CN113679831B - 一种注射用水包油型乳剂黏膜疫苗及其制备方法和应用 - Google Patents
一种注射用水包油型乳剂黏膜疫苗及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种注射用水包油型乳剂黏膜疫苗及其制备方法和应用,属于生物制药领域。所述注射用水包油型乳剂黏膜疫苗包括:初乳、全反式维甲酸、金属离子化合物、抗原。所述全反式维甲酸可以溶解在制备初乳的油相中,也可以分散在含pH缓冲液的初乳中,金属离子化合物吸附于初乳表面,抗原在制备的过程中加入并通过金属离子化合物吸附于初乳表面,实现抗原和疏水性全反式维甲酸的共同包载。本发明提供的水包油型乳剂黏膜疫苗,通过注射免疫的方式,与已有方法的单一激活相比,能同时实现体液免疫、细胞免疫和黏膜免疫的多重保护,具有广阔的应用前景。
Description
技术领域
本发明属于生物制药领域,具体涉及一种注射用水包油型乳剂黏膜疫苗及其制备方法和应用。
背景技术
黏膜免疫系统,包括肠黏膜相关淋巴组织、支气管黏膜相关淋巴组织、泌尿生殖道黏膜相关淋巴组织、肺黏膜相关淋巴组织和眼结膜相关淋巴组织。黏膜表面与外界有害病原体直接接触,是机体抵抗沙门氏菌、口蹄疫病毒、幽门螺杆菌、流感病毒等感染的第一道防线。疫苗接种是防控感染疾病的最有效方式。因此,如果在诱导体液免疫和细胞免疫的同时,激活黏膜免疫,能大大提高疫苗的防控效力,从而降低防治工作的人力成本。
分泌型免疫球蛋白(SIgA)是黏膜免疫中的主要抗体,充当着黏膜的第一道防线,在黏膜免疫中起到关键作用。SIgA主要分布在唾液、泪液、肠胃液、乳汁、生殖道分泌液及呼吸道分泌液中。
疫苗的免疫途径极大地影响了其引起的免疫类型。传统的疫苗接种方式如皮下注射、肌肉注射和皮内注射由于组织限制性已被反复证明无法实现黏膜免疫的激活。引发黏膜免疫应答最有效的方式就是经黏膜途径给药。然而,高浓度可溶性抗原在黏膜表面刺激容易诱导免疫耐受。除此之外,极端的pH、丰富的蛋白酶、上皮屏障和黏膜屏障等导致黏膜给药所诱导的免疫应答往往差强人意。目前为止,应用最广的上市黏膜疫苗为脊髓灰质炎口服疫苗和轮状病毒口服疫苗。但这两种疫苗均为减毒活疫苗,存在诸多问题,例如毒力返祖的潜在危险,且其不稳定,不易于保存和运输,同时也不合适免疫缺陷及免疫功能低下人群接种。
相比之下,亚单位疫苗、多肽疫苗和DNA疫苗具有纯度高、安全性好、可规模化生产等优势,从而得到广泛的开发。但是由于这些疫苗的免疫原性普遍较弱,因此常添加佐剂以增强其诱导免疫反应的能力。另外,利用疫苗递送载体,如脂质体、纳米粒等,能够显著改善疫苗的稳定性并提高免疫应答水平。然而,这些疫苗都通过注射的方式接种,不能引发有效的黏膜免疫应答,无法实现抗原特异性SIgA的分泌。因此,亟需开发一种注射用的黏膜疫苗,以期实现体液免疫应答、细胞免疫应答和黏膜免疫应答的多重保护。
全反式维甲酸(RA)别名维A酸,维甲酸等,是维生素A的一种重要活性代谢物,在体内由维生素A通过一系列的复杂过程转化而来。研究发现肠道中的派式结树突状细胞、肠系膜淋巴结树突状细胞和固有层树突状细胞能将维生素A代谢为全反式维甲酸。全反式维甲酸不仅调控固有免疫反应中的巨噬细胞、树突状细胞和固有淋巴样细胞,而且也调控获得性免疫应发中的T细胞和B细胞的功能。此外,全反式维甲酸也能诱导黏膜归巢受体在B细胞的表达,并将B细胞转换成产生IgA的浆细胞,促进SIgA的分泌,参与黏膜免疫应答。
由于全反式维甲酸是一种难溶于水的小分子,因此一般需要使用PEG400或植物油等溶剂进行溶解,但是高剂量且频繁的给药次数对其应用带来了极大的不便,适应性差。而本发明通过将RA包载于纳米粒中解决了上述问题,实现了诱导黏膜免疫应答的效果。本发明设计一种能同时高效包载抗原和疏水性全反式维甲酸的疫苗载体,实现了全反式维甲酸与抗原的共递送,诱导了更强的免疫应答,且通过常规的注射免疫方式,同时实现了体液免疫应答、细胞免疫应答和黏膜免疫应答的有效激活,具有重要的应用价值。
全反式维甲酸是诱导黏膜免疫的关键分子,其引起的免疫应答强度与RA的剂量有关,低剂量RA往往不能诱导有效的免疫应答。因此,为了提高RA的载药量,本发明通过将其溶解在制备初乳的油相中以及通过旋转蒸发法得到含RA的薄膜再分散于上述初乳中,大大提高了RA的载药量。
以MF59为代表的水包油型乳剂被美国食品药品监督局(FDA)批准为人用佐剂已有很长的历史,其被广泛用于人麻疹和疱疹病毒疫苗、流感病毒疫苗、丙型和乙型肝炎疫苗等。乳剂型佐剂能同时刺激机体产生Th1型和Th2型免疫应答。但乳剂型佐剂能包载的抗原量通常有限,此外,金属离子类佐剂具有较广的应用,其中以铝盐为代表的铝佐剂被认为是世界上最安全的佐剂。主要以磷酸铝、硫酸钾铝和氢氧化铝两种铝盐形式存在,该类佐剂通过增加抗原滞留、缓慢释放抗原,并在局部引起炎症反应而增强免疫应答。然而铝佐剂主要引起Th2型免疫应答而非Th1型免疫应答,不利于清除侵入宿主的病原体。本发明通过在水包油型乳剂中引入金属离子佐剂,一方面通过金属离子吸附抗原,大大提高了抗原的包载量;另一方面,金属离子的纳米化使得乳剂的免疫应答类型由Th2型向Th1型转化。
发明内容
针对现有技术的以上缺陷或改进需求,本发明提供了一种注射用水包油型乳剂黏膜疫苗及其制备方法和应用,其目的在于通过将RA溶解在制备初乳的油相中,同时将RA分散在含pH缓冲液的初乳中,再将金属离子化合物吸附在初乳表面,使其比表面积增大,从而提高乳剂吸附抗原和全反式维甲酸的能力,提高全反式维甲酸的包封率。
本发明的另一个目的是提供一种注射用水包油型乳剂黏膜疫苗及其制备方法和应用,通过注射免疫后,诱导系统性黏膜免疫应答的同时,诱导高效黏膜免疫应答,应用于通过黏膜入侵的病原体感染。
为实现上述目的,本发明的疫苗包括全反式维甲酸、初乳、抗原和金属离子化合物。本发明将全反式维甲酸、油相、水相、乳化剂通过超声、高压均质、高速搅拌、微流控等方法制备得到初乳,再将含有全反式维甲酸的薄膜分散在初乳中,将金属离子化合物吸附于初乳表面,再加入抗原制成疫苗。所述全反式维甲酸同时溶解在制备初乳的油相中和分散在含pH缓冲液的初乳中,所述金属离子化合物和抗原吸附在乳剂表面。
本发明的目的之一是提供一种注射用水包油型乳剂黏膜疫苗及其制备方法和应用,其特征在于,含有初乳、全反式维甲酸、金属离子化合物和抗原,其中全反式维甲酸溶解在制备初乳的油相中,金属离子化合物吸附于初乳表面,抗原通过金属离子化合物吸附于初乳表面。其中,所述初乳包含全反式维甲酸、油相、水相、乳化剂,各组分的重要百分比为:全反式维甲酸0.01%-10%,油相0.05%-50%,乳化剂0.005%-10%,金属离子化合物0.01%-10%,抗原0.0001%-8%。
本发明中,所述强疏水性全反式维甲酸溶解初乳及制备初乳的油相中。
优选地,本发明的水包油型乳剂黏膜疫苗,其粒径为10-2000nm,优选为50-200nm,粒径分布均一。
本发明的目的之一提供了一种注射用水包油型乳剂黏膜疫苗及其制备方法和应用,其所述油相为任何医药上可应用的油,包括但不限定于动物油、植物油、天然油、矿物油、合成及半合成衍生物油、碳氢油,包括但不限定于包括角鲨烯油、生育酚及其类似物、大豆油、蓖麻油、矿物油、C6-C30脂肪及其芳香酸及醇类、中链油、油酸、亚油酸、中链甘油三酯、水不溶性维生素、C12-15烷基乳酸酯、阿甘油、菜籽油、智利油、椰子油、玉米油、棉籽油、亚麻籽油、葡萄籽油、芥末油,橄榄油、棕榈油、棕榈仁油、花生油、松子油、红花油、茶油、杏(仁)油、葡萄油、澳洲坚果油、小麦胚芽油、杏仁油、菜籽油、芝麻油、玉米油、大麻油、博伊斯油、苦杏仁油、鳄梨油、核桃油、鱼油、浆果油、多香果油、杜松子油、种子油、杏仁籽油、茴香籽油、芹菜籽油、肉豆蔻籽油、叶油、罗勒叶油、月桂叶油、桂皮叶油、鼠尾草叶油、桉树叶油、柠檬草叶油、白千层叶油、牛至叶油、广藿香叶油、薄荷叶油、松针油、迷迭香叶油、留兰香叶油、茶树叶油、百里香叶油、冬绿叶油、花油、洋甘菊油、鼠尾草油、丁香油、天竺葵花油、牛膝草花油、茉莉花油、薰衣草花油、麦卢卡花油、马贺兰花油、桔子花油、玫瑰花油、依兰花油、树皮油、桂皮油、肉桂树皮油、檫树树皮油、木材油、樟木油、雪松木材油、玫瑰木油、檀香油、根茎动物油、中的任意一种或者至少两种的组合。
优选地,所述医药上可应用的油为角鲨烯、大豆油、蓖麻油或蓖麻油类似物中的任意一种或者至少两种的组合。
本发明的目的之一提供了一种注射用水包油型乳剂黏膜疫苗及其制备方法和应用,全反式维甲酸是诱导黏膜免疫的关键分子,其引起的免疫应答强度与RA的剂量有关,低剂量RA往往不能诱导有效的免疫应答。因此,为了提高RA的载药量和包封率,本发明通过将其溶解在制备初乳的油相中以及通过旋转蒸发法得到含RA的薄膜再分散于上述初乳,以解决全反式维甲酸载药量低的技术问题。
优选地,其所述有机溶剂为可以溶解全反式维甲酸且易旋转蒸发除去的溶剂,包括乙醇、甲醇、丙醇、异丙醇、丙酮、二氯甲烷、三氯甲烷、己烷、环己烷、乙腈、四氢呋喃、乙醚、乙酸乙酯中的任意一种或至少两种的组合。
所述溶剂更优选为乙醇。
本发明的目的之一提供了一种注射用水包油型乳剂黏膜疫苗及其制备方法及其应用,所述金属离子化合物的引入,使得乳剂表面的结合位点增加,从而大大提高对疏水性全反式维甲酸和抗原的包封率和载药量,且金属离子化合物的引入并不影响原有乳剂的稳定性。全反式维甲酸与抗原共递送能同时诱导较强的系统免疫应答和黏膜免疫应答,尤其是黏膜处分泌型IgA显著提高。
优选地,所述的金属离子化合物中为可溶性的无机盐。
优选地,所述无机盐的阳离子为铝、铁、亚铁、钙、锌、锰、镁、锆、铈、镉、钴、镓的任意一种或至少两种的组合,所述无机盐的阴离子为磷酸根离子、磷酸氢根离子、磷酸二氢根离子、硫酸根离子、氯离子或氢氧根离子。
优选地,所述金属离子化合物为氢氧化铝、磷酸铝、氯化铝、硫酸铝或磷酸钙的任意一种或至少两种的组合。
优选地,所述全反式维甲酸、抗原与金属离子化合物的质量比为(0.05-4):(0.01-6):1。
本发明的目的之一提供了一种注射用水包油型乳剂黏膜疫苗及其制备方法和应用,所述乳化剂选自非离子表面活性剂、阴离子表面活性剂、阳离子表面活性剂、两性离子表面活性剂中的任意一种或至少两种的组合。
优选地,所述乳化剂包括大豆卵磷脂、蛋黄卵磷脂、氢化大豆卵磷脂、泊洛沙姆中的任意一种或至少两种的组合。
本发明的目的之一是提供了一种注射用水包油型乳剂黏膜疫苗及其制备方法和应用,其特征在于,所述抗原包括蛋白、多肽、氨基酸衍生物、核酸、寡糖、多糖、肿瘤细胞膜、肿瘤细胞裂解液、病毒或细菌裂解物、细菌细胞膜、支原体细胞膜、衣原体细胞壁、螺旋体、立克次体微荚膜、病毒包膜外泌体、细菌外膜囊泡中的任意一种或至少两种的组合。
优选地,其所述抗原为鸡卵清白蛋白OVA和鼠伤寒沙门氏菌ATCC14028裂解液的一种或一种以上的组合物。
本发明的目的之一是提供了一种注射用水包油型乳剂黏膜疫苗及其制备方法和应用,其特征在于,所述疫苗还可加入不同的佐剂以进一步提高免疫应答,所述佐剂包括Toll样受体激动剂、RIG-1样受体激动剂、NOD样受体激动剂、C性凝集素受体、STING激动剂、细菌毒素及其衍生物、矿物盐、皂苷类、多糖、细胞因子、双十二烷基二甲基溴化铵、(2,3-二油酰基-丙基)-三甲胺中的任意一种或至少两种的组合;所述佐剂占总重量的0%-20%。
优选地,所述附加佐剂包载在乳剂中或吸附在乳剂表面。
优选地,所述附加佐剂为Toll样受体激动剂或咪喹莫特中的任意一种或至少两种的组合。
所述附加佐剂更优选为Toll样受体9激动剂CpG1826。
优选地,其所述的注射用水包油型乳剂黏膜疫苗,还可以进一步附加添加剂,如防腐剂、螯合剂、抗氧化剂、填充剂、着色剂、赋形剂的一种或多种,所述添加剂占总重量的0%-0.5%。
本发明通过注射用水包油型乳剂黏膜疫苗实现了抗原和疏水性全反式维甲酸共同包载,且注射后将其高效传递到皮肤引流淋巴结,并被淋巴结内滞留的抗原提呈细胞摄取,从而诱导免疫细胞上调黏膜归巢受体CCR9,通过淋巴结的放大效应,达到免疫细胞向黏膜迁移的目的。
本发明的目的之一提供一种注射用水包油型乳剂黏膜疫苗及其制备方法和应用,其特征在于所述注射免疫包括皮下注射、肌肉注射、皮内注射、淋巴结内注射中的一种或至少两种的组合。
本发明的目的之一提供一种注射用水包油型乳剂黏膜疫苗及其制备方法和应用,其特征在于所述黏膜疫苗可应用于对抗黏膜组织相关疾病的疫苗中的应用。
优选地,所述黏膜组织相关疾病包括肠道细菌感染、肠道病毒感染、柯萨奇病毒感染、口蹄疫病毒感染、脊髓灰质炎病毒、流感病毒感染、衣原体感染、人乳头病毒感染、诺如病毒感染、艾滋病、炭疽、霍乱的任意一种或多种。
本发明的目的之一提供一种注射用水包油型乳剂黏膜疫苗,其特征在于,包括以下步骤:
(1)将全反式维甲酸和亲油性乳化剂溶于油相中,亲水性乳化剂溶于水相中,将二者通过超声、均质高速搅拌或微流控等方法充分混匀后形成初乳;
(2)将全反式维甲酸溶解在有机溶剂中,旋转蒸发除去有机溶剂得到薄膜;
(3)将pH缓冲液加入到步骤(1)制得的初乳中;
(4)将步骤(3)制得的初乳加入到步骤(2)制得的薄膜中水化;
(5)将金属离子化合物加入到步骤(4)制得的混合物中;
(6)将抗原加入到步骤(5)制得的混合物中,混合均匀即得;
优选地,步骤(3)所述pH缓冲液包括氯化钠溶液、柠檬酸钠缓冲液、磷酸盐缓冲液、Hepes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pH缓冲液pH值在5.0-10.0之间;优选为6.0-8.0之间。
本发明提供的注射用水包油型乳剂黏膜疫苗,相比于现有技术,具有以下优点:
1.本发明提供的注射用水包油型乳剂黏膜疫苗,其将全反式维甲酸溶解在制备初乳的油相中,同时分散在初乳中,抗原吸附在乳剂表面,实现疏水性全反式维甲酸和抗原的共包载和递送。
2.本发明提供的注射用水包油型乳剂黏膜疫苗,其所述初乳先通过超声、高压均质或高速搅拌等混合方法制备初乳,然后再涡旋、搅拌或者微流控技术等温和的条件下加入金属离子化合物和抗原的混合物,可以有效地保护抗原的构象和活性,且能实现大规模生产,利于工业转化。
3.本发明提供的注射用水包油型乳剂黏膜疫苗,与传统乳剂相比,其抗原包载能力提高,金属离子化合物的加入使得免疫应答能力提高,且与传统铝佐剂疫苗相比,具有更高的免疫应答能力和储存稳定性。
4.本发明提供的注射用水包油型乳剂黏膜疫苗,实现了疏水性全反式维甲酸和抗原的包载和共递送,经注射免疫后,能通过淋巴管迅速迁移到皮肤引流淋巴结,并被淋巴结内的抗原提呈细胞摄取和呈递。全反式维甲酸与维甲酸受体作用后,上调黏膜归巢受体CCR9,通过淋巴结的放大效应,能使更多的免疫细胞迁移到黏膜处,从而提高SIgA的分泌。
5.本发明提供的注射用水包油型乳剂黏膜疫苗与传统注射类疫苗相比,经注射免疫后,能同时激活机体的体液免疫、细胞免疫和黏膜免疫应答,促进粪便、鼻腔、粪便、肠道及阴道等多种黏膜处SIgA的分泌,实现系统性免疫和黏膜免疫的双重保护,适用于黏膜相关感染病的预防和治疗,具有广阔的应用前景。
附图说明
图1是本发明实施例1制备的水包油型乳剂EMR的粒径数据。
图2是本发明实施例2制备的水包油型乳剂EMR的粒径数据。
图3是本发明实施例3制备的水包油型乳剂EMR的透射电镜图片。
图4是本发明实施例4制备的水包油型乳剂EMR的粒径数据。
图5是本发明实施例1制备的水包油型乳剂EMR和不含金属离子的乳剂MR中RA和OVA的包封率测定。
图6a、b是本发明实施例1制备的水包油型乳剂EMR在4℃的粒径变化图。
图7是本发明实施例1制备的通过将RA溶解在油相中及通过旋转蒸发得到薄膜分散在初乳中得到的水包油型乳剂EMR,和直接将RA溶解在油相中得到的水包油型乳剂EMR-中RA的载药量的测定。
图8是本发明实施例8制备的水包油型乳剂EMR@CpG的粒径数据。
图9是本发明实施例9制备的水包油型乳剂EMR@CpG凝胶电泳图,CpG为游离0.5μgCpG的凝胶电泳。
图10是本发明实施例10中制备的载鼠伤寒沙门氏菌(ATCC14028)裂解液抗原的水包油型乳剂EMR的粒径数据。
图11是实施例1制备的水包油型乳剂EMR被抗原提呈细胞摄取情况的考察。
图12a、b是实施例1制备的水包油型乳剂EMR和实施例8制备的EMR@CpG的被淋巴结内抗原提呈细胞摄取情况的考察。
图13a、b、c是实施例1制备的水包油型乳剂EMR和实施例8制备的EMR@CpG皮下免疫后在血清中IgG、IgG1和IgG2a抗体的检测。
图14是实施例1制备的水包油型乳剂EMR和实施例8制备的EMR@CpG诱导肠系膜淋巴结表达黏膜归巢因子CCR9的情况。
图15是实施例1制备的水包油型乳剂EMR和实施例8制备的EMR@CpG皮下免疫后在粪便中SIgA的情况。
图16是实施例1制备的水包油型乳剂EMR和实施例8制备的EMR@CpG皮下免疫后在阴道黏膜处SIgA分泌的情况。
图17是实施例1制备的水包油型乳剂EMR和实施例8制备的EMR@CpG在肠道黏膜处SIgA分泌的情况。
具体实施方式
实施例1
水包油型乳剂的制备:将0.05g吐温80和0.204g卵磷脂溶解在15ml注射用水中,将0.5g全反式维甲酸(RA)溶解于0.5g角鲨烯中并加入上述溶液中,经高压均质混合形成初乳。将RA溶于乙醇中制成浓度为1mg/ml的储备液,取0.8ml RA储备液通过旋转蒸发除去乙醇后,加入0.53ml上述初乳和1ml 100mM pH为8的Hepes缓冲液的混合物,加入6μL 1M氢氧化钠后逐滴加入40μL 75mM硫酸铝水溶液,涡旋30s。接着,向其中加入35μL 2mg/ml的OVA水溶液,涡旋30s,得到共载RA和抗原的水包油型乳剂(EMR),用马尔文Nano-ZS 90粒径仪检测粒径,结果参见图1。结果显示,粒径约为58nm,PDI 0.140,粒径分布均一。
实施例2
水包油型乳剂的制备:将0.05g吐温80和0.204g卵磷脂溶解在15ml注射用水中,将0.5g全反式维甲酸(RA)溶解于4.5g角鲨烯中并加入上述溶液中,经高压均质混合形成初乳。将RA溶于乙醇中制成浓度为1mg/ml的储备液,取0.8ml RA储备液通过旋转蒸发除去乙醇后,加入0.53ml上述初乳和1ml 100mM pH为8的Hepes缓冲液的混合物,涡旋下加入6μL1M氢氧化钠后逐滴加入40μL 75mM硫酸铝水溶液,涡旋30s。接着,向其中加入35μL 2mg/ml的OVA水溶液,涡旋30s,得到共载RA和抗原的水包油型乳剂EMR,用马尔文Nano-ZS 90粒径仪检测粒径,结果参见图2。结果显示,粒径约为55nm,PDI 0.203,粒径分布均一。
实施例3
水包油型乳剂的制备:将0.05g吐温80和0.204g卵磷脂溶解在15ml注射用水中,将0.5g全反式维甲酸(RA)溶解于0.4g大豆油中并加入上述溶液中,经高压均质混合形成初乳。将RA溶于乙醇中制成浓度为1mg/ml的储备液,取0.8ml RA储备液通过旋转蒸发除去乙醇后,加入0.53ml上述初乳和1ml 100mM pH为8的Hepes缓冲液的混合物,涡旋下加入6μL1M氢氧化钠后逐滴加入35μL 75mM硫酸铝水溶液,涡旋30s即得;接着向其中加入35μL 2mg/ml的OVA水溶液,涡旋30s,得到共载RA和抗原的水包油型乳剂EMR,取少量样品,用透射电镜检测,结果参见图3。结果显示粒径在50-80nm之间,粒径分布均一。
实施例4
水包油型乳剂的制备:将0.05g吐温80和0.204g卵磷脂溶解在15ml注射用水中,将0.5g全反式维甲酸(RA)溶解于6.0g大豆油中并加入上述溶液中,经高压均质混合形成初乳。将RA溶于乙醇中制成浓度为1mg/ml的储备液,取0.8ml RA储备液通过旋转蒸发除去乙醇后,加入0.53ml上述初乳和1ml 100mM pH为8的Hepes缓冲液的混合物,涡旋下加入6μL1M氢氧化钠后逐滴加入35μL 75mM硫酸铝水溶液,涡旋30s即得;接着向其中加入35μL 2mg/ml的OVA水溶液,涡旋30s,得到共载RA和抗原的水包油型乳剂EMR,取少量样品,用马尔文Nano-ZS 90粒径仪检测粒径,结果参见图4。结果显示,粒径约为55nm,PDI 0.168,粒径分布均一。
实施例5
水包油型乳剂对RA和抗原包封率和载药量的测定:将0.05g吐温80和0.204g卵磷脂溶解在15ml注射用水中,将0.5g全反式维甲酸(RA)溶解于0.5g角鲨烯中并加入上述溶液中,经高压均质混合形成初乳。将RA溶于乙醇中制成浓度为1mg/ml的储备液,取0.8ml RA储备液通过旋转蒸发除去乙醇后,加入0.53ml上述初乳和1ml 100mM pH为8的Hepes缓冲液的混合物,向其中加入35μL 2mg/ml的OVA水溶液,涡旋30s,得到不含金属离子的共载RA和抗原的乳剂MR。同时测定MR和实施例1制备的共载RA和抗原的水包油型乳剂EMR中RA和OVA的包封率。RA通过高效液相色谱HPLC测定,OVA通过BCA试剂盒测定。结果参见图5,结果显示不含金属离子的乳剂MR对RA和OVA的包封率仅为10%,含金属离子的EMR中RA和OVA的包封率约为89%和92%,可见,金属离子的加入能大大提高了RA和OVA的包封率。
实施例6
水包油型乳剂在4℃的稳定性:将实施例1制备得到的共载RA和OVA的水包油型乳剂EMR分别于不同时间用马尔文Nano-ZS 90粒径仪检测粒径,以观察金属离子的加入对乳剂稳定性的影响,结果参见图6。图a、b分别表示粒径和均一性。结果显示,加入金属离子的水包油型乳剂EMR在4℃的粒径无明显变化,说明其具有良好的稳定性。
实施例7
水包油型乳剂中RA的包封率和载药量测定:将0.05g吐温80和0.204g卵磷脂溶解在15ml注射用水中,将0.5g全反式维甲酸(RA)溶解于0.5g角鲨烯中并加入上述溶液中,经高压均质混合形成初乳。取0.53ml上述初乳和1ml100mM pH为8的Hepes缓冲液的混合物,加入6μL 1M氢氧化钠后逐滴加入40μL 75mM硫酸铝水溶液,涡旋30s。接着,向其中加入35μL2mg/ml的OVA水溶液,涡旋30s,得到水包油型乳剂(EMR-),与实施例1制备得到的水包油型乳剂EMR比较载药量。结果参见图7,EMR中RA的载药量为5%,EMR-中RA的载药量为2%,可见,将RA溶解在制备初乳的油相中同时制得薄膜后分散在乳剂中能大大提高RA的载药量(数据分析采用单因素方差法进行各组之间均值的比较,***:P<0.001)。
实施例8
水包油型乳剂的制备:将0.05g吐温80和0.204g卵磷脂溶解在15ml注射用水中,将0.5g全反式维甲酸(RA)溶解于0.5g角鲨烯中并加入上述溶液中,经高压均质混合形成初乳。将RA溶于乙醇中制成浓度为1mg/ml的储备液,取0.8ml RA储备液通过旋转蒸发除去乙醇后,加入0.53ml上述初乳和1ml 100mM pH为8的Hepes缓冲液,涡旋下加入6μL 1M氢氧化钠后逐滴加入40μL 75mM硫酸铝水溶液,涡旋30s。接着,向其中加入35μL 2mg/ml的OVA水溶液和7μL1mg/ml的CpG1826水溶液,涡旋30s得到共载RA、抗原和佐剂的水包油型乳剂EMR@CpG,取少量样品,用马尔文Nano-ZS 90粒径仪检测粒径,结果参见图8。结果显示,粒径约为66nm,PDI 0.182,粒径分布均一。
实施例9
水包油型乳剂对CpG的包载能力测定:取实施例7制备的水包油型乳剂EMR@CpG 20μL进行琼脂糖凝胶电泳实验以检测乳剂对核酸的包载能力,结果参见图9,结果说明20μL乳剂可以包载1μg CpG。
实施例10
水包油型乳剂的制备:将0.05g吐温80和0.204g卵磷脂溶解在15ml注射用水中,将0.5g全反式维甲酸(RA)溶解于0.5g角鲨烯中并加入上述溶液中,经高压均质混合形成初乳。将RA溶于乙醇中制成浓度为1mg/ml的储备液,取0.8ml RA储备液通过旋转蒸发除去乙醇后,加入0.53ml上述初乳和1ml 100mM pH为8的Hepes缓冲液,涡旋下加入6μL 1M氢氧化钠后逐滴加入40μL 75mM硫酸铝水溶液,涡旋30s。接着,向其中加入35μL 2mg/ml的鼠伤寒沙门氏菌ATCC14028裂解液的水溶液,涡旋30s即得。用马尔文Nano-ZS 90粒径仪检测粒径,结果参见图10。结果显示,粒径约为60nm,PDI 0.147,粒径分布均一。
实施例11
水包油型乳剂在DC2.4上的摄取:在12孔板中接种1×106/孔DC2.4细胞,4个复孔,每孔用1ml 1640完全培养基培养,细胞贴壁后更换400μL 1640不完全培养基,向其中加入100μL用FITC标记的OVA制备的载RA的乳剂(实施例1制备),其中EM为不含RA的对照组,37℃,5%CO2培养环境中摄取1h,PBS洗涤3次后,用流式细胞仪检测,结果参见图11,其中OVA为游离的FITC标记的OVA,实验显示,与游离OVA相比,本发明制备的水包油型乳剂能被DC2.4细胞高效摄取,(数据分析采用单因素方差法进行各组之间均值的比较,****:P<0.0001),为免疫应答的产生提供基础。
实施例12
水包油型乳剂的淋巴结靶向:6-8周雌性BALB/c小鼠尾根部皮下注射50μlFITC-OVA制备的OVA+RA+CpG(即为游离RA、OVA和CpG的混合组)、EM@CpG(即为不含RA,其余与实施例7相同)、EMR@CpG(即实施例7),在6h处死小鼠,取腹股沟淋巴结研磨后,用抗小鼠CD11和F4/80抗体染色后,流式检测,结果参见图12。图a和b分别代表OVA和CpG分别被淋巴结内树突状细胞和巨噬细胞摄取的量。由实验结果可知,水包油型乳剂注射后在体内能快速有效地传递到皮肤引流淋巴结,并被淋巴结滞留的抗原提呈细胞摄取,与游离OVA、RA和CpG的混合组相比,EMR@CpG组与EM@CpG被淋巴结内抗原提呈细胞摄取能力显著提高,(数据分析采用单因素方差法进行各组之间均值的比较,*:P<0.05;**:P<0.01;***:P<0.001;****:P<0.0001),且含RA的EMR@CpG组与EM@CpG组相比,EMR@CpG组被淋巴结内树突状细胞和巨噬细胞摄取能力显著提高,(数据分析采用单因素方差法进行各组之间均值的比较,*:P<0.05;***:P<0.001),为诱导免疫细胞黏膜归巢能力提供了可能。
实施例13
水包油型乳剂的皮下注射诱导系统免疫应答:6-8周雌性BALB/c小鼠随机分为7组,分组为PBS组、OVA+RA组、EMR(实施例1)组、OVA+RA+CpG组、EM@CpG组(不含RA,其余与实施例7相同)、EMR@CpG(实施例7)组,每组5只动物,对小鼠进行尾根部皮下给药,给药体积为100μL,每只小鼠OVA给药量为10μg,CpG给药量为1μg,RA给药量为33.7μg。分别于第0天、第14天和第28天各免疫一次。第35天对小鼠进行眼眶取血约400μL,4℃条件下放置2h以上,6000rpm离心10min,取血清用ELISA法检测血清中抗体的分泌,结果参见图13。图a、b、c分别代表106稀释倍数下血清的IgG、IgG1和IgG2a的检测结果,实验结果说明,与游离的OVA和RA的混合组相比,共载OVA和RA的水包油型乳剂EMR能显著提高血清中IgG和IgG1的分泌,(数据分析采用单因素方差法进行各组之间均值的比较,*:P<0.05;***:P<0.001)。当加入Th1型佐剂CpG后,乳剂提高三种抗体分泌的能力大大提升,且与游离的OVA、RA和CpG组相比差异显著,(数据分析采用单因素方差法进行各组之间均值的比较,*:P<0.05;***:P<0.001)。IgG1和IgG2a的分泌能从一定程度反映Th2型和Th1型免疫应答的强弱,实验结果说明皮下注射后,水包油型乳剂能诱导较强的细胞免疫应答和体液免疫应答,且EMR@CpG的抗体水平高于EM@CpG组,(数据分析采用单因素方差法进行各组之间均值的比较,*:P<0.05)。可见,加入RA能显著提高水包油型乳剂的免疫应答能力。
实施例14
水包油型乳剂诱导黏膜免疫应答:水包油型乳剂的淋巴结靶向诱导系统免疫应答:6-8周雌性BALB/c小鼠随机分为7组,分组为PBS组、OVA+RA组、EMR(实施例1)组、OVA+RA+CpG组、EM@CpG组(不含RA,其余与实施例7相同)、EMR@CpG(实施例7)组,每组5只动物,对小鼠进行尾根部皮下给药,给药体积为100μL,每只小鼠OVA给药量为10μg,CpG给药量为1μg,RA给药量为33.7μg。分别于第0天、第14天和第28天各免疫一次。第35天处死小鼠,分离肠系膜淋巴结并研磨后,用抗小鼠CCR9抗体染色后,流式细胞仪检测,结果参见图14。结果显示,EMR@CpG皮下注射后能显著上调肠系膜淋巴结中黏膜归巢因子CCR9的表达,而EM@CpG则无此现象,可见,加入RA并将其与抗原和佐剂共载制成水包油型乳剂可以上调CCR9,(数据分析采用单因素方差法进行各组之间均值的比较,***:P<0.001;****:P<0.0001),具有预料不到的技术效果。
实施例15
水包油型乳剂诱导黏膜免疫应答:水包油型乳剂的淋巴结靶向诱导系统免疫应答:6-8周雌性BALB/c小鼠随机分为7组,分组为PBS组、OVA+RA组、EMR(实施例1)组、OVA+RA+CpG组、EM@CpG组(不含RA,其余与实施例7相同)、EMR@CpG(实施例7)组,每组5只动物,对小鼠进行尾根部皮下给药,给药体积为100μL,每只小鼠OVA给药量为10μg,CpG给药量为1μg,RA给药量为33.7μg。分别于第0天、第14天和第28天各免疫一次。第28天收集小鼠的新鲜粪便,以2mL/g的浓度加入含0.05mM EDTA-Na2、1mM PMSF的10mM PBS溶液中,破碎后水浴超声10min,10000rpm离心10min,取上清后再以相同转速离心一次后取上清用ELISA法检测粪便SIgA的分泌,结果参见图15。其结果显示,EMR@CpG粪便中SIgA抗体水平最高,与游离OVA、RA和CpG相比,EMR@CpG乳剂能显著提高粪便中SIgA的抗体水平,(数据分析采用单因素方差法进行各组之间均值的比较,**:P<0.01);不含RA的乳剂EM@CpG组,其粪便中的SIgA的分泌情况与空白对照组PBS相当;EMR@CpG组SIgA分泌情况显著高于EM@CpG组,(数据分析采用单因素方差法进行各组之间均值的比较,***:P<0.001);可见,水包油型乳剂中RA的加入可以上调黏膜归巢受体CCR9,并通过淋巴结的放大效应,使更多的淋巴细胞迁移到肠系膜淋巴结,诱导黏膜免疫应答,从而提高粪便中SIgA的分泌,取得了预料不到的技术效果。
实施例16
水包油型乳剂诱导黏膜免疫应答:水包油型乳剂的淋巴结靶向诱导系统免疫应答:6-8周雌性BALB/c小鼠随机分为7组,分组为PBS组、OVA+RA组、EMR(实施例1)组、OVA+RA+CpG组、EM@CpG组(不含RA,其余与实施例7相同)、EMR@CpG(实施例7)组,每组5只动物,对小鼠进行尾根部皮下给药,给药体积为100μL,每只小鼠OVA给药量为10μg,CpG给药量为1μg,RA给药量为33.7μg。分别于第0天、第14天和第28天各免疫一次。第28天收集收集小鼠阴道灌洗液:用移液枪吸取含1%BSA、1mM PMSF的10mM PBS溶液,从小鼠阴道部位轻轻推入,但应注意避免推入溶液体积过多而流出,吸出后再次推入,重复多次,收集阴道灌洗液总体积为150μL,而后10000rpm离心10min,取上清用ELISA发检测阴道黏膜处SIgA的分泌,参见图16,其结果显示,EMR@CpG组阴道灌洗液中SIgA抗体水平最高,与游离OVA、RA和CpG相比,EMR@CpG乳剂能显著提高阴道灌洗液中SIgA的抗体水平,(数据分析采用单因素方差法进行各组之间均值的比较,**:P<0.01);不含RA的乳剂EM@CpG组,其阴道灌洗液中的SIgA的分泌情况与游离OVA、RA和CpG相比相当,可见,共载RA、OVA和CpG的水包油型乳剂中可以上调黏膜归巢受体CCR9,并通过淋巴结的放大效应,使更多的淋巴细胞迁移到肠系膜淋巴结,诱导黏膜免疫应答,从而提高阴道灌洗液中SIgA的分泌。
实施例17
水包油型乳剂诱导黏膜免疫应答:水包油型乳剂的淋巴结靶向诱导系统免疫应答:6-8周雌性BALB/c小鼠随机分为7组,分组为PBS组、OVA+RA组、EMR(实施例1)组、OVA+RA+CpG组、EM@CpG组(不含RA,其余与实施例7相同)、EMR@CpG(实施例7)组,每组5只动物,对小鼠进行尾根部皮下给药,给药体积为100μL,每只小鼠OVA给药量为10μg,CpG给药量为1μg,RA给药量为33.7μg。分别于第0天、第14天和第28天各免疫一次。第35天处死小鼠后取出整个肠段,加入4mL含1mM PMSF的1%PBS溶液中,剪碎至无明显组织块,4℃条件下10000rpm离心10min,离心2次,收集上清用ELISA法肠道内黏膜中SIgA的分泌,结果参见图17。结果显示EMR@CpG皮下免疫后能显著提高其在肠道黏膜处SIgA的分泌,(数据分析采用单因素方差法进行各组之间均值的比较,*:P<0.05);不含RA的乳剂EM@CpG组,其阴道灌洗液中的SIgA的分泌情况与游离OVA、RA和CpG相比相当,可见,共载RA、OVA和CpG的水包油型乳剂中可以上,而OVA+RA+CpG组与EM@CpG则无此现象,可见,加入RA并将其与抗原和佐剂共载制成水包油型乳剂可以上调黏膜归巢受体CCR9,并通过淋巴结的放大效应,诱导黏膜免疫应答,从而显著提高肠道中SIgA的分泌。
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上的限制,虽然本发明已以优选实施例揭露如上,然而并非用以限定本发明,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,但凡是未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,均仍属于本发明技术方案的范围内。
Claims (16)
1.一种注射用水包油型乳剂黏膜疫苗,其特征在于,含有油相、水相、乳化剂、全反式维甲酸、金属离子化合物和抗原,所述注射用水包油型乳剂黏膜疫苗的制备方法,包括以下步骤:
(1)将全反式维甲酸和亲油性乳化剂溶于油相中,亲水性乳化剂溶于水相中,将二者通过超声、均质高速搅拌或微流控方法充分混匀后形成初乳;
(2)将全反式维甲酸溶解在有机溶剂中,所述有机溶剂为可溶解全反式维甲酸且易旋转蒸发去除的溶剂,旋转蒸发除去有机溶剂得到薄膜;
(3)将pH缓冲液加到步骤(1)所得的初乳中;
(4)将步骤(3)制得的混合物加入步骤(2)所得薄膜中;
(5)将金属离子化合物加入到步骤(4)制得的混合物中;
(6)将抗原加入到步骤(5)制得的混合物中,混合均匀即得;
其中金属离子化合物吸附于初乳表面,全反式维甲酸溶解在制备初乳的油相中以及存在于除去有机溶剂得到的薄膜再分散在含pH缓冲液的初乳中,所述的金属离子化合物为可溶性的无机盐,所述无机盐的阳离子为铝,所述无机盐的阴离子为磷酸根离子、磷酸氢根离子、磷酸二氢根离子、硫酸根离子、氯离子或氢氧根离子,抗原通过金属离子化合物吸附于初乳表面。
2.根据权利要求1所述的注射用水包油型乳剂黏膜疫苗,其特征在于,所述注射用水包油型乳剂黏膜疫苗中组分的质量比为:全反式维甲酸0.01%-10%,油相0.05%-50%,乳化剂0.005%-10%,金属离子化合物0.01%-10%,抗原0.0001%-8%。
3.根据权利要求2所述的注射用水包油型乳剂黏膜疫苗,其特征在于,所述全反式维甲酸、抗原与金属离子化合物的质量比为(0.05-4):(0.01-6):1。
4.根据权利要求1所述的注射用水包油型乳剂黏膜疫苗,其特征在于,所述注射用水包油型乳剂黏膜疫苗的平均粒径为10nm-2000nm。
5.根据权利要求4所述的注射用水包油型乳剂黏膜疫苗,其特征在于,所述注射用水包油型乳剂黏膜疫苗的平均粒径为50nm-200nm。
6.根据权利要求1所述的注射用水包油型乳剂黏膜疫苗,其特征在于,所述金属离子化合物为氢氧化铝、磷酸铝、氯化铝、硫酸铝的任意一种或至少两种的组合。
7.根据权利要求1-6任一所述的注射用水包油型乳剂黏膜疫苗,其特征在于,所述油相为任何医药上可应用的油。
8.根据权利要求1所述的注射用水包油型乳剂黏膜疫苗,其特征在于,所述有机溶剂为乙醇、甲醇、丙醇、异丙醇、丙酮、二氯甲烷、三氯甲烷、己烷、环己烷、乙腈、四氢呋喃、乙醚、乙酸乙酯中的任意一种或至少两种的组合。
9.根据权利要求8所述的注射用水包油型乳剂黏膜疫苗,其特征在于,所述有机溶剂为乙醇。
10.根据权利要求1-6任一所述的注射用水包油型乳剂黏膜疫苗,其特征在于,所述抗原为蛋白、多肽、氨基酸衍生物、核酸、寡糖、多糖、肿瘤细胞膜、肿瘤细胞裂解液、病毒或细菌裂解物、细菌细胞膜、支原体细胞膜、衣原体细胞壁、螺旋体、立克次体微荚膜、病毒包膜外泌体、细菌外膜囊泡中的任意一种或至少两种的组合。
11.根据权利要求1-6任一所述的注射用水包油型乳剂黏膜疫苗,其特征在于,所述注射用水包油型乳剂疫苗含有佐剂,所述佐剂为Toll样受体9激动剂CpG1826,所述佐剂包载在乳剂中或吸附在乳剂表面,所述佐剂的质量百分比为0.01%-10%。
12.根据权利要求1所述的注射用水包油型乳剂黏膜疫苗,其特征在于,所述pH缓冲液为氯化钠溶液、柠檬酸钠缓冲液、磷酸盐缓冲液、Hepes缓冲液或Tris缓冲液中的任意一种或者至少两种的组合。
13.根据权利要求1所述的注射用水包油型乳剂黏膜疫苗,其特征在于,所述pH缓冲液pH值在5.0-10.0之间。
14.根据权利要求13所述的注射用水包油型乳剂黏膜疫苗,其特征在于,所述pH缓冲液pH值在6.0-8.0之间。
15.根据权利要求1-6中任一所述的注射用水包油型乳剂黏膜疫苗,其特征在于,所述注射用水包油型乳剂黏膜疫苗的免疫方式为注射免疫。
16.根据权利要求15所述的注射用水包油型乳剂黏膜疫苗,其特征在于,所述注射免疫为皮下注射、肌肉注射、皮内注射、淋巴结内注射中的一种或至少两种的组合。
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