CN117467127A - 具有佐剂作用的高分子衍生物及其制备方法与应用 - Google Patents
具有佐剂作用的高分子衍生物及其制备方法与应用 Download PDFInfo
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Abstract
本发明涉及一种具有佐剂作用的高分子衍生物及其制备方法与应用,本发明先制备得到糖衍生物,其既可以作为佐剂增强免疫,又可以作为疫苗递送系统,在多种递送途径中发挥作用。本发明制备方法操作简单,产物易提纯,收率高;通过将糖基与PEG或与PLGA‑PEG‑COOH、DSPE‑PEG‑COOH等结合,使得海藻糖、甘露糖等更容易掺入疫苗递送系统中,更好地发挥其靶向作用和协同增效的佐剂作用,可与减毒活疫苗、裂解疫苗、亚单位疫苗、重组疫苗和核酸疫苗等共同递送,产生对疫苗免疫效果的增强作用。本发明对药物载体制备和疾病预防治疗有重要意义,包括但不限于微针、微囊微球、纳米粒、脂质体、粉针和溶液等制剂形式。
Description
技术领域
本发明属于药物递送技术领域,具体地说是涉及一种具有佐剂作用的高分子衍生物及其制备方法与应用。
背景技术
疫苗接种仍然是抗击传染病最重要的医疗干预措施之一,可同时减少传播、发病率和死亡率。鉴于此,大量的研究工作集中在开发不同的疫苗技术和策略上,以诱导对危险病原体的免疫力。在疫苗开发的早期阶段,全病毒疫苗占主导地位,但其副作用频繁发生,耐受性较低。为了提高安全性,疫苗逐渐演变为仅包含部分微生物、亚基甚至纯化抗原的设计。但是随着抗原变得越来越简单纯化,它们的免疫刺激能力也随之下降。为了提高高度纯化的抗原的免疫原性,佐剂开始添加到疫苗中,其可以诱导对抗原的更强免疫应答,还可以降低对疫苗接种反应不佳的人群的疫苗剂量和生产成本。但从1926年Glenny首先发现硫酸铝钾(明矾,铝佐剂)能增强白喉毒素的免疫原性起,到1939年,铝佐剂被批准可用于白喉疫苗,虽然近百年来全世界被批准上市的疫苗含有佐剂的也已超过三十种,可是佐剂的多样性发展却非常缓慢。受限于副作用、稳定性、有效性等因素,目前除了铝佐剂外,只有MF59(含角鲨烯的水包油乳剂)、AS03(含角鲨烯、维素E和Tween80)、含单磷酰基脂质A(monophos-phoryl lipid A,MPL)AS01和AS02佐剂、以及胞嘧啶鸟嘌呤寡聚脱氧核苷酸(CpG oligo-nucleotide,CpG-ODN)等少数几种佐剂被批准应用于人类疫苗,并且这些佐剂多为欧美大企业专利垄断,限制了其普及使用。
在这些可用的佐剂中,铝盐是被最广泛使用的疫苗佐剂。然而,铝盐不能增强细胞介导的1型辅助性T细胞(Th1)或细胞毒性T淋巴细胞(CTL)反应,铝盐也不是黏膜免疫系统的良好诱导剂。由于铝佐剂不能刺激足够的抗原免疫,因此其积累可能导致肾功能不全并影响脑组织和骨组织,导致神经系统综合征和透析相关痴呆。如今,大多数兽用疫苗要么是用油基佐剂配制的灭活生物,要么是减毒活疫苗。然而,油基乳液佐剂也存在局部注射部位反应的问题。较粘稠的油助剂不利于注射。虽然基于矿物油的佐剂通常对兽用疫苗有效,但矿物油会引起严重的副作用,如局部肉芽肿、脓肿或发热等。
佐剂通过激活先天免疫细胞来增强疫苗的适应性免疫。根据作用机制,佐剂可分为免疫刺激剂和递送系统。免疫刺激剂,例如病原体相关分子模式(PAMP)、损伤相关分子模式(DAMP)和化学合成的Toll样受体(Toll-like receptor,TLR)的小分子激动剂等,可导致APC产生免疫响应信号。递送系统,如聚乳酸-羟基乙酸共聚物(PLGA)、糖类化合物等天然或合成聚合物及其纳米粒和脂质纳米颗粒(LNP)等,可模拟病原微生物的结构,诱导免疫细胞内吞行为,通过促进抗原在MHC分子上的呈递发挥作用。
疫苗佐剂中的糖类化合物因具有免疫调节特性、生物相容性、生物降解性和低毒性,因而广受关注。含有甘露糖、海藻糖、葡萄糖等糖基的糖类化合物通过结合APCs表面的特异性糖类结合受体,例如,TLRs、核苷酸结合寡聚化结构域2(NOD2)样受体(NLRs)和C型凝集素受体(CLRs)等来刺激抗原吞噬作用,协同增强了疫苗的免疫原性和免疫效果。特异糖类化合物与CLRs的相互作用触发了介导APC成熟的细胞内信号级联,这是初始B细胞和T细胞活化所必需的。此外,糖类化合物也可以通过电荷相互作用被APCs主动摄取和累积,并触发多种信号通路,从而刺激先天性和适应性免疫应答。重要的是,与其他佐剂相比,糖类化合物可以刺激平衡的促炎和抗炎反应,从而提高了其作为疫苗佐剂的综合效力和安全性。
甘露糖受体(MR)是最常见的疫苗接种目标,MR由五个片段组成:i)富含N端半胱氨酸的结构域(CR),ii)单个纤连蛋白II型(FN II)结构域,iii)一组八个C型凝集素样结构域,称为碳水化合物识别结构域(CRD),iv)跨膜结构域,以及v)细胞质尾巴。MRs与糖类化合物结合亲和力主要是由于位于第四和第八CRD之间的残基,所有CRD都结合富含甘露糖的多价低聚糖,然而,第四种CRD对单糖(例如甘露糖、岩藻糖、GalNAc)的亲和力最高。Mincle是巨噬细胞诱导的C型凝集素,是一种II型跨膜蛋白,其细胞外结构域包含一个C型碳水化合物识别结构域(CRD),其主要的含糖配体是海藻糖二霉菌酸盐,在分枝杆菌的外膜中发现的糖脂,以及真菌和酵母中的其他含甘露糖或葡萄糖的糖缀合物。海藻糖以及甘露糖与受体的相互作用可为设计佐剂提供基础。但游离的甘露糖或海藻糖等由于不能形成能够刺激疫苗抗原被内吞的空间结构联动特征,无法通过将其直接掺入到疫苗递送系统中发挥作用,因此它们通常需要与递送载体或抗原化学结合。然而,怎样一种结合方式和组装形式更有利于这些糖基获得被相应受体特异识别和有效结合并由此启动细胞内吞行为和其他免疫反应,尚难以简单判断,存在未知性和不确定性。
目前上市的甘露聚糖肽作为免疫增强剂发挥作用,主要从菌株培养液中提取,来源匮乏,且与多种成分的疫苗混合易发生配伍禁忌,安全性不高。同样的免疫调剂海藻糖二酯类,也存在类似情况,限制其作为疫苗佐剂的应用。
因此,开发更容易获得的、更加安全和高效的疫苗佐剂迫在眉睫,是本行业技术人员急需解决的难题。
发明内容
本发明提供了一种具有佐剂作用的高分子衍生物及其制备方法与应用,具体地,涉及了含有糖基的高分子系列化合物在制备药物递送载体中的应用。本发明提供了具有糖基的高分子衍生物以扩大其在药物递送方面应用,本发明先制备得到糖衍生物,该类物质既可以作为佐剂增强免疫,又可以作为疫苗递送载体材料,在多种递送途径中发挥独特作用。
本发明采用的技术方案为:
一种具有佐剂作用的高分子衍生物,结构式如(I)、(II)或(III)所示:
其中,R1、R2分别包括海藻糖基、甘露糖基、岩藻糖基、葡萄糖基、果糖基、或其相应的衍生基团,R3包括聚乙二醇(PEG)、聚乳酸-羟基乙酸共聚物(PLGA)、透明质酸(HA)、壳聚糖(CS)、以氨基酸为基本单元的聚合物(包括多肽、蛋白质、聚氨基酸等)、磷脂及其衍生物(包括磷酸乙醇胺磷脂DSPE等)、胆固醇及其衍生物。
在本发明中,R1和R2可以相同,也可以不同。
更为优选地,结构式(I)为海藻糖衍生物(海藻糖-PEG,PEG-Tre),其具体结构为:
结构式(II)为甘露糖衍生物(甘露糖-PEG,PEG-Man),其具体结构为:
结构式(III)为甘露糖衍生物PLGA-PEG-甘露糖(PLGA-PEG-Man)或DSPE-PEG-甘露糖(DSPE-PEG-Man),其具体结构为:
PLGA-PEG-Man:
DSPE-PEG-Man:
本发明还提供了上述具有佐剂作用的高分子衍生物的制备方法,结构式(I)通过下述方法制备得到:以包括海藻糖在内的糖类为起始原料,通过乙酰化、炔丁基取代、去乙酰化、炔基-叠氮环加成反应制备得到目标产物;
结构式(II)(III)分别通过下述方法制备得到:以包括甘露糖胺在内的氨基化糖类为起始原料,通过缩合反应制备得到目标产物。
本发明采用的点击化学反应具有产率高,应用范围广,具有立体选择性,副产物少,产物易于分离,反应溶剂易于除去等优点,非常适合生物医用材料等的开发。而酰胺化反应,条件温和,适用范围广,非常适合大生产。本发明应用点击化学成功制备了一种海藻糖衍生物,又应用酰胺反应成功制备了甘露糖衍生物。
作为优选,所述具有佐剂作用的高分子衍生物的制备方法,包括下述步骤:
(1)将海藻糖、乙酸酐、及无水乙酸钠依次加入反应器,进行乙酰化反应得到乙酰化海藻糖;
(2)将乙酰化海藻糖和3-丁炔-1-醇在酸性条件下,进行取代反应得到炔基乙酰海藻糖;
(3)将炔基乙酰海藻糖和甲醇钠,在甲醇溶剂中,于室温进行脱保护基得到炔基海藻糖;
(4)将二叠氮聚乙二醇(N3-PEG-N3)、炔基海藻糖、五水硫酸铜、抗坏血酸钠加入反应器,经炔基-叠氮环加成反应得到PEG-Tre。
其反应的总路线如下:
作为优选,所述具有佐剂作用的高分子衍生物的制备方法,包括下述步骤:将二乙酸聚乙二醇与甘露糖胺盐酸盐溶解在有机溶剂中,然后逐步加入4-二甲氨基吡啶(DMAP)和碳二亚胺(EDCI),将反应混合物在氮气室温条件下搅拌过夜,之后经沉淀、离心、干燥得产物PEG-Man。
其反应的总路线如下:
另外,本发明还提供了上述具有佐剂作用的高分子衍生物在制备药物递送载体中的应用。
作为优选,所述药物递送载体包括微针、脂质体、纳米粒、微囊微球、粉针剂和溶液。
作为优选,具有佐剂作用的高分子衍生物可与包括减毒活疫苗、裂解疫苗、亚单位疫苗、重组疫苗和核酸疫苗在内的疫苗共同递送,产生对疫苗免疫作用的协同增效。
作为优选,所述微针为海藻糖-PEG微针或PEG-甘露糖微针,其制备方法如下:
①微针阴模制备
将金属微针阳模阵列放在阳模容器中,将聚二甲基硅氧烷和固化剂以10:1的质量比充分混合后,注满容器;随后将其放入真空干燥箱中,在-80Kpa真空度下1h以完全除去气泡,接着将其放入70℃烘箱下2h固化,将聚二甲基硅氧烷阴模与微针阳模分离,得到微针阴模;
②基质溶液配制
称取基质材料,加入去离子溶解,静置12h消除气泡,后与70wt%的海藻糖-PEG或甘露糖-PEG共混备用,备用;其中,基质材料包括PLGA、聚乳酸(PLA)、聚乙烯醇(PVA)、聚乙烯吡咯烷酮(PVP)、CS、HA、普鲁兰糖(PL)、明胶、纤维素;基质材料与PEG-Tre或PEG-Man的体积比为1:1~30:1;
③微针的制备
取基质溶液于聚二甲基硅氧烷阴模中,随后将阴模放入离心机中,在25℃、4000rpm的条件下离心10min使基质溶液入模;离心结束后,将阴模在室温下干燥24h,将微针从模具中脱出,即得。
作为优选,DSPE-PEG-Man脂质体通过下述方法制备得到:
(1)将甘露糖胺与磷脂聚乙二醇羧酸(DSPE-PEG-COOH)通过缩合生成DSPE-PEG-Man;
(2)称取大豆卵磷脂、胆固醇,加入无水乙醇,水浴超声,直到固体充分溶解,得总脂质浓度为12.5mg/m L,作为油相;将20mg DSPE-PEG-Man溶解于2mL pH为7.4的PBS中,作为水相;取10mL油相于注射器中,除去管道中的死体积,以水相:油相的流速比为4:1,注入微流控设备中,先用油相将整个微流控管道充满,再通水相,即得DSPE-PEG-Man脂质体。
PLGA-PEG-Man纳米粒(PLGA-PEG-Man NPs)通过下述方法制备得到:
(1)将甘露糖胺与聚乳酸-羟基乙酸共聚物-羧酸(PLGA-PEG-COOH)反应,通过共聚物的羧基端与甘露糖胺的氨基端反应生成PLGA-PEG-Man;
(2)纳米粒的制备采用纳米沉淀法进行,将PLGA-PEG-Man溶于丙酮溶剂中呈有机相,在搅拌条件下,将有机相匀速滴加至水溶液中,然后进行挥发完全至除去有机溶剂得纳米粒悬浮液;其中PLGA-PEG-Man浓度为10~100mg/mL、有机相与水相的体积比为1:4~1:10。
作为优选,药物递送途径包括经皮肤递送、局部粘膜递送、口服递送、注射递送、植入递送。
本发明的有益效果在于:
本发明制备方法的原料易得、反应条件温和易于控制,操作简单,产物易提纯,收率高;PEG反应活性高,通过点击化法与缩合反应分别将糖基与PEG相结合,或将糖基与PLGA-PEG-COOH、DSPE-PEG-COOH等结合,使得海藻糖、甘露糖等更容易掺入疫苗递送系统中,更好地发挥其靶向作用和协同增效的佐剂作用,可与减毒活疫苗、裂解疫苗、亚单位疫苗、重组疫苗和核酸疫苗等共同递送,产生对疫苗免疫效果的增强作用。本发明对药物载体制备和疾病预防治疗有重要意义,包括但不限于微针、微囊微球、纳米粒、脂质体、粉针和溶液等制剂形式。
附图说明
图1是本发明实施例1中乙酰海藻糖的核磁氢谱图;
图2是本发明实施例1中炔基乙酰海藻糖的核磁氢谱图;
图3是本发明实施例1中炔基海藻糖的核磁氢谱图;
图4是本发明实施例1中海藻糖-PEG的核磁氢谱图;
图5是本发明实施例1中甘露糖-PEG的核磁氢谱图;
图6是本发明微针制备流程示意图;
图7是本发明多种基质与甘露糖-PEG共混微针的机械力测试结果图;
图8是本发明PLGA-PEG-Man核磁氢谱图;
图9是本发明PLGA-PEG NPs与ConA结合前后的粒径变化图(a),以及PLGA-PEG-ManNPs与ConA结合前后的粒径变化图(b);
图10是本发明PLGA NPs、PLGA-PEG NPs、PLGA-PEG-Man-NPs与不同浓度ConA孵育后在550nm的浊度变化图;
图11是利用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析本发明模型抗原在纳米粒的稳定性的电泳图;其中,包封在纳米粒中的模型蛋白(泳道1),模型蛋白BSA(泳道2),标准蛋白质分子Marker(泳道M);
图12是本发明PLGA NPs、PLGA-PEG NPs、PLGA-PEG-Man-NPs对大鼠肺泡巨噬细胞的细胞毒性结果图;
图13是本发明细胞摄取结果图;
图14是本发明给药程序图(a),以及免疫效价结果图(b);
图15是本发明DSPE-PEG-Man核磁谱氢图;
图16是本发明口服免疫效价结果图。
具体实施方式
下面通过实施例,对本发明的技术方案作进一步具体的说明,这些实施例是对本发明的说明而作,不是对本发明的限制。
实施例中所述实验方法,如无特殊说明,均为常规方法;涉及的百分比,如无特殊说明,均为质量百分比;所述试剂和材料,如无特殊说明,均可从商业途径获得。本发明采用的聚乙二醇的分子量为2000道尔顿。
实施例1
海藻糖(甘露糖)-PEG的合成
(1)PEG-Tre的合成
①乙酰海藻糖的合成
将海藻糖1g,乙酸酐10mL,无水乙酸钠1.07g依次加入三口瓶中,在90℃条件下进行乙酰化反应4h,加入10mL饱和NaHCO3水溶液后分层,再向其中加入30mL醋酸乙酯,搅拌后分层,取油层在80℃下旋蒸,旋干即可,得到乙酰海藻糖,为淡黄色透明油状产品,产率为99%。
将乙酰海藻糖溶于氘代氯仿中进行核磁表征,其核磁图如图1所示,1HNMR(600MHz,Chloroform-d)δ5.49(m,2H),5.29(m,4H),5.04(m,4H),4.23(dd,J=12.1,5.6Hz,2H),4.04(m,2H),4.01(d,J=12.2Hz,2H),2.08(overlapped,12H),2.05(s,6H),2.03(s,6H).
②炔基乙酰海藻糖的合成
将乙酰海藻糖(2g,5.1mmol),3-丁炔-1-醇(360mg,5.1mmol),二氯甲烷(20mL)依次加入100mL三口瓶,氮气保护,冰浴降温至4℃,滴入三氟化硼的乙醚溶液(1.25mL,10.2mmol),室温反应过夜。第二日加入饱和碳酸氢钠水溶液(30mL)淬灭反应,加入二氯甲烷(50mL)萃取,无水硫酸钠干燥,过滤,旋干。将此产物溶解于二氯甲烷中,于硅胶粉混合,质量比约1:2(产物:硅胶粉),室温干燥过夜。柱色谱分离,流动相为二氯甲烷和甲醇(150:1)。产率为85%。
将炔基乙酰海藻糖溶于氘代氯仿中进行核磁表征,其核磁图如图2所示,1H NMR(600MHz,Chloroform-d)δ5.49(t,J=9.8Hz,2H),5.29(d,J=3.8Hz,2H),5.04(m,4H),4.23(dd,J=12.1,5.7Hz,2H),4.11(q,J=7.1Hz,1H),4.04(ddd,J=10.3,5.7,2.2Hz,2H),4.01(dd,J=12.1,2.2Hz,2H),2.05(m,24H).
③炔基海藻糖的合成
将炔基乙酰海藻糖(2g,5mmol),甲醇(20mL)依次加入100mL三口瓶,氮气保护,然后加入甲醇钠(2.02g,37.5mmol),室温搅拌过夜。第二日加入冰乙酸(2.7mL,45mmol)搅拌10分钟,旋干,加入去离子水(5mL)溶解,用离子交换树脂纯化,取醇冲洗液,旋干得产品。产率为64%。
将炔基乙酰海藻糖溶于氘代甲醇中进行核磁表征,其核磁图如图3所示,1H NMR(600MHz,Methanol-d4)δ5.13(d,J=3.7Hz,2H),3.87–3.79(m,7H),3.71(dd,J=11.9,5.1Hz,2H),3.52(dd,J=9.8,3.7Hz,2H),2.08(s,2H)。
④PEG-Tre的合成
将N3-PEG-N3(1g,0.5mmol)溶于叔丁醇(8mL),加入到50mL单口瓶,然后加入炔基海藻糖(664mg,2.88mmol)的水(2mL)溶液,0.3mol/L的五水硫酸铜(180μL,0.05mmol),抗坏血酸钠(36mg,0.02mmol),50℃反应过夜。旋干后加入二氯甲烷(10mL)冰箱冷藏5h,过滤,滤液用葡聚糖凝胶色谱纯化,流动相为甲醇,旋干后用5mL水溶解,冷冻干燥得白色固体。产率为56%。
将PEG-Tre溶于氘代甲醇中进行核磁表征,其核磁图如图4所示,1H NMR(600MHz,Methanol-d4)δ3.80–3.74(m,1H),3.72–3.68(m,5H),3.58(dd,J=5.6,4.2Hz,1H),3.55–3.51(m,1H),3.40(t,J=4.9Hz,4H),3.33(p,J=1.6Hz,2H)。
(2)PEG-Man的合成
利用甘露糖胺与COOH-PEG-COOH共聚物的羧基端与甘露糖胺的氨基端共价偶联生成COOH-PEG-COOH。将6g COOH-PEG-COOH和3g甘露糖胺盐酸盐溶解在50mL DMF中。然后逐步加入0.36g DMAP和0.58g EDCI。将反应混合物在氮气室温条件下搅拌48h。之后加入400mL二氯甲烷(丙酮或乙酸乙酯)沉淀PEG-Man,离心分离沉淀,室温静置挥发溶剂后用去离子水溶解产物,冷冻干燥后称重为4.87g,收率为80.16%。之后将PEG-Man溶于重水中进行核磁表征,其核磁图如图5所示,1H NMR(600MHz,D2O)δ7.87(s,2H),6.82(d,2H),5.34-5.13(m,6H),4.12(dd,2H),3.95-3.49(m,14H),1.02(m,190H)。
海藻糖/甘露糖衍生物的急性毒性实验
实验动物:ICR小鼠,由浙江省实验动物中心提供,体重18-23g/只,每组10只,雌雄各半;在25℃实验室内适应性饲养3天后给药。
PEG-Man和PEG-Tre分别用5%葡萄糖溶液进行溶解配制,各按100mg/kg剂量单次尾静脉注射给药,连续观察7天,逐日记录小鼠的毒性反应和死亡小鼠的个数。试验结束,未发现有小鼠死亡,表明甘露糖-PEG和海藻糖-PEG静脉给药的半数致死量(LD50)大于100mg/kg,这说明PEG-Man和PEG-Tre具有非常好的生物安全性。
实施例2
海藻糖(甘露糖)-PEG在微针递送疫苗上的应用
皮肤中存在丰富的免疫细胞,常常被视为疫苗递送的高潜在靶标。而微针阵列可以将疫苗直接递送至皮肤上层,增强免疫原性。本发明探究了PEG-Man和PEG-Tre用于微针递送疫苗的潜能。
①海藻糖(甘露糖)-PEG微针的构建探究
预实验显示PEG-Man和PEG-Tre无法独立制备微针。于是我们将CS,PVA,HA,PVP,PL五种微针基质材料和PEG-Man或PEG-Tre混合后制备微针,通过测试微针机械力筛选最适合的共混基质。其制备方法如下:
a.微针阴模制备
将金属微针阳模阵列放在阳模容器中。将PDMS(聚二甲基硅氧烷)和固化剂甲基三乙氧基硅烷以10:1的质量比充分混合后,注满容器。随后将其放入真空干燥箱中,在-80Kpa真空度下1h以完全除去气泡,接着将其放入70℃烘箱下2h固化,用手术刀小心将PDMS阴模与微针阳模分离,得到微针阴模。
b.基质溶液配制
分别称取一定质量的CS,PVA,HA,PVP,PL,加入适量的去离子溶解,静置12h消除气泡,后与5%~20%的PEG-Man按1:1共混制备基质溶液备用。
c.微针的制备
取基质溶液于PDMS阴模中,随后将阴模放入离心机中,在25℃、4000rpm的条件下离心10min使基质溶液入模。离心结束后,将阴模在室温下干燥24h,将微针从模具中脱出,即得微针,待用,过程如图6所示。
②机械力测试
使用万能试验机进行上述微针轴向的压变性能考察。万能试验机的试验参数如下:采用压缩模式,下压速度为0.02mm/s,触发力为0.01N,当位移达到500μm时结束试验。将微针倒置于压盘上,针尖朝上放置,使其垂直于探头的平面,进行轴向应力测定。测定结果见图7。
以机械力为指标,筛选了五种共混基质,最终确定了机械力最优的PVP作为最佳共混基质。进一步筛选处方,以获得最优的机械性能。
③海藻糖(甘露糖)-PEG/PVP共混微针处方探究
在筛选出最佳的共混基质后,我们筛选了不同处方配比对微针机械性能的影响。在每个微针处方的基础上加入了伊红染料水溶液作为刺入指示剂。将制得的可溶性微针利用给药器以10N的力刺入离体猪耳皮肤1min后,取下微针观察离体皮肤的红点数量并计算其与微针针数的比例,比例越高则微针机械强度越好。不同处方对海藻糖(甘露糖)-PEG/PVP微针机械性能的影响结果如表1所示。
表1
No | 共混基质材料(wt%) | 成功刺入百分比(mean%±SD) |
1 | 90%PEG-Man+10%PVP | 70.70±1.35 |
2 | 80%PEG-Man+20%PVP | 91.08±0.97 |
3 | 70%PEG-Man+30%PVP | 96.09±0.75 |
4 | 90%PEG-Tre+10%PVP | 79.99±0.93 |
5 | 80%PEG-Tre+20%PVP | 94.17±1.01 |
6 | 70%PEG-Tre+30%PVP | 98.44±0.45 |
根据表1的海藻糖(甘露糖)-PEG/PVP微针机械性能数据可知:处方3和6刺入率最佳,可作为最佳处方进行动物免疫试验,其中PVP与海藻糖(甘露糖)-PEG体积比为1:1~30:1。
④负载流感疫苗的海藻糖(甘露糖)-PEG/PVP微针的免疫原性
在筛选出最优的微针处方后,需进一步探究海藻糖(甘露糖)-PEG/PVP微针用于疫苗递送的免疫效果。
为了探究海藻糖(甘露糖)-PEG/PVP微针的免疫原性,制备了不同处方的微针用于递送流感疫苗,分别为PVP空白组、甘露糖/PVP组、海藻糖/PVP组、PEG/PVP组、聚肌胞苷酸(poly(I:C))/PVP共混组、PEG-Man组和PEG-Tre组,其中佐剂与疫苗的比例为1:19,流感疫苗含量为6μg/片。于实验前一夜,使用宠物毛推剪和脱毛膏将wistar大鼠背部毛发除去。在第1天,使用微针专用给药器的3档将流感疫苗可溶性微针刺入大鼠皮肤,按压30s后,使用医用胶带将微针进行固定,50min后移除固定装置。于第14天进行加强免疫。于第28天取血,其血凝抑制实验结果如表2所示。
表2
从表中可以看出相比较共混组的微针,在同等载药量下,PEG-Man组与海PEG-Tre组效果均有显著提高,且与poly(I:C)/PVP组和i.m组效果无显著差异。因此,PEG-Man与PEG-Tre具备基质材料和疫苗佐剂这两种功能,且在一定程度上解决了由于微针体积狭小而影响载药量的问题。并且,因为佐剂的有效作用,可以使得给药剂量较低时也能获得令人满意的免疫保护效果,有利于减少疫苗用量,降低原料成本。
实施例3
PLGA-PEG-Man的制备
基于实施例1酰胺化反应的原理,将甘露糖胺与PLGA-PEG-COOH反应,通过共聚物的羧基端与甘露糖胺的氨基端反应生成PLGA-PEG-Man。制备得到的PLGA-PEG-Man核磁图如图8所示。
由图可知1.58和5.22ppm处的峰是乙醇酸链段的甲基和甲烷基团的特征,而4.82ppm处的峰归因于乳酸链段的亚甲基,这两者都是由PLGA链贡献的。在3.64ppm处观察到的峰对应于PEG段的亚甲基。甘露糖胺的峰与PEG的峰(3.62ppm)重叠,因此仅检测到一个4.2-4.3ppm左右处的小峰,并将其归因于甘露糖胺,由于甘露糖仅修饰在这种聚合物材料的一段,导致甘露糖胺的峰较低。
PLGA-PEG-Man NPs的制备
纳米粒的制备采用纳米沉淀法进行,将PLGA-PEG-Man溶于丙酮溶剂中呈有机相,在搅拌条件下,将有机相匀速滴加至水溶液中,然后进行挥发完全至除去有机溶剂得纳米粒悬浮液。在制备过程中,通过对PLGA-PEG-Man浓度10~100mg/mL、有机相与水相的体积比1:4~1:10进行筛选得最优处方。
通过激光粒度仪对纳米粒的粒径分布、多分散性及Zeta电位进行测定。纳米沉淀法制备PLGA-PEG-Man NPs的理化性质如表3、4所示。
表3
实验编号 | 聚合物浓度(mg/mL) | 平均粒径(nm) | PDI | 电位(mV) |
1 | 5 | 241 | 0.164 | -13.0 |
2 | 10 | 207 | 0.156 | -10.3 |
3 | 20 | 153 | 0.144 | -16.9 |
4 | 30 | 256 | 0.163 | -15.8 |
5 | 50 | 609 | 0.227 | -2.62 |
表4
实验编号 | V有机相:V水相 | 平均粒径(nm) | PDI | 电位(mV) |
1 | 1:4 | 165 | 0.182 | -10.2 |
2 | 1:6 | 153 | 0.144 | -16.9 |
3 | 1:8 | 191 | 0.193 | -20.5 |
4 | 1:10 | 197 | 0.236 | -23.7 |
PDI是用于指示颗粒尺寸分布的参数。PDI值等于0.05表示单分散体系,而PDI在0.08~0.7之间表示中等多分散性,>0.7表示样品具有非常宽的尺寸分布。所制备的纳米颗粒PDI均位于0.1~0.3范围,且随着聚合物浓度增加,PDI从0.164显著增加至0.227(p<0.05);随着水相体积增加PDI也增大,均归因于纳米颗粒的聚集。
Zeta电位值是显示粒子表面电荷特性的重要参数,它表面纳米颗粒之间的相互作用(吸引力和排斥力),这影响制剂的稳定性、颗粒与生物体液的相互作用,与细胞的相互作用。考虑到纳米粒系列理化性质对鼻黏膜给药的影响,选择聚合物浓度分别为10mg/mL、20mg/mL,有机相与水相比例分别为1:4、1:6进行下一步研究。
实施例4
PLGA-PEG-Man纳米粒作为佐剂用于疫苗递送的潜力
(1)抗原结合效率和载药量选用牛血清白蛋白(BSA)为模型蛋白,利用Bradford法测定抗原负载效率。
根据上述得到的结果,由于PLGA-PEG-Man浓度为10mg/mL,有机相与水相之比为1:6时,所制备的纳米粒粒径高于其他组,因此舍弃该处方。以BSA为模型抗原测定3种理化性质纳米粒的抗原封装效率和载药量。不同处方纳米粒的理化性质如表5所示。
表5
由上表可知,当聚合物浓度为20mg/mL有机相与水相之比为1:6时,纳米粒的包封率达到54.8%,载药量为2.2%,高于另外2组。基于此,本实验选择较优处方为聚合物浓度为20mg/mL,有机相与水相体积比为1:6制备纳米粒进行后面的实验。在同样的制备条件下,制备PLGA-PEG NPs、以及通过筛选粒径尺寸相近的PLGA NPs。三种类型纳米粒的理化性质及其包封率结果如表6所示。
表6
由上可知,由于PLGA本身的疏水性质,PLGANPs抗原封装效率和载药量均显著低于PLGA-PEG NPs、PLGA-PEG-Man NPs。甘露糖修饰提高了PLGA-PEG NPs与亲水蛋白的结合能力,聚合链中的甘露糖残基导致模型蛋白的吸附效率提高了8.9%。
(2)凝集素结合测定
使用刀豆球蛋白A(Con A)凝集素结合实验研究了体外配体特异性活性,以评估修饰纳米颗粒形成后与甘露糖配体的表面取向和可用性。将甘露糖功能化纳米颗粒(PLGA-PEG-Man NPs,1mg/mL)与Con A(0.7mg/mL)的PBS在37℃下轻轻搅拌孵育1小时,使用未甘露糖功能化纳米粒作为阴性对照。在4℃下10000rpm离心45分钟,用PBS洗涤两次,通过动态光散射评估聚集体的形成。将200μL甘露糖修饰纳米粒与未修饰的纳米颗粒稀释至1mL,加入1mL ConA(25μg/mL,50μg/mL)并在550nm出检测1小时内的浊度变化。
由于ConA常用于模型受体蛋白测定结合甘露糖配体的主要趋势。使用Con A凝集素结合实验,与动态光散射90评估修饰纳米颗粒形成后与甘露糖配体的表面取向和可用性,由图9(b)可知,ConA与甘露糖功能化纳米粒表面暴露的甘露糖基结合形成聚集体。PLGA-PEG-Man NPs与ConA孵育后,ConA诱导颗粒聚集,纳米粒平均尺寸由169nm增加至1600nm,相反,由图9(a)可知非甘露糖功能化纳米粒没有聚集。纳米颗粒与不同浓度凝集素孵育后,PLGA-PEG-Man NPs的吸光度随时间而增加,表明甘露糖残基结合于纳米粒表面且取向正确。如图10所示,50分钟左右,因为结合位点的饱和吸光度值达到平台未继续增加。而未进行甘露糖修饰的PLGA NPs、PLGA-PEG NPs没有显示任何浊度的增加。
(3)纳米粒包封抗原的完整性
通过SDS-PAGE评估了制备条件对模型蛋白BSA完整性的影响。电泳后,包封在纳米颗粒的BSA应与BSA溶液条带在同一水平线上。如图11所示,纳米颗粒中的BSA与未包封的蛋白质之间分子量一致且条带清晰,这表明在制备空白纳米颗粒过程中有机溶剂已经完全挥发,因此在制备制剂过程中BSA结构相对完整,没有因为纳米粒的影响聚集或者遭到破坏。
(4)体外细胞毒性研究
使用CCK-8法评估纳米载体悬浮液对大鼠肺泡巨噬细胞的细胞毒性。将处于对数生长期的大鼠肺泡巨噬细胞以1×104/孔的密度接种于96孔板上,培养48小时后,将纳米载体悬浮液加入培养基中孵育2小时,纳米材料在培养基中浓度分别为5、50、100、200和400μg/mL。用10μL CCK-8溶液替换悬浮液,并在37℃下孵育2小时,使用酶标仪在450nm处记录吸光度,以测量细胞活力。每个样品在5次重复后进行分析。抗原递送系统应安全无毒才能用于抗原的递送,因此需要评估纳米粒对细胞存活率的影响。CCK-8法是一种常用的细胞毒性的测定方法。本发明利用该方法测定不同种类纳米粒对大鼠肺泡巨噬细胞的细胞活力。结果见图12,在5~400μg/mL的纳米粒浓度范围内,细胞存活率均大于90%,说明该纳米材料具有相当好的细胞相容性。
(5)细胞摄取实验
在人气道上皮细胞系Calu-3研究了负载FITC-BSA的PLGA-PEG-Man纳米载体的细胞摄取,将Calu-3细胞以1×106cell/mL细胞接种于96孔板上,每孔加入细胞悬液100μL,培养板静置于37℃培养箱中24h。每孔加入负载FITC-BSA的PLGA-PEG-Man纳米载体50μL,孵育3h后,采用无菌DPBS清洗3遍后,弃去上清液,采用荧光显微镜观察Calu-3细胞摄取作用。
细胞摄取结果如图13所示,PLGA NPs组具有弱的细胞摄取。而PLGA-PEG NPs与PLGA-PEG-Man NPs具有PEG测链而拥有一定的粘液惰性因此绿色荧光信号明显更多。且甘露糖修饰纳米颗粒较未修饰的摄入量更多,可能使由于上述提到的糖基化增加纳米颗粒的载药量。
(6)体内研究
在优化研究之后,评估不同流感疫苗纳米颗粒比例制剂的免疫效果。将Wistar大鼠随机分配6组,每组6只大鼠,并提供食物和水。分别设置空白对照组(PBS),流感疫苗组、流感疫苗-PLGA NPs组、流感疫苗-PLGA-PEG NPs组、流感疫苗-PLGA-PEG-Man NPs组、流感疫苗肌肉注射组。每只鼠50μL制剂,共进行两次鼻腔免疫,接种间隔为2周,接种时间分别于第0天和第14天。第28天,取血进行抗体滴度分析,结果见图14。将PLGA-PEG-Man NPs与疫苗以适当的比例混合,并用作体内给药的流感疫苗滴鼻剂制剂。进行体内研究是为了进一步研究这些NP递送流感疫苗以诱导免疫反应的能力。疫苗直接鼻滴递送的流感疫苗的效力目前非常差。PLGA NPs递送疫苗组与单独施用疫苗相比,抗体滴度有所增加,但效果仍不令人满意。PLGA-PEG NPs由于其PEG链而具有较弱的粘蛋白结合能力,这增加了纳米粒与流感疫苗复合物的粘液渗透,并且更容易刺激粘膜免疫反应。与其他组相比,本发明新型PLGA-PEG-Man NPs可作为疫苗的合格经鼻递送系统,并产生更高的抗体滴度,即有效的体液免疫(p<0.0001)。这是由于适当的甘露糖修饰的NPs可以作为有效的载体,使得流感疫苗和NPs更容易被APC细胞识别和呈递,从而导致更好的免疫响应。
实施例5
DSPE-PEG-Man脂质体的制备
(1)DSPE-PEG-Man的制备
基于实施例1酰胺化反应的原理,将甘露糖胺与DSPE-PEG-COOH反应,通过羧基端与甘露糖胺的氨基端反应生成磷脂聚乙二醇甘露糖DSPE-PEG-Man,其核磁图如图15;
(2)DSPE-PEG-Man脂质体的制备
称取100mg大豆卵磷脂、25mg胆固醇加入10mL无水乙醇,水浴超声,直到固体充分溶解,得总脂质浓度为12.5mg/mL,作为油相;将20mg DSPE-PEG-Man溶解于2mL pH为7.4的PBS中为水相;取10mL油相于注射器中,除去管道中的死体积,以水相:油相的流速比为4:1(水相:1.2mL/min;油相:0.3mL/min),注入微流控设备中,先用油相将整个微流控管道充满,再通水相,即得DSPE-PEG-Man脂质体;
(3)按照体积比5:4与疫苗共混,得到包载疫苗的DSPE-PEG-Man脂质体。
DSPE-PEG-Man脂质体的测定
使用马尔文ZS90型激光粒度分析仪,动态光散射法测定各条件下制备样品的粒径及分布。在每次分析之前,将制备的DSPE-PEG-Man脂质体在蒸馏水中进行适当的稀释。取1ml样品置于样品池中,设定测量前样品25℃下平衡2min,选择测量模式为Automatic,并以25℃时水的折射率及黏度指数进行分析,每个样品平行测定3次。脂质体表征结果如表7所示。
表7
制剂 | 粒径(nm) | PDI | Zeta电位(mV) |
DSPE-PEG-Man脂质体 | 201 | 0.242 | ﹢5.24 |
从表7的数据可以看出,DSPE-PEG-Man脂质体粒径均一,分散性良好,可用于疫苗递送。
实施例6
DSPE-PEG-Man脂质体吸附疫苗的体内免疫效果
DSPE-PEG-Man脂质体疫苗吸附率测定
BSA标准曲线的建立:精密称取BSA20.0mg,以pH 7.4PBS溶解并定容到10mL容量瓶中,得到浓度为2.0mg/mL的标准溶液,置4℃冰箱保存。分别精密移取上述标准溶液1.0mL、2.0mL、3.0mL、4.0mL、5.0mL和6.0mL,分别置于10mL量瓶中,以pH 7.4PBS稀释并定容,摇匀,得到浓度分别为0.10、0.20、0.30、0.40、0.50、0.60mg/mL的系列标准溶液,在波长(λ)为290nm进行检测,平行测定两次,记录吸光度,计算平均值。以BSA浓度C(mg/mL)为横坐标,吸光度(A)为纵坐标,绘制标准曲线,计算线性回归方程为:y=0.2516x+0.1023,,计算得R2=0.9811,表明在该浓度范围内OVA浓度与吸光度线性关系良好。
采用离心法测定其吸附率。分别精密量取上述脂质体溶液1mL置于离心管中。在4℃条件下,置于离心机中10000rpm离心30min,取上清液少许,采用紫外-可见分光光度计测定游离疫苗含量。根据下列公式计算疫苗的吸附率(LE%)。
其中M0表示加入的疫苗总药物量;M1表示脂质体中游离的疫苗含量。
DSPE-PEG-Man脂质体疫苗吸附率测定结果如表8所示:
表8
浓度(mg/mL) | 吸光度(A) |
0.5 | 0.1871 |
1.0 | 0.3618 |
1.5 | 0.5174 |
2.0 | 0.6341 |
2.5 | 0.7349 |
3.0 | 0.8206 |
样品(扣除溶剂背景) | 0.2934 |
根据样品吸光度及y=0.2516x+0.1023式,计算得M1=9.82mg,进而根据计算得DSPE-PEG-Man脂质体吸附率为50.9%,与poly(I:C)脂质体吸附率相当,说明本发明制剂可以作为佐剂使用。
动物实验样品的制备和动物免疫
将20.00mg DSPE-PEG-Man、poly(I:C)脂质体各自溶于10mL 0.05MpH=7.4PBS溶液中;在生化培养箱中,之后各取250μL重与200μL疫苗混合,然后涡旋30s。用移液枪吸取150μL疫苗原液(0.167μg/μL),加入到250μL0.05M PBS溶液中,进行动物给药。为了免疫,将小鼠随机分为4组,每组6只动物。这些组分别为疫苗+DSPE-PEG-Man脂质体组、疫苗+poly(I:C)脂质体组、疫苗、PBS组,每只小鼠在第0天和第14天后腿肌内注射给药。28后取血,血凝抑制实验结果如表9所示:
表9
从实验结果来看,疫苗+DSPE-PEG-Man脂质体组所产生的抗体血凝滴度的几何平均效价GMT(813)要比不加佐剂的纯疫苗组的(456)显著更高,且优于传统佐剂poly(I:C)组的(575),因此可以得出DSPE-PEG-Man在疫苗肌肉注射领域突出的佐剂作用,拓宽了其作为佐剂作用的递送途径。
实施例7
糖基化衍生物用于疫苗口服递送的探究
实验样品的制备
称取100mg大豆卵磷脂、25mg胆固醇加入10mL无水乙醇,水浴超声,直到固体充分溶解,得总脂质浓度为12.5mg/mL,作为油相。分别将20mg PEG-Man,PEG-Tre,PLGA-PEG-Man,DSPE-PEG-Man和poly(I:C)溶解于2mL pH为7.4的PBS中为水相。取10mL油相于注射器中,除去管道中的死体积,以水相:油相的流速比为4:1(水相:1.2mL/min;油相:0.3mL/min),注入微流控设备中,先用油相将整个微流控管道充满,再通水相,然后按照疫苗20μg/200μL加入共混,分别得到相应的脂质体。
动物分组及免疫
为了免疫,将小鼠随机分为7组,每组6只动物。这些组分别为疫苗+DSPE-PEG-Man脂质体组、疫苗+PLGA-PEG-Man脂质体组,疫苗+PEG-Man脂质体组,疫苗+PEG-Tre脂质体组,poly(I:C)脂质体+疫苗组,PBS组,i.m组。免疫前12h禁食水,用7.5% NaHCO3和Hank液按1:4比例混合制备胃酸中和液,每只鼠灌胃300μL胃酸中和液中和胃酸,30min后将药物灌胃免疫。实验组小鼠200μL脂质体灌胃疫苗,对照组小鼠灌胃等量的生理盐水。每组小鼠免疫2次,每次间隔14d。28d后取血,血凝抑制实验结果如图16,从实验结果来看,通过糖基化修饰后的脂质体口服递送疫苗,比口服单纯疫苗组效果要好,且相较于传统佐剂poly(I:C)组,表现出不俗的抗体滴度。因此可以得出所合成的糖基化高分子衍生物在口服疫苗方面突出的佐剂作用,进一步拓宽了其作为佐剂作用的递送途径。
以上结合实施例对本发明进行了详细说明,但所述内容仅为本发明的较佳实施例,不能被认为用于限定本发明的实施范围。凡依本发明申请范围所作的均等变化与改进等,均应仍属于本发明的专利涵盖范围之内。
Claims (10)
1.一种具有佐剂作用的高分子衍生物,其特征在于结构式如(I)、(II)或(III)所示:
其中,R1、R2分别包括海藻糖基、甘露糖基、岩藻糖基、葡萄糖基、果糖基、或其相应的衍生基团,R3包括PEG、PLGA、PLA、透明质酸、壳聚糖、以氨基酸为基本单元的聚合物、磷脂及其衍生物、胆固醇及其衍生物。
2.一种权利要求1所述具有佐剂作用的高分子衍生物的制备方法,其特征在于结构式(I)通过下述方法制备得到:以包括海藻糖在内的糖类为起始原料,通过乙酰化、炔丁基取代、去乙酰化、炔基-叠氮环加成反应制备得到目标产物;
结构式(II)、(III)分别通过下述方法制备得到:以包括甘露糖胺在内的氨基化糖类为起始原料,通过缩合反应制备得到目标产物。
3.根据权利要求2所述具有佐剂作用的高分子衍生物的制备方法,其特征在于包括下述步骤:
(1)将海藻糖、乙酸酐、及无水乙酸钠依次加入反应器,进行乙酰化反应得到乙酰化海藻糖;
(2)将乙酰化海藻糖和3-丁炔-1-醇在酸性条件下,进行取代反应得到炔基乙酰海藻糖;
(3)将炔基乙酰海藻糖和甲醇钠,在甲醇溶剂中,于室温进行脱保护基得到炔基海藻糖;
(4)将二叠氮聚乙二醇、炔基海藻糖、五水硫酸铜、抗坏血酸钠加入反应器,经炔基-叠氮环加成反应得到海藻糖-PEG。
4.根据权利要求2所述具有佐剂作用的高分子衍生物的制备方法,其特征在于包括下述步骤:将二乙酸聚乙二醇与甘露糖胺盐酸盐溶解在有机溶剂中,然后逐步加入4-二甲氨基吡啶和碳二亚胺,将反应混合物在氮气室温条件下搅拌过夜,之后经沉淀、离心、干燥得产物甘露糖-PEG。
5.一种权利要求1所述具有佐剂作用的高分子衍生物在制备药物递送载体中的应用。
6.根据权利要求5所述的应用,其特征在于:所述药物递送载体包括微针、脂质体、纳米粒、微囊微球、粉针剂和溶液。
7.根据权利要求5和6所述的应用,其特征在于:具有佐剂作用的高分子衍生物可与包括减毒活疫苗、裂解疫苗、亚单位疫苗、重组疫苗和核酸疫苗在内的疫苗共同递送,产生对疫苗免疫作用的协同增效。
8.根据权利要求6所述的应用,其特征在于:所述微针为海藻糖-PEG微针或甘露糖-PEG微针,其制备方法如下:
①微针阴模制备
将金属微针阳模阵列放在阳模容器中,将聚二甲基硅氧烷和固化剂以10:1的质量比充分混合后,注满容器;随后将其放入真空干燥箱中,在-80Kpa真空度下1h以完全除去气泡,接着将其放入70℃烘箱下2h固化,将聚二甲基硅氧烷阴模与微针阳模分离,得到微针阴模;
②基质溶液配制
称取基质材料,加入去离子溶解,静置12h消除气泡,后与70wt%的海藻糖-PEG或甘露糖-PEG共混得备用;其中,基质材料包括PLGA、PLA、PVA、PVP、壳聚糖、透明质酸、普鲁兰糖、明胶、纤维素;基质材料与海藻糖-PEG或甘露糖-PEG的体积比为1:1~30:1;
③微针的制备
取基质溶液于聚二甲基硅氧烷阴模中,随后将阴模放入离心机中,在25℃、4000rpm的条件下离心10min使基质溶液入模;离心结束后,将阴模在室温下干燥24h,将微针从模具中脱出,即得。
9.根据权利要求6所述的应用,其特征在于:脂质体通过下述方法制备得到:
(1)将甘露糖胺与磷脂聚乙二醇羧酸通过反应生成磷脂聚乙二醇甘露糖,记为DSPE-PEG-Man;
(2)称取大豆卵磷脂、胆固醇,加入无水乙醇,水浴超声,直到固体充分溶解,得总脂质浓度为12.5mg/m L,作为油相;将20mg DSPE-PEG-Man溶解于2mL pH为7.4的PBS中,作为水相;取10mL油相于注射器中,除去管道中的死体积,以水相:油相的流速比为4:1,注入微流控设备中,先用油相将整个微流控管道充满,再通水相,即得DSPE-PEG-Man脂质体;
纳米粒通过下述方法制备得到:
(1)将甘露糖胺与聚乳酸-羟基乙酸共聚物-羧酸反应,通过共聚物的羧基端与甘露糖胺的氨基端反应生成PLGA-PEG-Man;
(2)纳米粒的制备采用纳米沉淀法进行,将PLGA-PEG-Man溶于丙酮溶剂中呈有机相,在搅拌条件下,将有机相匀速滴加至水溶液中,然后进行挥发完全至除去有机溶剂得纳米粒悬浮液;其中PLGA-PEG-Man浓度为10~100mg/mL、有机相与水相的体积比为1:4~1:10。
10.根据权利要求5所述的应用,其特征在于:药物递送途径包括经皮肤递送、局部粘膜递送、口服递送、注射递送、植入递送。
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