CN101066458A - 可逆转肿瘤细胞耐药性的细胞核靶向给药系统的应用 - Google Patents
可逆转肿瘤细胞耐药性的细胞核靶向给药系统的应用 Download PDFInfo
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Abstract
本发明提供一种细胞核靶向给药系统,在制备可逆转肿瘤细胞耐药性的分子靶标位于细胞核的抗肿瘤药物中的应用。细胞核靶向给药系统由细胞核靶向给药载体和被包封的抗肿瘤药物组成,细胞核靶向给药载体选用壳寡糖-脂肪酸嫁接物胶团。本发明提供的壳寡糖-脂肪酸嫁接物载药胶团,细胞毒性低,具有快速透过细胞膜、很少被细胞内溶酶体破坏、细胞核靶向的功能。通过嫁接物胶团自身的疏水性内核特性,可包封分子靶标位于细胞核的疏水性抗肿瘤药物,形成嫁接物载药胶团。在显著提高抗肿瘤药物疗效的同时,逆转耐药肿瘤细胞的耐药性。本发明为分子靶标位于细胞核的药物,提供一种靶向给药载体材料。
Description
技术领域
本发明属细胞核靶向给药系统的应用,涉及一种细胞核靶向给药系统在制备分子靶标位于细胞核并可逆转肿瘤细胞耐药性的抗肿瘤药物中的应用。
背景技术
肿瘤一直是直接威胁人类健康的重大疾病,肿瘤化疗由于药物本身缺乏分子靶向性,因而出现治愈率低、多药耐药性、毒副作用巨大等重大治疗问题。临床化疗失败的重要原因之一,是肿瘤细胞对化疗药物产生耐药性。多数肿瘤病人的死因,与肿瘤细胞对抗肿瘤药物的耐药直接或间接相关。因此,寻找并研究能够逆转耐药肿瘤细胞耐药性的新型给药系统,是肿瘤化疗研究与发展的重要策略与方向之一。
研究发现,有多种原因导致了肿瘤多药耐药性的产生,并且与P-糖蛋白(P-gp)、谷胱甘肽S转移酶(GST-л),以及拓扑异构酶(TOPO-II)的表达异常有关。P-糖蛋白(P-gp)主要分布于细胞膜,是依赖ATP酶的药物输出泵,也是一种Ca2+与Cl-的通道。P-gp能够与进入细胞浆的抗肿瘤药物结合,借助ATP水解释放出的能量,直接或间接将药物泵出细胞;或借P-gp本身的通道作用,将药物从细胞内泵出细胞外,导致肿瘤细胞内药物浓度降低、细胞毒作用减弱或丧失,进而使肿瘤产生耐药。
研究显示,聚合物载体可在一定程度上逆转肿瘤的多药耐药性。非离子型表面活性剂聚环氧乙烯-聚苯醚-聚环氧乙烯三聚体(PEO-PPO-PEO),包裹抗肿瘤药物后,当聚合物浓度接近临界胶团浓度时,可以发挥较好的抗肿瘤效果,并体现逆转多药耐药性倾向。聚合物载体可有效减少耐药细胞的ATP量,却不会引起敏感细胞的ATP量的变化。耐药细胞中ATP量的减少,直接减少了被耐药细胞泵出的肿瘤药物量,表现为对肿瘤耐药性的逆转。又如,聚左旋组氨酸(PolyHis),通过咪唑基团的“质子海绵体”机制,可实现核内体膜裂解活性。如果所设计的聚合物胶团,能够在早期的核内体中发生触发性释放并内在化,随后使成熟的核内体膜破裂,则可以使药物逃逸,并在细胞浆和细胞核(潜在的药物作用位点)中表现出更高的药物浓度,这样的给药系统将成为有效的克服多药耐药性的药物传递模型。因此,带有聚左旋组氨酸核心的聚合物胶团,也有望成为有效的肿瘤细胞多药耐药性的逆转剂。
大多数抗肿瘤药物的分子作用靶标位于细胞核,如:阿霉素、丝裂霉素C等。现有的肿瘤化疗技术,基本沿用了药物的非靶向给药模式,主要依靠药物自身的理化性质,分布到肿瘤组织。通过合适的载体技术,将药物直接靶向病变组织(器官)、细胞及亚细胞器,是解决癌症化疗治愈率低和毒副作用大的重要手段之一。目前,国内外科学家通过纳米载体技术,在抗肿瘤药物的组织(器官)和细胞靶向上取得了一定的进展,但并没有取得突破性的疗效,主要原因是纳米载体无法将药物大量输送到细胞内,尤其是细胞核内的药物分子作用位点。因此,针对肿瘤细胞内药物分子作用靶点(亚细胞器)的靶向性纳米载体材料技术的研究开发,是突破肿瘤化疗瓶颈技术的关键。
聚合物胶团(polymeric micelles,PMs)是近几年正在发展的一类新型的纳米载体,具有增溶、靶向、低毒和长循环的优点。聚合物胶团由两亲性的聚合物在水性环境中自发形成,具有独特的核-壳结构。其内核可为难溶性药物提供储库,亲水性外壳则可进行理化性质修饰。通过包封多肽蛋白类药物及基因药物,可达到体内靶向分布、逃避单核巨噬细胞的吞噬、提高生物膜转运等作用。聚合物胶团自身可通过“增强的透过及滞留效应”(enhanced permeabilityand retention effect)聚集到肿瘤组织。
壳聚糖是一类由氨基葡萄糖组成的阳离子聚合物,具有很好的生物相容性、低毒性和可生物降解性。壳聚糖被广泛应用于药物辅料、止血材料、组织工程支架等。尽管以壳聚糖为基本材料所制备的微粒和纳米粒,已广泛应用于药物载体材料的研究,但由于壳聚糖本身的亲水性结构,使它难以被细胞大量摄取,无法实现亚细胞器靶向。
发明内容
本发明的目的是提供一种细胞核靶向给药系统在制备逆转肿瘤细胞耐药性的分子靶标位于细胞核的抗肿瘤药物中的应用。
细胞核靶向给药系统由细胞核靶向给药载体和被包封的抗肿瘤药物组成,选用壳寡糖-脂肪酸嫁接物胶团作为细胞核靶向给药载体,当壳寡糖-脂肪酸嫁接物在水性介质中的浓度超过临界胶团浓度时,可自发形成嫁接物胶团,胶团具备被细胞快速摄取,并靶向细胞核的功能。本发明的嫁接物胶团的组成,已为专利“表面修饰疏水改性壳寡糖聚合物给药胶团及其制备方法”(专利申请号200610051601.0),和专利“一种非病毒基因转染载体及制备方法和用途”(专利申请号200610050516.2)所涵盖。
由于该类嫁接物载体毒性低、具有被细胞快速摄取,并靶向细胞核的功能。因此,可作为分子靶标位于细胞核的抗肿瘤药物靶向治疗,解决肿瘤细胞的耐药性问题,提高药物的疗效。
本发明提供的壳寡糖-脂肪酸嫁接物载药胶团,在显著提高敏感细胞抗肿瘤药效的同时,可完全逆转耐药肿瘤细胞的耐药性。
将含羧基的疏水性物质(如脂肪酸),与壳聚糖分子结构中的部分氨基嫁接,可增强壳聚糖分子结构的疏水性,制备得到的壳聚糖-脂肪酸嫁接物,能够在水性介质中通过自聚集形成嫁接物胶团。该嫁接物胶团的疏水性内核,可增溶疏水性的药物分子。通过控制壳聚糖分子量,以及嫁接物合成过程中的投料比例,可制备得到具有特定氨基取代度的嫁接物,并在水性介质中形成嫁接物胶团。这种胶团能够体现出被细胞快速摄取的特性,并显示出在细胞核高浓度聚集的靶向功能。将该类药物载体应用于分子靶标位于细胞核的抗肿瘤药物的传递,可在提高药物分子靶标区域(细胞核)药物浓度、增强抗肿瘤疗效的同时,逆转耐药肿瘤细胞的耐药性。
本发明的有益之处是为分子靶标位于细胞核的药物,提供一种靶向给药载体材料。壳寡糖-脂肪酸嫁接物胶团细胞毒性低,具有快速透过细胞膜、很少被细胞内溶酶体破坏、细胞核靶向的功能。通过嫁接物胶团自身的疏水性内核特性,可包封分子靶标位于细胞核的疏水性抗肿瘤药物,形成嫁接物载药胶团。在显著提高抗肿瘤药物疗效的同时,逆转耐药肿瘤细胞的耐药性。
附图说明
图1:共孵育3小时后,MCF-7细胞和MCF-7-adr细胞对FITC标记的壳寡糖-硬脂酸嫁接物胶团的摄取照片。
具体实施方式
本发明通过实施例和附图作进一步的说明。
实施例:壳寡糖-脂肪酸嫁接物载药胶团在抗肿瘤药效(乳腺癌细胞,MCF-7细胞)及逆转耐药细胞(乳腺癌耐药细胞,MCF-7-adr细胞)耐药性中的应用
1)低分子量壳聚糖(壳寡糖)制备
取市售分子量为450kDa的壳聚糖(脱乙酰度为92.5%)90g,加至3000mL1.25%(v/v)的盐酸水溶液中,55~60℃温度条件下,溶涨2小时后,加入5%的纤维素酶(w/w),在55~60℃温度条件下进行降解。控制反应温度和时间,得低分子量壳聚糖(壳寡糖)。所得降解反应液,使用50Kda和10Kda超滤膜(Biomax-10,Millipore Co.,USA)超滤分级后,取分子量10~50Kda超滤液冷冻干燥。凝胶渗透色谱法测定壳寡糖分子量,采用TSK-gel G3000SW色谱柱,0.1mol/L醋酸钠(pH6.0)为流动相,分别以分子量0.73,5.9,11.8,47.3,212.0,788.0Kda的葡聚糖标样制备洗脱曲线,用该洗脱曲线计算壳寡糖分子量。经测定,所得壳寡糖的重均分子量为18.4kDa。
2)壳寡糖-硬脂酸嫁接物制备及理化性质测定
取上述壳寡糖0.4g,精密称定,加30mL蒸馏水搅拌溶解后,加入配比量(壳寡糖与碳二亚胺的摩尔比为1∶100)的碳二亚胺(EDC),搅拌溶解。按照壳寡糖∶硬脂酸(摩尔比)1∶20,称取硬脂酸溶于20mL无水乙醇。将上述两种溶液的混合液,在400rpm磁力搅拌条件下,80℃恒温反应5小时,冷却至室温(25℃),继续搅拌6小时。将终反应液置透析袋中,蒸馏水透析48小时。透析液冷冻干燥后,以无水乙醇洗涤去除残留的硬脂酸,得壳寡糖-硬脂酸嫁接物。
采用三硝基苯磺酸法,测定嫁接物的氨基取代度。取不同量的壳寡糖(0.5~9mg),精密称定,分别溶于2mL的蒸馏水中,加入4%(w/v)的碳酸氢钠溶液2mL和0.1%(w/v)的三硝基苯磺酸2mL,37℃孵育2小时。加入2N盐酸2mL,摇匀,在344nm波长处测定吸收值,制备标准曲线。取上述的壳寡糖-硬脂酸嫁接物4mg,溶于2mL蒸馏水,同法操作,测定得到嫁接物的氨基取代度为14.8%。
采用芘荧光法,测定壳寡糖-硬脂酸的临界胶团浓度。制备不同浓度壳寡糖-脂肪酸嫁接物溶液各10mL,分别加入定量的芘(芘终浓度为7×10-7mol/L),室温水浴超声30min。扫描芘的激发光谱和发射光谱,测定在375nm和396nm荧光强度,并以测定I375/I396倾斜率的突然变化,确定该嫁接物的临界胶团浓度为0.03mg/mL。
采用微粒粒度及表面电位分析仪,测定壳聚糖-硬脂酸嫁接物胶团的粒径和表面电位。经测定,1mg/mL的壳寡糖-硬脂酸嫁接物胶团的粒径和表面电位,分别为57.6nm和36.8mV。
3)壳寡糖-硬脂酸嫁接物胶团细胞摄取
采用异硫氰基荧光素(Fluorescein isothiocyanate,FITC)标记壳寡糖-硬脂酸嫁接物,进行壳寡糖-硬脂酸嫁接物胶团的亚细胞器转运研究。取壳寡糖-硬脂酸嫁接物20mg,精密称定后溶解于2.4mL的50%甲醇溶液中。另取5.0mg异硫氰基荧光素,精密称定后溶解于5.0mL乙醇。取0.5mL FITC的乙醇溶液与0.5mL蒸馏水混合,在冰浴、避光与磁力搅拌条件下,将壳寡糖-硬脂酸嫁接物的甲醇溶液,滴加到FITC乙醇溶液中,继续反应24小时后,使用蒸馏水透析(MWCO 10,000)24小时,最终得FITC标记壳寡糖-硬脂酸嫁接物,避光保存备用。
分别取MCF-7和MCF-7-adr细胞,在含有10%小牛血清的RPMI 1640培养液中培养(5%CO2,37℃孵箱)。取对数生长期细胞,pH7.4的PBS润洗后,加入胰酶消化并用培养液稀释,按每孔1×105个细胞的密度,接种于24孔培养板内,孵箱内培养24小时。24孔培养板内细胞贴壁生长后,弃去旧培养液,PBS润洗2次后,分别加入新鲜培养液1mL及FITC标记壳寡糖-硬脂酸嫁接物胶团溶液(终浓度200μg·mL-1)。孵育一定时间后,用PBS冲洗细胞2次,置荧光倒置显微镜观察并拍照。分别与MCF-7和MCF-7-adr细胞孵育3小时后,嫁接物胶团的细胞摄取荧光显微镜照片见图1,其中A图为MCF-7细胞摄取照片,B图为MCF-7-adr细胞的摄取照片。
4)壳寡糖-硬脂酸嫁接物载药胶团制备
称取壳寡糖-硬脂酸嫁接物10mg,加水5ml溶解。于磁力搅拌下,逐滴滴入阿霉素二甲亚砜溶液适量,使阿霉素的投药量为1.5%。室温搅拌4小时后,转移至透析袋中,外相为水,透析24小时除去二甲亚砜。将透析液加适量水定容至10ml,使壳寡糖-硬脂酸嫁接物的终浓度为1mg/mL。将透析液继续以4000rpm离心10min,除去尚未增溶的阿霉素,得载药胶团。取载药胶团分散液适量,以微粒粒度与表面电位测定仪,测定载药胶团的粒径及表面电位。
经测定,壳寡糖-硬脂酸嫁接物载药胶团的粒径为76.1nm,表面电位为35.2mV。药物包封率为56.5%。
5)壳寡糖-硬脂酸嫁接物载药胶团的抗肿瘤药效及逆转耐药肿瘤细胞的耐药性
本发明以肿瘤细胞抑制率,评价壳寡糖-脂肪酸嫁接物载药胶团的抗肿瘤药效,以及对耐药肿瘤细胞耐药性的逆转效率。具体方法为:分别以乳腺癌细胞(MCF-7)及其耐药细胞(MCF-7-adr)为模型细胞,在96孔细胞培养板中,每孔加入100μL含5×103个MCF-7细胞或MCF-7-adr细胞的培养液,置37℃、5%CO2孵箱培养24小时。待细胞完全贴壁后,细胞孔中分别加入不同浓度的壳寡糖-硬脂酸嫁接物胶团溶液、壳寡糖-硬脂酸嫁接物载药胶团溶液和阿霉素溶液,以未经处理的空白细胞为对照,每孔设复孔。正常DMEM培养液继续培养48小时,每孔加5mg/mL噻唑兰(MTT)溶液20μL,置于37℃、5%CO2孵箱继续培养4小时后,弃上清液,每孔加入二甲基亚砜150μL,用多功能酶标仪测定吸光度,并计算细胞抑制率。细胞抑制率采用下式计算:
细胞抑制率%=(空白对照组吸光度-实验组吸光度)/空白对照组吸光度×100%
经计算,壳寡糖-硬脂酸嫁接物胶团溶液、阿霉素溶液,以及壳寡糖-硬脂酸嫁接物载药胶团溶液,对MCF-7敏感和耐药细胞的IC50(细胞半数致死率)值,见表1。
表1壳寡糖-硬脂酸嫁接物胶团溶液、阿霉素溶液和壳寡糖-硬脂酸嫁接物载药胶团溶液,对MCF-7敏感和耐药细胞的细胞毒性
处方组成 | IC50(μg·mL-1) | 毒性增加倍数 | ||
MCF-7 | MCF-7-adr | MCF-7 | MCF-7-adr | |
壳寡糖-硬脂酸嫁接物 | 285.74±3.24 | 271.32±4.26 | - | - |
阿霉素溶液 | 0.49±0.03 | 22.84±0.25 | 1 | 1 |
壳寡糖-硬脂酸嫁接物载药胶团 | 0.18±0.02 | 0.26±0.03 | 2.70 | 87.84 |
研究结果表明,壳寡糖-硬脂酸嫁接物为低细胞毒性材料,阿霉素经壳寡糖-硬脂酸嫁接物胶团包封后,可提高MCF-7敏感细胞2.7倍的抗肿瘤疗效,提高MCF-7耐药细胞87.8倍的抗肿瘤疗效,并完全逆转MCF-7耐药细胞的耐药性。
Claims (1)
1、一种细胞核靶向给药系统在制备逆转肿瘤细胞耐药性的分子靶标位于细胞核的抗肿瘤药物中的应用,细胞核靶向给药系统由细胞核靶向给药载体和被包封的抗肿瘤药物组成,细胞核靶向给药载体选用壳寡糖-脂肪酸嫁接物胶团。
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