CN117338716A - 一种共递送抗原与佐剂的皮克林乳剂及其制备方法和应用 - Google Patents
一种共递送抗原与佐剂的皮克林乳剂及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供一种皮克林乳剂,并提供其制备方法。本方法选用负载金属化合物的蛋白颗粒作为固体乳化剂,同时选择可生物代谢的油相作为乳剂内核,构建了可用于提高适应性免疫应答的皮克林乳剂递送系统。本发明中的皮克林乳剂能够高效地荷载蛋白抗原,同时显著提高亚单位疫苗的体液免疫应答与细胞免疫应答。
Description
技术领域
本发明涉及一种共递送抗原与佐剂的皮克林乳剂及其制备方法和应用,属于生物医药技术领域。
背景技术
淋巴结作为重要的外周免疫器官之一,富含多种免疫细胞,包括B细胞、T细胞、树突状细胞(DCs)和巨噬细胞,直接影响免疫应答的强弱。越来越多的研究显示,疫苗在体内的时空分布规律与免疫应答的强弱息息相关,如果将抗原与胞内发挥免疫刺激作用的佐剂(如TLR9激动剂、Sting激动剂等)同时共递送至淋巴结内抗原提呈细胞,则可实现抗原与佐剂发挥作用的一致性,进而显著提升疫苗激活适应性免疫应答的水平。
通过将抗原与佐剂借助特定的纳米载体递送至淋巴结,延长其在淋巴结内的滞留时间,能够有效增加抗原被淋巴结内抗原提呈细胞摄取的效率,进而诱导T细胞的活化,促进后续适应性免疫应答的激活。一般来说,粒径在20-200nm范围的内的纳米颗粒经皮下注射后可以通过毛细淋巴管的内皮细胞间隙进入淋巴循环,随淋巴液引流至淋巴结。
现有的技术多是借助纳米载体通过化学连接、静电作用以及疏水相互作用等作用力包载抗原和佐剂,但是其制备工艺复杂,同时存在抗原或佐剂泄露的风险,从而诱发毒副作用,因此寻找新的抗原佐剂共递送体系始终是当前疫苗的研发热点之一。
通过采用胶体颗粒代替表面活性剂分子稳定的乳液称为皮克林乳乳液,其具有以下优势:(1)充当乳化剂的胶体颗粒用量相比表面活性剂用量低,可以降低疫苗的生产成本;(2)乳剂体系中少了表面活性剂成分,对身体和环境更加友好;(3)胶体颗粒充当乳化剂,固体颗粒挣脱两相界面需要的热能更高,因而乳剂体系的稳定性要更强;(4)皮克林乳剂表面粗糙程度高,更有利于与细胞的相互作用。但目前,大多数皮克林乳剂普遍采用不具有生物相容性的固体颗粒或油相制备,因此其在生物医药领域的应用受到限制。例如,CN101445580A公开了一种乳液聚合制备聚乙烯/二氧化硅核壳结果复合材料的方法,利用二氧化硅纳米粒作为固体颗粒,但其中含有乙酸乙酯以及甲苯所溶解的催化剂,最后加入乙烯,得到聚乙烯/二氧化硅乳液,该体系中二氧化硅和甲苯在临床上应用都受到限制,因此,该乳液无法直接在生物医药领域应用。
金属免疫治疗是利用金属离子的免疫调节功能治疗疾病。越来越多的证据表明,金属离子在调节先天免疫感知和宿主防御入侵病原体方面都有重要作用,包括T细胞活化(Ca2+)和干细胞特性(K+),炎症小体(K+,Ca2+和Na+),病原体-宿主相互作用(Fe2+/3+,Zn2+,Mn2 +和Cu2+)和cGAS-STING信号(Zn2+和Mn2+)。最近的研究表明,Mn2+在DNA病毒感染期间使cGAS-STING通路对双链DNA增敏,并且与免疫检查点抑制剂、化疗、原位疫苗和光动力疗法有协同作用。类似Mn2+通过激活cGAS-STING通路促进CD8+T细胞活化的肿瘤疗法也被国内外多个实验室所发现或证实。
因此,提供一种根据疫苗佐剂的需求来进行设计、优化以最大化疫苗潜力的佐剂递送系统对本领域而言具有重要意义。这里,我们基于皮克林乳剂技术,利用蛋白抗原和负载金属化合物的蛋白颗粒共同作为乳剂的稳定剂,一步乳化即得可共递送抗原与佐剂的皮克林乳剂系统,制备工艺简单,易于放大生产,可快速实现靶向递送抗原以及激活体液和细胞免疫的多重功能。
发明内容
针对现有技术的不足,本发明的目的在于提供一种可共递送抗原与佐剂的皮克林乳剂系统及其制备方法和应用。所述皮克林乳剂直接采用蛋白抗原和负载金属化合物的蛋白颗粒作为固体乳化剂,稳定性好,安全性高;且所述乳剂由于其粗糙的表面和优越的抗原吸附能力,具备更强的激活适应性免疫应答的能力。
为了达到上述目的,本发明采用了如下技术方案:
第一方面,本发明提供一种采用蛋白抗原和负载金属化合物的蛋白颗粒作为稳定剂的皮克林乳剂,所述皮克林乳剂包括油相、水相、负载金属化合物的蛋白颗粒和蛋白抗原,其中,所述蛋白抗原和负载金属化合物的蛋白颗粒分布于油水界面。
在本发明中,负载金属化合物的蛋白颗粒的存在有两方面作用,一方面可以维持油水界面的稳定;另一方面可直接充当疫苗佐剂。其佐剂作用机制可归因于以下几个方面:1)纳微颗粒能够特异性激活抗原提呈细胞,增加其摄取量;2)用纳微颗粒包埋、吸附或偶联抗原,可持续释放抗原,延长细胞吸收和抗原表达时间;3)部分纳微颗粒(如带有正电荷的壳聚糖纳微颗粒),可以通过质子泵效应等实现抗原的溶酶体逃逸,实现抗原的交叉提呈,可促进机体细胞免疫反应;4)部分纳微颗粒还可募集炎症细胞,从而促进抗原与抗原提呈细胞之间的作用。
因此,采用固体颗粒替代表面活性剂制备皮克林乳剂,不仅可以避免表面活性剂对疫苗制剂的负面影响,而且可以通过固体颗粒和水包油乳液的免疫协同作用,获得更为全面、显著、持久的免疫保护效果。该皮克林乳剂中不含表面活性剂,避免了表面活性剂对抗原的影响,产品具有良好的安全性和稳定性,并可用于疫苗的不同接种途径。
本发明中所述的皮克林乳剂,负载金属化合物的蛋白颗粒中的蛋白具备生物相容性,其包括内源性蛋白和外源性蛋白。优选地,所述内源性蛋白为白蛋白、血红蛋白、甲状腺素转运蛋白和/或铁蛋白;所述外源性蛋白进一步优选为蛋白抗原,包括重组蛋白、微生物和/或植物来源的蛋白;优选地,所述蛋白为白蛋白,包括天然血清白蛋白和/或人工合成血清蛋白,进一步优选为人血清白蛋白、牛血清白蛋白或鼠血清白蛋白中的任意一种或者至少两种的组合;优选地,所述负载金属化合物的蛋白颗粒是蛋白经过修饰而成,所述修饰包括亲水修饰、疏水修饰、金属离子生物矿化、覆层或接枝改性中的任意一种或者至少两种的组合;优选地,所述负载金属化合物的蛋白颗粒为Mn2+矿化蛋白或氢氧化铝覆层的蛋白,进一步,优选地负载金属化合物的蛋白颗粒为Mn2+矿化的白蛋白或氢氧化铝覆层的白蛋白。
本发明中所述的皮克林乳剂中乳滴的平均粒径为50nm~1μm,例如50nm、100nm、300nm、600nm、1μm,所述皮克林乳剂中乳滴的平均粒径的选择,与其作用的组织相匹配,优选范围为50nm~300nm。
优选地,所述皮克林乳剂的油水两相体积比为1:(1~100),例如可以是1:1、1:5、1:10、1:15、1:20、1:25、1:30、1:35、1:40、1:45、1:50、1:55、1:60、1:65、1:70、1:75、1:80、1:85、1:90、1:95或1:100等,优选为1:(2~50)。
优选地,所述负载金属化合物的蛋白颗粒在水相中的质量浓度为0.1~20wt%,例如为0.1wt%、0.5wt%、5wt%、6wt%、7wt%、10wt%、15wt%或20wt%,优选为0.5~10wt%,所述负载金属化合物的蛋白颗粒在水相中的质量浓度为负载金属化合物的蛋白颗粒的质量除以负载金属化合物的蛋白颗粒和水相质量和的比值。
油相优选角鲨烯和生育酚中的一种或两种的混合物。所述角鲨烯是一种三萜类化合物,其英文名称是Squalene,分子结构为三十碳五十氢的异戊二烯,分子式为:2,3,10,15,19,23-六甲基-2,6,10,14,18,22-二十四碳六烯,CAS:111-02-4,分子质量:410.72,可来源于动物、植物提取或化学合成。角鲨烯是一种可代谢的油,因为它是胆固醇的生物合成的中间产物(Merk索引,第10版本,登记号8619)。这是一种所有高等生物,包括在人类身上(皮脂中可以找到)自然分泌的油脂。含有角鲨烯的乳剂(含表面活性)在动物实验和临床实验上表现出优异大的免疫增强作用。
所述生育酚是α-生育酚或其衍生物如α-生育酚琥珀酸酯(也称作维生素E琥珀酸酯)。α-生育酚在针对老年患者(如年龄大于60岁或更大的患者)的疫苗中,可以起到增强免疫应答的作用。存在的生育酚包括α、β、γ、δ、ε,ζ等多种生育酚,优选α-生育酚,尤其是DL-α-生育酚。优选地,所述油相与水相互不相容,还可以包括其它代谢的油。
为了使所述水包油乳液适合于疫苗或药物制剂,本发明中所述水包油乳液的油相为可代谢的油。术语“代谢的油”意思已为本领域所熟知。“可代谢的”可被定义为“能够通过代谢作用转变”(Dorland的医学词典解释,W.B.Sanders公司,第25版本(1974))。
示例性的可代谢的油可以是任何对受体无毒并可通过代谢作用转化的植物油、鱼油、动物油或合成油,包括但不限于大豆油、米格列醇(Miglyo1812)、中链油、鱼油、维生素E、维生素E琥珀酸酯、维生素E醋酸酯、红花油、玉米油、沙棘油、亚麻籽油、花生油、茶油、葵花籽油、杏仁油、薏仁油、月见草油、芝麻油、棉籽油、蓖麻油、低芥酸菜籽油、油酸乙酯、油酸、亚油酸乙酯、月桂酸异丙酯、肉豆蔻酸异丙酯、丁酸乙酯、乳酸乙酯、辛酸甘油三酯或癸酸甘油三酯中的任意一种或者至少两种的组合。坚果、种子和谷物是常见的植物油来源。
本发明中所述水包油乳液的水相优选为注射用水、磷酸盐缓冲液、柠檬酸缓冲液或Tris缓冲液中的任意一种或者至少两种的组合。所述组合例如注射用水和磷酸盐缓冲液的组合,柠檬酸缓冲液和Tris缓冲液的组合,注射用水、磷酸盐缓冲液和柠檬酸缓冲液的组合,Tris缓冲液、注射用水、磷酸盐缓冲液、柠檬酸缓冲液或Tris缓冲液的组合。
优选地,所述磷酸盐缓冲液、柠檬酸缓冲液或Tris缓冲液的pH值独立地为5.0~8.1,例如5.2,5.4,5.6,5.8,6,6.2,6.4,6.6,6.8,7,7.2,7.4,7.6,7.8或8,优选为6.0~8.0。
本发明中所述水相中还包括药用辅助物质,如pH调节剂或/和缓冲剂等,优选自乙酸钠、乳酸钠、氯化钠、氯化钾、氯化钙、人血清白蛋白、必需氨基酸、非必需氨基酸、L-精氨酸盐酸盐、蔗糖、无水D-海藻糖、甘露醇、甘露糖、淀粉或明胶中任意一种或者至少两种的组合。所述组合例如乙酸钠和乳酸钠的组合,氯化钠和氯化钾的组合,氯化钙、人血清蛋白和必须氨基酸的组合,非必需氨基酸、L-精氨酸盐酸盐、蔗糖和无水D-海藻糖的组合,甘露醇、甘露糖、淀粉和明胶的组合,乙酸钠、乳酸钠、氯化钠、氯化钾、氯化钙和人血清蛋白的组合,必须氨基酸、非必需氨基酸、L-精氨酸盐酸盐、蔗糖、无水D-海藻糖、甘露醇、甘露糖、淀粉和明胶的组合。
本发明中所述皮克林乳剂中还可以包含药用添加剂,所述药用添加剂包括例如稀释剂、稳定剂或防腐剂中的任意一种或者至少两种的组合。
本发明中所述皮克林乳剂中还可以包括以下佐剂但不限于:模式识别受体(例如Toll样受体、RIG-1和NOD样受体(NLR)的刺激剂,如含CPG基序的寡核苷酸或双链RNA或含回文序列的寡核苷酸或含聚(dG)序列的寡核苷酸)、肠细菌(例如大肠杆菌、明尼苏达沙门菌、鼠伤寒沙门菌、或弗式志贺菌)的单磷酰脂质A(MPLA)、皂苷类佐剂、脂质体和脂质体配置品(如AS01)、合成的或特别制备的微颗粒和微载体(如淋病奈瑟球菌)、沙眼衣原体和其它细菌的源于细菌的外膜泡(OMV)、多糖(如壳聚糖)可选择的病原相关分子模式(PAMPs)、小分子免疫增强剂(SMIP)、细胞因子和趋化因子。
第二方面,本发明提供一种如第一方面所述的皮克林乳剂的制备方法,本发明中所述的皮克林乳剂可采用多种方法制备。具体地,本发明中所述皮克林乳剂可采用下述方法制备,但不受下述制备方法的限制。
本发明中所述皮克林乳剂先将蛋白抗原与负载金属化合物的蛋白颗粒分散在水相中,然后将油相和水相混合后超声乳化,即可得到可共递送抗原与佐剂的皮克林乳剂。油相和水相的混合可以选择微流控、均质、超声、注射器双推乳化、喷雾、微射流、微通道、膜乳化、搅拌、振荡、倒转或手摇混合等多种方式。根据不同的需求,混合方式可以优选微流控、微通道或膜乳化等可以获得均一粒径分布乳剂的方式,也可以优选微射流、注射器双推乳化、均质、搅拌或振荡等便于规模化制备的混合方式。
第三方面,本发明还提供一种疫苗,所述抗原分布于皮克林乳剂的油水界面。所述抗原包括但不限于人类抗原、非人类动物抗原、植物抗原、细菌抗原、真菌抗原、病毒抗原、寄生虫抗原或肿瘤抗原中的任意一种或者至少两种的组合。所述组合例如人类抗原和非人类动物抗原的组合,植物抗原和细菌抗原的混合物,真菌抗原、病毒抗原和寄生虫抗原的组合,肿瘤抗原、人类抗原、非人类动物抗原、植物抗原、细菌抗原和真菌抗原的组合,病毒抗原、寄生虫抗原、肿瘤抗原、人类抗原、非人类动物抗原和植物抗原的组合,细菌抗原、真菌抗原、病毒抗原、寄生虫抗原和肿瘤抗原的组合。
所述抗原可以来自但不限于鸡胚培养、细胞培养、携带者体液、器官或组织中纯化分离所得、重组基因表达或化学合成所得,优选所述抗原包括但不限于减毒疫苗、灭活疫苗、裂解疫苗、亚单位疫苗或多糖结合疫苗等中的任意一种或者至少两种的组合。与已有技术相比,本发明具有如下有益效果:
本发明首次采用抗原佐剂共乳化技术,一次乳化即可制备具备淋巴结靶向能力的纳米疫苗,制备工艺简单快速,成本低,易于大规模生产;其次,采用固体乳化剂代替传统的表面活性剂作为乳剂的稳定剂,生物安全性高,对人体的副作用小;再者,抗原佐剂共递送技术可大幅度提高疫苗激活适应性免疫应答的水平,具备增强疫苗免疫原性、延长疫苗保护周期的潜力。
附图说明
图1为二价锰离子(Mn2+)经人血清白蛋白(HSA)生物矿化前后(HSA-Mn)的形态示意图和透射电镜图。
图2为氢氧化铝覆层的白蛋白颗粒的皮克林乳剂和Mn2+矿化白蛋白颗粒的皮克林乳剂的粒径分布图和透射电镜图。
图3为氢氧化铝覆层的白蛋白颗粒的皮克林乳剂和Mn2+矿化白蛋白颗粒的皮克林乳剂的粒径随时间变化的曲线图与抗原包封率检测结果。
图4为氢氧化铝覆层的白蛋白颗粒的皮克林乳剂和Mn2+矿化白蛋白颗粒的皮克林乳剂的体外细胞摄取和诱导BMDC成熟的检测结果。
图5为氢氧化铝覆层的白蛋白颗粒的皮克林乳剂和Mn2+矿化白蛋白颗粒的皮克林乳剂的淋巴结靶向检测结果。
图6为氢氧化铝覆层的白蛋白颗粒的皮克林乳剂和Mn2+矿化白蛋白颗粒的皮克林乳剂的体液免疫应答检测结果。
图7为氢氧化铝覆层的白蛋白颗粒的皮克林乳剂和Mn2+矿化白蛋白颗粒的皮克林乳剂的细胞免疫应答检测结果。
具体实施方式
下面结合附图并通过具体实施方式来进一步说明本发明的技术方案,但下述的实例仅仅是本发明的简易例子,并不代表或限制本发明的权利保护范围,本发明的保护范围以权利要求书为准。
实施例1
基于白蛋白制备二价锰离子矿化的白蛋白颗粒,具体制备方法如下(图1-a):
通过利用白蛋白天然的生物模板特性,二价锰离子可以在碱性环境下有序的排列于蛋白表面,完成生物矿化,具体步骤为:称量0.2695gMnCl2·4H20于50ml烧瓶中,加入15ml超纯水,边搅拌边溶解;加入HSA溶液(200mg/ml,5ml),室温搅拌5min;用1M氢氧化钠调节pH至10.00左右;转移至34℃油浴锅,搅拌反应2h;收集样品,10kDa透析袋透析两天;0.45或0.22μm滤头过滤后,冻干收集粉末;利用ICP-OES准确定量矿化粉末中的锰元素含量。
实施例2
基于甲状腺素转运蛋白(MSA)制备二价锰离子矿化的甲状腺素转运蛋白颗粒,具体制备如下:
利用甲状腺素转运蛋白MSA与HSA类似的生物模板特性,二价锰离子可以在碱性环境下有序的排列于蛋白表面,完成生物矿化,具体步骤为:称量0.2695gMnCl2·4H20于50ml烧瓶中,加入15ml超纯水,边搅拌边溶解;加入MSA溶液(200mg/ml,5ml),室温搅拌5min;用1M氢氧化钠调节pH至10.00左右;转移至34℃油浴锅,搅拌反应2h;收集样品,10kDa透析袋透析两天;0.45或0.22μm滤头过滤后,冻干收集粉末;利用ICP-OES准确定量矿化粉末中的锰元素含量。
实施例3
基于重组蛋白PcrV蛋白(PcrV蛋白即为铜绿假单胞菌细胞膜表面蛋白)制备二价锰离子矿化的蛋白,具体制备如下:
二价锰离子可以在碱性环境下有序的排列于蛋白表面,完成生物矿化,具体步骤为:称量0.2695gMnCl2·4H20于50ml烧瓶中,加入15ml超纯水,边搅拌边溶解;加入PcrV溶液(200mg/ml,5ml),室温搅拌5min;用1M氢氧化钠调节pH至10.00左右;转移至34℃油浴锅,搅拌反应2h;收集样品,10kDa透析袋透析两天;0.45或0.22μm滤头过滤后,冻干收集粉末;利用ICP-OES准确定量矿化粉末中的锰元素含量。
实施例4
采用透射电镜观察实施例1制备HSA-Mn形貌(图1-b),具体步骤为:取1mg冻干后的HSA-Mn溶于1ml无菌注射用水中,用毛细管蘸取适量悬液,置于覆有碳膜的铜网上,静置片刻后用滤纸吸走多余溶液。然后再用毛细管蘸取2%磷钨酸溶液,静置染色1-2min后,用滤纸吸走染液,置于红外灯下烘干,最后将其置于透射电子显微镜中观察。
HSA-Mn在电镜下的形貌如图1所示,呈现立方块样形貌。
实施例5
基于白蛋白HSA乳化角鲨烯(squalene)制备皮克林乳剂HSA-NE;基于氢氧化铝覆层的白蛋白乳化角鲨烯(squalene)制备皮克林乳剂HSA-AlNE;基于HSA-Mn乳化角鲨烯(squalene)制备皮克林乳剂HSA-MnNE;具体制备方法如下:
HSA-NE:将24mg HSA溶于2880μl HEPES(20mM,pH8.0)中,充分溶解后待用;6mg角鲨烯溶于150μl氯仿,滴加入上述溶有HSA的水相中;150W超声乳化5min,旋转蒸发除去有氯仿,即得HSA-NE。
HSA-AlNE:将24mg HSA溶于2880μl HEPES(20mM,pH8.0)中,充分溶解后待用;6mg角鲨烯溶于150μl氯仿,滴加入上述溶有HSA的水相中;150W超声乳化5min,旋转蒸发除去有氯仿,即得HSA-NE;取1ml制备好的HSA-NE,滴加100μl硫酸铝(10mM),即得HSA-AlNE。
HSA-MnNE:将12mgHSA-Mn溶于3000μl HEPES(50mM,pH7.2)中,充分溶解后待用;6mg角鲨烯溶于150μl氯仿,滴加入上述溶有HSA-Mn的水相中;150W超声乳化5min,旋转蒸发除去有氯仿,即得HSA-MnNE。
分别取1ml制备好的HSA-NE、HSA-AlNE、HSA-MnNE加入到样品池中,使用马尔文粒径仪进行测定,并通过透射电子显微镜表征形貌。
HSA-NE、HSA-AlNE与HSA-MnNE的粒径和形貌如图2所示,水合粒径分别为130nm、160nm、130nm左右,并呈现白色球形形貌,其中HSA-AlNE颗粒表面显示一层无规则外壳,为附着于颗粒表面的氢氧化铝,正因如此,其水合粒径要稍大于HSA-NE和HSA-MnNE。HSA-AlNE和HSA-MnNE在4℃存放期间的粒径变化情况如图3a所示,结果表明本发明制备的皮克林乳剂具备良好的稳定性。
实施例6
基于甲状腺素转运蛋白MSA乳化角鲨烯(squalene)制备皮克林乳剂MSA-NE;基于氢氧化铝覆层的MSA乳化角鲨烯(squalene)制备皮克林乳剂MSA-AlNE;基于MSA-Mn乳化角鲨烯(squalene)制备皮克林乳剂MSA-MnNE;具体制备方法如下:
MSA-NE:将24mg MSA溶于2880μl HEPES(20mM,pH8.0)中,充分溶解后待用;6mg角鲨烯溶于150μl氯仿,滴加入上述溶有MSA的水相中;150W超声乳化5min,旋转蒸发除去有氯仿,即得MSA-NE。
MSA-AlNE:将24mg MSA溶于2880μl HEPES(20mM,pH8.0)中,充分溶解后待用;6mg角鲨烯溶于150μl氯仿,滴加入上述溶有MSA的水相中;150W超声乳化5min,旋转蒸发除去有氯仿,即得MSA-NE;取1ml制备好的MSA-NE,滴加100μl硫酸铝(10mM),即得MSA-AlNE。
MSA-MnNE:将12mgMSA-Mn溶于3000μl HEPES(50mM,pH7.2)中,充分溶解后待用;6mg角鲨烯溶于150μl氯仿,滴加入上述溶有MSA-Mn的水相中;150W超声乳化5min,旋转蒸发除去有氯仿,即得MSA-MnNE。
MSA-NE、MSA-AlNE、MSA-MnNE的粒径和PDI分布如表1所示。
表1.基于MSA的皮克林乳剂粒径与PDI分布
实施例7
本实施例用于检测皮克林乳剂(HSA-NE、HSA-AlNE、HSA-MnNE)对模式抗原OVA的包封率,具体方法如下:
OVA/HSA-NE:将24mg HSA与360μl OVA(1mg/ml)溶于2520μl HEPES(20mM,pH8.0)中,充分溶解后待用;6mg角鲨烯溶于150μl氯仿,滴加入上述溶有HSA的水相中;150W超声乳化5min,旋转蒸发除去有氯仿,即得OVA/HSA-NE。
OVA/HSA-AlNE:首先参考上述方法制备OVA/HSA-NE,其次,取1ml制备好的OVA/HSA-NE,滴加100μl硫酸铝(10mM),即得OVA/HSA-AlNE。OVA/HSA-MnNE:将12mgHSA-Mn与300μl OVA(1mg/ml)溶于2700μl HEPES(50mM,pH7.2)中,充分溶解后待用;6mg角鲨烯溶于150μl氯仿,滴加入上述溶有HSA-Mn的水相中;150W超声乳化5min,旋转蒸发除去有氯仿,即得OVA/HSA-MnNE。
包封率检测:采用超滤法+荧光法进行检测。OVA的包封率是通过100kDa超滤管分离游离OVA的方法来测定的。原理:超滤管的滤膜只会截留分子量大于其截留孔径的物质,使其留在内管中,结果就是内管中的物质浓度越来越高,达到了浓缩的效果。而分子量小于其孔径的物质则能够自由通过超滤管的滤膜,最终使得其在内外管中的浓度一致。被包载于皮克林乳剂中OVA,由于纳米乳剂分子量远大于100kDa,被留在了内管,外管中的OVA则为未被包载的游离OVA。通过测量外管中的OVA便可推算出OVA的包封率。
分别将游离OVA、OVA/HSA-NE、OVA/HSA-AlNE和OVA/HSA-MnNE中的OVA替换为荧光标记的OVA(FITC-OVA),其余制备条件保持一致。分别取1ml制备好的含有荧光的乳剂,使用超滤法(100kDa,3000g*15min)收集滤液,进行荧光定量。
由于蛋白质在溶液中可能存在团聚的情况,需要首先考察100kDa超滤管对游离OVA的截留情况。将OVA溶解于HEPES缓冲液中(1mg/ml),通过600g*5min的离心超滤,部分溶液通过了超滤管的滤膜到达了外管中。通过荧光定量法定量外管中游离OVA的量,发现OVA的通过率只有83.3%。部分OVA可能团聚成了分子量大于100kDa的颗粒,没能通过滤膜。因此,我们以此通过率为校正系数为计算OVA的包封率,即:
包封率=(1-外管中OVA量/(校正系数*OVA总量))*100%
结果如图3b所示,皮克林乳剂对于模式抗原OVA的包封率均在97%以上,显示出卓越的抗原包载能力。
实施例8
本实施例用于检测按照实施例7制备的含抗原皮克林乳剂在DC细胞上的摄取差异以及诱导BMDC成熟的能力差异,具体方法如下:
DC细胞摄取:以Cy5荧光标记模式抗原OVA,将Cy5-OVA替换普通OVA制备OVA/HSA-AlNE、OVA/HSA-MnNE,制备方法如实施例7所示,从而探究乳剂在体外细胞水平上的行为。取长势良好的DC2.4铺12孔板,5E5 cells/孔,待细胞长至约90%时,移除孔中培养基,加入900μl不完全培养基,并每孔加入100μl荧光比较的样品(含10μgCy5-OVA),无血无抗摄取1h后,再次移除上清,用1mlPBS吹下细胞,400g*4min离心,再用1mlPBS洗一遍,最后沉淀用300μlPBS重悬,流式检测。结果如图4a所示:相比游离OVA组,所有的乳剂组均能够显著增强DC细胞的摄取,尤其是OVA/HSA-AlNE与OVA/HSA-MnNE组,表明使用负载金属化合物的蛋白颗粒制备皮克林乳剂更有利于细胞的摄取。
BMDC成熟诱导:将BMDCs铺于12孔板中,1.5E6 cells/孔,稳定4h后,每孔加入100μl样品(含10μlOVA),继续培养24h。同时,设置阴性对照(加入100μlPBS)和阳性对照(终浓度为10μg LPS/孔)。孵育结束后,用上清反复吹打孔中细胞,并转移到1.5ml EP管中,200g*10min,收集细胞沉淀,再用1mlPBS洗一遍,再用抗小鼠CD40-PE、CD80-FITC和CD86-APC流式抗体染色,流式检测。结果如图4b所示:相比OVA组,所有的乳剂组都能够显著活化BMDC,其中OVA/HSA-MnNE组诱导成熟的能力最佳,显示出金属锰不仅可用于修饰蛋白颗粒作为乳化剂,且锰同时还能发挥佐剂作用,具有较强的免疫刺激能力。
实施例9
本实施例用于比较实施例7提供的皮克林乳剂淋巴结迁移的能力差异,具体方法如下:
利用CY5-NHS染料标记模式抗原OVA,得到Cy5-OVA。将Cy5-OVA替换普通OVA制备OVA/HSA-AlNE、OVA/HSA-MnNE,制备方法如前实施例所述。
给药情况:6~8周健康的C57BL/6小鼠,尾根部皮下注射100μl OVA/HSA-AlNE、OVA/HSA-MnNE,并以游离组和铝凝胶组(Algel)作为对照,每只小鼠都给与10μgCy5-OVA。铝凝胶组样品的制备:上下反复颠倒混匀铝凝胶佐剂溶液,吸取适量体积后加入到预先混合的游离的OVA溶液中,然后用枪反复吹打溶液数十次以使游离OVA完全吸附到铝凝胶上。
分别在给药后3h、10h、17h和24h断颈处死小鼠,剖取腹股沟淋巴结置于70μm的细胞筛网上,加入1.5ml PBS溶液,在避光的环境中,使用1ml注射器柄反复研磨淋巴结,然后吸取淋巴细胞悬液于1.5mlEP管中,400g*5min离心,最后细胞沉淀用抗小鼠CD11c、F4/80流式抗体染色,细胞滤网过滤除去肉眼可见颗粒物后,流式检测。
结果如图5a-c所示:(1)在给药后的24h内,游离OVA和2%Alge-OVA组到达淋巴结的量较低,表明其主要还是通过局部的APC细胞摄取后,再由细胞迁移至淋巴结,该过程需要的时间较长,一般在72h左右;(2)相比之下,OVA/HSA-AlNE与OVA/HSA-MnNE两组在给药后10h,均在淋巴结内实现了最高水平的富集,显著高于游离OVA与2%Algel-OVA组,表明乳剂显著提高了抗原的淋巴结迁移能力;10h以后,荧光信号呈现下降趋势,表明抗原在淋巴结内逐步被降解提呈,导致信号减弱,与此同时,该荧光信号在淋巴结内驻留DC和驻留Macrophage上均保持一致的变化水平。
实施例10
本实施例用于考查皮克林乳剂亚单位疫苗诱导体液免疫的能力。
42只SPF级雌性C57BL/6小鼠(6-8周龄),随机分成7组,其中PBS组作为阴性对照组,阳性对照组2%Algel-OVA-CDN,采用临床用氢氧化铝佐剂+CDN佐剂作为阳性对照组。OVA、OVA/HSA-NE、OVA/HSA-AlNE、OVA/HSA-MnNE和OVA/HSA-MnNE-CDN作为实验组,其中CDN为STING激动剂,这里使用的是2,3-cGAMP。具体免疫信息如下所示(表2)。
表2.免疫方案
待第三次免疫结束后第7天,即Day28,分别取各个组小鼠的眼眶血,37℃静置2h,10,000g离心10min,收集血清,使用ELISA方法检测抗体水平。
抗体检测结果如图6所示:(1)相比游离OVA组,仅OVA/HSA-MnNE、OVA/HSA-MnNE-CDN及2%Algel-OVA-CDN显示出了明显的抗体优势,而此三组之间,OVA/HSA-MnNE和OVA/HSA-MnNE-CDN表现最为均衡,2%Algel-OVA-CDN诱导IgG2a的水平略显不足,充分表明含锰皮克林乳剂确实激活体液免疫应答的能力最强;(2)OVA/HSA-AlNE仅在IgG2a上强于游离OVA组,表明其更倾向于Th1型免疫应答;(3)阳性对照组,2%Algel-OVA-CDN仅在IgG与IgG1上表现出最高水平的抗体滴度,而在IgG2a上应答不足,表明即便加入了小分子STING激动剂,铝佐剂由于其很强的滞留能力,使得抗原和佐剂更多地滞留在给药部位,主要通过募集局部的APC细胞到达淋巴结,从而最终诱导了倾向于Th2型的免疫应答。
实施例11
本实施例用于考查皮克林乳剂亚单位疫苗诱导细胞免疫的能力。
按实施例6中免疫方案免疫各组小鼠。第28天,断颈处死一定数量的空白C57BL/6小鼠,取出脾脏,研磨成单细胞悬液,加入适量的ACK裂解液重悬细胞,室温裂解10min,400g×5min离心,收集细胞;将得到脾细胞悬液均分到两个细胞皿中,一皿加入SIINFEKL抗原肽溶液刺激(终浓度为2μg/ml)作为靶细胞,另一皿加入相同体积的灭菌PBS作为内标,然后补加一定量的培养基,混匀后放入细胞培养箱中孵育1h;收集细胞,其中靶细胞用含4μM CFSE染料的PBS重悬,记作CFSEhigh,内标管用含0.4μM CFSE染料的PBS重悬,记作CFSElow,将两管混匀后放入培养箱中避光染色20min,分别向各管中加入1/2原体积的FBS以终止染色,400g×5min离心,再用PBS洗涤一遍;分别用PBS重悬细胞沉淀,将细胞浓度调整为5×107cells/ml,然后将靶细胞管与内标管等体积混合,将混合后的细胞悬液通过尾静脉注射到待测的小鼠体内,200μl/只,即细胞注射量为1×107cells/只;待18-24h后,断颈处死各组小鼠,剖取脾脏,研磨成单细胞悬液,ACK裂解并重悬细胞,进行流式检测。特异性杀伤率的计算公式如下:
根据细胞毒性T细胞杀伤(CTL)结果(图7),OVA/HSA-AlNE、OVA/HSA-MnNE、OVA/HSA-MnNE-CDN均表现出了最强的靶细胞杀伤水平,均超过80%以上,证明了含铝皮克林乳剂与含锰皮克林乳剂能够诱导高水平的细胞免疫应答。
综上实施例结果,本发明中的皮克林乳剂能够发挥佐剂与递送系统的双重功能。其能够高效地(包封率大于97%)荷载蛋白抗原,同时显著提高亚单位疫苗的体液免疫应答与细胞免疫应答水平,且在不含小分子STING激动剂的情况,OVA/HSA-AlNE与OVA/HSA-MnNE已经能够发挥足够水平的免疫刺激能力和CTL杀伤水平,这提示本发明中所设计的皮克林乳剂具备制备简单、成本低廉、增强疫苗免疫原性、以及延长疫苗保护周期的潜力。
以上所述仅是本发明的优选实施例而已,并非对本发明做任何形式的限制,虽然本发明以优选实施例揭露如上,然而并非用以上限定本发明,任何熟悉本专业的技术人员,在不脱离本发明技术方案的范围内,当可利用上述揭示的技术内容做出些许更动或修饰为等同变化的等效实施例,但凡是脱离本发明技术方案的内容,依据本发明的技术实质对以上实施例所做的任何简单修改、等同变化与修饰,均扔属于本发明技术方案的范围内。
Claims (10)
1.一种共递送抗原与佐剂的皮克林乳剂系统,其特征在于,所述皮克林乳剂包括油相、水相、蛋白抗原以及负载金属化合物的蛋白颗粒,所述蛋白抗原和负载金属化合物的蛋白颗粒在油水界面。
2.根据权利要求1所述的皮克林乳剂系统,其特征在于,所述皮克林乳剂中乳滴的平均粒径为50nm~1μm,优选为50nm~300nm;优选地,所述皮克林乳剂的油水两相体积比为1:(1~100),优选为1:(2~50);优选地,所述负载金属化合物的蛋白颗粒在水相中的质量浓度为0.1~20wt%,优选为0.5~10wt%。
3.根据权利要求1~2任一项所述的皮克林乳剂系统,其特征在于,所述负载金属化合物的蛋白颗粒中,蛋白包括内源性蛋白和外源性蛋白,内源性蛋白进一步优选为白蛋白、血红蛋白、甲状腺素转运蛋白和/或铁蛋白;外源性蛋白进一步优选为蛋白抗原,包括重组蛋白、微生物和/或植物来源的蛋白;优选地,所述白蛋白包括天然血清白蛋白和/或人工合成血清蛋白,进一步优选为人血清白蛋白、牛血清白蛋白或鼠血清白蛋白中的至少一种。
4.根据权利要求1~2任一项所述的皮克林乳剂系统,其特征在于,所述负载金属化合物的蛋白颗粒中,金属化合物包括铝类化合物、钙类化合物、钡类化合物、过度金属化合物中的至少一种;优选地,所述铝类化合物为氢氧化铝或/和磷酸铝;优选地,过度金属化合物为氢氧化铁或/和含锰化合物;所述负载金属化合物的蛋白颗粒中,金属化合物的负载方式包括配位作用、覆层、生物矿化或接枝改性中的任意一种或至少两种的组合,进一步优选为覆层或/和生物矿化。
5.根据权利要求1~2任一项所述的皮克林乳剂系统,其特征在于,所述油相包括角鲨烯、生育酚、橄榄油、大豆油、维生素E、油酸乙酯、油酸、乳酸乙酯、二甲基硅油、月桂酸异丙酯或癸酸甘油三酯中的任意一种或者至少两种的组合,进一步优选为角鲨烯、生育酚或二甲基硅油中的任意一种或者至少两种的组合;优选地,所述水相包括纯化水、注射用水、磷酸盐缓冲液、柠檬酸缓冲液或Tris缓冲液中的任意一种或者至少两种的组合;优选地,所述磷酸盐缓冲液、柠檬酸缓冲液或Tris缓冲液的pH值独立地为5.0~8.1,优选为6.0~8.0。
6.根据权利要求1~2任一项所述的皮克林乳剂系统,其特征在于,所述皮克林乳剂还包括药用添加剂;优选地,所述药用添加剂包括稀释剂、稳定剂或防腐剂中的任意一种或者至少两种的组合。
7.根据权利要求1~2任一项所述的皮克林乳剂系统,其特征在于,所述水相中和/或油水界面上还包括免疫活性物质;优选地,所述免疫活性物质包括CPG、STING激动剂、单磷酰脂质A、皂苷或溶菌酶中任意一种或者至少两种的组合。
8.根据权利要求1~7任一项所述的皮克林乳剂系统的制备方法,其特征在于,所述制备方法包括如下步骤:将蛋白抗原和负载金属化合物的蛋白颗粒分散于水相,再将油相与水相混合,直接乳化得到所述皮克林乳剂;优选地,所述乳化的方法包括超声乳化。
9.一种疫苗,其特征在于,所述抗原位于权利要求1-7任一项所述的皮克林乳剂系统的油水界面,所述抗原包括人类抗原、非人类动物抗原、植物抗原、细菌抗原、真菌抗原、病毒抗原、寄生虫抗原或肿瘤抗原中的任意一种或者至少两种的组合;优选地,所述抗原来自鸡胚培养、细胞培养、携带者体液、器官或组织中纯化分离所得、重组基因表达或化学合成所得,优选所述抗原选自减毒疫苗、灭活疫苗、裂解疫苗、亚单位疫苗或多糖结合疫苗等中的任意一种或者至少两种的组合;优选地,所述疫苗的免疫接种方式包括静脉注射、脊椎腔注射、肌内注射、皮下注射、皮内注射、经呼吸道喷入或吸入、腹腔注射、经鼻给药、经眼给药、经口给药、直肠给药、阴道给药、局部给药或头皮给药中的任意一种或者至少两种的组合。
10.权利要求1~8任一项所述的皮克林乳剂系统在制备疫苗和/或药物中的应用。
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