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CN113368259B - 一种基于修饰细菌加载纳米药物的方法及其复合物和在治疗结肠癌中的应用 - Google Patents

一种基于修饰细菌加载纳米药物的方法及其复合物和在治疗结肠癌中的应用 Download PDF

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CN113368259B
CN113368259B CN202110692013.XA CN202110692013A CN113368259B CN 113368259 B CN113368259 B CN 113368259B CN 202110692013 A CN202110692013 A CN 202110692013A CN 113368259 B CN113368259 B CN 113368259B
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吴锡龙
吴云娣
吕超逸
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Abstract

本发明公开了一种基于修饰细菌加载纳米药物的方法，利用超分子反应在表面修饰金刚烷胺的减毒鼠伤寒沙门氏菌上加载经羟丙基环糊精包覆的熊果酸纳米药物，其中1×108CFU表面修饰金刚烷胺的减毒鼠伤寒沙门氏菌加载6.5‑16.5mg熊果酸纳米药物，熊果酸纳米药物包括摩尔比为2:1的熊果酸和羟丙基环糊精。利用本方法制备的新型复合药物不仅利用细菌的天然乏氧靶向型，将药物运输至肿瘤核心缺氧区，减少药物在体内运输中的损耗，显著提高了药物的利用度；且对肿瘤进行生物和化学双重治疗，相对于化疗药物+细菌物理混合治疗具有明显的协同疗效，可显著抑制肿瘤的生长且没有出现肿瘤复发情况。

Description

一种基于修饰细菌加载纳米药物的方法及其复合物和在治疗 结肠癌中的应用
技术领域
本发明涉及生物医药领域，尤其涉及一种基于修饰细菌加载纳米药物的方法及其复合物和在治疗结肠癌中的应用。
背景技术
恶性肿瘤是当今世界上最致命的癌症之一，而化疗是临床上治疗肿瘤的主要方法，但有些化疗药物无明显靶向性导致治疗效果有限，生物相容性差导致其副作用大。如熊果酸(Ursolic Acid，UA)在癌症治疗中具有显著安全性和有效性，但疏水性强，溶解性较差，新陈代谢快和生物相容性差限制其进一步的临床应用。
肿瘤部位缺氧是局部进展期实体瘤的一个独特特征，也是导致血液营养物质与肿瘤细胞的分散距离增加及血液氧气运输能力下降的主要原因。由于肿瘤缺氧使得实体肿瘤对依赖于氧气的放疗、化疗和光动力疗法更有抵抗力。有研究发现双歧杆菌，沙门氏菌及大肠杆菌等能够特异性靶向肿瘤部位，对癌细胞具有选择性细胞毒性。因此细菌将有可能作为潜在的抗肿瘤药得以应用。如Engineering of Bacteria for the Visualization ofTargeted Delivery of a Cytolytic Anticancer Agent和Salmonella typhimuriumSuppresses Tumor Growth via the Pro-Inflammatory Cytokine Interleukin-1β等文献表明了减毒鼠伤寒沙门氏菌(S.typhimuriumΔppGpp/lux)在小鼠结肠癌模型中具有靶向性，虽然具有抑制肿瘤生长作用，但剂量依赖性毒性导致其15天后复发率高。
目前有学者提出细菌与化疗药物联合用药的方案，但游离的化疗药物面临缺少靶向性、毒副作用大、体内输送过程中药物损耗且无法渗入实体瘤内部等问题，因此针对肿瘤乏氧微环境进行药物靶向输送并达到有效治疗目的，研究和开发出靶向载体药物势在必行。
发明内容
有鉴于此，本发明提供了一种基于修饰细菌加载熊果酸纳米颗粒的方法，解决了传统抗癌药物无法进肿瘤内部导致疗效不佳的问题。
本发明一方面提供一种基于修饰细菌加载纳米药物的方法，在表面修饰金刚烷胺的减毒鼠伤寒沙门氏菌上加载经羟丙基环糊精包覆的熊果酸纳米药物。
优选地，1×108CFU表面修饰金刚烷胺的减毒鼠伤寒沙门氏菌加载5.5-16.5mg熊果酸纳米药物。
优选地，1×108CFU表面修饰金刚烷胺的减毒鼠伤寒沙门氏菌加载11mg熊果酸纳米药物。
优选地，加载条件为在37℃下以180rpm转速震荡2h。
优选地，熊果酸纳米药物包括摩尔比为2:1的熊果酸和羟丙基环糊精。
优选地，表面修饰金刚烷胺的减毒鼠伤寒沙门氏菌由以下方法制备而得：在10mL水溶液中加入1×108CFU减毒鼠伤寒沙门氏菌和1mg金刚烷胺，在37℃下以180rpm震荡2h，后离心5min并弃去上清，得到表面修饰金刚烷胺的减毒鼠伤寒沙门氏菌。
本发明另一方面提供一种基于修饰细菌加载纳米药物的复合物及在治疗结肠癌中的应用。
综上所述，采用本发明所提供的一种基于修饰细菌加载熊果酸纳米颗粒的方法，先利用环糊精对熊果酸药物进行包覆成纳米颗粒，再通过超分子反应将熊果酸纳米颗粒接至金刚烷胺修饰后的细菌表面，方法简易，易于推广。
进一步地，利用本方法制备的新型复合药物将UA药物运输至肿瘤核心乏氧区后，UA药物的释放促进了肿瘤细胞的死亡，同时细胞代谢物可为SA细菌提供营养，导致SA细菌大量繁殖，进一步提高了SA细菌对肿瘤细胞的杀灭能力。
进一步地，本发明的新型复合药物生物相容性好，副作用小，不仅利用细菌的天然乏氧靶向型，减少药物在体内运输中的损耗，显著提高了药物的利用度，减少化疗药物用量；且可对肿瘤进行生物和化学双重治疗，相对于化疗药物+细菌混合治疗具有明显的协同疗效，可显著抑制肿瘤的生长且没有出现肿瘤复发情况。
附图说明
图1为实施例一的一种基于修饰细菌加载熊果酸纳米药物的制备示意图；
图2为实施例三的S、SA@H、SA@HU0.6、SA@HU1.2和SA@HU1.8的扫描电镜图；
图3为实施例三的S、SA@H、SA@HU0.6、SA@HU1.2和SA@HU1.8的细胞活力测试结果；
图4为基于修饰细菌加载熊果酸纳米颗粒的复合药物SA@HU对小鼠结肠癌细胞的毒性检测结果；
图5为基于修饰细菌加载熊果酸纳米颗粒的复合药物SA@HU对小鼠结肠癌细胞的的细胞活力凋亡测试结果；
图6为基于修饰细菌加载熊果酸纳米颗粒的复合药物SA@HU对结肠癌小鼠体内靶向给药测试的活体成像图；
图7为基于修饰细菌加载熊果酸纳米颗粒的复合药物SA@HU对结肠癌小鼠体内抗肿瘤效果图；
图8为图7中不同组老鼠在不同药物治疗后的肿瘤体积变化图；
图9为图7中不同组老鼠在不同药物治疗后血液中TNF-α浓度变化图；
图10为图7中不同组老鼠在不同药物治疗后的肿瘤部位经H&E染色图；
图11为图7中不同组老鼠在不同药物治疗后的主要脏器经H&E染色图(核酸被染为紫蓝色，细胞质和细胞外基质中的成分被染为红色)。
具体实施方式
以下对本发明的原理和特征进行描述，所举实施例只用于解释本发明，并非用于限定本发明的范围。
以下实施例所涉及的减毒鼠伤寒沙门氏菌为现有细菌，由韩国全南国立大学和顺医院分子成像和治疗学研究院提供；CT26细胞(小鼠结肠癌细胞)由美国模式培养物集存库(ATCC)提供。另本发明实施例所涉及简称的术语解释如表1。
表1本发明实施例所涉及简称的术语解释
Figure BDA0003126504650000041
实施例一：如图1，一种基于修饰细菌加载纳米药物的方法包括以下步骤：
(1)熊果酸纳米颗粒(HU)的制备
分别配置含有2mol/mL熊果酸(UA)的1、4-二氧六环(Dio)溶液和1mol/L羟丙基环糊精(HPCD)，将5mL熊果酸(UA)溶液缓慢滴加5mL羟丙基环糊精水溶液中避光搅拌24h，避光透析及冻干获得熊果酸纳米颗粒，其中熊果酸纳米颗粒平均粒径为220nm。
在另外一个实施例中，熊果酸和羟丙基环糊精的摩尔比为1:1或1:2。
(2)表面修饰金刚烷胺的减毒鼠伤寒沙门氏菌(SA)的制备
在10mL水溶液反应体系中加入1×108CFU的减毒鼠伤寒沙门氏菌与1mg金刚烷胺(AMA-NH2)，在37℃下以180rpm震荡2h，使AMA-NH2通过静电作用结合到减毒鼠伤寒沙门氏菌表面，后离心5min并弃去上清，得到表面修饰金刚烷胺的减毒鼠伤寒沙门氏菌(SA)。
(3)基于修饰细菌加载纳米药物
在10mL水溶液反应体系加入步骤(1)中的11mg HU和步骤(2)修饰后的3×108CFUSA，在37℃下以180rpm震荡2h后进行洗涤，得到基于修饰细菌加载纳米药物的复合药物(SA@HU1.2)。经计算本复合药物中每1×108CFU修饰后减毒鼠伤寒沙门氏菌表面负载1.2mg当量UA的熊果酸纳米颗粒。
实施例二：实施例二和实施例一的区别在于，熊果酸纳米颗粒(HU)和修饰后的减毒鼠伤寒沙门氏菌(SA)的投入比例不同，载药量不同如表2。
表2不同比例的熊果酸纳米颗粒和修饰后的减毒鼠伤寒沙门氏菌对载药量的影响
项目 SA@HU<sub>0.6</sub> SA@HU<sub>1.2</sub> SA@HU<sub>1.8</sub>
HU(mg) 5.5 11 16.5
SA(CFU) 3×10<sup>8</sup> 3×10<sup>8</sup> 3×10<sup>8</sup>
载药量(mg UA/10<sup>8</sup>CFU) 0.6 1.2 1.8
对比例一：对比例一和实施例一的区别在于，SA+HU由3×108CFU表面修饰金刚烷胺的减毒鼠伤寒沙门氏菌与11mg熊果酸纳米颗粒共混而得。
对比例二：对比例二和实施例一的区别在于，SA@H不含熊果酸，SA@H由3×108CFU表面修饰金刚烷胺的减毒鼠伤寒沙门氏菌与11mg羟丙基-β-环糊精共混而得。
实施例三：实施例一的基于修饰细菌加载熊果酸纳米颗粒的复合药物SA@HU的表征及测试
(1)利用扫描电镜对未修饰的减毒鼠伤寒沙门氏菌S、SA@H、SA@HU0.6、SA@HU1.2、SA@HU1.8进行形貌测试
结果如图2表明，经金刚烷胺修饰的减毒鼠伤寒沙门氏菌表面已经成功加载熊果酸纳米颗粒，且随着熊果酸纳米颗粒投入量的增多，其细菌表面的吸附量明显增多。
(2)利用细菌菌落总数(CFU法)对实施例一的未修饰的减毒鼠伤寒沙门氏菌(S)、SA@H、SA@HU0.6、SA@HU1.2、SA@HU1.8进行细胞活力测试
结果如图3表明，相对于未修饰的减毒鼠伤寒沙门氏菌(S)，经金刚烷胺修饰的减毒鼠伤寒沙门氏菌再加载熊果酸纳米颗粒，细胞活力变化不大，其中SA@HU1.2组可加载较多纳米颗粒，且纳米颗粒对细菌的毒性在20％以下，但加载熊果酸纳米颗粒为1.8mg时，细胞的活性明显下降，即毒性增大。
实施例四：实施例一的基于修饰细菌加载熊果酸纳米颗粒的复合药物SA@HU对小鼠结肠癌细胞的毒性检测和细胞活力凋亡测试
细胞毒性检测和细胞活力凋亡结果分别如图4和5，表明实施例一的SA@HU用于细胞实验中进行细胞活力凋亡及毒性测试，说明SA@HU促进肿瘤细胞凋亡，具有良好的肿瘤细胞抑制效果。
实施例五：实施例一的基于修饰细菌加载熊果酸纳米颗粒的复合药物SA@HU对结肠癌小鼠体内靶向给药测试
选用18只6-8周小鼠雌性小鼠后，将100μL浓度为107/mL的结肠癌肿瘤细胞溶液接种于小鼠右肋部皮下构建小鼠结肠癌模型，后分为6组，每组6只，S给药组、SA+HU给药组、SA@HU给药组。分别将药物溶液注射到结肠癌小鼠体内并观察细菌的体内分布情况。如图6结果表明将SA@HU(减毒鼠伤寒沙门氏菌表达Lux荧光)尾静脉注射至小鼠体内，相对于SA+HU给药组，SA@HU组有较多的细菌和药物定植在肿瘤部位，说明实施例一的基于修饰细菌加载熊果酸纳米颗粒的复合药物SA@HU对结肠癌小鼠的肿瘤细胞具有更好的靶向作用。
实施例六：实施例一的基于修饰细菌加载熊果酸纳米颗粒的复合药物SA@HU对结肠癌小鼠体内抗肿瘤效果测试
选用36只6-8周小鼠雌性小鼠(20g左右)后，将100μL浓度为107/mL的结肠癌肿瘤细胞溶液接种于小鼠右肋部皮下构建小鼠结肠癌模型，当小鼠皮下肿瘤体积达到100mm3，将老鼠分为6组，每组6只。其中A、对照组(未治疗组)；B、HU处理组(18mg·kg-1UA)；C、S处理组(3×107CFU细菌)；D、SA@H处理组(3×107CFU细菌)；E、S+HU处理组(18mg·kg-1UA+3×107CFU细菌)；F、SA@HU处理组(18mg·kg-1UA+3×107CFU细菌)。在结肠癌小鼠的尾静脉注射相应的药物溶液并每隔两天测量肿瘤体积变化，如图7结果表明：在治疗15天后，相对于单一未改性HU组(肿瘤体积为1100mm3)、单一未改性S组(肿瘤体积为440mm3)、改性SA@H处理组(肿瘤体积为500mm3)及改性S+HU物理混合组(肿瘤体积为255mm3)，SA@HU处理组可显著抑制肿瘤的生长(肿瘤体积为48mm3)且无复发现象。
另外分别在给药第1、4、7、14、21天测试小鼠血液中的TNF-α浓度。如图8结果表明，21天SA@HU产生更多的TNF-α(浓度为54pg/mL)，TNF-α浓度的增加进一步抑制结肠癌肿瘤细胞的生长。如图9的H&E染色结果表明，SA@HU组的细胞坏死个数最多，说明SA@HU复合药物显著抑制结肠癌肿瘤细胞的生长。
在治疗7天后，取小鼠主要脏器，心、肝、脾、肺、肾，做组织切片进行H&E染色。如图10结果表明，SA@HU均未造成其他脏器的病理损失和炎性反应，也无肿瘤细胞转移迹象，说明本SA@HU复合药物安全性高。
综上所述，采用本发明所提供的一种基于修饰细菌加载熊果酸纳米颗粒的方法，先利用环糊精对熊果酸药物进行包覆成纳米颗粒，再通过超分子反应将熊果酸纳米颗粒接至金刚烷胺修饰后的细菌表面，方法简易，易于推广。
进一步地，利用本方法制备的新型复合药物将UA药物运输至肿瘤核心乏氧区后，UA药物的释放促进了肿瘤细胞的死亡，同时细胞代谢物可为SA细菌提供营养，导致SA细菌大量繁殖，进一步提高了SA细菌对肿瘤细胞的杀灭能力。一方面利用细菌的天然乏氧靶向型，减少药物在体内运输中的损耗，显著提高了药物的利用度，减少化疗药物用量；另一方面实验表明SA@HU可显著抑制肿瘤的生长且没有出现肿瘤复发情况。利用本方法制备的新型复合药物对肿瘤实现了生物和化学双重治疗，解决了传统药物或纳米药物无法进肿瘤内部的问题，同时克服了单一细菌治疗易复发的情况。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明保护的范围之内。

Claims (8)

1.一种基于修饰细菌加载纳米药物的方法，其特征在于，在表面修饰金刚烷胺的减毒鼠伤寒沙门氏菌上加载经羟丙基环糊精包覆熊果酸得到的纳米药物。
2.根据权利要求1所述的一种基于修饰细菌加载纳米药物的方法，其特征在于， 1×108CFU表面修饰金刚烷胺的减毒鼠伤寒沙门氏菌加载5.5-16.5mg纳米药物。
3.根据权利要求2所述的一种基于修饰细菌加载纳米药物的方法，其特征在于， 1×108CFU表面修饰金刚烷胺的减毒鼠伤寒沙门氏菌加载11mg纳米药物。
4.根据权利要求1所述的一种基于修饰细菌加载纳米药物的方法，其特征在于，所述加载条件为在37℃下以180rpm转速震荡2h。
5.根据权利要求1所述的一种基于修饰细菌加载纳米药物的方法，其特征在于，所述纳米药物包括摩尔比为2:1的熊果酸和羟丙基环糊精。
6.根据权利要求1所述的一种基于修饰细菌加载纳米药物的方法，其特征在于，所述表面修饰金刚烷胺的减毒鼠伤寒沙门氏菌由以下方法制备而得：在10mL水溶液中加入1×108CFU减毒鼠伤寒沙门氏菌和1mg金刚烷胺，在37℃下以180rpm震荡2h，后离心5min并弃去上清，得到表面修饰金刚烷胺的减毒鼠伤寒沙门氏菌。
7.一种基于修饰细菌加载纳米药物的复合物，根据权利要求1-6 任一项方法制备而得。
8.权利要求7的一种基于修饰细菌加载纳米药物的复合物在制备治疗结肠癌药物中的应用。
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