CN114848608B - 一种蛋白或多肽递送载体及其制备方法与应用 - Google Patents
一种蛋白或多肽递送载体及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种蛋白或多肽药物/疫苗递送载体、制备方法及应用,属于医学免疫学和生物医药领域。一种蛋白或多肽药物/疫苗递送载体的制备方法,包括以下步骤:将蛋白或多肽结合在DMPE‑PEG‑NHS上,将所得产物与DSPE‑PEG混合,制成胶束;将制得胶束溶解后与外泌体共孵育,冷却后分离纯化,获得装载蛋白或多肽的外泌体。
Description
技术领域
本发明涉及医学免疫学和生物医药领域,具体涉及一种蛋白或多肽药物或蛋白或多肽疫苗递送载体、制备方法及应用。
背景技术
外泌体是一种直径小于200nm的小型细胞外囊泡,可由任意活细胞分泌并存在于多种体液当中。其中,红细胞外泌体具有易获取、储备多、成分差异小等特点,是优选的外泌体来源。研究证实,外泌体作为蛋白、核酸和小分子物质等的载体介导细胞之间对话和物质运输。由于其免疫原性低、可穿透生物屏障、无细胞毒性、天然稳定,常被用做药物递送研究的生物载体。如今,携带蛋白或多肽的外泌体疫苗已经是肿瘤和感染性疾病领域的一种新兴免疫治疗方法或预防方法,也出现了多种外泌体装载蛋白或多肽的方法,包括外泌体表面编辑和装载物编辑。然而现有方法除了需要对载体或载物进行编辑使操作复杂化之外,还可能会对其造成不必要的损伤。另外,现有方法局限于种类较少的、已知序列的蛋白多肽装载,缺乏一定普适性。因此,本发明利用自组装技术,成功构建了更为简便易操作的适用于装载多种随机蛋白或多肽的外泌体递送系统。
发明内容
针对现有技术的不足,本发明提出了一种蛋白或多肽药物/疫苗递送载体、制备方法及应用。
本发明的目的可以通过以下技术方案实现:
一种蛋白或多肽递送载体的制备方法,包括以下步骤:
将多种或单种的蛋白或多肽结合在DMPE-PEG-NHS上,将所得产物与DSPE-PEG混合,制成胶束;
将制得胶束溶解后与外泌体共孵育,冷却至4℃后立即用尺寸排阻色谱法分离纯化,收集第二个峰产物为成功装载蛋白或多肽的外泌体。
可选地,所述外泌体源自红细胞或其他人体细胞。
可选地,加入络合剂辅助蛋白或多肽与DMPE-PEG-NHS结合。
可选地,所述的蛋白多肽为SARS-CoV-2的5种蛋白的31条T细胞表位肽或其他蛋白或多肽。
另一方面,本发明还提出了一种上述的方法制得的药物递送载体。
再一方面,本发明提出了上述的药物递送载体的制备方法在制备蛋白疫苗、蛋白质药物、多肽疫苗及多肽药物中的应用。
本发明的有益效果:
1.本发明提供了可以同地向外泌体装载多种蛋白或多肽的方法,并且通过肽键连接,无需提前知晓蛋白或多肽的序列,无需提前对外泌体表面和装载物(蛋白/多肽)进行编辑或修饰,因此具有较为广泛的适用性;
2.本发明提供的装载多种随机蛋白或多肽的外泌体递送系统可以利用外泌体的生物载体功能将多种抗原肽递送至皮下抗原递呈细胞分布较多处,并且外泌体较低的免疫原性减少了对机体的不必要的损伤,这对COVID-19疫苗的研制具有一定的价值;
3.本发明制备方法简单、容易操作,红细胞简单易获取、储备充足,可量产。
附图说明
下面结合附图对本发明作进一步的说明。
图1为外泌体装载多肽的示意图;
图2为DMPE-PEG-NHS与BSA不同比例装载的效率图;
图3为红细胞外泌体的(A)粒径分布、(B)形态学和(C)WB指标的表征;
图4为装载BSA的外泌体递送系统产物(BSA-PEG-lipid-exoosme)的鉴定结果图:(A)BSA装载外泌体混合物尺寸排阻层析结果显示三个蛋白峰;(B)Western Blot检测出BSA蛋白分布在峰2和峰3,外泌体分布在峰1和峰2;(C)液体和磁(1H-NMR)检测出DMPE-PEG分布在峰2和峰3;(D-F)透射电镜展示峰1、2、3产物形态。
图5~8为装载SARS-CoV-2蛋白的31种T细胞表位肽的外泌体递送系统免疫HLA转基因健康小鼠的结果及其统计图:图5为IFN-γ酶联免疫斑点法(ELISPOT)检测的原始斑点图结果;图6为胞内荧光染色后流式细胞检测结果图;(图7A、7B)ELISPOT检测小鼠免疫后,淋巴细胞在多肽刺激下的活化情况;(图7C、7D)胞内荧光染色细胞流式检测小鼠免疫后,淋巴细胞在多肽刺激下CD3+/CD8+T细胞活化情况;图8为IFN-γELISPOT检测单肽免疫效果的统计结果。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其它实施例,都属于本发明保护的范围。
实施例1BSA-PEG-lipid胶束的合成
1.DMPE-PEG-NHS包被BSA
实验步骤如下:(DMPE-PEG-NHS与DSPE-PEG皆为上海芃硕生物科技有限公司合成)
(1)试剂溶解:DMPE-PEG-NHS 10mg+DMSO 100ul(100ug/ul)
BSA 1mg+DMSO 2ml(0.5ug/ul)
(2)如下表1所示分组进行不同比例包被,室温过夜;
表1优化BSA-PEG-DMPE的生成
2.BSA-PEG-DMPE与DSPE-PEG合成胶束
试剂溶解:DSPE-PEG 10mg+HEPES缓冲液(pH 7.4)100ul(100ug/ul);
向BSA-PEG-DMPE补加HEPES缓冲液至DMSO浓度不高于1%(V*100-V);
加DSPE-PEG各13.2ul(与DMPE-PEG-NHS摩尔比1:1),60℃15min;
超滤法(100KD MWCO)去除未结合的蛋白,至上层液体为1ml或者滤不动;
收集超滤管下层液体超滤(30KD MWCO)至1ml左右,用BCA法检测蛋白含量为未结和BSA含量,其与加入BSA总量的差值即为成功装载的BSA含量;
结果见图2,装载的BSA含量随比例增加逐渐上升,直到1:3之后开始下降,装载效率一直处于较高水平直到1:3之后开始下降,因此选用DMPE-PEG-HNS:BSA=1:3作为最佳装载比例;
收集超滤管上层液体为将胶束,放置4℃保存,2周内使用;
实施例2装载蛋白多肽的外泌体递送系统的构建与鉴定
1.红细胞来源外泌体的提取
(1)取健康人外周血:PBS冲洗滤盘,对倍稀释,后用人淋巴细胞分离液(Ficoll分离液)分离PBMC;
(2)小心弃去血浆层、PBMC层、Ficoll层和白细胞层,转移底层RBC沉淀至新50ml无菌离心管中;
(3)用PBS稀释25倍,3000rpm,10min,4℃;清洗2次后弃去上清,保留沉淀;
(4)RBC沉淀用PBS稀释50倍:即800ul沉淀补充PBS至40ml,加入1μM的钙离子载体之后封口,置4℃摇床48h;
(5)超速离心法提取外泌体:4℃离心2000g,20min,留取上清液,弃红细胞沉淀;4℃离心13500g,30min,取上清,弃小细胞碎片以及大囊泡,接着用0.22um滤膜过滤;滤后上清,超速离心100,000g 2h;小心弃去上清,用100ul PBS重悬,-80℃冻存。
图3所示为红细胞来源外泌体的鉴定结果,直径75%位于50-150nm之间,呈球形,并且表达人外泌体经典指标CD63、Alix和CD9。
2.BSA-PEG-lipid-exosome合成
(1)将胶束在60℃处理10min,使其重新溶解,然后用超声振荡仪振荡2次(10uM振幅5s/次)。目的是降低胶束的大小,有利于后续与EV的分离;
(2)将外泌体与胶束按照蛋白比1:1混合均匀,水浴40℃,2h;
(3)冷却至4℃,立即用Sepharose CL-4B凝胶柱(26/40型管)分离纯化:高压后PBS平衡凝胶柱,上样体积不超过6.5ml,后用PBS洗柱,收集到三个蛋白峰(280nm紫外检测仪),如图4A所示;
(4)收集各蛋白峰的洗脱液,用超滤管(峰1、2使用100KD MWCO;峰3使用10KDMWCO)离心浓缩,以供进一步鉴定。
3.装载BSA的外泌体递送系统的鉴定
(1)标志物检测:通过Western blot检测每个蛋白峰是否有BSA和外泌体表面标志蛋白,如图4B所示,峰2、3均有BSA存在,峰1、2均有外泌体标志物CD63存在,证明完整的BSA-PEG-lipid-exosome产物位于峰2;
(2)胶束成分检测:通过氢谱液体核磁共振检测三个蛋白峰中是否有DMPE-PEG-NHS,3.6ppm、2.6ppm和1.1ppm分别代表PEG、水解的NHS产物和DMPE,如图4C所示,峰2、3均有较多的DMPE-PEG-NHS,证明胶束多存在于此;
(3)形态检测:通过透射电镜观察三个蛋白峰产物形态,如图4D、E、F所示,分别为峰1、2、3产物,可见峰1有成堆体型较大囊泡装结构,峰2有100nm左右双层杯状结构,为正常外泌体形态,峰3产物中未见囊泡样结构;
本实施结果说明,蛋白可简便易操作地装载至外泌体上,无需提前编辑,并且未对外泌体形态和标志物造成损伤。蛋白峰1为凝集成团的囊泡,峰2为构建成功的装载BSA的外泌体递送系统,峰3为多余未装载的胶束。
实施例3装载SARS-CoV-2T细胞表位肽的外泌体递送系统的免疫效果验证1.构建peptide-PEG-lipid-exosome递送系统
将筛选出的31种HLA-A0201分子递呈的来自SARS-CoV-2蛋白的经验证的T细胞表位多肽(VEPs)分别溶解于DMSO(10μL/mg),并于使用前混合;
如实施例1、2所述方法,将DMPE-PEG-NHS与VEPs按照摩尔比1:3混合,加入1%TEA后室温过夜。接着按照摩尔比1:1加入DSPE-PEG在60℃共孵育15min。超滤去除未装载的肽,再溶解和超声之后,按照蛋白比1:1的比例与外泌体在40℃共孵育2h。冷却至4℃之后立即进行尺寸排阻层析,纯化并收集合成产物。
2.用装载VEPs的外泌体递送系统免疫HLA-A2/DR1转基因鼠
HLA-A0201+/+/DR1+/+/H-2-β2m-/-/I-Aβ-/-C57BL/6小鼠与普通C57BL/6小鼠杂交后的转基因杂交鼠被随机分成两组:给予递送系统+佐剂(peptide-PEG-lipid-exosome/PolyI:C)的疫苗组和给予生理盐水+佐剂的对照组。在Day 0、7和21行皮下注射,疫苗组的5只小鼠每次给药共1.64mg VEPs(每种多肽大约10μg/只)和500μg佐剂,对照组5只小鼠每次给药共500μg佐剂,末次注射一周后处死小鼠,取脾脏提取脾细胞与表位肽库共培养20小时后进行酶联免疫斑点法(ELISPOT)和胞内荧光染色流式检测,检测多肽疫苗在体内诱导特异性T细胞反应的能力。ELISPOT结果如图5所示,分别用多肽、无关肽(AFP)和PBS刺激小鼠淋巴细胞观察到T细胞活化并IFN-γ的生成情况;图7A、B为ELISPOT统计结果,疫苗组小鼠的脾细胞群中活化的淋巴细胞高于对照组10-82倍;胞内荧光染色流式检测结果见图6,展示了脾细胞群中被活化并分泌IFN-γ的CD8+T细胞的比例;图7C、D为流式统计结果,疫苗组小鼠的脾细胞群中VEPs特异性CD3+/CD8+T细胞的数量比对照组高13-65倍,证明装载VEPs的外泌体递送系统在转基因杂交鼠体内能诱导强劲的表位肽特异性T细胞免疫应答。为了明确31种表位肽种哪些表位肽在体内具有免疫原性,本实验进一步将31种表位肽分别与免疫小鼠的脾细胞共培养20小时,然后进行ELISPOT实验。图8为单种多肽刺激的IFN-γ阳性斑点数与PBS刺激斑点数的比较,结果证实:在装载到本递送系统的31种VEPs中,有28种多肽能有效诱导特异性T细胞免疫反应,只有R3、R11和R12在所有小鼠中刺激T细胞反应较弱,ELISPOT斑点数均未达PBS刺激组的两倍及以上。
上述的实验结果说明,本发明所制备的装载VEPs的外泌体递送载体可以用于同时递送多种蛋白多肽,并且可作为外泌体疫苗在体内诱导SARS-CoV-2特异性CD8+T细胞免疫应答。
上述实施例中的SARS-CoV-2的表位肽可以选自申请人提交的已公开的专利申请(申请号为2021102090657)序列表中所记载表位肽。
可以理解的是,上述的实施例中,虽然针对于SARS-CoV-2的表位肽的搭载与递送进行了说明,但本申请的方法中所制备的递送载体的应用并不仅限于SARS-CoV-2的疫苗的递送以及表位肽的递送。由于本申请所制备的递送载体的普适性,其他的蛋白质药物、蛋白疫苗以及多肽药物及疫苗的递送,也属于本申请的载体的应用范围。
在本说明书的描述中,参考术语“一个实施例”、“示例”、“具体示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中,对上述术语的示意性表述不一定指的是相同的实施例或示例。而且,描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。
Claims (6)
1.一种蛋白递送载体的制备方法,其特征在于,包括以下步骤:
将BSA结合在DMPE-PEG-NHS上,DMPE-PEG-NHS与BSA的摩尔比为1:3,将所得产物与DSPE-PEG混合,制成胶束;
将制得胶束溶解后与外泌体共孵育,冷却后分离纯化,获得装载蛋白的外泌体;
其中,所述胶束的合成步骤如下:
试剂溶解:向10mg DSPE-PEG中加入pH为7.4的HEPES缓冲液100μl;
向BSA-PEG-DMPE中补加HEPES缓冲液至DMSO浓度不高于1%;
再加入DSPE-PEG 13.2μl,DSPE-PEG与DMPE-PEG-NHS摩尔比1:1,60℃ 15min;
超滤法去除未结合的蛋白,超滤膜为100KD MWCO,至上层液体为1ml或者滤不动;
收集超滤管下层液体超滤至1ml,超滤膜为30KD MWCO,用BCA法检测蛋白含量为未结合BSA含量,其与加入BSA总量的差值即为成功装载的BSA含量;
收集超滤管上层液体即为胶束。
2.根据权利要求1所述的蛋白递送载体的制备方法,其特征在于,所述外泌体源自红细胞。
3.根据权利要求1所述的蛋白递送载体的制备方法,其特征在于,加入络合剂辅助蛋白与DMPE-PEG-NHS结合。
4.根据权利要求1所述的蛋白递送载体的制备方法,其特征在于,所述的分离纯化步骤包括:通过尺寸排阻色谱法分离纯化,收集第二个峰产物为装载蛋白的外泌体。
5.由权利要求1~4任一所述的方法制得的蛋白递送载体。
6.权利要求1~4任一所述的蛋白递送载体的制备方法在制备蛋白疫苗和蛋白质药物中的应用。
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