CN117323442A - 一种巨噬细胞靶向二氧化锰纳米系统的制备方法及其应用 - Google Patents
一种巨噬细胞靶向二氧化锰纳米系统的制备方法及其应用 Download PDFInfo
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Abstract
本发明公开了一种巨噬细胞靶向二氧化锰纳米系统的制备方法及其应用，属于生物医学技术领域。本发明公开的一种巨噬细胞靶向二氧化锰纳米系统的制备方法，包括MnO2NPs的制备、Rif@HA‑MnO2NPs的制备、Tuf‑Rif@HA‑MnO2NPs的制备。本发明利用巨噬细胞靶向二氧化锰材料的宿主细胞免疫调控功能和宿主细胞靶向药物递送功能以更有效杀伤和清除宿主细胞内的结核分枝杆菌，从而为更有效的结核病治疗提供新策略，为其他疾病的治疗提供新思路。
Description
技术领域
本发明涉及生物医学技术领域，更具体的说是涉及一种巨噬细胞靶向二氧化锰纳米系统的制备方法及其应用。
背景技术
近年来，功能纳米材料已被大量研究，将纳米技术和材料科学结合应用于医学生物学和疾病治疗使纳米药物越来越多地用于临床疾病的诊疗。纳米材料在传染病的预防和治疗方面也显示出巨大的潜力。如果能结合功能纳米材料的宿主细胞免疫调控功能和宿主细胞靶向药物递送功能，有望实现针对结核病等以巨噬细胞为宿主细胞传染病的更有效治疗，然而，目前仍无能有效结合宿主细胞免疫调控功能和宿主细胞靶向药物递送功能的抗结核系统。
锰(Mn)是一种无机微量元素，在动物机体的多种生理过程中具有重要的氧化调节作用和其他方面的活性。研究表明，宿主细胞抗肿瘤和抗病毒反应通路严重依赖于锰离子(Mn2+)，锰离子可以刺激cGAS/STING途径激活先天免疫和适应性免疫。然而，Mn2+非常不稳定，其生物医学应用受到严重限制。二氧化锰纳米颗粒(MnO2 NPs)是一种稳定且生物相容性良好的纳米粒子，可在不同配体修饰后作为靶向递送的药物载体进行药物递送，并且还能与细胞内谷胱甘肽(GSH)反应生成Mn2+，从而通过Mn2+的生物学功能激活cGAS/STING通路以调控机体免疫。MnO2 NPs在抗肿瘤和抗病毒治疗中显示出强大的潜力。综上所述，MnO2NPs是理想的药物递送载体及cGAS/STING途径激动剂。与其他金属化合物纳米粒子相比，MnO2 NPs相对来说安全性更高，易于代谢，便于修饰，且能作为优良的cGAS/STING激活剂。
结核病是由结核分枝杆菌(Mtb)感染引起的慢性传染病，世界上约四分之一的人口均为Mtb潜伏感染者，是全球传染范围最广且最致命的传染病之一。基于“高传染性”、“高致病性”的特点，全世界的结核发病率和死亡率居高不下。
肺泡巨噬细胞可以吞噬不同的宿主细胞杀菌免疫病原体，并通过抗原向T细胞递呈启动保护性适应性免疫反应，是抵抗结核分枝杆菌感染和停留的第一道免疫防线。但结核分枝杆菌已经发展出大量复杂的免疫逃逸策略—通过抑制巨噬细胞的一些关键抗结核免疫反应，如抑制Mtb与溶酶体融合、抑制巨噬细胞自噬、抑制巨噬细胞凋亡和抑制巨噬细胞M1抗菌极化，以及抑制抗原呈递和T细胞的抗结核细胞因子产生以逃避宿主细胞的抗菌机制。因此，探索抑制结核菌免疫逃逸并促进抗结核免疫的新策略有助于开发更有效的抗结核策略。
巨噬细胞的M1抗菌极化是其主要的抗结核功能之一，M1型巨噬细胞可通过分泌大量促炎因子(如TNF-α和IL-6等)以抑制胞内Mtb的生长，并激活T细胞分泌IFN-γ等杀伤性细胞因子，从而增强机体的抗结核免疫应答以更有效抑制胞内Mtb，另一方面，在结核菌感染过程中，被吞噬的结核菌可将DNA等信号分子释放到巨噬细胞的细胞质中，巨噬细胞的cGAS能感知DNA并被激活。而活化的cGAS则催化三磷酸腺苷酸和三磷酸鸟苷生成cGAMP，最终激活STING。这种激活的STING进一步结合并激活磷酸化干扰素调节因子IRF3的TBK1，提高IFN-β以及TNF-α的产生，激活细胞内自噬，从而更有效清除和控制Mtb感染。
因此，提供一种巨噬细胞靶向二氧化锰纳米系统的制备方法及其应用是本领域技术人员亟需解决的问题。
发明内容
有鉴于此，本发明提供了一种巨噬细胞靶向二氧化锰纳米系统的制备方法及其应用。本发明制备了巨噬细胞靶向装载利福平、表面修饰有Tuftsin(Tuf)多肽的二氧化锰的纳米系统(Tuf-Rif@HA-MnO2NPs)，从而获得有望结合宿主细胞免疫调控功能和宿主细胞靶向药物递送功能的新型纳米载药系统。本发明有望将其用于结核病的宿主靶向治疗及疫苗开发，并对其他疾病预防和治疗提供新思路。
为了实现上述目的，本发明采用如下技术方案：
(1)MnO2 NPs的制备
用水将37.4mg/ml PAH溶液稀释10倍后，加入40％体积分数的3.5mg/ml高锰酸钾(KMnO4)溶液，混合均匀后于室温下避光反应至所有高猛酸盐均转化为二氧化锰；得到的溶液离心，弃去沉淀，将得到的上清液透析过夜，收集透析袋内的溶液即为二氧化锰纳米颗粒(MnO2NPs)。
(2)Rif@HA-MnO2 NPs的制备
在MnO2 NPs溶液中加入20％体积分数的5mg/ml利福平溶液、4％体积分数的1mg/ml透明质酸(Hyaluronic Acid，HA)溶液、1％体积分数的1mg/ml EDC溶液、1％体积分数的1mg/mlNHS溶液，混合均匀后避光于4℃冰箱摇床反应过夜，反应后进行透析并收集透析袋内溶液即可得到Rif@HA-MnO2NPs。
(3)Tuf-Rif@HA-MnO2 NPs的制备
配置1mg/ml Tuftsin多肽溶液，配置好后向其中加入5％体积分数的1mg/ml EDC溶液、5％体积分数的1mg/mlNHS溶液，混匀后于4℃静置10min，10min后向其中加入与Tuftsin多肽溶液等体积的上述制备好的Rif@HA-MnO2 NPs溶液，混匀后于4℃静置反应过夜，过夜后进行透析并收集透析袋内溶液，即可得到Tuf-Rif@HA-MnO2 NPs。
进一步，利用上述方法制备得到的装载利福平、表面修饰有Tuftsin多肽的二氧化锰纳米系统Tuf-Rif@HA-MnO2 NPs。
进一步，所述的Tuf-Rif@HA-MnO2 NPs在靶向巨噬细胞中的应用。
进一步，所述的Tuf-Rif@HA-MnO2NPs在制备抑制结核分支杆菌药物中的应用。
进一步，所述的Tuf-Rif@HA-MnO2 NPs在制备治疗结核病药物中的应用。
进一步，所述的Tuf-Rif@HA-MnO2 NPs在制备结核病疫苗中的应用。
本发明具体为将二氧化锰纳米粒(MnO2 NPs)作为药物载体的制备方法以及其在结核菌宿主靶向治疗和疫苗开发中的潜在应用。MnO2 NPs具有制备方法简单、生物相容性高、易功能化等优点，作为载体表现出靶向递送的巨大潜力。利用MnO2 NPs的这些特性，对其进行药物装载后，进一步修饰巨噬细胞靶向多肽，从而构建巨噬细胞靶向的二氧化锰抗结核纳米系统。本发明将宿主细胞靶向药物治疗及免疫调控疗法联用，有望用于结核菌宿主靶向治疗以及结核病疫苗的开发，为结核病等以巨噬细胞为宿主细胞的传染病提供新的预防和治疗策略。
经由上述的技术方案可知，与现有技术相比，本发明公开提供了一种巨噬细胞靶向二氧化锰纳米系统的制备方法及其应用，具有以下有益效果：
(1)MnO2 NPs能高效地装载药物，并通过巨噬细胞靶向多肽的偶联使其具有巨噬细胞靶向性；
(2)Tuf-Rif@HA-MnO2 NPs具有良好的生物相容性；
(3)Tuf-Rif@HA-MnO2 NPs能有效杀伤和抑制胞内结核分枝杆菌。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据提供的附图获得其他的附图。
图1附图为本发明装载利福平、表面修饰有Tuftsin(Tuf)多肽的二氧化锰纳米系统(Tuf-Rif@HA-MnO2 NPs)的形态特征；
其中，A：从左到右分别为Rif@HA-MnO2 NPs的透射电镜(TEM)、水合粒径及Zeta电位分析；B：从左到右分别为Tuf-Rif@HA-MnO2 NPs的透射电镜(TEM)、水合粒径及Zeta电位分析；C：Tuf-Rif@HA-MnO2 NPs的TEM-EDS元素分析；数据代表三次独立实验的结果；
图2附图为为本发明Tuf-RhB@HA-MnO2 NPs的THP-1来源巨噬细胞靶向性；
其中，A：等量4μg/ml Tuf-RhB@HA-MnO2 NPs与RhB@HA-MnO2 NPs处理THP-1来源的巨噬细胞相应时间后的流式图；B：Tuf-RhB@HA-MnO2 NPs与RhB@HA-MnO2 NPs处理THP-1来源的巨噬细胞相应时间后的FITC阳性率(％)统计图(n＝3)；C：Tuf-RhB@HA-MnO2 NPs与RhB@HA-MnO2 NPs处理THP-1来源的巨噬细胞相应时间后的FITC平均荧光强度(MFI)(n＝3)；数据代表三次独立实验的结果并表示为平均值±标准误，***P<0.001。
图3附图为本发明Tuf-RhB@HA-MnO2 NPs的小鼠巨噬细胞靶向性；
其中，A：Tuf-RhB@HA-MnO2 NPs在小鼠脾脏细胞CD3+T细胞、CD19+B细胞及CD14+巨噬细胞中的荧光强度流式分布图；B：RhB@HA-MnO2 NPs在小鼠脾脏细胞CD3+T细胞、CD19+B细胞及CD14+巨噬细胞中的荧光强度流式分布图；C：Tuf-RhB@HA-MnO2 NPs与RhB@HA-MnO2 NPs在各类细胞中的FITC阳性率(％)统计图(n＝3)；D：Tuf-RhB@HA-MnO2 NPs与RhB@HA-MnO2NPs在各类细胞中的FITC平均荧光强度(MFI)统计图(n＝3)；数据代表三次独立实验的结果并表示为平均值±标准误，**P<0.01，***P<0.001；
图4附图为本发明Tuf-Rif@HA-MnO2NPs激活THP-1来源的巨噬细胞和小鼠骨髓源巨噬细胞(BMDM)中cGAS/STING通路及LC3介导的自噬；
其中，A：Tuf-Rif@HA-MnO2 NPs促进THP-1来源的巨噬细胞和小鼠骨髓源巨噬细胞(BMDM)中TBK1和IRF3的磷酸化表达并触发STING降解；B：Tuf-Rif@HA-MnO2 NPs激活THP-1来源的巨噬细胞和小鼠骨髓源巨噬细胞(BMDM)中自噬(LC3)；数据代表三次独立实验的结果。
图5附图为本发明Tuf-Rif@HA-MnO2NPs的胞内杀菌功能；
其中，A：MnO2 NPs,Rifampicin,Tuf-Rif@HA-MnO2 NPs对THP-1细胞以及小鼠骨髓来源巨噬细胞(BMDM)内H37Ra抑制作用的代表性菌落数图片；B：Tuf-Rif@HA-MnO2 NPs对THP-1细胞内H37Ra的杀伤抑制作用(n＝3)；C:Tuf-Rif@HA-MnO2 NPs对BMDM细胞内H37Ra的杀伤抑制作用(n＝3)；数据代表三次独立实验的结果并表示为平均值±标准误，*P<0.05，**P<0.01，***P<0.001。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
PAH、利福平、透明质酸：Sigma-Aldrich,USA；高锰酸钾：大翔，中国；EDC、NHS：Thermo Fisher Scientific,USA；Tuftsin多肽：肽谷生物，中国。小鼠来源的CD3+、CD19+及CD14+的荧光抗体：Tonbo Biosciences,USA。
实施例1Tuf-Rif@HA-MnO2 NPs的制备与表征
(1)MnO2 NPs的制备
将1ml 37.4mg/ml多环芳烃(Polycyclic aromatic hydrocarbons，PAH)溶液与9ml水混合后立即加入4ml 3.5mg/ml高锰酸钾(KMnO4)溶液，混合均匀后于室温下避光反应15min，至所有高猛酸盐均转化为二氧化锰。得到的溶液于3000rpm离心10min后，弃去沉淀，将得到的上清液透析过夜，收集透析袋内的溶液即为二氧化锰纳米颗粒(MnO2 NPs)。
(2)Rif@HA-MnO2 NPs的制备
在1ml MnO2 NPs中加入0.2ml 5mg/ml利福平(rifampin，Rif)溶液、0.04ml 1mg/ml透明质酸(Hyaluronic Acid，HA)溶液、0.01ml 1mg/ml EDC溶液、0.01ml 1mg/ml NHS溶液，混合均匀后避光于4℃冰箱摇床反应过夜，反应后进行透析并收集透析袋内溶液即可得到Rif@HA-MnO2 NPs。
(3)Tuf-Rif@HA-MnO2 NPs的制备
配置1ml 1mg/ml Tuftsin多肽溶液，配置好后向其中加入0.05ml 1mg/ml EDC、0.05ml 1mg/ml NHS，混匀后于4℃静置10min，10min后向其中加入1ml上述制备好的Rif@HA-MnO2 NPs，混匀后于4℃静置反应过夜，过夜后进行透析并收集透析袋内溶液，即可得到Tuf-Rif@HA-MnO2 NPs。
将以上制备得到的Rif@HA-MnO2 NPs、Tuf-Rif@HA-MnO2 NPs溶液直接上机进行激光粒仪分析两样本的水合粒径及Zeta表面电位，其水合粒径主要在80-200nm，平均Zeta电位分别为43.9mV、36mV，透射电镜显示其均为100-150nm左右的球形纳米材料，具有较稳定的结构。并且还对Tuf-Rif@HA-MnO2 NPs进行元素分析，可以看到纳米粒子的主要成分为Mn元素和O元素，证明Tuf-Rif@HA-MnO2 NPs已被制备成功(图1A-C)。
实施例2Tuf-Rif@HA-MnO2 NPs的巨噬细胞靶向性
以等量的带有FITC荧光的罗丹明(RhB)代替利福平制备RhB@HA-MnO2NPs、Tuf-RhB@HA-MnO2 NPs，其他步骤与实施例1保持一致。利用佛波醇12-十四酸酯13-乙酸酯(Phorbol 12-myristate 13-acetate，PMA)刺激THP-1细胞(1*106个/孔)于6孔板中分化为巨噬细胞并贴壁生长24h，后向每孔加入1ml 4μg/ml RhB@HA-MnO2 NPs/Tuf-RhB@HA-MnO2NPs，于37℃孵育1h、3h、6h，使纳米粒子被细胞摄取。
取小鼠脾脏，研磨并消化成单细胞悬液，以2*105个/孔转移至12孔板中，加入1ml4μg/ml RhB@HA-MnO2 NPs/Tuf-RhB@HA-MnO2 NPs处理6h，收集悬液并于1500g离心5min，弃去上清，加入PBS重悬细胞，以5μl/1*106个细胞的量加入小鼠来源的CD3+、CD19+及CD14+的荧光抗体，进行流式细胞术分析CD3+T细胞、CD19+B细胞及CD14+巨噬细胞的FITC阳性率(％)及每个细胞的平均荧光强度(MFI)。
结果显示，THP-1来源的巨噬细胞经RhB@HA-MnO2 NPs和Tuf-RhB@HA-MnO2 NPs处理1h、3h、6h后，Tuf-RhB@HA-MnO2 NPs处理后的细胞FITC阳性率(％)和每个细胞的平均荧光强度(MFI)都明显高于RhB@HA-MnO2 NPs处理组(图2A-C)。
在小鼠脾脏细胞中，RhB@HA-MnO2 NPs和Tuf-RhB@HA-MnO2 NPs处理后CD14+巨噬细胞的荧光信号强度明显高于CD3+T和CD19+B细胞。此外，与RhB@HA-MnO2 NPs相比，Tuf-RhB@HA-MnO2 NPs处理后CD14+巨噬细胞的FITC阳性率(％)和平均荧光强度(MFI)显著升高。这些结果表明HA-MnO2 NPs具有良好的巨噬细胞靶向性，而Tuf的修饰将进一步增强其巨噬细胞靶向能力，最终得到的Tuf-Rif@HA-MnO2 NPs纳米系统具有优良的巨噬细胞靶向性(图3A-D)。
实施例3Tuf-Rif@HA-MnO2 NPs激活cGAS/STING通路及自噬
利用PMA刺激使THP-1细胞(1*106个/孔)于6孔板中分化为巨噬细胞并贴壁生长24h，后向每孔加入1ml的H37Ra菌悬液(感染系数(MOI)＝2)作用24h以模拟Mtb感染，后加入1ml不同浓度(0μg/ml、1μg/ml、2μg/ml、4μg/ml、8μg/ml、16μg/ml)的Tuf-Rif@HA-MnO2 NPs于37℃孵育24h。24h后提取蛋白并进行Westernblot分析。
利用20ng/ml的重组小鼠集落因子蛋白(Mouse Colony-Stimulating Factor，M-CSF)刺激小鼠骨髓细胞分化成小鼠骨髓来源巨噬细胞(Bone marrow-derivedmacrophages，BMDM，1*106个/孔)于6孔板中分化为巨噬细胞并贴壁生长4天，后向每孔加入1ml的减毒的结合分支杆菌菌株-H37Ra悬液(MOI＝2)作用24h以模拟Mtb感染，后加入1ml不同浓度(0μg/ml、1μg/ml、2μg/ml、4μg/ml、8μg/ml、16μg/ml)的Tuf-Rif@HA-MnO2NPs于37℃孵育24h。24h后提取蛋白并进行Westernblot分析。
结果显示，Tuf-Rif@HA-MnO2 NPs处理组磷酸化TBK1和IRF3的表达水平更高，并且触发了STING降解，表明Tuf-Rif@HA-MnO2 NPs在体外激活了cGAS-STING信号通路。除此之外，Tuf-Rif@HA-MnO2NPs还激活了巨噬细胞的抗菌自噬(LC3)(图4A-B)。这些结果均表明Tuf-Rif@HA-MnO2NPs能有效激活巨噬细胞的cGAS信号通路介导的抗菌免疫功能。
实施例4Tuf-Rif@HA-MnO2NPs的抗结核功能
以H37Ra作为结核分支杆菌的模型菌，通过菌落计数评估Tuf-Rif@HA-MnO2 NPs对H37Ra的抑制作用：利用PMA刺激使THP-1和BMDM细胞(1*106个/孔)于6孔板中分化为巨噬细胞并贴壁生长24h和96h，后向每孔加入1ml的H37Ra菌悬液(MOI＝2)作用24h以模拟Mtb感染，后使用不同浓度(2000ng/ml、1000ng/ml、500ng/ml)的Tuf-Rif@HA-MnO2NPs作为处理组，不同浓度的MnO2 NPs(2000ng/ml、1000ng/ml、500ng/ml)、利福平(25ng/ml、12.5ng/ml、6.25ng/ml)作为对照组，加入RMPI-1640培养基作为空白对照组(Control)，每组均加入1.5ml相应的溶液并作用72h。72h后使用1％SDS对细胞进行裂解，收集裂解后的悬液并稀释成不同的浓度(10-1、10-2、10-3、10-4)，涂布至7H11培养基，于37℃培养3-4周后进行菌落计数。
结果显示，与空白对照组相比，Tuf-Rif@HA-MnO2 NPs能够显著抑制H37Ra的生长，且抗菌活性显著强于相同浓度利福平的杀菌活性(图5A-C)，这表明该材料对结核菌具有明显的杀伤作用。
综上所述，本发明证明了利用二氧化锰不仅可以作为抗结核药物的载体以实现巨噬细胞靶向的药物递送，而且能够通过激活cGAS/STING通路和自噬等抗结核机制以抑制胞内结核分枝杆菌，通过将宿主靶向药物治疗和宿主细胞抗结核免疫调控结合起来更有效杀伤和清除巨噬细胞内的结核分枝杆菌，为临床结核病的治疗提供一种新思路。
对所公开的实施例的上述说明，使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (6)
1.一种巨噬细胞靶向二氧化锰纳米系统的制备方法，其特征在于，具体步骤如下：
(1)MnO2 NPs的制备
用水将37.4mg/ml PAH溶液稀释10倍后，加入40％体积分数的高锰酸钾溶液，混合均匀后于室温下避光反应至所有高猛酸盐均转化为二氧化锰；得到的溶液离心，弃去沉淀，将得到的上清液透析过夜，收集透析袋内的溶液，获得MnO2 NPs溶液；所述高锰酸钾溶液的浓度为3.5mg/ml；
(2)Rif@HA-MnO2 NPs的制备
在步骤(1)获得的MnO2 NPs溶液中加入20％体积分数的利福平溶液、4％体积分数的透明质酸溶液、1％体积分数的EDC溶液、1％体积分数的NHS溶液，混合均匀后避光于4℃冰箱摇床反应过夜，反应后进行透析并收集透析袋内溶液，得到Rif@HA-MnO2 NPs溶液；所述利福平溶液的浓度为5mg/ml；所述透明质酸溶液、EDC溶液和NHS溶液的浓度均为1mg/ml；
(3)Tuf-Rif@HA-MnO2 NPs的制备
配置Tuftsin多肽溶液(1mg/ml)，配置好后向其中加入5％体积分数的EDC溶液、5％体积分数的NHS溶液，混匀后于4℃静置10min，10min后向其中加入与所述Tuftsin多肽溶液等体积的步骤(2)制备得到的Rif@HA-MnO2 NPs溶液，混匀后于4℃静置反应过夜，过夜后进行透析并收集透析袋内溶液，得到Tuf-Rif@HA-MnO2 NPs；所述EDC溶液和NHS溶液的浓度均为1mg/ml。
2.利用权利要求1所述的方法制备得到的装载利福平、表面修饰有Tuftsin多肽的二氧化锰纳米系统Tuf-Rif@HA-MnO2NPs。
3.权利要求2所述的Tuf-Rif@HA-MnO2NPs在靶向巨噬细胞中的应用。
4.权利要求2所述的Tuf-Rif@HA-MnO2 NPs在制备抑制结核分支杆菌药物中的应用。
5.权利要求2所述的Tuf-Rif@HA-MnO2 NPs在制备治疗结核病药物中的应用。
6.权利要求2所述的Tuf-Rif@HA-MnO2 NPs在制备结核病疫苗中的应用。
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