CN105797172B - 具有肿瘤靶向和示踪作用的atp敏感荧光探针脂质体及其制备方法与应用 - Google Patents
具有肿瘤靶向和示踪作用的atp敏感荧光探针脂质体及其制备方法与应用 Download PDFInfo
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- CN105797172B CN105797172B CN201610255250.9A CN201610255250A CN105797172B CN 105797172 B CN105797172 B CN 105797172B CN 201610255250 A CN201610255250 A CN 201610255250A CN 105797172 B CN105797172 B CN 105797172B
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Abstract
本发明所述ATP敏感荧光探针脂质体为将ATP敏感核苷酸双链封装在偶联有肿瘤靶向多肽的脂质双分子层膜内形成的纳米囊泡,所述ATP敏感核苷酸双链由偶联荧光染料的ATP敏感核苷酸单链和偶联荧光淬灭剂的ATP敏感核苷酸互补单链根据碱基互补配对原则自组装形成,所述偶联有肿瘤靶向多肽的脂质双分子层膜由肿瘤靶向多肽偶联的聚乙二醇化磷脂酰乙醇胺、磷脂和胆固醇组成,肿肿瘤靶向多肽偶联的聚二醇化磷脂酰乙醇胺、磷脂、胆固醇的摩尔比为(2~8):(42~78):(20~50)。本发明还提供了上述脂质体的制备方法。该荧光探针脂质体兼具肿瘤主动靶向性、ATP敏感智能荧光开光和体内长循环功能,并且制备工艺简单。
Description
技术领域
本发明属于影像试剂领域,具体涉及一种荧光探针脂质体。
背景技术
肿瘤已经成为威胁人类健康的重大疾病。目前肿瘤确诊时,绝大多数肿瘤患者处于中晚期,治疗方法主要以手术、放疗、化疗为主。而肿瘤中晚期患者治疗后极易出现复发、耐药、转移以及并发症,这是治疗失败和病人死亡的主要原因。因此,开展肿瘤早期诊断,对于提高治疗效果,减少肿瘤患者死亡率具有重大意义。荧光示踪技术是指将能发荧光的物质导入所要跟踪或显影的细胞或组织中,利用荧光特性提供被研究对象的信息。该技术具有高灵敏度和高稳定性等特点。然而目前的荧光探针存在缺乏肿瘤靶向性和智能开关荧光等问题,造成非肿瘤组织荧光强度高,干扰肿瘤组织荧光成像,难以实现早期/小体积肿瘤的准确示踪。
现有技术中报道了一种三磷酸腺苷(ATP)触发荧光开关的胶束,可作为荧光探针用于活细胞分子影像。该胶束的分子结构为5’-Lipid-(PEG)2-Dabycl(荧光淬灭剂)-GACCTG GGG GAG TAT TGC GGA AGG TT-(PEG)6-CCA GGT C-TMR(荧光分子)-3’,在细胞外低浓度ATP环境中呈环状,此时荧光分子和荧光猝灭剂距离很近,荧光被淬灭,处于关闭状态;在细胞内高浓度ATP环境中,胶束分子构型变化,环状解开,荧光分子和荧光淬灭剂距离增大,使荧光复原,荧光信号开启,实现荧光显像(Cuichen Wu et al.ACSNANO,2013,7:5724-5731)。该胶束作为荧光探针可在体内有效传递,进入细胞,并具有高信号-低背景比值,优良的选择性和生物相容性,但存在以下问题:1、仅依靠纳米尺寸的增强渗透和滞留(EPR)效应达到肿瘤部位,无主动靶向性,靶向效率差,文章没有提供体内靶向实验数据;2、PEG链太短,难以实现延长体内循环的效果;3、需要合成含寡核苷酸、聚乙二醇和二脂肪酰基脂质的杂化分子,合成步骤多,合成难度大,纯化操作复杂。
发明内容
本发明的目的在于针对现有技术的不足,提供一种具有肿瘤靶向和示踪作用的ATP敏感荧光探针脂质体及其制备方法与应用,该荧光探针脂质体兼具肿瘤主动靶向性、ATP敏感智能荧光开光和体内长循环功能,并且制备工艺简单。
本发明所述ATP敏感荧光探针脂质体为将ATP敏感核苷酸双链封装在偶联有肿瘤靶向多肽的脂质双分子层膜内形成的纳米囊泡,所述ATP敏感核苷酸双链由偶联荧光染料的ATP敏感核苷酸单链和偶联荧光淬灭剂的ATP敏感核苷酸互补单链根据碱基互补配对原则自组装形成,所述偶联有肿瘤靶向多肽的脂质双分子层膜由肿瘤靶向多肽偶联的聚乙二醇化磷脂酰乙醇胺、磷脂和胆固醇组成,肿肿瘤靶向多肽偶联的聚二醇化磷脂酰乙醇胺、磷脂、胆固醇的摩尔比为(2~8):(42~78):(20~50)。
上述具有肿瘤靶向和示踪作用的ATP敏感荧光探针脂质体,所述偶联荧光染料的ATP敏感核苷酸单链为偶联荧光染料cy3、cy5、cy5.5或cy7的ATP敏感核苷酸单链。
上述具有肿瘤靶向和示踪作用的ATP敏感荧光探针脂质体,其特征在于所述ATP敏感核苷酸单链的核苷酸序列为序列表中SEQ ID NO:1所述。
上述具有肿瘤靶向和示踪作用的ATP敏感荧光探针脂质体,所述偶联荧光淬灭剂的ATP敏感核苷酸互补单链为偶联荧光淬灭剂4-(4’-二甲基氨基偶氮苯基)苯甲酸的ATP敏感核苷酸互补单链。
上述具有肿瘤靶向和示踪作用的ATP敏感荧光探针脂质体,所述肿瘤靶向多肽偶联的聚乙二醇化磷脂酰乙醇胺为末端氨基酸为半胱氨酸的含精氨酸-甘氨酸-天冬氨酸环状多肽偶联的聚乙二醇化磷脂酰乙醇胺、末端氨基酸为半胱氨酸的人类表皮生长因子受体2多肽偶联的聚乙二醇化磷脂酰乙醇胺、末端氨基酸为半胱氨酸的转铁蛋白偶联的聚乙二醇化磷脂酰乙醇胺中的一种。
上述具有肿瘤靶向和示踪作用的ATP敏感荧光探针脂质体,所述肿瘤靶向多肽偶联的聚乙二醇化磷脂酰乙醇胺为肿瘤靶向多肽偶联的二硬脂酰磷脂酰乙醇胺-聚乙二醇2000、肿瘤靶向多肽偶联的二肉豆蔻酰磷脂酰乙醇胺-聚乙二醇2000、肿瘤靶向多肽偶联的二棕榈酰磷脂酰乙醇胺-聚乙二醇2000、肿瘤靶向多肽偶联的二月桂酰基磷脂酰乙醇胺-聚乙二醇2000中的一种。
上述具有肿瘤靶向和示踪作用的ATP敏感荧光探针脂质体,所述磷脂为卵磷脂、大豆磷脂、二硬脂酰磷脂胆碱中的一种。
本发明所述具有肿瘤靶向和示踪作用的ATP敏感荧光探针脂质体的制备方法,工艺步骤如下:
(1)将末端为半胱氨酸的肿瘤靶向多肽与磷脂酰乙醇胺-聚乙二醇2000-马来酰亚胺按摩尔比1:1溶于甲醇中得到混合溶液,甲醇的用量以所述混合溶液磷脂酰乙醇胺-聚乙二醇2000-马来酰亚胺的浓度为1~10mmol/L计,将所得混合溶液在搅拌下于室温反应20~28小时,反应结束后除去反应液中的甲醇,即得肿瘤靶向多肽偶联的聚乙二醇化磷脂酰乙醇胺;
(2)将步骤(1)所得肿瘤靶向多肽偶联的聚二醇化磷脂酰乙醇胺、磷脂、胆固醇按摩尔比为(2~8):(42~78):(20~50)溶于氯仿和甲醇组成的复合溶剂中,得到混合溶液,复合溶剂的用量以所得混合溶液中磷脂和胆固醇的浓度为1~10mmol/L计,减压蒸馏除去混合溶液中的复合溶剂得到偶联有肿瘤靶向多肽的脂质双分子层膜;
(3)将偶联荧光染料的ATP敏感核苷酸单链和偶联荧光淬灭剂的ATP敏感核苷酸互补单链按摩尔比1:1加入去离子水得混合液,去离子水的用量以所述混合液中ATP敏感核苷酸单链的浓度为0.1~10mmol/L计,在室温下搅拌10~60分钟,使偶联荧光染料的ATP敏感核苷酸单链和偶联荧光淬灭剂的ATP敏感核苷酸互补单链根据碱基互补配对原则自组装形成ATP敏感核苷酸双链;
(4)将步骤(3)所得ATP敏感核苷酸双链与步骤(2)所得偶联有肿瘤靶向多肽的脂质双分子层膜按照质量比1:(10~50)混合后于40~60℃水化一小时,得到磷脂和胆固醇的浓度为1~10mmol/L的悬液,将所得悬液水浴超声5~30分钟,再依次过400纳米、200纳米和100纳米的多聚碳酸酯膜,然后采用凝胶排阻层析柱分离纯化后即得到具有肿瘤靶向和示踪作用的ATP敏感荧光探针脂质体。
上述方法中,所述末端为半胱氨酸的肿瘤靶向多肽为末端为半胱氨酸的含精氨酸-甘氨酸-天冬氨酸的环状多肽(cRGD-cys)、末端为半胱氨酸的人类表皮生长因子受体2多肽(Her-2-cys)、末端为半胱氨酸的转铁蛋白(Transferrin-cys)中的一种;
所述磷脂酰乙醇胺-聚乙二醇2000-马来酰亚胺为二硬脂酰磷脂酰乙醇胺-聚乙二醇2000-马来酰亚胺(DSPE-PEG2000-Mal)、二肉豆蔻酰磷脂酰乙醇胺-聚乙二醇2000-马来酰亚胺(DMPE-PEG2000-Mal)、二棕榈酰磷脂酰乙醇胺-聚乙二醇2000-马来酰亚胺(DPPE-PEG2000-Mal)、二月桂酰基磷脂酰乙醇胺-聚乙二醇2000-马来酰亚胺(DLPE-PEG2000-Mal)中的一种;所述磷脂为卵磷脂、大豆磷脂、二硬脂酰磷脂胆碱中的一种;
所述磷脂为卵磷脂、大豆磷脂、二硬脂酰磷脂胆碱中的一种;
所述偶联荧光染料的ATP敏感核苷酸单链为偶联荧光染料cy3、cy5、cy5.5或cy7的ATP敏感核苷酸单链,所述ATP敏感核苷酸单链的核苷酸序列为序列表中SEQ ID NO:1所述;
所述偶联荧光淬灭剂的ATP敏感核苷酸互补单链为偶联荧光淬灭剂4-(4’-二甲基氨基偶氮苯基)苯甲酸的ATP敏感核苷酸互补单链。
上述方法中,氯仿和甲醇组成的复合溶剂的配比:氯仿与甲醇的体积比可以是任意比例,氯仿与甲醇的体积比优选3:1。
上述方法中,偶联荧光染料的ATP敏感核苷酸单链和偶联荧光淬灭剂的ATP敏感核苷酸互补单链,可通过固相亚磷酰胺化学方法,利用DNA合成仪(ABI 3400 synthesizer)制备ATP敏感核苷酸单链或ATP敏感核苷酸互补单链,再将制备得到的ATP敏感核苷酸单链或ATP敏感核苷酸互补单链分别从固相树脂上断裂下来并脱去保护基后,分散于含荧光染料或荧光淬灭剂的甲醇、叔丁胺和水以体积比1:1:2混合所得的混合溶液中,使混合溶液中荧光染料与ATP敏感核苷酸单链,或荧光淬灭剂与ATP敏感核苷酸互补单链的摩尔比为1:(1~10),于65℃反应4小时,即得偶联荧光染料的ATP敏感核苷酸单链或偶联荧光淬灭剂的ATP敏感核苷酸互补单链。也可通过市场购买。
本发明制备的具有肿瘤靶向和示踪作用的ATP敏感荧光探针脂质体的粒径范围为50~150nm。
本发明所述具有肿瘤靶向和示踪作用的ATP敏感荧光探针脂质体在制备体内或体外肿瘤示踪剂中的应用。
与现有技术相比,本发明具有以下有益效果:
1、本发明提供了一类具有肿瘤靶向和示踪作用的ATP敏感荧光探针脂质体,为肿瘤诊断与示踪提供了一种新的显像试剂。
2、本发明所述具有肿瘤靶向和示踪作用的ATP敏感荧光探针脂质体粒径约为100纳米,不仅能通过物理靶向,即肿瘤微血管增强渗透和滞留效应(EPR效应)到达肿瘤组织,并且由于偶联了具有高特异性和高稳定性的肿瘤靶向多肽,可以通过主动靶向到达肿瘤组织,即通过双重靶向作用到达肿瘤组织,因而显著提高了肿瘤靶向效率,可实现肿瘤的高特异性显像。
3、本发明所述具有肿瘤靶向和示踪作用的ATP敏感荧光探针脂质体利用胞内高浓度ATP作为荧光开关,静脉注射进入人体后在血液循环过程中探针信号处于“沉默”状态,进入肿瘤细胞后在高浓度ATP环境中核苷酸双链解开,荧光信号“开启”,荧光强度显著提高,大幅度提高肿瘤/正常组织以及血液间的信噪比,有利于早期/小体积肿瘤的准确示踪。
4、本发明所述具有肿瘤靶向和示踪作用的ATP敏感荧光探针脂质体含有聚乙二醇化磷脂,在脂质体表面可形成亲水性保护层,可降低体内内皮网状系统和单核巨噬细胞系统对其的清除,因而具有长循环功能,能实现延长体内循环时间的效果。
5、本发明所述具有肿瘤靶向和示踪作用的ATP敏感荧光探针脂质体的制备方法工艺简单,成本低廉,有利于工业化生产。
6、本发明所述具有肿瘤靶向和示踪作用的ATP敏感荧光探针脂质体中的核苷酸可以偶联具有不同发射波长的荧光分子,从而实现体内/体外肿瘤显像,满足不同荧光检测要求。
附图说明
图1为实施例1中的ATP敏感核苷酸双链的ATP响应曲线图。
图2为实施例1制备的荧光探针脂质体的透射电子显微镜图。
图3为实施例1制备的荧光探针脂质体的粒径分布图。
图4为实施例1~4制备的荧光探针脂质体细胞毒性实验结果图。
图5为实施例6中脂质体体外肿瘤细胞显像图(A为普通荧光脂质体,B为实施例1制备的荧光探针脂质体)。
图6为实施例7中脂质体体内肿瘤显像图(A为普通荧光脂质体,B为实施例2制备的荧光探针脂质体)。
图7为本发明所述具有肿瘤靶向和示踪作用的ATP敏感荧光探针脂质体的结构示意图。
具体实施方式
下面通过具体实施例对本发明所述具有肿瘤靶向和示踪作用的ATP敏感荧光探针脂质体及制备方法作进一步说明。
以下实施例中,二硬脂酰磷脂酰乙醇胺-聚乙二醇2000-马来酰胺(DSPE-PEG2000-Mal)、二肉豆蔻酰磷脂酰乙醇胺-聚乙二醇2000-马来酰胺(DMPE-PEG2000-Mal)、二棕榈酰磷脂酰乙醇胺-聚乙二醇2000-马来酰胺(DPPE-PEG2000-Mal)、二月桂酰基磷脂酰乙醇胺-聚乙二醇2000-马来酰胺(DLPE-PEG2000-Mal)购自美国Nanocs公司。cRGD-cys,Her2-cys和Transferrin-cys购自中科亚光科技有限公司。cy3、cy5、cy5.5和cy7,荧光淬灭剂dabcyl购自武汉博士德生物技术有限公司。卵磷脂、大豆磷脂、DOPC、胆固醇购自西安瑞禧生物科技有限公司。
实施例1
本实施例中所述具有肿瘤靶向和示踪作用的ATP敏感荧光探针脂质体的制备方法如下:
(1)将cRGD-cys与二硬脂酰磷脂酰乙醇胺-聚乙二醇2000-马来酰胺(DSPE-PEG2000-Mal)按摩尔比1:1溶于甲醇中得到混合溶液,甲醇用量以所述混合溶液中DSPE-PEG2000-Mal的浓度为1mmol/L计。将所得混合溶液在室温下磁力搅拌12小时进行反应,反应结束后减压旋转蒸发除去反应液中的溶剂,即得环状RGD偶联的二硬脂酰磷脂酰乙醇胺-聚乙二醇2000(cRGD-DSPE-PEG2000);
(2)将步骤(1)制备所得cRGD-DSPE-PEG2000、卵磷脂和胆固醇,按摩尔比为5:65:35溶于氯仿和甲醇以体积比3:1混合所得的复合溶剂中得到混合溶液,复合溶剂的用量以所得混合溶液中卵磷脂和胆固醇的浓度为1mmol/L计,减压旋转蒸发除去混合溶液中的溶剂得到脂质体薄膜;
(3)通过固相亚磷酰胺化学法,利用DNA合成仪(ABI 3400 synthesizer)制备ATP敏感核苷酸单链,再将制备的ATP敏感核苷酸单链从固相树脂上断裂下来并脱去保护基后,分散于含cy3的甲醇、叔丁胺和水以体积比1:1:2混合所得的混合溶液中,使混合溶液中cy3与ATP敏感核苷酸单链的摩尔比为1:2,于65℃反应4小时,即得偶联荧光染料cy3的ATP敏感核苷酸单链。同法制备ATP敏感核苷酸互补单链,将制备得到的ATP敏感核苷酸互补单链从固相树脂上断裂下来并脱去保护基后,分散于含dabcyl的甲醇、叔丁胺和水以体积比1:1:2混合所得的混合溶液中,使混合溶液中dabcyl与ATP敏感核苷酸互补单链的摩尔比为1:2,于65℃反应4小时,即得偶联荧光淬灭剂dabcyl的ATP敏感核苷酸互补单链。将制得的偶联荧光染料cy3的ATP敏感核苷酸单链和偶联荧光淬灭剂dabcyl的ATP敏感核苷酸互补单链按照摩尔比1:1加入去离子水得混合液,去离子水的用量以所述混合液中ATP敏感核苷酸单链的浓度为10mmol/L为限,磁力搅拌10min,使二者根据碱基互补配对原则自组装形成ATP敏感核苷酸双链;
(4)将步骤(3)所得ATP敏感寡核苷酸双链与步骤(2)所得脂质体薄膜按照质量比1:10混合后置于40℃下水化一小时,得到卵磷脂和胆固醇的浓度为1mmol/L的脂质体悬液,将所得脂质体悬液水浴超声5分钟,再依次过400nm、200nm和100nm的多聚碳酸酯膜,采用Sephadex G-50凝胶排阻层析柱,以PBS为洗脱液,分离除去游离药物,即得到粒径约100nm的具有肿瘤靶向和示踪作用的ATP敏感荧光探针脂质体。
检测制得的脂质体是否具有ATP敏感性荧光开关:
取100μL步骤(3)所得ATP敏感核苷酸双链加入石英比色皿中,置于荧光分光光度计中监测荧光强度,激发光波长为554纳米,发射光波长为570纳米。监测50秒后,加入等量8mmol/L的ATP溶液于上述ATP敏感核苷酸双链中混合均匀,继续在相同条件下监测荧光强度。结果如图1所示。从图1可知,ATP敏感核苷酸双链荧光强度很弱,加入ATP溶液后,荧光信号瞬间开启,荧光强度大幅增加。
用透射电子显微镜观察步骤(4)水化所得脂质体悬液,结果如图2所示,脂质体为圆形且粒径均一。用激光粒度仪检测步骤(4)水化所得脂质体悬液中脂质体的粒径,结果如图3所示,平均粒径为100nm。
实施例2
(1)将Her2-cys与二肉豆蔻酰磷脂酰乙醇胺-聚乙二醇2000-马来酰胺(DMPE-PEG2000-Mal)按摩尔比1:1溶于甲醇中得到混合溶液,甲醇用量以所述混合溶液中DMPE-PEG2000-Mal的浓度为10mmol/L计。将所得混合溶液在室温下磁力搅拌24小时进行反应,反应结束后减压旋转蒸发除去反应液中的溶剂,即得Her2偶联的二肉豆蔻酰磷脂酰乙醇胺-聚乙二醇2000(Her2-DMPE-PEG2000);
(2)将步骤(1)制备所得Her2-DMPE-PEG2000、大豆磷脂和胆固醇按摩尔比为2:78:20溶于氯仿和甲醇以体积比3:1混合所得的复合溶剂中得到混合溶液,复合溶剂用量以所得混合溶液中大豆磷脂和胆固醇的浓度为10mmol/L计,减压旋转蒸发除去混合溶液中的溶剂得到脂质体薄膜;
(3)通过固相亚磷酰胺化学法,利用DNA合成仪(ABI 3400 synthesizer)制备ATP敏感核苷酸单链,再将制备得到的核苷酸链从固相树脂上断裂下来并脱去保护基后,分散于含cy5.5的甲醇、叔丁胺和水以体积比1:1:2混合所得的混合溶液中,使混合溶液中cy5.5与ATP敏感核苷酸单链的摩尔比为1:10,于65℃反应4小时,即得偶联荧光染料cy5.5的ATP敏感核苷酸单链。同法制备ATP敏感核苷酸互补单链,将制备得到的核苷酸链从固相树脂上断裂下来并脱去保护基后,分散于含dabcyl的甲醇、叔丁胺和水以体积比1:1:2混合所得的混合溶液中,使混合溶液中dabcyl与ATP敏感核苷酸互补单链的摩尔比为1:10,于65℃反应4小时,即得偶联荧光淬灭剂dabcyl的ATP敏感核苷酸互补单链。将制得的偶联荧光染料cy5.5的ATP敏感核苷酸单链和偶联荧光淬灭剂dabcyl的ATP敏感核苷酸互补单链按照摩尔比1:1加入去离子水得混合液,去离子水的用量以所述混合液中ATP敏感核苷酸单链的浓度为5mmol/L为限,磁力搅拌60min,使二者根据碱基互补配对原则自组装形成ATP敏感核苷酸双链;
(4)将步骤(3)所得ATP敏感寡核苷酸双链与步骤(2)所得脂质体薄膜按照质量比1:50混合后置于60℃下水化一小时,得到大豆磷脂和胆固醇的浓度为1mmol/L的脂质体悬液,将所得脂质体悬液水浴超声30分钟,再依次过400nm、200nm和100nm的多聚碳酸酯膜,采用凝胶排阻层析柱分离纯化后即得到平均粒径约100nm的具有肿瘤靶向和示踪作用的ATP敏感荧光探针脂质体。
实施例3
(1)将Her2-cys与二棕榈酰磷脂酰乙醇胺-聚乙二醇2000-马来酰胺(DPPE-PEG2000-Mal)按摩尔比1:1溶于甲醇中得到混合溶液,甲醇用量以所述混合溶液中DPPE-PEG2000-Mal的浓度为5mmol/L计。将所得混合溶液在室温下磁力搅拌16小时进行反应,反应结束后减压旋转蒸发除去反应液中的溶剂,即得Her2偶联的二棕榈酰磷脂酰乙醇胺-聚乙二醇2000(Her2-DPPE-PEG2000);
(2)将步骤(1)制备所得Her2-DMPE-PEG2000、大豆磷脂和胆固醇按摩尔比为8:42:50溶于氯仿和甲醇以体积比3:1混合所得的复合溶剂中得到混合溶液,复合溶剂用量以所得混合溶液中大豆磷脂和胆固醇的浓度为5mmol/L计,减压旋转蒸发除去混合溶液中的溶剂得到脂质体薄膜;
(3)将从赛默飞世尔科技(中国)有限公司购买的偶联荧光染料cy5的ATP敏感寡核苷酸单链和偶联荧光淬灭剂dabcyl的ATP敏感寡核苷酸互补单链按摩尔比1:1加入去离子水得混合液,去离子水的用量以所述混合液中ATP敏感核苷酸单链的浓度为3mmol/L为限,磁力搅拌60min,使二者根据碱基互补配对原则自组装形成ATP敏感核苷酸双链;
(4)将步骤(3)所得ATP敏感寡核苷酸双链与步骤(2)所得脂质体薄膜按照质量比1:50混合后置于50℃下水化一小时,得到大豆磷脂和胆固醇的浓度为1mmol/L的脂质体悬液,将所得脂质体悬液水浴超声20分钟,再依次过400nm、200nm和100nm的多聚碳酸酯膜,采用凝胶排阻层析柱分离纯化后既得到平均粒径约100nm的具有肿瘤靶向和示踪作用的ATP敏感荧光探针脂质体。
实施例4
(1)将Her2-cys与二月桂酰基磷脂酰乙醇胺-聚乙二醇2000-马来酰胺(DLPE-PEG2000-Mal)按摩尔比1:1溶于甲醇中得到混合溶液,甲醇用量以所述混合溶液中DLPE-PEG2000-Mal摩尔浓度为5mmol/L计,将所得混合溶液在室温下磁力搅拌24小时进行反应,反应结束后减压旋转蒸发除去反应液中的溶剂,即得Her2偶联的二月桂酰基磷脂酰乙醇胺-聚乙二醇2000(Her2-DLPE-PEG2000);
(2)将步骤(1)制备所得Her2-DLPE-PEG2000、二硬脂酰磷脂胆碱和胆固醇按摩尔比为5:65:35溶于氯仿和甲醇以体积比3:1混合所得的复合溶剂中得到混合溶液,复合溶剂的用量以所得混合溶液中二硬脂酰磷脂胆碱和胆固醇的浓度为5mmol/L计,减压旋转蒸发除去混合溶液中的溶剂得到脂质体薄膜;
(3)将从赛默飞世尔科技(中国)有限公司订购的偶联荧光染料cy7的ATP敏感核苷酸单链和偶联荧光淬灭剂dabcyl的ATP敏感核苷酸互补单链按摩尔比1:1加入去离子水得混合液,去离子水的用量以所述混合液中ATP敏感核苷酸单链的浓度为1mmol/L为限,磁力搅拌60min,使二者根据碱基互补配对原则自组装形成ATP敏感核苷酸双链;
(4)将步骤(3)所得ATP敏感寡核苷酸双链与步骤(2)所得脂质体薄膜按照质量比1:30混合后置于50℃下水化一小时,得到二硬脂酰磷脂胆碱和胆固醇的浓度为1mmol/L的脂质体悬液,将所得脂质体悬液水浴超声20分钟,再依次过400nm、200nm和100nm的多聚碳酸酯膜,采用凝胶排阻层析柱分离纯化后既得到平均粒径约100nm的具有肿瘤靶向和示踪作用的ATP敏感荧光探针脂质体。
实施例5 细胞毒性实验
将密度为1×105个/mL的L929细胞(购自中国科学院细胞库)悬液接种于96孔培养板内,每孔接种0.1mL,并向每孔加入0.1mL完全培养基(DMEM培养基+10%胎牛血清+100g/mL链霉素),然后置于37℃、5%CO2、饱和湿度的细胞恒温培养箱中孵育24h。分别将实施例1~4所制备的荧光探针脂质体用完全培养基稀释作为四组试验组,各实验组均有脂质体终浓度分别为0.01mg/mL、0.05mg/mL、0.30mg/mL、1.0mg/mL的四个样品。以完全培养基为阴性对照组。各试验组和阴性对照组均设平行样品3个。将各试验组和阴性对照组置于37℃、5%CO2、饱和湿度的细胞恒温培养箱中培养48h后弃去每孔中的上清液,用PBS缓冲液(135mmol/L NaCl,2.7mmol/L KCl,1.5mmol/L KH2PO4,,8mmol/L K2HPO4,pH=7.4)洗涤2次,然后每孔依次加入200μL PBS缓冲液、20μL 5mg/mL的M TT液溶(将0.5g MTT溶于100mLPBS缓冲液中得到)继续培养4h,继后吸尽上清液,加入100μL二甲基亚砜(DMSO),振荡10min,用酶联免疫检测仪测定波长为570nm处的光密度值(OD),通过与阴性对照组的OD值比较得到各试验组的细胞存活率(细胞存活率%=实验组OD*100/阴性对照组OD)。细胞毒性试验结果见图4。从图4可以看出,实施例1~4制备的荧光探针脂质体均无明显细胞毒性。
实施例6 体外荧光显像
按照以下方法制备普通荧光脂质体:将DSPE-PEG2000、卵磷脂和胆固醇按摩尔比5:65:35的溶于氯仿和甲醇以体积比3:1混合所得的复合溶剂中得到混合溶液,向所得混合溶液中加入荧光分子cy3,复合溶剂用量以卵磷脂和胆固醇的浓度为1mmol/L计。利用旋转蒸发仪减压旋转蒸发除尽溶剂,形成脂质体薄膜。将磷酸缓冲液加入上述脂质体薄膜中,置于40℃水化一小时得到脂质体悬液,并使所得脂质体悬液中卵磷脂和胆固醇的浓度为1mmol/L,荧光分子浓度为10μg/ml。水浴超声30分钟,使脂质体悬液形成微小的脂质体囊泡,再分别过400nm、200nm和100nm的多聚碳酸酯膜,即得粒径在100nm左右的均一普通荧光脂质体。
将对数生长期的MDA-MB-435肿瘤细胞(人乳腺癌细胞)细胞用胰蛋白酶消化,用培养基稀释成3×104/ml的细胞悬液,将所得细胞悬液按100μl/孔均匀加入直径20mm的玻底皿中。将玻底皿置于37℃孵箱中,孵育24h,显微镜下观察可见细胞融合贴壁生长。将普通荧光脂质体和实施例1制得具有肿瘤靶向和示踪作用的ATP敏感荧光探针脂质体加入细胞培养基中,使荧光分子的终浓度均为0.25μg/ml,继续培养4小时后,吸去培养基,用新鲜磷酸盐缓冲液清洗两次。然后依次加入10μL 10mg/ml的Hoechst 33258试剂、200μL磷酸盐缓冲液共同孵育15分钟。孵育结束后弃去溶液,用新鲜磷酸盐洗涤一次,加入200μL新鲜磷酸盐缓冲液保持细胞润湿备测。激光共聚焦488nm激发,575-585nm发射光检测,结果见图5。由图5可见,实施例1制备的具有肿瘤靶向和示踪作用的ATP敏感荧光探针脂质体组细胞内荧光强于普通荧光脂质体组,这是因为实施例1制备的荧光探针脂质体具有肿瘤靶向配体,能够输送更多的荧光探针脂质体进入肿瘤细胞,从而显示出更强的荧光。因此,本发明所述具有肿瘤靶向和示踪作用的ATP敏感荧光探针脂质体具有更好的肿瘤细胞示踪和显像能力。
实施例7 活体荧光显像
按照以下方法制备普通荧光脂质体:将DMPE-PEG2000、卵磷脂和胆固醇按摩尔比5:65:35的溶于氯仿和甲醇以体积比3:1混合所得的复合溶剂中得到混合溶液,向所得混合溶液中加入荧光分子cy5.5,复合溶剂用量以卵磷脂和胆固醇的浓度为10mmol/L计。利用旋转蒸发仪减压旋转蒸发除尽溶剂,形成脂质体薄膜。将磷酸缓冲液加入上述脂质体薄膜中,置于60℃水化一小时得到脂质体悬液,并使所得脂质体悬液中卵磷脂和胆固醇的浓度为1mmol/L,荧光分子浓度为10μg/ml。水浴超声30分钟,使脂质体悬液形成微小的脂质体囊泡,再分别过400nm、200nm和100nm的多聚碳酸酯膜,即得粒径在100nm左右的均一普通荧光脂质体。
将对数生长期的HER2阳性的SKOV3肿瘤细胞(人卵巢癌细胞)用胰蛋白酶消化后用PBS(磷酸盐缓冲液)稀释成1X107个/mL。取6只BLB/C裸鼠,对每只BLB/C裸鼠于右后肢皮下注射100ul细胞悬,制作小鼠乳腺癌模型。当肿瘤体积增长至50~100mm3时,将荷瘤BLB/C裸鼠分为两组(每组3只),分别尾静脉注射普通荧光脂质体和实施例2制备的具有肿瘤靶向和示踪作用的ATP敏感荧光探针脂质体100uL,Cy5.5浓度为50nmol。注射后48h,裸鼠腹腔注射水合氯醛,利用Maestro in-vivo imaging system活体成像仪观察荧光物质的分布和在肿瘤部位的聚集,激发波长700~770,发射波长790,结果见图6。如图6所示,在裸鼠肿瘤部位,实施例2制备的荧光探针脂质体的荧光强度强于普通荧光脂质体;其他组织和血液中实施例2制备的荧光探针脂质体的荧光强度显著弱于普通荧光脂质体,说明采用本发明方法制备的具有肿瘤靶向和示踪作用的ATP敏感荧光探针脂质体与普通荧光脂质体相比具有良好的肿瘤靶向性,更强的肿瘤显像能力。
Claims (10)
1.具有肿瘤靶向和示踪作用的ATP敏感荧光探针脂质体,其特征在于所述ATP敏感荧光探针脂质体为将ATP敏感核苷酸双链封装在偶联有肿瘤靶向多肽的脂质双分子层膜内形成的纳米囊泡,所述ATP敏感核苷酸双链由偶联荧光染料的ATP敏感核苷酸单链和偶联荧光淬灭剂的ATP敏感核苷酸互补单链根据碱基互补配对原则自组装形成,所述偶联有肿瘤靶向多肽的脂质双分子层膜由肿瘤靶向多肽偶联的聚乙二醇化磷脂酰乙醇胺、磷脂和胆固醇组成,肿瘤靶向多肽偶联的聚二醇化磷脂酰乙醇胺、磷脂、胆固醇的摩尔比为(2~8):(42~78):(20~50)。
2.根据权利要求1所述具有肿瘤靶向和示踪作用的ATP敏感荧光探针脂质体,其特征在于所述偶联荧光染料的ATP敏感核苷酸单链为偶联荧光染料cy3、cy5、cy5.5或cy7的ATP敏感核苷酸单链。
3.根据权利要求2所述具有肿瘤靶向和示踪作用的ATP敏感荧光探针脂质体,其特征在于所述ATP敏感核苷酸单链的核苷酸序列为序列表中SEQ ID NO:1所述。
4.根据权利要求1至3中任一权利要求所述具有肿瘤靶向和示踪作用的ATP敏感荧光探针脂质体,其特征在于所述偶联荧光淬灭剂的ATP敏感核苷酸互补单链为偶联荧光淬灭剂4-(4’-二甲基氨基偶氮苯基)苯甲酸的ATP敏感核苷酸互补单链。
5.根据权利要求1至3中任一权利要求所述具有肿瘤靶向和示踪作用的ATP敏感荧光探针脂质体,其特征在于所述肿瘤靶向多肽偶联的聚乙二醇化磷脂酰乙醇胺为末端氨基酸为半胱氨酸的含精氨酸-甘氨酸-天冬氨酸环状多肽偶联的聚乙二醇化磷脂酰乙醇胺、末端氨基酸为半胱氨酸的人类表皮生长因子受体2多肽偶联的聚乙二醇化磷脂酰乙醇胺、末端氨基酸为半胱氨酸的转铁蛋白偶联的聚乙二醇化磷脂酰乙醇胺中的一种。
6.根据权利要求5所述具有肿瘤靶向和示踪作用的ATP敏感荧光探针脂质体,其特征在于所述肿瘤靶向多肽偶联的聚乙二醇化磷脂酰乙醇胺为肿瘤靶向多肽偶联的二硬脂酰磷脂酰乙醇胺-聚乙二醇2000、肿瘤靶向多肽偶联的二肉豆蔻酰磷脂酰乙醇胺-聚乙二醇2000、肿瘤靶向多肽偶联的二棕榈酰磷脂酰乙醇胺-聚乙二醇2000、肿瘤靶向多肽偶联的二月桂酰基磷脂酰乙醇胺-聚乙二醇2000中的一种。
7.根据权利要求1至3中任一权利要求所述具有肿瘤靶向和示踪作用的ATP敏感荧光探针脂质体,其特征在于所述磷脂为卵磷脂、大豆磷脂、二硬脂酰磷脂胆碱中的一种。
8.一种具有肿瘤靶向和示踪作用的ATP敏感荧光探针脂质体的制备方法,其特征在于工艺步骤如下:
(1)将末端为半胱氨酸的肿瘤靶向多肽与磷脂酰乙醇胺-聚乙二醇2000-马来酰亚胺按摩尔比1:1溶于甲醇中得到混合溶液,甲醇的用量以所述混合溶液中磷脂酰乙醇胺-聚乙二醇2000-马来酰亚胺的浓度为1~10mmol/L计,将所得混合溶液在搅拌下于室温反应20~28小时,反应结束后除去反应液中的甲醇,即得肿瘤靶向多肽偶联的聚乙二醇化磷脂酰乙醇胺;
(2)将步骤(1)所得肿瘤靶向多肽偶联的聚二醇化磷脂酰乙醇胺、磷脂、胆固醇按摩尔比为(2~8):(42~78):(20~50)溶于氯仿和甲醇组成的复合溶剂中,得到混合溶液,复合溶剂的用量以所得混合溶液中磷脂和胆固醇的浓度为1~10mmol/L计,减压蒸馏除去混合溶液中的复合溶剂得到偶联有肿瘤靶向多肽的脂质双分子层膜;
(3)将偶联荧光染料的ATP敏感核苷酸单链和偶联荧光淬灭剂的ATP敏感核苷酸互补单链按摩尔比1:1加入去离子水得混合液,去离子水的用量以所述混合液中ATP敏感核苷酸单链的浓度为0.1~10mmol/L计,在室温下搅拌10~60分钟,使偶联荧光染料的ATP敏感核苷酸单链和偶联荧光淬灭剂的ATP敏感核苷酸互补单链根据碱基互补配对原则自组装形成ATP敏感核苷酸双链;
(4)将步骤(3)所得ATP敏感核苷酸双链与步骤(2)所得偶联有肿瘤靶向多肽的脂质双分子层膜按照质量比1:(10~50)混合后于40~60℃水化一小时,得到磷脂和胆固醇的浓度为1~10mmol/L的悬液,将所得悬液水浴超声5~30分钟,再依次过400纳米、200纳米和100纳米的多聚碳酸酯膜,然后采用凝胶排阻层析柱分离纯化后即得到具有肿瘤靶向和示踪作用的ATP敏感荧光探针脂质体。
9.根据权利要求8所述具有肿瘤靶向和示踪作用的ATP敏感荧光探针脂质体的制备方法,其特征在于:
所述末端为半胱氨酸的肿瘤靶向多肽为末端为半胱氨酸的含精氨酸-甘氨酸-天冬氨酸的环状多肽、末端为半胱氨酸的人类表皮生长因子受体2多肽、末端为半胱氨酸的转铁蛋白中的一种;
所述磷脂酰乙醇胺-聚乙二醇2000-马来酰亚胺为二硬脂酰磷脂酰乙醇胺-聚乙二醇2000-马来酰亚胺、二肉豆蔻酰磷脂酰乙醇胺-聚乙二醇2000-马来酰亚胺、二棕榈酰磷脂酰乙醇胺-聚乙二醇2000-马来酰亚胺、二月桂酰基磷脂酰乙醇胺-聚乙二醇2000-马来酰亚胺中的一种;
所述磷脂为卵磷脂、大豆磷脂、二硬脂酰磷脂胆碱中的一种;
所述偶联荧光染料的ATP敏感核苷酸单链为偶联荧光染料cy3、cy5、cy5.5或cy7的ATP敏感核苷酸单链,所述ATP敏感核苷酸单链的核苷酸序列为序列表中SEQ ID NO:1所述;
所述偶联荧光淬灭剂的ATP敏感核苷酸互补单链为偶联荧光淬灭剂4-(4’-二甲基氨基偶氮苯基)苯甲酸的ATP敏感核苷酸互补单链。
10.权利要求1~7中任一权利要求所述ATP敏感荧光探针脂质体在制备体内或体外肿瘤示踪剂中的应用。
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Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1846691A (zh) * | 2005-04-11 | 2006-10-18 | 北京大学 | 注射用的整合素配体修饰的载抗癌药的长循环脂质体 |
CN103341180A (zh) * | 2013-05-08 | 2013-10-09 | 上海纳米技术及应用国家工程研究中心有限公司 | 用于肿瘤成像的靶向脂质体递药系统及制备方法和应用 |
CN103371975A (zh) * | 2012-04-16 | 2013-10-30 | 上海现代药物制剂工程研究中心有限公司 | 靶向长循环脂质体制剂及其制备方法 |
CN105055315A (zh) * | 2015-08-03 | 2015-11-18 | 四川大学 | 一种交联线粒体靶向阿霉素脂质体及其制备方法 |
-
2016
- 2016-04-21 CN CN201610255250.9A patent/CN105797172B/zh active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1846691A (zh) * | 2005-04-11 | 2006-10-18 | 北京大学 | 注射用的整合素配体修饰的载抗癌药的长循环脂质体 |
CN103371975A (zh) * | 2012-04-16 | 2013-10-30 | 上海现代药物制剂工程研究中心有限公司 | 靶向长循环脂质体制剂及其制备方法 |
CN103341180A (zh) * | 2013-05-08 | 2013-10-09 | 上海纳米技术及应用国家工程研究中心有限公司 | 用于肿瘤成像的靶向脂质体递药系统及制备方法和应用 |
CN105055315A (zh) * | 2015-08-03 | 2015-11-18 | 四川大学 | 一种交联线粒体靶向阿霉素脂质体及其制备方法 |
Non-Patent Citations (5)
Title |
---|
ATP-triggered anticancer drug delivery;Ran Mo et al.;《NATURE COMMUNICATIONS》;20140311;第5卷;第1-10页 |
Engineering of Switchable Aptamer Micelle Flares for Molecular Imaging in Living Cells;Cuichen Wu et al.;《ACSNANO》;20130607;第7卷(第7期);第5725-5726页结果和讨论部分第1段 |
Enhanced Anticancer Efficacy by ATP-Mediated Liposomal Drug Delivery;Ran Mo et al.;《Angew. Chem. Int. Ed.》;20140424;第53卷;第5815页右栏第1段,图1 |
Mild hyperthermia triggered doxorubicin release from optimized stealth thermosensitive liposomes improves intratumoral drug delivery and efficacy;Li Li et al.;《Journal of Controlled Release》;20130321;第168卷;第142-150页 |
克服肿瘤生理病理屏障的纳米药物递送系统的研究进展;苏志桂 等.;《中国药科大学学报》;20151231;第46卷(第1期);第28-39页 |
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