CN111569061A - 一种用于核酸疫苗增强剂的纳米材料 - Google Patents
一种用于核酸疫苗增强剂的纳米材料 Download PDFInfo
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Abstract
本发明提供了一种用于核酸疫苗增强剂的纳米材料,其特征在于,所述纳米材料通过以下方法制备获得:(1)制备A溶液:将CaCl2与Tris buffer溶于水中;(2)制备B溶液:将HEPES缓冲液与水混合;(3)将B溶液滴加到A溶液中,搅拌,制得CP纳米颗粒沉淀;(4)将帕米膦酸钠滴加到步骤(3)的AB混合液中,滴加人血丙种球蛋白,获得本发明的纳米材料。本发明的纳米材料显著提高核酸疫苗对靶细胞的转染效率,增加抗原表达,促进抗原提呈细胞成熟从而建立有效的免疫反应,实现核酸疫苗的增强或增效功能。
Description
技术领域
本发明属于纳米材料技术领域,具体涉及一种用于核酸疫苗增强剂的纳米材料。
背景技术
核酸疫苗是将编码某种抗原蛋白的外源基因(DNA或RNA)导入动物或人体细胞内,并通过宿主细胞的表达***合成抗原蛋白,诱导宿主产生对该抗原蛋白的免疫应答,以达到预防和治疗疾病的目的。核酸疫苗相比传统疫苗具有很多优点,目前已有核酸疫苗进入临床阶段甚至上市应用。但是,如何让核酸疫苗在进入机体后能够顺利到达靶细胞并有效表达和提呈抗原蛋白,一直是阻碍核酸疫苗快速发展和加速应用的瓶颈。
目前,因为核酸疫苗进入机体后,只有极少量的核酸疫苗(2%)能够自由进入细胞,而能到达靶细胞(抗原提呈细胞)的疫苗则更少,造成核酸疫苗免疫用量大,副作用较大,成本过高,且免疫效果不理想。核酸疫苗免疫机体后,必须在抗原提呈细胞中有效表达抗原蛋白,加工和提呈抗原并完成细胞成熟免疫反应才能建立,而如果核酸疫苗进入的是未成熟的抗原提呈细胞,则依然不能实现抗原蛋白的表达,造成免疫效率低下。有很多开发出的核酸疫苗不能最终用于临床,不是因为疫苗本身设计的问题,而是没有一种核酸疫苗增强剂来提高它对有效细胞的转染效率,无法建立有效的免疫反应,最终无奈放弃对该疫苗的进一步开发与推广应用。
虽然目前有一些零星研究用纳米材料辅佐核酸疫苗增强免疫反应,它们被称为疫苗佐剂,而且只针对抗原提呈某一环节。我们提出的核酸疫苗增强剂不同于佐剂,是从核酸疫苗整个体内环节包括,疫苗保护,促进进入抗原提呈靶细胞,提高抗原提呈靶细胞成熟,增强蛋白的转录表达和抗原加工提呈,全面提升核酸疫苗效率。
目前还没有核酸疫苗增强(效)剂的报道。本专利弥补了这一空白,可以极显著提高核酸疫苗对靶细胞的转染效率,增加抗原表达,促进抗原提呈细胞成熟从而建立有效的免疫反应,实现核酸疫苗的增强或增效功能。
发明内容
本发明的目的在于克服上述现有技术的不足之处而提供一种用于核酸疫苗增强(效)剂的纳米材料,可以更有效把核酸疫苗送入靶细胞并显著提高抗原蛋白的表达和提呈,因而从多个环节提高免疫反应。
为实现上述目的,本发明采取的技术方案为:一种用于核酸疫苗增强剂的纳米材料,通过以下方法制备获得:
(1)制备A溶液:将CaCl2与Tris buffer溶于水中;
(2)制备B溶液:将HEPES缓冲液与水混合;
(3)将B溶液滴加到A溶液中,搅拌,制得CP纳米颗粒沉淀;
(4)将帕米膦酸钠滴加到步骤(3)的AB混合液中,滴加人血丙种球蛋白,获得本发明的纳米材料。
优选地,CaCl2的浓度为1.0~2.0M;所述Tris buffer的浓度为10mM,pH值为10。
优选地,所述HEPES缓冲液包括以下组分:200~280mM NaCl、10~15mM Na2HPO4和24~50mM HEPES。本发明所述HEPES为4-(2-羟乙基)-1-哌嗪乙磺酸。
优选地,所述帕米膦酸钠(Pamidronate)的浓度为0.1-5mg/mL。
优选地,所述人血丙种球蛋白的浓度为0.04-0.8ug/mL。
与现有技术相比,本发明具有以下有益效果:
1、本发明的核酸疫苗纳米增强剂具有细胞选择性,可特异地与抗原提呈细胞相结合,具有核酸疫苗递送的细胞靶向性(抗原提呈细胞)。
2、细胞膜为磷脂双分子层,在膜的靠内外两侧各有一条厚约2.5nm的电子致密带,使得细胞膜的特性不利于大多数的核酸片段或质粒进入细胞,进而极大地降低了核酸进入细胞内的几率。本发明的核酸疫苗纳米增强剂为一种物、化相容性较好的纳米颗粒,可以有效地在核酸与细胞膜之间建立桥梁连接作用,极大地提高核酸与细胞膜的接触机率及进入细胞浆及细胞核的效率,并且该纳米颗粒可以刺激抗原提呈细胞的吞噬功能和细胞成熟并促进核酸疫苗的免疫反应。
3、携带核酸疫苗的纳米颗粒在被抗原提呈细胞吞噬后,能够改变吞噬溶酶体内的pH值,进而保护核酸不被吞噬溶酶体内的核酸酶降解破坏。
4、DNA只有进入细胞核后,才能通过转录和翻译,最终表达出抗原蛋白。本发明的核酸疫苗纳米增强剂具有提高核酸疫苗高效进入细胞核的能力。
5、未成熟的抗原提呈细胞即使摄取了核酸物质并进入细胞核后,仍不能呈递相关的表达抗原,只有这些细胞成熟后,才能实现把抗原有效呈递给T或B细胞,诱导免疫反应。本发明的核酸疫苗纳米增强剂自身成分可以显著促进未成熟的抗原提呈细胞向成熟的抗原提呈细胞转化,进而有效提高核酸疫苗的免疫效力。
附图说明
图1为本发明MCP纳米颗粒透射电镜图。
图2为4小时巨噬细胞RAW264.7对CP-Cy5、Liposome-Cy5、MCP-Cy5的效率摄取效率示意图。
图3为pGFP转染至巨噬细胞,MCP纳米颗粒体外的传递效率示意图,A:阳性细胞百分比,B:流式细胞仪结果。
具体实施方式
为了更加简洁明了的展示本发明的技术方案、目的和优点,下面结合具体实施例对本发明做进一步的详细描述。
实施例1
本实施例提供一种用于核酸疫苗增强剂的纳米材料,通过以下方法制备获得:
(1)制备A溶液:将0.42mL浓度为2.0M的CaCl2与0.84mL pH=10浓度为10mM的Trisbuffer溶于2mLddH2O中;
(2)制备B溶液:将0.43mL的HEPES缓冲液与0.3mL的ddH2O混合;其中HEPES缓冲液包含浓度为280mM的NaCl,15mM的Na2HPO4和50mM的HEPES。
(3)将B溶液滴加到A溶液中,在500rpm下搅拌30分钟,制得CP纳米颗粒沉淀;
(4)将0.1mg/mL的帕米膦酸钠(Pamidronate)滴加到步骤(3)的AB混合液中,滴加浓度为0.04ug/mL的人血丙种球蛋白(Humanγ-globulin),搅拌30分钟,获得本发明的纳米材料。
实施例2
本实施例与实施例1的唯一区别在于本实施例中的帕米膦酸钠浓度为1mg/mL。
实施例3
本实施例与实施例1的唯一区别在于本实施例中的帕米膦酸钠浓度为3mg/mL。
实施例4
本实施例与实施例1的唯一区别在于本实施例中的帕米膦酸钠浓度为5mg/mL。
实施例5
本实施例与实施例1的唯一区别在于本实施例中的人血丙种球蛋白浓度为0.08mg/mL。
实施例6
本实施例与实施例1的唯一区别在于本实施例中的人血丙种球蛋白浓度为0.15mg/mL。
实施例7
本实施例与实施例1的唯一区别在于本实施例中的人血丙种球蛋白浓度为0.3mg/mL。
实施例8
本实施例与实施例1的唯一区别在于本实施例中的人血丙种球蛋白浓度为0.6mg/mL。
实施例9
本实施例与实施例1的唯一区别在于本实施例中的人血丙种球蛋白浓度为0.8mg/mL。
实施例10
将本发明制得的MCP纳米颗粒进行电镜扫面,将纳米颗粒在ddH2O中稀释成终浓度为0.1mg/mL,进行透射电镜(TEM)成像,透射电镜为日立公司的7700电子显微镜。结果如图1所示,图1A显示MCP纳米颗粒为100nm左右园形球体,多个颗粒团聚在一起,图1B显示颗粒经蛋白修饰后,粒径变大180nm左右,其分散性较好。
实施例10MCP纳米颗粒的pDNA装载率
制备CP-pGFP和MCP-pGFP
DNA样品先在溶液A中预混合,然后与溶液b混合。将合成的纳米颗粒以15000×g离心10min,收集上清进行DNA负载测定。然后加入1毫升的ddH2O清洗颗粒。悬浮液以15000×g离心5min,小心除去上清。最后,将样品重新分散在ddH2O或细胞培养基中进行进一步测试。
每个样本固定10μg/毫升pGFP,CP纳米颗粒及MCP纳米颗粒数量调整以达到5%,10%,20%(wt%)加载。每个样本被收集到一个新的1.5mL的试管中,并以15,000g离心10分钟。收集上清液,使用Nanodrop 1000(Thermo scientific)对卸载的pGFP残渣进行定量。凝胶保留分析用于确认pDNA与纳米颗粒的结合。阳性对照是质粒准备在10μg/毫升水。对于每一个样本和阳性对照,均取20μL上层清液的混合4μL 6×加载缓冲区和加载1%琼脂糖凝胶GelRed染料。电泳使用PowerPac Basic(BIO-RAD),80V电泳1小时,凝胶图像由ChemiDoc MP(BIO-RAD)采集。
实施例11巨噬细胞对MCP纳米颗粒摄取效率。
装载50nM dsDNA-Cy5,合成CP NPs-dsDNA-Cy5(CP-Cy5)和MCP NPs-dsDNA-Cy5(MCP-Cy5)。使用50nM dsDNA-Cy5制备脂质体(OligofectamineTM试剂,ThermoFisher)-Cy5。利用FACS(CytoFLEX,Beckman Coulter)定量检测CP-Cy5、MCP-Cy5和脂质体-cy5的细胞摄取。在24孔板中以5×104/孔的密度接种RAW 264.7细胞,在37℃、5%CO2湿培养箱中培养24h。然后加入10μg/毫升MCP-Cy5培养4小时。之后,丢弃培养基,PBS清洗3次细胞。细胞经胰蛋白酶化,离心收集,在4%的PFA溶液中固定,用于FACS分析。同时,脂质体-cy5和CP-Cy5作为对照组,遵循相同的方案。结果如图2所示,与CP-Cy5、Liposome-Cy5组相比,巨噬细胞对MCP纳米颗粒摄取效率具有显著性差异。
实施例12体外DNA疫苗向巨噬细胞传递模型
我们使用pGFP作为功能模型来评价MCP纳米颗粒体外的传递效率。该pDNA在U2OS细胞株中使用流式细胞仪进行验证。具体用转染后GFP阳性细胞百分比%(图3A)和平均荧光强度MFI(图3B)来评估。比较脂质体(Liposome)-pGFP、电转(electroporation)、CP-pGFP、MCP-pGFP的转染效率。每个转染体系,使用1.0μg/mL pGFP孵育48小时。与脂质体和电转方法比较,MCP转染效率(百分比和MFI)都显著高。另外比类似calcium phosphate(CP)颗粒也都显著高。这些结果显示MCP颗粒具有优越的传递DNA疫苗的能力。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (5)
1.一种用于核酸疫苗增强剂的纳米材料,其特征在于,所述纳米材料通过以下方法制备获得:
(1)制备A溶液:将CaCl2与Tris buffer溶于水中;
(2)制备B溶液:将HEPES缓冲液与水混合;
(3)将B溶液滴加到A溶液中,搅拌,制得CP纳米颗粒沉淀;
(4)将帕米膦酸钠滴加到步骤(3)的AB混合液中,滴加人血丙种球蛋白,获得本发明的纳米材料。
2.如权利要求1所述的纳米材料,其特征在于,所述CaCl2的浓度为1.0~2.0M;所述Trisbuffer中的浓度为10mM,pH值为10。
3.如权利要求1所述的纳米材料,其特征在于,所述HEPES缓冲液包括以下组分:200~280mM NaCl、10~15mM Na2HPO4和24~50mM HEPES。
4.如权利要求1所述的纳米材料,其特征在于,所述帕米膦酸钠的浓度为0.1-5mg/mL。
5.如权利要求1所述的纳米材料,其特征在于,所述人血丙种球蛋白的浓度为0.04-0.8ug/mL。
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