CN115054577A - 纳米解毒剂及其在中和mrsa穿孔毒素的应用 - Google Patents
纳米解毒剂及其在中和mrsa穿孔毒素的应用 Download PDFInfo
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Abstract
本发明公开了一种纳米解毒剂及其在中和MRSA穿孔毒素的应用方案，其中纳米解毒剂由天然蛋黄卵磷脂6份、鞘磷脂3份、聚乙二醇二硬脂酰磷脂酰乙醇胺1份、聚乳酸‑羟基乙酸共聚物纳米粒10份构成，该组合物应用在中和MRSA穿孔毒素中。本发明能够实现各类细胞在MRSA PFTs刺激下的完全保护，让保护更全面。并且采用的脂质体为人工合成的纳米囊泡，具有较好的均一性与稳定性，且能够实现量产。
Description
技术领域
本发明涉及纳米解毒剂及其应用纳米解毒剂及其在中和MRSA穿孔毒素的应用。
背景技术
耐甲氧西林金黄色葡萄球菌(Methicillin-resistant Staphylococcus aureus，MRSA)是临床上最常见的细菌，因其表现出广谱耐药性，目前临床尚无特效药用于治疗MRSA感染。MRSA穿孔毒素(Pore-forming toxins，PFTs)能够与正常细胞的细胞膜发生结合，寡聚后在膜上产生孔洞从而导致细胞死亡。研究表明，中和细菌PFTs能够有效控制细菌对周围组织细胞的破坏，提高宿主免疫系统对细菌的清除效率。人工提取的红细胞膜具有天然的PFTs亲和能力，能够在体内外吸附PFTs从而保护正常细胞免受PFTs侵袭，该策略已经被用于MRSA PFTs的中和。然而，红细胞膜的提取依赖大量血液供应，不适用于工业化量产。脂质体是基于磷脂成分的一种人工合成的纳米囊泡，具有较好的均一性与稳定性，且能够实现量产，具有较好的中和PFTs潜力。有研究表明以鞘磷脂为主要成分的脂质体模拟了哺乳动物细胞膜，具有良好的MRSA(USA300)PFTs中和能力。然而，由于MRSA分泌蛋自中含有a-toxin，Panton-Valentine leukocidin(PVL)，Bi-componentγ-Haemolysin(Hlg)AB，Leukocidin(Luc)ED在内的多种PFTs，每种PFTs的作用靶点不尽相同，这给PFTs的中和带来了难题。另外，不同细胞对于PFTs的敏感性不同，必须优化脂质体配方从而实现MRSA PFTs的完全中和，保护各类细胞免受MRSAPFTs的破坏。
虽然现有技术中，专利号为CN113041346A的发明专利公开了一种细菌毒素疫苗及其在预防细菌感染中的用途，其采用了人工脂质体，并让该人工脂质体由胆固醇、磷脂酰胆碱、聚乙二醇二硬脂酰磷脂酰乙醇胺和/或鞘磷脂构成。但是该方案下的脂质体在只针对Hlα产生抗体，对MRSA分泌的蛋白中含有的多种PFTs并不能完全产生保护。因此现有技术的组合物中仍有不足。
发明内容
针对现有技术存在的不足，本发明的目的在于提供一种纳米解毒剂及其在中和MRSA穿孔毒素的应用，实现各类细胞在MRSA PFTs刺激下的完全保护，让保护更全面。
为实现上述目的，本发明提供了如下技术方案：
一种纳米解毒剂，包括
天然蛋黄卵磷脂6份；
鞘磷脂3份；
聚乙二醇二硬脂酰磷脂酰乙醇胺1份；
聚乳酸-羟基乙酸共聚物纳米粒10份。
作为本发明的优选，该解毒剂采用如下方式制备获得：
天然蛋黄卵磷脂、鞘磷脂、聚乙二醇二硬脂酰磷脂酰乙醇胺混合后溶解于三氯甲烷中，通过旋转蒸发去除三氯甲烷，使用10％蔗糖溶液水化脂质膜后形成脂质体混悬液，通过脂质体混悬液混合聚乳酸-羟基乙酸共聚物纳米粒的水溶液形成脂质体包裹PLGA-NPs的组合物。
进一步优选中，脂质体混悬液与聚乳酸-羟基乙酸共聚物纳米粒的水溶液混合后通过探头超声将脂质体包裹PLGA-NPs，获得组合物。
进一步优选中，水化脂质膜的温度为60℃。
本方案还将上述成分的纳米解毒剂用于中和MRSA穿孔毒素。
本发明的有益效果：
通过优化以脂质体为主要成分的纳米解毒剂的配方以实现各类细胞在MRSA PFTs刺激下的完全保护，采用的脂质体为人工合成的纳米囊泡，具有较好的均一性与稳定性，且能够实现量产。
附图说明
图1为本发明纳米解毒剂体外配方筛选数据图；
图2为本发明纳米解毒剂对HUVEC的解毒作用结果图；
图3为本发明纳米解毒剂对HUVEC与L929的安全性测定结果图；
图4为本发明纳米解毒剂对RBCs的保护作用示意图；
图5为本发明纳米解毒剂对L929细胞的解毒作用示意图；
图6为本发明纳米解毒剂的表征图；
图7为本发明纳米解毒剂对小鼠皮下注射MRSAPFTs的解毒作用结果图。
具体实施方式
下面将结合附图所给出的实施例对本发明做进一步的详述。
参照图1-7所示，
一种纳米解毒剂，包括
天然蛋黄卵磷脂6份；
鞘磷脂3份；
聚乙二醇二硬脂酰磷脂酰乙醇胺1份；
聚乳酸-羟基乙酸共聚物纳米粒10份。
上述份数表示为天然蛋黄卵磷脂(PC-98T)、鞘磷脂、聚乙二醇二硬脂酰磷脂酰乙醇胺(DSPE-PEG2000)、聚乳酸-羟基乙酸共聚物纳米粒(PLGA-NPs)的比例为6∶3∶1∶10(wt/wt)。
其中，
1.PLGA-NPs的制备
配制10mg/mL的PLGA丙酮溶液1mL，在玻璃瓶中加入2mL ddH2O，将1mL10mg/mLPLGA丙酮溶液打入2mL ddH2O中，搅拌挥发30min，最终液体2mL左右，即得到PLGA-NPs的水溶液。
2.纳米解毒剂的制备
利用薄膜水化法制备脂质体混悬液，将12mg PC-98T，6mg鞘磷脂，2mg DSPE-PEG2000充分溶解于三氯甲烷中，通过旋转蒸发充分除去有机溶剂，随后加入2mL无菌的10％蔗糖溶液，在60℃条件下水化洗下脂质膜，即得到10mg/mL的脂质体混悬液。随后将2mL10mg/mL的PLGA-NPs水溶液加入到上述脂质体悬液中，利用探头超声(80W，8min，2s开，3s关)使脂质体完全包裹PLGA-NPs，即得到纳米解毒剂(Lipo(PS)+PLGA)。将制备得到的纳米解毒剂置于4℃保存。
其中，制备获得的脂质体悬液直接利用超声探头(80W，8min，2s开，3s关)直接超声脂质体得到Lipo(PS)。
3.纳米解毒剂体外配方筛选
选用含10％胎牛血清的DMEM培养基培养HUVEC细胞，将细胞铺于96孔板中并置于37℃含5％CO2培养箱中培养。实验前将细胞置于DMEM无血清培养基中培养12h。将不同配方(不同胆固醇含量、不同PC含量、不同粒径PLGA)的纳米解毒剂(2mg/mL，0.1mL)与细胞孵育后立即加入MRSA PFTs(0.25，0.5，1，2，4μL/孔)，于37℃培养箱中孵育60min，PBS清洗3次后加入含10％CCK-8溶液的DMEM无血清培养基培养40min，于450nm处测定OD值，通过计算得出细胞存活率。
参照图1所示，在HUVECs细胞中进行MRSA PFTs解毒实验，优化结果显示配方中引入胆固醇(Ch，Cholesterol)会降低解毒效果(图1a)，引入软磷脂(PC)可在一定程度上增强解毒效果(图1b)，引入PLGA纳米粒基本不影响解毒效果(图1c)。结果显示，最终处方纳米粒对MRSAPFTs刺激下的HUVECs具有明显的解毒效果(图1d)。由此显示，背景技术中所提到的现有技术引入了胆固醇，在本方案中该成分会导致解毒效果降低，本方案显示的解毒效果优于现有技术。
上述成分组合构成的纳米解毒剂能够实现体外人脐静脉内皮细胞(HUVEC)、小鼠红细胞(mRBCs)、小鼠成纤维细胞(L929)在MRSA PFTs刺激下的完全保护。将该纳米解毒剂与MRSA PFTs混合后注射入小鼠皮下，不会造成明显的创伤。
相较于现有技术的组成来说，不仅仅局限于MRSA分泌的部分PFTs，例如背景技术中提到的Hlα。
以下，结合实验数据和过程予以说明：
1.纳米解毒剂对HUVEC的解毒作用
参照图2所示，选用含10％胎牛血清的DMEM培养基培养HUVEC细胞，将细胞铺于96孔板中并置于37℃含5％CO2培养箱中培养。实验前将细胞置于DMEM无血清培养基中培养12h。将不同浓度最终配方的纳米解毒剂(250，500，1000，2000μg/mL脂质)与细胞孵育后立即加入MRSAPFTs(0.3μL/孔)，于37℃培养箱中孵育60min，PBS清洗3次后加入含10％CCK-8溶液的DMEM无血清培养基培养40min，于450nm处测定OD值，通过计算得出细胞存活率。
图2中，固定Toxin(PFTs)加入的量的条件下，提高Lipo(PS)+PLGA的浓度可增强解毒效果，证明Lipo(PS)+PLGA的解毒具有剂量依赖性。
2.纳米解毒剂对HUVEC与L929的安全性测定
参照图3所示，选用含10％胎牛血清的DMEM培养基培养HUVEC与L929细胞，将细胞铺于96孔板中并置于37℃含5％CO2培养箱中培养。实验前将细胞置于DMEM无血清培养基中培养12h。将不同浓度最终配方的纳米解毒剂(250，500，1000，2000μg/mL脂质)与细胞孵育，于37℃培养箱中孵育60min，PBS清洗3次后加入含10％CCK-8溶液的DMEM无血清培养基培养40min，于450nm处测定OD值，与正常组进行对比，测定纳米解毒剂对HUVEC与L929细胞的安全性。
随着Lipo(PS)+PLGA的浓度升高，并不影响HUVEC细胞与L929细胞的细胞活性，证明Lipo(PS)+PLGA具有良好的生物相容性。
3.纳米解毒剂对RBCs的保护作用
参照图4所示，为了测定纳米解毒剂对MRSAPFTs刺激下RBCs溶血的保护作用，将不同体积的MRSA PFTs(0.25，0.5，1，2，4，v/v％)与PBS混合至固定体积50μL。然后将纳米解毒剂(2mg/mL)添加到100μL 5％(v/v)小鼠RBC中，在37℃下孵育30min。孵育后，将样品以13,000rpm离心5min，取上清液在540nm处测定OD值。图4中，相比对照组(Con)，Lipo(PS)+PLGA对红细胞具有良好的解毒效果。同一浓度毒素刺激下，可明显降低红细胞溶血率，证明Lipo(PS)+PLGA能够保护红细胞免受毒素破坏。
4.纳米解毒剂对L929细胞的解毒作用
参照图5所示，选用含10％胎牛血清的DMEM培养基培养L929细胞，将细胞铺于96孔板中并置于37℃含5％CO2培养箱中培养。实验前将细胞置于DMEM无血清培养基中培养12h。将纳米解毒剂(2mg/mL，0.1mL)与细胞孵育后立即加入MRSA PFTs(0.25，0.5，1，2，4μL/孔)，于37℃培养箱中孵育60min，PBS清洗3次后加入含10％CCK-8溶液的DMEM无血清培养基培养40min，于450nm处测定OD值，通过计算得出细胞存活率。相比对照组(Con)，Lipo(PS)+PLGA对L929细胞具有良好的解毒效果。同一浓度毒素刺激下，可明显提高L929细胞的存活率，证明Lipo(PS)+PLGA能够保护L929细胞。
5.纳米解毒剂的表征
使用Zetasizer Nano仪器(LitesizerTM 500，Anton Paar，Austria)测定纳米解毒剂的水化粒径与Zeta电位。如图6所示，Lipo(PS)的粒径为145.33nm，PLGA-NPs的粒径为122.47nm，经Lipo(PS)表面包裹后的Lipo(PS)+PLGA粒径增大至202.83nm。Lipo(PS)的电位为-3.76mV，PLGA-NPs的电位为-22.76mV，经Lipo(PS)表面包裹后的Lipo(PS)+PLGA电位增大至-8.17mV。通过粒径与电位结果可知，Lipo(PS)成功包裹至PLGA-NPs表面，形成Lipo(PS)+PLGA。
6.纳米解毒剂对小鼠皮下注射MRSA PFTs的解毒作用
参照图7所示，将100μL纳米解毒剂(10mg/mL)与MRSA PFTs(1.5μL)混合后于37℃水浴中孵育30min，随后将BalB/C小鼠(6-8w)后背部剃毛，异氟烷吸入麻醉小鼠，将孵育好的纳米解毒剂注射入小鼠皮下(100μL/只)，48h后观察小鼠皮肤处是否出现伤口及使用Image J软件进行伤口面积统计。
图7中，正常小鼠(Control)皮肤无伤口，经sMT(MRSA上清液，含PFT毒素)皮下注射后，48h在注射位置形成明显的皮肤溃烂伤口。经Lipo(PS)+PLGA注射后仍无明显伤口，证明Lipo(PS)+PLGA良好的生物相容性。将sMT与Lipo(PS)+PLGA混合孵育后注射入小鼠皮下，在48h后相较sMT组，伤口面积明显减小，证明Lipo(PS)+PLGA在体内具有良好的解毒效果。
以上所述仅是本发明的优选实施方式，本发明的保护范围并不仅局限于上述实施例，凡属于本发明思路下的技术方案均属于本发明的保护范围。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理前提下的若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (5)
1.一种纳米解毒剂，其特征在于，包括
天然蛋黄卵磷脂 6份；
鞘磷脂 3份；
聚乙二醇二硬脂酰磷脂酰乙醇胺 1份；
聚乳酸-羟基乙酸共聚物纳米粒 10份。
2.根据权利要求1所述的纳米解毒剂，其特征在于，天然蛋黄卵磷脂、鞘磷脂、聚乙二醇二硬脂酰磷脂酰乙醇胺混合后溶解于三氯甲烷中，通过旋转蒸发去除三氯甲烷，使用10％蔗糖溶液水化脂质膜后形成脂质体混悬液，通过脂质体混悬液混合聚乳酸-羟基乙酸共聚物纳米粒的水溶液形成脂质体包裹PLGA-NPs的组合物。
3.根据权利要求2所述的纳米解毒剂，其特征在于，脂质体混悬液与聚乳酸-羟基乙酸共聚物纳米粒的水溶液混合后通过探头超声将脂质体包裹PLGA-NPs，获得组合物。
4.根据权利要求2所述的纳米解毒剂，其特征在于，水化脂质膜的温度为60℃。
5.一种纳米解毒剂在中和MRSA穿孔毒素的应用，其特征在于，该纳米解毒剂为权利要求1～4任一一项所述的纳米解毒剂。
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