CN105727320B - 检测小细胞肺癌的靶向纳米微泡及其制备方法和应用 - Google Patents
检测小细胞肺癌的靶向纳米微泡及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供了一种检测小细胞肺癌的靶向纳米微泡及其制备方法和应用。靶向纳米微泡包括脂质双分子外壳和包裹在脂质双分子外壳内部的惰性气体,在脂质双分子外壳上连接有针对小细胞肺癌的抗前胃泌素释放肽单克隆抗体,纳米微泡平均粒径在300‑400nm。制备方法:通过薄膜水化法将二棕榈卵磷脂、二硬脂酰磷脂酰乙醇胺、二棕榈磷脂酸完全溶解于氯仿中制成脂质体,针对小细胞肺癌的抗前胃泌素释放肽单克隆抗体用巯基乙胺打开,与上述脂质体孵育,形成稳定的硫醚键,然后通入全氟丙烷气体,通过机械震荡的方法获得靶向小细胞肺癌的含有抗前胃泌素释放肽单克隆抗体靶向纳米微泡。该靶向纳米微泡可应用于彩色多普勒超声检测小细胞肺癌。
Description
技术领域
本发明涉及一种靶向纳米微泡,具体是一种检测小细胞肺癌的靶向纳米微泡的制备方法和应用。
背景技术
小细胞肺癌(small cell lung cancer,SCLC)约占肺癌20%,两年生存率不到5%,在肺癌中恶性度最高,发病原因不明,是一种可产生多种异位激素的神经内分泌肿瘤常规检查X线、CT及MRI对SCLC的诊断缺乏特异性,常在确诊时已到了SCLC的晚期(Kalemkerian GP,et a1.Small cell lung cancer.J Natl Compr Canc Netw 2013,11(1):78-98.)。
1981年,McDonald等发现SCLC细胞株能够分泌胃泌素释放肽(GRP)并含有GRP受体(McDonald TJ,et a1.A qualitative comparison of canine plasmagastroenteropancreatic hormone response to bombesin and the porcine gastrin-releasing peptide(GRP).Regul Pept,1981,2(5):293-304.),但其在血液中的半衰期仅两分钟,前胃泌素释放肽(proGRP)与GRP等摩尔分泌,在血液中相对稳定(Miyake Y,eta1.Progastrin-releasing peptide(3 1-98)is a specific tumor marker in patientswith small cell lung carcinoma[J].Cancer Res,1994,54(8):2136-2140.)。我们与中国辐射研究院合作,用ProGRP(31-98)作为抗原免疫Balb/c小鼠成功制备并筛选出抗-ProGRP单克隆抗体,并研究了其的纯化及体外对SCLC的活性(崔大伟,王伟,周小林,等人肌红蛋白基因的克隆、表达及其抗体制备.中国生物制品学杂志,2007,20(3):166-169;周小林等.抗前胃泌素释放肽单克隆抗体的纯化及体外对小细胞肺癌的活性。肿瘤研究与临床2011,7(23):467-470.)。
随着分子生物学技术的发展与延伸,超声造影不再局限于仅仅获取组织的血流灌注信息,而是逐渐应用于特异性的超声分子成像。高特异性靶向超声微泡的设计和构建是实现超声分子显像的基础和重要环节。靶向超声微泡是将特异性抗体或配体与微泡表面连接,静脉注射后,使其能够较长时间定向地聚积于靶组织或器官,使靶区超声图像特异性增强或进行靶向治疗(Wang D,et al.Combination of high-intensity focusedultrasound with nanoscale ultrasound contrast agent in treatment of rabbitbreast VX2 tumors:a pilot study.Clin Imaging.2012.36(6):717-723.)。超声微泡是一种气-液乳剂,目前用的微泡是微米级的,由脂质外壳包裹惰性气体六氟化硫构成,粒径1~8μm属于血池显影,它只能停留在血管腔内,这就限制了在细胞及分子水平上探查疾病的发生、发展及转化过程(Deshpande N,etal.Molecular ultrasound imaging:currentstatus and future directions.Clin Radiol.2010.65(7):567-581.);而肿瘤血管内皮间隙扩大,最大孔径可达780nm(Maeda H,etal.Vascular permeability in cancer andinfection as related to macromolecular drug delivery,with emphasis on the EPReffect for tumor-selective drug targeting.Proc Jpn Acad Ser B Phys BiolSci.2012.88(3):53-71.),因此纳米微泡(粒径小于700nm)可以穿透血管内皮实现血管外靶组织显像,反应真正的发病机制,大幅度提高影像诊断的灵敏性和准确性。因而研制针对肿瘤细胞自身表达的特异性抗原的靶向纳米超声微泡,实现肿瘤细胞特异性的分子成像,具有重要的意义。
目前国内外尚未见有关抗ProGRP单克隆抗体靶向的纳米微泡的构建、靶向分子显影的超声检测的研究。针对目前SCLC诊断中的问题和难点,我们构建了一种以抗-ProGRP单克隆抗体为靶向的纳米超声微泡,能穿过SCLC肿瘤血管,与SCLC细胞特异性靶向结合,为SCLC的靶向分子显影提供新的方法和研究基础,将有利于SCLC的早期诊断。
发明内容
本发明的目的在于提供一种能特异性靶向检测小细胞肺癌的纳米级超声微泡,并提供一种构建靶向检测小细胞肺癌的纳米级超声微泡的方法,以及该纳米微泡在医学诊断中的应用。
为实现上述目的,本发明提供的技术方案是:
一种检测小细胞肺癌的靶向纳米微泡,包括脂质双分子外壳和包裹在脂质双分子外壳内部的惰性气体,在脂质双分子外壳上连接有针对小细胞肺癌的抗前胃泌素释放肽单克隆抗体,纳米微泡平均粒径在300-400nm;所述的脂质双分子由二棕榈卵磷脂(DPPC)、二硬脂酰磷脂酰乙醇胺(DSPE)、二棕榈磷脂酸(DPPA)混合制成;所述的惰性气体为全氟丙烷气体(C3F8)。
一种检测小细胞肺癌的靶向纳米微泡的制备方法,包括如下步骤:
1)采用薄膜水化法,按质量17-20:2-3:1将二棕榈卵磷脂、二硬脂酰磷脂酰乙醇胺、二棕榈磷脂酸溶解于氯仿中;在通风橱中待氯仿自然挥发形成磷脂薄膜;在磷脂薄膜中加入体积比PBS:甘油=9:1的水化液,室温下摇床上水化60min形成脂质体;
2)取抗前胃泌素释放肽单克隆抗体用10mM PBS-EDTA(NaCl 8g/L,KCl 0.2g/L,Na2HPO4 1.44g/L)稀释;将巯基乙胺溶于PBST中;将上述抗体和巯基乙胺的PBS-EDTAT溶液混合,37℃孵育90min,用每PBS-EDTA溶液超滤3次,超滤条件:3000rpm,8min,4℃,得到单链抗体;
3)将单链抗体与脂质体孵育,4℃过夜,次日用PBS溶液超滤洗涤三次,去除没有链接在脂质体上的单链抗体,用PBS溶液重悬,通入全氟丙烷气体(C3F8)、用银汞胶囊调和器机械震荡45s,静置后弃去上层泡沫,所得乳白色悬液即靶向纳米微泡。
粒径测量:用Zeta电位及粒径分析仪对纳米微泡粒径进行测量(共测7次),靶向纳米微泡的粒径小于700nm。
其中所述的靶向抗体是针对小细胞肺癌的抗前胃泌素释放肽单克隆抗体,该抗体可与小细胞肺癌细胞株上的胃泌素释放肽受体结合。
本发明检测小细胞肺癌的靶向纳米微泡,其中所述的纳米微泡与抗前胃泌素释放肽单克隆抗体的连接是利用微泡表面的马来酰亚胺键和单克隆抗体打开后的巯基反应形成稳定的硫醚键,从而将单克隆抗体片段连接在微泡上,不仅使单克隆抗体的分子量减半,暴露保守的Fc段中的巯基,而且不影响抗体的抗原识别效能。
本发明检测小细胞肺癌的靶向纳米微泡,可以通过静脉注射使纳米微泡穿过小细胞肺癌肿瘤血管,进入肿瘤组织间隙,与小细胞肺癌细胞特异性结合,在肿瘤部位有效富集以及停留延长,通过聚集成像清晰区分病变组织和周围的正常组织,应用彩色多普勒超声,达到小细胞肺癌的靶向分子显影。
附图说明
图1纳米微泡超声造影剂的制备方法示意图
图2巯基乙胺打开单克隆抗体暴露Fc段的示意图
图3靶向纳米微泡粒径的测量值,其中:(A)靶向纳米微泡的粒径为(378.1±50.7)nm,(B)空白纳米微泡的粒径为(356.9±45.7)nm。
图4靶向纳米微泡中抗体链接的鉴定,其中:(A)靶向纳米微泡表面显示明亮的绿色荧光(B)空白纳米微泡表面未见明显绿色荧光,图中标尺为2μm。
图5靶向纳米微泡与小细胞肺癌(H446细胞)细胞的结合,其中:(A)H446细胞周围有靶向纳米微泡紧密黏附,且沿其细胞膜排列规则,细胞平均黏附率为(90.2±3.24)%(B)H446细胞周围仅有少量空白纳米微泡黏附,箭头指向纳米微泡。
图6靶向纳米微泡在小细胞肺癌裸鼠移植瘤中的增强显影能力,其中:(A)裸鼠皮下成瘤,移植瘤直径约(1.18±0.12)cm;(B)超声诊断仪测量移植瘤大小,大小约(1.15x0.61)cm;(C)裸鼠移植瘤靶向纳米微泡开始显影;(D)裸鼠移植瘤靶向纳米微泡达峰。(圈指移植瘤)
具体实施方式
实施例1检测小细胞肺癌的靶向纳米微泡的制备
1)采用薄膜水化法将二棕榈卵磷脂(DPPC)36mg、二硬脂酰磷脂酰乙醇胺(DSPE)7mg、二棕榈磷脂酸(DPPA)2mg完全溶解于8ml氯仿中。在通风橱中待氯仿自然挥发形成磷脂薄膜。上述成膜的培养皿中加入8mL水化液(PBS:甘油=9:1(体积比)),室温下摇床上水化60min。将洗下的磷脂膜与水化液混匀转移至50mL离心管内形成脂质体(如图1所示)。
2)取10μg抗前胃泌素释放肽单克隆抗体用10mM的PBS(Nacl 8g/L,Kcl 0.2g/L,Na2HPO4 1.44g/L)稀释至50μL,加入1μL的0.5M的EDTA溶液;用60mg巯基乙胺溶于500μLPBS中,加入10μL的0.5M的EDTA溶液,将上述抗体和PBS溶液混合,37℃孵育90min,用每500μL含的PBS-EDTA溶液超滤3次(3000rpm,8min,4℃),获得双链打开的抗体(scAB)(如图2所示);
3)将单克隆抗体scAB与500μL脂质体孵育,4℃过夜,次日用PBS超滤洗涤三次,去除没有链接在脂质体上的抗体,用PBS重悬,定容至0.5mL,通入10ml全氟丙烷气体(C3F8)、用银汞胶囊调和器机械震荡45s,静置后弃去上层泡沫,所得乳白色悬液即靶向纳米微泡。粒径测量:用Zeta电位及粒径分析仪对纳米微泡粒径进行测量(共测7次),靶向纳米微泡的粒径是(378.1±50.7)nm(如图3所示)。
实施例2靶向纳米微泡中抗体链接的鉴定
将普通纳米微泡和靶向纳米微泡各100ul与Dylight488标记的山羊抗小鼠IgG4ul混合,荧光显微镜下(1000×)可见靶向纳米微泡表面发出明亮的绿色荧光,而普通纳米微泡表面未见明显荧光(如图4所示)。
实施例3靶向纳米微泡与小细胞肺癌(H446细胞)细胞的结合实验
每孔1.5×104个细胞接种于铺有盖玻片的6孔板中,于孵箱内培养过夜,4%多聚甲醛室温固定,将爬片分别加入靶向纳米微泡和普通空白纳米微泡30ul,37℃孵箱反应1小时,1000倍显微镜下观察靶向纳米微泡与细胞的结合情况,计算黏附率。H446细胞周围有靶向纳米微泡紧密黏附,且沿其细胞膜较规则排列,细胞平均黏附率为(90.2±3.24)%;而普通空白纳米微泡与H446细胞未结合(如图5所示)。
实施例4靶向纳米微泡在小细胞肺癌裸鼠移植瘤中的超声检测
腹腔注射100ul的5%的水合氯醛麻醉裸鼠并固定使移植瘤充分暴露在超声探头下,用GE log e9超声诊断仪测量移植瘤大小,CDFI检测其血流信号。将空白纳米微泡按30ul/只剂量经裸鼠尾静脉注射后,进行皮下移植瘤增强显影实验,记录移植瘤开始显影时间,达峰时间,峰值强度及显影持续时间。待空白纳米微泡廓清后(约30分-60分钟),用等量靶向纳米微泡按上述方式操作。结果用TIC软件对数据进行定量分析,载有抗前胃泌素释放肽单克隆抗体的靶向纳米微泡在移植瘤中的峰值强度及显影持续时间明显高于空白纳米微泡。表明靶向纳米微泡较空白纳米微泡在小细胞肺癌组织中聚集性能更好,稳定性高,体内显影时间长(如图6所示)。
Claims (2)
1.一种检测小细胞肺癌的靶向纳米微泡,包括脂质双分子外壳和包裹在脂质双分子外壳内部的惰性气体,特征在于,在脂质双分子外壳上连接有针对小细胞肺癌的抗前胃泌素释放肽单克隆抗体,纳米微泡平均粒径在300-400nm;所述的脂质双分子由二棕榈卵磷脂、二硬脂酰磷脂酰乙醇胺、二棕榈磷脂酸混合制成;所述的惰性气体为全氟丙烷气体;所述的检测小细胞肺癌的靶向纳米微泡的制备方法,包括如下步骤:
1)采用薄膜水化法,按质量17-20:2-3:1将二棕榈卵磷脂、二硬脂酰磷脂酰乙醇胺、二棕榈磷脂酸溶解于氯仿中;在通风橱中待氯仿自然挥发形成磷脂薄膜;在磷脂薄膜中加入体积比PBS:甘油=9:1的水化液,室温下摇床上水化60min形成脂质体;
2)取抗前胃泌素释放肽单克隆抗体用PBS-EDTA稀释;将巯基乙胺溶于PBS-EDTA中;将上述抗体和巯基乙胺溶液混合,37℃孵育90min,用PBS-EDTA溶液超滤3次,超滤条件3000rpm,8min,4℃,得到单链抗体;
3)将单链抗体与脂质体孵育,4℃过夜,次日用PBS溶液超滤洗涤三次,去除没有链接在脂质体上的单链抗体,用PBS溶液重悬,通入全氟丙烷气体(C3F8)、用银汞胶囊调和器机械震荡45s,静置后弃去上层泡沫,所得乳白色悬液即靶向纳米微泡。
2.如权利要求1所述的靶向纳米微泡在制备彩色多普勒超声检测小细胞肺癌试剂中的应用。
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