CN114470237A - 一种类病毒结构基因载体、药物递送系统、其制备方法及其应用 - Google Patents
一种类病毒结构基因载体、药物递送系统、其制备方法及其应用 Download PDFInfo
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Abstract
本发明提供了一种类病毒结构基因载体,包括阳离子聚合物、接枝环糊精的聚(L‑谷氨酸)和带有金刚烷胺端基的亲水性聚合物,其用于负载基因药物时,带有负电的基因药物等与带有正电的阳离子聚合物通过静电作用形成阳离子内核;接枝有环糊精的聚(L‑谷氨酸)包裹在所述阳离子内核表面形成遮蔽层,能够稳定内核;亲水性聚合物的金刚烷胺端基通过金刚烷与环糊精的主客体作用装配到粒子表面,更好地保护内核,避免基因药物被降解,同时亲水性化合物可以赋予药物递送系统其他特性该类病毒结构的基因载体具有高转染效率且结构简单,可广泛用于担载DNA、mRNA、siRNA、microRNA等。本发明还提供了一种药物递送系统、其制备方法及其应用。
Description
技术领域
本发明涉及生物医药技术领域,具体是一种类病毒结构基因载体、药物递送系统、其制备方法及其应用。
背景技术
基因治疗是一种很有前景的治疗方法,主要是通过将基因转入人体细胞中来修复或替换致病基因而实现对疾病的治疗。基因治疗的关键问题是解决治疗核酸的递送问题,因为单独的基因若直接注入体内会被体液中的物质和酶类快速的降解和破坏,且负电性的大分子DNA自身很难穿过细胞膜进入到细胞中。因此,需要借助基因载体包载目的基因从而完成运输到靶细胞以及进入细胞核转染的过程。
目前,核酸的主要担载载体分为病毒载体和非病毒载体。病毒载体包括腺病毒、腺相关病毒等类型,其基因转染效率高,但其制备比较困难、基因容量小、特异性差、免疫反应较强、且存在较高的安全隐患。非病毒载体具有容量大、免疫反应性低、安全性高等优点,通常采用的非病毒核酸递送载体为阳离子脂质体或阳离子聚合物,其能够与带有负电荷的核酸经静电作用复合,形成具有一定尺寸的纳米颗粒。例如,阳离子脂质体纳米颗粒(LNP)、多种阳离子高分子聚合物如聚(L-赖氨酸)、聚乙烯亚胺、聚酰胺和聚(β-氨基酯)等常被用于基因转染,表现出出色的基因转染效果。然而,阳离子-基因复合物这种静电形成的复合物稳定性差、存在一定的毒性、且无体内靶向性,体内转染受到极大限制。
一种解决思路是采用外壳对阳离子-基因复合物内核进行遮蔽。例如,现有技术CN109152830A公开了一种脂质双层遮蔽的核壳结构mRNA递送载体,其能够稳定内核并促进抗原呈递细胞对复合物的巨胞饮作用;然而,脂质双层包覆的复合物静脉注射后会大量在肝脏部位聚积,无法实现对其他器官的靶向输送。现有技术CN 105906800A公开了一种含有pH敏感可断裂的遮蔽材料的基因传递系统,利用聚乙二醇对内核复合物进行遮蔽;然而,这一遮蔽设计的稳定性不佳,进入体内后易脱落。现有技术CN 112641952A公开了一种含有聚乙二醇和聚谷氨酸共聚物的遮蔽层的核壳结构基因递送系统,能够在体内获得显著的转染效率;然而,这一遮蔽层难以添加靶向基团,基因转染的靶向性差。综上,能够实现稳定遮蔽并具有良好体内靶向性的非病毒基因递送载体仍然匮乏。
发明内容
有鉴于此,本发明要解决的技术问题在于提供一种类病毒结构基因载体、药物递送系统、其制备方法及其应用,本发明提供的类病毒结构基因载体稳定性好,且易于连接靶向基团,实现良好体内靶向性。
本发明提供了一种类病毒结构基因载体,包括阳离子聚合物、接枝环糊精的聚(L-谷氨酸)和带有金刚烷胺端基的亲水性聚合物,所述金刚烷胺端基包合于环糊精内部。
进一步的,所述类结构病毒基因载体中,部分亲水性聚合物还带有靶向基团端基,即所述类病毒结构基因载体包括阳离子聚合物、接枝环糊精的聚(L-谷氨酸)、带有金刚烷胺端基的亲水性聚合物和同时带有金刚烷胺端基和靶向基团端基的亲水性聚合物,所述金刚烷胺端基包合于环糊精内部。
本发明还提供了一种药物递送载体,包括基因药物和上文所述的类结构病毒基因载体。
参见图1,图1为本申请提供的药物递送载体的结构示意图。其中,带有负电的基因药物例如DNA等与带有正电的阳离子聚合物例如鱼精蛋白、聚乙烯亚胺等通过静电作用形成阳离子内核;接枝有环糊精的聚(L-谷氨酸)包裹在所述阳离子内核表面形成遮蔽层,能够稳定内核;亲水性聚合物的金刚烷胺端基通过金刚烷与环糊精的主客体作用装配到表面,更好地保护内核,避免基因药物,例如核酸等被降解,同时亲水性化合物可以赋予药物递送系统其他特性,例如增加其长循环特性等;同时,亲水性聚合物另一端的靶向基团具有靶向作用,能够提高药物递送系统的靶向性,例如胺乙基茴香酰胺可以靶向肿瘤细胞,能够提高纳米粒子对肿瘤细胞的特异性摄取。
在一个实施例中,所述阳离子聚合物选自鱼精蛋白、聚乙烯亚胺或其衍生物、聚β氨酯或其衍生物、聚α-氨基酯、聚(L-赖氨酸)或其衍生物以及上述聚合物的混合物。在一个实施例中,所述阳离子聚合物选自鱼精蛋白、聚乙烯亚胺、聚β氨酯、聚α-氨基酯、聚(L-赖氨酸)以及上述聚合物的混合物。在一个实施例中,所述阳离子聚合物选自鱼精蛋白和/或聚乙烯亚胺。在一个实施例中,所述阳离子聚合物选自鱼精蛋白和聚乙烯亚胺。在一个实施例中,所述鱼精蛋白和聚乙烯亚胺的质量比为1~2:1~2。在一个实施例中,所述鱼精蛋白和聚乙烯亚胺的质量比为1:1。
在一个实施例中,所述接枝环糊精的聚(L-谷氨酸)中,L-谷氨酸的重复单元数为20~200。在一个实施例中,L-谷氨酸的重复单元数为60~140。在一个实施例中,L-谷氨酸的重复单元数为80~120。在一个实施例中,环糊精的接枝率为10%~50%;在一个实施例中,环糊精的接枝率为20%~30%。
在一个实施例中,所述亲水性聚合物选自聚乙二醇或其衍生物、聚甲基噁唑啉或其衍生物和聚乙基噁唑啉或其衍生物中的至少一种。在一个实施例中,所述亲水性聚合物选自聚乙二醇、聚甲基噁唑啉和聚乙基噁唑啉中的至少一种。
在一个实施例中,所述亲水性聚合物的一端为金刚烷胺端基,另一端可以选择性地修饰靶向基团端基。在一个实施例中,所述类病毒结构基因载体中包含带有金刚烷胺端基的亲水性聚合物和同时带有金刚烷胺端基和靶向基团端基的亲水性聚合物。在一个实施例中,带有金刚烷胺端基的亲水性聚合物和同时带有金刚烷胺端基和靶向基团端基的亲水性聚合物的质量比为1~2:1~2。在一个实施例中,带有金刚烷胺端基的亲水性聚合物和同时带有金刚烷胺端基和靶向基团端基的亲水性聚合物的质量比为1:1。
在一个实施例中,所述靶向基团为胺乙基茴香酰胺或其衍生物、叶酸或其衍生物、CD3、CD8、CD5抗体或其片段中的至少一种。
在一个实施例中,所述类病毒结构基因载体中,阳离子聚合物、接枝环糊精的聚(L-谷氨酸)和亲水性聚合物的质量比为3~8:2:2~10。在一个实施例中,阳离子聚合物、接枝环糊精的聚(L-谷氨酸)和亲水性聚合物的质量比为5:2:2~10。
本发明提供的类病毒结构基因载体可以用于负载基因药物,因此,本申请进一步提供了一种药物递送系统,包括基因药物和上述技术方案所述的类病毒结构基因载体;
其中,所述阳离子聚合物和基因药物形成内核;
所述接枝环糊精的聚(L-谷氨酸)包裹在所述内核表面形成外壳;
所述金刚烷胺端基包合于所述环糊精内部,亲水性聚合物及其靶向基团端基修饰在表面作为靶头。
在一个实施例中,所述基因药物选自DNA、mRNA、microRNA或siRNA。
在一个实施例中,所述基因药物、阳离子聚合物、接枝环糊精的聚(L-谷氨酸)和亲水性聚合物的质量比为1:3~8:2:2~10。在一个实施例中,所述基因药物、阳离子聚合物、接枝环糊精的聚(L-谷氨酸)和亲水性聚合物的质量比为1:5:2:2~10。
本发明还提供了上述技术方案所述的药物递送系统的制备方法,包括:
分别提供基因药物溶液、阳离子聚合物溶液,接枝环糊精的聚(L-谷氨酸)溶液,带有金刚烷胺端基的亲水性聚合物溶液;
在涡旋条件下,将基因溶液与阳离子聚合物溶液混合,获得混合液A;
将混合液A涡旋、静置后加入接枝环糊精的聚(L-谷氨酸)溶液,再次涡旋、静置,得到混合液B;
将混合液B与带有金刚烷胺端基的亲水性聚合物溶液混合,静置后得到药物递送系统。
本发明提供的类病毒结构基因载体包括阳离子聚合物、接枝环糊精的聚(L-谷氨酸)和带有金刚烷胺端基的亲水性聚合物,其用于负载基因药物时,带有负电的基因药物例如DNA等与带有正电的阳离子聚合物例如鱼精蛋白、聚乙烯亚胺等通过静电作用形成阳离子内核;接枝有环糊精的聚(L-谷氨酸)包裹在所述阳离子内核表面形成遮蔽层,能够稳定内核;亲水性聚合物的金刚烷胺端基通过金刚烷与环糊精的主客体作用装配到粒子表面,更好地保护内核,避免基因药物,例如核酸等被降解,同时亲水性化合物可以赋予药物递送系统其他特性,例如增加其长循环特性等。进一步的,亲水性聚合物另一端可以选择性的修饰具有靶向作用的靶向基团,提高药物递送系统的靶向性,例如胺乙基茴香酰胺可以靶向肿瘤细胞,能够提高纳米粒子对肿瘤细胞的特异性摄取。该类病毒结构的基因载体具有高转染效率且结构简单,可广泛用于担载DNA、mRNA、siRNA、microRNA等。
本发明提供的基因载体具有核壳结构和靶头,阳离子聚合物与核酸复合形成内核,聚(L-谷氨酸)-接枝环糊精修饰在内核表面作为外壳,金刚烷胺-聚乙二醇、金刚烷胺-聚乙二醇-靶向基团等修饰在纳米粒子表面作为靶头。本发明提供的基因载体框架的基因转染效果良好、模块化分级组装制备条件简单温和、稳定性好,毒性较低,具有肿瘤细胞靶向能力,能够引起强烈的抗肿瘤免疫响应,进而使得由本发明基因载体框架制得的癌症疫苗对肿瘤生长具有显著的抑制效果。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。
图1为本申请提供的药物递送载体的结构示意图;
图2为实施例1得到的PLG-g-CD的1H NMR图谱;
图3为得到的Ad-PEG的1H NMR图谱;
图4为实施例5制备得到的不同装配比例的PEI+Protamine/pDNA/PLG-g-CD/Ad-PEG+Ad-PEG-AEAA的粒径;
图5为实施例10制备得到PEI+Protamine/pDNA/PLG-g-CD/Ad-PEG+Ad-PEG-AEAA的基因转染效率;
图6为实施例11的PEI+Protamine/OX40L pDNA/PLG-g-CD/Ad-PEG+Ad-PEG-AEAA用于B16F10、MC38、CT26、E0771、3T3细胞转染后的流式表达结果;
图7为实施例12的PEI+Protamine/OX40L pDNA/PLG-g-CD/Ad-PEG+Ad-PEG-AEAA用于B16F10肿瘤模型的体内转染结果;
图8为实施例13的PEI+Protamine/OX40L pDNA/PLG-g-CD/Ad-PEG+Ad-PEG-AEAA的用于MC38肿瘤模型的治疗结果;
图9为实施例14的PEI-4BImi/OX40L pDNA/PLG-g-CD/Ad-PEG+Ad-PEG-AEAA的用于MC38肿瘤模型的治疗结果。
具体实施方式
本发明公开了一种基因载体、药物递送系统、其制备方法及其应用。本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合,来实现和应用本发明技术。
以下结合实施例对本发明进行进一步阐述:
实施例1聚(L-谷氨酸)-接枝环糊精(PLG-g-CD)的制备
将300mg的PLG(具有约120个L-谷氨酸重复单元)、64mg的N,N-二异丙基乙胺(DIPEA)和184mg的2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸盐(HATU)共同溶于10mL的N,N-二甲基甲酰胺(DMF)溶液中,室温搅拌直至固体物质全部溶解,将900mg的氨基环糊精(CD-NH2)溶解于3mL的DMF中,加入到上述DMF混合液中,室温反应48h。产物使用无菌注射用水透析纯化,冻干得到终产物PLG-g-CD。
对所述PLG-g-CD进行分析,结果参见图2,图2为实施例1得到的PLG-g-CD的1H NMR图谱。
实施例2金刚烷胺-聚乙二醇(Ad-PEG)的制备
将120mg的聚乙二醇(PEG,分子量2000Da)、12mg的DIPEA和32mg的HATU共同溶解于3mL的DMF溶液中,将16mg的氨基金刚烷胺(Ad-NH2)溶解于4mL的DMF中,并加入到上述DMF反应体系在,室温搅拌下反应48h。产物使用无菌注射用水透析纯化,冻干得到终产物Ad-PEG。
对所述Ad-PEG进行分析,结果参见图3,图3为得到的Ad-PEG的1HNMR图谱。
实施例3金刚烷胺-聚乙二醇-氨基乙基茴香酰胺(Ad-PEG-AEAA)的制备
首先制备氨基乙基茴香酰胺(AEAA):将410mg的2-溴乙胺和800mg的DIPEA溶解于4mL的乙腈中、300mg的对甲氧基苯乙酰氯溶于3mL乙腈中,将上述两种溶液混合,室温搅拌下反应6h。
接下来在双功能PEG的一侧键合AEAA:向上述反应体系中加入200mg的羟基聚乙二醇氨基(HO-PEG-NH2),在80℃搅拌反应12h。
最后将金刚烷胺(Ad)键合到双功能PEG的另一端:将60mg的N,N’-羰基二咪唑(CDI)溶解于2mL的乙腈中并缓慢加入到上述混合溶液中,50℃反应5h从而激活CDI,160mg的Ad-NH2溶解于4mL DMF中并注入上述反应体系中,50℃下再搅拌48h。产物使用无菌注射用水透析纯化,冻干得到终产物Ad-PEG-AEAA。
实施例4金刚烷胺-聚乙二醇-抗CD3片段(Ad-PEG-aCD3F(ab’)2)的制备
首先将Ad键合到α,ω-二羧基聚乙二醇(HOOC-PEG-COOH)的一端:将200mg的HOOC-PEG-COOH,60mg的1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDC·HCl),36mg的N-羟基琥珀酰亚胺(NHS)共同溶于10mL的二甲基亚砜(DMSO)中,室温搅拌下活化30min,160mg的Ad-NH2溶解于4mL DMF中并注入上述反应体系中,室温搅拌下反应72h。
然后将aCD3F(ab’)2键合到HOOC-PEG-COOH的另一端:加入aCD3F(ab’)2抗体溶液(4:1摩尔比)室温反应6小时,产物使用无菌注射用水透析纯化,然后用40k Zeba离心柱过滤,冻干得到终产物Ad-PEG-aCD3F(ab’)2。
实施例5
PEI+Protamine/pDNA/PLG-g-CD/(Ad-PEG+Ad-PEG-AEAA)基因载体的制备
使用无菌注射用水溶解聚乙烯亚胺(PEI10k)和鱼精蛋白(Protamine)混合溶液(质量比1:1),浓度为0.25mg/mL,使用无菌注射用水溶解编码OX40L的质粒pDNA,使用无菌用水溶解PLG-g-CD,浓度为0.5mg/mL,使用无菌注射用水溶解Ad-PEG和Ad-PEG-AEAA混合溶液(质量比1:1),浓度为0.5mg/mL,将PEI10k、Protamine混合溶液与pDNA溶液混合,涡旋30s,静置5min后向体系中加入PLG-g-CD溶液,继续涡旋30s,静置5min,最后向体系中加入Ad-PEG和Ad-PEG-AEAA混合溶液,涡旋30s,静置5小时后制备得到PEI+Protamine/pDNA/PLG-g-CD/(Ad-PEG:Ad-PEG-AEAA)基因载体。以不同的质量比制备了一系列纳米颗粒(PEI+Protamine/pDNA/PLG-g-CD/Ad-PEG+Ad-PEG-AEAA=5/1/0/0,5/1/2/0,5/1/2/2、5/1/2/4、5/1/2/6、5/1/2/8、5/1/2/10),粒径使用马尔文粒度仪测量,结果参见图4,图4为实施例5制备得到的不同装配比例的PEI+Protamine/pDNA/PLG-g-CD/Ad-PEG+Ad-PEG-AEAA的粒径。
实施例6PEI衍生物(PEI-4BImi)的制备
将50mg的PEI(Mw=10kDa)溶于DMSO溶液中,将25mg的苯并咪唑-7-羧酸,60mg的EDC·HCl,36mg的NHS共同溶于10mL的DMSO中,室温搅拌,活化30min,加入到PEI的DMSO溶液中,室温反应72h。产物使用无菌注射用水透析纯化,冻干得到终产物PEI-4BImi。
实施例7
PEI-4BImi/pDNA/PLG-g-CD/(Ad-PEG+Ad-PEG-AEAA)基因载体的制备
使用无菌注射用水溶解PEI-4BImi,浓度为1mg/mL,使用无菌注射用水溶解编码OX40L的质粒pDNA,使用无菌用水溶解PLG-g-CD,浓度为0.25mg/mL,使用无菌注射用水溶解Ad-PEG和Ad-PEG-AEAA混合溶液(质量比1:1),浓度为1mg/mL,将2体积的pDNA加入到5体积的PEI-4BImi溶液中,涡旋30s,静置5min后向体系中加入8体积的PLG-g-CD溶液,继续涡旋30s,静置5min,最后向体系中加入体积为6的Ad-PEG和Ad-PEG-AEAA混合溶液,涡旋30s,静置5小时后制备得到PEI-4BImi/pDNA/PLG-g-CD/(Ad-PEG:Ad-PEG-AEAA)基因载体。使用马尔文粒度仪测量其粒径。
实施例8PEI-4BImi/pDNA/PLG-g-CD/Ad-POx基因载体的制备
使用无菌注射用水溶解PEI-4BImi,浓度为1mg/mL,使用无菌注射用水溶解编码OX40L的质粒pDNA,使用无菌用水溶解PLG-g-CD,浓度为0.25mg/mL,使用无菌注射用水溶解Ad-POx溶液,浓度为1mg/mL,将2体积的pDNA加入到5体积的PEI-4BImi溶液中,涡旋30s,静置5min后向体系中加入8体积的PLG-g-CD溶液,继续涡旋30s,静置5min,最后向体系中加入体积为6的Ad-POx溶液,涡旋30s,静置5小时后制备得到PEI-4BImi/pDNA/PLG-g-CD/Ad-POx基因载体。粒使用马尔文粒度仪测量其粒径。
实施例9
PEI-4BImi/mRNA/PLG-g-CD/(Ad-PEG:Ad-PEG-aCD3F(ab’)2)基因载体的制备
使用无菌注射用水溶解PEI-4BImi,浓度为1mg/mL,使用无菌注射用水溶解编码OX40L的mRNA,使用无菌用水溶解PLG-g-CD,浓度为0.25mg/mL,使用无菌注射用水溶解Ad-PEG和Ad-PEG-aCD3F(ab’)2混合溶液(质量比1:1),将2体积的pDNA加入到5体积的PEI-4BImi溶液中,涡旋30s,静置5min后向体系中加入8体积的PLG-g-CD溶液,继续涡旋30s,静置5min,最后向体系中加入体积为6的Ad-PEG和Ad-PEG-aCD3F(ab’)2混合溶液,涡旋30s,静置5小时后制备得到PEI-4BImi/mRNA/PLG-g-CD/(Ad-PEG:Ad-PEG-aCD3F(ab’)2)基因载体。并使用马尔文粒度仪测量其粒径。
实施例10
PEI+Protamine/pDNA/PLG-g-CD/(Ad-PEG+Ad-PEG-AEAA)基因载体的转染效率测定
黑色素瘤细胞B16F10接种于96孔板,密度为每孔1×105个细胞。按照不同的组分质量比制备了一系列纳米颗粒(PEI+Protamine/pDNA/PLG-g-CD/Ad-PEG+Ad-PEG-AEAA=5/1/0/0,5/1/2/0,5/1/2/2、5/1/2/4、5/1/2/6、5/1/2/8、5/1/2/10)分别加入到不同的孔中,每孔中加入的pDNA(荧光素酶质粒)相同,均为10μg/mL。孵育48小时后,弃去培养基液体,每孔加入50μL细胞裂解液,负80℃放置10min,使用荧光素酶报告基因检测试剂盒测定样品的荧光强度,结果参见图5,图5为实施例10制备得到PEI+Protamine/pDNA/PLG-g-CD/Ad-PEG+Ad-PEG-AEAA的基因转染效率。由图5可知结果显示比例为5/1/2/6的纳米粒子转染效果最佳,其转染效率是PEI25k(基因转染的“黄金标准”)的1.5倍。
实施例11
PEI+Protamine/pDNA/PLG-g-CD/(Ad-PEG+Ad-PEG-AEAA)纳米粒子用于体外OX40L转染测定
B16F10,MC38,CT26,E0771和3T3细胞接种在12孔板中,密度为每孔5×104个细胞。PBS,比例为5/1/2/6的PEI+Protamine/pDNA/PLG-g-CD/(Ad-PEG+Ad-PEG-AEAA)分别加入到不同的孔中,每孔中加入的pDNA(OX40L质粒)相同,均为5μg/mL。在恒温培养箱孵育48小时后,用胰蛋白酶消化贴壁细胞,使用抗鼠的APC-OX40L流式抗体进行染色后进行流式分析。所有的操作均在冰上完成。结果参见图6,图6为实施例11的PEI+Protamine/OX40L pDNA/PLG-g-CD/Ad-PEG+Ad-PEG-AEAA用于B16F10、MC38、CT26、E0771、3T3细胞转染后的流式表达结果。由图6可知,纳米粒子PEI+Protamine/pDNA/PLG-g-CD/(Ad-PEG+Ad-PEG-AEAA)可以成功转染多种细胞表达OX40L蛋白,证明本发明的基因载体框架可以成功地负载质粒基因并实现其表达。
实施例12
PEI+Protamine/pDNA/PLG-g-CD/(Ad-PEG+Ad-PEG-AEAA)/纳米药物用于B16F10肿瘤模型的体内转染分析
实施例中所使用的纳米药物是由实施例5制备得到比例为5/1/2/6的PEI+Protamine/pDNA/PLG-g-CD/(Ad-PEG+Ad-PEG-AEAA)将1×106个细胞注射到C57BL/6小鼠右侧,建立皮下B16F10黑色素瘤模型。当肿瘤体积达到200mm3左右时,将小鼠随机分为3组,瘤内给药1)PPD(OX40L),2)PEI25k(OX40L),3)PPD(GFP)。pDNA的单次给药剂量为5μg每只小鼠(PPD为PEI+Protamine/pDNA/PLG-g-CD/(Ad-PEG+Ad-PEG-AEAA)的简写)。48h和96h后,分别将小鼠安乐死,获取其肿瘤组织,研磨并过滤以获得单细胞悬液。然后将单细胞悬液用APC-OX40L荧光偶联抗体进行染色后进行流式分析。所有的操作均在冰上完成。结果参见图7,图7为实施例12的PEI+Protamine/OX40L pDNA/PLG-g-CD/Ad-PEG+Ad-PEG-AEAA用于B16F10肿瘤模型的体内转染结果;由图7可知,本发明的基因载体框架PPD(OX40L)在给药后48h和96h在肿瘤组织中均有较强的OX40L蛋白表达,分别为11.1%和8.5%。
实施例13
PEI+Protamine/pDNA/PLG-g-CD/(Ad-PEG+Ad-PEG-AEAA)纳米药物用于MC38模型的抗肿瘤分析
实施例中所使用的纳米药物是由实施例5制备得到比例为5/1/2/6的PEI+Protamine/pDNA/PLG-g-CD/(Ad-PEG+Ad-PEG-AEAA)在MC38结肠癌模型的抗肿瘤分析中,6到8周龄的雌性C57小鼠皮下注射1×106个MC38肿瘤细胞。当肿瘤体积达到约80mm3左右时,将小鼠随机分为3组1)PBS,2)PPD(OX40L),3)aOX40组(PPD为PEI+Protamine/pDNA/PLG-g-CD/(Ad-PEG+Ad-PEG-AEAA)的简写)。在第0,4,8和12天,PPD(OX40L)组小鼠接受瘤内注射治疗。pDNA的单次给药剂量为5μg每只小鼠,aOX40组小鼠接受腹腔给药治疗,aOX40的单次给药剂量为40μg每只小鼠。肿瘤体积每两天测量一次,肿瘤体积计算公式为V=a×b2×0.5,其中a为肿瘤的长度,b为肿瘤的宽度。结果参见图8,图8为实施例13的PEI+Protamine/OX40L pDNA/PLG-g-CD/Ad-PEG+Ad-PEG-AEAA的用于MC38肿瘤模型的治疗结果;如图8所述,基因载体PPD(OX40L)治疗组的肿瘤抑制率达到78.1%,与高剂量的aOX40(肿瘤抑制率=72.4%)治疗效果相似。
实施例14
PEI-4BImi/pDNA/PLG-g-CD/(Ad-PEG+Ad-PEG-AEAA)纳米药物用于MC38模型的抗肿瘤分析
实施例中所使用的纳米药物是由实施例7制备得到的PEI-4BImi/OX40LpDNA/PLG-g-CD/(Ad-PEG+Ad-PEG-AEAA)。在MC38结肠癌模型的抗肿瘤分析中,6到8周龄的雌性C57小鼠皮下注射1×106个MC38肿瘤细胞。当肿瘤体积达到约80mm3左右时,将小鼠随机分为4组:1)PBS组,2)PEI10k/pDNA@PDAA,3)PEI-4BImi/pDNA@PDAA(15μg),4)PEI-4BImi/pDNA@PDAA(30μg)(@PDAA表示PLG-g-CD/Ad-PEG+Ad-PEG-AEAA外壳)。在第0,5和10天,每组小鼠接受瘤内注射治疗。治疗组2)和治疗组3)的pDNA的单次给药剂量为15μg每只小鼠,治疗组4)的pDNA给药剂量为每只30μg。肿瘤体积每两天测量一次,肿瘤体积计算公式为V=a×b2×0.5,其中a为肿瘤的长度,b为肿瘤的宽度。结果参见图9,图9为实施例14的PEI-4BImi/OX40L pDNA/PLG-g-CD/Ad-PEG+Ad-PEG-AEAA的用于MC38肿瘤模型的治疗结果。如图9所示,与PBS组相比,PEI10k/pDNA@PDAA,PEI-4BImi/pDNA@PDAA(15μg)和PEI-4BImi/pDNA@PDAA(30μg)均具有良好的抗肿瘤作用,肿瘤抑制率分别为58.1%、77.6%和81.5%。
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,根据本发明的技术方案及其发明构思加以等同替换或改变,都应涵盖在本发明的保护范围之内。
Claims (10)
1.一种类病毒结构基因载体,包括阳离子聚合物、接枝环糊精的聚(L-谷氨酸)和带有金刚烷胺端基的亲水性聚合物,所述金刚烷胺端基包合于环糊精内部。
2.根据权利要求1所述的类病毒结构基因载体,其特征在于,部分亲水性聚合物还带有靶向基团端基。
3.根据权利要求2所述的类病毒结构基因载体,其特征在于,带有金刚烷胺端基的亲水性聚合物和同时带有金刚烷胺端基和靶向基团端基的亲水性聚合物的质量比为1~2:1~2。
4.根据权利要求2所述的类病毒结构基因载体,其特征在于,所述靶向基团为胺乙基茴香酰胺或其衍生物、叶酸或其衍生物、CD3、CD8、CD5抗体或其片段中的至少一种。
5.根据权利要求1所述的类病毒结构基因载体,其特征在于,所述阳离子聚合物选自鱼精蛋白、聚乙烯亚胺或其衍生物、聚β氨酯或其衍生物、聚α-氨基酯、聚(L-赖氨酸)或其衍生物以及上述聚合物的混合物;
所述接枝环糊精的聚(L-谷氨酸)中,L-谷氨酸的重复单元数为20~200,环糊精的接枝率为10%~50%;
所述亲水性聚合物选自聚乙二醇或其衍生物、聚甲基噁唑啉或其衍生物和聚乙基噁唑啉或其衍生物中的至少一种。
6.根据权利要求1~5任意一项所述的类病毒结构基因载体,其特征在于,阳离子聚合物、接枝环糊精的聚(L-谷氨酸)和亲水性聚合物的质量比为3~8:2:2~10。
7.一种药物递送系统,包括基因药物和权利要求1~6任意一项所述的类病毒结构基因载体;
所述阳离子聚合物和基因药物形成内核;
所述接枝环糊精的聚(L-谷氨酸)包裹在所述内核表面;
所述金刚烷胺端基包合于所述环糊精内部。
8.根据权利要求7所述的药物递送系统,其特征在于,所述基因药物选自DNA、mRNA、microRNA或siRNA;
所述基因药物、阳离子聚合物、接枝环糊精的聚(L-谷氨酸)和亲水性聚合物的质量比为1:3~8:2:2~10。
9.权利要求7~8任意一项所述药物递送系统的制备方法,包括:
分别提供基因药物溶液、阳离子聚合物溶液,接枝环糊精的聚(L-谷氨酸)溶液,带有金刚烷胺端基的亲水性聚合物溶液;
在涡旋条件下,将基因溶液与阳离子聚合物溶液混合,获得混合液A;
将混合液A涡旋、静置后加入接枝环糊精的聚(L-谷氨酸)溶液,再次涡旋、静置,得到混合液B;
将混合液B与带有金刚烷胺端基的亲水性聚合物溶液混合,静置后得到药物递送系统。
10.权利要求1~6任一项所述的类病毒结构基因载体在负载DNA、mRNA、microRNA或siRNA中的应用。
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