CN110559280B - 治疗肺癌的载紫杉醇脂质体细菌吸入制剂 - Google Patents
治疗肺癌的载紫杉醇脂质体细菌吸入制剂 Download PDFInfo
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Abstract
本发明公开了一种载紫杉醇脂质体细菌吸入制剂，并且发现其用于治疗肺癌有特殊效果。该制剂是先制备紫杉醇脂质体后，通过电致孔技术导入细菌如大肠杆菌或干酪乳杆菌中，直接肺部给药，细菌携带药物直接靶向作用于肺部肿瘤，高效、安全。
Description
技术领域
本发明涉及医药领域，具体涉及治疗肺癌的载紫杉醇脂质体细菌吸入制剂。
背景技术
紫杉醇(Paclitaxel，PTX)是从短叶紫衫树皮中提取的一种复杂的化合物，分子量为853，为白色粉末，可溶于多种有机溶剂，如甲醇、乙醇、丙酮、二氯甲烷、三氯甲烷等；难溶于水，不溶于石油醚。
紫杉醇是抗微管药物，通过抑制细胞在进行有丝分裂时不能形成纺链体和纺键丝，从而抑制了细胞分裂和增殖，使细胞停滞在G2期和M期，发挥抗肿瘤作用。PTX现已广泛应用于临床，除晚期卵巢癌、肺癌、子宮癌外，血液病和各种实体瘤的临床化疗中也广泛采用紫杉醇。临床上，紫杉醇在小细胞肺癌治疗过程中多用于患者复发后给药，但其价格昂贵，经静脉吸收入血，到达病变的药物减少，且药物全身分布，具有严重的过敏反应、骨髓抑制、消化道反应、神经肌肉毒性以及心脏毒性等毒副作用。
脂质体(Liposomes)是由磷脂和胆固醇形成的双分子层结构的类圆形囊泡，能够包载亲水性和亲脂性的药物，亦可以包载生物大分子，其粒径在10～1000nm范围，具有类细胞膜结构、高生物相容性、低免疫原性、能保护药物或者活性基团、延长药物半衰期、增效减毒等一系列优势。
细菌中的革兰氏阴性菌如大肠杆菌，为埃希氏菌属代表菌，一般不致病，为人和动物肠道中的常居菌，在一定条件下可引起肠道外感染。某些血清型菌株的致病性强，引起腹泻，统称致病性大肠杆菌。大肠杆菌周生鞭毛，能运动，无芽孢，能发酵多种糖类产酸、产气，是人和动物肠道中的正常栖居菌。常见的革兰氏阳性菌如干酪乳杆菌，属于乳杆菌属，是常见的益生菌，不产芽孢，无鞭毛，不运动，兼性异型发酵乳糖，不液化明胶；最适生长温度为37℃；菌体长短不一，两端呈方形，常成链；菌落粗糙，灰白色，有时呈微黄色，能发酵多种糖。
细菌疗法是肿瘤治疗的新型技术。细菌能够靶向肿瘤，因而能更大强度的作用于肿瘤细胞，发挥抗肿瘤作用。细菌在肿瘤组织内可聚集以及发挥抗肿瘤活性，其抗肿瘤活性可能与下列因素有关。在肿瘤坏死区，肿瘤细胞内营养丰富：快速生长的肿瘤组织内容易形成低氧区和肿瘤坏死区，产生大量的中间代谢产物，使肿瘤组织内环境与正常组织不同，能提供细菌繁殖必需的营养，因此在相同的时间内，肿瘤内细菌的增殖速度远高于正常组织。细菌的兼性厌氧特性使其既能在有一定氧含量的小转移性瘤细胞内定居，也能在较大肿瘤组织内深度缺氧的中心进行定居。在肿瘤组织内，细菌的多个基因的启动子可都被优先激活。肿瘤细胞和基质细胞分泌肿瘤坏死因子-P或其他免疫抑制因子，抑制中性粒细胞的激活和浸润，同时肿瘤组织内的低氧环境导致巨噬细胞与中性粒细胞的杀菌作用受到抑制，在实体瘤内产生免疫抑制作用，使得细菌不容易被清除。细菌对免疫系统的刺激作用能够诱导炎性反应，释放炎症因子对肿瘤产生非特异性杀伤，比如TNF-α，可以对肿瘤的脉管系统产生直接的影响。细菌与肿瘤细胞竞争生长所需的营养抑制肿瘤的生长。细菌能在肿瘤细胞中持续增殖并引起核碎裂。
肺部给药是通过将药物经特殊给药装置传递至肺部，发挥全身或局部肺部疾病治疗，与传统口服或静脉相比，药物主要集中在肺部，其他部位分布较少。药物大部分沉积在肺部，可避免或减少其他组织的毒副作用。相反，通常口服或注射到达肺的实际药量较低，导致肺中药物与血浆中药物比例较低或直接导致治疗失败。肺部给药常用剂型有气雾剂、喷雾剂和粉雾剂。临床上肺部给药主要用于治疗哮喘和慢性阻塞性肺炎等。
癌症严重威胁人类的生命与健康，肺癌是我国最常见的恶性肿瘤，其发病率和死亡率均居恶性肿瘤首位。据统计，全球肺癌的发病占总数的11.6％，占全球癌症第一位，死亡率达到全部癌症的18.4％，占全球癌症死亡第一位。肺癌是指肺的恶性上皮性肿瘤起源于支气管上皮、支气管腺体、细支气管上皮和肺泡上皮。因为正常呼吸道上皮细胞类型多，在肿瘤的发生发展过程中，多能干细胞可以向不同方向分化，导致肺癌在组织学上的异质性，所以肺癌组织形态较复杂。从临床角度出发，目前肺癌主要分为两类：小细胞肺癌(Small Cell Lung Cancer，SCLC)和非小细胞肺癌(Non-Small Cell Lung Cancer，NSCLC)，其中80％左右为非小细胞肺癌。临床上手术、化疗与放疗是治疗非小细胞肺癌的主要手段，但中晚期患者远期疗效仍很差，主要死亡原因为局部控制困难和远处转移。近年来，传统的化疗主要为口服或静脉给药，多会发生药物的全身分布，造成到达肺癌部位有效药物量少，而到达正常组织的药物很可能引起毒副作用，这些严重的毒副作用和低治疗率给肺癌病人带来极大的痛苦。
发明内容
本发明公开了一种载紫杉醇脂质体细菌吸入制剂，并且发现其用于治疗肺癌有特殊效果。
载紫杉醇脂质体细菌吸入制剂可以采用以下步骤制备：
(1)制备紫杉醇脂质体；
(2)选择合适的细菌作为载体；
(3)通过电致孔作用将紫杉醇脂质体载入细菌，得到载紫杉醇脂质体细菌吸入制剂。
进一步地，载紫杉醇脂质体细菌吸入制剂还可以采用以下步骤制备：
(1)制备紫杉醇脂质体；
(2)将紫杉醇脂质体制备成紫杉醇脂质体固体；
(3)选择合适的细菌作为载体；
(4)将(2)中紫杉醇脂质体固体重新水化成脂质体混悬液，然后通过电致孔作用将紫杉醇脂质体载入细菌，得到载紫杉醇脂质体细菌吸入制剂。
紫杉醇脂质体的制备方法，可以参考相关专业书籍和文献制备得到，具体地，其制备方法选自薄膜分散法、复乳法、熔融法、注入法、表面活性剂处理法、离心法、前体脂质体法、加压挤出法、钙融合法等技术，优选的是薄膜分散法。本发明中的紫杉醇脂质体，其粒径为1～1000nm。紫杉醇脂质体中紫杉醇的量、辅料的量没有限制，优选的紫杉醇脂质体中除去水分外，含有0.1％～30％重量比的紫杉醇、70％～99.9％重量比的脂质、0％～50％重量比的其它在药学上可接受的辅料。
本发明中的紫杉醇脂质体，其中制备脂质体的脂质选自卵磷脂、磷脂酰乙醇胺、大豆磷脂、胆固醇、脑磷脂、胆固醇乙酰脂、β-谷甾醇、牛胆酸钠、蛋磷脂酰胆碱、二月桂酰磷脂酰胆碱、二肉豆蔻酰磷脂酰胆碱、二棕榈酰磷脂酰胆碱、二硬脂酰磷脂酰胆碱、二棕榈酰磷脂酰甘油、二硬脂酰磷脂酰甘油、二棕榈酰磷脂酸、磷脂酰丝氨酸、磷脂酰肌醇、神经鞘磷脂、鞘髓磷脂、二鲸蜡磷酸酯、硬脂酰胺，优选自卵磷脂、大豆磷脂、胆固醇、二月桂酰磷脂酰胆碱、二肉豆蔻酰磷脂酰胆碱、二棕榈酰磷脂酰胆碱、二硬脂酰磷脂酰胆碱，更优选自卵磷脂、大豆磷脂、胆固醇、二棕榈酰磷脂酰胆碱，最优选自大豆磷脂和胆固醇。当采用大豆磷脂和胆固醇制备紫杉醇脂质体时，大豆磷脂和胆固醇的摩尔比例选自50∶1～1∶1，优选自20∶1～2∶1，更优选自10∶1～4∶1。上述紫杉醇脂质体制备步骤中所述的其它在药学上可接受的辅料选自糖类、醇类、氨基酸类、肺源性表面活性物质、环糊精、高分子物质、助流剂、抗氧剂、柠檬酸及其盐、磷酸盐。
上述载紫杉醇脂质体细菌吸入制剂制备步骤中紫杉醇脂质体固体的制备方法选自喷雾干燥法、冷冻干燥法，优选自冷冻干燥法。冷冻干燥法制备紫杉醇脂质体固体时，一般是在紫杉醇脂质体中加入冻干保护剂，冷冻干燥制备得到。冻干保护剂可以在紫杉醇脂质体制备的同时或制备得到紫杉醇脂质体后加入。冻干保护剂选自糖类、醇类，具体选自蔗糖、乳糖、木糖醇、葡聚糖、甘露醇、海藻糖，优选自乳糖、甘露醇，最优选的是甘露醇。紫杉醇脂质体固体中脂质体与冻干保护剂的重量比没有严格规定，只要水化后得到的脂质体粒径小且均一就可以。
上述载紫杉醇脂质体细菌吸入制剂制备步骤中所述的细菌没有明确限制，从菌属方面，可选自革兰氏阴性菌、革兰氏阳性菌；从致病性方面，可选自致病菌、益生菌。革兰氏阴性菌选自大肠杆菌、沙门氏菌、变形杆菌、痢疾杆菌、肺炎杆菌、布氏杆菌、流感(嗜血)杆菌、副流感(嗜血)杆菌、卡他(摩拉)菌、不动杆菌属、耶尔森菌属、嗜肺军团菌、志贺菌属、巴斯德菌属、副溶血性杆菌、类志贺吡邻单胞菌，优选自大肠杆菌、沙门氏菌。革兰氏阳性菌选自葡萄球菌、链球菌、肺炎双球菌、炭疽杆菌、白喉杆菌、破伤风杆菌。致病菌选自肺炎链球菌、金黄色葡萄球菌、军团菌、绿脓杆菌、肺炎克雷伯菌、铜绿假单胞菌、厌氧菌、肠杆菌、克雷伯杆菌、流感嗜血杆菌、粘质沙雷菌、变形杆菌、无芽孢厌氧、肉毒杆菌、沙门氏菌、副溶血性弧菌。益生菌选自嗜酸乳杆菌、干酪乳杆菌、拉曼乳杆菌、长双歧杆菌、短双歧杆菌、卵形双歧杆菌、嗜热双歧杆菌、粪链球菌、乳球菌、中介链球菌、酵母菌、鼠李糖乳杆菌、副干酪乳杆菌，优选自干酪乳杆菌、嗜热双歧杆菌、乳球菌、鼠李糖乳杆菌。
制备载紫杉醇脂质体细菌吸入制剂时，制备方法可以采用化学法和物理法。化学法可以通过化学键将紫杉醇与细菌结合，可以将细菌制备成原生质体载体后将药物载入；也可以通过重组质粒将细菌与药结合；化学法往往会对细菌自身活性造成较大影响。物理法主要是离心法、超声法、电致孔法。本发明中使用电致孔法制备载紫杉醇脂质体细菌吸入制剂，细菌存活率较高，对紫杉醇脂质体包封率较高。
载紫杉醇脂质体细菌吸入制剂用于治疗各种肺癌。
细菌粒径较小，繁殖迅速，可选择性进入肿瘤组织快速增殖，并发挥抗肿瘤作用。将其作为活性载体提高了药物的利用率，还可解决许多药物水溶性差、吸收差、不稳定、毒副作用大等问题。将药物包裹其中，减少药物降解、提高稳定性；同时提高药物的靶向性，促进药物进入肿瘤细胞内，增加吸收。活菌载体用于抗肿瘤药物肺吸入给药有突出优势。载紫杉醇脂质体细菌吸入制剂在肺吸入后，可进入肺组织深部，直接趋向到肺部肿瘤中，并且在肿瘤部位迅速繁殖；所载药物在肺肿瘤组织的浓度大大增加，可快速发挥药效；同时在正常细胞组织的药物较少，减少药物所带来的毒副作用。载紫杉醇脂质体细菌吸入制剂的肺吸入方式可以是雾化后吸入，也可以直接结合呼吸机和肺灌洗装置灌入，也可以在冻干后，以粉雾剂方式吸入。
附图说明
图1.紫杉醇脂质体固体重新分散后照片
图2.紫杉醇脂质体粒径分布
图3.紫杉醇脂质体透射电镜照片
图4.电致孔对细菌的作用
图5.不同浓度紫杉醇脂质体对细菌的存活的影响
图6.电致孔对细菌存活的影响
图7.各配方对A549细胞的毒性。PTX：紫杉醇；LP：紫杉醇脂质体；LP/E：紫杉醇脂质体和大肠杆菌的物理混合物(没有电致孔)；LP/L：紫杉醇脂质体和干酪乳杆菌的物理混合物(没有电致孔)；LPE：载紫杉醇脂质体的大肠杆菌(电致孔作用后)；LPL：载紫杉醇脂质体的干酪乳杆菌(电致孔作用后)。*，p＜0.05；**，p＜0.01。
图8.流式检测各配方对A549细胞凋亡状况。图中代号意义见图7说明，图中百分数是细胞凋亡的百分数。
图9.各配方体外细胞吞噬的共聚焦显微镜图。LR：罗丹明脂质体；LR/E：罗丹明脂质体和大肠杆菌的物理混合物(没有电致孔)；LR/L：罗丹明脂质体和干酪乳杆菌的物理混合物(没有电致孔)；LRE：载罗丹明脂质体的大肠杆菌(电致孔作用后)；LRL：载罗丹明脂质体的干酪乳杆菌(电致孔作用后)。
图10.各组大鼠肺组织外观。图中代号意义见图7说明。
图11.各组大鼠肺组织HE染色病理切片照片。图中代号意义见图7说明。
图12.各组大鼠肺组织Caspase-3染色病理切片照片。图中代号意义见图7说明。
图13.Tunel法检测各组大鼠肺组织中细胞凋亡。图中代号意义见图7说明。
图14.大鼠各器官组织匀浆菌落的分布。图中代号意义见图7说明。
图15.免疫蛋白印迹检测肺组织Bcl-2与HIF-1α的蛋白水平。图中代号意义见图7说明。
图16.大鼠血中白细胞和中性粒细胞数量。图中代号意义见图7说明。
图17.各组大鼠肺组织大鼠肺组织中VEGF、TNF-α、IL-4、IFN-γ含量变化。图中代号意义见图7说明。
具体实施方式
实施例1.紫杉醇脂质体及其冻干固体
采用薄膜分散法制备紫杉醇脂质体：称取大豆磷脂0.553g和胆固醇0.05g于烧杯中，加入5mL乙醇超声溶解，精密称取0.04g紫杉醇加入上述乙醇溶液中溶解，转移至旋转蒸发仪上，在50℃旋转蒸发，得到均匀薄膜后，加入20mL水，继续在水浴条件下旋转分散均匀，得到紫杉醇脂质体。
在上述20mL紫杉醇脂质体中加入1.2g甘露醇，溶解，在冷冻干燥机中冻干后，得到疏松固体，过180目筛，得到白色粉末，作为紫杉醇脂质体固体。
取适量紫杉醇脂质体固体分散于pH7.0的磷酸盐缓冲液中，得到淡蓝色乳光的液体(图1)，室温放置后8小时后未产生沉淀。紫杉醇脂质体固体重混悬后得到紫杉醇脂质体(图2)，粒径较小，约为70nm，透射显微镜下为球形囊泡，粒度约为100nm(图3)。
实施例2.载紫杉醇脂质体细菌吸入制剂
在冰上解冻感受态大肠杆菌，添加实施例1中的紫杉醇脂质体固体，冰上孵育约5min，使其充分溶解和分散；对电致孔仪进行参数设置，包括r＝550V，d＝60ms，s＝50个，i＝100ms；对上述液体进行电致孔操作，共计20次，制备得到载紫杉醇脂质体细菌吸入制剂。
将上述制备过程中的大肠杆菌替换为干酪乳杆菌，也得到载紫杉醇脂质体细菌吸入制剂。
实验例1.载紫杉醇脂质体细菌吸入制剂的性质
样品：实施例1制备的紫杉醇脂质体；实施例2制备的载紫杉醇脂质体细菌吸入制剂；大肠杆菌(E.coli)；干酪乳杆菌(L.casei)。
在透射电镜下，电致孔作用后的细菌壁有明显孔道(图4)，可促进药物进入细菌内。
经高效液相检测，电致孔作用后载紫杉醇脂质体细菌的药物包封率均较高，在95％以上。
在体外环境中，紫杉醇脂质体对细菌的存活无明显影响，低浓度到高浓度(0.635～5mmol/L紫杉醇)药物浓度下，菌落数与空白对照组相比无明显差异，也就是说紫杉醇脂质体无抑菌作用(图5)。
对于大肠杆菌，电致孔作用后菌落数为4.38×106CFU/mL，空白对照组菌落数为4.73×106CFU/mL。对于干酪乳杆菌，电致孔作用后菌落数为4.31×106CFU/mL，空白对照组菌落数为4.76×106CFU/mL。大肠杆菌存活率为92.6％，大肠杆菌存活率为90.5％，电致孔对细菌的生长无明显影响(图6)。
实验例2.载紫杉醇脂质体细菌吸入制剂的细胞学研究
样品：人肺癌细胞株A549；紫杉醇；实施例1中的紫杉醇脂质体；实施例2中的载紫杉醇脂质体细菌吸入制剂；大肠杆菌(E.coli)；干酪乳杆菌(L.casei)。紫杉醇脂质体和大肠杆菌的物理混合物以及紫杉醇脂质体和干酪乳杆菌的物理混合物采用实施例2中步骤制备，但没有进行电致孔操作。具体步骤包括：将感受态的细菌冻存于-80℃冰箱，在冰上解冻感受态细菌，添加实施例1中的紫杉醇脂质体固体，冰上孵育约5min，使其充分溶解和分散，得到紫杉醇脂质体和细菌的物理混合物。
下面英文缩写分别代表各配方。PTX：紫杉醇；LP：紫杉醇脂质体；LP/E：紫杉醇脂质体和大肠杆菌的物理混合物(没有电致孔)；LP/L：紫杉醇脂质体和干酪乳杆菌的物理混合物(没有电致孔)；LPE：载紫杉醇脂质体的大肠杆菌(电致孔作用后)；LPL：载紫杉醇脂质体的干酪乳杆菌(电致孔作用后)。
A549细胞分别接种于96孔板，每孔细胞数为5×105个，孵育8h细胞贴壁，以不同浓度的PTX、LP、E.coli、LP/E、LPE、L.casei、LP/L、LPL(除纯细菌制剂外，分别含有PTX的浓度为0.625～5μM)加入含细胞培养孔中，孵育24h，加入Cell Counting Kit-8(CCK-8)溶液，继续孵育2h，450nm处检测吸光度值，并计算各组药物的细胞存活率。将A549细胞接种于6孔板中(5×105/孔)37℃下孵育8h。加入上述配方后作用24h，A549细胞凋亡按照Annexin V-FITC/PI试剂盒操作书方法检测。用流式细胞仪检测各配方对细胞凋亡的影响。
细胞吞噬作用：按照实施例1和实施例2，以及本实验例记录的方法，制备罗丹明(rhodamine)的脂质体及细菌制剂，分别得到LR：罗丹明脂质体；LR/E：罗丹明脂质体和大肠杆菌的物理混合物(没有电致孔)；LR/L：罗丹明脂质体和干酪乳杆菌的物理混合物(没有电致孔)；LRE：载罗丹明脂质体的大肠杆菌(电致孔作用后)；LRL：载罗丹明脂质体的干酪乳杆菌(电致孔作用后)。
利用激光共聚焦显微镜测定，5×105个A549细胞接种于2×2mm玻片，37℃培养8小时，控制菌浓度106CFU/mL和罗丹明浓度5μM，将上述配方加入细胞培养液中，在0.5、2、4h取出玻片，用磷酸盐缓冲液(PBS)洗3次，4％多聚甲醛固定15min，PBS洗3次后用DAPI染色10min，免疫荧光剂进行封片，激光共聚焦显微镜观察。
结果：
单独的E.coli和L.casei(106CFU/mL)对A549细胞具有较高的细胞毒性，细胞存活率分别为45.6％、51.9％。PTX对A549细胞有一定细胞毒性，相同浓度的LP毒性大于PTX作用，且具有浓度依赖性(图7)。这是由于脂质体有较好的细胞膜渗透性和易被细胞内吞，促进药物进入细胞。含有紫杉醇脂质体的细菌制剂的抗A549细胞作用均大于单独紫杉醇和紫杉醇脂质体组，其中载紫杉醇脂质体的细菌吸入制剂(LPE、LPL)对A549细胞的毒性均大于相应的物理混合组(LP/E、LP/L)。
用流式检测药物对细胞凋亡状况(图8)，单独细菌E.coli和L.casei(106CFU/mL)对A549细胞的凋亡率分别为7.7％和3.8％，单独LP对细胞凋亡率为13.6％，细菌与LP联合作用对A549细胞的凋亡率远远高于单独作用，LPE和LPL组对A549细胞凋亡率最高，远大于LP/E和LP/L，表明细菌作为药物载体后的细胞毒作用大大增加。
细胞吞噬实验显示，随着时间的增长，载罗丹明脂质体细菌制剂进入细胞质的药物量增多(图9)，但各时间段在载罗丹明脂质体细菌制剂(LRE、LRL)进入细胞的能力明显高于单独罗丹明脂质体及罗丹明脂质体与细菌混合组(LR/E、LR/L)，表面细菌载体能促进药物进入细胞发挥药效。
实验例3.治疗大鼠肺癌药效研究
将45只雄性大鼠(平均重量180±20g/只)随机分为9组，每组5只，乙醚麻醉后，除正常对照组通过气管给予生理盐水，其他组向气管内匀速注人0.1mL 10％三甲基胆蒽碘化油溶液(m/v)。分别饲养45天，在诱癌剂诱导下自发形成原发性肺癌，然后对其进行治疗。分组如下(a)正常组对照(normal)，气管插管给予生理盐水，0.2mL；(b)肺癌模型组(model)，生理盐水，0.2mL；(c)干酪乳杆菌，106CFU(L.casei)；(d)大肠杆菌，106CFU(E.coli)；(e)紫杉醇脂质体，紫杉醇1mg(LP)；(f)干酪乳杆菌和紫杉醇脂质体混合物，106CFU+紫杉醇1mg(LP/L)；(g)大肠杆菌和紫杉醇脂质体混合物，106CFU+紫杉醇1mg(LP/E)；(h)载紫杉醇脂质体干酪乳杆菌，106CFU/紫杉醇1mg(LPL)；(i)载紫杉醇脂质体大肠杆菌，106CFU+紫杉醇1mg(LPE)。给药方式均为通过气管喷入方式。给药两次，一周一次，每天同一时间进行大鼠气管注入给药，最后给药10天后，将大鼠处死前通过尾静脉取血20μL，适量稀释后于血细胞计数仪测定白细胞与中性粒细胞的含量。各组大鼠经10％水合氯醛1mL麻醉处死，解剖暴露气管，开胸，使主、支气管清楚地暴露在术野中，在主气管处剪一个T型小开口。用止血钳结扎右侧支气管。将自制导管前端嵌入直至左主气管。抽取2mL 4℃生理盐水，经导管缓缓注入左肺，可观察到左肺充盈，在肺中停留30s后，轻轻抽回灌注液体，再将该液体注入到肺中，反复抽注3次，将注入的液体全部抽回，转移至5mL离心管中，重复灌洗1次。合并2次灌洗液，共4mL，于3500rmp离心10min，吸取上清液，于-80℃保存。取右肺组织，肺上叶置于10％甲醛中固定，常规脱水、包埋、苏木素-伊红(HE)染色后同时免疫组化标记Casepase-3蛋白，光镜下观察肺组织病理形态变化及Casepase-3表达状况，肺中叶进行组织匀浆后ELISA检测肿瘤坏死因子TNF-α、肿瘤坏死因子VEGF、白介素-4IL-4与缺氧转录因子IFN-γ的检测，肺下叶则免疫蛋白印迹分析抑凋亡蛋白Bcl-2、缺氧转录因子HIF-1α。将大鼠各个组织器官匀浆培养，进行菌落数统计，观察细菌载组织中的分布。
结果：
从刚取下的肺组织外观可看出，正常肺组织红润有光泽，肺癌组有大量肺癌白色结节与出血，各治疗组的肺组织白色癌结节明显减少，LPL与LPE组症状明显减轻(图10)。健康大鼠给于生理盐水后，肺组织结构完整，肺泡腔清晰。肺癌模型组大鼠肺组织肺泡结构被破坏，有大量炎性组织增生。各治疗组均明显改善病理状况。载紫杉醇脂质体细菌组具有较好改善效果(图11)。
Caspase-3是细胞凋亡联级通路的最后一环，文献报道紫杉醇可促进Caspase-3表达。本研究中模型组几乎无黄染效果，各治疗组黄染增加，LPL与LPE组的效果最好(图12)。
用Tunel法检测肺组织中细胞凋亡。模型组中肺组织细胞凋亡较少，治疗组细胞凋亡增加，单独的L.casei与E.coli组细胞凋亡较少，LP/L与LP/E组较单独LP和单独细菌组明显增加，而LPL与LPE组细胞凋亡最多(图13)。
从各器官组织匀浆菌可看出，各治疗组肺组织中菌落数非常多，远高于其他器官(图14)。其他器官中的菌落数比较，其中心组织中有大量菌落存在，可能心脏与肺组织较近有关；肝组织中有少量细菌存在，这与肝的代谢功能有关；其他组织基本无细菌(图14)。可以看出载紫杉醇脂质体细菌吸入制剂具有明显的肺组织沉积效果，细菌在治疗肿瘤的同时，也可将化疗药带向肿瘤部位，发挥抗肿瘤效果。
肺癌发生时，肺癌细胞大量增殖，会抑制抗凋亡蛋白的表达，使凋亡减少。Bcl-2是一种抑凋亡蛋白，它能阻止线粒体内细胞色素C向外释放，抑制凋亡发生。HIF-1α是一种低氧调节转录因子，调控多种低氧诱导基因的表达。当肿瘤快速生长使肿瘤组织处于相对缺氧状态下，HIF-1α蛋白水平的表达增加。因此可以通过检测HIF-1α蛋白的含量来评估肿瘤组织的氧合状态。此外，低氧条件也会诱导VEGF的表达，从而增加组织渗透性和诱导血管形成。通过免疫蛋白印迹分别检测Bcl-2与HIF-1α的蛋白水平，发现各配方都促进Bal-2与HIF-1α水平下降，其中LPL、LPE组的下降最多，说明载紫杉醇脂质体细菌吸入制剂对肺癌细胞凋亡具有明显促进作用(图15)。
白细胞和中性粒细胞是典型的免疫功能细胞，可清除外来细菌、病毒和变异细胞。细菌制剂治疗后，血中白细胞和中性粒细胞明显升高，说明细菌作用后会激发免疫。LP组与正常组无显著性差异。各细菌制剂组作用后，白细胞和中性粒细胞量明显升高(图16)。因此，细菌在体内会激起体内固有免疫应答，从而发挥抗肿瘤作用。
TNF-α是细胞免疫介质，可直接杀死肿瘤细胞，不伤害正常细胞。IL-4是Th2的标志物细胞，在体液免疫反应中发挥重要作用。含细菌制剂治疗后，TNF-α、IL-4因子水平明显增高，其中LPL与LPE组表达高于单独细菌L.casei与E.coli组(图17)，即可明显诱导细胞因子分泌，产生免疫调节作用，增强抗肿瘤功效。γ干扰素(IFN-γ)是一类分泌性蛋白，具有广谱抗病毒、抗肿瘤和免疫调节功能。本研究中，模型组IFN-γ表达较低，各细菌制剂组均有增加。单独细菌给药组明显高于紫杉醇脂质体组。载紫杉醇脂质体细菌吸入制剂组较单独细菌给药组增加明显(图17)。细菌可显著促进CD8+T细胞对IFN-γ的分泌，延缓肿瘤的生长。这些结果都表明，细菌在肿瘤微环境中主要参与了由CD8+T细胞介导的抗肿瘤免疫应答。细菌能够通过提高肿瘤内的免疫反应水平进而提高肿瘤内CD8+T细胞与CD11b+单核-巨噬细胞的比例，诱导其产生肿瘤坏死因子TNF-α与IFN-γ，破坏肿瘤内免疫逃逸的环境，产生抗肿瘤效果。
VEGF是诱导血管生成的主要调节因素，能够促进多种肿瘤组织的生长和高密度血管化。本研究中，VEGF的表达肺癌模型组表达最高，各治疗组均较模型组明显减少，LPL与LP/L组比，以及LPE与LP/E组比较，前者进一步减少了VEGF的表达，具有显著性差异(图17)。
将化疗药紫杉醇制备成脂质体后，再与细菌结合，利用细菌作载体提高紫杉醇靶向性，通过肺吸入给药技术，实现局部给药的方式，极大地降低了其毒副作用。通过药效机制探索，载紫杉醇脂质体细菌吸入制剂可以下调Caspase-3、Bcl-2的表达，减少VEGF的表达，抑制肿瘤的增殖，增强抗肿瘤作用，以及促进TNF-α、IL-4、IFN-γ等因子的产生，通过免疫调节增强抗肿瘤作用，是肺癌治疗的新方法。
Claims (3)
1.一种载紫杉醇脂质体细菌吸入制剂，用于治疗肺癌，是通过电致孔作用将紫杉醇脂质体载入细菌得到，其中细菌选自大肠杆菌、干酪乳杆菌。
2.如权利要求1所述的载紫杉醇脂质体细菌吸入制剂，其中紫杉醇脂质体采用薄膜分散法制备；紫杉醇脂质体中除去水分外，含有0.1％～30％重量比的紫杉醇、70％～99.9％重量比的脂质、0％～50％重量比的其它在药学上可接受的辅料；其中脂质为大豆磷脂和胆固醇，大豆磷脂和胆固醇的摩尔比例为10∶1～4∶1。
3.如权利要求1所述的载紫杉醇脂质体细菌吸入制剂，采用以下步骤制备：
称取大豆磷脂0.553g和胆固醇0.05g于烧杯中，加入5mL乙醇超声溶解，精密称取0.04g紫杉醇加入上述乙醇溶液中溶解，转移至旋转蒸发仪上，在50℃旋转蒸发，得到均匀薄膜后，加入20mL水，继续在水浴条件下旋转分散均匀，得到紫杉醇脂质体；在上述20mL紫杉醇脂质体中加入1.2g甘露醇，溶解，在冷冻干燥机中冻干后，得到疏松固体，过180目筛，得到白色粉末，作为紫杉醇脂质体固体；在冰上解冻感受态大肠杆菌，添加紫杉醇脂质体固体，冰上孵育约5min，使其充分溶解和分散；对电致孔仪进行参数设置，包括r＝550V，d＝60ms，s＝50个，i＝100ms；对上述大肠杆菌和紫杉醇脂质体的混合液体进行电致孔操作，共计20次，制备得到载紫杉醇脂质体细菌吸入制剂。
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