CN117100857A - 一种抗体金纳米笼载药系统、制备方法和用途 - Google Patents
一种抗体金纳米笼载药系统、制备方法和用途 Download PDFInfo
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Abstract
本发明公开了一种抗体金纳米笼载药系统、制备方法和用途。先单独制备LA‑DOX‑mPEG和金纳米笼,两者反应得到DOX@GNC:再加入HS‑PEG‑COOH和抗体,反应得到抗体金纳米载药系统。本发明的金纳米笼载药系统除了现有技术中纳米药物递送系统的优点外,还可实现对肿瘤细胞的主动靶向能力,降低化疗药物对正常细胞的毒副作用;肿瘤酸性微环境pH响应性DOX药物释放,可提高其在肿瘤组织的药物蓄积。同时,近红外光照射下,金纳米笼将光能转换成热能,pH响应释药和高温使得金纳米笼运载的DOX快速脉冲释放,实现光热与化疗协同治疗。
Description
技术领域
本发明属于纳米药物技术领域,涉及一种抗体金纳米笼载药系统、制备方法和用途。
背景技术
恶性肿瘤严重威胁人类健康,由于肿瘤高度异质性和肿瘤微环境特殊性,使抗肿瘤药物难以在肿瘤病灶富集,无法深层扩散、渗透至肿瘤实质发挥作用从而限制其临床疗效。利用肿瘤微环境特点,构建靶向性强、副作用小并实现多种治疗联合的智能型抗肿瘤纳米药物,具有十分重要的临床意义。B7-H3在肿瘤及其相关血管等细胞上特异高表达,是肿瘤治疗的优良靶点。金纳米笼(GNCs)具有良好的生物相容性、表面可修饰性和高效的光热转换效率,中空结构使其成为优质的药物载体。纳米药物精准靶向肿瘤部位,光热治疗不仅能杀伤手术难以切除肿瘤细胞,光热/化疗协同治疗还能加速药物的细胞内化增强抗肿瘤疗效。
光热治疗使肿瘤组织暴露于高温环境下(41~47℃),肿瘤细胞由于高温不耐受使其蛋白质变性并且细胞膜被破坏,通过凋亡途径引起细胞死亡。此外,许多研究表明,光热治疗不仅具有直接杀灭肿瘤细胞的作用,还可以抑制肿瘤的转移。肿瘤局部的光热治疗也诱导肿瘤细胞的免疫原性死亡,该过程受死亡细胞释放的肿瘤抗原和热休克蛋白调控,随后被抗原呈递细胞捕获,激活体内免疫系统,识别和进攻远处转移肿瘤。例如,有研究发现对接受静脉注射金纳米壳的黑色素肿瘤模型裸鼠给予光热治疗,肿瘤生长被显著抑制甚至缩小,其未接受关照的皮下种植瘤部位也有CD4+辅助性T细胞和CD8+细胞毒T细胞浸润。但单一光热疗法无法有效的根除肿瘤,这是由于光热治疗可能上调肿瘤细胞内的热休克蛋白(Heat Shock Proteins,HSPs),增加癌细胞的热应激耐受性,导致产生的热量不足以杀死肿瘤细胞。常将光热疗法联合传统化疗,构建多模式联合治疗体系达到抗癌效果联合增效的目的,还能防止单独使用化疗药物后癌症复发和耐药性等问题。光热治疗产生的肿瘤局部高温能够抑制化疗后肿瘤细胞中DNA修复酶的活性,扰乱细DNA合成和修复,增强化疗药物的细胞毒性。另外,局部高温通过引发线粒体功能障碍来抑制ATP的生成,抑制多药耐药基因和多药耐药相关蛋白的表达,避免产生肿瘤耐药性。此外,还可以提高药物在肿瘤微环境的溶解度并加速热运动,促进药物与细胞DNA的交联反应,增强化疗效果。
此外,光热疗法产生的肿瘤局部高温能够改善肿瘤微环境的生理条件,对于纳米药物在肿瘤组织的渗透有积极作用。当肿瘤加热到43℃时(通常在肿瘤组织外围),肿瘤血管的血流量可以增加大约两倍,改善新生血管的无效血流现象,引起血管压力升高。并且热诱导的细胞骨架损伤引起内皮细胞损伤,使血管间隙开孔进一步扩大,这种变化促使纳米药物从血管渗入肿瘤组织。增强的血流灌注还增加肿瘤新生血管和细胞膜的渗透性,促进肿瘤细胞对纳米药物的摄取,使更多的纳米药物穿透到肿瘤中。有研究发现将金纳米棒静脉注射到小鼠肉瘤(S-180)肿瘤模型中,经过光热治疗对比不同温度下伊文思蓝染料进入肿瘤部位的含量,可观察到当肿瘤温度达到43℃或以上时,染料在肿瘤部位的含量增高(高达1.8倍),结果说明通过光热作用增加肿瘤部位药物渗透的可行性。
现有技术中最接近本发明的金纳米符合载体可克服化疗药物突释及单一化疗效果不佳等缺陷,但对肿瘤细胞缺乏主动靶向性。
发明内容
本发明的目的在于针对现有技术中的不足,提供一种可精准靶向肿瘤组织并在肿瘤微环境pH响应释放DOX化疗药物的金纳米笼载药系统,在金纳米笼表面修饰B7-H3抗体片段和智能响应性材料。在所述的金纳米笼表面修饰B7-H3抗体片段,赋予金纳米笼复合体对肿瘤组织的主动靶向性;通过对其表面B7-H3抗体片段的修饰,更好地实现特异性靶向肿瘤组织。当纳米药物在肿瘤局部富集。在近红外光照射下,金纳米笼因表面等离子体共振效应,将光能转换成热能,导致局部温度急剧升高(高温可有效杀伤肿瘤细胞)和加速DOX药物在肿瘤微环境热疗温度下的脉冲释药(肿瘤弱酸性微环境使修饰DOX的腙键断裂,并且金纳米笼的高温可加快化疗药物的释放,使其产生药物的脉冲释放),实现光热治疗与化疗的联合治疗作用。
为实现上述目的,本发明提供一种抗体金纳米载药系统的制备方法,其特征在于,包括如下步骤,
S1.合成LA-DOX-mPEG,所述功能性LA-DOX-mPEG中聚乙二醇的分子量为1kDa~5kDa;
S2.金纳米笼的制备:电化学置换法制备得到粒径为30~200nm的金纳米笼;
S3.S1所得LA-DOX-mPEG与S2所得金纳米笼溶液反应得到DOX@GNC:
S4.B7-H3抗体的修饰:将S3所得DOX@GNC和HS-PEG-COOH,抗体反应,得到单克隆抗体金纳米载药系统。
进一步,所述S1为,
先制备硫辛酸乙酯,再将硫辛酸乙酯溶于无水有机溶剂后与水合阱,乙酸乙酯溶液反应,经过萃取干燥离心浓缩分离,得到硫辛酸酰阱LA-NHNH2;再将其与盐酸多柔比星溶于无水有机溶剂后滴加三氟乙酸反应得到硫辛酸酰多柔比星;
聚乙二醇单甲醚和三乙胺分别溶于无水有机溶剂中,滴加含氯甲酸4-硝基苯酯的二氯甲烷溶液进行反应,洗涤,分离,得到mPEG-NPC;
将所得硫辛酸酰多柔比星与mPEG-NPC溶于无水二甲基甲酰胺中,加入TEA反应,再纯化得到LA-DOX-mPEG。
进一步,所述S2为,
乙二醇加热后依次加入硫化钠,聚乙烯吡咯烷酮的乙二醇溶液,硝酸银的乙二醇溶液反应,反应结束后洗涤得到银立方体;
将聚乙烯吡咯烷酮加入到上述所得银立方体溶液中,再在加热搅拌下加入氯金酸溶液反应,反应结束后离心,洗涤,重悬得到金纳米笼溶液即GNC溶液。
进一步,所述S3为,
S2步骤所得的金纳米笼溶液与mPEG-SH反应得到溶液A;
S1所得LA-DOX-mPEG与NaBH4反应得到溶液B;
将溶液B加入到溶液A中反应,离心,重悬获得DOX@GNC;
进一步,所述LA-DOX-mPEG与金纳米笼的摩尔比为1:20~1:80。
进一步,所述S4为,
向S3所得的DOX@GNC中加入HS-PEG-COOH搅拌,再加入N-羟基琥珀酰亚胺和1-(3-二甲氨基丙基)-3-乙基碳二亚胺后搅拌,离心,重悬,再加入抗体到重悬液中继续搅拌,离心得到单克隆抗体金纳米载药系统。
进一步,所述HS-PEG-COOH与金纳米笼的摩尔比为1:300~1:400,抗体与HS-PEG-COOH的摩尔比为1:2~1:20。
进一步,所述抗体为单克隆抗体、单链抗体、抗体的Fab或Fc抗体片段。
本发明还提供一种抗体金纳米载药系统的制备方法制备得到的抗体金纳米载药系统。
本发明还保护所述抗体金纳米载药系统用于制备癌症治疗药物的用途;优选的,癌症不限于血癌、骨癌、淋巴癌、肠癌、肝癌、胃癌、盆腔癌、肺癌、脑癌、神经癌、乳腺癌、食道癌、肾癌。
本发明所述的LA-DOX-mPEG,也就是文中所述的linker。
现有技术中最接近本发明的金纳米递药系统,一定程度上克服化疗药物突释及单一化疗效果不佳等缺陷,但对肿瘤及其相关细胞(肿瘤病灶)的靶向性不足。因此,本发明在此基础上进一步开放,借助B7-H3抗体和智能响应DOX对现有技术中最接近本发明的复合材料进行巧妙修饰。使其具有主动靶向肿瘤组织的能力。通过pH敏感的腙键可实现药物在肿瘤酸性微环境的控释,提供一种新型光热化疗联合治疗的金纳米笼载药系统的制备方法,实现肿瘤的光热/化疗协同治疗,为智能响应载体的设计和多模式联合治疗肿瘤提供了重要的科学依据与参考。目前还没有同时载有B7-H3抗体片段和DOX的金纳米笼载药系统的报道。
同时,本发明的金纳米笼载药系统除了现有技术中纳米药物递送系统的优点外,还验证了B7-H3抗体修饰可实现对肿瘤细胞的主动靶向能力,降低化疗药物对正常细胞的毒副作用;肿瘤酸性微环境pH响应性DOX药物释放,可提高其在肿瘤组织的药物蓄积。同时,近红外光照射下,金纳米笼将光能转换成热能,pH响应释药和高温使得金纳米笼运载的DOX快速脉冲释放,实现光热与化疗协同治疗。
附图说明
图1是LA-DOX-mPEG合成流程图;
图2为pH响应性LA-DOX-mPEG的FTIR和1H-NMR谱图;
图3为制备的银立方体、金纳米笼和金纳米笼载药系统的TEM图;
图4为Anti-B7-H3-scFv@GNC@DOX在不同pH条件下的体外药物累积释放曲线图;
图5为实施例2中采用FACS和CLSM观察肿瘤细胞对不同材料的摄取行为图;
图6为实施例3中不同浓度DOX、mPEG@GNC、Anti-B7-H3-scFv@GNC、DOX@GNC和Anti-B7-H3-scFv@GNC@DOX在有或无近红外光照射下的细胞存活率柱状图;
图7为实施例4中DOX、mPEG@GNC、Anti-B7-H3-scFv@GNC、DOX@GNC和Anti-B7-H3-scFv@GNC@DOX(1μg/mL)理细胞24h并行有或无近红外光照处理所得细胞周期分布流式细胞术结果图;
图8为实施例5中mPEG@GNC、Anti-B7-H3-scFv@GNC、DOX、DOX@GNC或Anti-B7-H3-scFv@GNC@DOX(1μg/mL)处理细胞24h并行有或无近红外光照处理所得细胞活性氧产生流式细胞术结果图。
图9为实施例6中DOX、DOX@GNC和Anti-B7-H3-scFv@GNC(DOX剂量为3mg/kg)在大鼠体内的血浆药物浓度-时间曲线图,及小鼠体内的抗肿瘤药效结果图。
图10为实施例6中DOX、DOX@GNC和Anti-B7-H3-scFv@GNC(DOX剂量为3mg/kg)的小鼠的心、肝、脾、肺和肾5大组织的病理损伤及肿瘤的新生血管结果图。
图11为通过Octet仪器测定鼠B7-H3抗体的结合能力图。
具体实施方式
下面详细描述本发明的实施例,所述实施例的示例在附图中示出,其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的,旨在用于解释本发明,而不能理解为对本发明的限制。实施例中未注明具体技术或条件者,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
本发明提供了一种新型光热化疗联合治疗的靶向控释金纳米笼载药系统,其包含具有光热转换效率的金纳米笼,pH智能响应DOX和B7-H3抗体修饰物。
实施例1Anti-B7-H3-scFv@GNC@DOX金纳米笼载药系统的制备
反应过程如图1所示。
(1)pH响应性LA-DOX-mPEG(即linker)的合成
1)LAOEt的合成及纯化:在氮气和避光条件下,将硫辛酸(LA)、乙醇(EtOH)、对二甲氨基吡啶(DAMP)溶于二氯甲烷(DCM)中得到溶液A;将N,N'-二环己基碳二亚胺(DCC)溶于二氯甲烷中得到溶液B,于-10~20℃将溶液B逐滴加入到溶液A中,搅拌1~3h后升至25~45℃继续反应12~48h,过滤,浓缩滤液,经柱层析分离得到黄色油状物硫辛酸乙酯LAOEt。
2)LA-NHNH2合成及纯化:在氮气和避光条件下,将LAOEt溶于无水甲醇中,30~60℃搅拌逐滴加入水合阱,反应8~24h后,加入乙酸乙酯溶液,用饱和氯化钠溶液萃取,无水硫酸镁干燥,离心,浓缩上清液,经柱层析分离得到淡黄色固体硫辛酸酰阱LA-NHNH2。
3)LA-DOX合成及纯化:在氮气和避光条件下,将LA-NHNH2和盐酸多柔比星(DOX)溶于无水甲醇,再滴加三氟乙酸,0~35℃搅拌4~12h,浓缩静置后离心,乙腈洗涤,干燥后得深红色粉末硫辛酸酰多柔比星LA-DOX。
4)mPEG-NPC合成及纯化:在氮气条件下,将聚乙二醇单甲醚和三乙胺分别溶于无水二氯甲烷中,在-10~15℃下搅拌,往其中滴加含氯甲酸4-硝基苯酯的二氯甲烷溶液。反应在-10~15℃下进行0.5~3h,之后继续室温反应12~48h。反应液用1~5倍体积当量二氯甲烷稀释,然后用饱和食盐水洗三次,收集有机相,用无水硫酸镁干燥,过滤,旋蒸除去多余溶剂,石油醚洗涤,凝胶柱Sephadex LH-20分离杂质,旋蒸得到黄油状产物mPEG-NPC。
5)LA-DOX-mPEG合成及纯化:在室温下于氮气和避光保护下将上述所得LA-DOX和mPEG-NPC溶于无水二甲基甲酰胺中,在该溶液中加入TEA,搅拌8~48h后,溶液在25~55℃旋蒸除去溶剂,柱层析分离纯化,冻干获得红色固体LA-DOX-mPEG。图2为pH响应性LA-DOX-mPEG的FTIR和1H-NMR谱图,其中(A)为pH响应性LA-DOX-mPEG的FTIR谱图,(B)为pH响应性LA-DOX-mPEG的1H-NMR谱图。从图2可以看出,合成目标化合物的红外官能团特征峰和氢谱数量及其特征峰与预期相符。
(2)金纳米笼的制备
1)乙二醇在磁力搅拌下,加热至120~180℃时加入0.5~0.8mg/mL硫化钠,5~25min再加入10~30mg/mL聚乙烯吡咯烷酮的乙二醇溶液,在搅拌的情况下逐渐加入30~50mg/mL硝酸银的乙二醇溶液,反应5~30min停止冷却至室温,丙酮洗涤,离心收集产物,并用去离子水洗涤后得到银立方体;银立方体的TEM图见图3的(A)。
2)聚乙烯吡咯烷酮加入到上述所得银立方体溶液中,再在70~130℃搅拌下逐滴加入氯金酸溶液,反应停止冷却至室温,离心移除上清液,加入饱和氯化钠溶液洗涤,再用去离子水洗涤后得到金纳米笼(即GNC);超声重悬于超纯水得到金纳米笼溶液(即GNC或GNCs溶液),其TEM图见图3的(B)。
3)在30μg/mL的GNC溶液中加入mPEG-SH使mPEG-SH的终浓度为100nmol/mL,室温搅拌4~6h,离心(10000rpm,10min)除去未结合的部分,重悬于等体积去离子水中,合成mPEG@GNC。(为保证实验对照组间以单因素变量进行组间对比,本步骤制备的mPEG@GNC即为实验组GNC)
4)在30μg/mL的GNC溶液中加入mPEG-SH使mPEG-SH的终浓度为0.5nmol/mL,室温搅拌4~6h得到溶液A;
再加入精密称取的适量LA-DOX-mPEG与1.5~2.5倍质量当量的NaBH4,在0℃搅拌1~3h得到溶液B;
将溶液B加入到溶液A中后继续室温搅拌4~8h,离心(10000rpm,10min)除去未结合的部分,将沉淀重悬于等体积去离子水中,制备获得DOX@GNC。
(3)B7-H3抗体的修饰
在上述制备得到的30~50μg/mL的金纳米笼溶液(即GNC溶液)中加入0.5~0.8nmol/mL的HS-PEG-COOH室温搅拌2-8h,加入20~30nmol/mL的N-羟基琥珀酰亚胺(NHS)和20~30nmol/mL的1-(3-二甲氨基丙基)-3-乙基碳二亚胺(EDC)后在0~30℃搅拌2-8h,离心后,将沉淀加入等体积去离子水重悬,再加入Anti-B7-H3-scFv单链抗体到重悬液中,继续在0~30℃搅拌2-8h,离心除去未结合的抗体得到Anti-B7-H3-scFv@GNC@DOX金纳米笼载药系统。Anti-B7-H3-scFv@GNC@DOX金纳米笼载药系统的TEM图见图3的(C)。
Anti-B7-H3-scFv单链抗体的制备过程:
一、抗B7-H3单抗(鼠源)的产生
1.1免疫小鼠
取8周龄大小的雌性Balb/c小鼠,用人B7-H3重组抗原蛋白(从上海YEASEN公司购买,产品货号93111ES60,100ug,RMB3185,Uniport No.Q5ZPR3.1)与福氏完全佐剂(CFA,Sigma)混合乳化液皮下多点接种免疫,每只小鼠80-100μg蛋白;3周后用蛋白与不完全佐剂(IFA,Sigma)乳化液进行加强免疫,共免疫3次,每只小鼠80-100μg蛋白。每次在小鼠免疫2周左右取血清测定抗体效价,血清效价用ELISA方法通过酶标仪SpectraMax i3测定。当血清效价达到足够高时,用100μg人B7-H3重组抗原蛋白溶于PBS中,腹腔注射加强免疫一次,3天后取出脾脏进行融合。
1.2制备和筛选杂交瘤
杂交瘤细胞的产生通过免疫小鼠的脾细胞与小鼠骨髓瘤细胞系(SP2/0-Ag14)在聚乙二醇(PEG1450,SIGMA)融合剂作用下进行融合,融合的细胞悬于含有HAT(SigmaAldrich)选择培养液加入96孔培养板培养11天左右,利用ELISA方法通过SpectraMax i3鉴定培养上清。挑选出的阳性克隆再经过3遍以上有限稀释法进行亚克隆选择,确保是单一来源的阳性克隆。
1.3抗B7-H3单抗(鼠源)的纯化
1.2所述亚克隆经常规杂交瘤细胞培养后,培养上清经HiTrap Protein G HP(购自GE Healthcare,货号为29048581)捕获后,以0.1M甘氨酸(pH 3.5)洗脱,收集洗脱峰,再以HiTrap Desalting(购自GE Healthcare,货号为29048684)柱脱盐,置换缓冲液为1×PBS(pH7.4)。于-20度保存待用。
二、抗B7-H3单抗(鼠源)的特性(特异性结合能力)
采用高表达B7-H3的SPC-A1、A549、CA-9-22(人口腔上皮癌细胞)细胞株、经过Alexa Fluor 488Microscale Protein Labeling Kit(购自Thermo,货号为A30006)标记的B7-H3/Fc of human IgG1(序列来源于人IgG1的Fc段,序列号:AAB2181.1,如SEQ ID NO:12所示,总共456个氨基酸,按照标准方法克隆进入载体,然后转让293T细胞表达B7-H3/IgG1-Fc,表达出来的B7-H3/IgG1-Fc用蛋白A柱子来纯化和按上述1.3纯化的抗B7-H3单抗(鼠源)充分混合,4℃孵育20~30分钟,洗涤后,在FACSVerse上机检测,结果如图11所示。图11为通过Octet仪器测定鼠B7-H3抗体的结合能力(分子互动仪器检测方法,采用的是生物层干涉技术),其中横轴表示抗体结合细胞上B7-H3抗原的时间过程(以秒为单位),前半段上升曲线表示抗体结合到抗原所需要的时间(Association),后半段平缓曲线表示抗体结合抗原之后的分离情况(Dissociation)。结合所需时间越短(曲线越陡)表示抗体结合力越强,结合后曲线平缓(不下降)表示抗体结合抗原之后不怎么分离。这个实验表明单链抗体FD3-34-scFv结合B7-H3抗原的能力良好,具体数据见(图11和表1)。
表1FD3-34-scfv单链抗体对B7-H3抗原的结合参数表
注:KD代表抗体的结合力;Kon代表抗体与抗原的结合常数;Kdis代表抗体与抗原分离的常数。
QVQLQESGPGLVRPSQTLSLTCTVSGFTFTDFYMNWVRQPPGRGLEWIGFIRDKAKGYTTEYNPSVKGRVTMLVDTSKN
QFSLRLSSVTAADTAVYYCAREGHTAAPFDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV
TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE
LLGKABATGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT
VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。SEQID NO:12。三、抗B7-H3抗体(鼠源)的序列测定
根据杂交瘤制备技术(Kohler和Milstein,1975)及《分子克隆实验指南》(第3版)【Molecular Cloning,J.萨姆布鲁克,D.W.拉塞尔著,黄培堂等译】中cDNA文库制备及其基因鉴定和DNA测序等章节操作。对UM1-51、UM1-56和UM1-69杂交瘤细胞提取总RNA,合成cDNA后进行测序。
首先利用RNA纯化试剂盒(RAeasy Mini Kit,Qiagen 74104,QIAshredder,Qiagen79654)从杂交瘤细胞提取和纯化总RNA。然后用试剂盒(SMARter RACE cDNAAmplification Kit,Clontech)并按公司试剂操作手册将RNA反转录合成第一链cDNA链。利用5’RACE技术,用试剂盒提供的通用引物UPM(universal primer)作为上游引物,和根据小鼠IgG1重链可变区和轻链κ链基因序列设计的基因特异引物GSP(gene specific primer)作为下游引物,以SMART第一链cDNA为模板进行PCR扩增。然后将PCR产物分别连接到T载体(Zero Blunt TOPO PCR Cloning Kit,Invitrogen K2875-J10)。挑选克隆进行序列测定和分析,分别得到抗体的轻链和重链可变区序列,再根据重链可变区序列和小鼠IgG1序列设计引物,通过PCR获得重链全长序列,利用IGBLAST分析工具(KABAT)对序列进行分析,确定轻、重链及其CDR区域,如下所示。
轻链序列如下:
其中,单下划线“__”代表CDR1(GASENIYGALN,SEQ ID NO:1)序列;双下划线代表CDR2(/>SEQ ID NO:2)序列;下波浪线/>代表CDR3(/>SEQ ID NO:3)序列。
重链序列如下:
其中,单下划线“__”代表CDR1(GFNIKDT,SEQ ID NO:4)序列;双下划线代表CDR2(/>SEQ ID NO:5)序列;下波浪线/>代表CDR3(/>SEQ ID NO:6)序列。
四、抗B7-H3抗体(鼠源)的人源化
为了使抗体能够更好的应用于临床病人治疗,需要对抗体序列进行人源化改造,使其保留对抗原的高亲和力和其它有利的生物学性质(如降低其在人体中的免疫原性)。为了达到这一目的,根据优选的方法,通过用母体和人源化序列的三维模型分析母体序列及各种概念上的人源化产物的方法来制备人源化抗体。三维免疫球蛋白模型一般是可利用的,并为本领域技术人员所熟悉。几乎所有鼠抗体都可通过CDR嫁接来人源化,从而保持抗原结合性。相关的技术文献可以参考Sims等,1987,J.Immunol.,151:2296;Chothia等,1987,J.Mol.Biol.,196:901;Presta等,1993,J.Immnol.,151:2623;Carter等,1992,Proc.Natl.Acad.Sci.USA,89:4285;Lo Benny K.C.等,载于Antibody Engineering:Methods and Protocols(抗体工程:方法和方案),第248卷,Humana Press,New Jersey,2004。
在前述工作基础上,优选对FD3-34-scFv进行人源化改造。首先将抗体重链恒定区换为人IgG4恒定区序列,同时把抗体轻链恒定区换成人的κ链恒定区,得到人鼠嵌合的母本抗体(Chimeric Ab)。在确认母本抗体的功能活性后对重链可变区和轻链可变区序列进行人源化改造,在CDR移植的基础上进行FR区关键氨基酸的回复突变。对于不能确定是否影响抗原和抗体结合的FR间氨基酸进行兼并合成,构建一系列IgG突变体。经过基于抗体亲和力和表达水平的筛选后,筛选到1个重链和1个轻链序列并使用常规分子克隆技术克隆到pcDNA3.1表达载体中,轻链或重链序列5’端携带Kozak序列及人IL-2信号肽:MYRMQLLSCIALSLALVTNS(SEQ ID NO:11),其相应的核苷酸序列为:
轻链序列如下:
其中,单下划线“__”代表CDR1(GASENIYGALN,SEQ ID NO:1)序列;双下划线代表CDR2(/>SEQ ID NO:2)序列;下波浪线/>代表CDR3(/>SEQ ID NO:3)序列。
重链序列如下:
其中,单下划线“__”代表CDR1(GFNIKDT,SEQ ID NO:4)序列;双下划线代表CDR2(/>SEQ ID NO:5)序列;下波浪线/>代表CDR3(/>SEQ ID NO:6)序列。
上述轻链及重链表达载体瞬时转染ExpiCHO-S(Invitrogen)细胞可得到1个人源化抗体(FD3-34-scFv),纯化后的抗体经ELISA筛选后经过分子互动法(BLI)为结合力最优人源化抗体(图11)。
从图3可以看出,金纳米笼周围呈现晕圈,表明成功修饰上pH响应性缀合物和主动靶向B7-H3抗体片段。
所构建的Anti-B7-H3-scFv@GNC@DOX金纳米笼载药系统,尺寸大小约为70nm左右(见图3),电势约为-23mV。
精密量取Anti-B7-H3-scFv@GNC@DOX金纳米笼载药系统1mL于透析袋(截留分子量=3500Da),采用透析法考察其在不同pH值释放介质的EP管中(pH 7.4、6.5、5.5)的体外释放行为。图4为Anti-B7-H3-scFv@GNC@DOX金纳米笼载药系统在不同pH条件下的体外药物累积释放曲线图;可以看出,DOX在不同pH介质下的释放速率有显著不同。在pH 7.4的正常生理条件下,多功能金纳米载体中的DOX释放缓慢且最终释放累积量仅在30%左右,表明在多功能金纳米载体到达肿瘤部位之前,不会在正常生理条件下快速释放药物,减弱药物对正常组织的伤害;当递药系统聚集于弱酸性肿瘤微环境,在pH 6.5时,累积释放量约为50%;在pH 5.5时,DOX的释放非常迅速,在24h内即累积释放50%以上,最终累积释放量更是超过90%。
本发明的靶向控释金纳米笼载药系统是由B7-H3抗体片段对肿瘤细胞、肿瘤血管内皮细胞和基质成纤维细胞特异性识别,实现药物对肿瘤组织的精准递送;智能响应性DOX实现递送系统在肿瘤微环境的DOX药物的缓控性释放。
本发明的光热化疗联合治疗的金纳米笼载药系统可应用于近红外光热治疗,金纳米笼在近红外光照射下将光能转换成热能,pH响应释药和高温使得金纳米笼运载的DOX快速脉冲释放,实现光热与化疗协同治疗。
实施例2
取对数生长期的NCI-H1299人非小细胞肺癌细胞(可购自ATCC),采用流式细胞技术考察肿瘤细胞对游离DOX(临床用DOX作为对照,即将DOX溶于PBS)、DOX@GNC和Anti-B7-H3-scFv@GNC@DOX三种纳米颗粒的细胞摄取差异,以及Anti-B7-H3-scFv@GNC@DOX在1、2、4、12和24h不同时间肿瘤细胞的摄取差异;采用激光共聚焦显微镜评价肿瘤细胞对DOX、Anti-B7-H3-scFv@GNC、DOX@GNC和Anti-B7-H3-scFv@GNC@DOX的摄取能力。结果见图5,图5为采用FACS和CLSM观察肿瘤细胞对不同材料的摄取行为图,其中control为PBS溶剂对照。其中(A)是不同递药系统孵育12小时后,细胞内的化疗药物DOX含量,在Anti-B7-H3-scFv@GNC@DOX处理的细胞中,其DOX荧光强度显著高于游离DOX(2.14倍;p<0.001)或DOX@GNC(1.72倍;p<0.01);(B)是不同时间肿瘤细胞对所构建Anti-B7-H3-scFv@GNC@DOX的摄取能力,12h后可达到最大摄取量;(C)是聚光共聚焦荧光显微镜检测多功能金纳米载体的细胞内定位和药物释放,DOX为红色荧光,Anti-B7-H3-scFv单链抗体与550处理显示绿色荧光,DAPI为蓝色荧光,用于标记细胞核位置。可以看出,Anti-B7-H3-scFv@GNC@DOX处理的细胞显示出DOX的红色荧光弥散于整个细胞,并且较DOX和DOX@GNC处理的细胞有更强的荧光,说明在抗体介导的内吞作用下,细胞能够更多的摄取多功能金纳米载体。
实施例3
取对数生长期的NCI-H1299人非小细胞肺癌细胞,采用MTT考察游离DOX、mPEG@GNC、Anti-B7-H3-scFv@GNC、DOX@GNC和Anti-B7-H3-scFv@GNC@DOX在相同DOX(0、0.25、0.5、1、2、4μg/mL的DOX当量),是否联合光热治疗的肿瘤细胞增殖抑制情况,GNCs的细胞增殖抑制实验表明其对未有显著细胞毒性,具有良好的生物相容性;在相同近红外光作用下,其光热效应与GNCs浓度呈正相关(本发明所有实验组中的mPEG@GNC与GNCs一致)。结果见图6。
图6为实施例3中不同浓度DOX、mPEG@GNC、Anti-B7-H3-scFv@GNC、DOX@GNC和Anti-B7-H3-scFv@GNC@DOX在有或无近红外光照射下的细胞存活率柱状图。可以看出,当DOX@GNC连接上Anti-B7-H3-scFv后,在B7-H3受体介导的胞吞作用下能较大幅度的增强对DOX的摄取力,显示出更强的细胞杀伤作用。当使用NIR激光处理细胞后,其细胞存活率相较于无光照组有较大改变,培养液中GNC含量越高,对细胞的杀伤力越强。其中Anti-B7-H3-scFv@GNC@DOX加NIR激光照射组有明显的细胞增殖抑制效果。
实施例4
取对数生长期的NCI-H1299人非小细胞肺癌细胞,利用双链DNA荧光染料碘化丙啶(PI)与DNS结合可产生荧光,并且荧光强度和双链DNA的含量成正比。考察DOX、mPEG@GNC、Anti-B7-H3-scFv@GNC、DOX@GNC和Anti-B7-H3-scFv@GNC@DOX(1μg/mL DOX当量)在一组执行光照(808nm激光,3min);另一组不执行光照,继续培养至24h,用0.25%胰蛋白酶(不含EDTA)消化细胞,加入完全培养液终止消化,稀释计数后1000rpm离心5min,小心吸掉上清液,用预冷的PBS润洗两次,再1000rpm离心2次,小心弃掉上清。加入1mL预冷的70%乙醇溶液,4℃固定12h,洗去乙醇,于细胞沉淀中加入100μL RNase A溶液,重悬细胞,37℃水浴30min,加入400μL PI溶液,4℃避光孵育30min,随后完成流式细胞术检测。考察光热化疗联合治疗对肿瘤细胞周期的影响。
图7为实施例4中DOX、mPEG@GNC、Anti-B7-H3-scFv@GNC、DOX@GNC和Anti-B7-H3-scFv@GNC@DOX(1μg/mL)处理细胞24h并行有或无近红外光照处理所得细胞周期分布流式细胞术结果图;Ctrl为PBS。可以看出,单纯GNCs不会对细胞周期及增殖产生影响,当细胞用Anti-B7-H3-scFv@GNC@DOX处理加NIR照射引起S和G2/M期的明显转变,其阻滞效果强于DOX处理的组别(G0/G1期比例下降10.33%,p<0.05),说明光热作用引起细胞周期(S和G2/M期)阻滞的机制不同于DOX(G2/M期)周期阻滞,增强细胞对化疗药物DOX的敏感程度,使其细胞周期显著阻滞于S和G2/M期。
实施例5
利用本身没有荧光的2',7-二氯荧光素二乙酸酯(DCFH-DA)可自由穿过细胞膜,入胞可被胞内酯酶水解生成DCFH,细胞内的活性氧可以氧化无荧光的DCFH生成有荧光的DCF的特性,来评价细胞内活性氧的水平。
取对数生长期的NCI-H1299人非小细胞肺癌细胞(源于ATCC),胰酶消化并用细胞计数板计数,用含20ng/mL IFN-γ的培养液稀释至细胞浓度为5×105/mL,500μL/孔均匀接种于24孔板中,培养箱中孵育10h。吸去旧培养液,替换为含有DOX、mPEG@GNC、Anti-B7-H3-scFv@GNC、DOX@GNC和Anti-B7-H3-scFv@GNC@DOX(1μg/mL DOX当量)的培养液,继续培养12h,一组执行光照(808nm激光,3min),另一组不执行光照,继续培养至24h,用0.25%胰蛋白酶消化细胞,加入完全培养液终止消化,稀释计数后1000rpm离心5min,小心吸掉上清液,用10μM新鲜制备的DCFH-DA溶液处理15min,并进行流式细胞术检测。结果见图8,图8为实施例5中mPEG@GNC、Anti-B7-H3-scFv@GNC、DOX、DOX@GNC或Anti-B7-H3-scFv@GNC@DOX(1μg/mL)处理细胞24h并行有或无近红外光照处理所得细胞活性氧产生流式细胞术结果图;Ctrl为PBS。可以看出,未进行光照处理时,GNCs、Anti-B7-H3-scFv@GNC处理细胞产生与阴性对照组相当的活性氧当量。使用近红外光照和mPEG@GNC、Anti-B7-H3-scFv@GNC处理的细胞增加活性氧的产生。当近红外光照与DOX共同处理细胞,由于光热疗和化疗联合作用,能极大增加ROS水平,DOX@GNC和Anti-B7-H3-scFv@GNC@DOX加近红外光照处理组细胞内ROS水平是DOX组的2.15倍(p<0.001)和2.43倍(p<0.001)。
实施例6
为揭示Anti-B7-H3-scFv@GNC@DOX改善体内药动学及药效学性质,利用LC-ESI-MS/MS方法测定大鼠血浆DOX药物浓度,通过SD大鼠评价DOX(即表中的Dox·HCl)、DOX@GNC(表中的DOX@GNCS)及Anti-B7-H3-scFv@GNC@DOX(表中的B7-H3/Dox@GNCs)的体内药代动力学。构建NCI-H1299异种移植瘤小鼠模型,揭示Anti-B7-H3-scFv@GNC@DOX相较于临床用DOX和非靶向DOX@GNC在肿瘤病灶蓄积及抗肿瘤活性的改善程度。
通过比较Anti-B7-H3-scFv@GNC@DOX、DOX@GNC和临床用DOX间的PK参数(表1),发明人发现Anti-B7-H3-scFv@GNC@DOX的AUC0-∞分别是DOX@GNC的2.23倍和DOX的15.58倍,表明所构建主动靶向递药系统的DOX吸收明显增加。与DOX@GNC和临床用DOX相比,Anti-B7-H3-scFv@GNC@DOX组DOX的MRT0-∞值较高,CL值较低,结果表明所构建递药系统的体内潴留时间显著增强,体内清除率显著降低。
表1单次静脉注射Dox·HCl、Dox@GNCs及B7-H3/Dox@GNCs的药动学参数表(Mean±SD,n=6)
不同药物组在体内的组织分布及抗肿瘤活性评价,在NCI-H1299异种移植瘤小鼠模型(当肿瘤体积达到平均80-100mm3时)成功构建后开展分组治疗。图9为实施例6中DOX、DOX@GNC和Anti-B7-H3-scFv@GNC(DOX剂量为3mg/kg)在大鼠体内的血浆药物浓度-时间曲线图,及小鼠体内的抗肿瘤药效结果图。如图9的A所示,Anti-B7-H3-scFv@GNC@DOX和DOX@GNC相较于DOX在小鼠体内的血药浓度显著提高,药物在体内的半衰期和滞留时间显著延长,其中Anti-B7-H3-scFv@GNC@DOX的药时曲线下面积(AUC)最高,其次为DOX@GNC,DOX在体内容易被快速清除。此外,图9的B所示,Anti-B7-H3-scFv@GNC@DOX相较于DOX@GNC和DOX在肿瘤组织的蓄积程度显著提高,而对其他正常脏器的药物蓄积减少,降低药物对小鼠非靶器官的毒副损伤,体重维持正常(图9的C);而GNCs与Control相似,相较于其他药物组的小鼠体重及肿瘤体积未有显著性差异,表明GNCs作为药物载体具有良好的生物相容性。因此,在近红外光(NIR)作用下肿瘤病灶主动靶向蓄积递药系统的光热/化疗协同治疗效果进一步提升,Anti-B7-H3-scFv@GNC@DOX在体内的抗肿瘤活性显著高于其他药物组(图9的D及E)。
图10为实施例6中DOX、DOX@GNC和Anti-B7-H3-scFv@GNC(DOX剂量为3mg/kg)的小鼠的心、肝、脾、肺和肾5大组织的病理损伤及肿瘤的新生血管结果图。可以看出治疗组小鼠心、肝、脾、肺、肾主要脏器组织的损伤情况,DOX药物组的心肌纤维紊乱,存在明显心肌损伤;Anti-B7-H3-scFv@GNC@DOX通过主动靶向高度蓄积于肿瘤病灶,减弱药物对正常组织的伤害,主要组织脏器正常未见明显损伤。而GNCs组小鼠的心、肝、脾、肺和肾5大组织脏器与Control组一致,未发现实质脏器损伤,肿瘤组织的新生血管密度也未呈现变化。Anti-B7-H3-scFv@GNC@DOX主动靶向肿瘤及其新生血管细胞,在化疗/光热协同治疗下肿瘤新生血管数量显著降低。
最后所应当说明的是,以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制,对于本领域的普通技术人员来说,在上述说明及思路的基础上还可以做出其它不同形式的变化或变动,这里无需也无法对所有的实施方式予以穷举。凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明权利要求的保护范围之内。
Claims (10)
1.一种抗体金纳米载药系统的制备方法,其特征在于,包括如下步骤,
S1.合成LA-DOX-mPEG,所述功能性LA-DOX-mPEG中聚乙二醇的分子量为1kDa~5kDa;
S2.金纳米笼的制备:电化学置换法制备得到粒径为30~200nm的金纳米笼;
S3.S1所得LA-DOX-mPEG与S2所得金纳米笼溶液反应得到DOX@GNC:
S4.B7-H3抗体的修饰:将S3所得DOX@GNC和HS-PEG-COOH,抗体反应,得到单克隆抗体金纳米载药系统。
2.如权利要求1所述抗体金纳米载药系统的制备方法,其特征在于,所述S1为,
先制备硫辛酸乙酯,再将硫辛酸乙酯溶于无水有机溶剂后与水合阱,乙酸乙酯溶液反应,经过萃取干燥离心浓缩分离,得到硫辛酸酰阱LA-NHNH2;再将其与盐酸多柔比星溶于无水有机溶剂后滴加三氟乙酸反应得到硫辛酸酰多柔比星;
聚乙二醇单甲醚和三乙胺分别溶于无水有机溶剂中,滴加含氯甲酸4-硝基苯酯的二氯甲烷溶液进行反应,洗涤,分离,得到mPEG-NPC;
将所得硫辛酸酰多柔比星与mPEG-NPC溶于无水二甲基甲酰胺中,加入TEA反应,再纯化得到LA-DOX-mPEG。
3.如权利要求1所述抗体金纳米载药系统的制备方法,其特征在于,所述S2为,
乙二醇加热后依次加入硫化钠,聚乙烯吡咯烷酮的乙二醇溶液,硝酸银的乙二醇溶液反应,反应结束后洗涤得到银立方体;
将聚乙烯吡咯烷酮加入到上述所得银立方体溶液中,再在加热搅拌下加入氯金酸溶液反应,反应结束后离心,洗涤,重悬得到金纳米笼溶液即GNC溶液。
4.如权利要求1所述抗体金纳米载药系统的制备方法,其特征在于,所述S3为,
S2步骤所得的金纳米笼溶液与mPEG-SH反应得到溶液A;
S1所得LA-DOX-mPEG与NaBH4反应得到溶液B;
将溶液B加入到溶液A中反应,离心,重悬获得DOX@GNC。
5.如权利要求4所述抗体金纳米载药系统的制备方法,其特征在于,所述LA-DOX-mPEG与金纳米笼的摩尔比为1:20~1:80。
6.如权利要求1所述抗体金纳米载药系统的制备方法,其特征在于,所述S4为,
向S3所得的DOX@GNC中加入HS-PEG-COOH搅拌,再加入N-羟基琥珀酰亚胺和1-(3-二甲氨基丙基)-3-乙基碳二亚胺后搅拌,离心,重悬,再加入抗体到重悬液中继续搅拌,离心得到单克隆抗体金纳米载药系统。
7.如权利要求6所述抗体金纳米载药系统的制备方法,其特征在于,所述HS-PEG-COOH与金纳米笼的摩尔比为1:300~1:400,抗体与HS-PEG-COOH的摩尔比为1:2~1:20。
8.如权利要求1所述抗体金纳米载药系统的制备方法,其特征在于,所述抗体为单克隆抗体、单链抗体、抗体的Fab或Fc抗体片段。
9.权利要求1-8任一所述抗体金纳米载药系统的制备方法制备得到的抗体金纳米载药系统。
10.权利要求9所述抗体金纳米载药系统用于制备癌症治疗药物的用途;优选的,癌症不限于血癌、骨癌、淋巴癌、肠癌、肝癌、胃癌、盆腔癌、肺癌、脑癌、神经癌、乳腺癌、食道癌、肾癌。
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