CN115120743B - 一种仿乙型脑炎病毒纳米药物及其制备和应用 - Google Patents
一种仿乙型脑炎病毒纳米药物及其制备和应用 Download PDFInfo
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Abstract
本发明构建了一种仿乙型脑炎病毒纳米药物(JEV‑mimicking nanodrugs,HCQ@aPDL1‑Viro)及其制备和应用,以脂质体为框架,所述脂质体表面修饰日本脑炎病毒膜蛋白和PDL1抗体,脂质体框架内部包封羟氯喹。本发明的纳米药物能够封锁免疫检查点重塑免疫抑制微环境,激活免疫细胞活性。同时该纳米载体还能够上调细胞自噬水平,通过膜融合机制释放抑制剂,阻断自噬通量,导致细胞失去稳态发生凋亡。该发明的纳米药物在封锁免疫检查点、上调自噬水平和阻断自噬通量的三重功效的协同作用下,显著提高了抗肿瘤疗效,并为肿瘤治疗提供了一种潜在的治疗策略。
Description
技术领域
本发明属于肿瘤免疫治疗技术领域,尤其是涉及一种仿乙型脑炎病毒纳米药物及其制备和应用。
背景技术
程序性死亡蛋白1(Programmed death protein 1,PD-1)是T细胞表面一种常见的免疫抑制蛋白,在下调免疫系统和提高自我耐受中发挥不可或缺的作用。而肿瘤细胞选择性过表达其配体PD-L1,会抑制T细胞的功能,使其无法杀死肿瘤细胞,导致肿瘤免疫逃逸。近年来,使用PD-1/PD-L1单克隆抗体阻断免疫检查点癌症免疫疗法已经彻底改变了多种恶性肿瘤的临床效果。然而,单独阻断PD-1/PD-L1信号往往不足以逆转肿瘤免疫抑制微环境,完全清除肿瘤细胞,从而导致该免疫治疗在很大一部分患者中是无效的。因此,急需将PD-1/PD-L1单克隆抗体与协同药物相结合,开发更有效的联合疗法,进一步提高肿瘤免疫治疗的有效性。
自噬是一种进化保守的细胞过程,通过将受损的细胞器、错误折叠的蛋白质和其他大分子运输到溶酶体降解和重复利用,在细胞生存、细胞代谢和免疫应答中发挥关键作用。其中,自噬体与溶酶体的融合是自噬通量中的一个关键步骤。自噬通量的阻断会导致异常蛋白聚集的积累和线粒体功能障碍,促进氧化应激和细胞凋亡。越来越多的证据表明,癌细胞可以通过自噬增强自身代谢,抑制细胞死亡途径,从而促进肿瘤细胞存活。研究发现PD-L1/PD-1封锁后,会刺激肿瘤细胞的自噬水平进一步提高,从而促进细胞生存能力,降低免疫治疗的有效性。因此,阻断PD-L1/PD-1相互作用,同时抑制肿瘤细胞通过自噬增强自身代谢途径(自噬通量),有望提高PD-1/PD-L1检查点封锁癌症免疫治疗的疗效。
乙型脑炎病毒(JEV,也称日本乙型脑炎或流行性乙型脑炎)是一种具有正链RNA基因组的包膜黄病毒,糖蛋白E介导JEV的感染进入,与细胞表面的受体结合,并在低pH下病毒包膜与熟化内体膜融合释放基因组。最近有报道发现,JEV感染可上调细胞自噬水平,促进细胞存活,延长繁殖和复制时间。值得注意的是,JEV可以逃避溶酶体降解而实现基因组胞质释放,这一机制可以使药物逃脱溶酶体降解的命运,最大限度地提高药物的递送效率,解决了一直以来药物递送面临溶酶体降解的难题。
发明内容
针对现有技术的不足,本发明旨在提出一种仿乙型脑炎病毒纳米药物及其制备和应用,纳米药物表面偶联的PDL1抗体为免疫检查点封锁剂,可以激活T细胞免疫活性,改善免疫抑制微环境;同时和病毒膜蛋白为自噬诱导剂,能够刺激肿瘤细胞自噬水平上调,有利于肿瘤细胞的生存;羟氯喹HCQ为自噬通量抑制剂,能够阻断刺激后肿瘤细胞中高水平的自噬通量,肿瘤细胞无法代谢,导致失去稳态,从而发生凋亡。总得来说,该纳米药物具有封锁免疫检查点、提高细胞自噬及阻断自噬通量的三重作用来增肿瘤治疗效果。
为达到上述目的,本发明的技术方案是这样实现的:
一种仿乙型脑炎病毒纳米药物,以脂质体为框架,所述脂质体表面修饰乙型脑炎病毒膜蛋白和PDL1抗体,同时内部包封有羟氯喹。
纳米药物的脂质体成分是哺乳动物细胞质膜(OPM)外层的脂质成分,脂质体为天然膜成分,具有优异的生物相容性,PDL1抗体为免疫检查点封锁剂,同时和乙型脑炎病毒膜蛋白为自噬诱导剂,羟氯喹HCQ为自噬通量抑制剂。
优选地,所述纳米药物的水合粒径为10-200nm,更优选地,所述纳米药物的水合粒径为120-160nm。
本发明的第二目的在于提供所述的纳米药物的制备方法,包括如下步骤:
(1)制备脂质薄膜;
(2)向得到的所述脂质薄膜中加入硫酸铵溶液,为形成不同pH的内外水相提供条件,同时加入乙型脑炎病毒膜蛋白,震荡水合化,然后挤压成型,透析;
(3)取出上述样品,加入羟氯喹混合均匀,置于恒温震荡箱中孵育,然后在上述溶液中加入PDL1抗体过夜孵育,得到纳米药物混合液;
(4)将得到的所述纳米药物混合液进行纯化,收集纳米药物备用。
优选地,所述步骤(1)中制备脂质薄膜包括如下具体步骤:按照1-棕榈酰基-2-油酰基卵磷脂(POPC):胆固醇(Chol):鞘磷脂(SM):单唾液酸神经节甘脂(GM1):二硬脂酰基磷脂酰乙醇胺-聚乙二醇-氨基(DSPE-PEG(2000)-NH2)=30-40:25-35:25-35:1-5:2的摩尔比例将各磷脂加入样品瓶中,旋干,得到脂质薄膜。
优选地,所述步骤(2)中硫酸铵的浓度为100-500mM,乙型脑炎病毒膜蛋白的浓度为0.1-0.5mg/mL,,振荡器震荡20-30分钟,挤压成型时聚碳酸酯膜的孔径为50nm-200nm,在氯化钠溶液中透析。
优选地,所述步骤(3)中羟氯喹的浓度为0.1-5mg/mL,恒温震荡箱温度为20-40℃,恒温震荡箱孵育时间为15-60分钟,过夜孵育温度为1-5℃。
本发明的第三目的在于提供所述的纳米药物在肿瘤免疫治疗的应用。
相对于现有技术,本发明具有以下有益效果:
本发明的纳米药物能够封锁免疫检查点,重塑免疫抑制性TME,上调肿瘤细胞自噬水平并阻断自噬通量,导致细胞失去稳态,导致细胞凋亡。凋亡的肿瘤细胞能够进一步激起树突细胞的成熟,并将肿瘤抗原呈递,激活T细胞分化,上调CD8+、CD4+T细胞对抗肿瘤细胞,该纳米药物的三重功效协同复配,显著增强了肿瘤治疗效果。另外,这种联合治疗的方法能够有效抑制肿瘤的转移,治疗后的小鼠肺部肿瘤结节数量显著减少,小鼠存活率时间显著升高。该纳米药物为其他纳米材料在肿瘤免疫治疗中的潜在应用提供了理论基础,也为进一步开发新型纳米药物提供了科学依据。
附图说明
图1:本发明制备的HCQ@aPDL1-Viro纳米药物的电镜图;
图2:本发明制备的HCQ@aPDL1-Viro纳米药物的水合粒径分布;
图3:本发明制备的HCQ@aPDL1-Viro纳米药物对细胞自噬水平影响的评估,其中a为细胞自噬水平的荧光图,b为细胞自噬水平荧光信号的统计数据;
图4:本发明制备的HCQ@aPDL1-Viro纳米药物对细胞自噬水平和自噬通路阻断的评估,其中a为细胞自噬水平及自噬通路变化的荧光图,b为细胞自噬水平和自噬通路荧光信号的统计数据;
图5:本发明制备的HCQ@aPDL1-Viro纳米药物对不同处理的细胞中自噬通路相关蛋白表达量的表征,其中a为自噬相关蛋白的westernblot图,b为a中蛋白表达量变化的统计数据;
图6:本发明制备的HCQ@aPDL1-Viro纳米药物对癌细胞杀伤能力的评估,其中a为不同组处理细胞的Calcein-AM/PI染色荧光图,b为不同组样品对癌细胞杀伤能力的统计;
图7:本发明制备的HCQ@aPDL1-Viro纳米药物在小鼠肿瘤模型(4T1)中的抗肿瘤效果;
图8:本发明制备的HCQ@aPDL1-Viro纳米药物治疗后的小鼠体内肺部组织的HE染色图;
图9:本发明中不同组样品对小鼠免疫治疗中免疫指标的评估,其中a为小鼠脾脏内树突细胞熟化水平流式图,b为脾脏内CD4+,CD8+T细胞变化水平的流式图。
具体实施方式
除有定义外,以下实施例中所用的技术术语具有与本发明所属领域技术人员普遍理解的相同含义。以下实施例中所用的试验试剂,如无特殊说明,均为常规生化试剂;所述实验方法,如无特殊说明,均为常规方法。
为进一步说明本发明,现通过具体实施实例对本发明进行详细阐述。
一、涉及到的主要原料及其来源
1-棕榈酰基-2-油酰基卵磷脂(POPC)、胆固醇(Chol)、鞘磷脂(SM)和单唾液酸神经节苷脂(GM1)购自西安瑞熙生物科技有限公司(中国西安)。
DSPE-PEG(2000)-NH2购自中国湖南华腾药业有限公司。
细胞计数试剂盒(CCK-8)、Calcein-AM/PI双染色试剂盒、羟氯喹(HCQ)购自国药化学试剂有限公司(中国上海)。
培养基(DMEM)和胎牛血清(FBS)购自Gibco有限公司(美国纽约州格兰德岛)。
JEV膜蛋白购自上海生工生物技术有限公司。
二、纳米药物的制备
(1)按照1-棕榈酰基-2-油酰基卵磷脂(POPC):胆固醇(Chol):鞘磷脂(SM):单唾液酸神经节苷脂(GM1):DSPE-PEG(2000)-NH2=35:30:30:3:2的比例将磷脂加入样品瓶中,将样品瓶固定在圆底烧瓶中,用旋转蒸发仪去除所有的有机试剂,得到脂质薄膜。
(2)加入1mL 250mM硫酸铵溶液,同时加入乙型脑炎病毒膜蛋白(脂质体的摩尔数/病毒膜蛋白的摩尔数为8000:1)于振荡器震荡30分钟。然后水浴超声1分钟,通过0.1μm的聚碳酸酯膜挤压21次。并将上述溶液封装在析袋中,置于2L的氯化钠溶液中搅拌透析24小时。
(3)向上述溶液中加入42.5μL1mg/mL的羟氯喹,对HCQ进行包裹,该过程于37℃恒温震荡箱中孵育15分钟,抑制剂HCQ的包封率为73.7%。
(4)用质量比为20:1的比例加入EDC/NHS活化2.5μg的PDL1抗体,活化20分钟后,然后加入到步骤(3)得到的溶液中,4℃过夜孵育,抗体与DSPE-PEG-NH2通过化学键反应得到纳米药物混合液。最后通过NAP-5脱盐柱将得到的纳米药物混合液进行纯化,去除未反应的物质,得到500μg/mL的纳米药物备用。
三、纳米药物的结果分析
试验例1
本发明中制备的纳米药物形貌和粒径的表征:通过负染法将纳米药物处理,并自然风干。用生物电镜对样品拍图,制备的纳米药物尺寸均一,纳米药物的粒径为128nm,在PBS中分散性好,结果如图1所示。然后将纳米药物均匀的溶解在PBS溶液中,并进行水合粒径测试,粒径大约150nm,结果如图2所示。本发明纳米药物形貌和载药稳定性良好。
试验例2
本发明制备的纳米药物对细胞自噬水平的影响具体操作步骤如下:首先,4T1细胞在含有10%胎牛血清的DMEM共聚焦培养皿中孵育12h。不同组样品进一步处理12小时后,细胞与1μM AO共孵育10min,PBS洗涤3次,共聚焦荧光显微镜观察。结果如图3所示,本发明制备的纳米药物HCQ@aPDL1-Viro能够显著上调细胞的自噬水平。
试验例3
本发明制备的纳米药物对细胞自噬水平和通路的影响具体操作步骤如下:4T1细胞接种于共聚焦培养皿,每孔5×105个细胞,培养至密度为60%。使用jetPRIME转染试剂(VWR,Radnor,PA,USA)瞬时转染细胞。培养12小时后,每孔加1ml新鲜培养基进一步成像处理,结果如图4所示,本发明制备的HCQ@aPDL1-Viro能够有效的上调自噬水平和自噬通路。
试验例4
本发明制备的纳米药物对细胞自噬相关蛋白表达的影响具体操作步骤如下:4T1细胞(每孔105个)接种于6孔板,37℃过夜培养。然后分别用不同组样品处理细胞24小时。接下来,用含有蛋白酶抑制剂的细胞裂解液清洗细胞并裂解细胞。低温离心10min收集裂解液。用SDS-PAGE(12%)分离后,将总细胞蛋白转移到PVDF膜上,然后在4℃与一抗孵育过夜,接着在室温下加入HRP偶联的二抗体1小时。最后,使用Bio-Rad ChemiDocTM(Hercules,CA,USA)成像系统分析蛋白信号。结果如图5所示,本发明制备的HCQ@aPDL1-Viro纳米药物能够上调LC3-II和p62的表达,结果与上述结果一致。
试验例5
本发明制备的纳米药物的体外肿瘤细胞杀伤性能实验操作步如下:将4T1细胞接种在6孔板中,加入含10%FBS的培养基培养24小时。然后去除培养基,加入纳米药物(400μg/mL),继续培养12小时后,去除培养基,加入用Calcein-AM/PI对细胞染色20分钟。染色后用PBS冲洗2-3次,在倒置荧光显微镜下通过绿色荧光(活细胞)和红色荧光(死细胞)的比例来判断肿瘤细胞杀伤效果。结果如图6所示,制备的HCQ@aPDL1-Viro纳米药物处理的细胞死亡率明显增高,肿瘤细胞的死亡率高达80.3%。此实验中以PBS为对照组。
试验例6
本发明制备的纳米药物的体内肿瘤细胞杀伤性能实验步骤如下:
(1)小鼠肿瘤模型的建立:将4T1细胞复苏稳定传代3次。取对数期生长的细胞,胰酶消化,用PBS重悬细胞(密度为1×106每毫升)。取6周龄左右的BALB/c小鼠30只,用1毫升注射器吸取20微升的细胞悬液,乳房垫注射癌细胞,注射完毕后将小鼠置于动物房中饲养。
小鼠分组及治疗方案:小鼠随机分为6组,每组5只,待小鼠肿瘤体积大约达到50mm3时,开始进行分组治疗。具体的治疗方案如下:
组1:尾静脉注射浓度为0.01M的PBS缓冲溶液
组2:尾静脉注射浓度为0.63mg/mL的羟氯喹(HCQ)
组3:尾静脉注射浓度为4mg/mL的Virosome
组4:尾静脉注射浓度为4mg/mL的HCQ@Liposome
组5:尾静脉注射浓度为4mg/mL的HCQ@Virosome
组6:尾静脉注射浓度为4mg/mL的HCQ@aPDL1-Viro
在第0,3,6,9天每只小鼠注射200微升的组1-组6溶液,从治疗开始,每两天测量小鼠的体重及肿瘤大小。并在27天解剖小鼠,对肿瘤拍照。结果如图7所示,纳米药物处理组小鼠的肿瘤得到了有效的抑制,治疗效果明显。最后对小鼠脾脏部位进行流式测试评估体内免疫治疗效果。结果如图9显示,纳米药物组免疫反应水平最高,肿瘤生长明显低于其他组,另外发现肺部转移结节数量最少,结果如图8所示。上述结果表明纳米药物有效的抑制了肿瘤生长及转移。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (3)
1.一种仿乙型脑炎病毒纳米药物,其特征在于:以脂质体为框架,所述脂质体表面修饰乙型脑炎病毒膜蛋白和PDL1抗体,同时内部包封有羟氯喹,
所述的纳米药物的制备方法,包括如下步骤:
(1)制备脂质薄膜;
(2)向得到的所述脂质薄膜中加入硫酸铵溶液,同时加入乙型脑炎病毒膜蛋白,震荡水合化,然后挤压成型,透析;
(3)取出上述样品,加入羟氯喹混合均匀,置于恒温震荡箱中孵育,然后在上述溶液中加入PDL1抗体过夜孵育,得到纳米药物混合液;
(4)将得到的所述纳米药物混合液进行纯化,收集纳米药物备用,
所述步骤(1)中制备脂质薄膜包括如下具体步骤:按照1-棕榈酰基-2-油酰基卵磷脂(POPC):胆固醇(Chol):鞘磷脂(SM):单唾液酸神经节甘脂(GM1):二硬脂酰基磷脂酰乙醇胺-聚乙二醇-氨基(DSPE-PEG-NH2)=30-40:25-35:25-35:1-5:2的摩尔比例将各磷脂加入样品瓶中,旋干,得到脂质薄膜,
所述步骤(2)中硫酸铵的浓度为100-500mM,乙型脑炎病毒膜蛋白的浓度为0.1-0.5mg/mL,振荡器震荡20-30分钟,挤压成型时聚碳酸酯膜的孔径为50nm-200nm,在氯化钠溶液中透析,
所述步骤(3)中羟氯喹的浓度为0.1-5mg/mL,恒温震荡箱温度为20-40℃,恒温震荡箱孵育时间为15-60分钟,过夜孵育温度为1-5℃。
2.根据权利要求1所述的仿乙型脑炎病毒纳米药物,其特征在于:所述纳米药物的水合粒径为10-200nm。
3.权利要求1或2所述的纳米药物在制备肿瘤免疫治疗药物中的应用。
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