CN111840567B - 一种免疫/光动力抗肿瘤功能干细胞及其制备方法 - Google Patents
一种免疫/光动力抗肿瘤功能干细胞及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种免疫/光动力抗肿瘤功能干细胞及其制备方法,采用本发明中的方法制得的功能干细胞具有肿瘤趋向性、免疫豁免性优势,可搭载多种治疗剂,并实现靶向递送,具有免疫/光动力抗肿瘤功能,可用于肿瘤治疗中的药物靶向递送和联合治疗。
Description
技术领域
本发明属于生物医药技术领域,涉及一种功能干细胞,具体涉及一种免疫/光动力抗肿瘤功能干细胞及其制备方法。
背景技术
在肿瘤临床治疗中,治疗方式仍然以手术、放疗、化疗为主,由于传统治疗方法存在一定的不良后果和局限性,因此,迫切需要研究开发新的肿瘤治疗方法和策略,随着医学和科技的发展,一些新的治疗方式和策略被尝试和应用。
肿瘤免疫治疗通过激活和增强机体自身的免疫活性发挥抗肿瘤效果,可高效抑制转移和消除肿瘤。此外,免疫治疗可使机体获取免疫记忆作用,有效防止肿瘤复发。肿瘤的细胞因子治疗是一种非特异性免疫治疗方式,其中白介素12(IL12)是一种多效细胞因子,由于其能活化先天性免疫和适应性免疫,成为了肿瘤免疫治疗的理想候选者。然而,高剂量的IL12直接使用存在严重的毒副作用,低剂量使用则疗效低于预期,这极大的限制了其临床应用。基因治疗方式的引入,可以显著提高白介素12的治疗功效,同时降低毒副作用,以克服临床应用困难。肿瘤基因治疗(Gene Therapy)是将具有矫正作用的基因或者具有治疗作用的基因导入细胞,从而实现肿瘤治疗的目的,RNA干扰沉默和治疗基因的表达是基因治疗两种主要方式。治疗基因的表达通过上调目标蛋白表达量而增强肿瘤治疗效果,白细胞介素(IL12)基因表达产物可以有效地在体内外抑制肿瘤生长或杀伤肿瘤细胞。
此外,与单一治疗相比,联合治疗能整合优势获得更好治疗效果。光动力治疗(PDT)在肿瘤治疗领域表现出了巨大的应用潜力,具有微创、高效、可控优势,可与多种治疗方式结合进行联合治疗。光动力治疗通过产生活性氧(Reactive oxygen species,ROS)选择性的损伤肿瘤组织而实现治疗目标。另外,光动力治疗处理会导致肿瘤相关抗原释放,从而引发相应的免疫应答反应,进一步增强免疫抗肿瘤效果,因此,免疫与光动力联合治疗将实现更高效的肿瘤治疗。
然而,不管是基因治疗还是光动力治疗,治疗剂的有效递送都是至关重要的,一方面要实现靶向递送,另一方面要有效保护治疗剂。这便需要递送载体的参与。细胞载体(cell carrier)一般是指存在于机体本身或在某些因素诱导下产生,可以将抗癌药物靶向运输到肿瘤细胞的一类细胞。该类细胞具有其他药物载体无法比拟的生物相容性和生物可降解性,它不但增加药物的稳定性,延长药物的半衰期,而且增强其生物靶向性;此外被包埋药物的缓慢、持续低剂量释放可以减轻药物的毒副作用。目前研究较多的是红细胞和细胞因子诱导杀伤细胞(CIK)。首先是红细胞作为药物载体的研究主要集中在抗肿瘤药物方面,放线菌素D是研究最早的红细胞包载的抗肿瘤药物之一,研究发现对其表面进行修饰后其具有一定的靶向性。其次是细胞因子诱导杀伤细胞,通常该细胞用于肿瘤的免疫治疗,最近有研究将其用于载药。这些工作为研究细胞作为抗肿瘤药物载体奠定了基础,但这些细胞载体存在靶向性低,规模化、标准化制备困难等问题。因此,需要更加高效的靶向治疗体系。而骨髓间充质干细胞(BMSC)作为一种新兴的生物载体,其表面表达的趋化因子受体CXCR-4能与肿瘤细胞高表达的基质衍生因子SDF-1特异性识别,从而趋向于肿瘤组织迁移,可实现治疗剂的生物靶向递送,将治疗剂高效递送至肿瘤部位。
因此,我们选取了骨髓间充质干细胞(BMSC)作为细胞载体应用于肿瘤治疗。BMSC具有易于获取和分离优势,在体外易于扩增等特性,且表现出低免疫原性,应用于机体无免疫排斥反应,以及其自身具有良好的肿瘤趋向性,综上所述,以BMSC作为载体的药物递送是一种更有希望的肿瘤治疗策略。
发明内容
针对上述现有技术,本发明创制了一种安全高效、递送治疗剂的功能化干细胞平台,能降低毒副作用,实现靶向递送,获得高效的肿瘤免疫/光动力联合治疗效果。该功能干细胞平台具有如下优势:(1)利用骨髓间充质干细胞(BMSC)本身具有的低免疫原性和肿瘤趋向性,实现了治疗剂的安全靶向输送;(2)将基因治疗和细胞因子的免疫治疗结合起来,将IL-12基因运载到干细胞内进行有效表达;(3)将光动力治疗和免疫治疗结合,光动力治疗后产生的肿瘤相关抗原释放,进一步促进肿瘤的免疫治疗效果;(4)可通过旁观者效应将治疗剂转运至邻近肿瘤细胞中,即功能化干细胞通过细胞间隙连接或产生微泡将治疗剂转运至其邻近的肿瘤细胞中,增强对邻近肿瘤细胞的杀伤力,进一步增强上述光动力和免疫治疗效果;(5)该功能纳米平台可以搭载多种治疗剂,如各种用于治疗肿瘤的基因、光敏剂、光热剂、化疗前药等,且载药量大,毒副作用低,在药物递送领域有着广泛的用途;(6)同一功能干细胞平台上实现基因治疗/光动力治疗/免疫治疗等多种方式的联合治疗,有利于增强肿瘤治疗效果。
为了达到上述目的,本发明所采用的技术方案是:提供一种免疫/光动力抗肿瘤功能干细胞的制备方法,包括以下步骤:
S1:制备M-MSN纳米粒子,并配制成浓度为3~6mg/mL的M-MSN纳米粒子溶液;
S2:将浓度为18~22mg/mL的PEI溶液与M-MSN纳米粒子溶液混合,混合溶液中PEI与M-MSN纳米粒子的质量之比为1~5:5;然后于室温下反应1.5~3h,得M-MSN/PEI纳米粒子;
S3:将M-MSN/PEI纳米粒子加入到MB溶液中,所加入的M-MSN/PEI纳米粒子与MB的质量之比为1:0.33~3;然后于室温下避光搅拌反应20~25h;得M-MSN(MB)/PEI纳米粒子;
S4:将M-MSN(MB)/PEI纳米粒子加入到与pIL12质粒溶液中,所加入的M-MSN(MB)/PEI纳米粒子与pIL12的质量之比为10~50:1;然后在室温下孵育1~2h,得M-MSN(MB)/PEI/pIL12纳米复合物;
S5:将M-MSN(MB)/PEI/pIL12纳米复合物加入到DMEM低糖培养基中,使其浓度为80~100μg/mL,得混合培养基;
S6:培养BMSC,当BMSC细胞培养至细胞铺满培养容器的70%后,去除旧培养基,加入S5所得混合培养基,将培养容器转移至恒温培养箱,并在培养容器下方放置磁体,于36~40℃下孵育5~8h,得免疫/光动力抗肿瘤功能干细胞MB/IL12-BMSC。
在上述技术方案的基础上,本发明还可以做如下改进。
进一步,M-MSN纳米粒子的制备包括以下步骤:
(1)配制浓度为0.1~0.4g/mL的十六烷基三甲基溴化铵溶液,然后将四氧化三铁纳米粒子添加进该溶液,使该溶液中四氧化三铁纳米粒子的浓度为3.75~37.5mg/mL;搅拌均匀后加入浓度为28%的氨水,所加氨水与十六烷基三甲基溴化铵溶液的体积比为1~5:100,然后于25~70℃下反应2h;
(2)将正硅酸乙酯和乙酸乙酯加入反应后的混合物中,所加正硅酸乙酯和乙酸乙酯与所述十六烷基三甲基溴化铵溶液的体积比分别为0.25~1:100和5:100,于40℃下反应6h,然后离心、洗涤;
(3)将洗涤后的产物加入浓度为0.01g/mL的硝酸铵的乙醇溶液中,混匀后于80℃下冷凝回流反应1h;然后离心、洗涤,得M-MSN纳米粒子。
进一步,S2中PEI溶液与M-MSN纳米粒子溶液混合后,混合溶液中PEI与M-MSN纳米粒子的质量之比为1:5;反应时间为2h。
进一步,S4中加入pIL12质粒溶液中的M-MSN(MB)/PEI纳米粒子与pIL12的质量之比为20~30:1。
进一步,S6中孵育温度为37℃,孵育时间为6~8h。
采用本发明中的方法可以制得一种能有效抑制肿瘤的生长、具备良好的抗肿瘤效果的免疫/光动力抗肿瘤功能干细胞。
本发明的有益效果是:
1、所述免疫/光动力抗肿瘤功能干细胞可搭载多种抗肿瘤制剂,且载药量大,毒副作用低。
2、所述免疫/光动力抗肿瘤功能干细胞具有免疫豁免性、不被轻易从机体循环中清除,可趋向于肿瘤组织迁移,实现治疗剂的生物靶向递送。
3、所述免疫/光动力抗肿瘤功能干细胞兼具免疫/光动力效应,可用于实施肿瘤免疫/光动力联合治疗,对于肿瘤细胞的杀伤力显著。
附图说明
图1为M-MSN/PEI纳米粒子的扫描电镜图;
图2为M-MSN/PEI纳米粒子的透射电镜图;
图3为功能干细胞MB/IL12-BMSC中纳米复合物内吞荧光图;
图4为功能干细胞MB/IL12-BMSC中纳米复合物的荧光定量图;
图5为功能干细胞MB/IL12-BMSC的免疫效应图;
图6为功能干细胞MB/IL12-BMSC的光动力效应图;
图7为功能干细胞MB/IL12-BMSC的DCFH-DA荧光定量图;
图8为功能干细胞MB/IL12-BMSC应用于小鼠EMT-6乳腺癌疾病模型中抗肿瘤效果图。
具体实施方式
下面结合实施例对本发明的具体实施方式做详细的说明。
实施例1:制备功能干细胞
一种免疫/光动力抗肿瘤功能干细胞,其经过以下步骤制得:
(1)用量筒取10mL去离子水倒入洁净的圆底烧瓶中,精确称取0.2g十六烷基三甲基溴化铵(CTAB)加入圆底烧瓶中,加入磁子于磁力搅拌器上搅拌至溶解,加入7.5mg四氧化三铁纳米粒子(Fe3O4)至圆底烧瓶内,超声使其分散均匀。随后,往圆底烧瓶中补充90mL去离子水,并迅速加入28%的氨水3mL,然后盖上玻璃瓶塞,并用封口膜密封,将圆底烧瓶置于40℃水浴锅中搅拌2h。
(2)用移液枪吸取0.5mL正硅酸乙酯(TEOS)和5mL乙酸乙酯(EA)加入上述圆底烧瓶中,盖上玻璃瓶塞,再次用封口膜密封,继续在40℃水浴锅中搅拌反应6h。反应结束后,将圆底烧瓶内液体转移至离心管进行离心分离,设置离心参数为10000rpm,10min,弃上清,保留管壁上产物,以去离子水洗离三次,再以无水乙醇洗离三次。
(3)精确称取0.5g硝酸铵(NH4NO3)置于离心管中,加入50mL无水乙醇,涡旋振荡使其充分溶解,与(2)中所得产物混合,转移至洁净圆底烧瓶中,超声处理使其均匀分散,置于80℃水浴锅中冷凝回流反应1h。
(4)反应结束后,将圆底烧瓶内混合物转移至离心管进行离心分离,设置离心参数为10000rpm,10min,弃上清,保留管壁上产物,以无水乙醇洗离三次,再以去离子水洗离三次,获得磁性介孔二氧化硅纳米粒子(M-MSN纳米粒子),产物分散于去离子水中置于4℃的冰箱中密封保存,或者用真空冷冻干燥机冻干保存。
(5)将M-MSN纳米粒子配制成5mg/mL的M-MSN纳米粒子溶液,然后将20mg/mL的聚乙烯亚胺(PEI)溶液与配制的M-MSN纳米粒子溶液加入洁净玻璃瓶中,混合溶液中PEI与M-MSN纳米粒子的质量之比为1:5;将装有混合溶液的玻璃瓶置于室温下搅拌反应2h,让PEI通过静电吸附作用接枝到M-MSN纳米粒子上。反应结束后,将玻璃瓶中混合物收集到离心管中,高速离心,设置离心参数为10000rpm,10min,弃上清,保留管壁上产物,以去离子水洗离三次,获得M-MSN/PEI纳米粒子。采用扫描电镜和透射电镜对制得的M-MSN/PEI纳米粒子的形态进行分析,结果如图1和图2所示。图1扫描电镜和图2透射电镜图显示,所合成纳米载体M-MSN/PEI是粒径约为150nm的均匀球形粒子,且分散性良好。
(6)将M-MSN/PEI纳米粒子加入到亚甲基蓝(MB)溶液中,所加入的M-MSN/PEI纳米粒子与MB的质量之比为1:2;室温条件下避光搅拌反应24h。反应结束后,将混合液收集至离心管,高速离心,弃上清,加入去离子水洗离三次,去除未加载到纳米粒子上的MB,所得产物即为M-MSN(MB)/PEI纳米粒子。
(7)将M-MSN(MB)/PEI纳米粒子加入到与pIL12质粒溶液中,所加入的M-MSN(MB)/PEI纳米粒子与pIL12的质量之比为30:1;在室温下孵育1h,将基因吸附至粒子上,获得纳米复合物M-MSN(MB)/PEI/pIL12(MBPIL12);
(8)用异硫氰酸荧光素(FITC)标记MBPIL12,用活细胞染色剂CM-Dil标记人骨髓间充质干细胞(BMSC),将标记后的MBPIL12加入到DMEM低糖完全培养基中,使其浓度为100μg/mL,得混合培养基。
(9)培养BMSC,当BMSC细胞培养至细胞铺满培养瓶的70%后,去除旧培养基,用PBS洗涤3次,将步骤(8)中制得的混合培养基加入培养瓶中,将培养瓶转移至恒温培养箱,并在培养瓶下方放置磁体,孵育6h,通过磁靶向作用促进MBPIL12内化至BMSC中,从而制备获得功能化干细胞MB/IL12-BMSC。通过共聚焦显微镜观察纳米复合物MBPIL12在BMSC中的内化情况,验证MBPIL12的加载,以说明功能化干细胞MB/IL12-BMSC的成功制备。
对制得的功能干细胞进行荧光检测,结果如图3和图4所示,其中图3为荧光图,图4为荧光定量图。从图3中可以看出,与对照组(Control,未加入MBPIL12)相比,在实验组(MBPIL12)中出现了较强的绿色荧光信号(标记MBPIL12的FITC荧光信号);在磁靶向组(MBPIL12+M)中,绿色荧光信号进一步增强。通过Image J软件对内吞图中FITC荧光信号强度进行定量分析,结果如图4所示,对照组未检测到荧光信号,实验组出现一定信号强度的荧光信号,靶向组的荧光信号强度高于实验组。表明纳米复合物MBPIL12能有效被BMSC摄入细胞内,并且可通过磁靶向作用促进MBPIL12内化至BMSC中。
实施例2:功能化干细胞MB/IL12-BMSC性能考察
(1)通过研究MB/IL12-BMSC的IL12表达情况,反映其免疫效应。
消化BMSC并重悬,调节细胞浓度为1×104个/mL,接种于6孔板中培养24h,待其贴壁后,去除培养基,加入PBS洗涤3次。加入混合有MBPIL12的DMEM低糖完全培养基,在培养皿下方放置磁体,转移至恒温培养箱孵育6h后,除去培养液,加入PBS洗涤3次,加入新鲜培养基,转移至恒温培养箱中培养。在24h、48h和72h分别收集各组培养基上清,用于酶联免疫吸附方法(ELISA)检测IL12的表达情况。
图5为酶联免疫吸附检测结果。从图中可以看出,仅携载质粒pIL12的单载质粒组和共载治疗剂组纳米粒子处理的BMSC的IL12表达量上调,其培养液上清液中IL12浓度明显上升,并且IL12浓度随BMSC培养时间延长而上升。表明制备的功能干细胞MB/IL12-BMSC表达IL12的水平较高,可通过IL12相关调控通路发挥免疫抗肿瘤潜能。
(2)通过研究MB/IL12-BMSC的活性氧产生情况,反映其光动力效应。
实验组为MB/IL12-BMSC,对照组为BMSC进行同样的实验处理。消化BMSC并重悬,调节细胞浓度为1×104个/mL,接种于培养皿中培养24h,待其贴壁后,去除培养基,加入PBS洗涤3次。加入混合有MBPIL12的DMEM低糖完全培养基,并在培养皿下方放置磁体,转移至恒温培养箱孵育6h后,除去培养液,加入PBS洗涤3次,加入新鲜培养基。用660nm激光器,设置功率密度为500mW/cm2,照光处理5min后,转移至恒温培养箱中,继续培养24h。清除旧培养基,并加入DCFH-DA工作液,置于恒温培养箱中孵育20min,加PBS洗涤3次,去除多余的DCFH-DA,再加入无血清培养基,用荧光倒置显微镜下观察细胞内DCFH-DA(λEx=488nm)荧光。
图6光动力效应图显示BMSC经光照处理后,仅呈现微弱的绿色荧光信号,而在MB/IL12-BMSC组出现了显著高于BMSC组的荧光信号,图7为Image J软件分析的DCFH-DA荧光定量图,结果意味着MB/IL12-BMSC组可产生更多的ROS,由此可以证明制备的功能干细胞MB/IL12-BMSC可以通过光动力效应发挥了更好的抗肿瘤作用。
实施例3:功能化干细胞MB/IL12-BMSC治疗效果考察
(1)构建皮下肿瘤模型:扩增EMT-6细胞,将细胞消化并进行计数,用生理盐水洗涤3次后重悬,调节细胞浓度5×106个/mL,实验组小鼠右侧大腿皮下注射100μL细胞悬液,待肿瘤体积约为60mm3进行后续实验。
(2)给药治疗:随机将小鼠分组为以下6组,给药方案如下表所示,每3天进行一次尾静脉注射,注射量为100μL(每只小鼠治疗剂用量为:MB=20μg,pIL12=3.4μg;对照组注射生理盐水,各治疗组注射对应的细胞悬液),共进行3次注射。并在每次给药后的第3天,使用660nm激光器进行照光处理,设置参数为1.25W/cm2、5min,共进行3次照光治疗处理,研究功能干细胞的抗肿瘤效果,以剖出肿瘤大小评价效果。表1给出了不同实验组的给药情况。
表1不同实验组的注药情况
图8为在治疗结束后刨出肿瘤进行拍摄比较荷瘤小鼠离体肿瘤大小。从图中可以看出,功能干细胞MB/IL12-BMSC联合治疗组的肿瘤显著小于其他治疗组,表明该功能化干细胞平台组能有效抑制肿瘤的生长,具备良好的抗肿瘤效果。
虽然结合实施例对本发明的具体实施方式进行了详细地描述,但不应理解为对本专利的保护范围的限定。在权利要求书所描述的范围内,本领域技术人员不经创造性劳动即可作出的各种修改和变形仍属本专利的保护范围。
Claims (6)
1.一种免疫/光动力抗肿瘤功能干细胞的制备方法,其特征在于,包括以下步骤:
S1:制备M-MSN纳米粒子,并配制成浓度为3~6mg/mL的M-MSN纳米粒子溶液;
S2:将浓度为18~22mg/mL的PEI溶液与M-MSN纳米粒子溶液混合,混合溶液中PEI与M-MSN纳米粒子的质量之比为1~5:5;然后于室温下反应1.5~3h,得M-MSN/PEI纳米粒子;
S3:将M-MSN/PEI纳米粒子加入到亚甲基蓝溶液中,所加入的M-MSN/PEI纳米粒子与MB的质量之比为1:0.33~3;然后于室温下避光搅拌反应20~25h;得M-MSN(MB)/PEI纳米粒子;
S4:将M-MSN(MB)/PEI纳米粒子加入到pIL12质粒溶液中,所加入的M-MSN(MB)/PEI纳米粒子与pIL12的质量之比为10~50:1;然后在室温下孵育1~2h,得M-MSN(MB)/PEI/pIL12纳米复合物;
S5:将M-MSN(MB)/PEI/pIL12纳米复合物加入到DMEM低糖培养基中,使其浓度为80~100μg/mL,得混合培养基;
S6:培养BMSC,当BMSC细胞培养至细胞铺满培养容器的70%后,去除旧培养基,加入S5所得混合培养基,将培养容器转移至恒温培养箱,并在培养容器下方放置磁体,于37℃下孵育5~8h,得负载有M-MSN(MB)/PEI/pIL12纳米复合物的BMSC。
2.根据权利要求1所述的制备方法,其特征在于,M-MSN纳米粒子的制备包括以下步骤:
(1)配制浓度为0.1~0.4g/mL的十六烷基三甲基溴化铵溶液,然后将四氧化三铁纳米粒子添加进该溶液,使该溶液中四氧化三铁纳米粒子的浓度为3.75~37.5mg/mL;搅拌均匀后加入浓度为28%的氨水,所加氨水与十六烷基三甲基溴化铵溶液的体积比为1~5:100,然后于25~70℃下反应2h;
(2)将正硅酸乙酯和乙酸乙酯加入反应后的混合物中,所加正硅酸乙酯和乙酸乙酯与所述十六烷基三甲基溴化铵溶液的体积比分别为0.25~1:100和5:100;于40℃下反应6h,然后离心、洗涤;
(3)将洗涤后的产物加入浓度为0.01g/mL的硝酸铵的乙醇溶液中,混匀后于80℃下冷凝回流反应1h;然后离心、洗涤,得M-MSN纳米粒子。
3.根据权利要求1所述的制备方法,其特征在于:S2中PEI溶液与M-MSN纳米粒子溶液混合后,混合溶液中PEI与M-MSN纳米粒子的质量之比为1:5;反应时间为2h。
4.根据权利要求1所述的制备方法,其特征在于:S4中加入pIL12质粒溶液中的M-MSN(MB)/PEI纳米粒子与pIL12的质量之比为20~30:1。
5.根据权利要求1所述的制备方法,其特征在于:S6中孵育温度为37℃,孵育时间为6~8h。
6.采用权利要求1~5任一项所述的制备方法制备的免疫/光动力抗肿瘤功能干细胞。
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