CN102138927B - 氯喹和阿霉素共载脂质体及其制备方法 - Google Patents
氯喹和阿霉素共载脂质体及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种氯喹和阿霉素共载脂质体,由药物、磷脂、胆固醇类化合物、内部缓冲系统和pH值调节剂制成;其中,药物、磷脂和胆固醇类化合物的重量比为1∶2~100∶1~35;所述的药物为氯喹类药物和阿霉素类药物。利用氯喹和阿霉素的协同作用,使氯喹和阿霉素共载脂质体具有抑制多药耐药性和降低用药毒性的特点。本发明还公开了一种氯喹和阿霉素共载脂质体的制备方法,制备工艺简单,控制的参数较少,反应条件较低,有利于降低生产成本,易于工业化生产。
Description
技术领域
本发明涉及脂质体类药剂技术领域,特别涉及一种氯喹和阿霉素共载脂质体及其制备方法。
背景技术
近50年来,癌症的防治已经有重大的进展,但是由于癌症早期诊断困难,以及缺少治疗癌症转移的有效技术方案,治愈癌症仍然是一个难题,因此,寻找癌症治疗的新方法显得非常重要和迫切。
阿霉素是癌症化疗常用的药物(Multidrug resistance in cancer:role ofATP-dependent transporter),上世纪60年代初由米兰Faroitalia研究实验室首次从一种链霉菌中分离而得,是继柔红霉素后被发现的第二个蒽环霉素类抗生素。阿霉素和柔红霉素的结构基本相同,差异只在一个羟基上,但柔红霉素只对急性白血病有效,而阿霉素具有广谱抗恶性肿瘤的治疗效果。自1974年阿霉素在美国获准临床使用以来,在过去的30年中己报道2000种以上的阿霉素类似物经历抗肿瘤作用的试验性筛选。但是,阿霉素等药物的大量使用容易使细胞产生耐药性,这是当下临床上成功地治疗癌症的一大障碍,为解决这些问题,目前主要尝试采用以下方法来逆转多药耐药性(MDR):1)MDR逆转剂;2)化疗药物的化学修饰;3)联用化疗敏感药物;4)运用纳米粒载体及后两者相结合。
一直以来氯喹(CQ)是作为一种安全、有效且价廉的预防和治疗疟原虫感染的药物使用,也常常用于基因转染实验中,以提高转染效率。已有国外文献报道,CQ可作为多药耐药蛋白1(MRP1)的抑制剂,与阿霉素联用,可降低细胞的多药耐药性,从而达到较好的抑癌效果(Reversal ofMRP-Mediated Doxorubicin Resistance with Quinoline-Based Drugs)。多药耐药蛋白1(MRP1)属于ATP结合盒(ABC)的转运家庭,它能够广泛性地将胞内的抗癌药物泵出细胞外,从而大大削弱药物的抗癌作用,使细胞产生耐药(Pharmacogenomics of ABC transporters and its role in cancerchemotherapy)。但早期研究已表明注射使用氯喹溶液毒性很大,甚至危及生命,还有阿霉素也具有较大的心脏毒性及对正常细胞的毒性作用,降低剂量又很难达到预期效果,氯喹与阿霉素联用也存在上述问题,迫切需要解决。
在过去的20年里,控制药物释放和肿瘤靶向的药物运载系统作为一种有吸引力的治疗细胞毒素的治疗窗口出现了。在运用的载体中,有纳米粒、白蛋白微球以及脂质体。脂质体作为一种化疗药物载体最近在牛身上被重新探讨,以此开始了脂质体在控制药物分散方面的使用,用来改进抗癌效率和减少对正常细胞的毒性。药物用脂质体包裹后,能靶向作用于病变部位,从而提高药物的治疗指数,同时还可以减少药物的治疗剂量,降低全身不良反应,提高用药安全性。目前,已批准临床使用或正在进行临床评价的阿霉素制剂主要是脂质体制剂,如用于再发性乳腺癌的联合治疗的产品名为Mycet的阿霉素脂质体,虽然一定程度上降低了阿霉素对心脏毒性及对正常细胞的毒性,但是仍存在着容易使细胞产生耐药性以致抗癌效果不佳的问题。
发明内容
本发明提供了一种载体选择面广、药物包封率高的氯喹和阿霉素共载脂质体,利用氯喹和阿霉素的协同作用,克服了由MRP1介导的多药耐药性并降低了阿霉素和氯喹以游离形式使用时不可忽视的毒性。
本发明还提供了一种氯喹和阿霉素共载脂质体的制备方法,采用pH梯度法将氯喹和阿霉素两者同时装载入脂质体制得氯喹和阿霉素共载脂质体,制备工艺简单,控制的参数较少,反应条件较低,有利于降低生产成本,易于工业化生产。
一种氯喹和阿霉素共载脂质体,由药物、磷脂、胆固醇类化合物、内部缓冲系统和pH值调节剂制成;其中,药物、磷脂和胆固醇类化合物的重量比为1∶2~100∶1~35;
所述的药物为氯喹类药物和阿霉素类药物。
优选的,所述的药物、磷脂和胆固醇类化合物的重量比为1∶10~40∶2.5~10;进一步优选为1∶25∶5。
所述的氯喹类药物和阿霉素类药物为主要的药效成分,氯喹类药物和阿霉素类药物的比例可以根据实际需要任意调整,氯喹类药物和阿霉素类药物的质量比可以是1~100∶1~100,优选为1~2∶1~5。
所述的氯喹类药物包括氯喹、氯喹与酸所成的盐中一种或多种;所述的氯喹与酸所成的盐可选用磷酸氯喹、盐酸氯喹、硫酸氯喹中一种或多种。
所述的阿霉素类药物包括阿霉素、阿霉素与酸所成的盐中一种或多种;所述的阿霉素与酸所成的盐可选用盐酸阿霉素。
所述的磷脂可选用天然磷脂、合成磷脂中的一种或两种;所述的天然磷脂为大豆磷脂、卵磷脂中的一种或两种;所述的合成磷脂为中性磷脂、负电性磷脂或聚乙二醇修饰磷脂中的至少一种,合成磷脂具体为二豆蔻酰磷脂酰胆碱(DMPC)、二棕榈酰磷脂酰胆碱(DPPC)、二硬脂酰磷脂酰胆碱(DSPC)、二棕榈酰磷脂酰甘油酯(DPPG)、磷脂酰乙醇胺(PE)、磷脂酰丝氨酸(PS)、二硬脂酰磷脂酰乙醇胺-聚乙二醇(DSPE-PEG)中的至少一种。
所述的胆固醇类化合物为普通胆固醇、聚乙二醇(PEG)修饰胆固醇中的一种或者两种,所述的聚乙二醇(PEG)修饰胆固醇如聚乙二醇单甲醚胆固醇琥珀酸酯(CHS-PEG)。
所述的内部缓冲系统可选用柠檬酸盐缓冲系统、磷酸盐缓冲系统或碳酸盐缓冲系统;所述的柠檬酸盐缓冲系统优选为0.05mol/L~0.5mol/L的柠檬酸-柠檬酸钠缓冲液。所述的内部缓冲系统主要是用于乳化磷脂和胆固醇得到脂质体,其用量并没有严格的限制,一般每1重量份的药物中添加5mL~15mL。
所述的pH值调节剂为碱液,可选用氢氧化钠水溶液、磷酸氢二钠水溶液、碳酸钠水溶液、碳酸氢钠水溶液等中的一种或多种,碱液的浓度一般为0.05mol/L~0.5mol/L,以方便调节pH值。所述的pH值调节剂用于调节氯喹和阿霉素共载脂质体的pH值在5.5~8.0,pH值太小则磷脂双份子层内外形成不了浓度差,药物进不了脂质体内部,pH过大则会产生脂质体破裂等问题,优选调节pH值为7。
所述的氯喹和阿霉素共载脂质体的制备方法,包括以下步骤:
1)将磷脂和胆固醇类化合物溶于有机溶剂中,再加入内部缓冲系统,混合均匀,除去有机溶剂,得到空白脂质体悬液;
或者,将磷脂和胆固醇类化合物溶于有机溶剂中,混合均匀,除去有机溶剂,再加入内部缓冲系统混合均匀,得到空白脂质体悬液;
2)将氯喹和阿霉素溶解在步骤1)制备的空白脂质体悬液中,用pH值调节剂调节pH至5.5~8.0,在30℃~50℃水浴孵育10min~60min,分离出游离药物,得到氯喹和阿霉素共载脂质体。
步骤1)中,所述的有机溶剂选用乙醚或者氯仿(三氯甲烷)。
步骤1)中,除去有机溶剂后或者除去有机溶剂再加入内部缓冲系统后,可通过高压匀乳、超声或微射流进行粉碎,得到粒径低于200nm的空白脂质体悬液。
步骤2)中,分离出游离药物的方法可采用Sephadex G-50凝胶柱层析法或透析法。
所述的氯喹和阿霉素共载脂质体可作为抗癌药物治疗癌症等疾病,对肿瘤细胞具有较高的杀伤力。
本发明采用动态光散射粒径仪(Malvern Zetasizer Nano-S90,英国)检测粒径。
本发明具有如下优点:
(1)磷脂选择面广,可使用天然卵磷脂或大豆磷脂,也可使用合成的中性磷脂或负电性磷脂;(2)通过本发明方法制得的氯喹和阿霉素共载脂质体药物包封率可达到90%以上,粒径可低于200nm,具有包封率高、稳定性好、成本低等优点;(3)本发明方法制得的氯喹和阿霉素共载脂质体,能够有效抑制由MRP1介导的多药耐药性,对因MRP1高表达而产生耐药性的细胞具有耐药逆转作用,逆转倍数达10倍左右,是阿霉素脂质体的3倍左右;(4)在氯喹的毒性范围内(<20μg/ml),氯喹和阿霉素共载脂质体随着氯喹的量的增多,IC50降低;(5)氯喹和阿霉素共载脂质体使氯喹和阿霉素协同作用,从而提高了对肿瘤细胞的杀伤力,并且氯喹和阿霉素的协同作用相对可以减少药物的使用量,减小了毒副作用;(6)利用阿霉素和氯喹的pKa值相近,都在4.0左右,采用独特的两步pH梯度法将两者同时装载入脂质体,制备氯喹和阿霉素共载脂质体,制备方法操作简便,可控性和重现性好。
具体实施方式
下面结合具体实施例对本发明作进一步说明,但不作为对本发明权利范围的限制。
空白脂质体的制备
实施例1
称取500mg大豆磷脂(磷脂酰胆碱纯度>76%)和100mg胆固醇溶解于20mL乙醚中,再加入10mLpH=4.0柠檬酸-柠檬酸钠缓冲液,混合均匀进行乳化,然后减压蒸去有机溶剂乙醚,用细胞粉碎仪超声5min,得到10mL空白脂质体悬液,备用,作为空白脂质体。测得该空白脂质体悬液的平均粒径(数均)为129nm。
实施例2
称取420mg二棕榈酰磷脂酰胆碱(DPPC)、60mg磷脂酰乙醇胺(PE)和100mg胆固醇,溶解于10mL乙醚中,混合均匀,将该溶液置于磨口圆底烧瓶中,于37℃恒温水浴上用旋转蒸发仪减压蒸发有机溶剂乙醚,使磷脂等成膜材料在烧瓶底部形成均匀脂膜;向脂膜中加入5mL 0.1mol/L柠檬酸-柠檬酸钠缓冲溶液(pH=3.6),在旋转蒸发仪上旋转,直至脂膜水化变成乳白色脂质体混悬液,超声粉碎10min减小粒径,得到空白脂质体悬液10mL,备用,作为空白脂质体。测得该空白脂质体悬液的平均粒径为133nm。
实施例3
称取500mg大豆磷脂(磷脂酰胆碱纯度>76%)、40mg胆固醇和聚乙二醇单甲醚(分子量为2000)胆固醇琥珀酸酯(CHS-PEG 2000)80mg,溶解于20mL乙醚中,混合均匀,然后加入10mL 0.1mol/L柠檬酸-柠檬酸钠缓冲溶液(pH=3.6),混合均匀成乳液,然后减压蒸去有机溶剂乙醚,用细胞粉碎仪超声5min,得到空白脂质体悬液10mL,备用,作为空白脂质体。测得该空白脂质体悬液的平均粒径为137nm。
实施例4
称取450mgDSPC、100mgDPPG和100mg胆固醇溶解于50mL乙醚中,混合均匀,然后加入20mL 0.1mol/L柠檬酸-柠檬酸钠缓冲溶液(pH=3.6),二者混合成乳液,将该乳液置于磨口圆底烧瓶中,于37℃恒温水浴上用旋转蒸发仪减压蒸发有机溶剂乙醚,直至变成乳白色脂质体混悬液,经微射流减小粒径,得到空白脂质体悬液40mL,备用,作为空白脂质体。测得该空白脂质体悬液的平均粒径为131nm。
实施例5
称取500mg二豆蔻酰磷脂酰胆碱(DMPC)、50mg磷脂酰丝氨酸(PS)、20mg胆固醇溶解于20mL乙醚中,混合均匀,然后加入10mL 0.1mol/L柠檬酸-柠檬酸钠缓冲溶液(pH=3.6),称取二者混合进行乳化,然后减压蒸去有机溶剂乙醚,用细胞粉碎仪超声5min,得到空白脂质体悬液10mL,备用,作为空白脂质体。测得该空白脂质体悬液的平均粒径为134nm。
实施例6
称取420mg二棕榈酰磷脂酰胆碱(DPPC)、60mg磷脂酰乙醇胺(PE)、100mg胆固醇,溶解于10mL乙醚中,混合均匀,将该溶液置于磨口圆底烧瓶中,于40℃恒温水浴上用旋转蒸发仪减压蒸发有机溶剂,使磷脂等成膜材料在烧瓶底部形成均匀脂膜;向脂膜中加入5mL 0.1mol/L柠檬酸-柠檬酸钠缓冲溶液(pH=3.6),在旋转蒸发仪上旋转蒸发除去乙醚,直至脂膜水化变成乳白色脂质体混悬液,超声粉碎5min减小粒径,得到空白脂质体悬液10mL,备用,作为空白脂质体。测得该空白脂质体悬液的平均粒径为200nm。
实施例7
称取500mg大豆磷脂、40mg胆固醇、聚乙二醇单甲醚(分子量为2000)胆固醇琥珀酸酯(CHS-PEG 2000)80mg,溶解于20mL乙醚中,混合均匀,然后加入10ml 0.1mol/L柠檬酸-柠檬酸钠缓冲溶液(pH=3.6),二者混合成乳液,然后减压蒸去有机溶剂乙醚,用细胞粉碎仪超声5min,得到空白脂质体悬液10mL,备用,作为空白脂质体。测得该空白脂质体悬液的平均粒径为95nm。
氯喹和阿霉素共载脂质体
实施例8
将氯喹与阿霉素质量比为1∶20的氯喹和阿霉素的混合物10.5mg溶解到实施例1的10mL空白脂质体悬液中,用0.2mol/L磷酸氢二钠水溶液调pH至7.0,之后在40℃水浴中孵育20min,冷却至室温后,用SephadexG-50凝胶柱层析法分离脂质体和未被包封在内的游离药物,得到氯喹和阿霉素共载脂质体25mL,测得氯喹和阿霉素的包封率分别为94%和99%,载药量分别为0.08%和1.6%。
实施例9
将氯喹与阿霉素质量比为1∶10氯喹和阿霉素的混合物12mg溶解到实施例1的10mL空白脂质体悬液中,用0.2mol/L Na2HPO4水溶液调pH至7.0,之后在40℃水浴中孵育20min,冷却至室温后,用SephadexG-50凝胶柱层析法分离脂质体和未被包封在内的游离药物,得到氯喹和阿霉素共载脂质体27mL,测得氯喹和阿霉素的包封率分别为95%和99%,载药量分别为0.3%和1.6%。
实施例10
将氯喹与阿霉素质量比为2∶1氯喹和阿霉素的混合物15mg溶解到实施例1的10mL空白脂质体悬液中,用0.2mol/L Na2HPO4水溶液调pH至7.0,之后在40℃水浴中孵育20min,冷却至室温后,用SephadexG-50凝胶柱层析法分离脂质体和未被包封在内的游离药物,得到氯喹和阿霉素共载脂质体24mL,测得氯喹和阿霉素的包封率分别为93%、99%,载药量分别为1.5%和0.8%。
实施例11
将氯喹与阿霉素质量比为1∶1氯喹和阿霉素的混合物20mg溶解到实施例1的10mL空白脂质体悬液中,用0.2mol/L Na2HPO4水溶液调pH至7.0,之后在40℃水浴中孵育20min,冷却至室温后,用SephadexG-50凝胶柱层析法分离脂质体和未被包封在内的游离药物,得到氯喹和阿霉素共载脂质体26mL,测得氯喹和阿霉素的包封率分别为96%、97%,载药量分别为1.5%和1.6%。
实施例12
将氯喹与盐酸阿霉素质量比为1∶2氯喹和盐酸阿霉素的混合物30mg溶解到实施例1的10mL空白脂质体悬液中,用0.2mol/L Na2HPO4水溶液调pH至7.0,之后在40℃水浴中孵育20min,冷却至室温后,用SephadexG-50凝胶柱层析法分离脂质体和未被包封在内的游离药物,得到氯喹和阿霉素共载脂质体27mL,测得氯喹和阿霉素的包封率分别为91%、94%,载药量分别为1.4%和3.0%。
实施例13
将磷酸氯喹与阿霉素质量比为1∶5磷酸氯喹和阿霉素的混合物18mg溶解到实施例1的10mL空白脂质体悬液中,用0.2mol/L Na2HPO4调pH至7.0,之后在40℃水浴中孵育20min,冷却至室温后,用SephadexG-50凝胶柱层析法分离脂质体和未被包封在内的游离药物,得到氯喹和阿霉素共载脂质体24mL,测得氯喹和阿霉素的包封率分别为94%、98%,载药量分别为0.5%和1.6%。
实施例14
将氯喹与盐酸阿霉素质量比为1∶2的氯喹和盐酸阿霉素的混合物30mg溶解到实施例2的10mL空白脂质体悬液中,用0.2mol/L Na2HPO4水溶液调pH至7.0,之后在40℃水浴中孵育20min,冷却至室温后,用SephadexG-50凝胶柱层析法分离脂质体和未被包封在内的游离药物,得到氯喹和阿霉素共载脂质体27ml,测得氯喹和阿霉素的包封率分别为91%、95%,载药量分别为1.4%和3.0%。
实施例15
将盐酸氯喹与阿霉素质量比为1∶1的盐酸氯喹和阿霉素的混合物20mg溶解到实施例3的10mL空白脂质体悬液中,用0.2mol/L Na2HPO4水溶液调pH至7.0,之后在40℃水浴中孵育20min,冷却至室温后,用SephadexG-50凝胶柱层析法分离脂质体和未被包封在内的游离药物,得到氯喹和阿霉素共载脂质体26mL,测得氯喹和阿霉素的包封率分别为95%、96%,载药量分别为1.5%和1.6%。
实施例16
将盐酸氯喹与阿霉素质量比为1∶1盐酸氯喹和阿霉素的混合物20mg溶解到实施例5的10mL空白脂质体悬液中,用0.2mol/L Na2HPO4水溶液调pH至7.0,之后在40℃水浴中孵育20min,冷却至室温后,用SephadexG-50凝胶柱层析法分离脂质体和未被包封在内的游离药物,得到氯喹和阿霉素共载脂质体26mL,测得氯喹和阿霉素的包封率分别为94%、96%,载药量分别为1.5%和1.6%。
实施例17
将氯喹与盐酸阿霉素质量比为1∶2氯喹和盐酸阿霉素的混合物30mg溶解到实施例4的40mL空白脂质体悬液中,用0.2mol/L Na2HPO4水溶液调pH至7.0,之后在40℃水浴中孵育20min,冷却至室温后,用SephadexG-50凝胶柱层析法分离脂质体和未被包封在内的游离药物,得到氯喹和阿霉素共载脂质体27mL,测得氯喹和阿霉素的包封率分别为92%、95%,载药量分别为1.4%和3.0%。
实施例18
将氯喹与盐酸阿霉素质量比为1∶2氯喹和盐酸阿霉素的混合物30mg溶解到实施例6的10mL空白脂质体悬液中,用0.2mol/L Na2HPO4水溶液调pH至7.0,之后在40℃水浴中孵育20min,冷却至室温后,用SephadexG-50凝胶柱层析法分离脂质体和未被包封在内的游离药物,得到氯喹和阿霉素共载脂质体27mL,测得氯喹和阿霉素的包封率分别为92%、95%,载药量分别为1.4%和3.0%。
实施例19
将氯喹与阿霉素质量比为1∶2氯喹和阿霉素的混合物30mg溶解到实施例7的10mL空白脂质体悬液中,用0.2mol/L Na2HPO4水溶液调pH至7.0,之后在40℃水浴中孵育20min,冷却至室温后,用SephadexG-50凝胶柱层析法分离脂质体和未被包封在内的游离药物,得到氯喹和阿霉素共载脂质体27mL,测得氯喹和阿霉素的包封率分别为93%、96%,载药量分别为1.4%和3.0%。
对比例1
将阿霉素10mg溶解到实施例1的10mL空白脂质体悬液中,用0.2mol/L磷酸氢二钠水溶液调pH至7.0,之后在40℃水浴中孵育20min,冷却至室温后,用SephadexG-50凝胶柱层析法分离脂质体和未被包封在内的游离药物,得到阿霉素脂质体(DOXL)23mL,阿霉素的包封率为96%,载药量为4.6%。
对比例2
将氯喹10mg溶解到实施例1的10mL空白脂质体悬液中,用0.2mol/L磷酸氢二钠水溶液调pH至7.0,之后在40℃水浴中孵育20min,冷却至室温后,用SephadexG-50凝胶柱层析法分离脂质体和未被包封在内的游离药物,得到氯喹脂质体(CQL)24mL,氯喹的包封率为96%,载药量为1.6%。
细胞毒性对比实验
将作为对照组1的游离盐酸阿霉素水溶液(FDOX)和作为对照组2游离磷酸氯喹水溶液(FCQ)、对比例制备的脂质体、实施例制备的各种氯喹和阿霉素不同比例的脂质体分别用于细胞实验,采用MTT(噻唑蓝)比色法进行实验,步骤如下:
对数生长期的的癌细胞用胰蛋白酶消化、PBS缓冲液(即:含0.05%吐温-20的pH7.4的磷酸盐缓冲液)洗涤、离心后将其制备成浓度为1×105/ml的细胞悬液,将该悬液按100μl/孔均匀加入96孔细胞培养板中,每孔细胞数为5000~10000个。将细胞板置于37℃孵箱中,孵育24h,显微镜下观察可见细胞融合贴壁生长。分别将载药胶束及阿霉素溶于生理盐水中,所有溶液以其中阿霉素的含量为基准,稀释成不同浓度,折算成阿霉素浓度分别为0.5μg/ml,1μg/ml,2μg/ml,5μg/ml,10μg/ml,100μg/ml,500μg/ml。向上述96孔细胞培养板中加入不同浓度的聚合物溶液25μl/孔,培养24h后,每孔加入31.5μl浓度为5mg/ml噻唑蓝(MTT)溶液,继续培养4h,吸出上清液,加入200μl二甲基亚砜(DMSO),然后放置于CO2培养箱15分钟。用酶标仪检测各孔570nm处的OD值,记录结果。上述实验,每组重复3次,每个浓度设4个复孔。
IC50即50%抑制浓度,是抑制半数癌细胞生长时的药物浓度,IC50值越低,说明细胞毒性作用越大。试验中IC50值由IC50.exe软件计算得到。并且实验结果显示,同一氯喹和阿霉素比例,不同种类的磷脂对于IC50几乎没有影响。
对照组1、对照组2,对比例1、对比例2及实施例8、9、10、11和13制备的脂质体对药物敏感细胞株MCF-7(人乳腺癌细胞)和耐药细胞株MCF-7/ADR的IC50(μg/ml)值,如表1所示;对照组1、对照组2,对比例1、对比例2及实施例8、9、10、11和13制备的脂质体对药物敏感细胞株HL60(急性白血病细胞)和耐药细胞株HL60/ADR IC50(μg/ml)值,如表2所示。
表1
表2
从表1和表2的结果可见,对照组2中游离磷酸氯喹水溶液(FCQ)以及对比例2氯喹脂质体(CQL)的IC50几乎均比对照组1中游离盐酸阿霉素水溶液(FDOX)和对比例1阿霉素脂质体(DOXL)的IC50大,表明游离的氯喹和氯喹脂质体相对游离的盐酸阿霉素和阿霉素脂质体的细胞毒性要小,各实施例组对耐药组细胞都起到了一定的抑制作用,并且随着氯喹比例的增加而加大,说明阿霉素和氯喹两者协同作用较好,而对HL60/ADR的作用比MCF-7/ADR的强。由此可见,本发明氯喹和阿霉素共载脂质体对细胞的耐药性有较好的逆转作用,对MRPl引起的耐药性逆转效果相对较好。
Claims (10)
1.一种氯喹和阿霉素共载脂质体,其特征在于,由药物、磷脂、胆固醇类化合物、内部缓冲系统和pH值调节剂制成;其中,药物、磷脂和胆固醇类化合物的重量比为1∶2~100∶1~35;
所述的pH值调节剂调节脂质体pH至5.5~8.0;
所述的药物为氯喹类药物和阿霉素类药物;
所述的氯喹类药物为氯喹、氯喹与酸所成的盐中一种或多种;
所述的阿霉素类药物为阿霉素、阿霉素与酸所成的盐中一种或多种;
所述的胆固醇类化合物为胆固醇、聚乙二醇修饰胆固醇中的一种或者两种。
2.根据权利要求1所述的氯喹和阿霉素共载脂质体,其特征在于,所述的药物、磷脂和胆固醇类化合物的重量比为1∶10~40∶2.5~10。
3.根据权利要求2所述的氯喹和阿霉素共载脂质体,其特征在于,所述的药物、磷脂和胆固醇类化合物的重量比为1∶25∶5。
4.根据权利要求1所述的氯喹和阿霉素共载脂质体,其特征在于,所述的氯喹与酸所成的盐为磷酸氯喹、盐酸氯喹、硫酸氯喹中一种或多种;
所述的阿霉素与酸所成的盐为盐酸阿霉素。
5.根据权利要求1所述的氯喹和阿霉素共载脂质体,其特征在于,所述的磷脂为天然磷脂、合成磷脂中的一种或两种;所述的天然磷脂为大豆磷脂、卵磷脂中的一种或两种;所述的合成磷脂为中性磷脂、负电性磷脂或聚乙二醇修饰磷脂中的一种或多种。
6.根据权利要求5所述的氯喹和阿霉素共载脂质体,其特征在于,所述的合成磷脂为二豆蔻酰磷脂酰胆碱、二棕榈酰磷脂酰胆碱、二硬脂酰磷脂酰胆碱、二棕榈酰磷脂酰甘油酯、磷脂酰乙醇胺、磷脂酰丝氨酸、二硬脂酰磷脂酰乙醇胺-聚乙二醇中的一种或多种。
7.根据权利要求1所述的氯喹和阿霉素共载脂质体,其特征在于,所述的内部缓冲系统为柠檬酸盐缓冲系统、磷酸盐缓冲系统或碳酸盐缓冲系统;所述的pH值调节剂为碱液。
8.根据权利要求7所述的氯喹和阿霉素共载脂质体,其特征在于,所述的柠檬酸盐缓冲系统为0.05mol/L~0.5mol/L的柠檬酸-柠檬酸钠缓冲液;
所述的碱液为氢氧化钠水溶液、磷酸氢二钠水溶液、碳酸钠水溶液、碳酸氢钠水溶液中的一种或多种。
9.根据权利要求1所述的氯喹和阿霉素共载脂质体,其特征在于,所述的氯喹类药物和阿霉素类药物的质量比为1~100∶1~100。
10.根据权利要求1~9任一项所述的氯喹和阿霉素共载脂质体的制备方法,包括以下步骤:
1)将磷脂和胆固醇类化合物溶于有机溶剂中,再加入内部缓冲系统,混合均匀,除去有机溶剂,得到空白脂质体悬液;
或者,将磷脂和胆固醇类化合物溶于有机溶剂中,混合均匀,除去有机溶剂,再加入内部缓冲系统混合均匀,得到空白脂质体悬液;
2)将氯喹和阿霉素溶解在步骤1)制备的空白脂质体悬液中,用pH值调节剂调节pH至5.5~8.0,在30℃~50℃水浴孵育10min~60min,分离出游离药物,得到氯喹和阿霉素共载脂质体;
步骤1)中,所述的有机溶剂为乙醚或者氯仿。
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