CN103275178B - 正十二酸-rgd-正十四醇介导利尼法尼靶向脂质体的制备及抗肿瘤活性评价 - Google Patents
正十二酸-rgd-正十四醇介导利尼法尼靶向脂质体的制备及抗肿瘤活性评价 Download PDFInfo
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Abstract
本发明公开了正十二酸-RGD-正十四醇介导利尼法尼靶向脂质体的制备及抗肿瘤活性评价,将该化合物加入到靶向脂质体中,可以增加抗肿瘤药物在靶部位的浓度并可减少其在非靶部位的毒副作用,提高药物的治疗指数。本发明以腹水瘤S180小鼠为模型评价了利尼法尼靶向脂质体制剂的抗肿瘤活性,结果显示利尼法尼靶向脂质体比各对照组具有更优异的抗肿瘤活性。
Description
正十二酸-RGD-正十四醇介导利尼法尼靶向脂质体的制备及抗肿瘤活性评价
技术领域
本发明涉及靶向释药载体,尤其涉及正十二酸-RGD-正十四醇介导靶向药物载体及其构建方法,本发明还涉及药物载体在制备靶向抗肿瘤药物脂质体中的应用,属于生物医药学领域。
背景技术
许多恶性肿瘤的生长和转移均与整合素表达异常或分子结构改变相关。整合素是一跨膜蛋白大家族,由α、β两种亚基构成异二聚体。目前发现α约有18种,β约有8种,至少有24种异二聚体的整合素形式。整合素在肿瘤的进展可能具有两重性:1)肿瘤发生早期,整合素表达降低可致瘤细胞与基底膜或ECM成分的粘附作用减弱,从而有利于肿瘤在局部生长与扩散;2)瘤细胞进入血循环后,整合素表达增高有利于瘤细胞粘附于血管内皮,继而定位增殖。整合素在肿瘤细胞表面的表达高于正常细胞是公认的事实。
已知,精氨酸-甘氨酸-天冬氨酸三肽序列(Arg-Gly-Asp,RGD)是整合素特异识别序列片段,RGD三肽及修饰物通过与整合素特异结合,具有阻止肿瘤细胞定位增殖、抗肿瘤新生血管生成作用。
脂质体(liposome)是直径50~1000nm的球型脂质双层,磷脂膜的组成成分灵活,可以制成种类、大小、表面特征不同的多种类型,作为生物活性物质的有效运输载体。亲水性药物可以包容在脂质体的水核内,脂溶性药物可以嵌入磷脂双层间,中性药物可以通过调整脂质体内的pH值或通过添加反相离子与药物形成分子复合物而稳定地结合于脂质体内。
普通脂质体不具有靶向性。如在脂质体表面结合抗体、配体,利用肿瘤细胞与正常细胞表面表达的抗原受体的差异,通过抗原、抗体和受体、配体的特异性作用增加脂质体的主动靶向性,这样可以提高治疗指数。
抗肿瘤药物的主动靶向性制剂将是改进抗肿瘤药物制剂的新趋向。为了使载体系统具有特异靶向性,可将各种活性物质耦联到载体表面。借助这种特异性相互作用可将载有各种抗肿瘤药物的载体系统靶向到含有特异性受体的器官、组织 或细胞;同时受体与配体结合可促进载体系统内药物释放到肿瘤细胞内。
发明内容
本发明涉及一种作为配体的两亲性化合物,其化学式如下:
CH3(CH2)10CO-Arg-Gly-Asp-OCH2(CH2)12CH3。
分子结构式如下:
进一步的,本发明还包含,上述配体化合物的制备方法,包括:将Arg-Gly-Asp与正十二酸进行缀合,再与正十四醇进行缀合,即得。
优选的,本发明的化合物的制备方法,包括以下步骤:
(1)按照常规液相接肽技术分别合成CH3(CH2)10CO–Arg(NO2)-Gly-OH和Boc-Asp(OBzl)-OCH2(CH2)12CH3;片段
(2)将带有保护基保护的CH3(CH2)10CO–Arg(NO2)-Gly-OH和Boc-Asp(OBzl)-OCH2(CH2)12CH3偶联,再脱去全部保护基,即得。
本发明还涉及一种抗肿瘤药物靶向脂质体,含有所述的作为配体的化合物。
进一步的,所述的抗肿瘤药物靶向脂质体,由以下各组分组成:抗肿瘤药物,磷脂,配体化合物。
进一步的,所述的抗肿瘤药物靶向脂质体,由以下各组分组成,下述百分比均为质量百分比:抗肿瘤药物2-10%,磷脂80-90%,配体化合物5-10%。
进一步的,抗肿瘤药物为:利尼法尼,紫杉醇,多西紫杉醇中的一种或多种。
进一步的,磷脂为:卵磷脂,大豆磷脂,二棕榈酰磷脂酰胆碱,二硬脂酰磷脂酰胆碱中的一种或多种。
本发明涉及一种制备上述抗肿瘤药物靶向脂质体的方法,包括:将卵磷脂,利尼法尼,CH3(CH2)10CO-Arg-Gly-Asp-OCH2(CH2)12CH3,用2ml含有乙醚70%~ 80%的乙醚甲醇混合溶剂溶解;缓慢注入55~65℃剧烈搅拌的磷酸盐缓冲液(pH=7.4),继续搅拌至有机溶剂挥发干净;探头超声3分钟,即得。
本发明涉及所述的具有两亲性的正十二酸-RGD-正十四醇缀合物在制备抗肿瘤靶向脂质体药物中的应用。
本发明涉及所述的靶向脂质体等各种药物载体靶向脂质体等载体用于制备抗肿瘤药物制剂的用途。
本发明的两亲性化合物是经过筛选获得的,其筛选方案包括将相似结构的烷酸进行排列组合,通过生物利用度实验和抗肿瘤活性实验,找到其中最优选的技术方案,即本发明的技术方案。
经过筛选,发明人认为,本发明的两亲性化合物最具有应用价值。
和现有技术相比,本发明的两亲性化合物具有更高的生物利用价值,特别表现在靶向性上和治疗效果上。
本发明以腹水瘤S180小鼠为模型,采用尾静脉给药的治疗方案评价所述的抗肿瘤靶向脂质体制剂的抗肿瘤活性,结果显示靶向脂质体比各对照组具有更优异的抗肿瘤活性。
本发明涉及以人体黑色素瘤A375细胞为模型,采用MTT法评价所述的抗肿瘤靶向脂质体制剂的抗肿瘤活性,结果显示靶向脂质体比各对照组具有更优异的抗肿瘤活性。
具体实施方式
为了进一步阐述本发明,下面给出一系列实施例。这些实施例完全是例证性的,它们仅用来对本发明进行具体描述,不应当理解为对本发明的限制。
实施例1CH3(CH2)10CO-Arg-Gly-Asp-OCH2(CH2)12CH3的制备
1)Boc-Arg(NO2)-Gly-OBzl的制备
将6.70g(21mmol)Boc-Arg(NO2)-OH,2.70g(20mmol)N-羟基苯并三氮唑(HOBt)溶于无水THF中。冰浴下再滴入5.36g(26mmol)二环己基羰二亚胺(DCC)的无水THF溶液,冰浴下搅拌20分钟,得到反应液(Ⅰ)。冰浴下把4.03g(20.0mmol)Hcl·Gly-OBzl悬浮于适量无水DMF中,然后加入数滴N-甲基吗啉(NMM),调pH至9,得到反应液(Ⅱ)。冰浴下把反应液(Ⅰ)滴加入反应液(Ⅱ)中,先冰浴下搅拌2h,再室温搅拌10h,TLC(氯仿/甲醇,10:1)显示Hcl·Gly-OBzl 消失,停止反应。滤除二环己基脲(DCU),滤液挥发DMF。残留物用150ml乙酸乙酯溶解。得到的溶液依次用5%NaHCO3水溶液、饱和NaCl水溶液、5%KHSO4水溶液、饱和NaCl水溶液、5%NaHCO3水溶液、饱和NaCl水溶液各洗三遍。乙酸乙酯层用无水Na2SO4干燥、过滤、滤液减压浓缩至干,得到6.71g(72%)标题化合物,为白色固体。
ESI-MS(m/z)467.8[M+1]+,489.8[M+23]+,955.4[2M+23]+。
2)HCl·Arg(NO2)-Gly-OBzl的制备
将8.00g(18mmol)Boc-Arg(NO2)-Gly-OBzl溶解在10ml4mol/l氯化氢-乙酸乙酯溶液中,室温搅拌2h,TLC(氯仿/甲醇)显示原料点消失,减压浓缩除去乙酸乙酯,残留物反复加少量乙醚进行减压浓缩以除去氯化氢气。最后加少量乙醚将残留物研磨成6.80g(98.5%)标题化合物,为白色固体,直接用于下一步反应。ESI-MS(m/z)367.4[M+1]+,389.4[M+23]+,733.8[2M+1]+。
3)CH3(CH2)10CO–Arg(NO2)-Gly-OBzl的制备
按照Boc-Arg(NO2)-Gly-OBzl的制备方法,由2.30g(11mmol)CH3(CH2)10COOH和4.32g(10mmol)HCl·Arg(NO2)-Gly-OBzl制得4.27g(78%)标题化合物,为白色固体。ESI-MS(m/z)549.7[M+1]+,571.7[M+23]+。
4)CH3(CH2)10CO–Arg(NO2)-Gly-OH的制备
将1.81g(3.3mmol)CH3(CH2)10CO-Arg(NO2)-Gly-OBzl溶于15ml甲醇。冰浴下将得到的溶液用NaOH(2N)水溶液调pH至12并搅拌2h,TLC(氯仿/甲醇,10:1)显示CH3(CH2)6CO-Arg(NO2)-Gly-OBzl消失。反应混合物用饱合KHSO4水溶液调pH至7,减压浓缩除甲醇。残留物用饱合KHSO4水溶液调pH至2,用乙酸乙酯萃取(50ml×3)。合并的乙酸乙酯相用饱和NaCl水溶液洗3次,无水Na2SO4干燥。过滤,滤液减压浓缩至干,得1.466g(97.6%)标题化合物,为白色固体。ESI-MS(m/z)457.7[M-1]-。
5)Boc-Asp(OBzl)-OCH2(CH2)12CH3的制备
按照Boc-Arg(NO2)-Gly-OBzl的制备方法,由2.14g(10mmol)CH3(CH2)13OH和4.20g(13mmol)Boc-Asp(OBzl)-OH制得3.84g(74%)标题化合物,为白色固体。ESI-MS(m/z)520.1[M+1]+,542.6[M+23]+。
6)HCl·Asp(OBzl)-OCH2(CH2)12CH3的制备
将1g(2mmol)Boc-Asp(OBzl)-OCH2(CH2)12CH3溶解在5ml4mol/l氯化氢-乙酸乙酯溶液中,室温搅拌2h,TLC(氯仿/甲醇)显示原料点消失,减压浓缩除去乙酸乙酯,残留物反复加少量乙醚进行减压浓缩以除去氯化氢气。最后加少量乙醚将残留物研磨成0.74g(84%)标题化合物,为白色固体,直接用于下一步反应。ESI-MS(m/z)420.7[M+1]+,839.1[2M+1]+。
7)CH3(CH2)10CO-Arg(NO2)-Gly-Asp(OBzl)-OCH2(CH2)12CH3的制备
按照3.1的制备方法,365mg(0.87mmol)HCl·Asp(OBzl)-OCH2(CH2)12CH3,418mg(0.91mmol)CH3(CH2)10CO-Arg(NO2)-Gly-OH制得标题化合物,纯化得白色固体,7.5mg(10%)。ESI-MS(m/z)860.0[M+1]+,882.5[M+23]+。
1HNMR(CDCl3,500Hz),δ(ppm):8.48(s,1H,CONH),7.81(s,1H,CONH),7.66(s,1H,CONH),7.36-7.32(m,5H,-C6 H 5),5.11(t,J=7.5HZ,2H,CH 2-C6H5),4.87(m,1H,CHNH),4.67(s,1H,CHNH),4.06(d,J=4HZ,2H,COCH 2),3.92(d,J=7.5HZ,2H,OCH 2),3.03(dd,J=5HZ,J=17HZ,2H,COCH 2),2.93(dd,J=4HZ,J=15HZ,2H,CH 2NH),2.23(t,J=10.0HZ,2H,COCH 2),2.00(s,2H),1.85(s,2H),1.55(m,4H),1.35(m,38H),0.85(m,6H,CH 3);
13CNMR(CDCl3,125Hz),δ(ppm):174.25(CO-O),170.72(CH2 CONH),170.63(CH CONH),169.01(CH2 CONH),159.52(C=NNH),135.32,128.59,128.49,128.43,128.19(C6H5),66.95(C=C-CH2O),66.28(CH2 CH2O),48.80(CHNH),42.95(CHCO),40.63(CH2NH),36.39(CH2CO-O),31.89(CH2CO),30.05(CH2NH),29.63-24.59(11C),14.1(CH3).
8)CH3(CH2)10CO–Arg-Gly-Asp-OCH2(CH2)12CH3的制备
258mg(0.3mmol)CH3(CH2)10CO-Arg(NO2)-Gly-Asp(OBzl)OCH2(CH2)12CH3置于250ml茄形瓶中、用乙醇溶解、加50mgPd/C(25%)、通H2(0.02Mba),室温搅拌至原料点消失。滤除Pd/C、滤液减压浓缩至干,残留物反复用石油醚研磨,得110mg(51%)标题化合物,为白色粉末;ESI-MS(m/z):725.9[M+1]+。
1HNMR(DMSO,500Hz),δ(ppm):7.95(s,3H,CONH),4.47(m,1H,CHNH),4.23(m,1H,CHCO),3.95(s,2H,COCH 2NH),3.70(m,2H,CH 2-O),3.15(s,2H),2.92(m,2H,CH 2COOH),2.60(m,2H,CH 2NH),2.30(s,2H,CH 2CO),2.09(s,3H,NH-C=N),1.93(s,1H),1.49-1.45(m,6H,CH 2),1.20(m,36H),0.82(s,6H,CH 3);
13CNMR(DMSO,125Hz),δ(ppm):175.00(C=OOH),172.90(CONH),171.64(COO),169.07(CH-CONH),157.44(C=NH),64.78(CH2-O-C=O),52.47(NHCHCO),49.01(COCHNH),31.74-22.51(5C),14.0(CH3).
实施例2.利尼法尼脂质体的制备
采用乙醚甲醇混合溶剂注入法制备。将卵磷脂,利尼法尼,用2ml含有乙醚70%~80%的乙醚甲醇混合溶剂溶解。缓慢注入55~65℃剧烈搅拌的磷酸盐缓冲液(pH=7.4),继续搅拌至有机溶剂挥发干净。探头超声3分钟,即得利尼法尼脂质体。
粒径180~240nm,Zeta-Potential–20~-40mV。
实施例3.膜材中含CH3(CH2)10CO-RGD-OCH2(CH2)12CH3的利尼法尼脂质体的制备
各组分的用量:利尼法尼2-10%(质量百分比),卵磷脂80-90%(质量百分比),和整合素受体靶向配体CH3(CH2)10CO-Arg-Gly-Asp-OCH2(CH2)12CH35-10%(质量百分比)。
将卵磷脂,利尼法尼,CH3(CH2)10CO-Arg-Gly-Asp-OCH2(CH2)12CH3,用2ml含有乙醚70%~80%的乙醚甲醇混合溶剂溶解。缓慢注入55~65℃剧烈搅拌的磷酸盐缓冲液(pH=7.4),继续搅拌至有机溶剂挥发干净。探头超声3分钟,即得利尼法尼靶向脂质体。
粒径180~230nm,Zeta-Potential–20~-40mV。
实验例4.抗肿瘤药物靶向脂质体对S180腹水瘤小鼠的治疗作用
取腹腔接种S180腹水瘤第八天的昆明种小鼠一只,脱颈椎处死,用75%酒精消毒后置于超净台内,用镊子夹起腹部中线偏右的皮肤并用小剪刀剪一小口至可见乳白色腹水流出。将吸管由开口处轻轻插入腹腔吸出腹水。吸得的腹水加到装有约3ml灭菌生理盐水的15ml的离心管中,使体积增至约10ml。用吸管轻轻吹气,使腹水与生理盐水混匀。试管加盖,1000转/分离心5分钟。弃去上清液后,加9ml灭菌生理盐水,1000转/分离心5分钟,弃去上清液。往试管底部的乳白色胶状物中加9ml灭菌生理盐水,用吸管吹均匀。取100μl该悬浮液加至9.9ml灭菌生理盐水中,混匀,加盖,放入冰中待用。取100μl上述瘤细胞稀释液置入Eppendoff小管中,加100μl台盼兰染液,混匀。取少许该混匀液加至计数板的计数池内,于显微镜下计算4个大格中被染上蓝色的存活瘤细胞的个 数。将原液中的存活瘤细胞稀释成1.3×107个/ml,用于接种。用2%碘酒棉球和75%酒精棉球在小鼠右侧腋下消毒,并注入0.2ml瘤细胞液,缓缓抽出针头。按此方法给每批昆明种小鼠接种,随机分组,放入动物室饲养。
药物配制:本实验共设6组:1.利尼法尼混悬液组;2.利尼法尼脂质体组;3.膜材中含有CH3(CH2)10CO-Arg-Gly-Asp-OCH2(CH2)12CH3的利尼法尼脂质体组;4.膜材中含有CH3(CH2)10CO-Arg-Gly-Asp-OCH2(CH2)12CH3的大剂量利尼法尼脂质体组;5.膜材中含有CH3(CH2)10CO-Arg-Gly-Asp-OCH2(CH2)12CH3的小剂量利尼法尼脂质体组;6.生理盐水溶液组。
药物混悬液制备:精密称取药物,于0.5%CMC-Na振荡均匀制成混悬液。阳性对照是利尼法尼的脂质体,阴性对照为生理盐水。1,2,3组给药剂量为2.5mg/kg/day,大剂量4组5mg/kg/day,小剂量5组1.5mg/kg/day。脂质体的制备方法如实施例3所述方法制得。
实验方法:将右侧腋下接种有腹水瘤的小鼠随机分组,每组6只,共6组。各组小鼠正常饲养,从接种后24h开始给药,每天一次,共7次,每次尾静脉注射上述药物0.2ml。7天后将各组腹水瘤小鼠处死,解剖,取瘤。并称体重和瘤重,按抑瘤率=[(阴性对照组平均瘤重-治疗组平均瘤重)/阴性对照组平均瘤重]×100%计算抑瘤率。获得的数据以(±SDg)表示,并做t检验。结果见表1。
由表1可见,含有RGD肽偶联物的利尼法尼脂质体有优越的抗肿瘤活性;通过统计学分析可看出其中3,4,5组抗肿瘤作用均高于利尼法尼混悬液组;且显著高于利尼法尼脂质体组。
表1.利尼法尼及其脂质体对小鼠肿瘤抑制作用(n=6)
全部对比于生理盐水溶液有显著差异(P<0.01)
*对比于利尼法尼混悬液组有意义(P<0.05)
#对比于利尼法尼靶向脂质体有显著意义(P<0.01)
实验例5.抗肿瘤药物靶向脂质体对人黑色素瘤细胞A375的抑制作用
细胞培养
人黑色素瘤细胞A375用含10%灭活的胎牛血清,青霉素浓度最终为100U/ml,硫酸链霉素的最终浓度为100ug/ml的DMEM培养液培养,置37℃5%CO2恒温培养箱中,每天换新培养液,每2-3天传代一次。
药物配制
实验组:参照实施例3制备含CH3(CH2)10CO-RGD-O(CH2)13CH3的抗肿瘤药物靶向脂质体。药物浓度分别为10μmol/L、70μmol/L、100μmol/L、150μmol/L、200μmol/L、250μml/L。
对照组:参照实施例2制备抗肿瘤药物普通脂质体。药物浓度分别为10μmol/L、70μmol/L、100μmol/L、150μmol/L、200μmol/L、250μml/L。
阴性对照组:用含10%胎牛血清培养的A375细胞,不加药。
空白组:含10%胎牛血清的培养液。
实验方法将对数生长期的A375细胞用胰蛋白酶消化后,培养液稀释,制成3×104/ml细胞悬液,接种于96孔培养板内,每孔100μl。实验设不同浓度实验组、对照组、阴性对照组及空白对照组。接种后培养24小时,分别加入以上各组6个浓度25μL,每个浓度重复3孔,37℃5%CO2培养48h,每孔加MTT5mg/ml100μl37℃培养箱中培养4h。将96孔板弃去上液,每孔加二甲亚砜25μL,振荡器振摇10min,使充分溶解,在酶标仪上以检测波长为570nm测定吸光度(OD)值。A375细胞存活率%=[(抗癌药物组OD值-空白对照组OD值)/(阴性对照组OD值-空白对照组OD值)]×100%,计算半数抑制浓度(IC50),见表2。实验结果表明抗肿瘤药物靶向脂质体组比普通脂质体组有更好的抗肿瘤活性。
表2.利尼法尼及其剂型对A375细胞48h的IC50值(N=3,n=3)
*对比于利尼法尼有意义(P<0.05)
#对比于利尼法尼靶向脂质体有显著差异(P<0.01) 。
Claims (1)
1.一种制备抗肿瘤药物靶向脂质体的方法,步骤如下:
将卵磷脂,利尼法尼,CH3(CH2)10CO-Arg-Gly-Asp-OCH2(CH2)12CH3,用2ml含有乙醚70%~80%的乙醚甲醇混合溶剂溶解,缓慢注入55~65℃剧烈搅拌的磷酸盐缓冲液,调节溶液pH=7.4,继续搅拌至有机溶剂挥发干净,探头超声3分钟,即得利尼法尼靶向脂质体,粒径180~230nm,Zeta-Potential –20~-40mV,
其中,各组分的用量:利尼法尼2-10%(质量百分比),卵磷脂80-90%(质量百分比),和整合素受体靶向配体CH3(CH2)10CO-Arg-Gly-Asp-OCH2(CH2)12CH3 5-10%(质量百分比)。
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CN102250205A (zh) * | 2011-05-18 | 2011-11-23 | 首都医科大学 | 脂肪酰-rgd引导多西紫杉醇靶向脂质体的制备及其抗肿瘤活性 |
CN102532262A (zh) * | 2012-03-02 | 2012-07-04 | 首都医科大学 | 脂肪酸-rgd-脂肪醇偶联物介导表阿霉素靶向脂质体制备及抗肿瘤活性评价 |
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CN101240030A (zh) * | 2007-02-07 | 2008-08-13 | 首都医科大学 | 正十一烷基和rgd肽构建的缀合物、它们的合成及在医学中的应用 |
CN101401941A (zh) * | 2008-11-21 | 2009-04-08 | 首都医科大学 | 肿瘤靶向载体材料rgd-脂肪醇系列化合物的制备及应用 |
CN102250205A (zh) * | 2011-05-18 | 2011-11-23 | 首都医科大学 | 脂肪酰-rgd引导多西紫杉醇靶向脂质体的制备及其抗肿瘤活性 |
CN102532262A (zh) * | 2012-03-02 | 2012-07-04 | 首都医科大学 | 脂肪酸-rgd-脂肪醇偶联物介导表阿霉素靶向脂质体制备及抗肿瘤活性评价 |
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