CN112870348B - 一种壳聚糖纳米硒颗粒及其制备方法和在疫苗中的应用 - Google Patents
一种壳聚糖纳米硒颗粒及其制备方法和在疫苗中的应用 Download PDFInfo
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Abstract
本发明公开了一种壳聚糖纳米硒颗粒及其制备方法和在疫苗中的应用,其中所述壳聚糖纳米硒通过壳聚糖和亚硒酸钠等原料制备,其可实现对抗原和疫苗佐剂的高效包裹。该壳聚糖纳米硒颗粒具有极好的生物安全性。该壳聚糖纳米硒颗粒具有良好的物理稳定性、热稳定性,适用于疫苗工业化制备。该壳聚糖纳米硒颗粒作为免疫载体或佐剂,能够促进抗原呈递细胞对抗原的吞噬、处理和呈递,诱导产生更高水平的抗原特异性IgG抗体效价,在疫苗开发领域具有重要的应用价值。
Description
技术领域
本发明涉及疫苗研制技术领域,更具体地,涉及一种壳聚糖纳米硒颗粒及其制备方法和在疫苗中的应用。
背景技术
疫苗的研发经历了数代技术的更新。减毒活疫苗和灭活疫苗被广泛用于预防包括黄热病、麻疹和脊髓灰质炎在内的严重病毒性疾病,但有培养困难、容易感染的缺点,且容易引起过激的免疫反应。亚单位疫苗,包括蛋白、多糖或其他病原体成分,如b型流感、白喉、破伤风、无细胞穿孔病、脑膜炎球菌和肺炎球菌等,但它们也需要佐剂来增强其免疫原性,并且在暴露于敌对的体内环境后经常出现早期降解的情况。核糖核酸或脱氧核糖核酸疫苗的感染风险极小,能够引发针对特定病原体的免疫反应,而且成本不高;但面临着分子的过早降解和不能转化为功能性免疫原的问题。
为了克服上述提到的这些障碍,需要一种有效的疫苗递送系统。采用纳米技术开发新一代的复合疫苗是一个很好的选择。基于纳米载体的递送系统可以保护疫苗免于过早降解,并且具有良好的佐剂特性,有助于将免疫原靶向递送至抗原呈递细胞(APCs)。疫苗抗原可以被包裹在纳米载体中或修饰在它们的表面。纳米粒子内的封装可以保护抗原免于过早的蛋白酶降解并可实现可持续的释放,而表面吸附的抗原促进了它们与免疫细胞表面受体的相互作用,例如抗原提呈细胞的Toll-like受体(TLRs)。
壳聚糖具有良好的固有免疫和特异性免疫的刺激能力,并且能诱导黏膜免疫,对抗原有高效保护性,能增强抗原免疫原性。壳聚糖制备成纳米颗粒可以作为疫苗载体精确负载抗原,靶向呈递给免疫细胞,并能作为佐剂增强抗原的免疫原性。壳聚糖与传统铝佐剂、弗氏佐剂相比有更好的抗原保护效果。其巨大的分子量及复杂的空间结构可以将抗原包裹其中,并且因携带大量正电荷故而有强有力的静电作用保护疫苗在运输过程中不易被酶解,疫苗结合壳聚糖会比直接裸露的疫苗有更强的稳定性。
本发明初次尝试了一种壳聚糖纳米硒颗粒作为疫苗载体,通过吸附和包裹抗原,增强疫苗的淋巴结递送,提高免疫效果。本发明所用的壳聚糖纳米硒生物安全性较好,壳聚糖纳米硒本身可以作为保健食品或食品添加剂成分应用于食品行业,而该壳聚糖纳米硒颗粒能够显著提高抗原的免疫原性,适用于疫苗的开发。与壳聚糖纳米颗粒相比,壳聚糖纳米硒无需加入额外的交联剂,通过硒还原反应形成纳米硒颗粒,具有更好的生物安全性。本发明进行了一系列验证和条件摸索,证实壳聚糖纳米硒颗粒可以作为蛋白抗原的载体或佐剂,产生针对蛋白抗原的高免疫反应。这一策略提供了一种新型的疫苗载体,有效提高了抗原的免疫原性。
发明内容
本发明的第一个目的是提供一种负载抗原和疫苗佐剂的壳聚糖纳米硒颗粒。
本发明的第二个目的是提供一种上述壳聚糖纳米硒颗粒的制备方法。
本发明的第三个目的是提供一种上述壳聚糖纳米硒颗粒作为抗原载体或疫苗佐剂的应用。
本发明的第四个目的是提供一种上述壳聚糖纳米硒颗粒在制备抗肿瘤药物中的应用。
为实现上述目的,本发明提供如下技术方案:
本发明的第一个方面提供了一种负载抗原和疫苗佐剂的壳聚糖纳米硒颗粒,利用带正电荷的壳聚糖,与亚硒酸钠还原反应形成壳聚糖纳米硒颗粒,所述壳聚糖纳米硒颗粒呈球形,具有实心的球核,粒径范围在20~200nm。
进一步地,所述壳聚糖纳米硒颗粒的PDI为0.1~0.4,Zeta电位为15~40mV。
本发明的第二个方面提供了一种上述壳聚糖纳米硒颗粒的制备方法,包括如下步骤:取一定浓度的壳聚糖的醋酸溶液,加入亚硒酸钠溶液并在室温下磁力搅拌一定时间,加入抗坏血酸溶液反应一定时间,随后静置老化,通过透析袋透析纯化,即得所述壳聚糖纳米硒颗粒。
进一步地,所述亚硒酸钠与抗坏血酸的浓度比为2:1~1:20。
进一步地,所述抗坏血酸溶液在反应液中的含量为0~0.50mol/L。
进一步地,所述壳聚糖的醋酸溶液的浓度为0.1~5mg/mL。
进一步地,所述静置的反应条件为:避光,反应温度为0~100℃。
本发明的第三个方面提供了一种上述壳聚糖纳米硒颗粒作为抗原载体或疫苗佐剂的应用,所述壳聚糖纳米硒颗粒与带负电的抗原通过静电吸附作用或化学共价偶联的方式形成疫苗颗粒。
进一步地,所述疫苗佐剂选自Toll样受体9的配体CpG-ODN、Toll样受体4的配体MPLA、铝佐剂中的一种。
进一步地,所述疫苗颗粒的粒径为1~1000nm,优选为20~300nm。
本发明负载抗原和疫苗佐剂的壳聚糖纳米硒颗粒具有靶向淋巴结的功能,提高了疫苗在淋巴结内的富集,提高了疫苗被抗原呈递细胞的摄取,提高了体液和细胞免疫应答,以及记忆性免疫应答,在癌症预防的应用中展现出了良好的效果。
因此,本发明还请求保护所述壳聚糖纳米硒颗粒在制备抗肿瘤药物中的应用,壳聚糖纳米硒颗粒与鸡卵清白蛋白(OVA)通过静电吸附作用形成纳米颗粒,其中,OVA作为一种模式蛋白,用于抗肿瘤疫苗的开发和应用。所述壳聚糖纳米硒颗粒与OVA模式蛋白结合后,具有较强的免疫刺激能力,且免疫刺激能力与壳聚糖纳米硒颗粒的粒径大小相关,粒径较小的壳聚糖纳米硒颗粒所引起的免疫反应较强。
进一步地,所述壳聚糖纳米硒颗粒与OVA蛋白结合后的粒径为50~300nm;PdI为0.1~0.2;Zeta电位为15-35mV。
本发明相较于现有技术的优势和有益效果在于:
1、本发明的壳聚糖纳米硒颗粒,通过壳聚糖和亚硒酸钠在抗坏血酸的还原下反应获得,无需加入额外的交联剂,具有更好的生物安全性,可以作为食品添加剂用于食品行业;此外,本发明的壳聚糖纳米硒颗粒能溶于中性的去离子水中,在水溶液中的物理稳定性和稳定性好;
2、本发明的壳聚糖纳米硒颗粒可以通过反应条件的控制,得到不同尺寸的纳米颗粒,能够靶向淋巴结,提高抗原物质在淋巴结内的富集以及抗原呈递细胞的摄取,从而提高抗原特异性的体液免疫和细胞免疫应答,提高免疫记忆能力;
3、本发明的壳聚糖纳米硒颗粒,因壳聚糖具有阳离子官能团而具有电正性,能够与带负电的蛋白抗原进行静电吸附,能够与细胞表面进行静电吸附,从而具有较好的抗原负载能力和细胞靶向能力;纳米硒颗粒本身既能刺激免疫细胞增殖,又能刺激细胞因子的产生,因此也能提高壳聚糖纳米硒颗粒在疫苗制剂中的作用;
4、本发明的壳聚糖纳米硒颗粒作为抗原的载体或佐剂,在疫苗的应用中展示出了良好的效果,具有极大的潜力应用于疫苗工业中。
附图说明
图1为实施例12制得的壳聚糖纳米硒颗粒的透射电镜图;
图2为小鼠血清中OVA特异性抗体水平;(a)第28天小鼠血清(稀释300倍)ELISA实验测IgG和IgM混合抗体水平;(b)第1、3组第7、14、21、28天小鼠血清(稀释300倍)ELISA实验测IgG和IgM混合抗体水平;(c)第1、3组第28天小鼠血清(稀释300倍)ELISA实验分别测IgG1、IgG2b、IgG3和IgM抗体水平;*表示第三组与第一组有显著性差异(P<0.05)。
具体实施方式
下面结合附图和实施例对本发明作更进一步的说明。根据下述实施例,可以更好的理解本发明。然而,本领域的技术人员容易理解,实施例所描述的具体的物料配比、工艺条件及其结果仅用于说明本发明,而不应当也不会限制权利要求书中所详细描述的本发明。
以下实施例中所涉及制剂来源如下:
冰醋酸、亚硒酸钠来源于国药集团化学试剂有限公司,L-抗坏血酸、壳聚糖来源于萨恩化学技术(上海)有限公司,对硝基苯磷酸二钠六水合物、鸡蛋清白蛋白、牛血清白蛋白来源于源叶生物有限公司,其他常用试剂来源于国药集团化学试剂有限公司。
实施例1~11
本发明的壳聚糖纳米硒的制备通过改变反应中亚硒酸钠(Na2SeO3)、抗坏血酸(Vc)、壳聚糖的比例,以及反应温度、反应时间等条件来制得不同粒径的壳聚糖纳米硒,具体制备方法如下所述:
按表1~3所示配比及反应条件,取壳聚糖的醋酸溶液2.50mL于10mL离心管中,加入40.00μL亚硒酸钠溶液并在室温下磁力搅拌6h,加入抗坏血酸溶液200.00μL,并补加醋酸溶液使终体积为5.00mL,充分混合后在室温或加热条件下避光静置12h。最后将反应完成的壳聚糖纳米硒溶液装在截留分子量为10000kD的透析袋透析中,在室温下去离子水中透析24h,每4h换一次水以除去未反应的原料,即得到实施例1~11的壳聚糖纳米硒颗粒。
表1改变Na2SeO3与Vc浓度比壳聚糖纳米硒的表征结果
从表1可以看出,实施例1~3制得的壳聚糖纳米硒的粒径随还原剂Vc在反应液中的含量增加而从196.0nm增大至398.3nm,表明Na2SeO3与Vc浓度比会影响壳聚糖纳米硒的粒径大小,且在一定范围内,还原剂Vc在反应液中的含量越高制备出的壳聚糖纳米硒的粒径越大。
表2改变壳聚糖浓度壳聚糖纳米硒的表征结果
从表2可以看出,在其他条件不变的情况下,实施例4~6的壳聚糖的醋酸溶液浓度从1mg/mL增加至而3mg/mL,其制得的壳聚糖纳米硒的粒径也随之从103.4nm增大至151.8nm,表明在一定范围内,壳聚糖浓度增大,壳聚糖纳米硒的粒径也会增大。
表3改变反应温度和时间壳聚糖纳米硒的表征结果
从表3可以看出,随着静置反应的温度升高,颗粒的粒径呈现变大的趋势。
实施例12~13
实施例12相较于实施例1、以及实施例13相较于实施例3的区别都仅在于将反应体系从5mL增大至250mL,其余反应条件均不变,从而得到实施例12和实施例13的壳聚糖纳米硒颗粒。
如图1所示为实施例12的壳聚糖纳米硒颗粒的透射电镜图,从图1可以看出实施例12的壳聚糖纳米硒颗粒呈球形,内有实心的核,粒径小于100nm,在80nm左右。
实施例12和实施例13的壳聚糖纳米硒颗粒所测得的Size、PdI、Zeta数值与5mL的反应体积的数值对比如下表4所示。
表4反应体系体积为5mL和250mL所制备的壳聚糖纳米硒
从表4可以看出,反应体系的体积大小对制备壳聚糖纳米硒有很大影响,250mL体系下粒子粒径大幅度减小,粒子粒径均一度明显提高,Zeta电位绝对值明显增大,粒子稳定性也明显提高。
性能测试
1、壳聚糖纳米硒的稳定性测试
1.1物理稳定性
以实施例2、3、4和13所制得的壳聚糖纳米硒颗粒为例,于常温下测试其稳定性,具体方法为:
将实施例2、3、4和13所制得的壳聚糖纳米硒颗粒分别在第0天(d0)、第7天(d7)、第30天(d30)、第60天(d60)时测量其粒径,结果如下表5所示。
表5壳聚糖纳米硒常温下稳定性
从表5可以看出,四个样品的壳聚糖纳米硒颗粒的稳定性较强,较长时间内不会产生聚成现象,大于150nm的样品颗粒会在一段时间内粒径轻微变小,小于150nm的样品颗粒会在一段时间内粒径轻微变大,但是总体粒径随时间变化较为稳定。
1.2热稳定性
以实施例12和13制得的壳聚糖纳米硒颗粒为例,进行热稳定性测试,具体方法为:
将实施例12和13制得的壳聚糖纳米硒颗粒在50℃水浴中放置一段时间测定其粒径大小,结果如下表6所示:
表6壳聚糖纳米硒50℃下热稳定性
从表6可以看出,实施例12和13制得的壳聚糖纳米硒颗粒在50℃下稳定性较好,粒径变化程度很小。
实施例14
由于实施例12和13制得的壳聚糖纳米硒颗粒质量最高,颗粒均一性较好,Zeta电位较高,因此,以实施例12和13制得的壳聚糖纳米硒颗粒为样品(分别标记为CS-SeNP-12、CS-SeNP-13)进行疫苗样品的制备和表征、小鼠免疫实验。
1、疫苗样品的制备和表征
第一组疫苗制备:OVA+壳聚糖+铝佐剂(疫苗1),称取2.8mg壳聚糖溶于0.01mol/L2mL稀醋酸溶液然后等浓度等体积的氢氧化钠溶液中和,然后用高浓度1M的盐酸调节PH到7.4,加入OVA,并1:1加入铝佐剂,混合均匀。
第二组疫苗制备:CS-SeNP-12(疫苗2),根据壳聚糖纳米硒溶液中壳聚糖的含量,调整溶液浓度至壳聚糖含量为350μg/mL。
第三组疫苗制备:OVA+CS-SeNP-12(疫苗3),壳聚糖纳米硒溶液中加入OVA,根据壳聚糖纳米硒溶液中壳聚糖的含量,调整溶液浓度至壳聚糖含量为350μg/mL,OVA含量为100μg/mL。
第四组疫苗制备:OVA+CS-SeNP-13(疫苗4),同第三组配制过程。
疫苗表征结果如表7所示,从表7可以看出,添加OVA后的粒子,Zeta电位减小,Zeta电位值减小比例大约为15%;粒径轻微变小,粒径变化比例在4%左右;PdI值都轻微减小。说明加入OVA后粒径更趋于均一。这些数值变化能说明OVA已经通过静电作用吸附壳聚糖纳米硒表面。
表7疫苗表征结果
2、小鼠免疫实验
小鼠免疫实验分四组,分别对应上述四组疫苗,四组疫苗都保持OVA含量为100μg/mL,壳聚糖含量为350μg/mL。每组5只小鼠,每组的各小鼠均在第1、7、14、21天在皮下注射相应组别的疫苗,每次注射疫苗0.1mL(即每只小鼠每次注射OVA 10μg、壳聚糖35μg)。分别于免疫前、第7、14、21、28天采集小鼠静脉血,分离血清保存于-80℃的存储箱中,用于免疫活性检测。
3、疫苗免疫活性结果
获得小鼠抗血清后,先进行酶联免疫吸附实验(ELISA)测定IgG和IgM混合抗体滴度,初步判定疫苗的免疫效果,进一步分别测IgG、IgM抗体滴度和IgG不同亚型的抗体滴度。用OVA作为铺板抗原,羊抗鼠碱性磷酸酶为二抗,对硝基苯磷酸二钠缓冲液为显色剂,室温显色30分钟,酶标仪415nm处测定吸光度,通过吸光度来表征小鼠血清中OVA特异性抗体水平。
抗体滴度水平如图2所示。从图2(a)中可以看出,小鼠免疫4次后,第28天,第1、3、4组都产生了较高水平的OVA特异性的抗体滴度,其中,第3组的抗体滴度水平最高,显著高于第1组。由此结果可以看出,本发明的壳聚糖纳米硒具有较强的免疫刺激能力,能够增强所负载的蛋白抗原的免疫原性,且粒径大小为100nm左右的壳聚糖纳米硒负载OVA抗原(第3组免疫结果)所激起的免疫反应显著高于用铝剂组的免疫水平。随着壳聚糖纳米硒的粒径增大,免疫效果略有降低。因此,选取100nm左右的壳聚糖纳米硒作为疫苗载体能够有效刺激免疫活性且优于广泛使用的铝剂。从图2(b)可以看出,第14、21、28天的小鼠血清中的抗体滴度,100nm左右的壳聚糖纳米硒组所引起的抗体滴度,均高于铝剂组所引起的抗体滴度,因此,壳聚糖纳米硒作为载体和佐剂,优于现有的铝剂。从图2(c)可以看出,壳聚糖纳米硒作为疫苗的载体和佐剂,产生抗体类型主要为IgG1和IgG2b,说明免疫反应产生较好的抗体类型,且都高于传统铝剂的水平。
综上,本发明所制备的壳聚糖纳米硒具有较强的免疫刺激能力,能够产生比传统佐剂铝剂更强的免疫反应,有望成为疫苗的佐剂或载体的候选物。且本发明发现100nm尺寸的壳聚糖纳米硒具有较好的效果,可以进一步用于疫苗的开发中。
以上所述仅为本发明的较佳实施例,但本发明的保护范围并不局限于此。凡依本发明申请专利范围未违背本发明涉及原则所做的均等变化、简化与修饰,皆应属本发明的涵盖范围。
Claims (8)
1.壳聚糖纳米硒颗粒作为蛋白抗原载体的应用,其特征在于,所述壳聚糖纳米硒颗粒由带正电荷的壳聚糖,与亚硒酸钠还原反应形成壳聚糖纳米硒颗粒,所述壳聚糖纳米硒颗粒呈球形,具有实心的球核,粒径为20~200nm;
所述壳聚糖纳米硒颗粒与带负电的蛋白抗原通过静电吸附作用形成疫苗颗粒。
2.根据权利要求1所述的壳聚糖纳米硒颗粒作为蛋白抗原载体的应用,其特征在于,所述壳聚糖纳米硒颗粒的PDI为0.1~0.4,Zeta电位为15~40mV。
3.根据权利要求1或2所述的壳聚糖纳米硒颗粒作为蛋白抗原载体的应用,其特征在于,壳聚糖纳米硒颗粒的制备方法包括如下步骤:取壳聚糖的醋酸溶液,加入亚硒酸钠溶液并在室温下磁力搅拌,加入抗坏血酸溶液反应,随后静置老化,通过透析袋透析纯化,即得所述壳聚糖纳米硒颗粒。
4.根据权利要求3所述的壳聚糖纳米硒颗粒作为蛋白抗原载体的应用,其特征在于,所述亚硒酸钠与抗坏血酸的浓度比为2:1~1:20;所述抗坏血酸溶液在反应液中的含量为0~0.50mol/L;所述壳聚糖的醋酸溶液的浓度为0.1~5mg/mL。
5.根据权利要求3所述的壳聚糖纳米硒颗粒作为蛋白抗原载体的应用,其特征在于,所述静置的反应条件为:避光,反应温度为0~100℃。
6.根据权利要求1所述的壳聚糖纳米硒颗粒作为蛋白抗原载体的应用,其特征在于,所述疫苗佐剂选自Toll样受体9的配体CpG-ODN、Toll样受体4的配体MPLA、铝佐剂中的一种。
7.根据权利要求1所述的壳聚糖纳米硒颗粒作为蛋白抗原载体的应用,其特征在于,所述疫苗颗粒的粒径为1~1000nm,
8.根据权利要求1所述的壳聚糖纳米硒颗粒作为蛋白抗原载体的应用,其特征在于,所述疫苗颗粒的粒径为20~300nm。
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