CN111840254B - 纳米雄黄复合药物及其制备方法和应用 - Google Patents
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Abstract
本发明提供了一种纳米雄黄复合药物及其制备方法和应用,纳米雄黄复合药物主要包括As4S4、BSA、IMA和FA。其制备方法为以As4S4‑乙二胺分子簇溶液为前驱体,利用酸碱反应,使As4S4在BSA的稳定下成核生长,形成纳米粒。同时,利用疏水作用力,将IMA载于BSA的疏水结构域中。该纳米复合药物还可进一步进行靶向配体修饰,增加对肿瘤细胞的靶向性和治疗指数。本发明的纳米雄黄复合药物能下调BCR‑ABL蛋白水平,从线粒体途径诱导慢性粒细胞白血病细胞凋亡,且以特定比例将两种药物输送至治疗部位,实现疗效最大化,降低潜在毒副作用。
Description
技术领域
本发明涉及纳米生物技术领域,特别涉及一种纳米雄黄复合药物及其制备方法和应用。
背景技术
慢性粒细胞白血病(chronicmyeloidleukemia,CML)是一种起源于骨髓多功能造血干细胞的恶性克隆增殖性造血系统疾病,占所有白血病的15-20%。遗传学研究表明,95%以上的CML患者具有费城染色体(ph),其发病机制是由位于9号染色体上的原癌基因abl和22号染色体上的bcr(断裂丛集区)相互易位而形成t(9;22)(q34;q11),根据bcr基因断裂位点,可形成三种bcr-abl融合蛋白:p210、p230、p190。bcr-abl融合蛋白是一种异常的受体型酪氨酸激酶(PTK),可显著提高信号传导通路下游的特定底物蛋白分子的磷酸化水平,正常情况下,PTK的活性受到严格的调控,但在CML患者肿瘤细胞内,bcr-abl融合蛋白与ATP结合后,为一系列底物蛋白提供结合位点,使其酪氨酸磷酸化水平大大提高,进而激活多条信号传导通路,改变核内基因的表达,干扰细胞的正常生理功能,从而引起细胞的恶性转化。因此,以PTK为靶点的抗肿瘤治疗药物,是CML对因治疗的理想选择。
伊马替尼(imatinib,IMA)是第一个人工合成的针对CML发生机制的分子靶向药物,IMA通过竞争性抑制ATP及底物蛋白与bcr-abl融合蛋白催化中心区域的结合,阻止bcr-abl融合蛋白的活化,抑制其PTK活性,干扰CML细胞生存所依赖的信号传导通路,诱导CML细胞凋亡,但是其在慢性期患者中,IMA也难以根除恶性祖细胞,且长期用药后多数患者出现耐药性且易复发。
而雄黄(As4S4)在CML的治疗中具有缓解率高、生存期长和毒副作用低等优势,具有良好的应用潜能,且可通过降低BCR-ABL融合蛋白的表达,从而抑制其PTK的活性。因此As4S4能增强IMA对bcr-abl阳性白血病细胞的选择性杀伤作用,可从细胞生物学水平逆转IMA耐药,两者联用具有协同增效的效果,因此,构建一种高效包载As4S4和IMA的肿瘤靶向纳米递送系统,是实现两种药物协同增效的关键,为As4S4的新剂型研究提供新思路。
发明内容
本发明提供了一种纳米雄黄复合药物及其制备方法和应用,其目的是为了利用雄黄与伊马替尼的协同增效减毒的特性,拟构建一种叶酸修饰的纳米雄黄复合药物,期望其可通过CML细胞表面的叶酸受体特异性结合,选择性的在肿瘤部位累积促进细胞吞噬,降低雄黄对正常细胞的毒副作用,实现有效的治疗作用。
为了达到上述目的,本发明提供了一种纳米雄黄复合药物,所述复合药物分子式As4S4/IMA@BSA-FANPs,其中,As4S4、IMA、FA和BSA分别为雄黄、伊马替尼、叶酸和牛血清白蛋白,所述复合药物中As4S4、IMA分别负载于FA修饰的BSA上。
优选地,所述复合药物中,As4S4和IMA的摩尔比为2~18:1,BSA和As4S4的质量比为2~24:1。
优选地,所述复合药物的粒径为40~140nm。
本发明还提供了上述纳米雄黄复合药物的制备方法,包括以下步骤:
步骤1:将FA溶解后加入到BSA溶液中。制备BSA-FA;
步骤2:将IMA溶液加入到步骤1得到的BSA-FA中,制得IMA@BSA-FA;
步骤3:将As4S4溶于乙二胺中,得到As4S4-乙二胺分子簇溶液,然后加入到步骤2得到的IMA@BSA-FA中,制得纳米雄黄复合药物:As4S4/IMA@BSA-FANPs。
优选地,所述步骤1具体过程为:
步骤11:将FA,EDC,NHS按照摩尔比为1~20:2:1溶于DMSO中避光搅拌10~240min,得到浓度为25~250mg/ml的FA-NHS溶液;
步骤12:按照FA与BSA的摩尔比为1:10~100的比例将步骤11得到FA-NHS溶液缓慢滴加到BSA碳酸盐缓冲溶液中,在室温下搅拌12~36h后,得到BSA-FA混合液;
步骤13:将步骤12得到的BSA-FA混合液于浓度为10mM,PH=7.4的PBS中透析12~72h,再于水中透析8~48h,收集冻干后即得BSA-FA。
优选地,所述步骤2的具体过程为:
步骤21:将步骤1得到的BSA-FA溶于超纯水,得到浓度为20~120mg/ml的BSA-FA溶液;
步骤22:将IMA溶于甲醇中,得到浓度为2~18mg/ml的IMA溶液;
步骤23:将所述IMA溶液按体积比为1:3~14滴加到所述BSA-FA溶液中搅拌2~12h,即得IMA@BSA-FA溶液。
优选地,所述步骤3具体过程为:
步骤31:将As4S4溶于乙二胺中,超声5~60min得As4S4-乙二胺分子簇溶液,将As4S4-乙二胺分子簇溶液进行离心处理后,取上清液;
步骤32:将步骤31的上清液按照体积比为1:4~16的比例分散于水中,然后加入到步骤2得到的IMA@BSA-FA,搅拌2~20min,加入HCl溶液,再搅拌5~60min后,得到As4S4/IMA@BSA-FA溶液;
步骤33:将步骤32得到的As4S4/IMA@BSA-FA溶液于水中透析4~16h,即得纳米雄黄复合药物:As4S4/IMA@BSA-FANPs。
优选地,所述步骤31中,As4S4-乙二胺分子簇溶液浓度为5~80mg/ml;离心转速为10000~20000rpm,离心时间为5~20min;所述步骤32中,HCl溶液浓度为2~9M,HCl与As4S4的比例为4~12:1。
优选地,所述透析使用MWCO=3500Da的透析袋。
本发明还提供了上述纳米雄黄复合药物在制备治疗慢性粒细胞白血病药物中的应用。
本发明的上述方案有如下的有益效果:
1、本发明提供了一种纳米雄黄复合药物,以内源性BSA为载体,共载As4S4和IMA,既能实现两种药物高效包封,也能延长药物的体内滞留时间,并以FA对纳米体系进行靶向修饰,将药物选择性输送至治疗部位,增加药物在肿瘤部位的累积量,降低对正常组织的毒副作用,实现疗效最大化。本发明国内外均未见报道。
2、本发明提供了一种纳米雄黄复合药物的制备方法,其制备过程简单可控,为雄黄的新剂型研究提供新思路。
3、本发明提供了一种纳米雄黄复合药物在慢性粒细胞白血病的应用,以纳米载体共递送机制互补的两种药物As4S4和IMA,通过促进慢性粒细胞白血病细胞凋亡抑制肿瘤的生长,实现治疗的增效减毒,可以为慢性粒细胞白血病的治疗提供新途径。
附图说明
图1为本发明实施例1~3得到的产物中BSA和As4S4的质量浓度比图。
图2为本发明实施例2~3得到的产物的粒径与电位图。
图3为本发明实施例3中得到的As4S4/IMA@BSA-FANPs的透射电镜图。
图4为本发明实施例1~3得到的产物中BSA,BSA-FA,FA,As4S4@BSA-FA NPs,As4S4/IMA@BSA-FANPs,IMA的紫外光谱图。
图5为本发明实施例2~3得到的产物在PBS、IMDM完全培养基中的粒径变化图。
图6为本发明实施例4中As4S4/IMA@BSA-FANPs在激光共聚焦显微镜下的成像图。
图7为本发明实施例5中IMA、As4S4@BSANPs、As4S4@BSA-FANPs和As4S4/IMA@BSA-FANPs的细胞存活率结果。
图8为本发明实施例5中IMA、As4S4@BSANPs、As4S4@BSA-FANPs和As4S4/IMA@BSA-FANPs的IC50结果。
图9为本发明实施例6中IMA、As4S4@BSANPs、As4S4@BSA-FANPs和As4S4/IMA@BSA-FANPs与K562细胞孵育48h后的细胞凋亡情况图。
图10为本发明实施例7中IMA、As4S4@BSANPs、As4S4@BSA-FANPs和As4S4/IMA@BSA-FANPs与K562细胞孵育48h后的线粒体跨膜电位的变化图。
图11为本发明实施例8中IMA、As4S4@BSANPs、As4S4@BSA-FANPs和As4S4/IMA@BSA-FANPs与K562细胞孵育48h后的BCR-ABL蛋白的表达情况。
图12为本发明实施例9中纳米雄黄复合药物的体内疗效评价结果。
具体实施方式
为使本发明要解决的技术问题、技术方案和优点更加清楚,下面将结合附图及具体实施例进行详细描述。
实施例1
一种纳米雄黄复合药物,包括雄黄、牛血清白蛋白。
本实施例的纳米雄黄复合药物采用以下方法制备得到:
将As4S4溶于乙二胺中,浓度为20mg/mL,超声15min,15000rpm离心5min,取上清液。将2mLAs4S4上清液分散于8mL水中,加入3ml,20mg/ml BSA溶液,搅拌5min,加入7mL4MHCl,搅拌30min,用MWCO=3500Da的透析袋于水中透析8h,即得纳米雄黄复合药物:As4S4@BSANPs。
实施例2
一种纳米雄黄复合药物,包括雄黄,牛血清白蛋白,叶酸。
本实施例的纳米雄黄复合药物采用以下方法制备得到:
(1)按FA,EDC,NHS的摩尔比:1:2:1溶于DMSO中避光搅拌30min,得到浓度为25mg/ml的FA,按照FA与BSA的摩尔比为1:50的比例将FA溶液缓慢滴加到BSA碳酸盐缓冲溶液中,室温搅拌24h后,用MWCO=3500Da的透析袋于PBS(10mm,PH=7.4)中透析2d,水中透析1d,收集冻干后即得BSA-FA。
(2)将As4S4溶于乙二胺中,浓度为20mg/mL,超声15min,15000rpm离心5min,取上清液。同时,将冻干的60mgBSA-FA用3ml超纯水溶解,再将2mLAs4S4上清液分散于8mL水中,再缓慢滴加BSA-FA溶液,搅拌5min,加入7mL4MHCl,搅拌30min,用MWCO=3500Da的透析袋于水中透析8h,即得纳米雄黄复合药物:As4S4@BSA-FANPs。
实施例3
一种纳米雄黄复合药物,包括雄黄,伊马替尼,牛血清白蛋白,叶酸。
本实施例的纳米雄黄复合药物采用以下制备方法制备得到:
(1)按FA,EDC,NHS的摩尔比:1:2:1溶于DMSO中避光搅拌30min,得到浓度为25mg/ml的FA,按照FA与BSA的摩尔比为1:50的比例将FA溶液缓慢滴加到BSA碳酸盐缓冲溶液中,室温搅拌24h后,用MWCO=3500Da的透析袋于PBS(10mm,PH=7.4)中透析2d,水中透析1d,收集冻干后即得BSA-FA。
(2)将1ml5mg/mlIMA溶于甲醇中,同时将冻干的60mgBSA-FA用3ml超纯水溶解,然后将溶解的IMA缓慢滴加到BSA-FA溶液中搅拌3h,即得IMA@BSA-FA溶液。
(3)将As4S4溶于乙二胺中,浓度为20mg/mL,超声15min,15000rpm离心5min,取上清液。将2mLAs4S4上清液分散于8mL水中,加入上述所得IMA@BSA-FA溶液,搅拌5min,加入7mL4MHCl,搅拌30min,用MWCO=3500Da的透析袋于水中透析8h,即得纳米雄黄复合药物:As4S4/IMA@BSA-FANPs。
实施例1~3所得产物的测试
一、质量浓度比:稳定剂BSA与As4S4的比例探索。如图1所示,当BSA与雄黄的质量浓度比达到2:1以上时,产物粒径趋于稳定,综合考虑,BSA与雄黄的质量浓度比优选为2~10:1。
二、粒径与电位:分别检测As4S4@BSA-FANPs和As4S4/IMA@BSA-FA NPs的粒径和电位,测量方法为:取样品溶液置于MarlvenNanoZS仪器,采用动态光激光散射法检测粒径和电位,测定池温度设定为25℃,每个样品平行操作3份。图2为As4S4@BSA-FANPs、As4S4/IMA@BSA-FANPs的粒径及电位变化图,从图的结果可知:As4S4@BSA-FANPs粒径为58.3±0.93nm,电位为-16.8±1.43mV,而As4S4/IMA@BSA-FA粒径增至68.0±0.93nm,电位降至-20.2±1.24mV。
三、形态:观察As4S4/IMA@BSA-FANP的形态,形态的检测方法:样品滴加在覆盖碳膜的400目铜网上,置于干燥器中,待其自然干燥后置于透射电镜TitanG2-F20下观察。图3为As4S4/IMA@BSA-FANPs的透射电镜图。
四、紫外光谱:分别对As4S4/IMA@BSA-FANPs,As4S4@BSA-FANPs,BSA,FA,BSA-FA进行紫外光谱扫描,测定方法为:以蒸馏水为空白对照液,测定As4S4@BSA-FANPs,As4S4/IMA@BSA-FANPs,BSA,FA,BSA-FA的紫外吸收图谱。图4A为As4S4/IMA@BSA-FANPs,As4S4/IMA@BSA-FANPs,BSA,FA,BSA-FA的紫外吸收图谱,从图中可知:FA成功修饰在BSA上,BSA成功包载As4S4。
IMA测定方法:以甲醇为空白对照液,将As4S4@BSA-FANPs,As4S4/IMA@BSA-FANPs冻干,用甲醇复溶,测定As4S4/IMA@BSA-FANPs,As4S4@BSA-FANPs,IMA的紫外吸收光谱。图4B为As4S4/IMA@BSA-FANPs,As4S4@BSA-FANPs,IMA的紫外吸收光谱,从图中可知,纳米雄黄复合药物成功包载IMA。
五、稳定性检测:将As4S4/IMA@BSA-FANPs和As4S4@BSA-FANPs分别在37℃下置于PBS和含10%胎牛血清(FBS)的IMDM完全培养基,在不同时间点测定二者的粒径。图5为As4S4/IMA@BSA-FANPs和As4S4@BSA-FA NPs在PBS、IMDM完全培养基中的粒径变化图,从图中可知:As4S4/IMA@BSA-FANPs和As4S4@BSA-FANPs粒径均无显著性变化,说明As4S4/IMA@BSA-FANPs和As4S4@BSA-FANPs在PBS溶液及血浆中的稳定性良好。
实施例4
实施例1、实施例2和实施例3的纳米雄黄复合药物对人慢性髓源白血病细胞的靶向作用:
(1)分别制备载荧光染料FITC的纳米雄黄复合药物As4S4/IMA@FITC-BSANPs和As4S4/IMA@FITC-BSA-FANPs。具体步骤为:
1.1、取FITC,EDC,NHS的摩尔比1:30:10溶于3mlDMSO中避光搅拌30min,缓慢滴加到BSA碳酸盐缓冲溶液中。室温搅拌24h后,用MWCO=3500Da的透析袋于PBS(10mm,PH=7.4)中透析2d,水中透析1d,收集冻干后即得FITC-BSA,再将IMA溶于甲醇中,浓度为5mg/ml,滴加到3ml20mg/mlFITC-BSA溶液中搅拌3h,即得IMA@FITC-BSA,
将As4S4溶于乙二胺中,浓度为20mg/mL,超声15min,15000rpm离心5min,取上清。将2mLAs4S4溶液上清分散于8mL水中,加入所得IMA@FITC-BSA溶液,搅拌5min,加入7mL4MHCl,搅拌30min,用MWCO=3500Da的透析袋于水中透析8h,即得纳米雄黄复合药物:As4S4/IMA@FITC-BSANPs。
1.2、取FITC,FA,EDC,NHS的摩尔比1:10:30:10溶于3mlDMSO中避光搅拌30min,缓慢滴加到BSA碳酸盐缓冲溶液中。室温搅拌24h后,用MWCO=3500Da的透析袋于PBS(10mm,PH=7.4)中透析2d,水中透析1d,收集冻干后即得FITC-BSA,再将IMA溶于甲醇中,浓度为5mg/ml,滴加到3ml20mg/mlFITC-BSA-FA溶液中搅拌3h,即得IMA@FITC-BSA-FA,再将As4S4溶于乙二胺中,浓度为20mg/mL,超声15min,15000rpm离心5min,取上清。将2mLAs4S4溶液上清分散于8mL水中,加入所得IMA@FITC-BSA-FA溶液,搅拌5min,加入7mL4MHCl,搅拌30min,用MWCO=3500Da的透析袋于水中透析8h,即得纳米雄黄复合药物:As4S4/IMA@FITC-BSA-FANPs。。
(2)取对数生长的K562细胞(人慢性髓源白血病细胞,购自中南大学湘雅医学实验中心),计数,适量无胎牛血清(FBS)的IMDM培养基稀释至2×105cells/mL的细胞悬液,每孔1mL接种于6孔板,总共接种4个孔。
(3)取一个孔加入1mL2mg/mL游离FA(无FBS的IMDM溶解),其余3孔加入1mL无FBS的IMDM。孵育4h后离心吸弃培基,PBS润洗3次。
(3)分别将As4S4/IMA@FITC-BSANPs、As4S4/IMA@FITC-BSA-FANPs用IMDM培养基(无FBS)稀释成1μg/ml(以As4S4计)的样品溶液。
(4)进行FA干预的一个孔加入2mLAs4S4/IMA@FITC-BSA-FANPs,其余三个孔分别加入2ml无FBS的IMDM、As4S4/IMA@FITC-BSANPs、As4S4/IMA@FITC-BSA-FANPs。37℃孵育4h后吸弃培基,PBS润洗3次,激光共聚焦显微镜下观察各孔荧光强弱。
图6为As4S4/IMA@BSA-FANPs的细胞摄取。其中,载荧光染料FITC的As4S4/IMA@BSANPs、As4S4/IMA@BSA-FANPs分别与K562细胞共孵育4h;2mg/mLFA预处理K562细胞4h,As4S4/IMA@BSA-FANPs再与细胞孵育4h,激光共聚焦显微镜观察。FITC通道表明NPs标记为绿色荧光,Merged表明叠加白场和FITC通道。FA表示游离FA预处理。标尺=50μm。
从图中可知:两种纳米制剂与k562细胞孵育4h,荧光显微镜下可见细胞内有明显的绿色荧光,表明纳米粒被细胞摄取,其中As4S4/IMA@FITC-BSA-FA NPs孔荧光较As4S4/IMA@FITC-BSANPs孔强。然而,游离FA预处理4h后,其绿色显著减少。结果说明叶酸修饰的纳米雄黄复合药物对慢性粒细胞白血病细胞具有主动靶向作用,叶酸修饰后能增强对纳米雄黄复合药物的摄取。
实施例5
实施例1、实施例2和实施例3的纳米雄黄复合药物对人慢性髓源白血病细胞K562的细胞毒性:
(1)取对数生长的K562细胞,用含10%胎牛血清的IMDM培养基稀释成密度为50000cells/mL细胞悬液,以每孔100μL接种到96孔培养板中。在二氧化碳培养箱内(37℃、5%CO2、饱和湿度)培养2h后离心移弃培养液。
(2)每孔加入100μL用培养基稀释至不同浓度的As4S4/IMA@BSA-FA,As4S4@BSA-FA,As4S4@BSA,IMA(浓度以As4S4的浓度计,分别为0、0.5、1、5、10、20、50μg/ml),同一浓度重复6个复孔,孵育48h。
(3)离心除去培基,每孔加入100μLMTT溶液(0.5mg/mL,IMDM稀释),继续孵育4h后终止培养,吸弃上清液。
(4)每孔加入DMSO溶液100μL,置摇床上低速振摇10min使结晶溶解完全,用酶标仪测定570nm波长处的吸光值(OD)。
图7为As4S4/IMA@BSA-FANPs,As4S4@BSA-FANPs,As4S4@BSANPs,IMA与细胞孵育48h后,用MTT法测定细胞存活率结果。从图7可以看出,本发明的As4S4/IMA@BSA-FANPs,As4S4@BSA-FANPs,As4S4@BSANPs的细胞存活率均为剂量依赖性。
图8为IC50结果。数据以均数±标准差表示(n=6);从IC50值可以看出,As4S4/IMA@BSA-FANPs对K562细胞的细胞毒性比As4S4@BSA-FANPs,As4S4@BSANPs大。
从毒性实验的结果可知:本发明实施例1、实施例2和实施例3的纳米雄黄复合药物能够抑制肿瘤细胞增殖,这说明本发明的纳米雄黄复合药物具有一定的体外抗慢性粒细胞白血病的疗效,可以作为用于抑制慢性粒细胞白血病生长的药物。
实施例6
纳米雄黄复合药物对人慢性髓源白血病细胞K562的细胞凋亡:
(1)取对数生长的K562细胞,计数,适量IMDM培养基稀释至106cells/mL的细胞悬液,每孔1mL接种于6孔板,总共接种5个孔。
(2)每孔加入1ml0.99μg/mlAs4S4/IMA@BSA-FANPs,As4S4@BSA-FA NPs,As4S4@BSANPs,IMA(以As4S4),重复3个复孔,孵育48h。
(3)用EP管收集细胞,1200rpm5min离心,用PBS洗两次,离心除去培基。
(4)用200μl1*的BindingBuffer重悬细胞,每管加入5μlAnnexin V-FITC和5μlPI,室温,避光,孵育15min,加入Buffer至300μL,轻轻混匀
(5)最后在1h内使用用流式细胞仪检测。
图9As4S4/IMA@BSA-FANPs,As4S4@BSA-FANPs,As4S4@BSANPs,IMA处理48h后K562细胞凋亡情况。从图中可以看出As4S4/IMA@BSA-FA的凋亡情况最严重,表明本发明的纳米雄黄复合药物会促进K562细胞早期凋亡,从而治疗慢性粒细胞白血病。
实施例7
纳米雄黄复合药物对人慢性髓源白血病细胞K562线粒体跨膜电位的影响:
(1)取对数生长的K562细胞,计数,适量IMDM培养基稀释至106cells/mL的细胞悬液,每孔1mL接种于6孔板,总共接种5个孔。
(2)每孔加入1ml0.99μg/mlAs4S4/IMA@BSA-FANPs,As4S4@BSA-FA NPs,As4S4@BSANPs,IMA(以As4S4),重复3个复孔,孵育48h。
(3)用EP管收集细胞,1200rpm5min离心,用PBS洗两次,离心除去培基。
(4)JC-1染色工作液的配制:六孔板每孔所需JC-1染色工作液的量为1ml,对于细胞悬液每50-100万细胞需0.5mlJC-1染色工作液。取适量JC-1(200X),按照每50μlJC-1(200X)加入8ml超纯水的比例稀释JC-1。剧烈Vortex充分溶解并混匀JC-1。然后再加入2mlJC-1染色缓冲液(5X),混匀后即为JC-1染色工作液。
(5)阳性对照的设置:把试剂盒中提供的CCCP(10mM)推荐按照1:1000的比例加入到细胞培养液中,稀释至10μM,处理细胞20分钟。随后按照下述方法装载JC-1,进行线粒体膜电位的检测。对于大多数细胞,通常10μM CCCP处理20分钟后线粒体的膜电位会完全丧失,JC-1染色后观察应呈绿色荧光;而正常的细胞经JC-1染色后应显示红色荧光。
(6)取10-60万细胞,重悬于0.5ml细胞培养液中,细胞培养液中可以含血清和酚红。加入0.5mlJC-1染色工作液,颠倒数次混匀。细胞培养箱中37℃孵育20分钟。
(7)在孵育期间,按照每1mlJC-1染色缓冲液(5X)加入4ml蒸馏水的比例,配制适量的JC-1染色缓冲液(1X),并放置于冰浴,37℃孵育结束后,1000rpm4℃离心3-4分钟,沉淀细胞。弃上清,注意尽量不要吸除细胞。
(8)用JC-1染色缓冲液(1X)洗涤2次:加入1mlJC-1染色缓冲液(1X)重悬细胞,1000rpm4℃离心3-4分钟,沉淀细胞,弃上清。再加入1mlJC-1染色缓冲液(1X)重悬细胞,1000rpm4℃离心3-4分钟,沉淀细胞,弃上清。
(9)再用适量JC-1染色缓冲液(1X)重悬后,用荧光显微镜或激光共聚焦显微镜观察。
图10为As4S4/IMA@BSA-FANPs,As4S4@BSA-FANPs,As4S4@BSANPs,IMA处理48h后K562细胞的线粒体跨膜电位情况。从图中可以看出As4S4/IMA@BSA-FANPs的电位下降最严重,表明本发明的纳米雄黄复合药物可通过降低线粒体跨膜电位来促进细胞凋亡。
实施例8
纳米雄黄复合药物对人慢性髓源白血病细胞K562的细胞效应:
(1)取对数生长的K562细胞,计数,适量IMDM培养基稀释至106cells/mL的细胞悬液,每孔1mL接种于6孔板,总共接种5个孔。
(2)每孔加入1ml0.99μg/mlAs4S4/IMA@BSA-FANPs,As4S4@BSA-FA NPs,As4S4@BSANPs,IMA(以As4S4),重复3个复孔,孵育48h。
(3)使用Western裂解液裂解K562细胞,收集细胞中的蛋白样品,测定蛋白样品的蛋白浓度。
(3)配制SDS-PAGE凝胶,在收集的蛋白样品中加入适量浓缩的SDS-PAGE蛋白上样缓冲液,100℃或沸水浴加热3-5分钟,以充分变性蛋白。
(4)冷却至室温后把蛋白样品直接上样到SDS-PAGE胶加样孔内进行电泳,溴酚蓝到达胶的底端处附近停止电泳。
(5)选用PVDF膜进行转膜,使用Bio-Rad的标准湿式转膜装置,转膜完毕后,加入5%脱脂牛奶室温封闭1h。
(6)吸尽封闭液,加入稀释好的一抗,室温孵育过夜。回收一抗,加入Western洗涤液,洗涤3次。
(7)按照适当比例用Western二抗稀释液稀释辣根过氧化物酶(HRP)标记的二抗。吸尽洗涤液,加入稀释好的二抗,室温孵育1h。洗涤3次。
(8)最后使用BeyoECLPlus(P0018)等ECL类试剂检测蛋白。
图11为浓度分别为As4S4/IMA@BSA-FANPs,As4S4@BSA-FANPs,As4S4@BSANPs,IMA与K562细胞孵育48h后,BCR-ABL蛋白表达情况。从图中可以看出As4S4可降低BCR-ABL蛋白的表达,而IMA不能,但两者联用后降低更甚,表明本发明的纳米雄黄复合药物可降低BCR-ABL蛋白的表达,协同治疗慢性粒细胞白血病。
实施例9
纳米雄黄复合药物的体内抗肿瘤活性:
(1)建立荷瘤裸鼠模型:收集对数生长的K562细胞分散于PBS中,细胞密度为1×107/100μL,等体积与基质胶混合,注射于BALB/c裸鼠(雌性,6周)的腋下部位。雌性BALB/c裸鼠,6周龄,购自常州卡文斯实验动物有限公司。
(2)待荷瘤小鼠肿瘤长至200mm3左右时,将小鼠随机分成5组(n=6),每组在第0、3、6、9天分别注射PBS,IMA,As4S4/IMA@BSA-FANPs,As4S4@BSA-FANPs,As4S4@BSANPs(As4S4,10mg/kg),每两天给小鼠称重并用游标卡尺测量肿瘤的体积至第14天,通过各组肿瘤的相对体积比较各组抗肿瘤效率。肿瘤体积计算公式:V=长×宽2/2。
图12为分别在第0、3、6、9天尾静脉注射PBS,IMA,As4S4/IMA@BSA-FA NPs,As4S4@BSA-FANPs,As4S4@BSANPs后的肿瘤体积变化曲线。从图中可知,与PBS组相比IMA,As4S4/IMA@BSA-FANPs,As4S4@BSA-FANPs,As4S4@BSANPs均具有一定的抗肿瘤效果,其中As4S4@BSA-FANPs肿瘤抑制效果稍强于As4S4@BSANPs组,As4S4/IMA@BSA-FANPs肿瘤抑制效果稍强于每组。结果表明,纳米雄黄复合药物可显著抑制肿瘤生长,协同治疗慢性粒细胞白血病。
以上所述是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明所述原理的前提下,还可以作出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (10)
1.一种纳米雄黄复合药物,其特征在于,所述复合药物分子式为As4S4/IMA@BSA-FANPs,其中,As4S4、IMA、FA和BSA分别为雄黄、伊马替尼、叶酸和牛血清白蛋白,所述复合药物中As4S4、IMA分别负载于FA修饰的BSA上。
2.根据权利要求1所述的纳米雄黄复合药物,其特征在于,所述复合药物中,As4S4和IMA的摩尔比为2~18∶1,BSA和As4S4的质量比为2~24∶1。
3.根据权利要求1所述的纳米雄黄复合药物,其特征在于,所述复合药物的粒径为40~140nm。
4.一种制备权利要求1~3任一项所述纳米雄黄复合药物的方法,其特征在于,包括以下步骤:
步骤1:将FA溶解后加入到BSA溶液中,制备BSA-FA;
步骤2将IMA溶液加入到步骤1得到的BSA-FA中制得IMA@BSA-FA;
步骤3:将As4S4溶于乙二胺中,得到As4S4-乙二胺分子簇溶液,然后加入到步骤2得到的IMA@BSA-FA中,制得纳米雄黄复合药物:As4S4/IMA@BSA-FA NPs。
5.根据权利要求4所述的纳米雄黄复合药物的制备方法,其特征在于,所述步骤1具体过程为:
步骤11:将FA,EDC,NHS按照摩尔比为1~20∶2∶1溶于DMSO中避光搅拌10~240min,得到浓度为25~250mg/ml的FA-NHS溶液;
步骤12:按照FA与BSA的摩尔比为1∶10~100的比例将步骤11得到FA-NHS溶液缓慢滴加到BSA碳酸盐缓冲溶液中,在室温下搅拌12~36h后,得到BSA-FA混合液;
步骤13:将步骤12得到的BSA-FA混合液于浓度为10mM,PH=7.4的PBS中透析12~72h,再于水中透析8~48h,收集冻干后即得BSA-FA。
6.根据权利要求4所述的纳米雄黄复合药物的制备方法,其特征在于,所述步骤2的具体过程为:
步骤21:将步骤1得到的BSA-FA溶于超纯水,得到为20~120mg/ml BSA-FA溶液;
步骤22:将IMA溶于甲醇中,得到浓度为2~18mg/ml的IMA溶液;
步骤23:将所述IMA溶液按体积比为1∶3~14滴加到所述BSA-FA溶液中搅拌2~12h,即得IMA@BSA-FA溶液。
7.根据权利要求4所述的纳米雄黄复合药物的制备方法,其特征在于,所述步骤3具体过程为:
步骤31:将As4S4溶于乙二胺中,超声5~60min得As4S4-乙二胺分子簇溶液,将As4S4-乙二胺分子簇溶液进行离心处理后,取上清液;
步骤32:将步骤31的上清液按照体积比为1∶4~16的比例分散于水中,然后加入到步骤2得到的IMA@BSA-FA,搅拌2~20min,加入HCl溶液,再搅拌5~60min后,得到As4S4/IMA@BSA-FA溶液;
步骤33:将步骤32得到的As4S4/IMA@BSA-FA溶液于水中透析4~16h,即得纳米雄黄复合药物:As4S4/IMA@BSA-FA NPs。
8.根据权利要求7所述的纳米雄黄复合药物的制备方法,其特征在于,所述步骤31中,As4S4-乙二胺分子簇溶液浓度为5~80mg/ml;离心转速为10000~20000rpm,离心时间为5~20min;所述步骤32中,HCl溶液浓度为2~9M,HCl与As4S4的比例为4~12∶1。
9.根据权利要求5或7任一项所述的纳米雄黄复合药物的制备方法,其特征在于,所述透析使用MWCO=3500Da的透析袋。
10.一种根据权利要求1~3任一项所述的纳米雄黄复合药物或由权利要求4~9任一项所述的方法制备而成的纳米雄黄复合药物在制备治疗慢性粒细胞白血病药物中的应用。
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