CN116999524B - 一种可口服的杂化膜囊泡及其制备方法和抗菌应用 - Google Patents
一种可口服的杂化膜囊泡及其制备方法和抗菌应用 Download PDFInfo
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Abstract
本发明属于药物制剂领域，具体涉及一种可口服的杂化膜囊泡及其制备方法和抗菌应用。本发明提供了一种杂化膜囊泡是由姜黄衍生外泌体(EVs)和幽门螺杆菌的细菌膜囊泡(DMVs)杂化组成，在超声作用下发挥强大抗菌作用。姜黄衍生外泌体和幽门螺杆菌双层膜囊泡组成的杂化膜仿生囊泡，结合了两种膜的优势，实现在感染部位的靶向滞留，并通过超声响应实现声动力高效杀菌的功能。由于制备简单，产量大，生物安全性良好等特点，为幽门螺杆菌感染提供一种有前景的治疗方法，具有转化到临床应用的潜力。
Description
技术领域
本发明属于药物制剂领域，具体涉及一种可口服的杂化膜囊泡及其制备方法和抗菌应用。
背景技术
幽门螺杆菌是一种螺旋形、微厌氧、对生长条件十分苛刻的革兰氏阴性细菌。幽门螺杆菌在鞭毛泳动的作用下专门感染胃部并定植于上皮细胞粘液层中，能够抵抗胃部酸性的极端环境。自从1983年发现幽门螺杆菌以来，越来越多的研究证明其与胃炎、胃溃疡、胃癌密切相关，至少90％的胃癌与幽门螺杆菌有关。
现行治疗幽门螺杆菌的方法主要是抗生素为主的三联或四联疗法，但由于抗生素耐药性的激增，抗生素疗效下降，导致抗生素疗法都难以根治病原感染。此外，抗生素无法实现抗菌作用还有以下三个原因：首先是抗生素在胃液酸性环境不稳定；二是药物在细菌感染区域浓度低；三是抗生素胃部时间停留短。提高药物稳定性，增加药物在胃部滞留时间和富集于感染部位，从而实现高效的抗菌与良好的生物安全性，是提高抗生素疗效的重要思路。
植物外泌体是指由植物细胞分泌出来的大小在10-1000nm之间的膜囊结构，包含一定量的蛋白质、核酸、代谢产物和脂质等成分。外泌体具有抵御消化系统酶作用，可以满足天然活性成分在体内的输送，或者作为体内递药系统。植物外泌体携带药物有利于人体吸收，提高生物利用率，在治疗疾病方面有巨大的潜力，同时不易引起机体炎症反应[1]。姜黄外泌体当中含有姜黄素，是姜黄中的主要抗炎活性成分，研究发现其治疗炎症疼痛、肿瘤、细菌感染都有效果[2，3]。无论是革兰氏阴性菌还是革兰氏阳性菌，姜黄素都表现出较强的抗菌活性和很少的副作用[4]。同时，姜黄素是一种天然声敏剂，在超声的作用下能够释放活性氧，具有显著灭活微生物的作用[5]。但是姜黄素不溶于水，单独使用难以滞留胃部并富集于感染区域，故需要将其搭载在递药系统上。相较于姜黄素，外泌体在水中分散性更好。
外膜囊泡(OMVs)是革兰氏阴性细菌在正常生长过程中由外膜分泌的球形纳米囊泡，其结构相对稳定，尺寸在20-300nm不等，主要包含着来自细菌外膜和细胞质的成分。OMVs来自细胞外膜，膜上必然也包含外膜成分，例如脂蛋白、脂多糖、外膜蛋白等，这些成分都是病原相关分子能够被宿主识别，从而起到靶向作用。同时，OMVs是一种多功能容器，可以容纳各种不同的分子，例如脂质、蛋白质、核酸，是一种理想的靶向递送系统[6]。这表明OMVs作为生物药物递送系统具有潜在意义，不仅能够穿过生物屏障，而且能够针对性进入受体细胞，逃避免疫的同时远距离输送大分子药物[7]。研究表明，口服OMVs能够在小鼠胃部停留24h以上进入胃上皮细胞，在人胃腺癌细胞至少稳定12h，在人胃癌高转移细胞至少稳定72h[8]。但OMVs产量较低，是临床转化率极低的障碍之一。同OMVs类似，双层膜囊泡(DMVs)来自革兰氏阴性菌的整个外膜，其提取方法是在低温高压环境下破坏细胞，悬浮液离心后得到DMVs。DMVs具有靶向宿主细胞的病原相关分子，并且具有足够的稳定性[9]。与合成纳米材料作为药物输送载体相比，OMVs和DMVs这类细菌衍生囊泡与靶细胞相互作用更加强大，生物相容性更好[10]。同时，由于DMVs具有制备简易且产量大的特性更易于转化。
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发明内容
为了解决上述问题，本发明提供了一种由姜黄衍生外泌体(EVs)和幽门螺杆菌双层膜囊泡(DMVs)组成的杂化膜仿生纳米囊泡，DMVs与母细菌相似的膜组成和结构，使得其具有与靶菌株的外膜融合，将其包载的物质递送进细菌内部的能力。姜黄衍生外泌体经证明可以在超声条件下产生活性氧，具有一定抗菌作用。姜黄衍生外泌体和幽门螺杆菌双层膜囊泡组成的杂化膜仿生囊泡，结合了两种膜的优势，实现在感染部位靶向滞留和超声响应的声动力高效杀菌的功能。
一方面，一种杂化膜囊泡，包括革兰氏阴性菌外膜囊泡和姜黄衍生外泌体，所述的革兰氏阴性菌外膜囊泡和姜黄衍生外泌体的质量比可以是≤1：1。
优选地，所述的革兰氏阴性菌外膜囊泡和姜黄衍生外泌体的质量比可以是1：1。
具体地，所述的革兰氏阴性菌包括但不限于：幽门螺杆菌，金黄色葡萄球菌、鲍曼不动杆菌、铜绿假单胞菌或大肠杆菌中的至少一种。
具体地，所述的姜黄衍生外泌体中姜黄素的含量可以为2％-6％；所述的杂化膜囊泡的水合粒径可以是170nm-260nm。
优选地，所述的姜黄衍生外泌体中姜黄素的含量可以为3％-4％；所述的杂化膜囊泡的水合粒径可以是170nm-220nm。
进一步优选地，所述的姜黄衍生外泌体中姜黄素的含量可以为3.64％-3.68％；所述的杂化膜囊泡的水合粒径可以是180-210nm。
另一方面，本发明提供了杂化膜囊泡的制备方法包括以下步骤：
(1)获得前述的革兰氏阴性菌外膜囊泡和姜黄衍生外泌体；
(2)将步骤(1)的革兰氏阴性菌外膜囊泡和姜黄衍生外泌体进行杂化。
具体地，步骤(1)或(2)所述的革兰氏阴性菌可以是幽门螺杆菌。
具体地，步骤(2)所述的杂化可以在超声中进行的。
进一步具体地，所述的超声在冰浴条件下完成；超声功率可以为10-20％；所述的超声可以为间断式超声；所述的间断式超声可以设置为开1-3s，关2-4s；所述的超声时间可以为8-15min。
优选地，所述的超声功率为15％；所述的间断式超声设置为开2s，关3s；所述的超声时间为10min。
又一方面，本发明提供了前述的杂化膜囊泡或利用前述的制备方法制备的杂化膜囊泡在制备抗菌药物中的应用。
具体地，所述的药物可以与超声结合进行杀菌。
具体地，所述的药物的剂型包括但不限于：片剂、液体剂、胶囊剂、散剂、栓剂和颗粒剂。
具体地，所述的药物中还包括其他药学上可以接受的载体。
进一步具体地，所述的载体包括但不限于：缓释剂、赋形剂、填充剂、粘合剂、润湿剂、崩解剂、吸收促进剂、吸附载体、表面活性剂或润滑剂中的任意一种或多种。
又一方面，本发明提供了一种抗菌药物，所述的药物包括前述的杂化膜囊泡或利用前述的制备方法制备的杂化膜囊泡。
具体地，所述的药物可以与超声结合进行杀菌。
具体地，所述的药物的剂型包括但不限于：片剂、液体剂、胶囊剂、散剂、栓剂和颗粒剂。
具体地，所述的药物中还包括其他药学上可以接受的载体。
进一步具体地，所述的载体包括但不限于：缓释剂、赋形剂、填充剂、粘合剂、润湿剂、崩解剂、吸收促进剂、吸附载体、表面活性剂或润滑剂中的任意一种或多种。
本发明所取得的的技术效果：
(1)具有较好的膜融合性和靶向性。荧光共振能量转移和荧光共定位的结果表明杂化膜融合成功并能够靶向幽门螺杆菌。
(2)酸性条件下稳定性强。在人工胃液中模拟胃部酸性环境，杂化膜囊泡的粒径和电位随时间无明显变化。
(4)抗菌能力强。在中性环境和模拟胃液中均具有优异的抗菌作用，抗菌率可达99.9％以上。
(4)生物安全性高。体内实验证明血液生化指标与对照组无显著性差异，具有良好的生物安全性。
附图说明
图1为姜黄衍生外泌体(EVs)与细菌外膜囊泡(OMVs)或者双层膜囊泡(DMVs)形成的杂化膜EVs-OMVs和EVs-DMVs的制备方法与超声作用下的靶向抗菌示意图。
图2为杂化膜纳米囊泡EVs-OMVs和EVs-DMVs的制备与表征，其中A为外泌体、衍生囊泡和杂化膜的透射电镜图像，比例尺：100nm；B为EVs-OMVs和EVs-DMVs融合前后水合粒径和Zeta电位。
图3为杂化膜纳米囊泡EVs-OMVs和EVs-DMVs的制备与表征，其中A为EVs-OMVs的共聚焦荧光共定位图像；B为EVs-DMVs的共聚焦荧光共定位图像；比例尺：10μm。
图4为杂化膜纳米囊泡EVs-OMVs和EVs-DMVs的制备与表征，其中A为共振能量转移(FRET)研究EVs与DMVs的融合情况；B为杂化膜在pH＝2.2模拟胃液中8h粒径；C为Zeta电位变化。
图5为EVs-OMVs、EVs-DMVs与幽门螺杆菌共孵育的共聚焦显微镜图像，比例尺：10μm。
图6为杂化膜纳米囊泡的细菌摄取和单线态氧释放研究，其中A为单线态氧探针1,3-二苯基异苯并呋喃(DPBF)溶液与外泌体孵育后超声0-8min的紫外-可见吸收光谱；B为与杂化膜EVs-OMVs孵育后超声0-8min的紫外-可见吸收光谱；C为与EVs-DMVs孵育后超声0-8min的紫外-可见吸收光谱。
图7为杂化膜纳米囊泡的细菌摄取和单线态氧释放研究，其中A为单线态氧荧光探针SOSG溶液与外泌体孵育后超声0-2min的荧光光谱；B为与杂化膜EVs-OMVs孵育后超声0-5min的荧光光谱；C为与EVs-DMVs孵育后超声0-5min的荧光光谱。
图8为杂化膜纳米囊泡的体外抗菌研究，其中A为中性环境下不同样品与细菌混合后进行超声(US)处理前后的细菌涂板图像；B为菌落计数结果(n＝3)，检测限为102CFU/mL；C为酸性环境(pH＝2.2)，不同样品与细菌混合后超声前后的细菌涂板图像；D为菌落计数结果(n＝3)，检测限为102CFU/mL；P值计算通过two-tailed Student’s t-test:**p<0.01；****p<0.0001。
图9为杂化膜纳米囊泡EVs-DMVs体内对幽门螺杆菌的抗感染作用和生物安全性，其中A为Balb/c小鼠体内幽门螺杆菌感染和治疗的示意图；B为各组小鼠(PBS：磷酸盐缓冲盐水，US：灌胃PBS后胃部超声，EVs：姜黄外泌体，EVs+US：灌胃EVs后进行胃部超声，EVs-DMVs：姜黄外泌体和幽门螺杆菌双层囊泡杂化膜，EVs-DMVs+US：灌胃EVs-DMVs后进行胃部超声，OAC：奥美拉唑、阿莫西林和克拉霉素联合)治疗结束提取胃部进行菌落计数(n＝5)。
图10为杂化膜纳米囊泡EVs-DMVs体内对幽门螺杆菌的抗感染作用和生物安全性，其中A为各组小鼠治疗结束后，胃部研磨液通过ELISA检测肿瘤坏死因子-α(TNF-α)水平；B为白细胞介素-6(IL-6)水平；C为健康小鼠口服给药PBS(Control组)或EVs-DMVs(EVs-DMVs组)并进行胃部超声后，血液生化指标丙氨酸转移酶(ALT)、天冬氨酸转移酶(AST)、尿素(UREA)和肌酐(CREA)指数；P值计算通过two-tailed Student’s t-test:ns，无显著性差异；*p<0.05；**p<0.01；***p<0.001；****p<0.0001。
具体实施方式
下面结合具体实施例，对本发明作进一步详细的阐述，下述实施例不用于限制本发明，仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明，实施例中未注明具体条件的实验方法，通常按照常规条件，下述实施例中所使用的材料、试剂等，如无特殊说明，均可从商业途径得到。
材料：姜黄购于安庆乐图商贸有限公司。
菌株：本实验使用的幽门螺旋杆菌菌株编号为ATCC 43504，来源为American TypeCulture Collection(ATCC，美国)。
实验动物：本研究采用Balb/c小鼠(6-8周，雌性，体重18-22克，SPF级)作为动物实验对象，购买自深圳市迈科生物科技有限公司，动物生产许可证号：SCXK(粤)2020-0051，动物合格证编号：44822700020213。
实施例1杂化膜纳米囊泡的制备与表征
1、制备步骤
(1)获得外膜囊泡(OMVs)的方法：
选取正常生长的幽门螺杆菌混匀于10mL BHI培养基中，加入10％ FBS，使得菌液OD600值在0.1左右，放入三气培养箱中，在恒定转速(120rpm)下培养48-72h。10mL菌液倒入200mL BHI中加入10％ FBS，37℃微需氧条件下以120rpm恒定转速培养48-72h。完成培养的幽门螺杆菌菌悬液按照35mL每管分装，在6500×g，4℃条件下离心10min。收集上清，在超速离心管中配平，于100000×g，4℃条件下超速离心1.5h。倾倒上清，沉淀用1mLPBS重悬收集，在6500×g，4℃条件下离心10min，收集上清，重复离心至没有沉淀产生，取30μL液体涂布平板确定除菌完全。
(2)获得双层膜囊泡(DMVs)的方法：
将生长在哥伦比亚血平板上的菌落分散于10mL PBS中，使得OD600值在1.0左右，菌液在低温超高压连续流细胞破碎仪进行破碎，破碎后的液体在6500×g，4℃条件下离心30min，收集上清。
(3)获得EVs的方法
将姜黄(购于安庆乐图商贸有限公司)表面附着的泥渍洗净，削皮称重，按1mL/g加入PBS，榨汁，用无菌纱布过滤榨汁所得汁液，除去植物粗纤维，得到姜黄匀浆液。先低速离心(3000g，4℃，30min)，取上清液。再离心(10000g，4℃，1h)，取上清液超速离心(100000g，4℃，1h)，收集沉淀，重悬于PBS中。随后通过蔗糖梯度溶液进行纯化，用保存在4℃的20mM，pH7.4Tris-HCl配置8％、30％、45％蔗糖梯度溶液，超离管中按照蔗糖浓度从低到高的顺序，依次从底部加入蔗糖溶液，最上层沿管壁加入1mL超速离心所得样品溶液，超速离心(100000g，4℃，1h)，收集不同密度分界处的样品，得到8-30％、30-45％上下两层EVs1和EVs2。将两层中的EVs组分转移至100kDa超滤管中离心(4℃，3000g，30min)，除去蔗糖，用PBS收集姜黄衍生外泌体(EVs)，姜黄素含量为3.68％(EVs1)和3.64％(EVs2)，-80℃保存，后续研究均使用EVs2。
(4)取蛋白质质量比为1:1的EVs和DMVs，加入600μL PBS，在超声波细胞粉碎机中于15％功率、超声开2s关3s的设定下冰浴超声工作10min，完成EVs-DMVs杂化。EVs-OMVs杂化膜制备同理。
2、膜融合验证
(1)利用荧光共振能量转移验证膜融合
选择1,1'-二十八烷基-3,3,3'，3'-四甲基碳菁高氯酸盐(DiI染料，激发波长551nm，发射波长569nm)和3,3'-二十八烷基沙碳氰酸高氯酸盐(DiO染料，激发波长483nm，发射波长501nm)染料作为FRET荧光染料对，两种染料加入EVs，37℃、300rpm条件下孵育40min。按照1:0、1:1、1:2、1:3的蛋白质质量比将EVs与DMVs进行融合。用荧光分光光度计测定，488nm作为激发波长，检测500-650nm发射荧光强度。
(2)利用荧光共定位检测膜融合
用DiO将EVs进行染色，DiI对OMVs、DMVs进行染色，孵育完毕在膜融合条件下制备杂化膜，并将没有杂化的囊泡作为对照组。样品用激光共聚焦显微镜观察融合效果。
透射电镜图像显示，杂化形成的两种囊泡形态相似，具有膜结构(图2中的A)。外泌体、细菌衍生囊泡以及两种杂化膜的水合粒径和Zeta电位测定结果显示：EVs、OMVs、DMVs、EVs-OMVs和EVs-DMVs的水合粒径分别为225.6nm、202.5nm、178.2nm、190.7nm和200.7nm。EVs、OMVs、DMVs、EVs-OMVs和EVs-DMVs的Zeta电位分别为-12.5mV、-24.3mV、-15.4mV、-22.7mV和-17.7mV(图2中的B)。此外，通过共聚焦显微镜(图3)观察膜融合情况，用DiI标记细菌膜囊泡DMVs，DiO标记姜黄外泌体EVs，超声形成的杂化膜的红色和绿色荧光重叠形成黄色区域，说明膜融合成功。由图4中的A可知随着DMVs所占比例的增加，DiO的荧光发射增加，DiI的荧光发射减少，这是由于DMVs的插入使EVs上的染料对距离变大，FRET特性减弱，证明DMVs和EVs的成功融合。
3、杂化膜酸性环境稳定性实验
将EVs和杂化膜加入pH＝2.2的模拟胃液(SGF，北京雷根生物技术有限公司，货号：CZ0211)中，每隔2h取100μL样品于900μL纯水中，混匀后用动态光散射在25℃测量粒径和Zeta电位。
研究表明杂化膜的粒径和电位大小在8h的测量时间中都没有明显的变化(图4中的B、C)，说明外泌体和杂化膜在模拟胃液中具有良好的稳定性，不容易被降解或破坏。
实施例2杂化膜纳米囊泡的细菌摄取和单线态氧释放研究
由于膜成分相似，OMVs和DMVs对幽门螺杆菌具有靶向作用。为了验证杂化膜是否保留了衍生囊泡这一特性，将杂化膜(DiO标记)与幽门螺杆菌进行孵育。共聚焦激光扫描显微镜(CLSM)图像显示(图5)，与没有杂化的物理混合对照组相比，标有绿色荧光的EVs与标有蓝色荧光的幽门螺杆菌重合数量更多，荧光强度更强，说明幽门螺杆菌对杂化膜有更多的摄取，因此衍生囊泡具有带领外泌体靶向幽门螺杆菌的功能。
外泌体中的姜黄素在超声作用下能够释放单线态氧起到杀菌作用。为了验证杂化膜是否保留了外泌体这一特性，研究选用两种探针进行实验。1,3-二苯基异苯并呋喃(DPBF)是一种检测单线态氧的指示剂，其在420nm特征吸收峰强度的下降与单线态氧生成增加成正比。单线态氧荧光探针SOSG的激发波长为504nm，发射波长为525nm，荧光强度随单线态氧释放而增强。通过DPBF单线态氧指示探针(图6)和SOSG单线态氧绿色荧光探针(图7)表明在超声条件下，杂化膜可以产生活性氧，为进一步的抗菌作用提供支持。
实施例3体外抗菌研究
(1)中性环境的体外抗菌验证
将EVs、EVs-OMVs和EVs-DMVs与幽门螺杆菌在pH为中性(pH＝7.4)PBS中共孵育，然后进行超声，随后稀释不同倍数进行涂板，培养72-96h后，对血平板上细菌菌落拍照和计数来考察不同样品对浮游幽门螺杆菌的抗菌活性(图8中的A、B)。与超声后的EVs组相比，EVs-OMVs组和EVs-DMVs组引起了幽门螺杆菌菌落大幅度的减少，EVs的抗菌率为61.27％，而EVs-OMVs和EVs-DMVs的抗菌率分别达到99.97％和99.98％，这表明杂化膜相对于EVs具有更强的体外抗游离菌作用。其中，与超声后的EVs-OMVs组相比，EVs-DMVs组抗菌效果稍佳。
(2)酸性环境的体外抗菌验证
保持组别不变，将pH为中性(pH＝7.4)PBS替换为pH＝2.2的模拟胃液，并在其中加入尿素保证幽门螺杆菌的存活，在酸性环境下样品与幽门螺杆菌共孵育，然后进行超声，随后稀释不同倍数进行涂板，培养72-96h后，对血平板上细菌菌落拍照和计数来考察不同样品对浮游幽门螺杆菌的抗菌活性(图8中的C、D)。与超声后的EVs组相比，杂化膜抗菌效果能够达到检测下限，即抗菌率超过99.99％。这一结果表明胃部酸性环境并不影响杂化膜的抗菌效果，即在胃酸中杂化膜仿生纳米系统依然能够在超声作用下释放单线态氧起到抗菌作用。
实施例4体内治疗研究
每只BALB/c小鼠灌胃200μL的幽门螺杆菌菌液(1×108CFU/mL)，连续4天，感染两周后继续体内抗菌实验。将感染的小鼠随机分为PBS组(磷酸盐缓冲盐水，0.01M，200μL/只)、US组(灌胃PBS后胃部超声)、EVs组(灌胃姜黄外泌体浓度1.5mg/mL，200uL/只)、EVs+US组(姜黄外泌体浓度1.5mg/mL，200uL/只，EVs灌胃后进行胃部超声)、EVs-DMVs组(姜黄外泌体浓度1.5mg/mL，200uL/只，杂化膜囊泡灌胃)、EVs-DMVs+US组(姜黄外泌体浓度1.5mg/mL，200uL/只，EVs-DMVs灌胃后进行胃部超声)和OAC组(奥美拉唑400μmol/kg、阿莫西林28.5mg/kg、克拉霉素14.3mg/kg联合)共7组，每组5只小鼠。通过灌胃给药，每次200μL，每天1次，连续3天。以灌胃PBS的小鼠作为阴性对照。三联疗法(OAC)组，给予小鼠质子泵抑制剂(奥美拉唑)30min后灌胃阿莫西林与克拉霉素，以中和胃酸，防止潜在的抗生素降解。需要超声的治疗组，每次灌胃后用超声治疗仪(1.0MHz,50％占空比，1.5W cm-2)对小鼠胃进行3次超声，共6min。治疗结束后第二天处死小鼠，取胃组织，采用胃部组织匀浆涂板法评价各组治疗效果。在涂板法中，将胃组织置于2.8mL PBS中匀浆，连续稀释后置于哥伦比亚血琼脂平板上，用含有5％无菌脱纤维羊血和多种抗生素(10μg/mL万古霉素、5μg/mL头孢磺啶钠、5μg/mL乳酸甲氧苄氨嘧啶、5μg/mL两性霉素B)对匀浆液进行涂板，然后在37℃的微氧条件下(5％ O2、10％ CO2、85％ N2)孵育4天。并通过菌落计数的方法评价体内抑菌活性。
小鼠胃部研磨液涂板统计结果显示(图9中的B)，尽管EVs+US组能实现了一定程度的细菌清除(相对于PBS组其菌落数降低1.11-lg CFU)，但EVs-DMVs+US组显示出更强的抗菌能力(相对于PBS组其菌落数降低1.87-lg CFU)，表明EVs-DMVs在体内也能发挥显著声动力抗菌功能。EVs-DMVs相较于单独的姜黄衍生纳米囊泡EVs具有更强的体内杀菌能力，这主要是由于其中DMVs的靶向作用。EVs-DMVs+US体内抗菌与OAC类似，说明对于药物敏感菌株，EVs-DMVs+US能实现临床药物的治疗效果。以上结果证明，将EVs与DMVs杂化后形成的杂化膜纳米囊泡针对体内幽门螺杆菌造成的胃部感染具有较好的治疗效果。进一步，对细胞炎性因子测定结果表明(图10中的A、B)，尽管EVs+US组能一定程度降低肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)的水平，但EVs-DMVs+US组显示出更强的抑制这两种促炎因子的能力。与OAC处理组相比，EVs-DMVs+US组同样表现出出显著的抗炎作用，说明EVs-DMVs不仅能有效治理幽门螺杆菌感染，同时能降低小鼠炎症反应，抑制感染的进一步发展。
此外，对杂化膜体内生物安全性进行评价。连续4天对健康小鼠灌胃EVs-DMVs，并对胃部进行3轮共6min超声，血液生化指标显示与对照组相比没有显著性差异，证实了超声下EVs-DMVs具有良好的生物安全性(图10中的C)。
Claims (4)
1.一种抗幽门螺杆菌的杂化膜囊泡，其特征在于，由革兰氏阴性菌外膜囊泡和姜黄衍生外泌体组成，所述的革兰氏阴性菌外膜囊泡和姜黄衍生外泌体的质量比≤1：1；
所述的革兰氏阴性菌为幽门螺杆菌；
所述的姜黄衍生外泌体中姜黄素的含量为2%-6%；所述的杂化膜囊泡的水合粒径为170nm-260nm；
所述的杂化膜囊泡的制备方法包括以下步骤：
（1）获得革兰氏阴性菌外膜囊泡和姜黄衍生外泌体；
（2）将步骤（1）的革兰氏阴性菌外膜囊泡和姜黄衍生外泌体进行杂化；
所述步骤（2）中的杂化是在超声中进行的；
所述的超声在冰浴条件下完成；超声功率为10-20%；所述的超声为间断式超声；所述的间断式超声设置为开1-3s，关2-4s；所述的超声时间为8-15min。
2.根据权利要求1所述的杂化膜囊泡，其特征在于，所述的超声功率为15%；所述的间断式超声设置为开2s，关3s；所述的超声时间为10min。
3.权利要求1-2任一项所述的杂化膜囊泡在制备抗幽门螺杆菌的药物中的应用。
4.根据权利要求3所述的应用，其特征在于，所述的药物与超声结合进行杀菌。
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