CN112386711B - 一种多功能外壳材料pmtph及其制备方法和用途 - Google Patents
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Abstract
本发明提供了一种多功能外壳材料PMTPH及其制备方法和用途,其由以下重量配比的原料制备而成:基质金属蛋白酶10~15份,甲氧基聚乙二醇马来酰亚胺30~35份,羧基被活化后的透明质酸40~45份,氨基聚乙二醇马来酰亚胺3~8份,穿膜肽15~20份。该外壳材料与阳离子聚合物基因载体结合后,能使其带弱负电荷,通过静脉注射用于体内治疗;克服了现有阳离子聚合物基因载体进入血液后造成溶血等强毒副作用的问题。同时,该外壳材料不会降低阳离子聚合物基因载体的转染效率。该外壳材料没有免疫原性,安全性好;能对肿瘤微环境的酶敏感产生响应,利于肿瘤细胞吞噬;该外壳材料带有穿膜肽,利于携带基因进入细胞内。该外壳材料在制备用于体内治疗的基因载体中具有良好的应用前景。
Description
技术领域
本发明属于医药领域,具体涉及一种多功能外壳材料PMTPH及其制备方法和用途。
背景技术
基因治疗是指将治疗基因通过载体运送到靶器官或组织以修复受损或缺失基因,发挥治疗效果的一种新型疗法。目前基因治疗主要策略包括导入抑癌基因、自杀基因、siRNA、耐药基因、免疫基因、CRISPR-Cas9系统等外源性基因。但是外源性基因无法主动被靶细胞纳入,需要依靠基因载体运载进入细胞。因此,低毒性高效率的载体是基因治疗的关键。
目前广泛使用的基因载体主要分为病毒类载体和非病毒类载体。病毒类载体包括反转录病毒、腺病毒、腺相关病毒等,其基因转染效率高,但存在免疫原性和基因插入突变两个严重的安全性问题。近年来随着药剂学、材料学等多学科的迅速发展,大量合成材料及天然材料涌现,非病毒载体备受关注,包括阳离子脂质体或脂类复合物和阳离子高分子聚合物,它们作为载体的优点是,能够递送大尺寸的核酸;有明确的化学结构,易于商品化等。
但是,阳离子聚合物和基因结合后带有大量的正电荷,这些正电荷进入血液后会与血液中的负电蛋白结合,造成溶血等强毒副作用,因此,将其作为基因载体只能在体外进行细胞实验,不能静脉注射,用于体内基因治疗。材料用于体内的治疗,需要带一定的弱负电性或者中性,避免与血液中的负电蛋白结合,造成溶血等毒副作用。因此,研究一种低毒性高效率,且能用于体内治疗的基因载体对于促进基因治疗的发展有重要意义。
发明内容
为了解决上述问题,本发明提供了一种多功能外壳材料PMTPH及其制备方法和用途。
本发明提供了一种多功能外壳材料PMTPH,它是由以下重量配比的原料制备而成:基质金属蛋白酶10~15份,甲氧基聚乙二醇马来酰亚胺30~35份,羧基被活化后的透明质酸40~45份,氨基聚乙二醇马来酰亚胺3~8份,穿膜肽15~20份。
进一步地,前述的多功能外壳材料PMTPH是由以下重量配比的原料制备而成:基质金属蛋白酶13.6份,甲氧基聚乙二醇马来酰亚胺33.5份,羧基被活化后的透明质酸42.3份,氨基聚乙二醇马来酰亚胺6.7份,穿膜肽18.6份。
进一步地,所述基质金属蛋白酶为基质金属蛋白酶9;和/或,所述甲氧基聚乙二醇马来酰亚胺的分子量为2000;和/或,所述氨基聚乙二醇马来酰亚胺的分子量为600;所述羧基被活化后的透明质酸为20%羧基被活化后的透明质酸;所述穿膜肽为穿膜肽Cys-TATs,其氨基酸序列为YGRKKRRQRRRC(SEQ ID NO.1)。
进一步地,所述羧基被活化后的透明质酸的制备方法为:
取透明质酸、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N羟基丁二酰亚胺,在MES缓冲液中活化反应,即得。
进一步地,所述透明质酸、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N羟基丁二酰亚胺的质量比为42~43:2~2.5:1.4;所述透明质酸和MES缓冲液的质量体积比为20~45:25(mg/mL);和/或,所述活化反应的反应条件为室温中反应2h;
优选地,所述透明质酸、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N羟基丁二酰亚胺的质量比为42.3~42.4:2.2~2.3:1.4;所述透明质酸和MES缓冲液的质量体积比为21.2~42.3:25(mg/mL)。
本发明还提供了一种前述的多功能外壳材料PMTPH的制备方法,它包括如下步骤:
(1)按前述的重量配比称取原料;
(2)将基质金属蛋白酶和甲氧基聚乙二醇马来酰亚胺溶于溶剂中反应,得产物A;
(3)将步骤(2)所得产物A、氨基聚乙二醇马来酰亚胺加入羧基被活化后的透明质酸中反应,得产物B;
(4)将穿膜肽加入步骤(3)所得产物B中反应,得产物C,即得。
进一步地,
步骤(2)中,所述溶剂与基质金属蛋白酶的体积质量比为4:13.6(mL/mg);
优选地,步骤(2)中,所述溶剂为PBS溶液。
进一步地,所述羧基被活化后的透明质酸的制备方法为:
取透明质酸、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N羟基丁二酰亚胺,在MES缓冲液中活化反应,即得。
进一步地,所述透明质酸、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N羟基丁二酰亚胺的质量比为42~43:2~2.5:1.4;所述透明质酸和MES缓冲液的质量体积比为20~45:25(mg/mL);和/或,所述活化反应的反应条件为室温中反应2h;
优选地,所述透明质酸、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N羟基丁二酰亚胺的质量比为42.3~42.4:2.2~2.3:1.4;所述透明质酸和MES缓冲液的质量体积比为21.2~42.3:25(mg/mL)。
进一步地,
步骤(2)中,所述反应为氮气保护下室温反应45~50h,优选为48h;
和/或,步骤(3)中,所述反应为室温反应45~50h,优选为48h;
和/或,步骤(4)中,所述反应为室温反应45~50h,优选为48h。
进一步地,
步骤(2)中,所述反应后对产物A进行纯化,所述纯化为透析;
和/或,步骤(3)中,所述反应后对产物B进行纯化,所述纯化为透析;
和/或,步骤(4)中,所述反应后对产物C进行纯化,所述纯化为透析,然后冻干;
优选地,
步骤(2)中,所述透析为将产物A置于1000Da的透析袋中,在PBS溶液中透析48h;
和/或,步骤(3)中,所述透析为将产物B置于3500Da透析袋中,在PBS溶液中透析48h;
和/或,步骤(4)中,所述透析为将产物C置于3500Da透析袋中,在PBS溶液中透析48h。
本发明还提供了前述的多功能外壳材料PMTPH在制备基因载体中的用途。
进一步地,所述多功能外壳材料PMTPH作为基因载体的外壳材料。
进一步地,所述基因载体为阳离子聚合物非病毒基因载体。
本发明中,室温是指25±5℃,过夜是指12±2h。
本发明非病毒外壳材料PMTPH与载基因的阳离子聚合物基因载体结合后,能够屏蔽其正电荷,使基因载体带有弱负电荷,带弱负电荷的基因载体能够通过静脉注射用于体内治疗,克服了现有阳离子聚合物基因载体与血液中的负电蛋白结合,造成溶血等强毒副作用的问题;同时,与该外壳材料结合后,阳离子聚合物基因载体的转染效率不会降低。此外,本发明非病毒外壳材料PMTPH没有免疫原性,安全性好;能够对肿瘤微环境的酶敏感产生响应,更有利于肿瘤细胞吞噬;该外壳材料带有穿膜肽Cys-TATs,更加有利于携带基因进入细胞内。本发明外壳材料PMTPH在制备用于体内治疗的基因载体中具有良好的应用前景。
显然,根据本发明的上述内容,按照本领域的普通技术知识和惯用手段,在不脱离本发明上述基本技术思想前提下,还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式,对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1为本发明非病毒外壳材料PMTPH及其原料的红外光谱图(A)和核磁共振氢谱图(B)。
图2为本发明非病毒外壳材料PMTPH对HEK293细胞和HELA细胞生长的影响。
图3为不同条件下本发明非病毒外壳材料PMTPH的粒径和zeta电位。
图4为不同纳米粒的的TEM形态图;A代表PF34纳米粒的透射电镜图,B代表PMTPHC纳米粒的透射电镜图,C代表PMTPHC纳米粒经过基质金属蛋白酶消化后产生的纳米粒的透射电镜图,D代表PMTPHC纳米粒经过透明质酸酶消化后产生的纳米粒的透射电镜图。
图5为各基因载体的转染效率。
具体实施方式
本发明具体实施方式中使用的原料、设备均为已知产品,通过购买市售产品获得。主要材料如下:
NH2-MMP9-Cys:基质金属蛋白酶9(上海强耀生物有限公司);
MPEG2000-maleimide:分子量为2000的甲氧基聚乙二醇马来酰亚胺(上海拓旸生物有限公司);
HA:透明质酸(山东福瑞达生物科技有限公司);
EDCI:1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(上海麦克林生化科技有限公司);
NHS:N羟基丁二酰亚胺(上海麦克林生化科技有限公司);
NH2-PEG600-maleimide:分子量为600的氨基聚乙二醇马来酰亚胺(上海拓旸生物有限公司);
穿膜肽Cys-TATs:氨基酸序列为YGRKKRRQRRRC(上海强耀生物有限公司)。
实施例1、本发明非病毒外壳材料PMTPH的制备
第一步:将13.6mg NH2-MMP9-Cys和33.5mgMPEG2000-maleimide溶于4mL的PBS溶液中,在氮气保护下室温反应48h;将产物置于1000Da的透析袋中,在PBS溶液中透析48h。
第二步:将42.3mg透明质酸、2.3mg EDCI和1.4mg NHS溶于25mL MES缓冲液中,室温中活化透明质酸上的羧基2h。
第三步:将第一步制备得到的产物和6.7mg NH2-PEG600-maleimide加入42.3mg第二步羧基被活化后的透明质酸中,室温反应48h,将产物置于3500Da的透析袋中,在PBS溶液中透析48h。
第四步:将18.6mg Cys-TATs加入第三步的产物中,室温反应48h,将最终的产物置于3500Da的透析袋中,在PBS溶液中透析48h,冻干,即得本发明非病毒外壳材料PMTPH(聚乙二醇2000-MMP9/TATS-PEG600-HA)。
实施例2、本发明非病毒外壳材料PMTPH的制备
第一步:将13.6mg NH2-MMP9-Cys和33.5mgMPEG2000-maleimide溶于4mL的PBS溶液中,在氮气保护下室温反应48h;将产物置于1000Da的透析袋中,在PBS溶液中透析48h。
第二步:将21.2mg透明质酸、1.1mg EDCI和0.7mg NHS溶于25mL MES缓冲液中,室温中活化透明质酸上的羧基2h。
第三步:将第一步制备得到的产物和6.7mg NH2-PEG600-maleimide加入第二步活化后的42.3mg透明质酸中,室温反应48h,将产物置于3500Da的透析袋中,在PBS溶液中透析48h。
第四步:将18.6mgCys-TATp加入第三步的产物中,室温反应48h,将最终的产物置于3500Da的透析袋中,在PBS溶液中透析48h,冻干,即得本发明非病毒外壳材料PMTPH(聚乙二醇2000-MMP9/TATS-PEG600-HA)。
以下通过具体的试验例证明本发明的有益效果。
试验例1、本发明非病毒外壳材料PMTPH的结构表征
采用傅立叶变换红外光谱(FTIR)和核磁共振氢谱(1H NMR)对本发明实施例1制备的非病毒外壳材料PMTPH及其原料的化学结构进行确认,结果如图1所示。
由图1可知:经过比对特征峰在图中的特定位置(羧基:3442cm-1;醚键:1162cm-1,1075cm-1;胍基:1636cm-1),本发明成功合成了非病毒外壳材料PMTPH。
试验例2、本发明非病毒外壳材料PMTPH的细胞毒性验证
用MTT实验检测PMTPH的细胞毒性。将处于对数期的人正常细胞系HEK293(293T)细胞和HELA细胞铺于96孔板中(细胞密度为5000个/孔),培养过夜使细胞完全贴壁。用不同浓度(0,3,6,12.5,25,50,100和200μg/mL)的本发明实施例1制备的非病毒外壳材料PMTPH处理HEK293细胞48小时,用不同浓度(0,2,5,12.5,25,50,100和200μg/mL)的本发明实施例1制备的非病毒外壳材料PMTPH处理HELA细胞48小时。然后用每孔加入20μL的MTT(5mg/m L),继续培养4小时。最后弃去培养基,每孔加入150μL DMSO溶解甲瓒沉淀,在570nm出测定吸光度。每个实验做3个复孔。PMTPH对HEK293细胞和HELA细胞活性的影响如图2所示。
由图2可知:即便使用高浓度的PMTPH,细胞活性也可以达到80%以上,说明PMTPH对细胞毒性非常低。
试验例3、本发明非病毒外壳材料PMTPH多功能性验证
1、阳离子基因载体PF34的制备
按照专利CN106188537A中记载的方式(实施例1)制备阳离子基因载体PF34。
2、本发明非病毒外壳材料PMTPH多功能性验证
将阳离子基因载体PF34和质粒(EGFP质粒)以2:1的质量比混合,孵育20分钟,形成PF34/pDNA复合物。然后在PF34/pDNA复合物中分别加入本发明实施例1制备的PMTPH(PMTPH和PF34/pDNA的质量比为20:1)和透明质酸钠(HA和PF34/pDNA的质量比为20:1),培养25分钟,分别形成PMTPHC和HAC。通过动态光散射(DLS)分别检测PF34/pDNA复合物、PMTPHC和HAC的粒径分布和zeta电位,并用透射电子显微镜(TEM)观察其形貌特征。
PF34/pDNA复合物、PMTPHC和HAC的粒径分布和zeta电位结果分别如图3a和图3b所示。试验结果表明:PF34/pDNA复合物、HAC和PMTPHC的粒径分别为64±8nm、109±6nm和177±2nm(见图3a);PF34/pDNA复合物、HAC和PMTPHC的zeta电位分别为+15mV、-39mV和-33mV(见图3b)。可见,本发明外壳材料PMTPH可以有效屏蔽阳离子基因载体的负电荷。
3、模拟MMP-9酶和HAase(透明质酸酶)高表达的肿瘤微环境:
在PMTPHC中加入5mL浓度为3.5μg/ml的MMP-9酶溶液,PMTPHC的浓度为1mg/ml,25℃共孵育2小时。通过酶降解的方式,对PMTPHC最外层进行剪切,形成TPHC。检测TPHC(PMTPHC+MMP-9)的粒径分布和zeta电位。结果如图3c和3d所示。
在PMTPHC中加入2mL浓度为100μg/ml的HAase溶液,PMTPHC的浓度为1mg/ml,25℃共孵育2小时。对孵育后的PMTPHC(PMTPHC+HAase)的粒度分布、zeta电位和形态特征进行了分析。结果如图3c、图3d和图4D所示。
试验结果表明:在MMP-9酶溶液中培养后,PMTPHC的粒径从177±2nm减小到138±3nm(n=3),PMTPHC的zeta电位从-33mV变为-34mV(n=3)。在HAase溶液中培养后,PMTPHC的粒径从177±2nm减小到72±2nm(n=3),PMTPHC的zeta电位从-33mV变为+24mV(n=3)。
粒径逐渐变小,更有利于肿瘤细胞的内吞,电位变化,有利于促进细胞内吞和促进内涵体/溶酶体逃逸,保护质粒在溶酶体中不被降解,释放入胞浆,进一步入核发挥基因编辑作用。试验结果表明本发明外壳材料PMTPH修饰的基因载体具有逐步多层释放的功能。
阳离子聚合物带大量正电荷,不能用于静脉注射。在外面加一层PMTPH外壳后能屏蔽内核的正电荷,带上负电荷,能够用于体内静脉注射给药。PMTPH外壳具有多功能性,能够在肿瘤内高浓度MMP9酶作用下,进行第一步降解断裂,暴露出穿膜肽Cys-TATs,促进细胞内吞。在血液循环中,未到达肿瘤部位之前,能够有效遮蔽穿膜肽Cys-TATs,避免穿膜肽Cys-TATs对其他正常细胞的穿膜,导致毒副作用。
试验例4、本发明非病毒外壳材料PMTPH作为基因载体外壳后该基因载体在黑色素瘤细胞株中的转染效率
研究本发明非病毒外壳材料PMTPH(实施例1制备的非病毒外壳材料PMTPH)作为基因载体外壳后该基因载体在无血清条件下在黑色素瘤细胞株B16F10中的转染效率。
1、试验方法
在六孔板中分别加入含有2×105个B16F10细胞的2mL DMEM培养基(含10%胎牛血清)培养过夜。第二天进行换液,加入DMEM无血清培养基,然后每个孔分别加入100μL的本发明非病毒外壳材料PMTPH作为基因载体外壳的PF34质粒复合物(质粒和PF34的比例采用最佳转染比例),PF34质粒复合物(质粒和PF34的比例采用最佳转染比例),PEI 10K质粒复合物(质粒和PEI 10K的比例采用最佳转染比例),PEI 25K质粒复合物(质粒和PEI25K的比例采用最佳转染比例),培养5-6小时后进行换液,更换成新的DMEM培养基(含10%胎牛血清),24小时后用流式细胞仪进行分析。
2、试验结果
本发明非病毒外壳材料PMTPH作为基因载体外壳后该基因载体在黑色素瘤细胞株中的转染效率如图5所示。由图5可知:PEI 10K质粒复合物对黑色素瘤基本无转染效率,PEI25K质粒复合物对黑色素瘤的转染效率在约40%,而相比于PEI 25K,PF34质粒复合和本发明非病毒外壳材料PMTPH作为基因载体外壳的PF34质粒复合物对黑色素瘤的转染效率均能达到约90%,能够明显提高材料对黑色素瘤的转染效率。试验结果说明增加本发明外壳材料PMTPH后不会降低阳离子基因载体的转染效率。
综上,本发明非病毒外壳材料PMTPH与载基因的阳离子聚合物基因载体结合后,能够屏蔽其正电荷,使基因载体带有弱负电荷,带弱负电荷的基因载体能够通过静脉注射用于体内治疗,克服了现有阳离子聚合物基因载体与血液中的负电蛋白结合,造成溶血等强毒副作用的问题;同时,与该外壳材料结合后,阳离子聚合物基因载体的转染效率不会降低。此外,本发明非病毒外壳材料PMTPH没有免疫原性,安全性好;能够对肿瘤微环境的酶敏感产生响应,更有利于肿瘤细胞吞噬;该外壳材料带有穿膜肽Cys-TATs,更加有利于携带基因进入细胞内。本发明外壳材料PMTPH在制备用于体内治疗的基因载体中具有良好的应用前景。
SEQUENCE LISTING
<110> 四川大学华西医院
<120> 一种多功能外壳材料PMTPH及其制备方法和用途
<130> GYKH1094-2019P016582CC
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 12
<212> PRT
<213> 人工序列(artificial sequence)
<400> 1
Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Cys
1 5 10
Claims (11)
1.一种多功能外壳材料PMTPH,其特征在于:它是由以下重量配比的原料制备而成: 基质金属蛋白酶9 13.6份,分子量为2000的甲氧基聚乙二醇马来酰亚胺33.5份,20%羧基被活化后的透明质酸42.3份,分子量为600的氨基聚乙二醇马来酰亚胺6.7份,穿膜肽18.6份;所述穿膜肽为穿膜肽Cys-TATs,其氨基酸序列为YGRKKRRQRRRC;
所述制备包括如下步骤:
(1)称取各原料;
(2)将所述基质金属蛋白酶9和分子量为2000的甲氧基聚乙二醇马来酰亚胺溶于PBS溶液中,氮气保护下室温反应48h,得产物A;所述PBS溶液与基质金属蛋白酶9的体积质量比为4:13.6(mL/mg);
(3)将步骤(2)所得产物A、分子量为600的氨基聚乙二醇马来酰亚胺加入20%羧基被活化后的透明质酸中,室温反应48h,得产物B;
(4)将所述穿膜肽加入步骤(3)所得产物B中室温反应48h,得产物C,即得。
2.根据权利要求1所述的多功能外壳材料PMTPH,其特征在于:
步骤(2)中,所述反应后对产物A进行纯化,所述纯化为透析;
和/或,步骤(3)中,所述反应后对产物B进行纯化,所述纯化为透析;
和/或,步骤(4)中,所述反应后对产物C进行纯化,所述纯化为透析,然后冻干。
3.根据权利要求2所述的多功能外壳材料PMTPH,其特征在于:
步骤(2)中,所述透析为将产物A置于1000Da的透析袋中,在PBS溶液中透析48h;
和/或,步骤(3)中,所述透析为将产物B置于3500Da透析袋中,在PBS溶液中透析48h;
和/或,步骤(4)中,所述透析为将产物C置于3500Da透析袋中,在PBS溶液中透析48h。
4.根据权利要求1~3任一项所述的多功能外壳材料PMTPH,其特征在于:所述20%羧基被活化后的透明质酸的制备方法为:
取透明质酸、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N羟基丁二酰亚胺,在MES缓冲液中活化反应,即得;所述透明质酸、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N羟基丁二酰亚胺的质量比为42.3~42.4:2.2~2.3:1.4;所述透明质酸和MES缓冲液的质量体积比为21.2~42.3:25(mg/mL)。
5.一种权利要求1~4任一项所述的多功能外壳材料PMTPH的制备方法,其特征在于:它包括如下步骤:
(1)称取各原料;
(2)将所述基质金属蛋白酶9和分子量为2000的甲氧基聚乙二醇马来酰亚胺溶于PBS溶液中,氮气保护下室温反应48h,得产物A;所述PBS溶液与基质金属蛋白酶9的体积质量比为4:13.6(mL/mg);
(3)将步骤(2)所得产物A、分子量为600的氨基聚乙二醇马来酰亚胺加入20%羧基被活化后的透明质酸中,室温反应48h,得产物B;
(4)将所述穿膜肽加入步骤(3)所得产物B中室温反应48h,得产物C,即得。
6.根据权利要求5所述的制备方法,其特征在于:
步骤(2)中,所述反应后对产物A进行纯化,所述纯化为透析;
和/或,步骤(3)中,所述反应后对产物B进行纯化,所述纯化为透析;
和/或,步骤(4)中,所述反应后对产物C进行纯化,所述纯化为透析,然后冻干。
7.根据权利要求6所述的制备方法,其特征在于,步骤(2)中,所述透析为将产物A置于1000Da的透析袋中,在PBS溶液中透析48h;
和/或,步骤(3)中,所述透析为将产物B置于3500Da透析袋中,在PBS溶液中透析48h;
和/或,步骤(4)中,所述透析为将产物C置于3500Da透析袋中,在PBS溶液中透析48h。
8.根据权利要求5~7任一项所述的制备方法,其特征在于:所述20%羧基被活化后的透明质酸的制备方法为:
取透明质酸、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N羟基丁二酰亚胺,在MES缓冲液中活化反应,即得;所述透明质酸、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N羟基丁二酰亚胺的质量比为42.3~42.4:2.2~2.3:1.4;所述透明质酸和MES缓冲液的质量体积比为21.2~42.3:25(mg/mL)。
9.权利要求1~4任一项所述的多功能外壳材料PMTPH在制备基因载体中的用途。
10.根据权利要求9所述的用途,其特征在于:所述多功能外壳材料PMTPH作为基因载体的外壳材料。
11.根据权利要求10所述的用途,其特征在于:所述基因载体为阳离子聚合物非病毒基因载体。
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