CN109350750A - 一种靶向多功能纳米粒及其制备方法和应用 - Google Patents
一种靶向多功能纳米粒及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及一种靶向多功能纳米粒及其制备方法和应用,属于生物医药技术领域,采用超声双乳化法制备靶向多功能纳米粒,利用支化PEI与PLGA进行共价结合,将原本带负电荷的PLGA纳米粒修饰为携带正电荷的阳离子纳米粒,能够有效的吸附质粒,在循环系统中保证质粒不会因为物理作用而脱落,进入细胞后被胞内酶降解的机率也降低。该纳米粒中还含有IR780,能够靶向于肿瘤细胞线粒体,同时,IR780的强荧光和光敏剂特性使纳米粒成为一个很好的荧光和光声成像介质,方便观察肿瘤。该纳米粒中可以载药,以便联合转基因治疗,不但更有利于消灭肿瘤细胞,还能减少化疗药物的毒副作用。该靶向多功能纳米粒制备方法简单,易操作,适合扩大化生产。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种靶向多功能纳米粒及其制备方法和应用。
背景技术
小动物基因转染多数采用尾静脉直接注射质粒,但这种方法没有转染部位的特异性和精准性,以及存在注射后的质粒不能很好的进入细胞,且易被降解,转染效率低等缺陷,因而不适用于肿瘤的基因治疗。以纳米粒作为载体,由于肿瘤的EPR效应,纳米粒本身就有一种在肿瘤部位聚集吞噬的趋势,并且常用作药物缓释载体,因此可以将质粒包载到纳米粒中内水相里面,但是目前将质粒包载于纳米粒中内水相里面还存在两个弊端,一个是包载的过程中,合成纳米粒需要用到声震仪,这对于质粒是一种破环,使其出现开环的情况;其次,包载于内水相的质粒进入细胞后,其释放效率相比于吸附于表面而言会低很多。因此,急需一种释放效率高、靶向性好的多功能纳米粒。
发明内容
有鉴于此,本发明的目的之一在于提供一种靶向多功能纳米粒的制备方法;目的之二在于提供一种靶向多功能纳米粒;目的之三在于提供靶向多功能纳米粒在制备肿瘤基因治疗药物中的应用。
为达到上述目的,本发明提供如下技术方案:
1、一种靶向多功能纳米粒的制备方法,所述方法包括如下步骤:
(1)将分子量为7000-12000的聚乳酸-羟基乙酸共聚物和七甲川花菁类荧光小分子IR780溶于二氯甲烷中,然后加入内水相,冰浴条件下超声处理,再加入聚乙烯醇水溶液,再次超声后加入异丙醇水溶液,获得混合液Ⅰ,搅拌所述混合液Ⅰ2.5-3.5h后去除所述二氯甲烷,最后离心获得沉淀Ⅰ,将所述沉淀Ⅰ洗涤后以MES缓冲液重悬获得重悬液,将所述重悬液放置于4-8℃下,备用;所述混合液Ⅰ中聚乳酸-羟基乙酸共聚物、七甲川花菁类荧光小分子IR780、内水相、聚乙烯醇和异丙醇的质量体积比为50:0.5-3:0.025-0.3:80-240:0.04-0.2,所述质量体积比的单位为mg:mg:mL:mg:mL;
(2)将N-羟基硫代琥珀酰亚胺和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐溶于MES缓冲液中,然后加入步骤(1)中获得的重悬液中,获得混合液Ⅱ,搅拌所述混合液Ⅱ2-2.5h,所述N-羟基硫代琥珀酰亚胺和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐与步骤(1)中聚乳酸-羟基乙酸共聚物的质量比为15-25:60-75:50;
(3)将分子量≤25000的支化聚乙烯亚胺溶于MES缓冲液中,然后加入经步骤(2)处理后的混合液Ⅱ中,获得混合液Ⅲ,搅拌所述混合液Ⅲ2-2.5h后进行稀释,最后离心获得沉淀Ⅱ,将所述沉淀Ⅱ洗涤后冷冻干燥,即可;所述支化聚乙烯亚胺与步骤(1)中聚乳酸-羟基乙酸共聚物的质量比为40-50:1。
优选地,步骤(1)中,所述内水相为水、化疗药物的水溶液或相变材料中的一种。
优选地,所述水为双蒸水;所述化疗药物的水溶液为盐酸多柔比星水溶液或硫酸长春新碱水溶液中的一种;所述相变材料为全氟正戊烷或全氟己烷中的一种。
优选地,步骤(1)中,两次超声的条件为:超声功率为135w,超声频率为20kHz,每间隔5s超声一次,每次超声时间为5s,共超声14-17次。
优选地,步骤(1)、步骤(2)和步骤(3)中,所述搅拌的速度为40-100r/min。
优选地,步骤(1)和步骤(3)中,所述离心的速度为10000-11500r/min,离心的时间为5-8min。
优选地,步骤(1)、步骤(2)和步骤(3)中,所述MES缓冲液的浓度为0.1M,pH值为5-6。
优选地,步骤(1)中,所述聚乳酸-羟基乙酸共聚物和MES缓冲液的质量体积比50:0.8;步骤(2)中,所述1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和MES缓冲液的质量体积比60:0.8;步骤(3)中,所述支化聚乙烯亚胺和MES缓冲液的质量体积比2:8;所述质量体积比的单位均为mg:mL。
2、由所述的方法制备的靶向多功能纳米粒。
3、所述靶向多功能纳米粒在制备肿瘤基因治疗药物中的应用。
本发明的有益效果在于:本发明提供了一种靶向多功能纳米粒及其制备方法和应用,采用超声双乳化法制备靶向多功能纳米粒,利用支化聚乙烯亚胺与聚乳酸-羟基乙酸共聚物进行共价结合,将原本带负电荷的聚乳酸-羟基乙酸共聚物纳米粒修饰为携带正电荷的阳离子纳米粒,能够有效的吸附质粒,在循环系统中保证质粒不会因为物理作用而脱落,进入细胞后被胞内酶降解的机率也降低。该纳米粒中还含有七甲川花菁类荧光小分子IR780,能够靶向于肿瘤细胞线粒体,可以更加精准的将纳米粒导入肿瘤细胞内,减少非靶向部位的转染带来的副作用,同时,IR780的强荧光和光敏剂特性使纳米粒成为一个很好的荧光和光声成像介质,方便观察肿瘤。加之,并发明在制备该靶向多功能纳米粒的过程中,通过限定各原料的用量,可以实现在保证最终制备的纳米粒的粒径及产品得率的同时,减少原料的浪费。另外,本发明中纳米粒中可以载药,以便联合转基因治疗,起到一个协同效应,不但有利于消灭肿瘤细胞,还减少了化疗药物的用量,进而减少化疗药物的毒副作用。本发明中靶向多功能纳米粒制备方法简单,易操作,适合扩大化生产。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚,本发明提供如下附图进行说明:
图1为实施例1中制备的靶向多功能纳米粒的扫描电镜图;
图2为实施例1中制备的靶向多功能纳米粒的透射电镜图;
图3为实施例1中制备的靶向多功能纳米粒的zeta电位测试图;
图4为实施例1中制备的靶向多功能纳米粒对质粒的吸附能力测试结果图;(a为质粒的激光扫描图,b为靶向多功能纳米粒的激光扫描图,c为吸附了质粒的靶向多功能纳米粒的激光扫描图)
图5为实施例1中制备的靶向多功能纳米粒对质粒的吸附量测试结果图;
图6为实施例1中制备的靶向多功能纳米粒对癌细胞的靶向作用测试结果图;
图7为实施例1中制备的靶向多功能纳米粒介导的基因表达情况测试结果图;
图8为LIFU对实施例1中制备的靶向多功能纳米粒与转基因的增效作用测试结果荧光图;
图9为LIFU对实施例1中制备的靶向多功能纳米粒与转基因的增效作用测试结果柱状图;
图10为经实施例1中制备的靶向多功能纳米粒转染基因后的肿瘤细胞培养过程中划痕变化图;
图11为经实施例1中制备的靶向多功能纳米粒转染基因后的肿瘤细胞的迁移能力测试结果图。
具体实施方式
下面将对本发明的优选实施例进行详细的描述。
实施例1
制备靶向多功能纳米粒
(1)将50mg分子量为12000的聚乳酸-羟基乙酸共聚物(PLGA)和2mg七甲川花菁类荧光小分子IR780溶于1mL二氯甲烷中,然后加入200uL浓度为15g/L的盐酸多柔比溶液,在超声功率为135w、超声频率为20Hz,冰浴条件下利用声震仪超声处理,处理时每间隔5s超声一次,每次超声时间为5s,共超声16次,再加入4mL浓度为40g/L的聚乙烯醇水溶液,再次在超声功率为135w、超声频率为20Hz条件下利用声震仪超声处理,处理时每间隔5s超声一次,每次超声时间为5s,共超声16次后加入6mL体积分数为2%的异丙醇水溶液,获得混合液Ⅰ,以60r/min的速度搅拌该混合液Ⅰ3h后去除二氯甲烷,最后以10500r/min的速度离心6min,获得沉淀Ⅰ,用浓度为0.1M,pH值为5.1的MES缓冲液将该沉淀Ⅰ洗涤两次,每次洗涤完后均以10500r/min的速度离心6min,再以800μL浓度为0.1M,pH值为5.1的MES缓冲液重悬获得重悬液,将该重悬液放置于4℃下,备用;
(2)将20mg N-羟基硫代琥珀酰亚胺(sulfo-NHS)和60mg 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)溶于800μL浓度为0.1M,pH值为5.1的MES缓冲液中,然后加入步骤(1)中获得的重悬液中,获得混合液Ⅱ,以60r/min的速度搅拌该混合液Ⅱ2h;
(3)将2g分子量为25000的支化聚乙烯亚胺(PEI)溶于8mL浓度为0.1M,pH值为5.1的MES缓冲液中,然后加入经步骤(2)处理后的混合液Ⅱ中,获得混合液Ⅲ,以60r/min的速度搅拌该混合液Ⅲ2h后用双蒸水进行稀释,最后以11000r/min的速度离心6min获得沉淀Ⅱ,将该沉淀Ⅱ用浓度为0.1M,pH值为5.1的MES缓冲液洗涤三次,每次洗涤完后均以11000r/min的速度离心5min,再冷冻干燥后制得靶向多功能纳米粒。
利用扫描电镜对实施例1中制备的靶向多功能纳米粒进行检测,检测结果见图1,由图1可知,纳米粒成球性良好,粒径分布均小于400纳米。
将实施例1中制备的靶向多功能纳米粒约2mg与200μl质粒(浓度约为295ng/μl)混合,室温下在80r/min速度的摇床内孵育3h,放置4℃冰箱过夜,以10000r/min的速度离心4min后双蒸水重悬至液体几乎无色,利用透射电镜观察,观察结果见图2,由图2可知,纳米粒表面吸附了不等量的质粒。
利用激光粒度仪对实施例1中制备的靶向多功能纳米粒进行检测,检测结果见图3,由图3可知,纳米粒的zeta电位约为+37~39mV。
实施例2
制备靶向多功能纳米粒
(1)将50mg分子量为7000的聚乳酸-羟基乙酸共聚物(PLGA)和3mg七甲川花菁类荧光小分子IR780溶于1mL二氯甲烷中,然后加入25uL双蒸水,在超声功率为135w、超声频率为20Hz,冰浴条件下利用声震仪超声处理,处理时每间隔5s超声一次,每次超声时间为5s,共超声14次,再加入4mL浓度为20g/L的聚乙烯醇水溶液,再次在超声功率为135w、超声频率为20Hz条件下利用声震仪超声处理,处理时每间隔5s超声一次,每次超声时间为5s,共超声14次后加入2mL体积分数为2%的异丙醇水溶液,获得混合液Ⅰ,以100r/min的速度搅拌该混合液Ⅰ2.5h后去除二氯甲烷,最后以10000r/min的速度离心8min,获得沉淀Ⅰ,用浓度为0.1M,pH值为6的MES缓冲液将该沉淀Ⅰ洗涤两次,每次洗涤完后均以10000r/min的速度离心8min,再以800μL浓度为0.1M,pH值为6的MES缓冲液重悬获得重悬液,将该重悬液放置于8℃下,备用;
(2)将15mg N-羟基硫代琥珀酰亚胺(sulfo-NHS)和75mg 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)溶于800μL浓度为0.1M,pH值为6的MES缓冲液中,然后加入步骤(1)中获得的重悬液中,获得混合液Ⅱ,以100r/min的速度搅拌该混合液Ⅱ2h;
(3)将2.5g分子量为15000的支化聚乙烯亚胺(PEI)溶于8mL浓度为0.1M,pH值为6的MES缓冲液中,然后加入经步骤(2)处理后的混合液Ⅱ中,获得混合液Ⅲ,以100r/min的速度搅拌该混合液Ⅲ2h后用双蒸水进行稀释,最后以10000r/min的速度离心8min获得沉淀Ⅱ,将该沉淀Ⅱ用浓度为0.1M,pH值为6的MES缓冲液洗涤三次,每次洗涤完后均以10000r/min的速度离心8min,再冷冻干燥后制得靶向多功能纳米粒。
实施例3
制备靶向多功能纳米粒
(1)将50mg分子量为9000的聚乳酸-羟基乙酸共聚物(PLGA)和0.5mg七甲川花菁类荧光小分子IR780溶于1mL二氯甲烷中,然后加入300uL全氟正戊烷,在超声功率为135w、超声频率为20Hz,冰浴条件下利用声震仪超声处理,处理时每间隔5s超声一次,每次超声时间为5s,共超声17次,再加入4mL浓度为60g/L的聚乙烯醇水溶液,再次在超声功率为135w、超声频率为20Hz条件下利用声震仪超声处理,处理时每间隔5s超声一次,每次超声时间为5s,共超声17次后加入10mL体积分数为2%的异丙醇水溶液,获得混合液Ⅰ,以40r/min的速度搅拌该混合液Ⅰ3.5h后去除二氯甲烷,最后以11500r/min的速度离心5min,获得沉淀Ⅰ,用浓度为0.1M,pH值为5的MES缓冲液将该沉淀Ⅰ洗涤两次,每次洗涤完后均以11500r/min的速度离心5min,再以800μL浓度为0.1M,pH值为5的MES缓冲液重悬获得重悬液,将该重悬液放置于6℃下,备用;
(2)将25mg N-羟基硫代琥珀酰亚胺(sulfo-NHS)和65mg 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)溶于800μL浓度为0.1M,pH值为5的MES缓冲液中,然后加入步骤(1)中获得的重悬液中,获得混合液Ⅱ,以40r/min的速度搅拌该混合液Ⅱ2.5h;
(3)将2.25g分子量为8000的支化聚乙烯亚胺(PEI)溶于8mL浓度为0.1M,pH值为5的MES缓冲液中,然后加入经步骤(2)处理后的混合液Ⅱ中,获得混合液Ⅲ,以40r/min的速度搅拌该混合液Ⅲ2.5h后用双蒸水进行稀释,最后以11500r/min的速度离心5min获得沉淀Ⅱ,将该沉淀Ⅱ用浓度为0.1M,pH值为5的MES缓冲液洗涤三次,每次洗涤完后均以11500r/min的速度离心6min,再冷冻干燥后制得靶向多功能纳米粒。
实施例4
测试本发明中靶向多功能纳米粒对质粒的吸附作用
(1)提取质粒,然后加入1μLGel-red,静置15min,加入1mg实施例1中制备的靶向多功能纳米粒,4℃冰箱孵育过夜,离心倒掉上清,取沉淀,将沉淀洗涤后离心两次,每次均以10000r/min的速度离心4min,双蒸水轻微重悬至液体几乎无色透明后滴至载玻片上,覆盖盖玻片后使用激光扫描共聚焦观察,结果如图4所示,图中红色为质粒(如图4中a所示),绿色为纳米粒(如图4中b所示),由图4可知,质粒和纳米粒的图像重合,证实二者之间有吸附作用(如图4中c所示)。
(2)提取质粒,测定核酸浓度,乘以体积计算质粒总量(结果见图5),称取3mg实施例1中制备的靶向多功能纳米粒,双蒸水重悬,加入质粒溶液(记录两种液体体积之和),室温下在80r/min速度的摇床内孵育3h,放置在4℃冰箱过夜后以11000r/min离心速度离心6min,取上清液Ⅰ,测定上清液Ⅰ中核酸浓度,乘以上清液Ⅰ的体积得出未吸附于纳米粒的质粒量(结果见图5),将沉淀用浓度为1M的氢氧化钠溶液进行解离后离心,获得上清液Ⅱ,测上清液Ⅱ中核酸浓度(结果见图5),由图5可知,上清液Ⅰ中质粒量与上清液Ⅱ中质粒量之和与质量总量基本相等,说明实施例1中制备的靶向多功能纳米粒能够大量的吸附质粒。
实施例5
测试本发明中靶向多功能纳米粒对癌细胞的靶向作用
将乳腺癌细胞MD-MB-231消化并铺于共聚焦皿,培养贴壁生长至约70%满,取3mg实施例1中制备的靶向多功能纳米粒用全培养基重悬,使每0.5mL培养基含有1μg纳米粒,共聚焦皿用无菌PBS缓冲液洗涤后,加入含有纳米粒的培养基0.5毫升,培养60min。LIFU(低强度聚焦超声)参数设定为:功率1W,脉冲模式为间歇4s,涂抹超声耦合剂,每个加入了含有纳米粒的培养基的共聚焦皿经过120s的LIFU作用,放入培养箱继续培养,第二天换液,培养两天后,使用DiI染细胞膜,多聚甲醛固定样品,洗涤,少量PBS缓冲液湿润样品,在激光扫描共聚焦下观察,通道选择红色DiI,绿色EGFP,结果如6所示,由图6可知,孵育20min便有纳米粒进入细胞内,随着时间增加,40min胞内纳米粒浓度增加,到60min几乎饱和。
实施例6
测试本发明中靶向多功能纳米粒介导的基因表达情况
将细胞用胰酶消化后,以培养基重悬滴入共聚焦皿,密度以细胞单个散在为佳,培养过夜使贴壁,1mL培养基加入含约1μl质粒的纳米粒,替换共聚焦皿中的培养基,孵育1h后利用LIFU作用(功率为1W,脉冲模式为间歇8s)80s后换液,继续培养两天,按试剂盒操作标准进行DiI细胞膜染色,然后多聚甲醛固定,利用激光共聚焦进行观察,结果如图7所示,图7中DiI是红色细胞膜染料,EGFP是质粒携带的绿色荧光蛋白,该绿色荧光蛋白随着质粒中目的基因的表达而同时表达,Merge是左右两张图的叠加图,由图7可知,绿色荧光蛋白确实是在细胞内表达,说明由靶向多功能纳米粒介导的基因在细胞内进行了表达。
实施例7
测试本发明中LIFU(低强度聚焦超声)对靶向多功能纳米粒与转基因的增效作用
以实施例6中同法铺皿加纳米粒,一组使用LIFU驱动转基因,一组单纯加入载基因纳米粒。利用激光共聚焦观察,结果如图8所示,图8中上排三张图分别是非LIFU驱动转基因皿中取的三个不同观察视野,下排三张图是LIFU驱动转基因皿中取的三个不同的观察视野。利用imageJ软件分别进行荧光强度定量分析绘制得出柱状图9,由图9可知,利用LIFU驱动转基因的细胞生长,表达目的蛋白的量明显高于没有LIFU驱动的组。
实施例8
测试经本发明中靶向多功能纳米粒转染基因后的肿瘤细胞的迁移能力
准备六孔板,细胞种板,培养至80%满,上下两排分为LIFU转染组(即每孔加入3μg吸附了质粒的实施例1中制备的靶向多功能纳米粒,用LIFU驱动)和LIFU组(即每孔仅加入3μg实施例1中制备的靶向多功能纳米粒,用LIFU驱动)。孵箱培养至第二天,倒掉培养基,PBS缓冲液洗涤后,用200μL无菌枪头笔直划痕,再次PBS缓冲液清洗,然后每孔加入1mL浓度为1%的胎牛血清的DMEM高糖培养基培养,每日观察划痕情况并拍照记录,持续三天,结果如图10和图11所示,由图10和图11可知,经本发明中靶向多功能纳米粒转染的细胞,其迁移能力确实受到抑制,细胞无向划痕中央迁移的倾向,三天的划痕宽度无明显差异,非转染组划痕宽度逐渐减少,细胞有迁移倾向。
最后说明的是,以上优选实施例仅用以说明本发明的技术方案而非限制,尽管通过上述优选实施例已经对本发明进行了详细的描述,但本领域技术人员应当理解,可以在形式上和细节上对其作出各种各样的改变,而不偏离本发明权利要求书所限定的范围。
Claims (10)
1.一种靶向多功能纳米粒的制备方法,其特征在于,所述方法包括如下步骤:
(1)将分子量为7000-12000的聚乳酸-羟基乙酸共聚物和七甲川花菁类荧光小分子IR780溶于二氯甲烷中,然后加入内水相,冰浴条件下超声处理,再加入聚乙烯醇水溶液,再次超声后加入异丙醇水溶液,获得混合液Ⅰ,搅拌所述混合液Ⅰ2.5-3.5h后去除所述二氯甲烷,最后离心获得沉淀Ⅰ,将所述沉淀Ⅰ洗涤后以MES缓冲液重悬获得重悬液,将所述重悬液放置于4-8℃下,备用;所述混合液Ⅰ中聚乳酸-羟基乙酸共聚物、七甲川花菁类荧光小分子IR780、内水相、聚乙烯醇和异丙醇的质量体积比为50:0.5-3:0.025-0.3:80-240:0.04-0.2,所述质量体积比的单位为mg:mg:mL:mg:mL;
(2)将N-羟基硫代琥珀酰亚胺和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐溶于MES缓冲液中,然后加入步骤(1)中获得的重悬液中,获得混合液Ⅱ,搅拌所述混合液Ⅱ2-2.5h,所述N-羟基硫代琥珀酰亚胺和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐与步骤(1)中聚乳酸-羟基乙酸共聚物的质量比为15-25:60-75:50;
(3)将分子量≤25000的支化聚乙烯亚胺溶于MES缓冲液中,然后加入经步骤(2)处理后的混合液Ⅱ中,获得混合液Ⅲ,搅拌所述混合液Ⅲ2-2.5h后进行稀释,最后离心获得沉淀Ⅱ,将所述沉淀Ⅱ洗涤后冷冻干燥,即可;所述支化聚乙烯亚胺与步骤(1)中聚乳酸-羟基乙酸共聚物的质量比为40-50:1。
2.如权利要求1所述的方法,其特征在于,步骤(1)中,所述内水相为水、化疗药物的水溶液或相变材料中的一种。
3.如权利要求2所述的方法,其特征在于,所述水为双蒸水;所述化疗药物的水溶液为盐酸多柔比星水溶液或硫酸长春新碱水溶液中的一种;所述相变材料为全氟正戊烷或全氟己烷中的一种。
4.如权利要求1所述的方法,其特征在于,步骤(1)中,两次超声的条件为:超声功率为135w,超声频率为20kHz,每间隔5s超声一次,每次超声时间为5s,共超声14-17次。
5.如权利要求1所述的方法,其特征在于,步骤(1)、步骤(2)和步骤(3)中,所述搅拌的速度为40-100r/min。
6.如权利要求1所述的方法,其特征在于,步骤(1)和步骤(3)中,所述离心的速度为10000-11500r/min,离心的时间为5-8min。
7.如权利要求1所述的方法,其特征在于,步骤(1)、步骤(2)和步骤(3)中,所述MES缓冲液的浓度为0.1M,pH值为5-6。
8.如权利要求7所述的方法,其特征在于,步骤(1)中,所述聚乳酸-羟基乙酸共聚物和MES缓冲液的质量体积比50:0.8;步骤(2)中,所述1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和MES缓冲液的质量体积比60:0.8;步骤(3)中,所述支化聚乙烯亚胺和MES缓冲液的质量体积比2:8;所述质量体积比的单位均为mg:mL。
9.由权利要求1-8任一项所述的方法制备的靶向多功能纳米粒。
10.权利要求9中所述靶向多功能纳米粒在制备肿瘤基因治疗药物中的应用。
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