外周DPP/DOX通过DSPE-PEG结合在磷脂层的表面，会在在酸性环境中断裂释放PAMAM粒子。
实施例1
DSPE-PEG-PAMAM合成
将DPSE-PEG2000-CHO 10mg和PAMAM-NH2(G4)3.3mg按照单体摩尔比1:1.5投入5mlDMSO溶液中，40℃加热溶解配成溶液。向溶液中加入20μl 0.1M的NaOH溶液维持碱性，并在40℃水浴下磁力搅拌反应24h。将反应完的溶液转移到切割分子量为3500Da的透析袋中，40℃纯水透析24h(3L*3)，然后进行冷冻干燥即得到酸性敏感的DSPE-PEG-PAMAM(DPP)材料。图2是DSPE-PEG-PAMAM核磁氢谱，可以看出核磁结果同时含有PAMAM乙二胺骨架亚甲基特征峰(δ2.57～2.23ppm附近)以及DSPE-PEG-CHO上磷脂末端甲基特征峰(δ0.88～0.87ppm附近)，表明目标化合物DSPE-PEG-PAMAM合成成功。根据PAMAM亚甲基和DSPE-PEG-CHO末端甲基积分面积可以计算出DSPE-PEG-PAMAM接枝率为25.7％。
实施例2
DSPE-PEG-PAMAM/DOX纳米粒构建及性质研究
(1)DSPE-PEG-PAMAM/DOX纳米粒的制备
取3mg DPP粉末溶解在3ml 40℃纯水中配成1mg/ml溶液，取2.4mg盐酸阿霉素和120μl三乙胺加入1ml甲醇中溶解。接着将阿霉素甲醇溶液逐滴加入搅拌的DPP溶液中，并在室温下避光敞口搅拌24h使纳米粒负载阿霉素并挥发甲醇。结束后将溶液在4℃下用12000rpm离心10min并将上清液用0.22μm的水系微孔滤膜过滤去除没负载的阿霉素，然后用3000Da的超滤管进行超滤(3000rpm30min)去除游离的阿霉素。收集超滤管内物质加纯水混合即得到酸性敏感DSPE-PEG-PAMAM/DOX纳米粒溶液。
(2)DSPE-PEG-PAMAM/DOX纳米粒的性质研究
用动态光散射(Dynamic Light Scattering,DLS)测量制备的纳米粒溶液粒子粒径，结果如图3所示，DSPE-PEG-PAMAM/DOX纳米粒粒径为31.77±4.38nm。通过透射电子显微镜(Transmission Electron Microscope,简称TEM)观察粒子微观结构，如图4所示发现实际纳米粒大小检测的粒径相近，而且纳米粒呈现不规则条状与理论上未修饰PAMAM球形状态不同，也进一步证明成功制备出DSPE-PEG-PAMAM/DOX纳米粒。
实施例3
rHDL/R837纳米粒构建及性质研究
(1)rHDL/R837纳米粒的制备
称取10mg大豆磷脂、2mg胆固醇、1mg胆固醇酯、1mg R837溶解在5ml混合溶剂(VMethanol:VChloroform＝1:4)中，并转移入圆底烧瓶中，40℃下水浴减压旋蒸。待溶剂挥发后，脂质在烧瓶底部形成一层均一的薄膜，将其置于真空干燥器中干燥6h以上完全除去有机溶剂。干燥结束后向烧瓶中加入3ml的蒸馏水，并在40℃下水化30min得到脂质混悬液。在冰浴下，利用超声探头(工作2s关闭2s，工作功率200w)分散15min得到脂质纳米粒。然后在4℃下与1mg ApoA-Ⅰ蛋白孵育24h。最后将得到的纳米粒依次在0.45μm和0.22μm微孔滤膜(水系)下过滤，得到rHDL/R837纳米粒溶液。
(2)rHDL/R837纳米粒性质研究
用动态光散射(DLS)测量制备的纳米粒溶液粒子粒径，结果如图5所示，纳米粒平均有效粒径为126.4±7.9nm。通过透射电子显微镜(TEM)观察粒子微观结构，如图6所示，实际纳米粒大小检测的粒径相近，制备的纳米粒圆润完整且有脂质纳米粒特有的结构，粒径分布约100nm与粒径结果相近，说明成功制备出rHDL/R837纳米粒。
实施例4
DPP/DOX@M2pep-rHDL/R837纳米复合物制备及性质研究
(1)DPP/DOX@M2pep-rHDL/R837纳米复合物制备
将含3.5mg rHDL/R837纳米粒溶液和含1.3mg DSPE-PEG-PAMAM/DOX纳米粒溶液在室温下磁力搅拌混合孵育(200rpm)12h。然后2-8℃下与0.07mgC18-M2pep孵育0.5h，即得到纳米复合物溶液。
(2)DPP/DOX@M2pep-rHDL/R837纳米复合物性质研究
用动态光散射(DLS)测量制备的纳米粒溶液粒子粒径，结果如图7所示，纳米粒平均有效粒径为176.6±8.5nm。通过透射电子显微镜(TEM)观察粒子微观结构，如图8所示发现实际纳米粒大小检测的粒径相近，制备的纳米粒电镜图中有rHDL纳米粒类似的纹路且表面也有类似DPP纳米粒大小的黑色点。与rHDL相比纳米复合物边缘更加粗糙，说明DPP成功负载在表面，成功制备出DPP/DOX@M2pep-rHDL/R837纳米粒。
实施例5
DPP/DOX@M2pep-rHDL/R837纳米复合物pH敏感性研究
按照制备方案新制备一批纳米复合物，并取含0.4mg纳米复合物的纳米粒溶液分别加入2mlpH6.4～7.6PBS缓冲液中，搅拌稀释后用动态光散射(DLS)测量制备的纳米粒溶液粒子粒径。结果如图9所示，在不同pH缓冲液中纳米粒粒径发生改变，并在pH达到6.8时发生明显粒径变化，与肿瘤微环境pH6.5～6.8相对应，说明纳米粒在肿瘤酸性环境中可以发生尺寸的变换。
实施例6
DPP/DOX@M2pep-rHDL/R837纳米粒复合物体外对于4T1肿瘤细胞毒性研究
取生长状态良好且处于对数生长期的4T1肿瘤细胞和RAW264.7巨噬细胞接种，将RAW264.7巨噬细胞用以5×103个/孔的浓度接种于TRANSWELL小室(孔径0.4μm)中，同时在小室下层接种2×104个/孔的4T1细胞。待细胞贴壁后，接着用含PBS、rHDL/R837、DPP/DOX、DPP/DOX@M2pep-rHDL/R837的DMEM培养基培养24h。然后更换成新鲜的完全培养基，继续培养24h后移除TRANSWELL小室，每孔加入5mg/ml的MTT溶液20μL，于5％CO2、37℃细胞培养箱中孵育6h。弃去上清液，每孔加入150μL的二甲基亚砜，震荡60s，用酶联免疫测定仪于490nm测定各孔的吸光度值，计算细胞存活率，计算公式如下：
细胞存活率＝(ASample-APBS)/(Acontrol-APBS)×100％
其中ASample代表该纳米复合物处理后的细胞组的吸光度；APBS代表PBS溶液的吸光度；Acontrol代表未经制剂处理的细胞组的吸光度。
结果如图10所示，最终的纳米复合物对于4T1肿瘤细胞杀伤效果最好也验证了本发明所制备的纳米粒递药系统在体外有良好的杀伤肿瘤作用。
实施例7
纳米复合物对于巨噬细胞极化效果研究
取生长状态良好且处于对数生长期的RAW264.7巨噬细胞以5×105个/孔接种于6孔板中，待细胞贴壁后更换含IL-4 20ng/ml的DMEM完全培养基继续培养36h以极化细胞为M2型巨噬细胞。结束后更换含有PBS、R837、DPP/DOX@M2pep-rHDL/R837纳米复合物的DMEM培养基培养24h。然后弃去上清，并将巨噬细胞消化并吹打下来与CD86-PE和CD206-APC抗体孵育，接着将细胞至于流式细胞仪上进行分析。
结果如图11所示，最终的纳米复合物中Q1和Q2区域的细胞比例更大，说明纳米粒成功使得巨噬细胞复极化成M1表型，可以改善肿瘤免疫抑制的环境，并刺激分泌相关细胞因子抑制肿瘤生长；且相较于游离的R837药物纳米粒效果更好，说明地本发明所制备的纳米粒递药系统可以更好将药物递送进入细胞内。

Claims (9)

1.一种重组高密度脂蛋白-聚酰胺-胺纳米复合物，其特征在于，所述纳米复合物主要由硬脂酰基磷脂酰乙醇胺-聚乙二醇（DPSE-PEG）、聚酰胺-胺型树枝状高分子（PAMAM）、阿霉素、磷脂、胆固醇、胆固醇酯、载脂蛋白、靶向肽、硬脂酸和咪喹莫特组成，所述纳米复合物为核壳结构，其核层为由磷脂、胆固醇、胆固醇酯、载脂蛋白和咪喹莫特组成的负载咪喹莫特的靶向重组高密度脂蛋白纳米粒，磷脂层壳层为由阿霉素和DSPE-PEG-PAMAM制得的负载阿霉素的聚酰胺-胺型树状纳米粒（DPP/DOX），所述靶向肽通过硬脂酸插入磷脂层的表面，所述靶向肽为C18-M2pep，序列为C18-YEQDPWGVKWWY，所述DSPE-PEG-PAMAM是硬脂酰基磷脂酰乙醇胺-聚乙二醇（DPSE-PEG）修饰的聚酰胺-胺型树枝状高分子（PAMAM），所述硬脂酰基磷脂酰乙醇胺-聚乙二醇为DPSE-PEG2000-CHO，所述聚酰胺-胺型树枝状高分子为PAMAM-NH2，所述DPSE-PEG2000-CHO和PAMAM-NH2的单体摩尔比为1:1.2~1:2。
2.根据权利要求1所述的纳米复合物，其特征在于，所述DSPE-PEG2000-CHO的末端为醛基封端；所述PAMAM为G4-PAMAM，其末端为氨基封端。
3.根据权利要求1所述的纳米复合物，其特征在于，所述重组高密度脂蛋白-聚酰胺-胺纳米复合物的磷脂层壳层是将DSPE-PEG-PAMAM通过疏水作用结合阿霉素，形成聚酰胺-胺型树状纳米粒（DPP/DOX），所述核层与磷脂层壳层通过疏水作用结合。
4.根据权利要求1所述的纳米复合物，其特征在于，所述磷脂为大豆卵磷脂、蛋黄卵磷脂、二肉豆蔻酰卵磷脂、二棕榈酰卵磷脂或二硬脂酰卵磷脂；所述胆固醇酯为胆固醇与脂肪酸缩合成的胆固醇脂肪酸酯；所述载脂蛋白为ApoA-Ⅰ。
5.权利要求1所述的重组高密度脂蛋白-聚酰胺-胺纳米复合物的制法，其特征在于，包括以下步骤：
（1）制备负载阿霉素的聚酰胺-胺型树状纳米粒：将DPSE-PEG2000-CHO和PAMAM-NH2添加到DMSO 溶液中；调节溶液为碱性，冷冻干燥得到DSPE-PEG-PAMAM（DPP）；滴加阿霉素的甲醇溶液，搅拌离心，过滤，即得负载阿霉素的聚酰胺-胺型树状纳米粒（DPP/DOX）；
（2）制备负载咪喹莫特的靶向重组高密度脂蛋白纳米粒rHDL/R837：将磷脂、胆固醇、胆固醇酯、咪喹莫特溶解在有机溶剂中，旋蒸，加水搅拌，超声分散，加入载脂蛋白ApoA-Ⅰ孵育，过滤，即得负载咪喹莫特的靶向重组高密度脂蛋白纳米粒；
（3）制备重组高密度脂蛋白-聚酰胺-胺纳米复合物：将负载咪喹莫特的靶向重组高密度脂蛋白纳米粒和负载阿霉素的聚酰胺-胺型树状纳米粒溶解在水中，调节溶液pH为碱性，搅拌孵育并加入靶向肽，即得所述重组高密度脂蛋白-聚酰胺-胺纳米复合物。
6.根据权利要求5所述的制法，其特征在于，步骤（1）中，DPSE-PEG2000-CHO和PAMAM-NH2的质量比为10:3~10:4。
7.根据权利要求5所述的制法，其特征在于，步骤（1）中，所述阿霉素与DPP质量比为1:10~8:10。
8.根据权利要求5所述的制法，其特征在于，步骤（3）中，负载咪喹莫特的靶向重组高密度脂蛋白纳米粒、负载阿霉素的聚酰胺-胺型树状纳米粒和靶向肽的质量比为50:15:1~50:34:3。
9.权利要求1所述的重组高密度脂蛋白-聚酰胺-胺纳米复合物的应用，其特征在于，所述纳米复合物在制备改善肿瘤免疫抑制环境和抑制肿瘤生长的药物的应用。
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