CN115804841A - 一种基于聚谷氨酸共轭光敏剂的肿瘤靶向载药纳米制剂 - Google Patents
一种基于聚谷氨酸共轭光敏剂的肿瘤靶向载药纳米制剂 Download PDFInfo
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Abstract
本发明属于肿瘤靶向纳米制剂领域,提供了一种基于聚谷氨酸共轭光敏剂的肿瘤靶向载药纳米制剂。所述聚谷氨酸共轭光敏剂采用PGA与常见的第二代光敏剂Ce6通过化学键共轭偶联,所述肿瘤靶向纳米制剂采用聚谷氨酸共轭光敏剂包载疏水性化疗药,表面进行肿瘤细胞膜包覆。采用不同机制的化疗药物共同治疗,可以有效提高肿瘤杀伤效果。
Description
技术领域
本发明属于肿瘤靶向纳米制剂技术领域,具体涉及一种聚谷氨酸共轭光敏剂的制备以及相关肿瘤靶向纳米制剂的制备方法、包含所述肿瘤纳米制剂的药物组合物及其在抗肿瘤药物制备中的应用。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解,而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
光动力疗法(PDT)是一种相对新颖的肿瘤无创治疗方法。光敏剂在特定波长光源照射后可产生活性氧(ROS)损伤肿瘤。第一代光敏剂主要是血卟啉类,第二代光敏剂为二氢卟吩类,目前应用较多的第二代光敏剂有二氢卟吩e6(Ce6)。第三代光敏剂,则是第二代的光敏剂偶联上不同的靶向分子如单克隆抗体,以提高其靶向性,降低PDT治疗过程中的不良反应。相比与传统肿瘤疗法,PDT能够发挥局部治疗优势,无全身毒副作用,更重要的是PDT能够激活机体的抗肿瘤免疫反应,如实现肿瘤细胞免疫原性死亡,产生细胞因子等。尽管这种治疗方法具有很多优点,但由于光敏剂的自身特性大大限制了其作用的完全发挥。例如:(1)光敏剂在水中分散性差,在溶液中易聚集产生荧光淬灭效应;(2)光敏剂肿瘤特异性差,静脉注射后易被网状内皮系统捕获清除,无法到达肿瘤部位;(3)第三代光敏剂经过单克隆抗体等结构修饰提高了靶向性,但同时带来了高昂的成本,使得PDT的施用距离普通临床市场越来越远。
聚谷氨酸(PGA)是一种具有良好生物相容性的绿色环保高分子材料,由谷氨酸的α-胺基和γ-羧基通过酰胺键交联组成。PGA的主链分子上含有大量羧基,能够共轭连接水溶性较差的小分子药物、基因药物、单克隆抗体等,在作为药物输送平台上有着巨大的应用前景。同时有研究表明,PGA能引起高效率的免疫应答,是一种安全有效的疫苗佐剂应用于肿瘤免疫治疗。例如:(1)PGA与紫杉醇直接连接形成的前药CT2103,目前已经进入非小细胞肺癌的Ⅲ期临床研究。(2)PGA分子中侧链羧基上的氢取代顺铂(CDDP)分子中的氯原子,形成有活性的、相对稳定的CDDP-PGA复合物,该复合物有较高的动力学稳定性和对正常细胞较低的毒性。(3)将PGA与难溶性10-羟基喜树碱(CPT)或9-氨基喜树碱与偶联形成PGA-CPT复合物后水溶性大为增加,且保持较高的抗肿瘤活性。现有PGA应用于抗肿瘤方向主要是直接或通过linker与化疗药相连接,但少有与光敏剂共轭连接的。尽管浙江大学CN104491863A公布了一种基于聚谷氨酸与四氯四碘荧光素键合物的抗肿瘤药物及其制备方法和应用,但其药物的组合方式仍有待改进。
光动力疗法虽然能损伤肿瘤,但往往造成肿瘤消除不彻底。其主要原因就是肿瘤自身存在修复机制。聚ADP核糖聚合酶(PARP)是一种DNA修复酶,可以结合到受损伤的DNA的单链损伤位点,修复DNA损伤。正常细胞同时拥有同源重组修复和PARP两种修复机制,而三阴性乳腺癌细胞有着较高的同源重组修复缺陷几率。
用细胞膜伪装纳米粒子是一种成熟的仿生方法。已经针对不同的应用场景开发了各种细胞膜包被策略。红细胞膜用于逃避免疫清除以增加长循环,血小板膜用于靶向损伤部位等。癌细胞膜因其独特的肿瘤归巢作用和携带肿瘤抗原的特性而备受关注。
发明内容
为了解决上述问题,本发明提供一种基于聚谷氨酸共轭光敏剂的肿瘤靶向载药纳米制剂。采用不同机制的化疗药物共同治疗,可以有效提高肿瘤杀伤效果。其中,所述聚谷氨酸共轭光敏剂采用PGA与常见的第二代光敏剂Ce6通过化学键共轭偶联,所述肿瘤靶向纳米制剂采用聚谷氨酸共轭光敏剂包载疏水性化疗药,表面进行肿瘤细胞膜包覆。
为了实现上述目的,本发明采用如下技术方案:
本发明的第一个方面,提供了一种基于聚谷氨酸共轭光敏剂的肿瘤靶向纳米制剂,包括:
聚谷氨酸共轭光敏剂;
所述聚谷氨酸共轭光敏剂包载有疏水性化疗药;
所述聚谷氨酸共轭光敏剂包覆有肿瘤细胞膜。
本发明首次将聚谷氨酸共轭光敏剂与化疗药组合施用,引入PARP抑制剂阻止肿瘤的DNA修复,协同治疗以期实现彻底消除肿瘤,同时,还通过包覆肿瘤细胞膜,提高同型原发肿瘤的特异性靶向。
本发明的第二个方面,提供了一种基于聚谷氨酸共轭光敏剂的肿瘤靶向纳米制剂的制备方法,包括:
将光敏剂通过共价键偶联、静电吸附方法连接到PGA分子上,得到聚谷氨酸共轭光敏剂;
通过疏水作用力、静电吸附、π-π共轭方式在所述聚谷氨酸共轭光敏剂包载疏水性化疗药,得到纳米制剂;
将所述纳米制剂采用肿瘤细胞的细胞膜包覆,即得。
本发明的有益效果
(1)本发明将光敏剂通过化学反应共轭偶联PGA,保留了其产生活性氧的特性,并增强其水中分散性、生物相容性与渗透性,同时由于linker胱胺的存在能实现肿瘤内部高GSH环境的响应性释放;
(2)将发明的这种能损伤肿瘤细胞DNA的聚谷氨酸光敏剂作为药物载体,与具有阻止DNA修复功能的PARP抑制剂相组合形成纳米制剂,实现协同抗肿瘤治疗。
(3)将上述纳米制剂进一步用肿瘤细胞的细胞膜包覆,实现同源肿瘤的特异性靶向,以构建仿生靶向纳米制剂。
(4)本发明制备方法简单、实用性强,易于推广。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解,本发明的示例性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。
图1,聚谷氨酸共轭光敏剂PC的合成过程。
图2,PGA、PGA-Cys、PC的1HNMR图谱。
图3,PGA、PGA-Cys、PC的FT-IR图谱。
图4,Ce6、PGA-Cys、PC的UV-Vis图谱及PC在水中产生的丁达尔现象。
图5,使用DPBF检测Ce6、PC的单线态氧产生能力。
图6,PC聚合物在水中的TEM图像。
图7,PCO、MPCO以及MPCO+GSH的TEM及粒径图像。
图8,PCO、4T1细胞膜、MPCO以及4T1细胞全蛋白的SDS-PAGE凝胶电泳图像。
图9,一周内PCO、MPCO纳米粒在PBS中的粒径及电位变化。
图10,不同释放介质条件下MPCO纳米粒对于Ola的药物释放曲线。
图11,4T-1细胞对于游离Ce6和MPCO纳米粒摄取的流式细胞术结果图。
图12,游离Ola、游离Ce6、PC聚合物、PCO和MPCO纳米粒在无光照和有光照条件下的细胞毒性结果图。
图13,Ce6、PCO和MPCO在荷瘤小鼠体内2小时、4小时、8小时和12小时的生物分布荧光图像和静脉注射后24小时肿瘤和主要器官的离体荧光成像。
具体实施方式
应该指出,以下详细说明都是示例性的,旨在对本发明提供进一步的说明。除非另有指明,本发明使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同含义。
术语解释
本申请中,“奥拉帕利”全称Olaparib,缩写Ola。
一种基于聚谷氨酸共轭光敏剂的肿瘤靶向纳米制剂,包括:
聚谷氨酸共轭光敏剂;
所述聚谷氨酸共轭光敏剂包载有疏水性化疗药;
所述聚谷氨酸共轭光敏剂包覆有肿瘤细胞膜。
在一些实施例中,所述聚谷氨酸共轭光敏剂的制备方法为:将光敏剂通过共价键偶联、静电吸附方法连接到PGA分子。
在一些实施例中,所述光敏剂为二氢卟吩e6、吲哚菁绿、叶绿素-a、脱镁叶绿酸a、焦脱镁叶绿酸-a、焦脱镁叶绿酸a己醚中至少一种。
在一些实施例中,所述疏水性化疗药为PARP抑制剂、紫杉醇、多西紫杉醇、多柔比星、奥沙利铂、槲皮素、姜黄素中至少一种。
在一些实施例中,所述PARP抑制剂为奥拉帕利、尼拉帕利、氟唑帕利、帕米帕利中至少一种。
在一些实施例中,肿瘤为黑色素瘤、肠癌、肺癌、胃癌、卵巢癌、前列腺癌、胰腺癌、乳腺癌、卵巢癌、肝癌、头颈部癌、淋巴瘤、肉瘤、慢性淋巴细胞白血病、甲状腺癌和睾丸癌、肛门癌中至少一种。
一种基于聚谷氨酸共轭光敏剂的肿瘤靶向纳米制剂的制备方法,包括:
将光敏剂通过共价键偶联、静电吸附方法连接到PGA分子上,得到聚谷氨酸共轭光敏剂;
通过疏水作用力、静电吸附、π-π共轭方式在所述聚谷氨酸共轭光敏剂包载疏水性化疗药,得到纳米制剂;
将所述纳米制剂采用肿瘤细胞的细胞膜包覆,即得。
在一些实施例中,聚谷氨酸共轭光敏剂的制备方法包括:
将分子量为5000-200000的PGA溶于溶液中,加入EDC·HCl和NHS进行活化,再加入胱胺二盐酸盐,室温下进行反应,反应完成后,透析、冻干,得聚谷氨酸-胱胺聚合物;
将光敏剂溶于溶液中,加入EDC·HCl和NHS进行活化,再加入所述聚谷氨酸-胱胺聚合物进行反应,反应完成后,透析、冻干,即得。
在一些实施例中,所述纳米制剂的制备方法包括:将PC和Ola溶于溶液中,混合均匀,旋转蒸发,得到薄膜,然后,注入PBS水化,超声分散,采用水系滤膜除去未包载的Ola,即得。
在一些实施例中,细胞膜包覆的具体步骤包括:将肿瘤细胞,加入裂解液进行反复冻融5-10次,并通过探头超声20-40min使细胞完全破裂;低温低速离心后,取上清,再高速离心,收集沉淀,即得肿瘤细胞膜碎片;
将肿瘤细胞膜与纳米制剂混合超声,并采用聚碳酸酯膜过滤器挤出10-30个来回,即得。
在一些实施例中,为了合成聚谷氨酸共轭光敏剂PC(PGA-Cys-Ce6,PC),将200mg的分子量为75000的PGA溶于10-30mL(优选20ml)蒸馏水中,加入1.8g的EDC·HCl和630mgNHS搅拌10-30min(优选20min),充分活化PGA的羧基。加入100-500mg(优选300mg)胱胺二盐酸盐,室温反应6-24h(优选12h)。用截留分子量为3500Da的透析袋透析48h,除去未反应的EDC,NHS与胱胺。冻干聚合物溶液,得聚谷氨酸-胱胺(PGA-Cys)聚合物。称取20mg Ce6溶于3-8mL(优选5ml)的DMF中,加入37mgEDC·HCl和26mgNHS搅拌10-30min(优选20min),充分活化Ce6的羧基。加入含30-80mg(优选50mg)的PGA-cys水溶液3-8mL(优选5ml),室温反应6-48h(优选12h)。用截留分子量为3500Da的透析袋,在DMF:H2O(1:1)中透析两天,在超纯水中透析两天。冻干聚合物溶液,得PC,4℃避光保存。最终产物分别用核磁共振氢谱、红外光谱和紫外光谱进行表征。
在一些实施例中,为了获得载PARP抑制剂纳米粒,采用薄膜分散法进行制备。将含有摩尔比为(1-20):(1-5)(优选的10:3)的PC和Ola溶于甲醇,并混合搅拌0.5-4h(优选1h),将混合溶液注入茄型瓶中旋转蒸发除去甲醇得到薄膜。注入2-10mL(优选4mL)PBS水化,超声1-30min(优选5min)后通过0.22μm的水系滤膜除去未包载的Ola。
在一些实施例中,为了得到肿瘤细胞膜包覆的载药纳米制剂,收集4T-1肿瘤细胞,加入裂解液在液氮罐反复冻融5-10次(优选7次),并通过探头超声20-40min(优选30min)使细胞完全破裂。在4℃条件下以500-1000g(优选700g)低速离心10min取上清,再以12000-16000(优选14000g)高速离心30min收集沉淀,即得4T-1肿瘤细胞膜碎片。将4T-1细胞膜与PCO混合超声,并通过200-800nm(优选400nm)的聚碳酸酯膜过滤器挤出10-30个(优选20个)来回,得到包裹4T-1细胞膜的PC/Ola聚合物纳米粒(MPCO)。
下面结合具体的实施例,对本发明做进一步的详细说明,应该指出,所述具体实施例是对本发明的解释而不是限定。
实施例1
1.聚谷氨酸共轭光敏剂PC的合成及表征
为了合成聚谷氨酸共轭光敏剂PC(PGA-Cys-Ce6,PC),将200mg的分子量为75000的PGA溶于20ml蒸馏水中,加入1.8g的EDC·HCl和630mgNHS搅拌20min,充分活化PGA的羧基。加入300mg胱胺二盐酸盐,室温反应12h。用截留分子量为3500Da的透析袋透析48h,除去未反应的EDC,NHS与胱胺。冻干聚合物溶液,得聚谷氨酸-胱胺(PGA-Cys)聚合物。称取20mgCe6溶于5ml的DMF中,加入37mgEDC·HCl和26mgNHS搅拌20min,充分活化Ce6的羧基。加入含50mg的PGA-cys水溶液5ml,室温反应12h。用截留分子量为3500Da的透析袋,在DMF:H2O(1:1)中透析两天,在超纯水中透析两天。冻干聚合物溶液,得PC,4℃避光保存。最终产物分别用核磁共振氢谱、红外光谱和紫外光谱进行表征。为了评估溶液中1O2的产生效率,使用1,3-二苯基异苯并呋喃(DPBF)作为指示剂。简而言之,10μL DPBF(在DMSO中为3mg/mL)和2ml游离Ce6或PC溶液(相当于5μg/mL Ce6)。然后,在用660nm激光(200mW/cm)照射不同时间后,用紫外-可见分光光度计记录DPBF在417nm的吸光度。DPBF水溶液用作对照。ROS产生效率以DPBF残留率评价,其计算公式为RemainDPBF=At/A0×100%,其中At和A0分别为t秒和0秒照射后残留DPBF在417nm处的吸光度。
2.载药纳米制剂PCO的合成及表征
为了获得载PARP抑制剂纳米粒,采用薄膜分散法进行制备。将含有摩尔比为10:3的PC和Ola溶于甲醇,并混合搅拌1h,将混合溶液注入茄型瓶中旋转蒸发除去甲醇得到薄膜。注入4mLPBS水化,超声5min后通过0.22μm的水系滤膜除去未包载的Ola。通过马尔文激光粒度仪和透射电镜对纳米制剂的粒径分布、电位和外观形态进行表征。
3.肿瘤细胞膜包覆纳米制剂MPCO的合成及表征
为了得到肿瘤细胞膜包覆的载药纳米制剂,收集4T-1肿瘤细胞,加入裂解液在液氮罐反复冻融7次,并通过探头超声30min使细胞完全破裂。在4℃条件下以700g低速离心10min取上清,再以14000g高速离心30min收集沉淀,即得4T-1肿瘤细胞膜碎片。将4T-1细胞膜与PCO混合超声,并通过400nm的聚碳酸酯膜过滤器挤出20个来回,得到包裹4T-1细胞膜的PC/Ola聚合物纳米粒(MPCO)。通过马尔文激光粒度仪和透射电镜对纳米制剂的粒径分布、电位和外观形态进行表征。
进行SDS-PAGE分析以鉴定PCO、4T1细胞膜、包被制剂MPCO和4T1全细胞的蛋白质组分。简而言之,将来自不同样品的等量蛋白质(用BCA蛋白质测定试剂盒定量)加入10% SDS聚丙烯酰胺凝胶中,以分离不同分子量的蛋白质。然后,用考马斯亮蓝法处理凝胶并成像。
4.肿瘤细胞膜包覆纳米制剂MPCO的体外释放实验
用透析法评价了Ola在不同条件下从MPCO中的释放行为。简而言之,将1mL MPCO密封在透析袋(MWCO=3500Da)中,并浸入20mL含有或不含GSH的释放介质(10mM)中,置于水浴恒温振荡器(37℃,100rpm)内。在预定的时间点,收集1mL释放介质并加入1mL新鲜释放介质。通过HPLC测定释放的Ola的浓度。此外,还研究了游离Ola的释放行为以进行比较。
5.不同治疗组的细胞摄取实验
通过流式细胞术检测不同制剂组对于细胞摄取的影响,将4T1细胞以1*105/孔接种至12孔板孵育12小时,加入Ce6浓度为5μg/mL的游离Ce6和MPCO并分别孵育1小时、2小时和4小时。用冷PBS洗涤细胞3次后用胰酶消化,用PBS重悬后流式检测。
6.不同治疗组的细胞毒性实验
为了检测体外细胞毒性实验,采用CCK8法检测细胞活力。将处于指数生长期的4T-1细胞消化后,以每孔8000个细胞的密度接种到96孔板中,在细胞孵育箱中孵育24h后加入100μL的Ola、Ce6、PC、PCO、MPCO含药培养基,其中Ola浓度分别为0.06,0.13,0.32,0.64,1.29μg/mL,Ce6浓度为0.05,0.1,0.25,0.5,1.0μg/mL。孵育12h后,对于光照处理组,换用100μL新鲜培养基,用660nm激光对每孔进行照射(100mW,1min);对于非光照处理组,换用100μL新鲜培养基,不予NIR照射。继续孵育12h后,加入10μLCCK8试剂,在孵育箱中继续孵育1h左右,移至酶标仪测定各孔在450nm处的吸光度,计算细胞存活率,所有实验独立重复3次。
7.不同治疗组的原位乳腺癌小鼠体内生物分布荧光成像
为建立荷瘤小鼠模型,雌性Balb/c小鼠(6-8周龄)在右侧第四乳腺脂肪垫皮下注射1×106个细胞。植入后约7天,将肿瘤大小为100mm3的小鼠随机分组。在静脉注射Ce6、浓度为4mg/kg的PCO和MPCO纳米粒后,使用体内成像系统仪器成像。注射后24小时,杀死小鼠,收集肿瘤和主要器官(心脏、肝脏、脾脏、肺和肾脏)用于离体成像。
实施例2
实施例1通过两步酰胺反应制备了聚谷氨酸共轭光敏剂PC,linker是含二硫键的胱胺(图1)。核磁共振氢谱3.0左右的质子峰,证明胱胺成功连接;8.0左右的质子峰证明Ce6的成功连接(图2)。红外图谱1734处为Ce6结构中羰基伸缩振动峰,表明Ce6成功接枝(图3)。合成的PC易分散于水、PBS、生理盐水以及细胞培养基等水性介质中,在水中呈棕绿色且分散均匀。UV-Vis图谱表明接枝聚合物在水中有着和Ce6接近的吸收峰,并且显示出明显的丁达尔效应,同时利用紫外建立Ce6的方法学并计算出PC中Ce6的含量为18.79±0.06%(图4)。通过DPBF检测了在激光照射条件下单线态氧生成,结果表明接枝聚谷氨酸共轭光敏剂PC具有和Ce6类似的单线态氧生成能力(图5)。PC是一种两亲性聚合物,TEM图像显示其在水中可自组装形成纳米囊泡(图6),外层为亲水性的PGA高分子,内层为接枝的光敏剂,且每个PC纳米粒都存在较大空间的疏水性空腔,这一结构特点为发展载药纳米制剂奠定了基础。
通过薄膜分散法制备了载Ola的纳米粒载药纳米粒PCO,筛选最佳药质比为10:3,载药量和包封率分别高达19.52%和80.86%。通过超声挤出法包覆4T1肿瘤细胞膜。使用DLS、TEM测定了粒径电位和形态(图7)。PCO纳米粒为均匀圆形,其腔内包载了疏水药物Ola,粒径为191.30±1.50nm,zeta电位为-9.54±0.96mV。MPCO形成了包覆状阴影,表明4T-1细胞膜的成功包覆,粒径为202.43±1.50nm,zeta电位为-17.5±0.52mV。使用SDS-PAGE凝胶电泳检测了包膜制剂的膜蛋白,MPCO保留了4T1细胞膜蛋白的完整性特征(图8)。考察了PBS中PCO和MPCO的粒径和电位的稳定性,结果表明7天内PCO和MPCO均能保持稳定(图9)。
通过透析法模拟不同的生理环境来研究MPCO的释放行为(图10)。pH 7.4的PBS用于模拟正常体液环境,而含有10mM GSH的pH 5.0的PBS用于模拟肿瘤细胞中的酸性和高GSH条件。如释放曲线所示,游离Ola在前6小时几乎完全释放。然而,在pH 7.4时,MPCO纳米粒在2天内释放了不到20%的Ola,这表明它可以在体液运输过程中保持其完整性,并且药物在到达肿瘤部位之前不会泄漏。形成鲜明对比的是,当MPCO在酸性和高GSH条件下孵育时,Ola的释放大于50%。其原因可能是由于MPCO纳米粒在高谷胱甘肽条件下半胱氨酸中的二硫键断裂,实现智能响应肿瘤微环境释放。
采用流式细胞术观察游离Ce6和MPCO纳米粒的细胞摄取(图11)。结果表明,在1h时,MPCO组的平均荧光强度比Ce6组高近一个数量级。4h时,MPCO纳米粒被摄入最多。研究表明,4T1细胞膜修饰的纳米颗粒可以被多种分子介导的细胞表面相互作用同源靶向,包括肿瘤特异性结合蛋白,如TF抗原和E-钙粘蛋白。可能由于肿瘤细胞膜介导的归巢效应,MPCO纳米粒显著增强了细胞的药物摄取能力。
CCK-8试验用于评估细胞毒性(图12)。在相同的Ola浓度下,由于不存在ROS损伤DNA的初始事件,单独的游离Ola几乎没有细胞毒性。非激光处理组的四种制剂具有较高的生物相容性,激光照射后细胞毒性显著增加。PC(+)获得了比Ce6(+)组更大的肿瘤杀伤效果,这可能是由于使用PGA缀合物改善了Ce6的分散性和细胞摄取。PCO(+)组和MPCO(+)组的IC50分别为0.4898μg/mL和0.4736μg/mL。
在BALB/c小鼠脂肪垫注射4T1肿瘤细胞,建立原位乳腺癌肿瘤模型,并用IVIS成像评估游离药和纳米制剂的生物分布(图13)。静脉注射游离Ce6、PCO或MPCO纳米粒后,游离Ce6代谢迅速,在12小时几乎没有荧光信号。然而,MPCO纳米粒组在12小时仍有较高的肿瘤蓄积,这归因于长的血液循环和由肿瘤细胞膜介导的肿瘤归巢机制。基于上述结果,在之后的动物实验中,选择给药后12h进行激光照射,以获得最佳的PDT效果。24小时后,处死小鼠,收集主要器官和肿瘤以检查荧光强度。离体器官荧光成像结果与活体成像结果一致,MPCO纳米粒实现了最强的肿瘤区域富集。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种基于聚谷氨酸共轭光敏剂的肿瘤靶向纳米制剂,其特征在于,包括:
聚谷氨酸共轭光敏剂;
所述聚谷氨酸共轭光敏剂包载有疏水性化疗药;
所述聚谷氨酸共轭光敏剂包覆有肿瘤细胞膜。
2.如权利要求1所述的基于聚谷氨酸共轭光敏剂的肿瘤靶向纳米制剂,其特征在于,所述聚谷氨酸共轭光敏剂的制备方法为:将光敏剂通过共价键偶联、静电吸附方法连接到PGA分子。
3.如权利要求1所述的基于聚谷氨酸共轭光敏剂的肿瘤靶向纳米制剂,其特征在于,所述光敏剂为二氢卟吩e6、吲哚菁绿、叶绿素-a、脱镁叶绿酸a、焦脱镁叶绿酸-a、焦脱镁叶绿酸a己醚中至少一种。
4.如权利要求1所述的基于聚谷氨酸共轭光敏剂的肿瘤靶向纳米制剂,其特征在于,所述疏水性化疗药为PARP抑制剂、紫杉醇、多西紫杉醇、多柔比星、奥沙利铂、槲皮素、姜黄素中至少一种。
5.如权利要求4所述的基于聚谷氨酸共轭光敏剂的肿瘤靶向纳米制剂,其特征在于,所述PARP抑制剂为奥拉帕利、尼拉帕利、氟唑帕利、帕米帕利中至少一种。
6.如权利要求4所述的基于聚谷氨酸共轭光敏剂的肿瘤靶向纳米制剂,其特征在于,肿瘤为黑色素瘤、肠癌、肺癌、胃癌、卵巢癌、前列腺癌、胰腺癌、乳腺癌、卵巢癌、肝癌、头颈部癌、淋巴瘤、肉瘤、慢性淋巴细胞白血病、甲状腺癌和睾丸癌、肛门癌中至少一种。
7.一种基于聚谷氨酸共轭光敏剂的肿瘤靶向纳米制剂的制备方法,其特征在于,包括:
将光敏剂通过共价键偶联、静电吸附方法连接到PGA分子上,得到聚谷氨酸共轭光敏剂;
通过疏水作用力、静电吸附、π-π共轭方式在所述聚谷氨酸共轭光敏剂包载疏水性化疗药,得到纳米制剂;
将所述纳米制剂采用肿瘤细胞的细胞膜包覆,即得。
8.如权利要求7所述的基于聚谷氨酸共轭光敏剂的肿瘤靶向纳米制剂的制备方法,其特征在于,聚谷氨酸共轭光敏剂的制备方法包括:
将分子量为5000-200000的PGA溶于溶液中,加入EDC·HCl和NHS进行活化,再加入胱胺二盐酸盐,室温下进行反应,反应完成后,透析、冻干,得聚谷氨酸-胱胺聚合物;
将光敏剂溶于溶液中,加入EDC·HCl和NHS进行活化,再加入所述聚谷氨酸-胱胺聚合物进行反应,反应完成后,透析、冻干,即得。
9.如权利要求7所述的基于聚谷氨酸共轭光敏剂的肿瘤靶向纳米制剂的制备方法,其特征在于,所述纳米制剂的制备方法包括:将PC和Ola溶于溶液中,混合均匀,旋转蒸发,得到薄膜,然后,注入PBS水化,超声分散,采用水系滤膜除去未包载的Ola,即得。
10.如权利要求7所述的基于聚谷氨酸共轭光敏剂的肿瘤靶向纳米制剂的制备方法,其特征在于,细胞膜包覆的具体步骤包括:将肿瘤细胞,加入裂解液进行反复冻融5-10次,并通过探头超声20-40min使细胞完全破裂;低温低速离心后,取上清,再高速离心,收集沉淀,即得肿瘤细胞膜碎片;
将肿瘤细胞膜与纳米制剂混合超声,并采用聚碳酸酯膜过滤器挤出10-30个来回,即得。
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