CN115054704A - 一种纳米组合物、制备方法及应用 - Google Patents
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Abstract
本发明公开了一种纳米组合物、制备方法及应用,涉及肿瘤治疗技术领域。其包括内核和静电吸附于内核外周的外壳,内核包括腺相关病毒载体,外壳的包裹使得纳米组合物整体具有免疫逃逸的结果,并且能够延长其在血液中的循环时间,从而避免了裸露的病毒注射到人体内由于中和抗体的存在降低了转染效果,有利于提升基因治疗效果。另外,发明人在外壳上连接有靶向剂,使得整个纳米组合物实现对目标组织或细胞的靶向效果。本发明提供的纳米组合物可以满足静脉注射的需求,避免了原位注射对患者带来创伤性手术的损伤和风险,无创性的注射能够更好的利用腺相关病毒优秀的转染效率。
Description
技术领域
本发明涉及肿瘤治疗技术领域,具体而言,涉及一种纳米组合物、制备方法及应用。
背景技术
腺相关病毒(Adeno-associated virus,AAV)是微小病毒科(Parvoviridae)家族的成员之一,是一类微小的,无被膜及其具有二十面体结构的病毒。病毒颗粒直径在20-26nm之间,含有大小在4.70kb左右的线状单链DNA(single-stranded DNA,ssDNA)。腺相关病毒的基因组全长在4.70kb左右,两端为145bp的反向末端重复序列(inverted terminalrepeat,ITR)。在ITR序列中间有两个开放阅读框(open reading frame,ORF),左侧的ORF编码4种Rep蛋白,右侧ORF编码3种Cap蛋白,嵌入部分1种组装激活蛋白(assembly-activating protein,AAP)。
基因组中的ITR序列是AAV病毒的复制、整合、拯救和包装所必需的顺式作用元件,同时还具有转录启动子的活性。
而Rep基因编码至少4种非结构蛋白:Rep78,Rep68,Rep52,Rep40。其中Rep78和Rep68是多功能蛋白,与AAV基因表达的正负调控有关。Rep52和Rep40参与了病毒的装配,它们在病毒双链DNA(dsDNA)合成中是不需要的,但在ssDNA和病毒颗粒的累积中则是必需的。在没有副辅助病毒和刺激因素的条件下,Rep蛋白只有微量的表达,这种表达能负反馈抑制其基因的进一步表达。Rep蛋白不仅与复制有关,还与AAV基因表达的自我调控、异原性启动子的抑制、癌基因放大的抑制等有关。
而Cap基因编码衣壳蛋白,编码三个衣壳蛋白,分别是VP1,VP2,VP3。这三个蛋白在成熟病毒颗粒中的比例约为1∶1∶10。病毒二十面体蛋白外壳每个面由3个衣壳蛋白分子组成,可以全部为VP3,也可以由VP1,VP2,VP3共同组成。
AAP主要定位于细胞核内,不但能帮助VPs进入细胞核,并可能为VPs装配提供支架,或协助VPs进行正确的折叠。已有研究表明没有AAP病毒蛋白则不能组装成病毒颗粒。
AAV的生活周期如下:AAV在培养正常细胞中不发生产毒性感染。以前病毒的形式整合到宿主细胞上,病毒DNA随宿主细胞染色体一起复制。AAV的另一种生活周期是在有辅助病毒感染时发生的,辅助病毒的感染可以在AAV感染之前、同时或之后进行。此时发生产毒性感染,AAV在宿主细胞内复制、装配并释放到细胞外。但某些损伤基因的因素如紫外线照射、γ射线照射等处理某些哺乳动物细胞,可以使AAV在无辅助病毒存在的情况下能够发生产毒性感染。所以说AAV不是真正意义上的缺陷性病毒。
重组腺相关病毒(recombination adeno-associated virus,rAAV)是在非致病性的野生型AAV基础上进一步改造而成的新型基因载体,由于其安全性好、宿主细胞范围广、免疫原性低、在体内表达外源基因的时间长等特点,在基因治疗的过程中被当作载体使用。将基因包封于腺相关病毒中,注射到人体中,腺相关病毒感染靶细胞基因释放到细胞中,表达出受体蛋白和分泌蛋白。从而达到对相关疾病的基因治疗效果。
目前,由于腺相关病毒在80%的成年人体内都存在中和抗体,当裸露的病毒注射到人体内时,会因为预先存在的中和抗体的作用而使得转染效果变低。此外,腺相关病毒存在的血清型很多,不同的血清型拥有不同的组织嗜性,即不同的血清型具有对不同组织的靶向性。目前只有通过腺相关病毒的衣壳工程来改变其衣壳特征,从而使得它能够有不同的靶向性。目前腺相关病毒载体治疗脑部疾病所采用的方式为原位注射,患者存在受创伤性手术所造成的伤害和风险。
鉴于此,特提出本发明。
发明内容
本发明的目的在于提供一种纳米组合物、制备方法及应用以解决上述技术问题。
本发明是这样实现的:
本发明提供了一种纳米组合物,其包括内核和静电吸附于内核外周的外壳,内核包括腺相关病毒载体,外壳的原料包括带正电单体、肿瘤微环境敏感性分子和丙烯酸酯聚乙二醇琥珀酰羧甲酯,其中,带正电单体静电吸附于腺相关病毒载体的蛋白外壳,丙烯酸酯聚乙二醇琥珀酰羧甲酯通过肿瘤微环境敏感性分子与正电单体连接,丙烯酸酯聚乙二醇琥珀酰羧甲酯上还连接有靶向剂以靶向目标细胞。
本发明在腺相关病毒的外部包裹上外壳,外壳的包裹使得纳米组合物整体具有免疫逃逸的结果,并且能够延长其在血液中的循环时间,从而避免了裸露的病毒注射到人体内由于中和抗体的存在降低了转染效果,有利于提升基因治疗效果。
另外,发明人在外壳上连接有靶向剂,使得整个纳米组合物携带有靶向剂,从而达到对目标组织的靶向效果。本发明提供的纳米组合物可以满足静脉注射的需求,避免了原位注射对患者带来创伤性手术的损伤和风险,无创性的注射能够更好的利用腺相关病毒优秀的转染效率。
由于腺相关病毒载体的蛋白外壳带负电,因此可以与带正电单体通过静电吸附。为了接枝连接丙烯酸酯聚乙二醇琥珀酰羧甲酯,发明人特设置交联剂,且该交联剂为肿瘤微环境敏感性分子,便于在生物体中降解暴露出包裹的腺相关病毒载体。丙烯酸酯聚乙二醇琥珀酰羧甲酯可以与靶向剂共价连接,从而靶向目标细胞。加入丙烯酸酯聚乙二醇琥珀酰羧甲酯是为了增加生物相容性。
在本发明应用较佳的实施方式中,上述腺相关病毒载体为重组腺相关病毒。在其他实施方式中,病毒外壳携带负电荷也在本发明的保护范围之内。
重组腺相关病毒的血清型可以是腺相关病毒所有已知的血清型,例如可以是AAV2,AAV5,AAV9等。
优选地,重组腺相关病毒携带有肿瘤治疗基因或神经退行性疾病治疗基因;优选地,肿瘤治疗基因选自肿瘤坏死因子超家族、抑癌基因、细胞因子基因、促细胞凋亡基因、血管抑制基因、自杀基因或其他基因。神经退行性疾病治疗基因选自Tau基因和/或SNCA基因(α-突触核蛋白基因(SNCA基因))及其它能够导致神经退行性疾病发生的基因。
优选地,肿瘤坏死因子超家族选自TRAIL;肿瘤坏死因子基因:肿瘤坏死因子超家族中的一员TRAIL,与细胞表面受体结合后,启动细胞凋亡途径,并选择性的促使肿瘤细胞凋亡。
抑癌基因选自p53、PTEN、Rb、NF1、VHL或APC。抑癌基因能够抑制肿瘤细胞的生长。
PTEN基因(人第10号染色体缺失的磷酸酶及张力蛋白同源的基因)是一种新发现的抑癌基因。其表达的PTEN蛋白在细胞的生长、凋亡、黏附、迁徙、浸润等方面具有重要作用。PTEN基因可以调控某种特殊的蛋白合成,而体内几乎所有组织都含有这种蛋白质。但是在脑胶质瘤中,PTEN表达是缺失的。这种蛋白质作为一种肿瘤抑制因子,通过组织细胞生长、分裂的速度过快或者分裂不受控制的方式,进而调控细胞的分裂周期。PTEN酶参与化学通路的传到,可以把信号传导给细胞,使细胞停止分裂并进入程序性死亡(细胞凋亡)。
在其他的实施方式中,上述的腺相关病毒所携带的基因可以更换为任何腺相关病毒可以装载的基因,以使研究人员可以达到自己想要的效果。
细胞因子基因选自IL-2、IL-12、IL-24、GM-CSF、IFN-a、IFN-β或IFN-Y。细胞因子具有杀伤肿瘤细胞,激活免疫细胞,增加造血功能等。
促细胞凋亡基因选自TRAIL、Bax、Caspase或Smac;细胞凋亡是多细胞生物生命活动的重要途径,细胞凋亡途径的异常是机体肿瘤发生的一个重要机制;抑制了细胞凋亡,肿瘤势必要发生;促细胞凋亡基因可加速肿瘤细胞的凋亡,是基因治疗肿瘤的有效基因。
血管抑制基因选自血管生成抑素基因(angiostatin)、sflt-l或血管内皮抑素基因(endostatin);血管抑制基因能干涉新生血管形成,可阻断肿瘤细胞的营养供应,肿瘤因营养不足而萎缩、死亡。
自杀基因选自大肠杆菌胞嘧啶脱氨酶基因(cd)或tk;例如疱疹病毒的脱氧胸腺嘧啶核苷激酶基因(HSV-tk)。
其他基因选自sflt-1、Omi、Bax或Eorf4;血管内皮生长因子可溶性受体sflt-1基因可竞争性抑制血管内皮生长因子发挥作用。
优选地,Caspase选自Caspase-3或Caspase-7;
angiostatin选自angiostatin kl、angiostatin k2、angiostatin k3、angiostatin k4或angiostatin k5。
在本发明应用较佳的实施方式中,上述带正电单体为胍基丙烯酸酯;优选地,肿瘤微环境敏感性分子选自还原敏感性分子、酸敏感性分子和ROS响应分子中的至少一种。
酸敏感性分子可以选自聚酸类的分子,如聚赖氨酸和柠康酸酐。ROS响应分子可以选自β-丁内酯。
优选地,肿瘤微环境敏感性分子选自还原敏感性分子;优选地,还原敏感性分子为包含二硫键的分子;优选地,还原敏感性分子为N,N’-双(丙烯酰)胱胺。
在一种实施方式中,丙烯酸酯聚乙二醇琥珀酰羧甲酯的相对分子量可以是2000。
优选地,靶向剂为载脂蛋白E肽(ApoE):LRKLRKRLLLRKLRKRLLC。载脂蛋白E肽可以靶向脑胶质瘤细胞,在胶质瘤细胞表面高水平表达的LRP-1,LRP-2和LRP-3的受体,从而到达很好的靶向效果。靶向剂增加了病毒对特定组织器官的靶向能力,同时也增加了其穿越血脑屏障(BBB)的能力。ApoE的相对分子量为2373.22。
在其他实施方式中,可以根据实验以及现实使用的需要,可以用聚乙二醇接上不同的靶向剂从而达到不同的靶向效果。只要能实现目的组织或细胞的靶向均可行。
在本发明应用较佳的实施方式中,上述目的细胞为脑胶质瘤、非小细胞肺癌或宫颈癌;优选地,目的细胞为脑胶质瘤,更优选地,目的细胞为胶质母细胞瘤。
在本发明应用较佳的实施方式中,上述纳米组合物中,带正电单体、丙烯酸酯聚乙二醇琥珀酰羧甲酯和肿瘤微环境敏感性分子的摩尔比为1-3:1-3:1-3,优选为1:1:1;腺相关病毒载体与丙烯酸酯聚乙二醇琥珀酰羧甲酯的质量比为1:200-1:220,例如1:210或1:220。
在上述摩尔比条件下,具有较优的细胞靶向效果和肿瘤抑制效果。
在本发明应用较佳的实施方式中,上述靶向剂与丙烯酸酯聚乙二醇琥珀酰羧甲酯的摩尔比为1:3。
本发明提供了一种纳米组合物的制备方法,其包括:将腺相关病毒载体、带正电单体、肿瘤微环境敏感性分子和丙烯酸酯聚乙二醇琥珀酰羧甲酯混合,反应制得纳米组合物。
在本发明应用较佳的实施方式中,上述混合包括先将腺相关病毒载体与胍基丙烯酸酯混合,在丙烯酸酯聚乙二醇琥珀酰羧甲酯上修饰靶向剂,然后将修饰有靶向剂的丙烯酸酯聚乙二醇琥珀酰羧甲酯、腺相关病毒载体与胍基丙烯酸酯的结合体、肿瘤微环境敏感性分子混合进行聚合反应。
聚合反应还包括加入催化剂和加速剂;优选地,催化剂为过硫酸铵,加速剂为四甲基乙二胺。
在一种实施方式中,过硫酸铵和四甲基乙二胺的添加量为1%。
优选地,腺相关病毒载体与胍基丙烯酸酯的混合是在HPEPS缓冲液中进行,反应温度为0-4℃。
优选地,聚合反应在隔绝氧气的环境下进行。可选的,通过向反应装置中通入氮气来达到除氧的作用。
丙烯酸酯聚乙二醇琥珀酰羧甲酯上修饰靶向剂是在37-40℃下避光反应,反应时间为18-24h;优选地,反应后进行透析。
本发明提供了一种纳米组合物在制备脑部疾病治疗药物中的应用。
在本发明应用较佳的实施方式中,上述脑部疾病为脑部原发性肿瘤、脑部继发性肿瘤、脑部血管性疾病;
优选地,脑部原发性肿瘤为脑胶质瘤;脑部继发性肿瘤为肺癌、乳腺癌、前列腺癌或结直肠癌发生的脑部转移;脑部血管性疾病为颅内动脉瘤或高血压性脑出血。
本发明具有以下有益效果:
本发明提供了一种纳米组合物,其包括内核和静电吸附于内核外周的外壳,内核包括腺相关病毒载体,本发明在腺相关病毒的外部包裹上外壳,外壳的包裹使得纳米组合物整体具有免疫逃逸的结果,并且能够延长其在血液中的循环时间,从而避免了裸露的病毒注射到人体内由于中和抗体的存在降低了转染效果,有利于提升基因治疗效果。
另外,发明人在外壳上连接有靶向剂,使得整个纳米组合物实现对目标组织或细胞的靶向效果。本发明提供的纳米组合物可以满足静脉注射的需求,避免了原位注射对患者带来创伤性手术的损伤和风险,无创性的注射能够更好的利用腺相关病毒优秀的转染效率。
附图说明
为了更清楚地说明本发明实施例的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,应当理解,以下附图仅示出了本发明的某些实施例,因此不应被看作是对范围的限定,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他相关的附图。
图1为纳米组合物制备过程图;
图2示出通过动态激发光散射(DLS)和透射电镜照片来检测裸露的病毒(AAV)、包裹上胶囊的病毒(AAV-NCs)、接上靶向剂并包裹上胶囊的病毒(TAAV-NCs)的粒径;
图3为过流式细胞仪检测PBS,AAV,AAV-NCs,TAAV-NCs组在体外进入细胞的能力;
图4为共聚焦显微镜表征细胞内吞结果;
图5为通过共聚焦显微镜表征在细胞内部的转染能力;
图6为细胞凋亡实验结果图;
图7为细胞毒性实验结果图;
图8为PTEN通路示意图和体外验证PTEN表达蛋白免疫印迹(Western Blot,WB)实验结果;
图9为BBB模型体外穿透模拟实验结果;
图10为体外肿瘤球(3D肿瘤球)穿透实验结果;
图11为体内BBB跨越作用及靶向实验结果;
图12为具有AAV抗体的免疫小鼠构建示意图和抗体产生情况检测结果;
图13为体内BBB跨越作用及靶向性实验结果;
图14为具有中和抗体的情况下体内BBB跨越作用及靶向性实验结果;
图15为生物体内抗肿瘤效果结果图;
图16为血生化血常规实验检测结果以及对肾脏和肝脏的几个炎症因子的实时荧光定量PCR的分析结果。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将对本发明实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
以下结合实施例对本发明的特征和性能作进一步的详细描述。
实施例1
本实施例提供了一种纳米组合物,其制备方法如下:
(1)靶向剂连接丙烯酸酯聚乙二醇琥珀酰羧甲酯(本实施例中带正电单体胍基丙烯酸酯)的步骤包括:
称取ACLT-PEG-SCM(23.5mg,0.0105mmol,MW=2227.23Da)、ApoE-NH2(50mg,0.0211mmol,MW=2373.32Da)和TEA(4.7μL,3.42mg,0.03376mmol,ρ=0.728g/mL,MW=101.19)。溶解于2mL的DMSO中,在37℃下,避光800rpm/min搅拌反应24h。3000Da透析袋先DMSO后纯水避光透析24h,而后冻干、称重、计算产率。合成为靶向链,用BCA法检测ApoE连接效率。可以采用BCA法检测ApoE连接效率。靶向剂ApoE连接上PEG之后,可以直接使用。以使病毒的胶囊系统拥有靶向到特定组织的能力。
(2)纳米胶囊的制作过程:在10mM的HPEPS缓冲液中进行反应,反应要求温度为0℃左右,即整个反应需要在冰上进行。先按照腺相关病毒的滴度,来求得其物质的量。然后根据摩尔比来进行后续的材料加入。
本实施例中,腺相关病毒的摩尔量:丙烯酸胍的摩尔量=1:220,丙烯酸胍的摩尔量:N,N'-双(丙稀酰)胱胺的摩尔量:ACLT-PEG-SCM的摩尔量=1:1:1。
向HPEPS缓冲液中加入腺相关病毒(AAV2),然后加入丙烯酸胍,搅拌(300r/min)10min。然后加入步骤(1)连接上丙烯酸酯聚乙二醇琥珀酰羧甲酯的靶向剂。搅拌5min后加入N,N'-双(丙稀酰)胱胺。最后通过加入催化剂过硫酸铵(1mg/mL)和等体积的1%(v/v)引发剂N,N,N',N'-四甲基乙二胺引发聚合反应。制得包裹有外壳的靶向纳米组合物(TAAV-NCs)。整个反应需要隔绝氧气,通过向反应装置中通入氮气来达到除氧的作用。纳米组合物制备过程以及需要的原料参照图1所示。
实施例2
非靶向性的AAV-NCs的合成:向HEPES缓冲液中加入腺相关病毒(AAV),然后加入丙烯酸胍,搅拌(300r/min)10min。然后加入甲氧基聚乙二醇丙烯酰胺(mPEG-NH2),MW:2000(购买自芃硕)。制得的AAV-NCs不具备靶向性。不能被人脑胶质瘤细胞高效吞噬。其余步骤同实施例1。区别仅在于将丙烯酸酯聚乙二醇琥珀酰羧甲酯替换成了甲氧基聚乙二醇胺。
实验例1
本实验例对实施例1制备的纳米组合物(TAAV-NCs)进行表征。结果参照图2所示,通过动态激发光散射仪(DLS)和透射电镜来看,裸露的病毒(AAV)的粒径在20nm左右,包裹上外壳的病毒(AAV-NCs)的粒径在25nm左右,接上靶向剂的纳米组合物(TAAV-NCs)粒径在50nm左右。纳米组合物具有很多病毒自身所不存在的优点,比如使其拥有了免疫逃逸的能力,通过靶向剂来增加病毒对特定组织器官的靶向能力,同时也增加了其穿越血脑屏障(BBB)的能力。
实验例2
本实验例通过流式细胞仪检测PBS,AAV,AAV-NCs,TAAV-NCs组在体外进入细胞的能力。在流式细胞仪检测中,将U87MG细胞培养在六孔板中(1×106细胞/孔),在37℃培养24h后,加入相关PBS,AAV,AAV-NCs,TAAV-NCs(MOI=105)。孵育4h后,吸走样品,用500μL胰蛋白酶消化细胞。得到的细胞悬液用1,000×g离心3min,用PBS洗两次,再次分散在500μLPBS,1h内进行流式细胞仪(BD FACS Calibur,Becton Dickinson,USA)测试,用Cell Quest软件圈取10000个细胞获得。
结果参照图3所示,细胞流式实验图结果表明,TAAV-NCs组的内吞效果最好,为AAV组的2倍。
实验例3
本实验例通过共聚焦显微镜表征细胞内吞。
将U87MG细胞铺于含显微镜载玻片的24孔细胞培养板里(1×105细胞/孔)培养24h后,加入TAAV-NCs,AAV-NCs,AAV和PBS。(MOI=105,Cy5浓度为10μg/mL)。孵育4h后,将培养基移除,用PBS清洗两遍。用DAPI染细胞核10min后清洗两次。荧光图片由CLSM(ZeissLSM800)拍摄获得。
图4所示,CLSM观察到TAAV-NCs在孵育4h后,U87MG细胞内部有更强的Cy5荧光,这充分证明了TAAV-NCs具有较强的进入细胞的能力。
将U87MG细胞铺于含显微镜载玻片的24孔细胞培养板里(1×105细胞/孔)培养24h后,加入TAAV-NCs,AAV-NCs,AAV和PBS。(MOI=105)。所加入的TAAV-NCs,AAV-NCs,AAV携带GFP基因,可以在细胞内表达。孵育4h后,将培养基移除,然后更换培养基并且接着孵育68h,一共培养细胞72h。培养结束之后用PBS清洗两遍。用DAPI染细胞核10min后清洗两次。荧光图片由CLSM(Zeiss LSM800)拍摄获得。
图5中示出通过共聚焦显微镜表征在细胞内部的转染能力。CLSM观察到TAAV-NCs在孵育4h后更换培养基,一共培养72h后,其表达的mcherry的量最多。表明TAAV-NCs具有更好地进入细胞的能力并且具有显著性的转染效果。
实验例4
本实验例进行细胞凋亡实验。将携带有PTEN基因的TAAV-NCs(PTEN),AAV-NCs(PTEN),AAV(PTEN)和不携带PTEN基因的TAAV-NCs(inactive),加入到U87MG细胞中,细胞培养于6孔细胞培养板。(1×105细胞/孔)培养24h,然后加入相关实验组,并且于加入后4h更换培养,接着孵育到72h。用500μL胰蛋白酶消化细胞。得到的细胞悬液用1,000×g离心3min,用PBS洗两次,将细胞进行计数。取5×105-1×106个细胞。用凋亡试剂盒(购自碧云天)进行染色。用PI对早凋细胞进行染色,用Annexin V对晚凋细胞进行染色。染色结束后避光,室温孵育0.5h。孵育结束后,在1h内用1h内进行流式细胞仪(BD FACS Calibur,BectonDickinson,USA)测试。
从所得的结果(图6)可以看出,TAAV-NCs(PTEN)对细胞造成的凋亡的效果最好,约40%。
实验例5
本实验例进行细胞毒性实验。U87MG细胞铺在96孔板内(1×103细胞/孔),24h后,培养基吸走,换上90μL新鲜培养基10μL TAAV-NCs(PTEN)或者TAAV-NCs(inactive)(MOI=105)。孵育72h后,加入10μL的CCK-8溶液(5mg/mL)孵育40min后,用酶标仪检测隔空在450nm处的吸收,以加了CCK-8的培养基孔为零点。每个实验数据平行做五组(n=5)。
从检测结果(参照图7)可以看出,在不同病毒滴度的情况下,TAAV-NCs组总是能够对U87MG细胞造成显著性的杀伤作用,这也和细胞凋亡实验相吻合。同时在病毒滴度为5×104时,效果最为显著。
实验例6
本实验例进行体外验证PTEN表达蛋白免疫印迹(Western Blot,WB)实验。
PTEN通过调节PI3K-PAKT通道来对肿瘤细胞进行一个杀伤性的作用。WB实验:将U87MG细胞接种于6孔板中,每孔2×105个细胞并孵育24h。然后分别加入TAAV-NCs(PTEN),AAV-NCs(PTEN),AAV(PTEN),TAAV-NCs(inactive)和PBS。在培养4h后吸去含有纳米粒子的培养基,更换新鲜的培养基,然后一直培养到72h。吸取培养基,用1×PBS缓冲液洗涤3次,随后胰蛋白酶消化并收集细胞。采用含有1mM PMSF的RIPA(强)细胞裂解液(Beyotime,中国)裂解收集的细胞,然后用BCA蛋白浓度检测试剂盒(Beyotime,中国)检测各组蛋白浓度,确定蛋白上样体积大小。通过电泳(SDS聚丙烯酰胺凝胶)分离裂解物,并将其转移到PDVF膜上(Beyotime,中国)。将PDVF膜分别与抗p4EBP1-Thr37/46,Ppras40-Thr246,PFoxo3a,PAKT-Ser473和PTEN的一抗(Abcam),4℃孵育过夜。IRDye800CW二抗孵育1h,显示蛋白条带(Licor,USA)。使用Image J软件分析蛋白质条带。本实验以GAPDH作为内参。
结果参照图8所示,由图可知,在体外成功验证PTEN基因的作用通路PI3K-pAKT。靶向组TAAV-NCs相对于其它非靶向组以及裸露的AAV组、PBS组和AAV(inactive)组能够明显的提高PTEN的表达,并且其它的下游蛋白所表现出的情况也符合该通路。
实验例7
本实验例进行BBB模型体外穿透模拟实验。
对TAAV-NCs的血脑屏障穿透能力通过体外血脑屏障模型进行评估。简而言之,将hCMEC/D3细胞(5×104/孔)接种在培养插入物(美国纽约州康宁市)的上室,该插入物被放入聚碳酸酯24孔透孔膜中。下室装有800微升DMEM培养基,含1%(体积/体积,100μL青霉素和100μL链霉素)和10%胎牛血清和5%二氧化碳,温度为37℃。培养基每2天更新一次。跨内皮电阻(TEER)仪器(世界精密仪器公司,美国佛罗里达州萨拉索塔)用于监测细胞单层的紧密度。以下实验仅在hCMEC/D3细胞单层的TEER值高于200ω·cm 2时进行。TAAV-NCs、AAV-NCs和AAV被加入上室(Cy5-AAV)。转孔在摇动培养箱(50转/分,37℃)中培养。在1小时、2小时、每2小时直至10小时,从基底外侧隔室收集500微升等分试样,并用等体积的新鲜培养基替换。在实验结束时,再次测量TEER,以监测hCMEC/D3细胞单层的完整性。AAV纳米胶囊的转运率(%)通过使用荧光分光光度计(美国热科学公司)测定样品的荧光来测量,并计算为单层中TAAV-NCs的累积量与od初始量之比。
从结果(图9)可以得出,TAAV-NCs具有很强的穿透BBB的能力,并且能够和AAV-NC、AAV产生显著性的优势。
实验例8
本实验例进行体外肿瘤球(3D肿瘤球)穿透实验以验证TAAV-NCs在体外穿透肿瘤的效果。将细胞1×105个细胞培养在3D肿瘤球板中,培养72h后观察肿瘤球的长成情况。然后加入TAAV-NCs,AAV-NCs和Naked AAV(Cy5-AAV)。培养4h后,取出3D肿瘤球,用1×PBS洗三遍。用4%多聚甲醛固定15Min。再用1×PBS洗三遍。荧光图片由CLSM(Zeiss LSM800)拍摄获得。
由结果(图10)分析可以看出,TAAV-NCs在90μm时仍然对肿瘤球有一定的穿透效果。相对于其它两组,对肿瘤球的穿透只停留在表层。
实验例9
本实验例进行体内BBB跨越作用及靶向性验证实验。我们将TAAV-NCs,AAV-NCs和AAV(Cy5-AAV)通过小鼠尾静脉注射到荷原位人脑胶质瘤U87MG裸鼠体内,由小动物成像仪(IVIS III)跟踪纳米药物在体内不同时间点的分布情况,并重点考察在脑部肿瘤位置的积累及滞留,通过和未添加靶向剂的病毒胶囊及未包裹胶囊的裸露的病毒进行定量和定性的比较,对体内BBB的跨越效率及纳米胶囊系统对体内肿瘤的靶向能力的验证。
由小动物成像仪(IVIS III)跟踪纳米胶囊系统在体内不同时间点的分布情况(图11)可以看出,TAAV-NCs在脑部的积累效果要强于其它两组对照组,说明具有靶向效果的纳米胶囊系统具有很好的靶向性,能够将AAV更多的递送到脑部肿瘤位置。
实验例10
本实验例进行具有AAV抗体的免疫小鼠构建示意图和抗体产生情况检测。
裸露的病毒(剂量:5.56×1010/只)于0、7、14天经尾静脉注射入免疫小鼠(Babl/c小鼠)。通过反复刺激自身免疫系统,使小鼠产生抗AAV的中和抗体。在第21天,可以开始向动物体内注射胶囊,该胶囊为具有靶向性的TAAV-NCs。其合成方法同上。具有胍基丙烯酸酯。从小鼠的眼眶静脉丛中收集300微升血液,在室温下放置4小时,然后用4000g离心5分钟。取上层无色透明的血清。将血清与携带绿色荧光蛋白基因的病毒一起孵育,然后感染细胞,观察病毒携带的绿色荧光蛋白基因的表达,以验证病毒中和抗体的产生。绿色荧光蛋白的表达与病毒产生的中和抗体数量成反比。
从结果(图12)可以看出从注射后第7天开始,具有免疫能力的小鼠体内产生了病毒抗体,并且和PBS对照组产生了显著性的效果。其产生的抗体对病毒的转染造成了显著性的抑制效果。证明免疫小鼠模型构建成功。
实验例11
本实验例进行体内BBB跨越作用及靶向性实验。
将TAAV-NCs,AAV-NCs和AAV(Cy5-AAV)分别通过小鼠尾静脉注射到荷原位鼠脑胶质瘤GL-261黑鼠体内,由小动物成像仪(IVIS III)跟踪纳米药物在体内不同时间点的分布情况,并重点考察在脑部肿瘤位置的积累及滞留,通过和未添加靶向剂的病毒胶囊及未包裹胶囊的裸露的病毒进行定量和定性的比较,对体内BBB的跨越效率及纳米胶囊系统对体内肿瘤的靶向能力的验证。
由小动物成像仪(IVIS III)跟踪纳米胶囊系统在体内不同时间点的分布情况(图13)可以看出,TAAV-NCs在脑部的积累效果要强于其它两组对照组,说明具有靶向效果的纳米胶囊系统具有很好的靶向性,能够将AAV更多的递送到脑部肿瘤位置。
将TAAV-NCs,AAV-NCs和AAV(Cy5-AAV)通过小鼠尾静脉注射到预先构建具有AAV中和抗体的荷原位鼠脑胶质瘤GL-261黑鼠(购自北京斯贝福公司)体内,由小动物成像仪(IVIS III)跟踪纳米药物在体内不同时间点的分布情况,并重点考察在脑部肿瘤位置的积累及滞留,通过和未添加靶向剂的病毒胶囊及未包裹胶囊的裸露的病毒进行定量和定性的比较,对体内BBB的跨越效率及纳米胶囊系统对体内肿瘤的靶向能力的验证。
由小动物成像仪(IVIS III)跟踪纳米胶囊系统在体内不同时间点的分布情况(图14)可以看出,TAAV-NCs在脑部的积累效果要强于其它两组对照组,说明即使在具有中和抗体的情况下,具有靶向效果的纳米胶囊系统仍然展现出很好的靶向性,能够将AAV更多的递送到脑部肿瘤位置。
实验例12
本实验例进行生物体内抗肿瘤效果验证实验。
U87MG脑胶质瘤原位模型的建立是在BALB/c裸鼠(18-20g,6-8周龄)脑部移植肿瘤组织。在脑部肿瘤生物荧光强度为1×105-5×105时进行肿瘤抑制实验。
以荧光素酶标记的人脑胶质瘤细胞(U87MG-Luc)建立原位模型,通过尾静脉注射方式多剂量给药,由IVIS III定性及定量跟踪肿瘤生长情况。在治疗过程中,通过肿瘤生物荧光强度、小鼠体重变化及生存率来评估纳米药物的系统毒副作用及抗肿瘤活性。治疗结束后,由H&E和TUNEL等组织学染色方法分析纳米药物治疗后小鼠各正常器官健康状况及肿瘤组织凋亡情况。
TAAV-NCs在荷瘤U87MG-Luc的BALB/C裸鼠中的治疗实验结果显示,在经过四次尾静脉给药之后(参照图15中的a图),每次剂量为5.56×1010/只,表现出最有的抗胶质瘤效果,能够显著性的抑制肿瘤的增长(图15b)。同时从小鼠的体重变化(图15d),生物发光定量结果也能同样证明TAAV-NCs对肿瘤生长的抑制效果要显著性的优于其它对照组。从Kaplan-Merier生存曲线(图15c和f)可以看出,TAAV-NCs能够极大的延长荷瘤小鼠的寿命,极显著的长于其它对照组。并且有两只小鼠存活超过120天。
从对切片免疫组化染色的结果进行定量分析后(图15g、h和i)可以得出Cleavedcaspase 3(cc3)凋亡蛋白表达量增多,表明TAAV-NCs可以有效促进肿瘤细胞凋亡。从PTEN的含量看出,TAAV-NCs能够显著性的提高肿瘤内部的PTEN含量。Ki67为细胞增殖因子,从结果可以看出,TAAV-NCs的Ki67表达量减少,证明TAAV-NCs可以有效的抑制肿瘤的增值。
实验例13
本实验例进行血生化血常规实验检测并对肾脏和肝脏的几个炎症因子的实时荧光定量PCR检测。TAAV-NCs的细胞毒性和体内生物相容性评估,AAV,PBS。血液化学检查、体重评估。
血常规检查包括白细胞(WBC)、红细胞(RBC)、血小板(PLT)、血浆丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)、白蛋白(ALB)、碱性磷酸酶(ALP)、尿素(UREA)、肌酐(CREA)、尿酸(UA)。数据表示为平均标准差(n=3)。在第2天和第14天进行AAAV-NCs和AAV治疗后,评估肝脏和肾脏中的核心促炎细胞因子,如肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)和白细胞介素-1β(IL-1β)。PBS用作对照。数据表示为平均标准差(n=3)。小鼠以8.1×1011GCs/kg的剂量静脉注射。
结果参照图16所示,由图可知,通过血生化和血常规实验对图16的相应指标惊醒检测,从所得到结果来看,TAAV-NCs没有对小鼠产生明显的毒副作用。图16(i-q图)对小鼠肝脏和肾脏的三种验证因子进行RT-PCR实验,可以判断TAAV-NCs是否会产生炎症反应,从结果可以得到TAAV-NCs不会产生明显的炎症反应。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种纳米组合物,其特征在于,其包括内核和静电吸附于所述内核外周的外壳,所述内核包括腺相关病毒载体,所述外壳的原料包括带正电单体、肿瘤微环境敏感性分子和丙烯酸酯聚乙二醇琥珀酰羧甲酯,其中,所述带正电单体静电吸附于所述腺相关病毒载体的蛋白外壳,所述丙烯酸酯聚乙二醇琥珀酰羧甲酯通过所述肿瘤微环境敏感性分子与所述正电单体连接,所述丙烯酸酯聚乙二醇琥珀酰羧甲酯上还连接有靶向剂以靶向目标细胞。
2.根据权利要求1所述的纳米组合物,其特征在于,所述腺相关病毒载体为重组腺相关病毒;
优选地,所述重组腺相关病毒携带有肿瘤治疗基因或神经退行性疾病治疗基因;优选地,所述肿瘤治疗基因选自肿瘤坏死因子超家族、抑癌基因、细胞因子基因、促细胞凋亡基因、血管抑制基因、自杀基因或其他基因;所述其他基因选自sflt-1、Omi、Bax或Eorf4;所述神经退行性疾病治疗基因选自Tau基因和/或SNCA基因及其它能够导致神经退行性疾病发生的基因;
优选地,所述肿瘤坏死因子超家族选自TRAIL;
所述抑癌基因选自p53、PTEN、Rb、NF1、VHL或APC;所述细胞因子基因选自IL-2、IL-12、IL-24、GM-CSF、IFN-a、IFN-β或IFN-Y;所述促细胞凋亡基因选自TRAIL、Bax、Caspase或Smac;所述血管抑制基因选自angiostatin、sflt-l或endostatin;所述自杀基因选自cd或tk;
优选地,所述Caspase选自Caspase-3或Caspase-7;
所述angiostatin选自angiostatin kl、angiostatin k2、angiostatin k3、angiostatin k4或angiostatin k5。
3.根据权利要求2所述的纳米组合物,其特征在于,所述带正电单体为胍基丙烯酸酯;优选地,所述肿瘤微环境敏感性分子选自还原敏感性分子、酸敏感性分子和ROS响应分子中的至少一种;
优选地,所述肿瘤微环境敏感性分子选自还原敏感性分子;优选地,所述还原敏感性分子为包含二硫键的分子;优选地,所述还原敏感性分子为N,N’-双(丙烯酰)胱胺;
优选地,所述靶向剂为载脂蛋白E肽。
4.根据权利要求1所述的纳米组合物,其特征在于,所述目的细胞为脑胶质瘤、非小细胞肺癌或宫颈癌;优选地,所述目的细胞为脑胶质瘤,更优选地,所述目的细胞为胶质母细胞瘤。
5.根据权利要求3所述的纳米组合物,其特征在于,所述纳米组合物中,所述带正电单体、丙烯酸酯聚乙二醇琥珀酰羧甲酯和肿瘤微环境敏感性分子的摩尔比为1-3:1-3:1-3,优选为1:1:1;所述腺相关病毒载体与所述丙烯酸酯聚乙二醇琥珀酰羧甲酯的质量比为1:200-1:220。
6.根据权利要求3所述的纳米组合物,其特征在于,所述靶向剂与所述丙烯酸酯聚乙二醇琥珀酰羧甲酯的摩尔比为1:3。
7.一种如权利要求1-6任一项所述的纳米组合物的制备方法,其特征在于,其包括:将腺相关病毒载体、带正电单体、肿瘤微环境敏感性分子和丙烯酸酯聚乙二醇琥珀酰羧甲酯混合,反应制得所述纳米组合物。
8.根据权利要求7所述的纳米组合物的制备方法,其特征在于,所述混合包括先将腺相关病毒载体与胍基丙烯酸酯混合,在丙烯酸酯聚乙二醇琥珀酰羧甲酯上修饰靶向剂,然后将修饰有靶向剂的丙烯酸酯聚乙二醇琥珀酰羧甲酯、腺相关病毒载体与胍基丙烯酸酯的结合体、肿瘤微环境敏感性分子混合进行聚合反应;
所述聚合反应还包括加入催化剂和加速剂;优选地,所述催化剂为过硫酸铵,所述加速剂为四甲基乙二胺;
优选地,腺相关病毒载体与胍基丙烯酸酯的混合是在HPEPS缓冲液中进行,反应温度为0-4℃;
优选地,所述聚合反应在隔绝氧气的环境下进行;
所述丙烯酸酯聚乙二醇琥珀酰羧甲酯上修饰靶向剂是在37-40℃下避光反应,所述反应时间为18-24h;优选地,反应后进行透析。
9.一种如权利要求1-6任一项所述的纳米组合物在制备脑部疾病治疗药物中的应用。
10.根据权利要求9所述的应用,其特征在于,所述脑部疾病为脑部原发性肿瘤、脑部继发性肿瘤、脑部血管性疾病;
优选地,所述脑部原发性肿瘤为脑胶质瘤;所述脑部继发性肿瘤为肺癌、乳腺癌、前列腺癌或结直肠癌发生的脑部转移;所述脑部血管性疾病为颅内动脉瘤或高血压性脑出血。
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