CN114099696B - 纳米硒海藻酸钠复合凝胶及制备方法和应用 - Google Patents
纳米硒海藻酸钠复合凝胶及制备方法和应用 Download PDFInfo
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- CN114099696B CN114099696B CN202111343680.3A CN202111343680A CN114099696B CN 114099696 B CN114099696 B CN 114099696B CN 202111343680 A CN202111343680 A CN 202111343680A CN 114099696 B CN114099696 B CN 114099696B
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- sodium alginate
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Abstract
本专利提供纳米硒海藻酸钠复合凝胶及制备方法和应用,制备方法包括步骤(1)制备透明质酸修饰的纳米硒,将透明质酸加水搅拌形成透明质酸溶液与Na2SeO3溶液、维生素C混合进行反应,透析,得到所述透明质酸修饰的纳米硒粒子;(2)将海藻酸钠溶液与所述透明质酸修饰的纳米硒粒子混合,然后将混合液逐滴加入到氯化钙溶液中,经过交联后形成以所述透明质酸修饰的纳米硒粒子为核,海藻酸钠凝胶为外壳的纳米硒海藻酸钠复合凝胶。所述纳米硒海藻酸钠复合凝胶能很好调控肠道菌群,提高治疗慢性肠道炎症性疾病的效果。
Description
技术领域
本专利涉及医药技术领域,特别涉及可口服纳米硒凝胶及其制备方法,以及可应用于调控肠道菌群。
背景技术
肠道菌群与我们的健康息息相关,肠道菌群失调可导致肥胖、自身免疫力下降、血压调节不良、过敏和癌症等多种疾病,因此,调节肠道菌群具有非常重要的意义。炎症性肠病(IBD)是一种常见的慢性肠道炎症性疾病,肠道细菌可能是参与IBD的发病和持续恶化的主要因素。肠道细菌及其代谢物会刺激肠黏膜免疫系统,诱发这些具有IBD易感性人群肠黏膜免疫系统功能紊乱,产生异常的免疫反应,导致IBD发病。正常情况下,肠道菌群的失衡会导致肠道益生菌的减少和致病菌的增加,致病菌及其释放的内毒素,如脂多糖(LPS)、肽聚糖、脂蛋白等能激活肠黏膜免疫系统,会导致肠道黏膜屏障受损,通透性增加,诱发IBD。
IBD的血性腹泻、腹痛和其他并发症可严重影响生活质量,目前一线治疗药物5-氨基水杨酸(5-ASA)和糖皮质激素由于其非特异性抗炎特性而引起多种副作用。尽管英夫利昔单抗等新型生物疗法已经开发出来,但这些生物疗法不仅价格昂贵,而且无应答率为30%,耐药率每年增加20%,并可能导致不良副作用如感染等,进而限制了其在IBD治疗中的应用。
而与以上的治疗方法不同的是,作为一种可调节肠道免疫微生物群的精准有效的药物输送方法,口服给药具有方便、安全、直接作用于局部粘膜等特点,是治疗慢性胃肠道疾病的首选给药方法,在临床转型方面具有巨大潜力。然而,由于整个消化道内广泛的pH环境(pH 1.2至8)、腹泻引起的快速药物清除、消化液中的大量药物降解以及系统暴露和吸收降低了药物的生物利用度。同时,增加频率和剂量以维持治疗效果会导致副作用增强。
因此,生物材料,如透明质酸(HA),在实验室显示出治疗IBD的潜力,但在体内未能达到预期效果,透明质酸在消化液中扩散和降解的敏感性限制了其口服应用。透明质酸(HA)颗粒不可控的全身扩散降低了生物利用度,并可能导致不良副作用,从而限制了其在IBD治疗中的应用。而硒(Se)是一种重要的微量元素,以无机和有机形式存在。据有关报道,硒作为第21种氨基酸,即硒代半胱氨酸,共翻译合并到硒蛋白中,其功能是调节炎症相关途径。流行病学研究表明,硒水平与炎症性肠病(IBD)之间存在负相关关系,炎症性肠病包括克罗恩病(CD)和溃疡性结肠炎(UC),这些疾病有可能发展为结肠癌。有研究表明亚硒酸钠(硒的一种形式)能减轻右旋糖酐硫酸钠(DSS)诱导的C57BL/6小鼠急性结肠炎,同时硒对小鼠肠癌的发病有一定预防作用。现有技术中目前没有发现基于HA修饰的纳米硒颗粒在IBD治疗中的应用。
现有技术中没有一种很好的凝胶复合物用以包裹具有抑制IBD的纳米硒药物,保护纳米硒药物不在消化液中被过早暴露和消融,因为消化液中的大量纳米硒药物降解以及系统暴露和吸收降低了纳米硒的生物利用度,不能很好调控肠道菌群,提高治疗慢性肠道炎症性疾病的效果。
发明内容
针对现有技术的不足,本发明的首要目的是提供纳米硒海藻酸钠复合凝胶用于调控肠道菌群,进而抑制炎症性肠炎(IBD)的应用。
本发明的另一目的在于提供一种纳米硒海藻酸钠复合凝胶及其制备方法。
本发明的再一目的在于提供一种抑制炎症性肠炎的药物。
在发明人在实验研究中,发现经过透明质酸修饰后的纳米硒,即HA@SeNPs具有体外抗炎的作用。因此,本发明人是把纳米硒(SeNPs)和透明质酸(HA)作为抑制炎症性肠病(IBD)的核心治疗药物,采用合理的制备方法将纳米硒通过HA修饰形成HA@SeNPs纳米粒子后,再用海藻酸钠(SA)经过交联后与HA@SeNPs纳米粒子混合形成凝胶。
海藻酸钠凝胶SA发挥对炎症肠道细胞靶向的作用,纳米硒(SeNPs)和透明质酸(HA)协同发挥抑制炎症性肠病(IBD)的作用,并且在纳米硒和透明质酸表面包覆一层海藻酸钠微珠。微珠外壳可以保护纳米硒和透明质酸不被胃酸降解,在肠道中性环境下海藻酸钠水凝胶膨胀而释放纳米硒,同时,凝胶体系的缓释效应可以使HA@SeNPs纳米颗粒在肠道停留的时间更长,能更好调控肠道菌群,提高治疗慢性肠道炎症性疾病的效果。
本发明方案提供纳米硒海藻酸钠复合凝胶的制备方法,包括步骤
(1)制备透明质酸修饰的纳米硒,将透明质酸(HA)加水搅拌形成透明质酸溶液与Na2SeO3溶液、维生素C混合进行反应,透析,得到所述透明质酸修饰的纳米硒(HA@SeNPs)粒子;
(2)将海藻酸钠溶液与所述透明质酸修饰的纳米硒(HA@SeNPs)粒子混合,然后将混合液逐滴加入到氯化钙溶液中,经过交联后形成以所述透明质酸修饰的纳米硒粒子为核,海藻酸钠凝胶为外壳的纳米硒海藻酸钠复合凝胶(SA@HA@SeNPs)。
进一步,步骤(1)中所述的Na2SeO3溶液的终浓度为1-5mM;维生素C溶液的终浓度为11-12mmol/L。
进一步,步骤(1)中所述透明质酸的添加量为终浓度5-10mg/mL;所述的维生素C和Na2SeO3按摩尔比2-4:1进行配比;所述透析为采用透析袋进行透析,透析的时间为10-48小时。
进一步,步骤(2)中所述海藻酸钠溶液的终浓度为2-3%(w/v),所述氯化钙溶液的终浓度为2-3%(w/v)。所述步骤(2)中交联时间为1-2h,交联温度为36-37℃。
本发明另一个方案是提供一种纳米硒海藻酸钠复合凝胶,是以所述透明质酸修饰的纳米硒粒子为核,海藻酸钠凝胶为外壳的纳米硒海藻酸钠复合凝胶。
本发明还提供一种抑制炎症性肠炎的药物,是以所述透明质酸修饰的纳米硒粒子为核,海藻酸钠凝胶为外壳的纳米硒海藻酸钠复合凝胶制成的可口服药物。
进一步,所述外壳中还包裹有调控肠道菌群的药物,抑制炎症性肠炎。当然所述纳米硒海藻酸钠复合凝胶可以作为载体,负载现有的治疗肠胃炎的药物粉末,外壳能保护药物不在消化液中被过早暴露和溶解,然后在中性环境下海藻酸钠水凝胶会膨胀而释放药物,提高治疗效果。
本发明还提供一种纳米硒海藻酸钠复合凝胶在调控肠道菌群、抑制炎症性肠炎中的应用,是以所述透明质酸修饰的纳米硒粒子为核,海藻酸钠凝胶为外壳的纳米硒海藻酸钠复合凝胶在调控肠道菌群、抑制炎症性肠炎中的应用。所述纳米硒海藻酸钠复合凝胶通过清除体外活性氧,以及下调免疫细胞相关炎症因子,用于抑制炎症性肠炎。
本发明相对于现有技术具有如下的优点及效果:
(1)本发明公开了纳米硒海藻酸钠复合凝胶(SA@HA@SeNPs)作为调节肠道菌群并可进一步抑制炎症性肠炎,在体外细胞实验中能有效抑制炎症性的巨噬细胞Raw264.7中的活性氧,以及下调相关炎症因子的表达,具有高效的抑制炎症的效果。试验结果显示,实验比较对照物如吐温80修饰的纳米硒Tw80@SeNPs,聚乙二醇修饰的纳米硒、聚乙烯吡咯烷酮修饰的纳米硒、壳聚糖修饰的纳米硒、茯苓多糖修饰的纳米硒与海藻酸钠形成的凝胶体系SA@PEG@SeNPs、SA@PVP@SeNPs、SA@CS@SeNPs、SA@CMP@SeNPs被巨噬细胞RAW264.7细胞的摄入量均明显低于SA@HA@SeNPs,均仅约为SA@HA@SeNPs摄入量的20-30%,且他们对该细胞的活性氧清除效果和体外抗炎效果也远远低于SA@HA@SeNPs,大约是SA@HA@SeNPs效果的10-20%;
实验比较对照物如硒酸钠、硒代蛋氨酸SeMet与海藻酸钠形成的凝胶体系SA@Na2SeO4、SA@SeMet,他们被巨噬细胞RAW264.7细胞的摄入量均明显低于SA@HA@SeNPs,他们的细胞摄入量均仅约为SA@HA@SeNPs摄入量的10-12%,且他们对该细胞的活性氧清除效果和体外抗炎效果也远远低于SA@HA@SeNPs,约SA@HA@SeNPs的5-10%;
实验比较对照物如亚硒酸钠、硒代半胱氨酸SeC与海藻酸钠形成的凝胶体系SA@Na2SeO3、SA@SeC,他们被巨噬细胞RAW264.7细胞的摄入量均明显低于SA@HA@SeNPs,他们的细胞摄入量均仅约为SA@HA@SeNPs摄入量的8-10%,且他们对该细胞的活性氧清除效果和体外抗炎效果也远远低于SA@HA@SeNPs,约SA@HA@SeNPs的5-8%。故相比于其他纳米硒或含硒海藻酸钠凝胶体系,SA@HA@SeNPs清除巨噬细胞RAW264.7胞内活性氧和抑制胞内炎症因子如IL-6和TNF-α表达水平的能力更强。因此,纳米硒海藻酸钠复合凝胶(SA@HA@SeNPs)可作为肠道性疾病抑制剂进行开发,能很好调控肠道菌群,提高治疗慢性肠道炎症性疾病的效果。
(2)目前,现有的口服给药治疗炎症性肠炎引起了很大的关注,但是这类生物材料易在消化液中扩散和降解,降低了生物利用度,并可能导致不良副作用,进而限制了其在IBD治疗中的应用。而本发明的纳米硒海藻酸钠复合凝胶中的纳米硒(SeNPs)和透明质酸(HA)协同发挥抑制炎症性肠病(IBD)的作用,HA通过与免疫细胞膜上的CD44相互作用和调节巨噬细胞分化发挥抗炎作用。
进一步所述纳米硒海藻酸钠复合凝胶还可以作为载体,负载现有的治疗肠炎的药物,比如包裹双歧杆菌酪酸活菌等,外壳能保护药物不在消化液中被过早暴露和溶解。作为药物递送体系的外壳,它能保护药物不在消化液尤其是胃液(pH=1.8-5.0)中被过早暴露,然后在中性环境下海藻酸钠水凝胶膨胀而释放药物,大大提高调节肠道菌群、抑制炎症性肠炎的效果,具有广阔的医学药物应用前景。
(3)本发明方案提供一种可口服纳米硒凝胶的制备方法,所得可口服纳米硒复合凝胶的原料廉价易得,合成和纯化步骤可操作性强,可通过优化工艺,马上扩大合成规模,实现药物的商业化和应用。
附图说明
图1为透明质酸修饰的纳米硒(HA@SeNPs)的TEM形貌表征图。
图2为海藻酸钠包裹的HA@SeNPs纳米粒子(SA@HA@SeNPs)的荧光显微镜图。
图3为海藻酸钠包裹的HA@SeNPs纳米粒子(SA@HA@SeNPs)的XPS分析图。
图4为巨噬细胞RAW264.7对海藻酸钠包裹的HA@SeNPs纳米粒子(SA@HA@SeNPs)和吐温80修饰的纳米硒Tw80@SeNPs的靶向摄取图。
图5A为海藻酸钠包裹的HA@SeNPs纳米粒子(SA@HA@SeNPs)对巨噬细胞RAW264.7胞内活性氧随时间变化的荧光强度图;
图5B为用DCFH-DA荧光法分析海藻酸钠包裹的HA@SeNPs纳米粒子(SA@HA@SeNPs)和吐温80修饰的纳米硒Tw80@SeNPs对巨噬细胞RAW264.7胞内活性氧的清除效果荧光图。
图6A为透明质酸修饰的纳米硒(HA@SeNPs)下调炎症性巨噬细胞RAW264.7的炎症因子的表达;
图6B为透明质酸修饰的纳米硒(HA@SeNPs)处理炎症性巨噬细胞RAW264.7的共聚焦显微镜图像。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述,但本发明的实施方式不限于此。
实施例一,本发明方案提供纳米硒海藻酸钠复合凝胶的制备方法,包括步骤:
(1)制备透明质酸修饰的纳米硒(HA@SeNPs),在室温下,将200-300mg透明质酸(HA)加入46.5mL水放入100mL烧杯中搅拌约2h,再加入1mL的Na2SeO3溶液(终浓度1-5mM)放入上述溶液中混合,缓慢加入2.5mL维生素C,所述的维生素C和Na2SeO3按摩尔比2-4:1进行配比,继续在室温下反应2-24小时,用透析袋在超纯水中透析10-48小时,得到所述透明质酸修饰的纳米硒(HA@SeNPs);
(2)取200-300mg海藻酸钠与10mL的HA@SeNPs溶液中混合,所述海藻酸钠溶液的终浓度为2-3%(w/v)搅拌过夜,然后将混合液逐滴加入到2%(w/v)的氯化钙溶液中,经过交联1-2h,交联温度为36℃后形成海藻酸钠微珠,即得到以HA@SeNPs纳米粒子为核,海藻酸钠凝胶为外壳的SA@HA-SeNPs复合凝胶体系。
参见图1-3,按照上述制备方法得到以所述透明质酸修饰的纳米硒粒子为核,海藻酸钠凝胶为外壳的纳米硒海藻酸钠复合凝胶。
本发明实施例还提供一种抑制炎症性肠炎的药物,是以所述透明质酸修饰的纳米硒粒子为核,海藻酸钠凝胶为外壳的纳米硒海藻酸钠复合凝胶制成的可口服药物。
所述外壳中还包裹有调控肠道菌群的药物,抑制炎症性肠炎。当然所述纳米硒海藻酸钠复合凝胶可以作为载体,负载现有的治疗肠胃炎的药物粉末,外壳能保护药物不在消化液中被过早暴露和溶解,然后在中性环境下海藻酸钠水凝胶会膨胀而释放药物,提高治疗效果。
作为实验比较对照物,再合成吐温80修饰的纳米硒Tw80@SeNPs、其他聚合物或多糖修饰的纳米硒(包括聚乙二醇修饰的纳米硒PEG@SeNPs,聚乙烯吡咯烷酮修饰的纳米硒PVP@SeNPs,壳聚糖修饰的纳米硒CS@SeNPs,茯苓多糖修饰的纳米硒CMP@SeNPs中的一种)与海藻酸钠形成纳米硒海藻酸钠复合凝胶体系以及硒酸钠、硒代蛋氨酸SeMet、亚硒酸钠、硒代半胱氨酸SeC与海藻酸钠形成含硒海藻酸钠复合凝胶体系SA@Na2SeO4、SA@SeMet、SA@Na2SeO3、SA@SeC作为对照。
Tw80@SeNPs的制备,在室温下,将200-300mg Tw80加入46.5mL水放入100mL烧杯中搅拌约2h,再加入1mL的Na2SeO3溶液(浓度1-5mM)放入上述溶液中混合,缓慢加入2.5mL Vc(Vc与Se的摩尔比为2-4:1),继续在室温下反应10小时,用透析袋在超纯水中透析10-48小时,即得产物Tw80@SeNPs。
SA@PEG@SeNPs的制备,在室温下,将PEG(终浓度为0.5-10mg/mL)加水搅拌形成溶液,与Na2SeO3溶液(终浓度为4-6mmol/L)、维生素C(终浓度为11-12mmol/L)混合进行反应,所述的维生素C和Na2SeO3按摩尔比2-4:1进行配比,采用透析袋进行透析,透析的时间为10-48小时,得到所述聚乙二醇修饰的纳米硒(PEG@SeNPs)粒子;再将海藻酸钠溶液与所述PEG修饰的纳米硒(PEG@SeNPs)粒子混合,所述海藻酸钠溶液的浓度为2%(w/v),所述聚乙二醇修饰的纳米硒粒子与海藻酸钠的体积比约为15%,然后将混合液逐滴加入到浓度为2%(w/v)的氯化钙溶液中,经过交联1-2h,交联温度为36℃后形成以所述聚乙二醇修饰的纳米硒粒子为核,海藻酸钠凝胶为外壳的纳米硒海藻酸钠复合凝胶(SA@PEG@SeNPs)。
SA@PVP@SeNPs的制备,在室温下,将PVP(终浓度为0.5-10mg/mL)加水搅拌形成溶液,与Na2SeO3溶液(终浓度为4-6mmol/L)、维生素C(终浓度为11-12mmol/L)混合进行反应,所述的维生素C和Na2SeO3按摩尔比2-4:1进行配比,采用透析袋进行透析,透析的时间为10-48小时,得到所述聚乙烯吡咯烷酮修饰修饰的纳米硒(PVP@SeNPs)粒子;再将海藻酸钠溶液与所述PVP修饰的纳米硒(PVP@SeNPs)粒子混合,所述海藻酸钠溶液的浓度为2%(w/v),所述聚乙烯吡咯烷酮修饰的纳米硒粒子与海藻酸钠的体积比约为15%,然后将混合液逐滴加入到浓度为2%(w/v)的氯化钙溶液中,经过交联1-2h,交联温度为36℃后形成以所述聚乙烯吡咯烷酮修饰的纳米硒粒子为核,海藻酸钠凝胶为外壳的纳米硒海藻酸钠复合凝胶(SA@PVP@SeNPs)。
SA@CS@SeNPs的制备,在室温下,将CS(终浓度为0.5-10mg/mL)加水搅拌形成溶液,与Na2SeO3溶液(终浓度为4-6mmol/L)、维生素C(终浓度为11-12mmol/L)混合进行反应,所述的维生素C和Na2SeO3按摩尔比2-4:1进行配比,采用透析袋进行透析,透析的时间为10-48小时,得到所述壳聚糖修饰的纳米硒(CS@SeNPs)粒子;再将海藻酸钠溶液与所述CS修饰的纳米硒(CS@SeNPs)粒子混合,所述海藻酸钠溶液的浓度为2%(w/v),所述壳聚糖修饰的纳米硒粒子与海藻酸钠的体积比约为15%,然后将混合液逐滴加入到浓度为2%(w/v)的氯化钙溶液中,经过交联1-2h,交联温度为36℃后形成以所述壳聚糖修饰的纳米硒粒子为核,海藻酸钠凝胶为外壳的纳米硒海藻酸钠复合凝胶(SA@CS@SeNPs)。
SA@CMP@SeNPs的制备,在室温下,将CMP(终浓度为0.5-10mg/mL)加水搅拌形成溶液,与Na2SeO3溶液(终浓度为4-6mmol/L)、维生素C(终浓度为11-12mmol/L)混合进行反应,所述的维生素C和Na2SeO3按摩尔比2-4:1进行配比,采用透析袋进行透析,透析的时间为10-48小时,得到所述茯苓多糖修饰的纳米硒(CMP@SeNPs)粒子;再将海藻酸钠溶液与所述CMP修饰的纳米硒(CMP@SeNPs)粒子混合,所述海藻酸钠溶液的浓度为2%(w/v),所述茯苓多糖修饰的纳米硒粒子与海藻酸钠的体积比约为15%,然后将混合液逐滴加入到浓度为2%(w/v)的氯化钙溶液中,经过交联1-2h,交联温度为36℃后形成以所述茯苓多糖修饰的纳米硒粒子为核,海藻酸钠凝胶为外壳的纳米硒海藻酸钠复合凝胶(SA@CMP@SeNPs)。
SA@Na2SeO4的制备,在室温下,取200-300mg海藻酸钠与10mL Na2SeO4溶液(终浓度为4-6mmol/L)混合,搅拌过夜后逐滴加入到2%(w/v)的氯化钙溶液中,经过交联后形成海藻酸钠微珠SA@Na2SeO4。
SA@SeMet的制备,在室温下,取200-300mg海藻酸钠与10mL SeMet溶液(终浓度为4-6mmol/L)混合,搅拌过夜后逐滴加入到2%(w/v)的氯化钙溶液中,经过交联后形成海藻酸钠微珠SA@SeMet。
SA@Na2SeO3的制备,在室温下,取200-300mg海藻酸钠与10mL Na2SeO3溶液(终浓度为4-6mmol/L)混合,搅拌过夜后逐滴加入到2%(w/v)的氯化钙溶液中,经过交联后形成海藻酸钠微珠SA@Na2SeO3。
SA@SeC的制备,在室温下,取200-300mg海藻酸钠与10mL SeC溶液(终浓度为4-6mmol/L)混合,搅拌过夜后逐滴加入到2%(w/v)的氯化钙溶液中,经过交联后形成海藻酸钠微珠SA@SeC。
作为实验空白对照物,将纯海藻酸钠水凝胶SA作为空白对照,即取200-300mg海藻酸钠与10mL超纯水混合,搅拌过夜后逐滴加入到2%(w/v)的氯化钙溶液中,经过交联后形成海藻酸钠微珠SA。
参见图4-6,分别将本发明实施例一的制备方法得到的纳米硒海藻酸钠复合凝胶(SA@HA@SeNPs),与Tw80@SeNPs进行检测它们对炎症性巨噬细胞RAW264.7胞内活性氧的清除效果与体外抗炎效果。
实验比较,巨噬细胞RAW264.7对SA@HA@SeNPs和Tw80@SeNPs的靶向摄取。
取对数生长期的RAW264.7细胞以1万/mL加入24孔板中培养24h,在加药前分为两组:SA@HA@SeNPs和Tw80@SeNPs两组,然后加入5μg/mL、10μg/mL、20μg/mL的SA@HA@SeNPs或Tw80@SeNPs分别处理4-6h后,抽去培养基,用PBS洗3次,每个皿加入2mL胰酶消化2min,终止消化收集细胞,用消化仪消解后测量药物吸收的量。(ex:466nm,em:504nm)。用Bio-tek荧光酶标仪读取吸光值用以计算巨噬细胞RAW264.7对SA@HA@SeNPs和Tw80@SeNPs的摄取量(见图4)。这表明,巨噬细胞RAW264.7摄取SA@HA@SeNPs相对Tw80@SeNPs粒子更多,表明有更多HA和SeNPs进入细胞发挥作用,更能发挥HA@SeNPs的活性氧清除效果和体外抗炎效果。值得注意的是,前期实验曾证实,实验比较对照物聚乙二醇修饰的纳米硒、聚乙烯吡咯烷酮修饰的纳米硒、壳聚糖修饰的纳米硒、茯苓多糖修饰的纳米硒与海藻酸钠形成的凝胶体系SA@PEG@SeNPs、SA@PVP@SeNPs、SA@CS@SeNPs、SA@CMP@SeNPs被巨噬细胞RAW264.7细胞的摄入量均明显低于SA@HA@SeNPs,均仅约为SA@HA@SeNPs摄入量的20-30%;
实验比较对照物如硒酸钠、硒代蛋氨酸SeMet与海藻酸钠形成的凝胶体系SA@Na2SeO4、SA@SeMet,他们被巨噬细胞RAW264.7细胞的摄入量均明显低于SA@HA@SeNPs,他们的细胞摄入量均仅约为SA@HA@SeNPs摄入量的10-12%;
实验比较对照物如亚硒酸钠、硒代半胱氨酸SeC与海藻酸钠形成的凝胶体系SA@Na2SeO3、SA@SeC,他们被巨噬细胞RAW264.7细胞的摄入量均明显低于SA@HA@SeNPs,他们的细胞摄入量均仅约为SA@HA@SeNPs摄入量的8-10%。
实验比较,SA@HA@SeNPs和Tw80@SeNPs对巨噬细胞RAW264.7胞内活性氧的清除效果。
应用DCF法检测细胞内ROS水平。首先取呈对数生长期的RAW264.7细胞以密度为20×104cells/mL(100μL)接种于96孔板中,使其贴壁生长24小时。然后去除旧的培养基,加入100μL的DCFH-DA探针,使其终浓度达10μM,并于37℃培养箱中孵育30min。接着加入不同浓度(5μg/mL、10μg/mL、20μg/mL)的SA@HA@SeNPs和Tw80@SeNPs粒子,并以脂多糖LPS作为阳性对照,立即在荧光酶标仪下连续2小时检测各孔吸光值,设置激发和发射波长分别为:488nm,525nm。计算处理组和对照组吸光值的比值,分析细胞经SA@HA@SeNPs和Tw80@SeNPs粒子处理后胞内ROS随时间的荧光变化情况(见图5A)。同时用荧光显微镜实时监测细胞中DCF的荧光强度(见图5B)。图5A说明,随着海藻酸钠包裹的HA@SeNPS的浓度增加,清除RAW264.7胞内活性氧ROS的能力越强,体现了SA@HA@SeNPs的效果存在剂量效应。图5B说明,RAW264.7胞内产生的活性氧ROS越多,检测到的DCF荧光强度越强。而随着加入的海藻酸钠包裹的HA@SeNPs的浓度增加,RAW264.7胞内活性氧ROS的荧光强度得到明显减弱,体现了SA@HA@SeNPs的清除活性氧的优势。
实验比较,HA@SeNPs对炎症性巨噬细胞RAW264.7的体外抗炎效果。
RAW264.7细胞(24孔培养板中每孔1×105个细胞)经贴壁生长24h后,用LPS(1μg/mL)刺激)处理24小时和48小时后,收集上清液。通过ELISA试剂盒测量培养基中RAW264.7细胞分泌的炎症因子IL-6和TNF-α的浓度(见图6A)。图6A说明,阳性对照组的LPS能促进炎症因子IL-6和TNF-α的过高表达,因此有促进炎症的作用。而不同浓度的HA@SeNPs能逆转炎症因子IL-6和TNF-α过高表达的状况,因此将其下调至趋于正常水平,体现了HA@SeNPs体外抗炎效果存在剂量效应。
同时,经过各种处理后,RAW264.7细胞在4%多聚甲醛中固定。用5%牛血清白蛋白(BSA)封闭细胞。随后,用一级抗体对细胞进行免疫染色,包括TNF-α(Proteintech,60291-1Ig,小鼠,1:50)和IL-6(Proteintech,16806-1-AP,兔,1:50)。在4℃下培养过夜后,将细胞进一步与相应的Alexafluor 594共轭山羊抗小鼠IgG(Proteintech,SA00013-3,1:500)或Alexafluor 488共轭山羊抗兔IgG(Proteintech,SA00013-2,1:500)在室温下培养1小时。最后,用4′,6-二氨基-2-苯基吲哚(DAPI,Sigma)对细胞核进行复染。使用共焦显微镜拍摄所有图像(见图6B)。图6B说明,RAW264.7胞内产生的炎症因子浓度越高,检测到的不同颜色的荧光强度越强。而随着海藻酸钠包裹的HA@SeNPs的加入,胞内炎症因子如IL-6、TNF-α浓度得到明显降低,表明HA@SeNPs能下调炎症因子IL-6、TNF-α的表达,体现了HA@SeNPs的体外抗炎的优势。
值得注意的是,前期实验曾证实,实验比较对照物聚乙二醇修饰的纳米硒、聚乙烯吡咯烷酮修饰的纳米硒、壳聚糖修饰的纳米硒、茯苓多糖修饰的纳米硒与海藻酸钠形成的凝胶体系SA@PEG@SeNPs、SA@PVP@SeNPs、SA@CS@SeNPs、SA@CMP@SeNPs他们对RAW264.7胞内活性氧ROS的清除效果和体外抗炎效果也远远低于SA@HA@SeNPs,约SA@HA@SeNPs效果的10-20%;
实验比较对照物如硒酸钠、硒代蛋氨酸SeMet与海藻酸钠形成的凝胶体系SA@Na2SeO4、SA@SeMet对RAW264.7胞内活性氧ROS的清除效果和体外抗炎效果也远远低于SA@HA@SeNPs,约SA@HA@SeNPs的5-10%;
实验比较对照物如亚硒酸钠、硒代半胱氨酸SeC与海藻酸钠形成的凝胶体系SA@Na2SeO3、SA@SeC对RAW264.7胞内活性氧ROS的清除效果和体外抗炎效果也远远低于SA@HA@SeNPs,约SA@HA@SeNPs的5-8%。故相比于其他纳米硒或含硒海藻酸钠凝胶体系,SA@HA@SeNPs清除巨噬细胞RAW264.7胞内活性氧和抑制胞内炎症因子如IL-6和TNF-α表达水平的能力更强。
通过实验证明,本发明纳米硒海藻酸钠复合凝胶作为调节肠道菌群并可进一步抑制炎症性肠炎,在体外细胞实验中能有效抑制炎症性的巨噬细胞Raw264.7中的活性氧(ROS),以及下调相关炎症因子的表达,具有高效的抑制炎症的效果。试验结果显示,纳米硒海藻酸钠复合凝胶(SA@HA@SeNPs)可作为肠道性疾病抑制剂进行开发,能很好调控肠道菌群,提高治疗慢性肠道炎症性疾病的效果。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (8)
1.纳米硒海藻酸钠复合凝胶的制备方法,其特征在于,包括步骤
(1)制备透明质酸修饰的纳米硒,将透明质酸(HA)加水搅拌形成透明质酸溶液与Na2SeO3溶液、维生素C混合进行反应,透析,得到所述透明质酸修饰的纳米硒(HA@SeNPs)粒子;
(2)将海藻酸钠溶液与所述透明质酸修饰的纳米硒(HA@SeNPs)粒子混合,然后将混合液逐滴加入到氯化钙溶液中,经过交联后形成以所述透明质酸修饰的纳米硒粒子为核,海藻酸钠凝胶为外壳的纳米硒海藻酸钠复合凝胶;
步骤(2)中所述海藻酸钠溶液的终浓度为2-3%(w/v),所述氯化钙溶液的终浓度为2-3%(w/v);所述步骤(2)中交联时间为1-2h,交联温度为36-37℃。
2.根据权利要求1所述纳米硒海藻酸钠复合凝胶的制备方法,其特征在于,步骤(1)中所述的Na2SeO3溶液的终浓度为1-5mM;维生素C溶液的终浓度为11-12mmol/L。
3.根据权利要求1所述纳米硒海藻酸钠复合凝胶的制备方法,其特征在于,步骤(1)中所述透明质酸的添加量为终浓度5-10mg/mL;所述的维生素C和Na2SeO3按摩尔比2-4:1进行配比;所述透析为采用透析袋进行透析,透析的时间为10-48小时。
4.一种纳米硒海藻酸钠复合凝胶,其特征在于,通过权利要求1-3任一种方法制备得到。
5.一种抑制炎症性肠炎的药物,其特征在于,是以权利要求4的纳米硒海藻酸钠复合凝胶制成的可口服药物。
6.根据权利要求5所述一种抑制炎症性肠炎的药物,其特征在于,所述外壳中还可包裹有调控肠道菌群的药物,抑制炎症性肠炎。
7.一种权利要求4所述纳米硒海藻酸钠复合凝胶在制备调控肠道菌群、抑制炎症性肠炎药物中的应用。
8.根据权利要求7所述纳米硒海藻酸钠复合凝胶在制备调控肠道菌群、抑制炎症性肠炎药物中的应用,其特征在于,所述纳米硒海藻酸钠复合凝胶通过清除体外活性氧,以及下调免疫细胞相关炎症因子,用于抑制炎症性肠炎。
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