CN106729700B - 表面胍基修饰的纳米佐剂材料及其制备方法与应用 - Google Patents
表面胍基修饰的纳米佐剂材料及其制备方法与应用 Download PDFInfo
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Abstract
本发明涉及一种表面胍基修饰的纳米佐剂材料及其制备方法与应用,其组成为:聚乙二醇‑b‑聚己内酯‑g‑聚(胍基‑乙基‑甲基丙烯酸酯),其中ε‑CL(ε‑己内酯)与BMPCL(γ‑(2‑溴‑2‑甲基丙酸酯)‑ε‑己内酯)的聚合度范围分别为30~35和3~3.5,胍基‑乙基‑甲基丙烯酸酯的聚合度为3~105。该纳米佐剂由三嵌段共聚物自组装形成。将抗原与纳米佐剂混合即获得纳米疫苗体系。本发明聚合物纳米佐剂具有良好的稳定性与较强的免疫原性,可以有效促进抗原被树突状细胞等抗原呈递细胞的摄取与交叉呈递。体内免疫该纳米疫苗能够显著提高抗原特异性的体液与细胞免疫应答。本发明合成与纳米疫苗制备方法简单、操作简便、重复性好,适合于大规模生产。
Description
技术领域
本发明属于免疫学领域,特别涉及一种表面胍基修饰的纳米佐剂材料及其制备方法与应用。
背景技术
疫苗是一种主要由蛋白、多糖或核酸等成分基于传统方法或基因工程等生物技术加工而成的生物制品,用于人类疾病的预防和治疗。根据制备疫苗的技术和疫苗成分,疫苗分为传统疫苗和新型疫苗或高技术疫苗。传统疫苗包括灭活、减毒活疫苗和从微生物及其衍生物分离提取的亚单位疫苗,如蛋白疫苗和多糖疫苗。新型疫苗包括基因工程亚单位疫苗、重组载体活疫苗、核酸疫苗、基因缺失活疫苗、遗传重配疫苗及合成肽疫苗。疫苗的最终目的是促进机体产生长期、有效的抗病原体感染的能力,然而当前的众多疫苗其免疫效力差强人意,因此需要佐剂增强其体液和细胞免疫应答。
纳米材料作为一种新型佐剂,能够模拟大多数生物活性物质如病毒,细菌及其它病原体等的天然球形结构,在高效包载抗原及免疫增强剂,增强抗原稳定性,促进抗原递呈细胞(antigen presenting cell,APC)对抗原的摄取与加工,调控抗原在细胞内的递送,促进抗原的交叉呈递,引起更强的体液免疫和细胞免疫反应等方面具有重要的作用。因此,基于纳米生物材料的疫苗佐剂将成为突破传统疫苗瓶颈的有力手段,促进重大疾病相关的疫苗研制与免疫治疗的发展与进步。然而,常规的纳米佐剂仍然存在免疫原性弱、抗原负载率低、生物安全性不明确、纳米佐剂的物理化学性质与疫苗免疫应答之间的关系不明确等问题。
中国专利CN201410519465.8公开了一种阳离子聚合物纳米免疫佐剂的制备方法,引入带正电核的聚乙烯亚胺(分子量为2000 g/mol),利用疏水相互作用形成mPEG-PCL-PEI三嵌段聚合物纳米体系。关于表面胍基修饰的纳米佐剂材料及其制备方法未见报道。
发明内容
本发明的目的是提供一种表面胍基修饰的纳米佐剂材料及其制备方法与应用。该纳米佐剂可以负载各类蛋白、多肽或核酸抗原。本发明具有免疫原性强、抗原负载率高、稳定性强、体内外免疫应答强等优点,可用于免疫疫苗的制备。
本发明提供的一种表面胍基修饰的纳米佐剂材料(含有胍基的共聚物)为:
聚乙二醇-b-聚己内酯-g-聚(胍基-乙基-甲基丙烯酸酯),其中ε-CL (ε-己内酯)与BMPCL (γ-(2-溴-2-甲基丙酸酯)-ε-己内酯)的聚合度范围分别为30~35和3~3.5,胍基-乙基-甲基丙烯酸酯的聚合度为3~105。
所述共聚物为三嵌段共聚物,主链结构为聚乙二醇与聚己内酯嵌段共聚物,聚己内酯侧链含有胍基。
本发明的纳米佐剂材料的制备方法包括以下步骤:
1)将干燥的聚乙二醇单甲醚(mPEG)加入Schlenk管中,称取定量的ε-己内酯(ε-CL)、γ-(2-溴-2-甲基丙酸酯)-ε-己内酯(BMPCL)与辛酸亚锡加入反应管中,在氮气保护下于120-130℃油浴中,搅拌反应10-20小时。待反应冷却后,加入二氯甲烷溶解产物,然后将溶液滴加至冷乙醚中,过滤,取沉淀,真空干燥,得到聚乙二醇与聚己内酯嵌段共聚物大分子引发剂mPEG-b-P(CL-co-BMPCL)。
2)将步骤1)制备的mPEG-P(CL-co-BMPCL),单体2-[(叔丁氧基羰基)氨基]乙基-甲基丙烯酸酯(tBMA),以及二联吡啶溶于丁酮。将上述混合物反复进行三次除氧后,加入溴化亚铜,在无氧和60℃条件下反应24小时。在纯水中反复透析提纯,冷冻干燥获得聚乙二醇-b-聚己内酯-g-聚(2-[(叔丁氧基羰基)氨基]乙基-甲基丙烯酸酯)(mPEG-b- PCL-g-PtBMA)。
3)将mPEG-b-PCL-g-PtBMA溶于三氟乙酸,室温搅拌2-5小时。在真空条件下将三氟乙酸旋蒸掉,然后加入N,N-二甲基甲酰胺溶解产物,将所获溶液滴加到冷乙醚中,过滤,取沉淀。用无水乙醚洗涤两次,真空干燥获得聚乙二醇-b-聚己内酯-g-聚(氨基-乙基-甲基丙烯酸酯)(mPEG-b-PCL-g-PAEM)。
4)将PEG-b-PCL-g-PAEM溶于碳酸氢钠水溶液中,加入S-乙基异硫脲氢溴酸盐,室温下搅拌反应48-96小时。然后在纯水中透析提纯,冷冻干燥后获得聚乙二醇-b-聚己内酯-g-聚(胍基-乙基-甲基丙烯酸酯)(mPEG-b-PCL-g-PGEM,PECG)。
聚乙二醇单甲醚的分子量为2000 g/mol,聚己内酯的分子量范围为3420-3990 g/mol,聚(γ-(2-溴-2-甲基丙酸酯)-ε-己内酯(BMPCL))的分子量范围为834-973 g/mol,聚(胍基-乙基-甲基丙烯酸酯)的分子量范围为516-18060 g/mol。
本发明所述的纳米佐剂材料的制备步骤1)中,单体ε-CL与聚乙二醇单甲醚的摩尔比为30~35:1;BMPCL与ε-CL的摩尔比为1:10;辛酸亚锡的投料量为单体摩尔总数的0.05%。
本发明所述的纳米佐剂材料的制备步骤2)中,单体tBMA与大分子引发剂mPEG-b-P(CL-co-BMPCL)的摩尔比为3~105:1。
本发明所述的纳米佐剂材料的制备步骤3)中,三氟乙酸的体积(mL)与mPEG-b-PCL-g-PtBMA的质量(g)比例为5:1。
本发明所述的纳米佐剂材料的制备步骤4)中,S-乙基异硫脲氢溴酸盐与mPEG-b-PCL-g-PAEM的摩尔比为6~7:1。
本发明所述的表面胍基修饰的纳米佐剂为聚乙二醇-b-聚己内酯-g-聚(胍基-乙基-甲基丙烯酸酯)共聚物在水溶液中自组装形成的纳米颗粒。
本发明所述的表面胍基修饰的纳米佐剂的应用形式为纳米佐剂与蛋白抗原、多肽抗原、核酸、免疫增强剂中任意一种或几种的复合物溶液或冻干粉制剂。
本发明提供了所述的表面胍基修饰的纳米佐剂具有积极的突出的有益效果:
本发明所述的纳米佐剂能够高效负载蛋白、多肽、核酸类抗原和免疫增强剂,所形成的抗原与纳米佐剂的复合物具有长期的稳定性。冷冻干燥后能够均匀的分散于水、PBS、或氯化钠溶液中。
本发明所述的纳米佐剂可以有效的刺激小鼠骨髓来源树突状细胞(BMDC)成熟,促进其表面CD80,CD86,CCR7等标志物分子的表达。
本发明所述的纳米佐剂,能够促进外源抗原被抗原递呈细胞的交叉呈递效果。
本发明所述的纳米佐剂经皮内免疫接种可引起较强的免疫反应,表现为淋巴结中的相关免疫细胞因子大量分泌。
本发明所述的纳米佐剂的材料合成与纳米疫苗制备方法简单、操作简便、重复性好,适合于大规模生产。
附图说明:
图1:PECG共聚物的合成路线图。
图2:PECG共聚物核磁共振氢谱图。
图3:PECG纳米佐剂的粒径(a)与形貌图(b)。
图4:PECG自组装纳米粒促BMDCs成熟图。
图5:PECG纳米佐剂促抗原交叉呈递效果图。
图6:PECG纳米佐剂体内皮下免疫后淋巴结中细胞因子表达情况。
具体实施方式
下面结合具体实施例,进一步详细阐述本发明。实施例中未注明具体条件的实验方法,通常按照常规条件以及手册中所述的条件,或按照制造厂商所建议的条件;所用的通用设备、材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1:mPEG-b-P(CL-co-BMPCL)的制备
将1.0 g无水聚乙二醇单甲醚(分子量2000 g/mol)、18.24 g ε-己内酯、4.87 gγ-(2-溴-2-甲基丙酸酯)-ε-己内酯,以及58 μL辛酸亚锡加入容器中,氮气保护下,于130℃搅拌反应12小时。冷却后,加入二氯甲烷溶解,滴加至冰乙醚中沉淀,过滤、干燥得到共聚物mPEG-b-P(CL-co-BMPCL)。
实施例2:mPEG-b-PCL-g-PtBMA的制备
将mPEG-b-P(CL-co-BMPCL)(0.626 g),2-[(叔丁氧基羰基)氨基]乙基-甲基丙烯酸酯(1.52 g)以及二联吡啶(0.0365 g)溶于3 mL丁酮中。将上述混合溶液在液氮中冷却,反复充氮气与抽真空三次,然后加入溴化亚铜(0.0144 g),进一步抽真空后,于60 ℃条件下搅拌反应12小时。将产物封装到透析袋中(截留分子量3500 Da),在超纯水中透析,冷冻干燥后得到mPEG-b-PCL-g-PtBMA共聚物。
实施例3:mPEG-b-PCL-g-PAEM的制备
将mPEG-b-PCL-g-PtBMA(1.0 g)溶于5 mL三氟乙酸,室温搅拌5小时。在真空条件下将三氟乙酸旋干,然后加入5 mL N,N-二甲基甲酰胺溶解,将所获溶液滴加到冷乙醚中,过滤,真空干燥获得mPEG-b-PCL-g-PAEM。
实施例4:mPEG-b-PCL-g-PGEM的制备
将mPEG-b-PCL-g-PAEM(1.0 g)溶于10 mL 0.1M碳酸氢钠溶液中,加入摩尔数为胍基两倍的S-乙基异硫脲氢溴酸盐,上述反应室温搅拌48小时。将所获溶液封装到透析袋中,透析(截留分子量3500 Da) 72小时,冷冻干燥得到mPEG-b-PCL-g-PGEM,其核磁谱图如图2所示。
实施例1-4的制备过程见图1。
实施例5:mPEG-b-PCL-g-PGEM(PECG)纳米佐剂的自组装
称量20 mg PECG共聚物溶解于2 mL三氟乙醇,逐滴加入置于磁力搅拌器上内含10mL PBS (pH=7.2, 0.01 M)的烧杯中。持续搅拌24 h,挥发掉三氟乙醇,将溶液体积补加到10 mL。通过马尔文激光粒度仪和透射电子显微镜分别检测PECG组装体的粒径大小与形貌(图3)。
实施例6:PECG纳米佐剂促BMDCs成熟
将1 mL BMDCs(密度为2×106/mL)悬液加入6孔板,然后加入2 mL cRPMI1640培养基。将PECG纳米粒加入铺有小鼠骨髓来源树突状细胞 (bone marrow-derived cells,BMDCs)的孔板中,最终浓度为20 μg/mL。将6孔板转移至5% CO2,37℃细胞培养箱中培养24h。收集各组细胞后离心(1500 rpm,5 min),取上清,置于-20 ℃保存备用。PBA(含有0.1%BSA的PBS溶液)洗细胞(1200 rpm,5 min)两次,1 mL/次。收集细胞,使用1 mL PBA重悬各组细胞,在离心管中分别加入荧光素标记抗体兔抗鼠CCR7-PE单克隆抗体,兔抗鼠CD86-PE单克隆抗体,兔抗鼠CD40-PE单克隆抗体及其同型对照各1 μL,置于冰浴中30 min。PBA洗细胞(1200 rpm,5 min)两次,各离心管中加入1.0 mL 2%多聚甲醛溶液,冰浴中固定30 min。PBA洗细胞(1200 rpm,5 min)两次,1.0 mL PBA重悬细胞后,流式细胞仪检测各组BMDCs活化相关信号分子CD86,CD40,CCR7的表达,结果如图4所示。
实施例7:PECG纳米佐剂促进抗原体外交叉呈递
将BMDCs以6×104/孔的密度铺于U型底96孔板中,置于5% CO2,37℃培养箱中培养过夜。次日,将裸抗原(鸡卵清蛋白OVA)或封装有OVA的PECG纳米粒溶液加入96孔板中,浓度为50 μg/mL,以SINFEKL肽段及培养基分别作为阳性与阴性对照。继续培养5小时后,使用DPBS轻柔洗细胞3次,然后将培养于含55 μM β-巯基乙醇,1 mM丙酮酸的cRPMI164培养基中的B3Z细胞(密度,5×105)加入96孔板中。共培养24小时后,离心(500 rcf,7 min),弃上清,每孔加入150 μL CPRG裂解缓冲液(0.15 M氯酚红-β-D-吡喃半乳糖,0.1% Trion-X-100,9mM氯化镁,100 μM巯基乙醇),避光孵育20小时。而后,将U型96孔板中的液体转移至平底96孔板中,测定570 nm波长处的吸光值,结果如图5所示。
实施例8:PECG纳米佐剂的体内免疫应答
以6-8周,雌性BALB/C小鼠为动物模型,皮下注射PECG纳米粒,间隔7天注射一次,共免疫三次。第三次免疫2周后,使用密度梯度离心法分离小鼠回流淋巴结中的淋巴细胞,与PECG纳米粒共孵育48小时后,使用ELISA试剂盒检测细胞上清中细胞因子IFN-γ、TNF-α、IL-10、IL-6的浓度,结果如图6所示。
Claims (9)
1.一种表面胍基修饰的纳米佐剂材料的制备方法,其特征在于包括以下步骤:
1) 将干燥的聚乙二醇单甲醚mPEG加入Schlenk管中,称取定量的ε-己内酯ε-CL、γ-(2-溴-2-甲基丙酸酯)-ε-己内酯BMPCL与辛酸亚锡加入反应管中,在氮气保护下于120-130℃油浴中,搅拌反应10-20小时;待反应冷却后,加入二氯甲烷溶解产物,然后将溶液滴加至冷乙醚中,过滤,取沉淀,真空干燥,得到聚乙二醇与聚己内酯嵌段共聚物大分子引发剂mPEG-b-P(CL-co-BMPCL);
2) 将步骤1)制备的mPEG-b-P(CL-co-BMPCL),单体2-[(叔丁氧基羰基)氨基]乙基-甲基丙烯酸酯tBMA,以及二联吡啶溶于丁酮;将上述混合物反复进行三次除氧后,加入溴化亚铜,在无氧和60℃条件下反应24小时;在纯水中反复透析提纯,冷冻干燥获得聚乙二醇-b-聚己内酯-g-聚(2-[(叔丁氧基羰基)氨基]乙基-甲基丙烯酸酯)mPEG-b-PCL-g-PtBMA;
3) 将mPEG-b-PCL-g-PtBMA溶于三氟乙酸,室温搅拌2-5小时;在真空条件下将三氟乙酸旋蒸掉,然后加入N,N-二甲基甲酰胺溶解产物,将所获溶液滴加到冷乙醚中,过滤,取沉淀;用无水乙醚洗涤两次,真空干燥获得聚乙二醇-b-聚己内酯-g-聚(氨基-乙基-甲基丙烯酸酯)mPEG-b-PCL-g-PAEM;
4) 将mPEG-b-PCL-g-PAEM溶于碳酸氢钠水溶液中,加入S-乙基异硫脲氢溴酸盐,室温下搅拌反应48-96小时;然后在纯水中透析提纯,冷冻干燥后获得聚乙二醇-b-聚己内酯-g-聚(胍基-乙基-甲基丙烯酸酯)mPEG-b-PCL-g-PGEM。
2.根据权利要求1所述的方法,其特征在于所述的聚乙二醇单甲醚的分子量为2000 g/mol;所述的聚己内酯的分子量范围为3420-3990 g/mol;所述的聚(γ-(2-溴-2-甲基丙酸酯)-ε-己内酯BMPCL的分子量范围为834-973 g/mol;所述的聚(胍基-乙基-甲基丙烯酸酯)的分子量范围为516-18060 g/mol。
3.根据权利要求1所述的方法,其特征在于步骤1)中,单体ε-CL与聚乙二醇单甲醚的摩尔比为30~35:1;BMPCL与ε-CL的摩尔比为1:10;辛酸亚锡的投料量为单体摩尔总数的0.05%。
4.根据权利要求1所述的方法,其特征在于步骤2)中,单体tBMA与大分子引发剂mPEG-b-P(CL-co-BMPCL)的摩尔比为3~105:1。
5.根据权利要求1所述的方法,其特征在于步骤3)中,三氟乙酸的体积以毫升计与mPEG-b-PCL-g-PtBMA的质量以克计比例为5:1。
6.根据权利要求1所述的方法,其特征在于步骤4)中,S-乙基异硫脲氢溴酸盐与mPEG-b-PCL-g-PAEM的摩尔比为6~7:1。
7.权利要求1-6任一所述的方法得到的表面胍基修饰的纳米佐剂材料。
8.权利要求7所述的表面胍基修饰的纳米佐剂材料与蛋白抗原、多肽抗原、核酸、免疫增强剂中任意一种或几种的复合物溶液或冻干粉制剂。
9.权利要求7所述的表面胍基修饰的纳米佐剂材料用于制备免疫疫苗。
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