CN114712521A - 一种靶向cd44受体的药物及其制备方法和应用 - Google Patents
一种靶向cd44受体的药物及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及药物化学技术领域,具体涉及一种靶向CD44受体的药物及其制备方法和应用。本发明提供的靶向CD44受体的药物包括受体靶向区域、交联区域和酶响应区域;其中,所述受体靶向区域包括能够靶向CD44的高分子聚合物;所述交联区域包括能够与金属离子配位结合的分子;所述酶响应区域包括能够被细胞膜上表达的酶剪切的分子。该靶向药物能够有效抑制肿瘤细胞的EMT进程,并抑制肿瘤细胞的转移,同时具有生物相容性高、特异性强的优势,对抗肿瘤药物的开发和肿瘤的临床治疗具有重要意义。
Description
技术领域
本发明涉及药物化学技术领域,具体涉及一种靶向CD44受体的药物及其制备方法和应用。
背景技术
近年来,癌症患者的数量呈明显增加趋势,而且癌症年龄标准化发病率的年平均变化率也呈现上升趋势。肿瘤转移是导致癌症死亡率高、预后差、易复发的主要原因,给患者的身体带来很大的痛苦,同时也增加了治疗的经济压力。
上皮-间充质转化(Epithelial mesenchymal transition,EMT)是肿瘤转移的重要环节。EMT是由众多调节因子经过各种调节通路导致上皮细胞特性消失而表达间充质细胞特性的细胞转化过程。EMT的典型特征是上皮细胞标记物下调,如E-钙黏蛋白(E-cadherin)、紧密连接(ZO1)、桥粒(desmoplakin)、细胞角蛋白(cytokeratins)、层黏连蛋白(laminin)、上皮蛋白基底细胞粘附分子(BCAM)、细胞粘附分子4(CADM4),同时伴随间质标记物表达的升高,如N-钙黏蛋白(N-cadherin)、前转移分化簇109(CD109)、编码波形蛋白(VIM)、编码纤维连接蛋白(FN1)、血管内皮生长因子(VEGF)。EMT是一个动态的连续的过程,一旦EMT程序被激活,癌细胞就失去了很多上皮特征,包括上皮细胞连接和顶端-基底极性的存在,取而代之的是间充质属性,如细长的成纤维细胞样形态,以及增加的迁移和侵袭能力。从EMT过程在癌症中的生理结果来看,癌细胞的侵袭性在EMT进程中不断增强,致瘤能力和耐药性在EMT的中等水平达到顶峰。因此,从细胞的EMT过程入手,找到合适的手段抑制细胞EMT进程,从而抑制肿瘤的迁移与侵袭,是一种前景较好的治疗手段。
近年来,纳米药物得到快速发展,纳米药物有着诸多优势,例如,尺寸小,渗透能力强,血液循环时间长等。此外,越来越多的研究报道显示,许多类型的肿瘤细胞相对于正常细胞,可以摄入更多的纳米颗粒。因此,纳米药物有望为抑制肿瘤细胞EMT进程提供有效的解决方案。
然而,目前大部分的研究报道都集中在纳米药物如何杀伤肿瘤细胞,很少有研究关注如何在提高安全性的同时,增强药物的有效性并有效抑制EMT过程和肿瘤转移。
发明内容
本发明的目的是提供一种靶向CD44受体的药物及其制备方法和应用。
本发明以开发能够有效抑制肿瘤细胞的EMT过程、抑制肿瘤转移的纳米药物为目的,经大量筛选和验证,开发出一种靶向CD44受体的药物,该药物靶向肿瘤细胞膜上的CD44受体,不进入肿瘤细胞内部,能够在肿瘤细胞外靶向聚集并原位形成纳米网络,发挥阻碍细胞EMT进程、抑制肿瘤迁移的作用,同时,该纳米药物毒性明显降低,具有较高的生物相容性,有利于提高患者的生存质量。
具体地,本发明提供以下技术方案:
本发明提供一种靶向CD44受体的药物,所述药物包括受体靶向区域、交联区域和酶响应区域;
其中,所述受体靶向区域包括能够靶向CD44的高分子聚合物;
所述交联区域包括能够与金属离子配位结合的分子;
所述酶响应区域包括能够被细胞膜上表达的酶剪切的分子。
以上所述的靶向CD44受体的药物的作用机制如下:受体靶向区域靶向CD44过表达的肿瘤细胞,酶响应区域被肿瘤细胞膜上的酶剪切,引发交联区域与金属离子(如铁离子等)等发生交联,进而抑制肿瘤细胞EMT进程,阻止肿瘤细胞转移。靶向CD44受体的药物的结构示意图如图1所示,其作用机制的示意图如图2所示。
上述靶向CD44受体的药物中,受体靶向区域与交联区域连接,交联区域与酶响应区域连接。
以上所述的连接方式包括共价键连接。所述共价键包括但不限于酰胺键等。
优选地,以上所述的能够靶向CD44的高分子聚合物为选自透明质酸、聚乙二醇、壳聚糖、葡聚糖、黄原胶中的一种或多种。
以上所述的能够与金属离子配位结合的分子为选自含有邻苯二酚结构的分子、含有羟基的分子中的一种或多种。优选为选自多巴胺、肾上腺素、去甲肾上腺素、酪氨酸中的一种或多种。
以上所述的细胞膜上表达的酶包括碱性磷酸酶。优选地,所述能够被细胞膜上表达的酶剪切的分子为磷酸分子。
作为本发明的一种优选实施方式,所述受体靶向区域为透明质酸,所述交联区域为多巴胺,所述酶响应区域为磷酸分子。
优选地,所述受体靶向区域与所述交联区域通过酰胺键连接。进一步优选地,所述受体靶向区域的羧基端与所述交联部分的氨基端以酰胺键连接。
上述由透明质酸、多巴胺和磷酸分子连接形成的靶向药物分子能够特异性地靶向肿瘤细胞膜上的CD44受体,酶响应区域在肿瘤细胞膜表达的碱性磷酸酶的作用下发生剪切,引发多巴胺发挥高效的交联作用,抑制细胞的EMT进程和肿瘤细胞迁移。透明质酸在与多巴胺连接后,能够高效地在肿瘤细胞外原位聚集形成纳米网络,进一步显著增强了抑制细胞EMT进程和肿瘤细胞迁移的作用。
优选地,所述靶向CD44受体的药物的化学结构式如式(I)所示:
其中,n为25000-100000的整数。
本发明还提供以上所述的靶向CD44受体的药物的制备方法,所述方法包括:将交联区域与酶响应区域连接后,再与受体靶向区域连接。
优选地,所述方法包括:将交联区域磷酸化得到磷酸化的交联区域,将受体靶向区域在基团活化剂的作用下与磷酸化的交联区域连接。
作为本发明的一种实施方式,所述方法包括如下步骤:
(1)将交联区域的分子与磷酸和五氧化二磷混合反应,将交联区域的分子进行磷酸化,得到磷酸化的交联区域;
(2)将受体靶向区域的高分子聚合物与水混合、溶解,得到高分子聚合物溶液。
(3)将步骤(2)的高分子聚合物溶液与基团活化剂混合后,再与步骤(1)得到的磷酸化的交联区域混合,搅拌反应。
上述步骤(3)中,在反应结束后,还可包含将反应所得溶液经透析和冷冻干燥,得到所述靶向CD44受体的药物。
对于化学结构式如式(I)所示的靶向CD44受体的药物,优选采用包括如下步骤的方法制备:
(1)将五氧化二磷和80-90%的磷酸按照1:(1-1.5)的质量比混合得到混合物,再将混合物与多巴胺按照(1.1-1.3):1的质量比混合,于75-85℃条件下搅拌20-30小时;反应结束后,将得到的琥珀色液体与水混合,继续在75-85℃的条件下加热25-35分钟,反应结束后,将所得溶液冷却后用正丁醇稀释,于0℃条件下放置2.5-3.5小时后,经过滤得到白色沉淀物,用水、乙醇、乙醚依次洗涤,得到白色粉末,即得到具有酶响应功能的交联区域;
(2)将透明质酸溶解于水中,得到浓度为0.2-0.4mM的透明质酸溶液,将透明质酸溶液与0.1-0.2mM的基团活化剂N-羟基琥珀酰亚胺和10-12mM的1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐混合,搅拌10-30分钟后,加入步骤(1)制得的具有酶响应功能的交联区域(0.4-0.6mM),在氮气保护下搅拌18-30小时,得到与具有酶响应功能的交联区域连接的受体靶向区域的溶液;
(3)将步骤(2)制得的与具有酶响应功能的交联区域连接的受体靶向区域的溶液进行透析,将透析所得溶液进行冷冻干燥,即得靶向CD44受体的药物。
基于本发明提供的靶向CD44受体的药物的功能,本发明提供所述靶向CD44受体的药物在制备用于抑制肿瘤细胞的上皮-间充质转化的药物中的应用;
以及所述靶向CD44受体的药物在制备用于抑制肿瘤转移的药物中的应用;
以及所述靶向CD44受体的药物在制备抗肿瘤药物中的应用。
本发明提供一种药物组合物,所述药物组合物包括所述靶向CD44受体的药物。
上述药物组合物可仅以靶向CD44受体的药物作为活性成分,也可包括其他具有抗肿瘤活性的药物。
上述药物组合物中还可包括药学领域允许的辅料。
本发明还提供一种抑制肿瘤细胞迁移或抑制肿瘤细胞EMT进程的方法,所述方法包括:将以上所述的靶向CD44受体的药物与肿瘤细胞共培养。
优选地,所述共培养体系中,所述靶向CD44受体的药物的浓度为10-600μM,更优选为20-500μM,可以是20μM、30μM、40μM、50μM、60μM、70μM、80μM、90μM或100μM等,但不仅限于所列举的数值,该范围内其他未列举的数值同样适用。更优选的浓度为100-200μM。
优选地,所述共培养的时间为0.5-24h,例如可以是0.5h、1h、2h、4h、6h、8h、10h、12h、14h、16h、18h、20h、22h或24h等,但不仅限于所列举的数值,该范围内其他未列举的数值同样适用。更优选的共培养时间为4-8h。
本发明具有以下有益效果:本发明的靶向CD44受体的药物包括受体靶向部分、酶响应部分和交联部分,该靶向药物能够有效抑制肿瘤细胞的EMT进程,并抑制肿瘤细胞的转移,同时具有生物相容性高、特异性强的优势,对抗肿瘤药物的开发和肿瘤的临床治疗具有重要意义。
附图说明
图1为本发明的靶向药物的结构示意图。
图2为本发明的靶向药物在细胞水平发挥作用的示意图,其中ALP代表碱性磷酸酶,crosslink代表交联,Dephosphorylate代表去磷酸化。
图3为本发明实施例1中靶向药物HDP分子在水溶液中的透射图像。
图4为本发明实施例1中靶向药物HDP分子酶响应前后的核磁图谱。
图5为本发明实施例1中靶向药物HDP分子经酶剪切后与金属离子络合的紫外吸收变化图。
图6为本发明实施例1中靶向药物HDP分子经酶剪切后与金属离子络合的透射电镜图。
图7为本发明实施例2中靶向药物HDP加入表达CD44受体的MDA-MB-231细胞,HDP与CD44受体的生物电镜透射图像。
图8为本发明实施例3中靶向药物HDP加入表达CD44受体的MDA-MB-231细胞,HDP与CD44受体的免疫荧光共定位共聚焦图像。
图9为本发明实施例4中靶向药物HDP与MDA-MB-231细胞共孵育后CCK-8细胞毒检测结果图。
图10为本发明实施例5中靶向药物HDP与MDA-MB-231细胞共孵育后进行划痕实验的结果图,其中,PBS代表磷酸缓冲盐溶液,HA代表透明质酸,即无酶响应和无交联功能的对照组,HDP代表靶向CD44受体的纳米药物。
图11为本发明实施例6中靶向药物HDP分子抑制MDA-MB-231细胞转移的Westernblot细胞毒检测电泳图和灰度统计结果,其中,PBS代表磷酸缓冲盐溶液,HA代表透明质酸,即无酶响应和无交联功能的对照组,HDP代表靶向CD44受体的纳米药物。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。
以下实施例中,基团活化剂的配方为N-羟基琥珀酰亚胺:(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)=1:1(m/m)。其余原料和试剂如无特殊说明,均可从商业途径获得。
实施例1
本实施例以CD44受体为靶向受体设计靶向药物HDP,其由受体靶向区域、交联区域和酶响应区域组成,其中,受体靶向区域含有透明质酸分子,交联区域含有多巴胺分子,酶响应区域含有磷酸基团。靶向药物HDP的化学结构式如式(I)所示,其中n为25000-100000的整数:
以上所述的靶向药物HDP的制备方法的步骤如下:
(1)在配有磁力搅拌器和加热器的圆底烧瓶(100mL)中,加入新鲜的五氧化二磷(10.0g)和85%的磷酸(13.0g),在摇床上震荡均匀,随后加入多巴胺(2.0g),继续摇匀。
(2)将步骤(1)得到的混合物在80℃条件下搅拌24小时。
(3)将步骤(2)得到的琥珀色液体中加入纯净水(30mL),并继续在80℃的条件下加热30分钟。
(4)将步骤(3)得到的溶液冷却至室温,用正丁醇(650mL)稀释,置于0℃中3小时。
(5)将步骤(4)得到的溶液经过滤得到白色细沉淀物,用冰水、乙醇、乙醚依次洗涤,得到白色粉末,即得到具有酶响应功能的交联部分,于-20℃保存待用。
(6)在玻璃瓶中称取透明质酸(150万~250万,0.3mM),溶于纯净水中,搅拌过夜。
(7)在步骤(6)所得的溶液中加入基团活化剂N-羟基琥珀酰亚胺(0.1mM)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(0.1mM),搅拌20分钟后加入步骤(5)制得的具有酶响应功能的交联部分(0.5mM),在氮气保护下搅拌过夜。
(8)将步骤(7)所得的溶液置于透析袋中透析。
(9)将步骤(8)透析所得溶液冷冻干燥,得到白色絮状粉末,将粉末进行核磁氢谱、磷谱鉴定正确后,即得到具有靶向CD44受体功能的靶向药物,将药物置于-20℃保存待用。
上述制得的靶向药物HDP分子在水溶液中的透射图像如图3所示,HDP分子酶响应前后的核磁图谱如图4所示,HDP分子经酶剪切后与金属离子络合的紫外吸收变化如图5所示,HDP分子经酶剪切后与金属离子络合的透射电镜图如图6所示。上述结果表明成功制备得到靶向药物HDP。
实施例2
本实施例对实施例1得到的靶向药物HDP进行功能鉴定,具体地,采用生物透射电镜技术对靶向药物HDP进行靶向功能的验证,方法和结果如下:
将靶向药物HDP与MDA-MB-231细胞共孵育8小时,置于生物透射电镜下观察,观察结果如图7所示,靶向药物HDP可以与细胞膜上的CD44受体结合,能够作为CD44受体的靶向药物使用。
实施例3
本实施例对实施例1得到的靶向药物HDP进行免疫荧光共定位分析,方法和结果如下:
在保持37℃和5%CO2的条件下,在含有10%胎牛血清(FBS)的DMEM培养基中,培养MDA-MB-231细胞。
向约105个CD44受体高表达的MDA-MB-231细胞中加入1mL、200μM的靶向药物HDP,于37℃、5%的CO2细胞培养箱中孵育4h后,用4%多聚甲醛固定细胞30min,随后进行免疫荧光检测。
结果如图8所示,可以看出靶向药物HDP在细胞膜上能够进行很好的共定位,表明靶向药物HDP具有靶向能力。
实施例4
本实施例对实施例1得到的靶向药物HDP进行细胞毒性分析,方法和结果如下:
在保持37℃和5%CO2的条件下,在含有10%胎牛血清(FBS)的DMEM培养基中,培养MDA-MB-231细胞。然后用0.05%的胰蛋白酶-EDTA分离细胞。将通过细胞计数器计算的4×103个细胞接在96孔板中,并于37℃含5%CO2的培养箱中培养。
24小时后,将每孔含有细胞的培养基换成分别含有500μM、300μM、200μM、100μM、50μM、25μM、12.5μM、6.25μM靶向药物HDP的培养基,然后在37℃含5%CO2的培养箱中培养48小时。随后在每孔加入100μL的细胞计数试剂(Cell counting kit-8,CCK-8),并培养2小时,再使用酶联免疫检测仪测定450nm处的吸光度。
检测结果如图9所示,添加上述各浓度的靶向药物HDP均未导致细胞存活率显著下降,表明靶向药物HDP具有优异的生物相容性。
实施例5
本实施例对实施例1得到的靶向药物HDP进行细胞划痕实验,方法和结果如下:
向6孔板中加入约5×105个CD44受体高表达的MDA-MB-231细胞,贴壁后用枪头垂直于细胞平面划线。划痕完成后用无菌PBS洗细胞3次,洗去未贴壁的细胞使得划痕清晰可见。随后加入2mL、200μM/mL的靶向药物HDP,于37℃、5%的CO2细胞培养箱中孵育24h。然后用4%多聚甲醛固定细胞30min,0.1%的结晶紫染色,最后在显微镜下观察并测量划痕宽度。
结果如图10所示,归一化后HDP组的划痕宽度为1,PBS对照组的划痕宽度是HDP组的40%,HA组的划痕宽度是HDP组的31%,,表明靶向药物HDP具有较好的抑制肿瘤细胞迁移的能力。
实施例6
本实施例对实施例1得到的靶向药物HDP进行蛋白免疫印迹实验,分析肿瘤细胞迁移能力下降的机制,方法如下:
1、全蛋白提取
(1)在5mL冷的RIPA裂解液中加入50μL、100mM PMSF储存液,混匀,放置在冰上备用;
(2)将贴壁细胞弃去培养基,用冷的PBS洗两次,加入计算好的细胞裂解液(每107细胞需要裂解液1mL);在冰上用细胞刮刀进行刮离细胞;
(3)将刮离的细胞裂解液移入Ep管中,颠倒裂解25min;
(4)裂解完毕后,4℃12000rpm离心30min,取上清,装入新的Ep管中。
2、蛋白定量
(1)配制蛋白定量工作液:BCA试剂与Cu试剂比例为50:1,在5mL BCA试剂中加入100μL Cu试剂,配置成工作液;
(2)利用BSA标准品分别配制0mg/mL、0.25mg/mL、0.5mg/mL、1mg/mL、2mg/mL和3mg/mL的标准溶液,用来绘制标准曲线;
(3)将标准溶液和样品加入到工作液中,并转移到96孔板进行孵育,孵育条件为37℃,30min。用酶标仪测定450nm处吸收波长;
(4)根据标准曲线计算出样品的蛋白浓度,并用稀释液(PBS)进行蛋白浓度一致性调节;
(5)加入5×上样缓冲液,并用金属浴煮沸10min进行蛋白变性。
3、蛋白电泳
(1)将15%预制胶取出装入电泳槽中后加入1×电泳液,双手分别拿住梳子两边,向上拉起梳齿;
(2)将20μL蛋白样品注入到蛋白上样孔中;
(3)调节电泳电压为90V,时间设定为90min,进行蛋白电泳;
(4)电泳完毕后,利用湿转方法对蛋白进行转膜,转膜条件为300mA,1.5h;
(5)转膜完毕后将膜取出,用5%脱脂奶粉常温孵育封闭2h。
4、抗原抗体免疫反应
(1)将目的条带裁剪分开,加入对应的一抗,4℃孵育16h;
(2)回收一抗,用TBST进行洗膜5次,每次7min;
(3)常温孵育二抗2h;
(4)回收二抗,用TBST进行洗膜5次,每次7min;
(5)加入ECL发光剂孵育膜,并用曝光仪检测目的条带。
结果如图11所示,与对照组相比,靶向药物HDP处理过的MDA-MB-231细胞的E-Cadherin蛋白含量提高,Vimentin蛋白含量下降,说明细胞的运动性降低,EMT进程被抑制。
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
Claims (10)
1.一种靶向CD44受体的药物,其特征在于,所述药物包括受体靶向区域、交联区域和酶响应区域;
其中,所述受体靶向区域包括能够靶向CD44的高分子聚合物;
所述交联区域包括能够与金属离子配位结合的分子;
所述酶响应区域包括能够被细胞膜上表达的酶剪切的分子。
2.根据权利要求1所述的靶向CD44受体的药物,其特征在于,所述受体靶向区域与所述交联区域连接,所述交联区域与所述酶响应区域连接。
3.根据权利要求1或2所述的靶向CD44受体的药物,其特征在于,所述能够靶向CD44的高分子聚合物为选自透明质酸、聚乙二醇、壳聚糖、葡聚糖、黄原胶中的一种或多种;
和/或,
所述能够与金属离子配位结合的分子为选自含有邻苯二酚结构的分子、含有羟基的分子中的一种或多种;优选为选自多巴胺、肾上腺素、去甲肾上腺素、酪氨酸中的一种或多种;
和/或,
所述细胞膜上表达的酶包括碱性磷酸酶;优选地,所述能够被细胞膜上表达的酶剪切的分子为磷酸分子。
4.根据权利要求3所述的靶向CD44受体的药物,其特征在于,所述受体靶向区域为透明质酸,所述交联区域为多巴胺,所述酶响应区域为磷酸分子;
优选地,所述受体靶向区域与所述交联区域通过酰胺键连接。
6.权利要求1~5任一项所述的靶向CD44受体的药物的制备方法,其特征在于,所述方法包括:将所述交联区域与所述酶响应区域连接后,再与所述受体靶向区域连接;
优选地,所述方法包括:将所述交联区域磷酸化得到磷酸化的交联区域,将所述受体靶向区域在基团活化剂的作用下与磷酸化的交联区域连接。
7.权利要求1~5任一项所述的靶向CD44受体的药物在制备用于抑制肿瘤细胞的上皮-间充质转化和/或抑制肿瘤转移的药物中的应用。
8.权利要求1~5任一项所述的靶向CD44受体的药物在制备抗肿瘤药物中的应用。
9.一种药物组合物,其特征在于,所述药物组合物包括权利要求1~5任一项所述的靶向CD44受体的药物。
10.一种非治疗目的的抑制肿瘤细胞迁移或抑制肿瘤细胞EMT进程的方法,所述方法包括:将权利要求1~5任一项所述的靶向CD44受体的药物与肿瘤细胞共培养。
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