CN105617380A - 一种竹红菌甲素-转铁蛋白靶向给药体系的制备方法及其在光动力疗法中的应用 - Google Patents
一种竹红菌甲素-转铁蛋白靶向给药体系的制备方法及其在光动力疗法中的应用 Download PDFInfo
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Abstract
本发明公开了一种竹红菌甲素-转铁蛋白靶向给药体系的制备方法及其在光动力疗法中的应用。所述的方法是,采用全铁转铁蛋白作为载体,利用尿素、β-巯基乙醇将全铁转铁蛋白失活,使其三维结构松散露出Fe离子配位点后,将过量竹红菌甲素加入,其与铁配位后,透析除掉未结合的HA及尿素、β-巯基乙醇以还原全铁转铁蛋白三维结构,获得竹红菌甲素-转铁蛋白靶向给药体系。本发明方法制备简单,应用前景良好;该靶向给药体系可制成冻干粉保存,溶解后制成适合静脉注射的制剂;因该体系中转铁蛋白的肿瘤靶向作用,该靶向给药体系可显著增强竹红菌甲素光敏抗肿瘤活性。
Description
技术领域
本发明属于具有光动力活性的光敏剂技术领域,涉及一种通过金属键螯合偶联竹红菌甲素光敏剂与靶向分子的方法,以及通过该方法制备得到的竹红菌甲素-转铁蛋白靶向给药体系在光动力疗法中的应用。
背景技术
光动力疗法(photodynamictherapy,简称PDT)是近年来发展起来的一种新型肿瘤临床疗法,因其抗瘤谱广、无严重副反应、微创以及经济方便等独特的优点受到广泛关注。尤其对于那些属于癌症晚期和由于年龄、体质等原因不适合使用传统治疗技术的病人,光动力疗法仍然是一种理想的治疗手段。光动力疗法主要通过使用适当波长的光源激发光敏剂产生活性氧,活性氧通过一系列生理生化反应最终导致肿瘤细胞死亡。
竹红菌甲素(HypocrellinA,简称HA)具有光敏产生各种活性氧的能力。与目前已经商品化的光敏剂(如血卟啉类衍生物)相比,其具有易得、易纯化、化学修饰性好、三重态量子产率高、单重态氧量子产率高、光毒性高、体内代谢快等优点,在光动力抗艾滋病毒、治疗肿瘤和微血管类疾病等方面有广泛的潜在应用价值。
然而,理想的光动力疗法药物除需具备较强的活性氧产生能力外,还需具备一定的肿瘤组织靶向能力,这样才可以将光疗药物有效的运送至靶组织、靶器官、靶细胞以及细胞内的特定部位,提高治疗效果,同时可以保护机体正常组织和细胞不受伤害。
在众多靶向系统中,受体介导的主动靶向系统因其具有高特异性、高选择性、饱和性、亲合力强和生物效应明显等特点而受到广泛关注。利用配体为药物的载体,通过受体介导作用,增加病灶局部药物浓度,提高疗效,降低毒副作用,达到靶向治疗目的。
转铁蛋白-转铁蛋白受体(Transferrin-TransferrinReceptor,Tf-TfR)受体介导的肿瘤细胞靶向给药体系是近年来的研究热点。转铁蛋白(Tf)是人体血液中主要的含铁蛋白质,负责体内铁的运输。铁是细胞生长增殖及机能活动所必需的微量元素之一,而肿瘤细胞主要是通过高表达转铁蛋白受体(TfR)来摄取铁。肿瘤细胞表面大量表达的转铁蛋白受体为转铁蛋白作为药物载体提供了可能。转铁蛋白与药物结合在一起,在组织中的分配性好,延长了药物在胞浆中的半衰期,同时还可达到控释目的。
传统的转铁蛋白-药物给药体系(Tf-Drug)的构建主要是通过两种途径,一是通过偶联反应形成药物与蛋白之间的西佛碱从而形成一个稳定的给药体系,但该化学反应过程会在一定程度上影响转铁蛋白的构象及活性;而且,稳定化学键可能影响后期药物的释放过程。另外一种方法是通过药物与蛋白之间简单的相互作用力,如静电力、疏水键、氢键等将蛋白与药物连接,这种方法反应体系简单、对蛋白构象及活性影响小,但因为以上三种作用力强度不高,因此该方法得到的给药体系在给药过程中往往发生所载药物从蛋白上脱落,造成给药体系的崩解。
发明内容
本发明的目的在于克服竹红菌甲素不具有主动的肿瘤组织靶向的缺点,提供一种竹红菌甲素靶向给药体系的制备方法,利用竹红菌甲素易与金属离子螯合的特点,通过金属键将竹红菌甲素与转铁蛋白偶联,制备出具有临床应用前景的竹红菌甲素靶向给药体系,并将该靶向给药体系应用于光动力疗法领域。
为实现上述发明目的,本发明采用的技术方案是:
一种竹红菌甲素-转铁蛋白靶向给药体系的制备方法,其特征在于,采用全铁转铁蛋白作为载体,在生理缓冲溶液中,使用尿素、β-巯基乙醇将全铁转铁蛋白失活,使其三维结构松散露出Fe离子配位点后,将过量竹红菌甲素加入(优选按HA与Tf按2:1比例加入),4℃恒温震荡10-24h,透析除掉未结合的竹红菌甲素(HA)及尿素、β-巯基乙醇,还原全铁转铁蛋白三维结构,制得竹红菌甲素-转铁蛋白靶向给药体系。
上述竹红菌甲素-转铁蛋白靶向给药体系,是一种适合静脉注射的制剂,样品经冷冻干燥后可获得冻干粉,使用之前用水溶解,溶解后可制成适合静脉注射的制剂。
本发明针对传统方法的缺陷,利用转铁蛋白的独特性质及竹红菌甲素的特性,在经典的蛋白变性-复性体系(尿素、β-巯基乙醇)中,利用变性剂将转铁蛋白螺旋结构打开后,进行其携带的铁离子与竹红菌甲素进行配位,而后通过透析除掉变性剂后,蛋白恢复构象,将竹红菌素装载于其内部,构建通过竹红菌甲素中的酚羟基与醌羰基与转铁蛋白中Fe3+螯合进而将苝醌类光敏剂紧密结合在转铁蛋白上的方法来实现竹红菌素的靶向给药。竹红菌甲素非常容易与金属离子螯合,这种螯合的作用力强度又远大于普通的作用力,如静电力、疏水键、氢键等,而且这种螯合是物理作用并没有发生化学反应,因此对蛋白的活性和构象都没有太大的影响。本发明采用的利用蛋白变性复性体系实现转铁蛋白药物装载的方法从未见报道;因此通过金属螯合键将竹红菌甲素与转铁蛋白偶联既不影响蛋白活性及构象,又能实现竹红菌甲素靶向给药,该类载体在光动力疗法领域具有极大的潜力。
完成本发明第二项发明任务的方案是:所述方法制得的竹红菌甲素-转铁蛋白靶向给药体系在制备光动力疗法药物中的应用。
本发明的一种竹红菌甲素-转铁蛋白靶向给药体系的制备方法所制得的竹红菌甲素-转铁蛋白靶向给药体系具有如下的优点:
1.本发明的靶向给药体系制备方法简单、条件温和,有效地保持了转铁蛋白的构象,对其靶向功能的维持非常重要;
2.与单独存在的竹红菌甲素相比,该竹红菌甲素-转铁蛋白靶向给药体系不但水溶性高、分散性好,且在生理环境稳定分散性质佳;
3.竹红菌甲素-转铁蛋白靶向给药体系具有肿瘤细胞靶向功能,体外癌细胞实验结果表明,该给药体系能有效增加肿瘤细胞对竹红菌甲素光敏剂的摄取进而增强其光动力活性。
附图说明
图1为竹红菌甲素(HA)装载入转铁蛋过程中,转铁蛋白构象变化及还原过程;其中
(A)Tf、Tf+尿素+β-巯基乙醇+HA透析前及Tf+尿素+β-巯基乙醇+HA透析后圆二色信号的叠加图;
(B)Tf圆二色信号以及软件计算得到的蛋白各种二级结构组分含量;
(C)Tf+尿素+β-巯基乙醇+HA透析前圆二色信号以及软件计算得到的蛋白各种二级结构组分含量;
(D)Tf+尿素+β-巯基乙醇+HA透析后圆二色信号以及软件计算得到的蛋白各种二级结构组分含量。
图2为竹红菌甲素-转铁蛋白螯合物(HA-Tf)及竹红菌甲素(HA)水溶液稳定性比较。
图3为竹红菌甲素-转铁蛋白螯合物(HA-Tf)及竹红菌甲素(HA)肿瘤细胞摄取能力比较;
图4为竹红菌甲素-转铁蛋白螯合物(HA-Tf)及竹红菌甲素(HA)光敏杀伤肿瘤细胞能力比较。
具体实施方式
实施例1
一种竹红菌甲素靶向给药体系的制备方法,使用全铁转铁蛋白制备竹红菌甲素-转铁蛋白螯合物,方法如下:
在生理缓冲溶液中,使用尿素、β-巯基乙醇将全铁转铁蛋白失活,使其三维结构松散露出Fe离子配位点后,将过量竹红菌甲素加入(按HA与Tf按2:1比例加入),4℃恒温震荡24h,透析除掉未结合的HA及尿素、β-巯基乙醇以还原全铁转铁蛋白三维结构,获得竹红菌甲素-转铁蛋白靶向给药体系,样品经冷冻干燥获得冻干粉,使用之前用纯净水溶解。
竹红菌甲素-转铁蛋白靶向给药体系表征:
本实施例的靶向给药体系制备过程中蛋白构象变化及复原过程、稳定性、肿瘤摄取及光敏抗肿瘤活性以圆二色谱(Chriascan,AppliedPhotophysics)、紫外可见光谱及MTT比色法检测。
(1)靶向给药体系制备过程中蛋白构象变化及复原过程
圆二色光谱仪通过测量生物大分子的圆二色光谱从而得到生物大分子的二级结构,广泛用于蛋白质折叠及蛋白质构象研究。因此,在靶向给药体系制备过程中蛋白构象变化及复原过程使用圆二色谱检测。尿素及β-巯基乙醇是经典的可诱导蛋白质空间构象解旋剂。如图1A所示,在向Tf溶液中加入尿素、β-巯基乙醇及HA反应24h后,Tf的圆二色信号发生显著改变,通过软件计算可知,其体系内包括α-螺旋、β-转角及无规则卷曲等结构均发生显著改变,且无规则卷曲含量增加,说明此时蛋白空间构象发生改变,整体结构趋向解旋。空间结构松散有利于暴露其铁离子,进而易于与HA发生配位。透析除掉尿素及β-巯基乙醇,发现其圆二色信号恢复,与原始Tf基本相同,经软件计算其α-螺旋、β-转角及无规则卷曲等结构也均与原始Tf基本相同,说明Tf构象恢复,且HA的载入对Tf构象影响很小,这对其保留肿瘤靶向功能非常重要。
(2)靶向给药体系水相中稳定性分析
药物在水相中的稳定性非常重要,若在储存或给药过程中,发生药物剧集沉降,可能会影响药物活性。蛋白质水溶性佳,生理环境分散性质优,因此,与转铁蛋白形成螯合物后,其水相稳定分散性质应有显著提升。使用紫外可见光谱法监测竹红菌甲素-转铁蛋白螯合物(HA-Tf)及竹红菌甲素(HA)在水相中的稳定分散性质。同浓度样品置于比色皿中,4℃保存及测试,连续3天测试上清液紫外可见吸收光谱,若样品发生聚集沉降,其紫外可见光谱吸收强度将发生显著下降,以此判断样品在水相中的稳定分散性质。如图2所示,3天后,竹红菌甲素-转铁蛋白螯合物(HA-Tf)几乎没有发生沉降,而竹红菌甲素样品则发生显著沉降,说明该复合体系具有优良的水相稳定分散性质。
(3)竹红菌甲素-转铁蛋白螯合物(HA-Tf)及竹红菌甲素(HA)肿瘤细胞摄取能力比较
据文献报道,乳腺癌细胞HeLa表面高表达Tf受体,因此,研究HeLa细胞对Tf修饰药物的摄取科学合理。竹红菌甲素-转铁蛋白螯合物(HA-Tf)及竹红菌甲素(HA)溶液加入培养有乳腺癌细胞HeLa的96孔细胞培养板中,使用紫外可见吸收光谱监测培养孔内培养液中所含药物浓度变化规律,判断肿瘤细胞对二者的吸收能力,如图3所示,在不同药物孵育时间范围内,HeLa细胞对竹红菌甲素-转铁蛋白螯合物(HA-Tf)均强于HA,说明体系具有肿瘤靶向能力。
(4)光敏抗肿瘤能力研究
使用MTT法研究竹红菌甲素-转铁蛋白螯合物(HA-Tf)及竹红菌甲素(HA)的光敏抗肿瘤能力,研究结果表明,470nmLED光照后,竹红菌甲素-转铁蛋白螯合物(HA-Tf)杀伤肿瘤细胞的能力强于竹红菌甲素(HA)。
Claims (3)
1.一种竹红菌甲素-转铁蛋白靶向给药体系的制备方法,其特征在于,采用全铁转铁蛋白作为载体,在生理缓冲溶液中,使用尿素、β-巯基乙醇将全铁转铁蛋白失活,使其三维结构松散露出Fe离子配位点后,将过量竹红菌甲素加入,4℃恒温震荡10-24h,透析除掉未结合的HA及尿素、β-巯基乙醇,还原全铁转铁蛋白三维结构,制得竹红菌甲素-转铁蛋白靶向给药体系。
2.根据权利要求1所述的竹红菌甲素-转铁蛋白靶向给药体系的制备方法,其特征在于,所述的反应物之间的比例关系是全铁转铁蛋白:竹红菌甲素=1:2。
3.一种权利要求1所述的方法制得的竹红菌甲素-转铁蛋白靶向给药体系在制备光动力疗法药物中的应用。
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CN111110871A (zh) * | 2020-01-08 | 2020-05-08 | 中国科学院上海硅酸盐研究所 | 一种高效跨越血脑屏障且靶向原位脑胶质瘤的纳米诊疗剂材料及其制备方法 |
CN113552368A (zh) * | 2021-07-21 | 2021-10-26 | 苏州立禾生物医学工程有限公司 | 肿瘤坏死因子α检测试剂盒及其检测方法 |
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