CN110559448A - 靶向递送siRNA仿生纳米颗粒、其制备方法及应用 - Google Patents
靶向递送siRNA仿生纳米颗粒、其制备方法及应用 Download PDFInfo
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Abstract
本发明提供了一种一种靶向递送siRNA仿生纳米颗粒,所述靶向递送siRNA仿生纳米颗粒为癌细胞膜包裹的PBAE/siRNA复合纳米颗粒,还提供了其制备方法和应用。本发明所述方法采用仿生纳米颗粒,高效地实现Plk1基因的沉默,诱导癌细胞凋亡,抑制癌细胞的增殖。本发明所述方法构建了一种可实现siRNA高效递送的体系。它能特异性识别同型癌细胞,并在癌细胞中蓄积,进入细胞后,成功从溶酶体逃逸,高效沉默目标基因,抑制目标基因的表达,导致癌细胞凋亡,从而抑制肿瘤的生长。
Description
技术领域
本发明属于医药领域,具体涉及一种靶向递送siRNA仿生纳米颗粒,及其制备方法和应用。
背景技术
基于对肿瘤发病机制的新发现,基因疗法被认为是一种潜在的癌症治疗方法。然而,成功的基因治疗,如广泛使用的RNA干扰(RNAi),很大程度上依赖于高效转染。毫无疑问,病毒载体被认为是该领域最有效的工具。但病毒固有的毒性,免疫原性和致瘤性等缺点引起了人们对其安全性的担忧。基于非病毒载体在设计方便,反应性智能和细胞毒性低等优良特性,它们在基因递送方面具有很好的前景,包括脂质体、聚合物和无机材料在内的非病毒载体可能成为病毒的良好替代品。许多已有载体在体外显示出高效的转染能力,但当它们在体内遇到复杂的生理环境时性能被严重削弱。为了有效地将治疗核酸递送到癌细胞中,基因载体必须克服细胞外和细胞内的屏障,例如(i)能避免被免疫系统俘获并到达病灶;(ii)被癌细胞摄取后,防止溶酶体对治疗性核酸的降解。
细胞膜工程化为纳米颗粒提供了一种自上而下的修饰方法,已成为改善材料界面的前沿策略。相对于极其困难的自下而上合成法,简便的细胞膜工程化赋予纳米颗粒高度复杂的功能。细胞膜工程化纳米颗粒显示出长循环、免疫逃逸和特异性靶向等特性。目前,来源于癌细胞、干细胞、红细胞和血小板等细胞膜已被用于改造纳米颗粒,并赋予它们类似细胞的功能。对于癌症治疗,癌细胞膜伪装纳米颗粒已被用于同型靶向递送小分子化合物、光敏剂或酶。已有研究表明,包括N-钙粘蛋白、半乳糖凝集素-3或上皮细胞粘附分子等与癌细胞膜上的同型粘附结构域可能有助于细胞集落的形成。经癌细胞膜伪装后,纳米颗粒完全复制表面抗原多样性,获得同型靶向性。
现有技术中采用仿生红细胞膜包裹全氟化碳缓解肿瘤部位的缺氧环境,纳米药物主要依赖于EPR效应被动靶向蓄积于肿瘤,实现改善缺氧环境和放疗协同治疗肿瘤。现有技术还采用癌细胞膜修饰负载紫杉醇的纳米颗粒,而同类的化疗药物往往具有较大毒性。现有技术采用癌细胞膜修饰负载化疗药物的磁性纳米颗粒,阿霉素同样具有较大毒性,并且磁纳米颗粒的负载效率很难达到高水平。
癌细胞膜工程化在同型靶向递送中展现出重要性,但鲜有研究利用这一特性来递送siRNA,这可能归因于一些技术瓶颈。首先,治疗性核酸通常显示出较高的负电性,采用带负电的细胞膜包裹,难以达到理想的包封效率。其次,需要合适的材料用于结合核酸。具有伯胺的阳离子聚合物如聚(L-赖氨酸)(PLL)或聚(乙烯亚胺)(PEI)可结合治疗性核酸,并将其压缩成具有正电荷的纳米颗粒。它们促进了细胞对纳米颗粒的摄取,随后是通过“质子海绵”效应,高效地从内涵体/溶酶体逃逸。然而,由于它们具有高密度正电荷,这些阳离子材料具有较高的细胞毒性。聚(β-氨基酯)(PBAEs)是一类广泛研究的材料。伯胺、仲胺和叔胺,以及可水解断裂的酯键可被设计成分子骨架。有趣的是,PBAE在pH=7.0的环境中呈疏水性,并在pH<7.0的环境中转化为亲水性。该性质可用于触发酸性环境中的药物释放,例如内涵体或溶酶体环境。此外,有研究表明,PBAE在生理条件下降解半衰期仅为半小时。这些化学特性赋予材料低毒性和高转染效率,PBAE成为基因递送的理想候选者。
发明内容
因此,本发明的目的在于克服现有技术中的缺陷,提供一种靶向递送siRNA仿生纳米颗粒,及其制备方法和应用。
在阐述本发明的技术方案之前,定义本文中所使用的术语如下:
术语“PBAE”是指:聚(β-氨基酯)。
术语“CCM”是指:癌细胞膜。
术语“CCMPP”是指:CCM修饰PBAE/siPlk1。
术语“PBS溶液”是指:磷酸盐缓冲溶液。
为实现上述目的,本发明的第一方面提供了一种靶向递送siRNA仿生纳米颗粒,所述靶向递送siRNA仿生纳米颗粒为癌细胞膜包裹的PBAE/siRNA复合纳米颗粒。
根据本发明第一方面的仿生纳米颗粒,其中,所述siRNA选自以下一种或多种:siPlk1、、siHsp27、siVEGF、siBcl-2、siClusterin、siBcl-xl、siXIAP、siSurvivin、siHras,siC-myc,siFGFR,siVEGF,siKi-67或siEPHB4,优选为siPlk1;和/或所述癌细胞膜选自以下一种或多种:NCI-H1299、A549、BEAS-2B、NCI-H596、NCI-H1770、NCI-H1975、NCI-H1650、NCI-H2106、H69PR、SNU-475、C3A、SNU-449、PLC/PRF/5、SNU-387、SK-HEP-1、SNU-423、UACC-812、UACC-893、UACC-3199、UACC-3133、UACC-1179、UACC-732、UACC-2087、MDA-MB-231、MCF-7、SNU-C1、SK-CO-1、SW1116、SW948、T84、LS123、LoVo、SW837、Hela细胞膜,优选为NCI-H1299细胞膜。
根据本发明第一方面的仿生纳米颗粒,其中,所述PBAE/siRNA复合纳米颗粒中PBAE与siRNA的质量配比为30:1~80:1,优选为40:1~80:1,更优选为40:1~50:1,最优选为50:1;和/或
所述PBAE/siRNA复合纳米颗粒的粒径为100~500nm。
根据本发明第一方面的仿生纳米颗粒,其中,所述癌细胞膜与所述PBAE/siRNA复合纳米颗粒的质量配比为1:1~30:1,优选为5:1~20:1,最优选为10:1。
本发明的第二方面提供了第一方面所述的仿生纳米颗粒的制备方法,所述方法包括以下步骤:
(1)合成PBAE;
(2)提取癌细胞膜;
(3)混合步骤(1)所制备的PBAE与siRNA得到PBAE/siRNA复合纳米颗粒,将步骤(2)所得癌细胞膜与所述PBAE/siRNA复合纳米颗粒混合并挤压得到所述仿生纳米颗粒。
根据本发明第二方面的制备方法,其中,所述步骤(1)中合成PBAE包括以下步骤:
(A)1,4-丁二醇二丙烯酸酯与4-氨基-1-丁醇混合加热,通过迈克尔加成反应聚合;优选地,所述1,4-丁二醇二丙烯酸酯与4-氨基-1-丁醇的比例为1~2:1,最优选为1.1:1;
(B)将步骤(A)所得产物溶于无水四氢呋喃,向溶液中加入封端基团化合物反应;优选地,所述封端基团化合物选自以下一种或多种:1-(3-氨基丙基)-4-甲基哌嗪、1,3-戊烷-二胺、2-羟乙胺丙胺,优选为1-(3-氨基丙基)-4-甲基哌嗪;
(C)乙醚洗涤步骤(B)所得产物,真空干燥得到所述PBAE。
根据本发明第二方面的制备方法,其中,所述步骤(2)中提取癌细胞膜的方法为反复冻融法。
根据本发明第二方面的制备方法,其中,所述步骤(3)中,所述挤压步骤通过聚碳酸酯膜进行;
优选地,所述聚碳酸酯膜的孔径为50~400nm,优选为80~200nm,最优选为100nm。
本发明的第三方面提供了一种递送系统,所述递送系统包括第一方面的仿生纳米颗粒和/或按照第二方面的制备方法而制得的仿生纳米颗粒。
本发明的第四方面提供了第一方面的仿生纳米颗粒在制备用于癌症靶向基因治疗的药物和/或医疗产品中的应用;优选地,所述癌症为肺癌;最优选地,所述癌症为非小细胞肺癌。
本发明人构建一种可实现siRNA高效递送的体系。它能特异性识别同源癌细胞,并在癌细胞中蓄积,进入细胞后,高效沉默目标基因,抑制目标基因的表达,导致癌细胞凋亡,从而抑制肿瘤的生长。
本发明的目的在于构建一种可实现siRNA高效递送的体系。它能特异性识别同源癌细胞,并在癌细胞中蓄积,进入细胞后,成功实现溶酶体逃逸,高效沉默目标基因,抑制目标基因的表达,导致癌细胞凋亡,从而抑制肿瘤的生长。
为达到目的,本发明采用以下技术方案:
本发明涉及一种基于同源细胞膜修饰纳米颗粒并递送siRNA的方法和应用。具有细胞膜的伪装赋予纳米颗粒类似细胞的功能,例如特异性识别,长期血液循环和免疫逃逸。对于癌症治疗,来自同源细胞的癌细胞膜伪装的纳米颗粒显示出同型靶向递送小分子化合物、光敏剂或酶到肿瘤。然而,由于核酸的特性,有效的基因递送遇到了这种方法的困难。在这里,本发明人构建了一种用于基因传递的新型人工癌细胞,其由优异的聚合物聚(β-氨基酯)(PBAE)配制以将siRNA(靶向Plk1基因)缩合成纳米颗粒(PBAE/siPlk1)作为核心,并进一步伪装癌细胞膜(CCM)。这种新型仿生纳米粒子(CCMPP)表现出对同型癌细胞的高度特异性靶向,有效下调PLK1水平,并诱导癌细胞凋亡。基于CCM上的同型结合粘附分子,细胞内化和同型靶向对肿瘤的积累明显改善。CCMPP在体外和体内均诱导癌细胞的高效凋亡,并导致显着的肿瘤抑制。具有同型特性的人工癌细胞可以用作癌症靶向基因治疗的仿生递送系统。具体流程如图1所示。
本发明提供了一种可特异性靶向Plk1 mRNA的siRNA:
本发明中细胞膜,PBAE的比例固定。
本发明中细胞膜源于NCI-H1299非小细胞肺癌细胞。
本发明的靶向递送siRNA仿生纳米颗粒可以具有但不限于以下有益效果:
本发明所述方法采用仿生纳米颗粒,高效地实现Plk1基因的沉默,诱导癌细胞凋亡,抑制癌细胞的增殖。本发明所述方法构建了一种可实现siRNA高效递送的体系。它能特异性识别同型癌细胞,并在癌细胞中蓄积,进入细胞后,成功从溶酶体逃逸,高效沉默目标基因,抑制目标基因的表达,导致癌细胞凋亡,从而抑制肿瘤的生长。
附图说明
以下,结合附图来详细说明本发明的实施方案,其中:
图1示出了具有同型特性的人工癌细胞可以用作癌症靶向基因治疗的仿生递送系统的流程图。
图2A示出了不同质量比下PBAE/siPlk1的粒径;图2B示出了不同质量比下PBAE/siPlk1的Zeta电位;图2C示出了不同质量比下PBAE/siPlk1的凝胶阻滞分析;图2D示出了不同质量比下CCMPP的粒径;图2E示出了不同质量比下CCMPP的Zeta电位。
图3 A示出了PBAE/siPlk1的透射电镜图片;图3 B示出了DLS分析PBAE/siPlk1;图3 C示出了CCMPP的透射电镜图片,图3 D示出了DLS分析CCMPP,图3 E示出了CCMPP表面膜蛋白分布情况;图3 F示出了CCMPP中siRNA的释放曲线图;图3 G示出了活/死细胞染色试剂盒标记的NCI-H1299细胞。
图4 A、B、C分别示出了试验例1中给药后的肿瘤图片、肿瘤体积的变化及体重变化情况;图4 D示出了治疗后Plk1 mRNA水平;图4 E示出了治疗后PLK1蛋白表达水平;图4 F示出了治疗后肿瘤的凋亡情况;图4G示出了CCMPP(siRNA采用Cy5染料标记)在荷瘤小鼠体内的分布情况。
图5示出了试验例2中细胞对CCMPP的摄入情况评估结果;其中,
图5A示出了不同浓度下细胞对CCMPP的摄入情况,图5B示出了不同时间细胞对CCMPP的摄入情况。
图6A示出了试验例3中溶酶体逃逸的激光共聚焦显微镜和流式细胞仪分析结果;图6B示出了体外细胞的特异性选择激光共聚焦显微镜和流式细胞仪分析结果。
图7A示出了Plk1 mRNA的表达水平;图7B图示出了WB分析CCMPP对细胞作用后,PLK1的表达水平;图7C图示出了细胞的凋亡情况。
具体实施方式
下面通过具体的实施例进一步说明本发明,但是,应当理解为,这些实施例仅仅是用于更详细具体地说明之用,而不应理解为用于以任何形式限制本发明。
本部分对本发明试验中所使用到的材料以及试验方法进行一般性的描述。虽然为实现本发明目的所使用的许多材料和操作方法是本领域公知的,但是本发明仍然在此作尽可能详细描述。本领域技术人员清楚,在上下文中,如果未特别说明,本发明所用材料和操作方法是本领域公知的。
以下实施例中使用的试剂和仪器如下:试剂:
1,4-丁二醇二丙烯酸酯,4-氨基-1-丁醇,四氢呋喃,1-(3-氨基丙基)-4-甲基哌嗪,乙醚,PBS溶液,蛋白酶抑制剂,siPlk1,购自宝日医生物技术(北京)有限公司;
NCI-H1299细胞(ATCC),细胞膜和胞浆蛋白质提取试剂盒,CCM,聚碳酸酯膜,H9C2细胞(ATCC)、hUV-MSCs细胞(ATCC)、Raw 264.7细胞(ATCC),活/死细胞染色试剂盒(赛默飞世尔科技(中国)有限公司)。
仪器:
透射电镜,购自日本电子株式会社(JEOL)、型号JEM-1400 TEM;Zeta电位-粒度分析仪,购自英国马尔文仪器有限公司、型号Nano ZS90;
激光共聚焦显微镜,购自德国卡尔.蔡司(Carl zeiss)公司、型号Zeiss 880;
流式细胞仪,购自默克密理博公司、型号ImageStreamX Imaging FlowCytometer。
实施例1:同型靶向仿生纳米颗粒的制备
(1)PBAE的合成。将1,4-丁二醇二丙烯酸酯与4-氨基-1-丁醇混合,在90℃通过迈克尔加成反应聚合24小时。单体丙烯酸酯-胺的比例是1.1:1。生成的产物溶于无水四氢呋喃,浓度为100mg/mL。向溶液中加入胺浓度为0.2M的过量小分子1-(3-氨基丙基)-4-甲基哌嗪用作聚合物封端基团,在室温中搅拌反应1小时。反应结束后,本发明人将乙醚加入到产物中,洗去过量的单体。收集PBAE,乙醚反复洗涤,并在真空中干燥。
(2)NCI-H1299细胞膜的提取。通过反复冻融法获得NCI-H1299细胞膜。首先,收集NCI-H1299细胞并用含有蛋白酶抑制剂的PBS溶液洗涤3次。向细胞中加入细胞膜和胞浆蛋白质提取试剂盒A试剂和不含1×EDTA的蛋白酶抑制剂,在4℃裂解细胞30分钟,每10分钟涡旋一次。将细胞悬浮液在-80℃下冷冻2小时并在室温下解冻,裂解物以10,000g离心30分钟。将细胞膜悬浮在水中并以60W的功率超声处理5分钟,并储存在-80℃。
(3)CCMPP的制备及条件筛选。为了获得PBAE/siPlk1,将PBAE与siPlk1分别以1、5、10、20、30、40、50、60、70和80的质量比混合。将混合物充分涡旋30秒。最后,将CCM与PBAE/siPlk1混合,并通过100纳米聚碳酸酯膜反复挤压11次。
如图2所示,本发明人筛选了PBAE/siPlk1和制备CCMPP的最佳条件。首先,本发明人将PBAE与siPlk1以不同的质量比混合。PBAE与siPlk1的质量比分别为1、5、10、20、30、40、50、60、70和80。本发明人发现,PBAE与siRNA的质量比超过30,通过正负电荷相互作用形成粒径相对均匀的纳米颗粒(粒径范围从~100到~500nm,PDI小于0.4),Zeta电位为~25mV。显然,比率为50的样品表现出优异的性能(粒径,110.2±4.68nm;PDI,0.11±0.03;Zeta电位,27.43±1.06)。凝胶阻滞分析表明,质量比超过40时,PBAE的负载效率约为95%;质量比为50时,负载效率最为理想,约为99%。本发明人选用最佳的条件合成纳米颗粒,进行后续实验。本发明人采用CCM修饰PBAE/siPlk1(CCM/PBAE/siPlk1,CCMPP),质量比范围为1至30。其中,质量比为10的CCMPP性能最佳(粒径,164±32nm;PDI,0.2±0.02;Zeta电位,-18±1.9mV)。本发明人在后续实验中以(CCM/PBAE/siPlk1=500/50/1)构建CCMPP。
图3示出了实施例1所得到的纳米颗粒的电镜图及各项性能图。A图为PBAE/siPlk1的透射电镜图片;B图为DLS分析PBAE/siPlk1;C图为CCMPP的透射电镜图片,D图为DLS分析CCMPP,E图CCMPP表面膜蛋白分布情况;F为CCMPP中siRNA的释放曲线图;G为活/死细胞染色试剂盒标记的NCI-H1299细胞。
试验例1:CCMPP抑瘤效果评估
如实施例1方法合成纳米颗粒,小鼠尾静脉给药。图4是与其它治疗方法对比。CCMPP是实验组。A、B、C图分别表示给药后的肿瘤图片、肿瘤体积的变化及体重变化情况;D图为治疗后Plk1 mRNA水平;E图为治疗后PLK1蛋白表达水平;F图表示治疗后肿瘤的凋亡情况;G图表示CCMPP(siRNA采用Cy5染料标记)在荷瘤小鼠体内的分布情况。每只小鼠给药量等价于20μg siRNA,体积为200μL。
试验例2:细胞对CCMPP的摄入情况评估
如实施例1方法合成纳米颗粒,激光共聚焦显微镜和流式细胞仪分析细胞对CCMPP高效摄入。图5所示,随着浓度和孵育时间的增加,CCMPP在NCI-H1299细胞中的蓄积增加。摄入率最高可达100%。图5A为37℃,5%CO2,孵育6小时;图5B为Cy5-siRNA等价于150nM的纳米颗粒于37℃孵育。
试验例3:溶酶体逃逸和体外细胞的特异性选择情况
如图6所示,A图激光共聚焦显微镜分析显示,CCMPP被细胞摄入1小时后,可能会到达溶酶体(溶酶体示踪剂和CCMPP的荧光重合);在6小时,siRNA成功从溶酶体逃逸(溶酶体示踪剂和CCMPP的荧光分离)。H1299细胞与Cy5-siRNA等价于150nM的纳米颗粒在37℃,5%CO2的细胞培养箱中分别孵育1小时和6小时。吸取培养液,PBS缓冲液洗涤3遍,加入含有200nM的溶酶体染料(赛默飞世尔科技(中国)有限公司),再孵育30分钟,去除染液,PBS缓冲液洗涤3遍,4%多聚甲醛固定。此外,B图激光共聚焦显微镜分析显示,非同型细胞株,如H9C2(大鼠心肌细胞)、hUV-MSCs(人脐带静脉间充质干细胞)和Raw 264.7细胞(小鼠巨噬细胞)对CCMPP的摄入量很小。Cy5-siRNA等价于150nM的纳米颗粒,37℃,5%CO2,孵育6小时。去掉固定液,PBS缓冲液洗涤3次,0.1%曲拉通X-100常温孵育5分钟。吸去孵育液,PBS缓冲液洗涤1次,1%牛血清白蛋白溶液孵育15分钟。去掉孵育液后,5μM FITC-鬼笔环肽(赛默飞世尔科技(中国)有限公司)孵育30分钟。吸取染液,PBS缓冲液洗涤3次,5μM DAPI染液常温中染色15分钟。
试验例4:体外细胞的Plk1基因的沉默情况
如实施例1方法合成纳米颗粒,NCI-H1299细胞与CCMPP(siPlk1等价于150nM)在37℃,5%CO2培养箱中共孵育6小时,如图7所示,A图为Plk1 mRNA的表达水平;B图为WB分析CCMPP对细胞作用后,PLK1的表达水平;C图为细胞的凋亡情况。如下图所示,与商业化试剂相比,CCMPP能更有效诱导细胞凋亡。CCMPC即为CCM/PBAE/siControl,其制备方法如实施例1:同型靶向仿生纳米颗粒的制备,siControl的随机序列(正向序列:5’-UUCUCCGAACGUGUCACGUdTdT-3’;反向序列:5’-ACGUGACACGUUCGGAGAAdTdT-3’)。CCM/PE125kD/siPlk1,制备方法如实施例1:同型靶向仿生纳米颗粒的制备,PEI25kD(CAS号:9002-98-6)购自西格玛奥德里奇中国。
尽管本发明已进行了一定程度的描述,明显地,在不脱离本发明的精神和范围的条件下,可进行各个条件的适当变化。可以理解,本发明不限于所述实施方案,而归于权利要求的范围,其包括所述每个因素的等同替换。
Claims (10)
1.一种靶向递送siRNA仿生纳米颗粒,其特征在于,所述靶向递送siRNA仿生纳米颗粒为癌细胞膜包裹的PBAE/siRNA复合纳米颗粒。
2.根据权利要求1所述的仿生纳米颗粒,其特征在于,所述siRNA选自以下一种或多种:siPlk1、siHsp27、siVEGF、siBcl-2、siClusterin、siBcl-xl、siXIAP、siSurvivin、siHras,siC-myc,siFGFR,siVEGF,siKi-67或siEPHB4,优选为siPlk1;和/或
所述癌细胞膜选自以下一种或多种:NCI-H1299、A549、BEAS-2B、NCI-H596、NCI-H1770、NCI-H1975、NCI-H1650、NCI-H2106、H69PR、SNU-475、C3A、SNU-449、PLC/PRF/5、SNU-387、SK-HEP-1、SNU-423、UACC-812、UACC-893、UACC-3199、UACC-3133、UACC-1179、UACC-732、UACC-2087、MDA-MB-231、MCF-7、SNU-C1、SK-CO-1、SW1116、SW948、T84、LS123、LoVo、SW837、Hela细胞膜,优选为NCI-H1299细胞膜。
3.根据权利要求1或2所述的仿生纳米颗粒,其特征在于,所述PBAE/siRNA复合纳米颗粒中PBAE与siRNA的质量配比为30:1~80:1,优选为40:1~80:1,更优选为40:1~50:1,最优选为50:1;和/或所述PBAE/siRNA复合纳米颗粒的粒径为100~500nm。
4.根据权利要求1至3中任一项所述的仿生纳米颗粒,其特征在于,所述癌细胞膜与所述PBAE/siRNA复合纳米颗粒的质量配比为1:1~30:1,优选为5:1~20:1,最优选为10:1。
5.根据权利要求1至4中任一项所述的仿生纳米颗粒的制备方法,其特征在于,所述方法包括以下步骤:
(1)合成PBAE;
(2)提取癌细胞膜;
(3)混合步骤(1)所制备的PBAE与siRNA得到PBAE/siRNA复合纳米颗粒,将步骤(2)所得癌细胞膜与所述PBAE/siRNA复合纳米颗粒混合并挤压得到所述仿生纳米颗粒。
6.根据权利要求5所述的方法,其特征在于,所述步骤(1)中合成PBAE包括以下步骤:
(A)1,4-丁二醇二丙烯酸酯与4-氨基-1-丁醇混合加热,通过迈克尔加成反应聚合;优选地,所述1,4-丁二醇二丙烯酸酯与4-氨基-1-丁醇的比例为1~2:1,最优选为1.1:1;
(B)将步骤(A)所得产物溶于无水四氢呋喃,向溶液中加入封端基团化合物反应;优选地,所述封端基团化合物选自以下一种或多种:1-(3-氨基丙基)-4-甲基哌嗪、1,3-戊烷-二胺、2-羟乙胺丙胺,优选为1-(3-氨基丙基)-4-甲基哌嗪;
(C)乙醚洗涤步骤(B)所得产物,真空干燥得到所述PBAE。
7.根据权利要求5或6所述的方法,其特征在于,所述步骤(2)中提取癌细胞膜的方法为反复冻融法。
8.根据权利要求5至7中任一项所述的方法,其特征在于,所述步骤(3)中,所述挤压步骤通过聚碳酸酯膜进行;
优选地,所述聚碳酸酯膜的孔径为50~400nm,优选为80~200nm,最优选为100nm。
9.一种递送系统,其特征在于,所述递送系统包括如权利要求1至4中任一项所述的仿生纳米颗粒和/或按照权利要求5至8中任一项的制备方法而制得的仿生纳米颗粒。
10.权利要求1至4中任一项所述的仿生纳米颗粒在制备用于癌症靶向基因治疗的药物和/或医疗产品中的应用;优选地,所述癌症为肺癌;最优选地,所述癌症为非小细胞肺癌。
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