CN114470188B - 一种枸杞多糖超大介孔二氧化硅纳米佐剂的制备方法及用途 - Google Patents
一种枸杞多糖超大介孔二氧化硅纳米佐剂的制备方法及用途 Download PDFInfo
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Abstract
本发明公开了一种枸杞多糖超大介孔二氧化硅纳米佐剂的制备方法及用途，包括如下步骤：(1)将CTAC溶液、TEA和去离子水混合均匀制成水相；(2)取氯苯和TEOS混合均匀制成油相；(3)将油相滴加入水相后水浴，匀速搅拌，离心收集沉淀，清洗，箱干，产物研磨后煅烧，得UCMS；(4)称取LBP和UCMS，分散于去离子水中，超声处理后常温搅拌，得LBP‑UCMS溶液。本发明首次通过结合枸杞多糖和药物递送载体(超大介孔二氧化硅)两者的优势构建新型免疫佐剂，体现交叉学科的优势。与常规介孔二氧化硅相比，超大介孔二氧化硅可以装载更多的多糖发挥更佳的免疫作用。
Description
技术领域
本发明涉及免疫佐剂制备方法及用途，特别涉及一种枸杞多糖超大介孔二氧化硅纳米佐剂的制备方法及用途。
背景技术
枸杞，甘、平，是一味药食同源的中药材。归肝、肺、肾经；滋肾，润肺，补肝，明目。枸杞中含有的枸杞多糖(LBP)具有促进免疫、抗衰老、抗肿瘤、清除自由基、抗疲劳、抗辐射、保肝、生殖功能保护和改善等多种作用。然而LBP临床应用中存在代谢快，靶向性差和用量大等缺点。因此，亟需寻求有效的方法解决这一问题。
药物递送系统是目前研究的热点。介孔二氧化硅(MSNs)就是一种很有希望克服传统佐剂缺点的新型纳米递送载体(诸如注射部位炎症、疼痛以及严重的急性毒性等)。它拥有高比表面积、大孔体积、生物相容性好、可功能化修饰等良好的性质，比传统的药物输送系统(如聚合物纳米颗粒，脂质体等)更灵活、更坚固。此外，MSNs制造工艺相对简单，成本显著降低，这对于满足未来的临床需求和商业化非常重要。目前介孔二氧化硅已被广泛作为亚单位疫苗的靶向递送载体，而超大介孔二氧化硅(UCMS)可以更容易吸附负载抗原、只需简单混合吸附即可，也可装载更多的抗原并抵抗胃肠液的破坏。
巨噬细胞是机体防御病原体的第一道防线，它既是一类重要的抗原提呈细胞，也是重要的炎症和免疫效应细胞，具有识别、吞噬、抗感染和免疫调节等多种免疫功能，在特异性免疫和非特异性免疫中起关键作用。鉴于巨噬细胞在机体免疫系统中的重要地位，
发明内容
发明目的：本发明目的是提供一种枸杞多糖超大介孔二氧化硅纳米佐剂的制备方法。
技术方案：所述的枸杞多糖超大介孔二氧化硅纳米佐剂的制备方法，包括如下步骤：
(1)将CTAC溶液、TEA和去离子水混合均匀制成水相；
(2)取氯苯和TEOS混合均匀制成油相；
(3)将油相滴加入水相后水浴，匀速搅拌，离心收集沉淀，清洗，箱干，产物研磨后煅烧，得UCMS；
(4)称取LBP和UCMS，分散于去离子水中，超声处理后常温搅拌，得LBP-UCMS溶液。
进一步地，所述步骤(3)采用无水乙醇、去离子水清洗。
进一步地，所述步骤(1)中将4.8mL 25wt％CTAC溶液、0.04g TEA和7.2mL去离子水混合均匀制成水相。
进一步地，所述步骤(2)中将3.5mL氯苯和0.5mL TEOS混合均匀制成油相。
所述方法制备得到的枸杞多糖超大介孔二氧化硅纳米佐剂在中药多糖新型免疫佐剂的研制及动物疫病药物中的用途。
本发明采用两相界面合成法制备UCMS并物理搅拌装载LBP，成功制备出LBP-UCMS。本发明以RAW264.7为研究的靶细胞，通过评估巨噬细胞的吞噬能力、细胞因子的mRNA含量和表面共刺激分子的表达量，测定LBP-UCMS对RAW264.7细胞的免疫调节作用。制备出的UCMS可装载更多的多糖，发挥更佳的免疫效果。产物LBP-UCMS可显著提高巨噬细胞分泌细胞因子的含量和表面标记分子的表达量，且更有效地刺激巨噬细胞，增强巨噬细胞的吞噬能力，将为中药多糖新型免疫佐剂的研制及动物疫病的防控提供参考。
有益效果：本发明与现有技术相比，具有如下优势：
1.首次通过结合枸杞多糖和疫苗递送载体(超大介孔二氧化硅)两者的优势构建新型免疫佐剂，体现交叉学科的优势。
2.与常规介孔二氧化硅相比，超大介孔二氧化硅可以装载更多的多糖发挥更佳的免疫作用。
附图说明
图1LBP、UCMS、LBP-UCMS固体外观图；
图2UCMS、LBP-UCMS的透射电镜图；
图3LBP、UCMS、LBP-UCMS红外光谱图；
图4LBP-UCMS对RAW264.7细胞因子分泌的影响；
图5LBP-UCMS对RAW264.7表面共刺激分子的影响；
图6LBP-UCMS对RAW264.7吞噬能力的影响。
具体实施方式
实施例1：制备UCMS并物理装载LBP
准确将4.8mL 25wt％CTAC溶液、0.04g TEA和7.2mL去离子水混合均匀制成水相。其次，准确量取3.5mL氯苯和0.5mL TEOS混合均匀制成油相。将油相缓慢滴加入水相中后60℃水浴，500r/min下匀速搅拌12h。高速离心收集沉淀后，用无水乙醇、去离子水各清洗3遍，置于60℃烘箱中风干。得到的产物研磨后于马弗炉550℃煅烧5h，得UCMS。准确称取20mgLBP和5mg UCMS，分散于2mL去离子水中。超声处理后常温搅拌12h，得LBP-UCMS溶液。
实施例2：表征测定
(1)透射电镜结果分析
制备的LBP-UCMS固体粉末如图1所示。透射电子显微镜(TEM)观察到LBP-UCMS的形态如图2所示。在电子高倍镜下观察可看到UCMS尺寸匀一，粒径在80-100nm左右，有褶皱状孔道直通内部，皱襞较薄，为物理吸附装载多糖提供更多的空间。LBP-UCMS纳米粒也呈球形，均匀分布，且UCMS的介孔间隙被填满，说明LBP成功被装载于UCMS的介孔间隙内。
(2)粒径、Zeta电位分析
如表1所示，通过动态光散射仪(DLS)测得UCMS在装载LBP后，粒径有所增大。LBP-UCMS的平均粒径为493.4±14.17nm(n＝3)，与通过TEM拍摄的纳米粒大小基本一致，且PDI均在0.1-0.35范围内。LBP、UCMS和LBP-UCMS的电势均为负数，而LBP-UCMS的Zeta电势绝对值比LBP、UCMS的值更大。
表1 LBP-UCMS的申势、粒径和PDI(n＝3)
(3)傅里叶红外光谱结果分析
样品LBP、UCMS和LBP-UCMS与KBr粉末混合研磨压片，在4000～400cm-1波长范围内进行红外扫描后，其红外光谱结果见图3。LBP在800-1200cm-1、1450-1800cm-1、2900-3000cm-1和3200-3600cm-1处有明显的红外吸收峰。LBP在3401cm-1处有一个典型的羟基(-OH)峰，2929cm-1处的峰值是C-H键拉伸振动产生的。1641cm-1处出现的峰值对应C＝C键和C＝O基团的吸收。UCMS在3435cm-1的峰值是H-O-H键伸缩振动产生的；804cm-1和1086cm-1出现的峰值分别对应的是Si-O-Si键对称伸缩振动和Si-O-Si键非对称伸缩振动产生的。LBP-UCMS中包含LBP、UCMS共同的红外特征峰。上述结果说明UCMS纳米材料是由SiO2组成的，且LBP-UCMS成功物理装载LBP。
实施例3：性能测试
(1)LBP-UCMS对RAW264.7细胞因子分泌的影响
植物多糖能够活化巨噬细胞，并通过促进巨噬细胞增殖、提高吞噬能力以及产生TNF-α、IL-6、和IL-1β等细胞因子来发挥免疫调节作用，从而提高免疫能力。qRT-PCR结果如图4所示，LBP-UCMS(250μg/mL)刺激巨噬细胞分泌的TNF-α含量与Control组差异显著(P＜0.001)，但与LBP组相比，略高于LBP组但无显著差异。LBP-UCMS(250μg/mL)刺激巨噬细胞分泌IL-1β、IL-6的含量显著高于LBP组(P＜0.0001)。表明与LBP单独刺激相比，LBP-UCMS可更多地刺激巨噬细胞产生相应的炎性因子。
(2)LBP-UCMS对RAW264.7表面共刺激分子的影响
外源性抗原经吞噬或吞饮作用，将抗原分解为抗原肽，并与内质网合成的MHC-II类分子结合形成抗原肽-MHC-II类分子复合物。该复合物表达于抗原呈递细胞表面，同时增加共刺激分子(如CD86、CD80)表达，激活特异性T和B淋巴细胞效应机制(适应性免疫)。通过流式细胞术检测细胞表面刺激分子结合的荧光量来反映其表达量的多少。结果如图5所示，浓度分别为125μg/mL、250μg/mL的LBP-UCMS作用细胞24h后，MHC-II、CD80、CD86的表达量都显著高于LBP组和细胞对照组。250μg/mL的LBP-UCMS组的MHC-II的平均表达量达到65.18％，而同浓度下的LBP组MHC-II的平均表达量仅为27.6％，并低于125μg/mL LBP-UCMS组的平均表达量。LBP-UCMS(250μg/mL)组CD80+的平均表达量达到48.91％，而同浓度的LBP组CD80+的平均表达量为16.84％，250μg/mL LBP-UCMS组CD80+的平均表达量是LBP组的2.9倍。LBP-UCMS(250μg/mL)组CD86+的平均表达量为54.05％，而同浓度的LBP组CD86+的平均表达量为21.77％，LBP-UCMS(250μg/mL)组CD86+的平均表达量是LBP组的2.48倍。试验结果表明LBP经过UCMS装载后对小鼠巨噬细胞表面分子的表达有显著的促进作用。
(3)LBP-UCMS对RAW264.7吞噬能力的影响
吞噬能力是反应巨噬细胞免疫防御能力的一个重要指标，通过吞噬作用，杀死病原体或参与炎症反应来引导抵抗病原体感染，本试验通过激光共聚焦检测巨噬细胞吞噬荧光量(FITC-葡聚糖)的多少来反映巨噬细胞的吞噬功能。如图6所示，与Control组相比，LPS组的FITC-葡聚糖分布更多，吞噬能力较强，但发生形态改变的细胞较多。在125μg/mL、250μg/mL浓度LBP-UCMS的作用下，巨噬细胞内分布的绿色荧光明显多于Control、LBP、UCMS组，吞噬能力明显增强。而125μg/mL的LBP-UCMS吞噬能力略高于250μg/mL浓度。说明UCMS装载LBP后，能够更有效的刺激巨噬细胞，增强巨噬细胞的吞噬能力。
Claims (2)
1.一种枸杞多糖超大介孔二氧化硅纳米佐剂的制备方法，其特征在于：包括如下步骤：
（1）将4.8 mL 25 wt% CTAC溶液、0.04 g TEA和7.2 mL去离子水混合均匀制成水相；
（2）将3.5 mL氯苯和0.5 mL TEOS混合均匀制成油相；
（3）将油相滴加入水相后水浴，匀速搅拌，离心收集沉淀，采用无水乙醇、去离子水清洗，烘干，产物研磨后煅烧，得超大介孔二氧化硅；
（4）称取枸杞多糖和超大介孔二氧化硅，分散于去离子水中，超声处理后常温搅拌，得枸杞多糖超大介孔二氧化硅纳米佐剂溶液。
2.权利要求1所述方法制备得到的枸杞多糖超大介孔二氧化硅纳米佐剂在制备中药多糖免疫佐剂或动物疫病药物中的用途。
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