CN111744010B - 一种纳米-细菌的杂合体系的制备方法 - Google Patents
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Abstract
本发明公开了一种纳米‑细菌杂合体系的制备方法,特点是包括将带有氨基基团的荧光纳米材料溶液和多糖配体溶液混合振荡反应后,加入硼氢化钠溶液,于室温下振荡反应过夜得到多糖配体偶联荧光纳米材料复合物溶液的步骤;将多糖配体偶联荧光纳米材料复合物溶液和光热剂溶液混合后振荡反应得到荧光纳米探针溶液的步骤;最后将荧光纳米探针溶液和工程菌悬液混合后置于摇床中培养一段时间后,得到纯化的纳米‑细菌杂合体系的步骤;优点是兼具肿瘤靶向及光诱导程序性肿瘤治疗功能,有效结合了纳米材料和细菌治疗肿瘤的优势,且不会对细菌本身活性产生影响,肿瘤治疗效率高。
Description
技术领域
本发明涉及一种纳米-细菌的杂合体系的制备方法,尤其是涉及一种兼具肿瘤靶向及光诱导程序性肿瘤治疗功能的纳米-细菌杂合体系的制备方法。
背景技术
近年来,在肿瘤治疗领域,以细菌靶向介导肿瘤治疗的研究掀起了热潮。研究发现部分厌氧菌(如双歧杆菌和芽孢杆菌)和兼性厌氧菌(如沙门氏菌和大肠杆菌)会优先靶向进入肿瘤内部并大量繁殖。细菌治疗相比于传统肿瘤治疗方法存在一定的优势,除了细菌本身无需修饰即可靶向多种肿瘤及转移瘤外,基因工程技术的引入还可将细菌功能化使其表达抗癌分子(如细胞毒性分子、细胞因子和肿瘤抗原等)治疗肿瘤。但是,传统的细菌治疗也存在一定的劣势,如细菌本身的潜在毒性、工程菌表达产物的不可控性以及单一化的细菌治疗疗效有限等问题,有待进一步研究改进。
另一方面,纳米材料载体具有载药率高和可控释药等优点,可在病灶部位实现多模态成像,并且能结合其他肿瘤疗法实施联合治疗。但纳米材料也存在一定问题,如需额外修饰针对特定肿瘤类型的特异性靶向性分子,并且受其本身尺寸、几何形状、成分及表面配体等的影响,纳米药物在肿瘤内部的穿透力有限,致使肿瘤治疗效果欠佳。目前已有研究报道通过将纳米材料聚沉或生长到细菌表面构建完整的“纳米-细菌”杂合体系,但是将纳米材料修饰到细菌表面的做法会在一定程度上损害细菌本身的活性和功能,影响最终的治疗效果。因此,亟需解决米材料有效联合并构建稳定的纳米-细菌杂合体系用于肿瘤治疗的问题。
发明内容
本发明所要解决的技术问题是提供一种兼具肿瘤靶向及光诱导程序性肿瘤治疗功能的纳米-细菌杂合体系的制备方法,该杂合体系有效结合了纳米材料和细菌治疗肿瘤的优势,且不会对细菌本身活性产生影响,肿瘤治疗效率高。
本发明解决上述技术问题所采用的技术方案为:一种纳米-细菌杂合体系的制备方法,包括下述步骤:
(1)将浓度为25mg/mL的带有氨基基团的荧光纳米材料溶液和浓度为10mg/mL的多糖配体溶液按体积比3:2混合,并于70℃振荡反应4~6小时后,加入2倍混合液体积的浓度为10μg/mL的硼氢化钠溶液,于室温下振荡反应过夜,超滤离心除去多余的未反应上的多糖配体分子,制备得到多糖配体偶联荧光纳米材料复合物溶液;
(2)将步骤(1)制备得到的多糖配体偶联荧光纳米材料复合物溶液和浓度为200 μg/mL的光热剂溶液按体积比2:1混合后,于室温下振荡反应12~24小时,通过超滤离心除去未反应上的光热剂分子,得到荧光纳米探针溶液;
(3)将可在特定温度下表达肿瘤坏死因子TNFα的质粒(pBV220-TNFα)按质粒转化方法转化入细菌中,得到可表达抗肿瘤分子的工程菌,将工程菌活化清洗后溶于生理盐水制成浓度为1.0×107-1.0×108CFU/mL的工程菌悬液;
(4)将步骤(2)制备得到的荧光纳米探针溶液和步骤(3)得到的工程菌悬液按体积比1:5混合后,置于摇床中培养一段时间后,超滤离心去除未结合的荧光纳米探针后,采用0.9wt%的NaCl溶液对细菌离心清洗,得到纯化的纳米-细菌杂合体系。
步骤(1)中所述的荧光纳米材料包括荧光硅纳米颗粒、复合荧光二氧化硅纳米颗粒、II-IV族量子点、荧光纳米微球和荧光纳米碳点中的任一种,所述的多糖配体包括麦芽糖糊精、直链淀粉和葡萄糖聚合物中的任一种。
步骤(2)中所述的光热剂包括吲哚菁绿(indocyanine green,ICG)、IR780和花菁类染料中的任一种。
步骤(2)中所述的超滤离心条件为采用3K的超滤管,于7500rpm下离心处理15min。
步骤(3)中所述的抗肿瘤分子包括细胞毒性分子、细胞因子和肿瘤抗原中的任一种;所述的细菌包括大肠杆菌、沙门氏菌和芽孢杆菌中的任一种。
步骤(4)中所述的培养时间为1.5-2.5小时,温度为37℃,摇床转速为200rpm;所述的超滤离心条件为8000rpm下离心处理10min。
与现有技术相比,本发明的优点在于:本发明一种纳米-细菌杂合体系的制备方法,一方面,纳米材料为表面修饰有多糖配体和光热剂的纳米探针(多糖配体小分子以共价键方式连接在荧光纳米颗粒表面,光敏剂小分子通过静电作用吸附到荧光纳米材料表面),其中多糖配体会引导整个纳米探针体系通过细菌膜上的糖特异性转运通道进入细菌内部,光热剂可在近红外激光照射下进行局部升温。另一方面,细菌为可在特定温度下表达抗癌分子的基因工程菌。纳米材料可在其上修饰的多糖配体的引领下进入构建的基因工程菌内部,制备完整的纳米-细菌杂合体系。该纳米-细菌杂合体系可在活体内主动靶向到肿瘤部位,并能在外界近红外激光照射下,借助瘤内细菌内部的纳米探针的光热效应逐步升高肿瘤患处温度,激发工程菌表达抗癌分子进行肿瘤治疗。
综上所述,本发明一种纳米-细菌杂合体系的制备方法,以细菌内吞纳米材料的方式所构建的纳米-细菌杂合体系克服了将纳米材料吸附到细菌表面构建杂合体系带来的潜在的细菌本身活性下降的问题,而细菌活性下降会直接影响到最终的肿瘤治疗效果。本发明所构建的纳米-细菌杂合体系可有效抑制肿瘤生长,肿瘤治疗效果显著。
附图说明
图1为本发明纳米-细菌杂合体系的制备方法及作用机理图,其中,A为GP-ICG-SiNPs 探针的合成步骤;B为TNFα基于pBV220载体的表达机制。其中抑制物cI857蛋白为温度敏感性蛋白,其可通过抑制启动子pR-pL活性禁止蛋白表达,但在环境温度升至42℃时即失去活性从而丧失抑制启动子的功能,促使蛋白表达;C是基于纳米-基因工程菌杂合体系的体外光诱导程序性癌症治疗策略;
图2为本发明纳米-细菌杂合体系的光热升温曲线图,分别是PBS、TNFα@EC、 GP-ICG-SiNPs和TNFα@EC@GP-ICG-SiNPs接受近红外激光照射的光热曲线(808nm, 1W/cm2);
图3为本发明纳米-细菌杂合体系活体内主动靶向到肿瘤部位的分析数据,其中,a为静脉注射mCherry@EC 1、3、5、7和15天的老鼠的主要器官和肿瘤部位的荧光成像图; b为荷4T1肿瘤小鼠接受不同处理后(分别注射有PBS、EC、GP-ICG-SiNPs和EC+ GP-ICG-SiNPs)的活体荧光成像图;
图4为本发明纳米-细菌杂合体系用于体外抗癌能力考查的分析数据,其中a为接受或未接受近红外激光(808nm)照射的TNFα@EC和TNFα@EC@GP-ICG-SiNPs对4T1癌细胞的毒性测试;b为与a相应的活死细胞荧光成像图,图中比例尺为100μm,**代表p< 0.01,***代表p<0.001;
图5为实施例4中各处理组小鼠的肿瘤体积生长曲线,其中V和V0分别代表处理后和处理前的小鼠肿瘤体积,**代表p<0.01,***代表p<0.001;
图6为实施例4中各处理组小鼠在治疗期末的肿瘤图片。
具体实施方式
以下结合附图实施例对本发明作进一步详细描述。
实施例1
取300μL浓度为25mg/mL的光化学法得的绿色荧光硅纳米颗粒(SiNPs)(表面拥有氨基)溶液,向其中混入200μL的浓度为10mg/mL葡萄糖多聚体(GP)溶液,将混合溶液置于振荡仪上70℃振荡反应4-6个小时后,加入1mL 10μg/mL的硼氢化钠溶液,于室温下振荡反应过夜。反应完毕后,为了除去多余的未反应上的GP分子,以3K的超滤管7500rpm每次离心15分钟,直至离心下层溶液中几乎不含GP分子,取上层GP-SiNPs的溶液;取100μL的反应得到的GP-SiNPs的溶液,向其中加入50μL浓度为200μg/mL的吲哚菁绿(ICG)溶液,将混合溶液置于振荡仪上室温下振荡反应12-24个小时;反应完毕后,为了除去多余的未吸附上的ICG分子,以3K的超滤管8000rpm每次离心10分钟,直至离心下层溶液中几乎不含ICG分子,最终制备得到本发明的GP-ICG-SiNPs荧光纳米探针,最终制备得到本发明的GP-ICG-SiNPs荧光纳米探针。
将可在特定温度下表达肿瘤坏死因子TNFα的质粒(pBV220-TNFα)按照常规的质粒转化方法转化入大肠杆菌中构建得到TNFα@EC工程菌。取200μL浓度为10mg/mL的 GP-ICG-SiNPs溶液加入活化清洗后的1mL的浓度为1.0×107-1.0×108CFU/mL的 TNFα@EC细菌悬液中,置于摇床(200rpm,37℃)中培养2h后离心(8000rpm,10min) 除去未结合的GP-ICG-SiNPs后,并以0.9wt%的NaCl溶液将细菌离心清洗2到3次,最终得到纯化的TNFα@EC@GP-ICG-SiNPs(纳米-细菌)杂合体系。
本发明纳米-细菌杂合体系的制备方法及作用机理图如图1所示,将细菌靶向性GP分子和光热剂吲哚菁绿(ICG)分子修饰到SiNPs表面,构建了GP-ICG-SiNPs纳米探针 (图1(A))。另一方面,分别构建了红色荧光大肠杆菌(mCherry@EC)用于细菌活体内示踪以及可在特定温度下(42℃)表达肿瘤坏死因子(tumor necrosis factor,TNFα)的大肠杆菌工程菌(TNFα@EC)用于肿瘤治疗。商用质粒载体pBV220为λpL/pR-cI857温度敏感型原核细胞表达系统,在细菌体内可在42℃环境下诱导细菌表达其上的蛋白(图 1(B))。具体原理如下:cI857是一种温度敏感性蛋白,在外界温度小于42℃时,它可通过抑制启动子(pR-pL)的活性从而抑制蛋白表达;当外界温度升至42℃时,cI857 便会失活从启动子上解离下来,促使启动子恢复启始蛋白表达的功能。在该策略中,首先将TNFα@EC静脉注射于体内,使其靶向到肿瘤部位后,再静脉注射构建好的 GP-ICG-SiNPs纳米探针,此时GP-ICG-SiNPs会在细菌靶向性分子GP的引领下通过细菌细胞膜表面的糖特异性ABC转运蛋白进入瘤内细菌内部,并于瘤内构成 TNFα@EC@GP-ICG-SiNPs杂合体系。在治疗第一阶段,通过体外808-nm激光照射,利用瘤内细菌内部的GP-ICG-SiNPs的光热效应使得肿瘤患处温度升至42℃,此时 TNFα@EC可表达TNFα因子杀死肿瘤细胞。在治疗第二阶段,需再次通过激光照射提升肿瘤患处温度至55℃引入光热疗法,进一步杀死残余肿瘤细胞及瘤内全部细菌,保证细菌不长时间滞留于体内产生安全隐患(图1(C))。
将TNFα@EC@GP-ICG-SiNPs置于808-nm激光器(1W/cm2)下照射5分钟,联合热成像仪测定光热曲线。结果如图2所示,从中可以看出TNFα@EC@GP-ICG-SiNPs和单独的GP-ICG-SiNPs一样具备很好的光热效应,可在5min时升温接近至55℃(NIR:808nm, 1W/cm2),而PBS及单独的TNFα@EC无光热效应。
除上述实施例外,荧光纳米材料还可以为表面拥有氨基的复合荧光二氧化硅纳米颗粒、II-IV族量子点、荧光纳米微球或者荧光纳米碳点;多糖配体还可以为麦芽糖糊精或者直链淀粉。光热剂还可以为IR780和花菁类染料。抗肿瘤分子可以为细胞毒性分子、细胞因子或者肿瘤抗原中的任一种;细菌还可以为沙门氏菌或者芽孢杆菌。
实施例2
1、将载有红色荧光蛋白(mCherry)基因的质粒(pRSETB-mCherry)按照常规的质粒转化方法转化入大肠杆菌中构建得到具备红色荧光的mCherry@EC工程菌。将 mCherry@EC菌液(1.0×108CFU/mL,200μL)尾静脉注射于小鼠体内,分别在第1、3、 5、7和15天时,将小鼠处死取出其心脏、肝脏、脾脏、肺脏、肾脏以及肿瘤置于小动物成像仪里进行mCherry荧光成像,研究细菌在器官及肿瘤内部的分布情况。
由图3a)像图可知,mCherry@EC的荧光信号在对照组小鼠体内始终主要聚集在肝脏部位,随着天数的增加,其肝脏部位的荧光信号也在逐渐消失,在第15天时已基本检测不到荧光信号。结果表明mCherry@EC在健康小鼠体内主要滞留在肝脏部位,后随着天数的增加逐渐被代谢,且在两周时基本被排出体外。而在荷瘤小鼠组中,在第1天时可在肝脏和肿瘤部位皆检测到荧光信号,后随着天数的增加,其肝脏部位的荧光信号迅速减少,肿瘤部位荧光信号逐渐聚集增加。在第7天时,可在肿瘤部位检测到大量荧光信号,接下来一周内,肿瘤内荧光信号会逐渐减小,至15天时,肿瘤部位只残存微弱的信号。表明mCherry@EC在荷瘤小鼠体内起初主要会滞留于肝脏和肿瘤部位,之后肝脏部位的细菌会被代谢排出体外,而肿瘤部位的细菌荧光信号则会因细菌大量增殖而不断增加。
2、在研究了细菌注射入活体内的生物分布行为及验证了其肿瘤靶向功能后,进一步考察构建的GP-ICG-SiNPs是否具备靶向瘤内细菌的功能。GP分子会引领整个 GP-ICG-SiNPs纳米探针通过细菌细胞膜上表达的糖特异性ABC转运通道进入细菌内部,因此GP-ICG-SiNPs可通过靶向进入瘤内细菌内部滞留在肿瘤部位。
将200μL浓度为10mg/mL的GP-ICG-SiNPs溶液尾静脉注射于小鼠体内,注射24h 后小鼠置于小动物成像仪中对小鼠肿瘤部位进行荧光成像(λex=720nm,λem=780 nm),即检测GP-ICG-SiNPs在肿瘤处富集的荧光信号。活体成像结果如图3b)所示,在单独的PBS组、EC注射组及仅注射GP-ICG-SiNPs的组中皆没有检测到GP-ICG-SiNPs的荧光,而只在瘤内存有细菌的小鼠肿瘤内部检测到了GP-ICG-SiNPs的信号。此结果证明 GP-ICG-SiNPs本身不会靶向到肿瘤部位,瘤内细菌的存在是GP-ICG-SiNPs可定位到肿瘤内部的关键,GP-ICG-SiNPs可通过靶向进入瘤内细菌内部从而随瘤内细菌一起滞留在肿瘤患处。
实施例3
TNFα@EC@GP-ICG-SiNPs体外抗癌实验主要利用transwell(带孔径为400nm的聚碳酸酯膜)及808-nm激光器完成。首先将鼠源乳腺癌4T1细胞接种于带transwell的24孔板中培养24h。以300μL的含10wt%胎牛血清的RPMI-1640培养基重悬 TNFα@EC@GP-ICG-SiNPs菌体,将菌液直接加入铺有4T1细胞的24孔板的transwell上层小室中。将此24孔板置于808-nm激光器下,通过调节激光器功率使其温度稳定在42℃,照射30min,后置于培养箱中稳定培养24h。收集transwell下层4T1细胞,利用MTT法测定细胞存活率。将细胞以碘化丙啶(染死细胞,λex=543nm,λem=560-620nm)和钙黄绿素(染活细胞,λex=488nm,λem=500-545nm)染液重悬,室温下染色5min后即可于荧光共聚焦显微镜下进行活死细胞成像分析。继续将新的 NFα@EC@GP-ICG-SiNPs菌液加入铺有4T1细胞的24孔板的transwell上层小室中,将其置于808-nm激光器下,通过调节激光器功率使其温度稳定在55℃左右,照射10min后进行和上述内容一致的活死细胞分析,结果如图4所示,接受或未接受近红外激光(808nm) 照射的TNFα@EC和TNFα@EC@GP-ICG-SiNPs对4T1癌细胞的毒性测试,图 4 a),以及相应的活死细胞荧光成像图,图 4 b)。
由图4a)可知,在TNFα@EC@GP-ICG-SiNPs处理组中,当温度上升至42℃时,4T1 癌细胞的存活率降至60%,而其余对照组细胞的存活率皆为95%及以上。此结果说明在 42℃环境的诱导作用下,TNFα@EC@GP-ICG-SiNPs表达出了TNFα蛋白并成功杀死了部分4T1癌细胞。进一步调节激光器功率使得TNFα@EC@GP-ICG-SiNPs菌液升温至55℃,持续照射10min,显示4T1癌细胞的存活率骤降至10%,表明残余4T1细胞也皆已死亡。上述结果证明构建的TNFα@EC@GP-ICG-SiNPs具备良好的体外光诱导程序性升温抗癌功能。
图4b)为图4a)的相应组的活死细胞染色结果,由图4b)荧光成像结果可知,在温度达到42℃后,已有大部分癌细胞发生死亡(死细胞会发出红色荧光),而在其基础之上继续升温至55℃时,癌细胞的死亡率更高,表明残存的癌细胞也已基本死亡。
实施例4
尾静脉注射TNFα@EC菌液(1.0×108CFU/mL,200μL)于相应组别小鼠体内,在注射细菌后的第3天,尾静脉注射200μL浓度为10mg/mL的GP-ICG-SiNPs溶液,在注射完GP-ICG-SiNPs 24h后,将小鼠肿瘤部位置于808-nm激光器下联合热成像仪通过调节激光器功率使肿瘤处的温度稳定在42℃,照射30min。在注射细菌后的第9天,再次尾静脉注射GP-ICG-SiNPs(2mg/kg的ICG)。并于给药后的24h时,将小鼠肿瘤部位置于 808-nm激光器下联合热成像仪通过调节激光器功率使肿瘤处的温度上升至55℃左右,照射5min。全程治疗过程中,记录小鼠体重及肿瘤体积。其中肿瘤体积计算方法为:分别量取肿瘤的最大直径a和最小直径b,根据标准计算公式计算肿瘤体积V:V=ab2/2。相对肿瘤体积为V/V0,V0是肿瘤初始体积,结果如图5和图6所示.
由图5提供的肿瘤体积生长曲线(V/V0)可知,注射了PBS、EC及EC+GP-ICG-SiNPs但并未接受光照的小鼠的肿瘤体积在持续上升,表明肿瘤在持续恶化。在治疗的第一阶段,小鼠注射完GP-ICG-SiNPs并首次光照(42℃)后(注射细菌后的第4天),光照组小鼠的肿瘤体积相比于其他组别小鼠肿瘤体积有明显抑制现象(显著性分析结果为 p<0.01),说明小鼠肿瘤内部的TNFα@EC@GP-ICG-SiNPs在42℃环境诱导下表达出的 TNFα因子起了一定的抗肿瘤效果。在治疗的第二阶段,再次给小鼠注射完GP-ICG-SiNPs 并二次光照(55℃)后(注射细菌后的第10天),光照组小鼠的肿瘤体积进一步削减约至零,肿瘤治疗效果显著(显著性分析结果为p<0.001),同时55℃的热环境会促使瘤内细菌发生死亡,使其可在治疗期结束时被尽快排出体外。
图6是各组小鼠在接受治疗处理后的解剖出的肿瘤图片,从中也可明显看出光照组小鼠的肿瘤体积要明显小于其余组小鼠肿瘤体积。
上述说明并非对本发明的限制,本发明也并不限于上述举例。本技术领域的普通技术人员在本发明的实质范围内,做出的变化、改型、添加或替换,也应属于本发明的保护范围。
Claims (6)
1.一种纳米-细菌杂合体系的制备方法,其特征在于包括下述步骤:
(1)将浓度为25mg/mL的带有氨基基团的荧光纳米材料溶液和浓度为10mg/mL的多糖配体溶液按体积比3:2混合,并于70℃振荡反应4~6小时后,加入2倍混合液体积的浓度为10μg/mL的硼氢化钠溶液,于室温下振荡反应过夜,超滤离心除去多余的未反应上的多糖配体分子,制备得到多糖配体偶联荧光纳米材料复合物溶液;
(2)将步骤(1)制备得到的多糖配体偶联荧光纳米材料复合物溶液和浓度为200μg/mL的光热剂溶液按体积比2:1混合后,于室温下振荡反应12~24小时,通过超滤离心除去未反应上的光热剂分子,得到荧光纳米探针溶液;
(3)将可在特定温度下表达肿瘤坏死因子TNFα的质粒pBV220-TNFα按质粒转化方法转化入细菌中,得到可表达抗肿瘤分子的工程菌,将工程菌活化清洗后溶于生理盐水中制成浓度为1.0×107-1.0×108CFU/mL的工程菌悬液;
(4)将步骤(2)制备得到的荧光纳米探针溶液和步骤(3)得到的工程菌悬液按体积比1:5混合后,置于摇床中培养一段时间后,超滤离心去除未结合的荧光纳米探针后,采用0.9wt%的NaCl溶液对细菌离心清洗,得到纯化的纳米-细菌杂合体系。
2.根据权利要求1所述的一种纳米-细菌杂合体系的制备方法,其特征在于:步骤(1)中所述的荧光纳米材料包括荧光硅纳米颗粒、复合荧光二氧化硅纳米颗粒、II-IV族量子点、荧光纳米微球和荧光纳米碳点中的任一种,所述的多糖配体包括麦芽糖糊精、直链淀粉和葡萄糖聚合物中的任一种。
3.根据权利要求1所述的一种纳米-细菌杂合体系的制备方法,其特征在于:步骤(2)中所述的光热剂包括吲哚菁绿(indocyanine green,ICG)、IR780和花菁类染料中的任一种。
4.根据权利要求1所述的一种纳米-细菌杂合体系的制备方法,其特征在于:步骤(2)中所述的超滤离心条件为采用3K的超滤管,于7500rpm下离心处理15min。
5.根据权利要求1所述的一种纳米-细菌杂合体系的制备方法,其特征在于:步骤(3)中所述的抗肿瘤分子包括细胞毒性分子、细胞因子和肿瘤抗原中的任一种;所述的细菌包括大肠杆菌、沙门氏菌和芽孢杆菌中的任一种。
6.根据权利要求1所述的一种纳米-细菌杂合体系的制备方法,其特征在于:步骤(4)中所述的培养时间为1.5-2.5小时,温度为37℃,摇床转速为200rpm;所述的超滤离心条件为8000rpm下离心处理10min。
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