CN111249471A - 一种基因递送的聚乙烯亚胺纳米粒微泡复合物的制备方法 - Google Patents
一种基因递送的聚乙烯亚胺纳米粒微泡复合物的制备方法 Download PDFInfo
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Abstract
本发明公开了一种基因递送的聚乙烯亚胺纳米粒微泡复合物的制备方法,包括如下步骤:制备生物素化脂质微泡;制备亲和素化脂质微泡;制备生物素化PEG‑SS‑PEI纳米粒;制备聚乙二醇‑聚乙烯亚胺纳米粒微泡复合物。本发明的优点有:通过生物素‑亲和素法将脂质微泡与聚乙二醇‑聚乙烯亚胺纳米粒偶联起来,制备了一种聚乙二醇‑聚乙烯亚胺纳米粒微泡复合物,它既弥补了脂质微泡作为基因载体载基因量的不足,又解决了聚乙二醇‑聚乙烯亚胺纳米粒靶向性不强的缺点。
Description
技术领域
本发明涉及药物/基因递送载体技术领域,尤其涉及纳米粒微泡复合物的制备技术。
背景技术
微泡(microbubble,MB)是一类能够显著增强超声回声检测信号的超声造影剂。自从1968年,Gramiak首次提出了超声造影剂显像的概念。目前的超声造影剂已发展至第四代多功能靶向超声造影剂,前三代已从微泡的内含气体及壳膜材料进行改良,使得微泡变得更加稳定,体内存留时间更持久,可用于大多数器官显影,商业化的超声造影剂(比如:SonoVue)已在临床诊断疾病上广泛使用。多功能靶向超声造影剂是指携载药物、基因等治疗物质,或在微泡表面连接靶向修饰物实现靶向治疗的超声造影剂,其在临床治疗方面有很大的发展潜力。微泡不仅可改善超声图像质量,提高超声显影效果,还可以作为装载基因和药物的载体,在超声作用下促进释放,实现基因及药物的靶向递送,为临床疾病治疗提供一种新的治疗模式,真正意义上实现疾病的诊疗一体化。
脂质微泡是最常用的超声造影剂,因为脂质壳膜较软,低能量超声激励微泡可产生较强的空化作用,但脂质微泡也存在一定的缺陷,脂质微泡载药不稳定,所载药物可分布在壳膜表面或是镶嵌在脂质双层膜之间,同时由于脂质微泡内含气体,导致药物载药量及包封率均不高;另外,基因/药物载体静脉注射后在体内的肿瘤部位浓度不高是由于生物体内具有天然的生理屏障:血液循环中具有大量的核酸酶(可快速降解DNA、RNA);肝脾的内皮网状系统可吞噬基因/药物载体;血管内皮屏障(肿瘤的血管内皮间隙较大,约380nm-780nm)阻碍基因/药物载体漏入肿瘤组织间质;细胞膜天然的屏障;细胞内的内涵体-溶酶体吞噬、降解及胞吐;细胞核膜屏障(核孔直径),微米级粒径的脂质微泡,单纯依靠EPR效应进入肿瘤组织的能力有限,不能达到药物聚集于肿瘤区域及长效缓释的目的。如何构建具有靶向能力及时空控释能力的药物载体是目前治疗肿瘤研究的热点之一。
发明内容
本发明的目的在于提供一种基因递送的聚乙烯亚胺纳米粒微泡复合物的制备方法。
为了达到上述目的本发明采用如下技术方案:
(1)制备生物素化脂质微泡;
(2)制备亲和素化脂质微泡;
(3)制备生物素化PEG-SS-PEI纳米粒;
(4)制备聚乙二醇-聚乙烯亚胺纳米粒微泡复合物PSP@MB。
进一步地,所述步骤(1)的操作过程是将成膜材料溶于氯仿中,水浴,蒸发溶剂形成脂膜,水浴,真空抽气,充入C3F8,振荡,离心,得到分层的生物素化脂质微泡;
成膜材料是二棕榈酰磷脂酰胆碱(DPPC)、二硬脂酰磷脂酰甘油(DSPG)、生物素-磷脂-聚乙二醇2000(Biotin-DSPE-PEG2000);或者是二棕榈酰磷脂酰胆碱(DPPC)、生物素-磷脂-聚乙二醇2000(Biotin-DSPE-PEG2000)、三甲基铵丙烷(DPTAP)。
进一步地,所述步骤(1)的详细操作过程是:将成膜材料溶于1ml氯仿中,用超声波清洗仪分散溶解5min后,再分装在4个Vial瓶,打开瓶盖,60℃水浴2小时后,置于旋转蒸发仪24h后形成脂膜;超声波分散5min分散脂膜,60-65℃水浴15min使脂膜完全溶解,用真空泵抽气3min,后充入C3F8,于银汞调和器振荡45s,离心(6000rpm,3min),获得分层生物素化脂质微泡(上层为白色泡沫状液体,下层为稍浑浊液体);其余Vial瓶盖好瓶盖,-20瓶盖保存;
成膜材料及其用量比例是:二棕榈酰磷脂酰胆碱(DPPC)10mg、二硬脂酰磷脂酰甘油(DSPG)4mg、生物素-磷脂-聚乙二醇2000(Biotin-DSPE-PEG2000)2mg;或者是:二棕榈酰磷脂酰胆碱(DPPC)9mg、生物素-磷脂-聚乙二醇2000(Biotin-DSPE-PEG2000)2mg、三甲基铵丙烷(DPTAP)1mg。
进一步地,所述步骤(2)的操作过程是:将生物素化脂质微泡与链霉亲和素按比例混合,振荡使充分连接,离心,取下层液体,去除未连接的链霉亲和素,获得亲和素化脂质微泡。
进一步地,所述步骤(2)的详细操作过程是:将生物素化脂质微泡与链霉亲和素按质量比2:1比例在vial瓶混合,轻柔振荡30min使其充分连接,离心(2000rcf,5min),使用1ml胰岛素针抽取下层液体,去除未连接的链霉亲和素,获得亲和素化脂质微泡。
进一步地,所述步骤(3)的操作过程是:将PEI溶液,加入Biotin-PEG-SS-NHS溶液50ml(10mg/ml),室温搅拌,过夜反应,获得生物素化PEG-SS-PEI纳米粒。
进一步地,所述步骤(3)的详细操作过程是:将50ml浓度为1mg/ml的25kDaPEI溶液,加入浓度为10mg/ml Biotin-PEG-SS-NHS溶液50ml,室温搅拌,过夜反应;获得生物素化PEG-SS-PEI纳米粒,使用截留分子量3500的透析袋在双蒸水中透析3天,使用0.22μm滤头过滤生物素化PEG-SS-PEI纳米粒除菌,将生物素化PEG-SS-PEI纳米粒浓度调整至1mg/mL,4℃冰箱保存。
进一步地,所述步骤(4)的操作过程是:将步骤(3)的产物加入步骤(2)的产物中,振荡使充分连接,离心,抽取下层液体,去除未连接的生物素化PEG-SS-PEI纳米粒,使用缓冲液置换,获得聚乙二醇-聚乙烯亚胺纳米粒微泡复合物。
进一步地,所述步骤(4)的详细操作过程是:将制备的生物素化PEG-SS-PEI纳米粒按质量比1:1比例加入亲和素化脂质微泡的vial瓶,轻柔振荡30min使其充分连接,2000rcf,离心5min,使用1ml胰岛素针抽取下层液体,去除未连接的生物素化PEG-SS-PEI纳米粒,使用体积分数0.2%甘油PBS混合液置换,获得聚乙二醇-聚乙烯亚胺纳米粒微泡复合物。
本发明的优点有:通过生物素-亲和素法将脂质微泡与聚乙二醇-聚乙烯亚胺纳米粒偶联起来,制备了一种聚乙二醇-聚乙烯亚胺纳米粒微泡复合物,它既弥补了脂质微泡作为基因载体载基因量的不足,又解决了聚乙二醇-聚乙烯亚胺纳米粒靶向性不强的缺点。
附图说明
此处所说明的附图用来提供对本发明的进一步理解,构成本申请的一部分,并不构成对本发明的不当限定,在附图中:
图1本发明中所述PSP纳米粒的粒径及GSH处理后PSP二硫键断裂的粒径;
图2本发明中所述PSP纳米粒的电位及GSH处理后PSP二硫键断裂的电位;
图3本发明中所述PSP纳米粒的核磁氢谱图及GSH处理后PSP二硫键断裂的核磁氢谱图;
图4本发明中所述PSP纳米粒的红外光谱图及GSH处理后PSP二硫键断裂的红外光谱图;
图5E是本发明中所述PSP@MB纳米粒微泡复合物的荧光图;
图5F是本发明中所述PSP@MB纳米粒微泡复合物的透射电镜图;
图6是本发明中所述脂质微泡及PSP@MB纳米粒微泡复合物的粒径;
图7是本发明中所述PSP@MB纳米粒微泡复合物的不同N/P比的凝胶阻滞实验;
图8是本发明中所述脂质微泡及PSP@MB纳米粒微泡复合物的电位。
具体实施方式
下面将结合附图以及具体实施例来详细说明本发明,在此以本发明的示意性实施例及说明用来解释本发明,但并不作为对本发明的限定。
本发明的成功制备微泡复合物(PSP@MB)。使用核磁氢谱、红外光谱表征聚乙二醇-聚乙烯亚胺纳米粒(PSP),马尔文激光粒度仪检测粒径、电位。对比多种脂质微泡及微泡复合物的基因转染率后,选择PSP@MB作为基因递送载体,联合超声进行下一步的实验验证。
超声靶向递送技术的发展与超声造影剂的研发息息相关。为了提高超声造影剂在体内的稳定性、增强细胞对超声造影剂的吸收及提高超声造影剂基因/药物携载量,本发明使用二硫键链接的聚乙二醇-聚乙烯亚胺纳米粒(PSP),通过生物素-亲和素连接法(Biotin-avidin system)与生物素化微泡(Biotin-MB)链接,获得聚乙二醇-二硫键-聚乙烯亚胺纳米粒-微泡复合物(PEG-SS-PEI@MB,PSP@MB),该基因/药物递送载体可有效响应超声及肿瘤高还原内环境,达到快速释放基因/药物的效果。并在体内外实验里证实了PSP@MB具有良好的生物安全性及肿瘤部位的超声造影效果。
PSP@MB载体设计的理念源于以下三点:第一,如何提高载体载基因/药物的携载量;第二,如何提高基因/药物递送载体在体内的稳定性;第三,如何增强细胞对基因/药物的释放与吸收,以下将逐一进行阐述。
首先,如何提高载体载基因/药物的携载量。针对这个问题,我们引入了分子量25kDa聚乙烯亚胺(Polyethyleneimine,PEI),25kDa PEI被认为是非病毒基因递送载体中的“金标准”,基因转染率很高,可与商业化的Lipofectamine2000相媲美,这是由于表面正电荷高,可通过静电作用络合pDNA及由于“质子海绵效应”产生内涵体逃逸。但25kDa PEI也是因为其表面正电荷高,产生的细胞毒性也大,并且PEI分子量越大,转染效率越高,其细胞毒性越大,并且25kDaPEI的表面高正电荷在血液循环中容易与带负电的蛋白结合,被内皮网状系统吞噬。因此限制了25kDaPEI在体内动物实验及临床的使用。而PEG化修饰PEI有效克服PEI上述的缺点,降低PEI的细胞毒性。
其次,如何提高基因/药物递送载体在体内的稳定性。本发明在载体PSP@MB的纳米粒及微泡部分均加入了聚乙二醇。聚乙二醇(polyethyleneglycol,PEG)化可屏蔽PEI表面过高的正电荷,利于增强PEI/质粒DNA的生物相容性以及水溶性;减少巨噬细胞的吞噬,延长PEI/质粒DNA体内循环的时间;可减少PEI/质粒DNA对蛋白质及酶的吸附,减少DNA酶的降解,同时也降低了PEI的细胞毒性;但PEG化修饰会使PEI入胞效率降低及内涵体逃逸困难,原因有两个:第一,PEG可屏蔽基因载体表面的荷电性质;第二,PEG在载体表面会形成空间位阻。如何使PEG在体内肿瘤组织内与PEI分离成为提高转染率的关键,可通过修饰响应肿瘤微环境的化学键来解决这个问题。
第三,如何增强细胞对基因/药物的释放与吸收。PSP@MB的设计引入了可响应肿瘤高还原环境的二硫键。二硫键在普通细胞外的低还原性环境(谷胱甘肽含量低)下可稳定存在,而进入肿瘤细胞内,在肿瘤细胞内的髙还原性环境下,由于肿瘤细胞内较高的还原型谷胱甘肽(g-glutamyl-cysteinyl-glycine,GSH)浓度,载体上的二硫键被GSH降解,PEG与PEI/pDNA分离,PEI恢复表面正电荷,产生“质子海绵效应”使PEI/pDNA从内涵体逃逸,避免pDNA的溶酶体降解及促进pDNA释放,有利于基因转染及药物递送。
PSP@MB是通过生物素-亲和素连接法联接PSP纳米粒及脂质微泡,亲和素和生物素之间具有很强的亲和力,可在极性条件下可保证稳定连接,并且生物素化不会改变物质本身的生物活性和生理特性。
超声作为一种机械波,安全性高、临床应用广泛,联合超声造影剂具有可视化及时空靶向控释的优点。首先,超声可视化,纳米粒-脂质微泡在体内血液循环中可使用超声造影跟踪,实现基因/药物在生物体内的运输过程的可视化。其次,超声时空靶向性,使用超声靶向微泡破坏技术在超声辐照部位实现基因/药物的控释,称之为超声的时空靶向性。利用聚焦超声的时空靶向性,纳米粒-微泡复合物形成被动靶向基因递送系统,保证了所附着的纳米颗粒存在于超声辐照的靶部位。第三,采用超声靶向脂质微泡的方法,可在超声辐照部位靶向沉积纳米粒和质粒DNA。超声靶向微泡破坏产生空化效应,可分为惯性空化及稳态空化,作用于细胞膜时,可产生微小可逆性声孔,称之为声孔效应,声孔效应能够提高靶组织的血管内皮通透性,促进基因/药物渗入靶区的血管和细胞中,实现基因/药物的靶向递送;第四,聚焦超声所产生的声辐射力可增加纳米粒-微泡复合物在超声辐照区域的浓度,促进PSP纳米粒通过EPR效应进入细胞,提高基因转染率及药物递送效率。本发明构建的PSP@MB平均粒径为502±75nm,Zeta电位为13±4.4mV。将PSP纳米粒与10mm GSH孵育2小时后,PSP纳米粒的粒径从100±53nm增加到210±101nm。超声辐照PSP@MB,微泡破裂后剩下PSP/pDNA,其粒径大小有利于产生EPR效应,进入肿瘤组织间质。PSP@MB与脂质微泡相比,PSP载基因后纳米粒粒径100nm左右,PSP@MB联合超声,MB破裂产生的空化效应有利于促进PSP纳米粒的EPR效应,聚集于肿瘤区域。产生EPR效应有三个关键的指标,纳米粒的粒径与血管内皮细胞的孔径大小,研究发现,肿瘤血管内皮细胞的孔隙大约在380~780nm之间,而PSP纳米粒的粒径100nm左右,并且超声微泡产生的空化效果可有助于打开血管内皮细胞的紧密联接,有助于增强PSP@MB的时空控释基因/药物能力,从而达到肿瘤靶向治疗的目的。常规的脂质微泡,不能透过血管内皮细胞间隙,只能作为血池超声造影剂,而小于100nm的纳米造影剂,有非靶向性渗透的风险,而本发明构建的PSP@MB纳米粒微泡复合物,可响应超声,超声辐照后微泡破裂,粒径变小,有效促进EPR效应,可提高肿瘤区域的超声被动靶向效果,结合肿瘤的高还原环境情况,可有效控释基因/药物,使其特异性地在肿瘤靶区长时间滞留,提高肿瘤的治疗效果。
超声靶向递送的参数优化有利于基因/药物治疗肿瘤,参数优化包括(超声参数:声强、频率、占空比、辐照时间、超声辐照装置及辐照方式)、微泡浓度、质粒量、药物量、细胞密度、细胞周期、培养基量、辐照后换液时间及荧光观察时间等),优化多组参数的组合及有效提高超声靶向递送的效果。多学科的交叉融合,更有利于医学的发展,本发明将医学(肿瘤干细胞及肿瘤)与物理(超声)、化学(药物/基因递送载体)、分子生物学(RNA干扰),纳米技术(PSP纳米粒)有效结合考虑,构建PSP@MB,促进超声靶向基因/药物递送治疗肿瘤技术的发展。
本发明成功制备了PSP@MB声学响应性基因/药物递送载体,主要分为两部分,脂质微泡可响应超声,产生空化效应,具有时空靶向控释的特点,另一部分为PEG化修饰的PEI,PEG与PEI由响应肿瘤高还原环境的二硫键连接,PEI具有良好的基因络合能力,PEG具有循环长时间的优势,PEG-SS-PEI纳米粒与脂质微泡之间使用生物素-亲和素相连,成功制备出具有双重响应性的基因/药物递送载体。
实施例一、
1.生物素化脂质微泡(Biotin-MB)的制备
采用薄膜水化法制备生物素化阴离子脂质微泡、生物素化阳离子脂质微泡。
取阴离子脂质微泡成膜材料:二棕榈酰磷脂酰胆碱(DPPC)10mg、二硬脂酰磷脂酰甘油(DSPG)4mg、生物素-磷脂-聚乙二醇2000(Biotin-DSPE-PEG2000)2mg;将阴离子脂质微泡成膜材料溶于1ml氯仿中,用超声波清洗仪分散溶解5min后,再分装在4个Vial瓶,打开瓶盖,60℃水浴2小时后,置于旋转蒸发仪24h后形成脂膜;取其中1个Vial瓶中加800μl含甘油PBS缓冲液;旋转蒸发含甘油PBS缓冲液;超声波分散5min分散脂膜,60-65℃水浴15min使脂膜完全溶解,用真空泵抽气3min,用真空泵抽气3min,后充入C3F8,于银汞调和器振荡45s,得到白色泡沫状液体,离心(6000rpm,3min),上层为白色泡沫状液体即是生物素化脂质微泡,下层为稍浑浊液体;其余3个Vial瓶盖好瓶盖,-20℃冰箱保存。
取阳离子脂质微泡成膜材料:二棕榈酰磷脂酰胆碱(DPPC)9mg、生物素-磷脂-聚乙二醇2000(Biotin-DSPE-PEG2000)2mg、三甲基铵丙烷(DPTAP)1mg,制备方法参照生物素化阴离子脂质微泡的制备过程。
采用生物素化阴离子脂质微泡或者生物素化阳离子脂质微泡进行后续的实验。
2.亲和素化脂质微泡(Avidin-MB)制备:
将Biotin-MB与链霉亲和素(FITC-Streptavidin)按质量比2:1比例在vial瓶混合,轻柔振荡30min使其充分连接,离心(2000rcf,5min),使用1ml胰岛素针抽取下层液体,去除未连接的链霉亲和素,获得Avidin-MB。
3.PSP纳米粒制备
生物素化PEG-SS-PEI(Biotin-PSP)纳米粒的制备:
利用酰胺键连接25kDaPEI的胺基与Biotin-PEG-SS-NHS的活化羧基。首先,将50ml浓度为1mg/ml的25kDa PEI溶液,加入浓度为10mg/ml的Biotin-PEG-SS-NHS(西安瑞禧生物科技有限公司)溶液50ml,室温搅拌,过夜反应。获得生物素化PEG-SS-PEI纳米粒,使用截留分子量3500的透析袋在双蒸水中透析3天。使用0.22μm滤头(Millex-LG,Millipore Co.,USA)过滤生物素化PEG-SS-PEI纳米粒除菌,最后,将生物素化PEG-SS-PEI纳米粒浓度调整至1mg/mL,4℃冰箱保存。
4、聚乙二醇-聚乙烯亚胺纳米粒微泡复合物(PSP@MB)的制备:
将制备的生物素化PEG-SS-PEI纳米粒按质量比1:1比例加入Avidin-MB的vial瓶,轻柔振荡30min使其充分连接,离心(2000rcf,5min),使用1ml胰岛素针抽取下层液体,去除未连接的生物素化PEG-SS-PEI纳米粒,使用体积比0.2%甘油PBS混合液置换,获得聚乙二醇-聚乙烯亚胺纳米粒微泡复合物(PSP@MB)混合物。采用荧光显微镜观察两者的连接情况,并拍照,试验结果如图5E所示。
实施例二
使用马尔文激光粒度仪检测本发明实施例一步骤3制备的PSP纳米粒的粒径及GSH处理后二硫键断裂的PSP的粒径,试验结果如图1所示;
使用马尔文激光粒度仪检测本发明中实施例一步骤3制备的PSP纳米粒的电位及GSH处理后二硫键断裂的PSP的电位,试验结果如图2所示;
使用核磁氢谱表征实施例一步骤3的PSP纳米粒及GSH处理后二硫键断裂的PSP,GSH可使二硫键裂解,伴随着化学位移从2.68-2.94至2.45ppm。试验结果如图3所示;
使用红外光谱表征实施例一步骤3的PSP纳米粒及GSH处理后二硫键断裂的PSP,试验结果如图4所示;
对PSP@MB纳米粒微泡复合物进行荧光处理,试验结果如图5E所示;
对PSP@MB纳米粒微泡复合物进行透射电镜观察,结果如图5F所示;
使用马尔文激光粒度仪检测阳离子脂质微泡及阳离子PSP@MB纳米粒微泡复合物的粒径,试验结果如图6所示;
采用不同N/P比的凝胶阻滞实验测试阳离子PSP@MB纳米粒微泡复合物,试验结果如图7所示;
使用马尔文激光粒度仪检测阳离子脂质微泡及阳离子PSP@MB纳米粒微泡复合物的电位,试验结果如图8所示。
以上实验是针对涉及阳离子脂质微泡的技术方案进行的,当然经过实验证明,涉及阴离子脂质微泡的技术方案的实验效果水平与涉及阳离子脂质微泡的技术方案相当,此处不再赘述。
以上这些试验应该都是本领域技术人员能够根据本说明书公开的内容以及公知常识进行适当调整就能理解并实现的,因此本文不在此一一赘述。
以上对本发明实施例所提供的技术方案进行了详细介绍,本文中应用了具体个例对本发明实施例的原理以及实施方式进行了阐述,以上实施例的说明只适用于帮助理解本发明实施例的原理;同时,对于本领域的一般技术人员,依据本发明实施例,在具体实施方式以及应用范围上均会有改变之处,综上所述,本说明书内容不应理解为对本发明的限制。
Claims (9)
1.一种基因递送的聚乙烯亚胺纳米粒微泡复合物的制备方法,其特征在于:
包括如下步骤:
(1)制备生物素化脂质微泡;
(2)制备亲和素化脂质微泡;
(3)制备生物素化PEG-SS-PEI纳米粒;
(4)制备聚乙二醇-聚乙烯亚胺纳米粒微泡复合物PSP@MB。
2.根据权利要求1所述的基因递送的聚乙烯亚胺纳米粒微泡复合物的制备方法,其特征在于:
所述步骤(1)的操作过程是将成膜材料溶于氯仿中,水浴,蒸发溶剂形成脂膜,水浴,真空抽气,充入C3F8,振荡,离心,得到分层的生物素化脂质微泡;
成膜材料是二棕榈酰磷脂酰胆碱(DPPC)、二硬脂酰磷脂酰甘油(DSPG)、生物素-磷脂-聚乙二醇2000(Biotin-DSPE-PEG2000);或者是二棕榈酰磷脂酰胆碱(DPPC)、生物素-磷脂-聚乙二醇2000(Biotin-DSPE-PEG2000)、三甲基铵丙烷(DPTAP)。
3.根据权利要求2所述的聚乙二醇-聚乙烯亚胺纳米粒微泡复合物及其制备方法,其特征在于:
所述步骤(1)的详细操作过程是:将成膜材料溶于1ml氯仿中,用超声波清洗仪分散溶解5min后,再分装在4个Vial瓶,打开瓶盖,60℃水浴2小时后,置于旋转蒸发仪24h后形成脂膜;取其中1个Vial瓶中加800μl含甘油PBS缓冲液,旋转蒸发含甘油PBS缓冲液;超声波分散5min分散脂膜,60-65℃水浴15min使脂膜完全溶解,用真空泵抽气3min,后充入C3F8,于银汞调和器振荡45s,获得分层白色泡沫状液体,即是生物素化脂质微泡,其余3个Vial瓶盖好瓶盖,-20℃冰箱保存。
成膜材料及其用量比例是:二棕榈酰磷脂酰胆碱(DPPC)10mg、二硬脂酰磷脂酰甘油(DSPG)4mg、生物素-磷脂-聚乙二醇2000(Biotin-DSPE-PEG2000)2mg;或者是:二棕榈酰磷脂酰胆碱(DPPC)9mg、生物素-磷脂-聚乙二醇2000(Biotin-DSPE-PEG2000)2mg、三甲基铵丙烷(DPTAP)1mg。
4.根据权利要求1所述的基因递送的聚乙烯亚胺纳米粒微泡复合物的制备方法,其特征在于:
所述步骤(2)的操作过程是:将生物素化脂质微泡与链霉亲和素按比例混合,振荡使充分连接,离心,取下层液体,去除未连接的链霉亲和素,获得亲和素化脂质微泡。
5.根据权利要求4所述的基因递送的聚乙烯亚胺纳米粒微泡复合物的制备方法,其特征在于:
所述步骤(2)的详细操作过程是:将生物素化脂质微泡与链霉亲和素按质量比2:1比例在vial瓶混合,轻柔振荡30min使其充分连接,离心(2000rcf,5min),使用1ml胰岛素针抽取下层液体,去除未连接的链霉亲和素,获得亲和素化脂质微泡。
6.根据权利要求1所述的基因递送的聚乙烯亚胺纳米粒微泡复合物的制备方法,其特征在于:
所述步骤(3)的操作过程是:将PEI溶液,加入Biotin-PEG-SS-NHS溶液50ml(10mg/ml),室温搅拌,过夜反应,获得生物素化PEG-SS-PEI纳米粒。
7.根据权利要求6所述的基因递送的聚乙烯亚胺纳米粒微泡复合物的制备方法,其特征在于:
所述步骤(3)的详细操作过程是:将50ml浓度为1mg/ml的25kDa PEI溶液,加入浓度为10mg/ml Biotin-PEG-SS-NHS溶液50ml,室温搅拌,过夜反应;获得生物素化PEG-SS-PEI纳米粒,使用截留分子量3500的透析袋在双蒸水中透析3天,使用0.22μm滤头过滤生物素化PEG-SS-PEI纳米粒除菌,将生物素化PEG-SS-PEI纳米粒浓度调整至1mg/mL,4℃冰箱保存。
8.根据权利要求1所述的基因递送的聚乙烯亚胺纳米粒微泡复合物的制备方法,其特征在于:
所述步骤(4)的操作过程是:将步骤(3)的产物加入步骤(2)的产物中,振荡使充分连接,离心,抽取下层液体,去除未连接的生物素化PEG-SS-PEI纳米粒,使用缓冲液置换,获得聚乙二醇-聚乙烯亚胺纳米粒微泡复合物。
9.根据权利要求8所述的基因递送的聚乙烯亚胺纳米粒微泡复合物的制备方法,其特征在于:
所述步骤(4)的详细操作过程是:将制备的生物素化PEG-SS-PEI纳米粒按质量比1:1比例加入亲和素化脂质微泡的vial瓶,轻柔振荡30min使其充分连接,2000rcf,离心5min,使用1ml胰岛素针抽取下层液体,去除未连接的生物素化PEG-SS-PEI纳米粒,使用体积分数0.2%甘油PBS混合液置换,获得聚乙二醇-聚乙烯亚胺纳米粒微泡复合物。
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