CN113616803A - 一种gsh响应型吉西他滨纳米粒子及其制备方法与应用 - Google Patents
一种gsh响应型吉西他滨纳米粒子及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种GSH响应型吉西他滨聚合物,由亲水性聚合物、含两个羧基的二硫键化合物以及吉西他滨反应制备而成。其中,含两个羧基的二硫键化合物与吉西他滨一步反应合成含二硫键的吉西他滨前药,封闭固定吉西他滨药物结构的活性基团,提高药物的稳定性,减小药物与正常细胞DNA结合机率。本发明还提供了一种由GSH响应型吉西他滨聚合物、二硬脂酰基磷脂酰乙醇胺‑聚乙二醇2000组成的GSH响应型吉西他滨纳米粒子。所述GSH响应型吉西他滨纳米粒子的血液相容性良好,能够抑制B细胞淋巴瘤细胞增殖,且具有氧化还原响应性,能够在高浓度GSH肿瘤微环境的刺激下可控释放吉西他滨,提高药物对肿瘤的疗效,减小药物对机体毒副反应。
Description
技术领域
本发明涉及生物医药技术领域,具体地,涉及一种GSH响应型吉西他滨纳米粒子及其制备方法与应用。
背景技术
吉西他滨是一种亲水性的胞嘧啶核苷衍生物,进入人体内后由脱氧胞嘧啶激酶活化,由胞嘧啶核苷脱氨酶代谢,其主要代谢物在细胞内掺入DNA,主要作用于DNA合成期的肿瘤细胞。因此,吉西他滨是一种破坏肿瘤细胞复制的二氟核苷类抗代谢物抗癌药。
近年来,基于肿瘤微环境(如高浓度谷胱甘肽(GSH),低pH)的独特性质,一系列功能化的聚合物药物偶联物已被开发用于肿瘤微环境响应给药,在药物靶向递送、控制释放、改善疗效等方面取得了很大的的进展。人们将治疗肿瘤的药物通过肿瘤微环境响应型化学键与药物载体连接到一起,制备得到的肿瘤微环境响应型纳米载药系统对肿瘤组织刺激十分敏感,能够选择性地在肿瘤处释放出药物,不仅能够提高药物疗效,而且能够有效降低药物对机体正常细胞的毒副作用。在肿瘤微环境响应型纳米载药系统的研究中,构建氧化还原响应递药系统是研究的热点之一。现有的研究结果表明,肿瘤细胞内GSH浓度(0.5~10mmol/L)显著高于正常细胞的GSH浓度(2~20μmol/L),二硫键在高浓度GSH的作用下快速裂解,已被广泛用作氧化还原响应递药系统的理想触发器。目前,关于氧化还原响应递药系统负载吉西他滨药物用于肿瘤治疗的研究颇多,例如:Chen等人构建了一种用于细胞内给药的还原响应交联吉西他滨前药胶束,所述还原响应交联吉西他滨前药胶束是聚(乙二醇)甲基丙烯酸酯(PEGMA)、7-(2-甲基丙烯酰乙氧基)-4-甲基香豆素(CMA)和丙烯酸2-((2-羟乙基)二硫烷基)乙酯-吉西他滨共聚合成的共聚物(HSEA-GEM),具有“隐形”表面、光交联特性和还原敏感,且能够有效抑制BxPC-3胰腺癌细胞细胞的增殖(Chen X,et al.One-steppreparation of reduction-responsive cross-linked gemcitabine prodrug micellesfor intracellular drug delivery[J].Colloids and Surfaces B:Biointerfaces,2019,181:94-101);Sun等人构建了一种基于吉西他滨的小型载体共递送三联药物,具体为:以氧化还原响应的吉西他滨(GEM)-缀合聚合物PGEM作为肿瘤穿透纳米载体来共载免疫调节剂(NLG919,吲哚胺2,3-双加氧酶1(IDO1)的抑制剂)和用于免疫化疗组合疗法的化疗药物(紫杉醇(PTX),实验结果表明,该小型载体共递送三联药物能够深入肿瘤渗透达到胰腺肿瘤的核心,显示出较好的抗肿瘤活性(Sun J,et al.Triple Drugs Co-Delivered bya Small Gemcitabine-Based Carrier for Pancreatic Cancer Immunochemotherapy[J].Acta Biomaterialia,2020,106:289-300)。然而,活性小分子化疗药物吉西他滨进入机体与正常细胞的DNA结合很可能会对机体的正常组织产生毒副作用,因此,如何改善纳米载药系统中的吉西他滨在体循环中的稳定性以抑制化疗药物吉西他滨在正常组织部位与DNA结合仍是一个需要解决的难题。
发明内容
本发明的目的在于克服现有技术中存在的上述缺陷和不足,提供一种GSH响应型吉西他滨聚合物。
本发明的第二个目的在于提供上述GSH响应型吉西他滨聚合物的制备方法。
本发明的第三个目的在于提供一种GSH响应型吉西他滨纳米粒子。
本发明的第四个目的在于提供上述GSH响应型吉西他滨纳米粒子的制备方法。
本发明的第五个目的在于提供上述制备方法制备得到的GSH响应型吉西他滨纳米粒子在制备抗肿瘤药物中的应用。
本发明的上述目的是通过以下技术方案给予实现的:
一种GSH响应型吉西他滨聚合物,由亲水性聚合物、含两个羧基的二硫键化合物以及吉西他滨反应制备而成;所述GSH响应型吉西他滨聚合物的结构式如式(I)所示:
式(I);
其中,R2选自CH2、CH2-CH2或CH2-CH2-CH2;R1、R3或R4为亲水性聚合物或吉西他滨或羟基,且R1、R3或R4中至少一个为亲水性聚合物;所述亲水性聚合物为PEG或mPEG-OH、mPEG-NH2。
在GSH响应型吉西他滨聚合物(GEM-S-S-PEG)中,含两个羧基的二硫键化合物与吉西他滨的氨基和/或羟基反应,封闭固定吉西他滨分子,增强吉西他滨在体内的稳定性,降低其在体内与正常组织的DNA结合的概率。此外,含两个羧基的二硫键化合物与亲水性聚合物发生酯化或缩合反应,进一步封闭固定了吉西他滨分子,提高吉西他滨的生物相容性。本发明提供的GEM-S-S-PEG通过上述双重封锁固定作用,有效抑制了吉西他滨在正常组织部位的释放和积累,从而降低了其对正常组织的毒副作用,所述吉西他滨聚合物的二硫键能够在机体内可控裂解,当聚合物到达肿瘤微环境时,吉西他滨聚合物结构中二硫键在高浓度的GSH环境下快速裂解,释放吉西他滨药物,达到高效治疗肿瘤的效果。
优选地,所述含两个羧基的二硫键化合物包括2,2'-硫代二乙酸、3,3'-硫代二丙酸、4,4'-硫代二丁酸中任意一种。
优选地,所述GEM-S-S-PEG的数均分子量为3000~10000。
优选地,所述GEM-S-S-PEG的数均分子量为3500~6000。
优选地,所述亲水性聚合物的数均分子量为500~2000。
更优选地,所述亲水性聚合物的数均分子量为2000。
优选地,所述亲水性聚合物、含两个羧基的二硫键化合物、吉西他滨的摩尔比为(0.1~0.5):(1~4.5):1。
本发明所述亲水聚合物的摩尔量为亲水聚合物的质量与数均分子量的比值。
本发明还提供了上述任一所述GSH响应型吉西他滨聚合物的制备方法,包括以下步骤:
S1.将吉西他滨、含两个羧基的二硫键化合物HOOC-R2-S-S-R2-HOOC、DCC以及DMAP混合,溶于有机溶剂中,反应18~36h,得到含二硫键的吉西他滨前药溶液;所述R2选自CH2、CH2-CH2或CH2-CH2-CH2;
S2.在步骤S1的含二硫键的吉西他滨前药溶液中加入亲水性聚合物、DCC、HOBT,反应24~48h,过滤产物,取滤液,透析,再经过旋蒸、冰乙醚沉淀、抽滤、真空干燥得到GEM-S-S-PEG粉末。
优选地,步骤S1所述含两个羧基的二硫键化合物、DCC、DMAP的摩尔比为1:(2~2.4):(0.1~0.6)。
优选地,步骤S2所述亲水性聚合物、HOBT、DCC的摩尔比为1:(0.74~1.1):(0.45~0.61)。
优选地,步骤S2所述透析的步骤为:首先在DMF透析3天,然后在水中透析3天。
本发明还提供了一种GSH响应型吉西他滨纳米粒子(GSP NPs),由上述任一所述的GSH响应型吉西他滨聚合物、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000组成,所述GSH响应型吉西他滨聚合物和二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000的质量比为1:(0.1~0.8)。
本发明还提供上述GSH响应型吉西他滨纳米粒子的制备方法,包括以下步骤:将上述GSH响应型吉西他滨聚合物溶于溶剂中,充分溶解后加入二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000(DSPE-PEG2000),混匀后滴加到水中,搅拌,过滤,离心,除杂,即得。
优选地,所述溶剂为DMSO。
作为优选的可实施方案,一种GSH响应型吉西他滨纳米粒子的制备方法,包括以下步骤:
S1.将2.84mmol吉西他滨和4.04mmol 3,3′-二硫代二丙酸加入9.11mmol DCC和1.95mmol DMAP的混合溶液中,反应18h,得到含二硫键的吉西他滨前药溶液;
S2.将步骤S1合成的含二硫键的吉西他滨前药溶液中加入0.4mmol数均分子量为2000的mPEG-OH、0.37mmol HOBT以及0.24mmol DCC,反应24h,过滤产物,取滤液,在DMF中透析3天(期间更换三次DMF),后继续置水中透析3天,再经过旋蒸、冰乙醚沉淀、抽滤、真空干燥得到GEM-S-S-PEG粉末;
S3.将步骤S2的GEM-S-S-PEG固体粉末充分溶解于DMSO内,制备得到浓度为20mg/mL的GEM-S-S-PEG溶液,取300μL GEM-S-S-PEG溶液、60μL浓度为20mg/mL的DSPE-PEG2000溶液和240μL DMSO溶液混匀,逐滴滴入18mL超纯水中,磁力搅拌器搅拌1min。过滤,在2500rpm下离心15min,重复离心3次,除杂,得到GSP NPs。
本发明还提供上述制备方法制备得到的GSH响应型吉西他滨纳米粒子在制备抗肿瘤药物中的应用。
优选地,所述肿瘤为B细胞淋巴瘤。
与现有技术相比,本发明具有以下有益效果:
(1)本发明提供的GSH响应型吉西他滨聚合物,由亲水性聚合物、含两个羧基的二硫键化合物以及吉西他滨制备而成,其中,含两个羧基的二硫键化合物的作用是作为连接吉西他滨分子间的“桥梁”,二者反应得到含二硫键的吉西他滨前药,封闭固定吉西他滨药物结构的活性基团,提高吉西他滨药物的稳定性,减小其与正常细胞DNA结合的机率;另外,含两个羧基的二硫键化合物与吉西他滨分子反应剩余的羧基进一步与亲水性聚合物发生酯化或缩合反应,进一步封闭固定了吉西他滨分子,提高吉西他滨的生物相容性。
(2)本发明还提供了一种GSH响应型吉西他滨聚合物、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000组成的GSH响应型吉西他滨纳米粒子,所述纳米粒子的血液相容性良好,能够抑制淋巴肿瘤细胞增殖,且其具有的氧化还原响应性能够可控地在高浓度GSH肿瘤微环境的刺激下释放药物,减小药物对机体毒副反应,提高药物对肿瘤的疗效。
附图说明
图1为GEM-S-S-PEG的核磁共振氢谱1H-NMR图。
图2为吉西他滨的核磁共振氢谱1H NMR图。
图3为GEM-S-S-PEG的质谱图。
图4为GSP NPs的粒径分布图。
图5为GSP NPs的Zeta电位图。
图6为GSP NPs的TEM图。
图7为GSP NPs在不同溶剂中的粒径变化。
图8为GSP NPs在不同溶剂中PDI变化。
图9为GSP NPs的溶血率(C+:阳性,C1:250μg/mL,C2:125μg/mL,C3:62.5μg/mL,C4:31.25μg/mL,C5:15.625μg/mL,C6:7.8125μg/mL)。
图10为CCK8法测定吉西他滨和GSP NPs对A20细胞的体外细胞活性。
图11为游离吉西他滨和GSP NPs诱导A20细胞凋亡结果。
图12为NPs@DiR和游离DiR在A20荷瘤小鼠体内的生物分布情况。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明,以下实施例所用试剂和材料均为市购。
实施例1GSP NPs的制备
S1.将2.84mol吉西他滨和4.04mol 3,3′-二硫代二丙酸加入9.11mmol DCC和1.95mmol DMAP的混合溶液中,反应18h,得到含二硫键的吉西他滨前药溶液。
S2.将步骤S1合成的含二硫键的吉西他滨前药溶液中加入0.4mmol数均分子量为2000的mPEG-OH、0.37mmol HOBT以及0.24mmol DCC,反应24h,过滤产物,取滤液,在DMF中透析3天(期间更换三次DMF),后继续置水中透析3天,再经过旋蒸、冰乙醚沉淀、抽滤、真空干燥得到GEM-S-S-PEG。
所述GEM-S-S-PEG的结构式如下:
S3.将步骤S2的GEM-S-S-PEG固体粉末充分溶解于DMSO内,制备得到浓度为20mg/mL的GEM-S-S-PEG溶液,取300μL GEM-S-S-PEG溶液、60μL浓度为20mg/mL的DSPE-PEG2000溶液和240μL DMSO溶液混匀,逐滴滴入18mL超纯水中,磁力搅拌器搅拌1min。过滤,在2500rpm下离心15min,重复离心3次,除杂,得到GSP NPs。
实施例2GSP NPs的制备
S1.将1mmol吉西他滨和2mmol 3,3′-二硫代二丙酸加入4mmol DCC和0.2mmolDMAP的混合溶液中,反应18h,得到含二硫键的吉西他滨前药溶液。
S2.将步骤S1合成的含二硫键的吉西他滨前药溶液中加入0.2mmol分子量(数均)为2000的mPEG-OH和0.15mmol HOBT以及0.1mmol DCC,反应24h,过滤产物,取滤液,在DMF中透析3天(期间更换三次DMF),后继续置水中透析3天,再经过旋蒸、冰乙醚沉淀、抽滤、真空干燥得到GEM-S-S-PEG。
S3.将步骤S2的GEM-S-S-PEG固体粉末充分溶解于DMSO内,制备得到浓度为20mg/mL的GEM-S-S-PEG溶液,取200μL GEM-S-S-PEG溶液、160μL浓度为20mg/mL的DSPE-PEG2000溶液和240μL DMSO溶液混匀,逐滴滴入18mL超纯水中,磁力搅拌器搅拌2min。过滤,在2500rpm下离心15min,重复离心3次,除杂,得到GSP NPs。
实施例3GSP NPs的制备
S1.将2mmol吉西他滨和3mmol 3,3′-二硫代二丙酸加入7.2mmol DCC和1.8mmolDMAP的混合溶液中,反应36h,得到含二硫键的吉西他滨前药溶液。
S2.将步骤S1合成的含二硫键的吉西他滨前药溶液中加入0.3mmol分子量(数均)为2000的mPEG-OH、0.3mmol HOBT以及0.14mmol DCC,反应48h,过滤产物,取滤液,在DMF中透析3天(期间更换三次DMF),后继续置水中透析3天,再经过旋蒸、冰乙醚沉淀、抽滤、真空干燥得到GEM-S-S-PEG。
S3.将步骤S2的GEM-S-S-PEG固体粉末充分溶解于DMSO内,制备得到浓度为20mg/mL的GEM-S-S-PEG溶液,取100μL GEM-S-S-PEG溶液、60μL浓度为20mg/mL的DSPE-PEG2000溶液和120μL DMSO溶液混匀,逐滴滴入9mL超纯水中,磁力搅拌器搅拌3min。过滤,在2500rpm下离心15min,重复离心3次,除杂,得到GSP NPs。
实施例4GSP NPs的制备
仅将实施例1步骤S1的“3,3′-二硫代二丙酸”替换为“2,2'-硫代二乙酸”,其余步骤与实施例1相同,制备GSP NPs。
实施例5GSP NPs的制备
仅将实施例1步骤S1的“3,3′-二硫代二丙酸”替换为“4,4'-硫代二丁酸”,其余步骤与实施例1相同,制备GSP NPs。
实施例6GSP NPs的制备
仅将实施例1步骤S2的“mPEG-OH”替换为“mPEG-NH2”,其余步骤与实施例1相同,制备GSP NPs。
实施例7GSP NPs的制备
仅将实施例1步骤S1的“3,3′-二硫代二丙酸”替换为“2,2'-硫代二乙酸”以及步骤S2的“mPEG-OH”替换为“mPEG-NH2”,其余步骤与实施例1相同,制备GSP NPs。
测试例1GSP NPs的表征
1.核磁共振氢谱(1H-NMR)分析
利用核磁共振氢谱对实施例1步骤S1制备得到的含二硫键的吉西他滨和步骤S2制备得到的GEM-S-S-PEG进行表征。
结果分析:最终产物GEM-S-S-PEG的1H NMR图如图1所示,吉西他滨的1H NMR图如图2所示。由图1、2可以看出,GEM-S-S-PEG的1H NMR谱图中的d和f处的峰发生了位移,d处的峰位从δ4.20~4.26ppm(图2)变为δ5.42~5.58ppm(图1),f处的峰位从δ3.60~3.70ppm(图2)变为δ4.30~4.50ppm(图1)。f和d位点的邻-OH峰完全消失,证明最终产物中没有游离的吉西他滨。为了进一步确认新形成的结构,结果显示,在GEM-S-S-PEG的1H NMR谱图(图1)中,δ7.40~7.48ppm处(图2)的吉西他滨结构上的-NH2峰消失了,出现了新的-NHCO-峰(δ8.20~8.30ppm),表明吉西他滨的三个不同位点都发生了反应。图1中还出现了-CH2-S-峰(δ0.80~1.82ppm),无-COOH峰,证实3,3′-二硫代二丙酸成功接枝到产物中,没有残留的游离3,3′-二硫代二丙酸。
2.基质辅助激光解吸/电离飞行时间质谱(MALDI-TOF-MS)分析
利用基质辅助激光解吸/电离飞行时间质谱仪检测实施例1的步骤S2制备得到的GEM-S-S-PEG。
结果分析:GEM-S-S-PEG的质谱图如图3所示,由图3可知,GEM-S-S-PEG的分子量分布在3500~4600之间,根据质子吸收峰的强度计算出GEM-S-S-PEG中的吉西他滨的载药率是24.6%。
测试例2GSP NPs的粒径分布、电位与稳定性
1.GSP NPs的粒径分布和电位
利用动态光散射粒度仪(DLS)测定实施例1制备得到的GSP NPs的粒径分布和Zeta电位,并用透射电镜(TEM)对GSP NPs进行表征。
结果分析:GSH响应型吉西他滨纳米粒子(GSP NPS)的粒径分布和电位分别如图4、5所示,GSP NPs的平均粒径为83.25nm,电位约-19.4mV。图6为GSP NPs的TEM图,从图中可看出,与DLS测得的GSP NPs尺寸相比,部分纳米粒子经TEM测量得到的尺寸略小(约50nm),这可能是因为在制备TEM测试样品的过程中需要对样品进行脱水处理,纳米粒子的mPEG亲水性壳层收缩,从而导致纳米粒子的粒径偏小。
2.GSP NPs的稳定性
为了探究GSP NPs的稳定性,选择不同的溶剂对浓缩的GSP NPs进行稀释。具体步骤为:将质量浓度为500μg/mL的GSP NPs分别用超纯水、1×PBS、含10%胎牛血清的1×PBS稀释至50μg/mL,并用DLS测定其粒径分布和聚合物分散指数(PDI),结果分别如图7、8所示。由图7可知,在超纯水、1×PBS、含10%胎牛血清的1×PBS中稀释5天后,GSP NPs的粒径无显著变化,表明GSP NPs的稳定性良好。从图8可以看出,在不同溶剂中稀释5天后的GSP NPs的PDI小于0.3,其粒径分布较均一。
测试例3GSP NPs的体外生物性能评价
1.溶血实验
实验步骤:从健康SD大鼠处取新鲜血液,3000rpm离心10分钟,然后将离心获得的红细胞用生理盐水进一步洗涤,直至上清液清澈。接着,用生理盐水稀释GSP NPs,分别得到250,125,62.5,31.25,15.625,7.8125μg/mL六个不同浓度的GSP NPs,依次记为C1~C6组,与等体积的红细胞悬液混合。同时,以含红细胞悬液的生理盐水为阴性对照(溶血率0%),以用超纯水稀释的等效红细胞悬液为阳性对照(溶血率100%,记为C+组)。所有样品放置于恒温振荡器中,37℃、100rpm振荡孵育2h。振荡完毕后,3000rpm离心10分钟,拍照,收集上清液,用酶标仪在436nm处测定各组吸光度,分析溶血率。实验一式三份。
结果分析:GSP NPs的溶血率测试结果如图9所示。从图中可以看出,与阳性对照组相比,GSP NPs处理组红细胞悬液的颜色并没有随着药物浓度的增加而出现明显的溶血现象。当浓度高达250μg/mL时,GSP NPs的溶血率仍然低于5%。以上结果表明,GSP NPs具有良好的血液相容性。
2.小鼠B细胞淋巴瘤细胞A20细胞毒性
使用CCK8试剂盒检测游离吉西他滨和GSP NPs的细胞毒性。A20细胞(1.5×104细胞/孔)接种于96孔板,分别加入不同浓度(0.005、0.02、0.1、2、10、50μg/mL)的游离吉西他滨、GSP NPs,置于37℃、5%CO2培养箱中培养24h。孵育结束后,每孔加入10μL CCK8溶液。4小时后,使用酶标仪在450nm处分析每个孔的吸光度值。空白培养基为零设置组,未加药处理细胞组为对照组,每组实验设置三个平行组。
结果分析:CCK8法测定吉西他滨和GSP NPs对A20细胞的体外细胞活性结果如图10所示。游离吉西他滨和GSP NPs均能抑制细胞增殖,随着药物浓度的增加,GSP NPs对A20细胞的增殖抑制性逐渐增强,说明两种药物的体外抗增殖活性均具有浓度和时间依赖性。实验结果发现,当药物浓度达到50μg/mL时,与游离吉西他滨相比,GSP NPs对A20细胞活性的抑制效果更佳。
3.A20细胞凋亡
使用AnnexinV-FITC/PI凋亡试剂盒检测GSP NPs诱导的细胞凋亡。A20细胞(3.0×104细胞/孔)接种于6孔板中,然后分别用游离吉西他滨、GSP NPs(吉西他滨等价浓度2.5μg/mL)处理,未加药处理细胞组为对照组。在24h后,弃去培养基,用PBS洗涤细胞,收集细胞;然后用500μL的Binding Buffer悬浮细胞,加入5μL Annexin V-FITC和10μL PropidiumIodide,混匀后室温下避光孵育5~15min;最后进行流式细胞仪检测。
结果分析:游离吉西他滨和GSP NPs诱导A20细胞凋亡结果如图11所示。如图所示,在作用48h后,GSP NPs组A20细胞的总凋亡率稍高于游离吉西他滨组A20细胞的总凋亡率。说明GSP NPs能有效诱导A20细胞凋亡。
测试例4GSP NPs的体内生物分布
GSP NPs在体内的生物分布通过小动物活体成像系统进行测定。用A20荷瘤小鼠研究GSP NPs的生物分布。雌性BALB/c Nude小鼠(8周,20g)后肢皮下注射A20细胞悬液100μL(细胞数5.0×106)。待肿瘤体积增大到300~400mm3后,A20荷瘤小鼠随机分为两组,分别静脉给予游离DiR、包载荧光素DIR的GSP NPs(NPs@DiR,DiR等价浓度0.5mg/kg)。然后对所有A20荷瘤小鼠通过腹腔注射1%戊巴比妥钠溶液(50mg/kg)进行全身麻醉。麻醉成功后,将A20荷瘤小鼠放置在小动物活体成像系统中,分别在静脉注射游离DiR、NPs@DiR后1、4、8、12、24、48和72小时观察荧光分布情况。
结果分析:NPs@DiR和游离DiR在A20荷瘤小鼠体内的生物分布情况如图12所示。荷瘤小鼠在注射游离DiR后,肿瘤部位的荧光成像信号弱,而注射NPs@DiR的荷瘤小鼠肿瘤部位则显示出较强的荧光强度,在注射后1h能够观察到肿瘤部位有荧光富集,且随着时间的延长,肿瘤部位荧光强度逐步增强,并在12h时达到最强,此时NPs@DiR在肿瘤部位的富集水平最高。以上结果表明,与游离DiR相比,NPs@DiR在肿瘤部位具有更强的富集能力。
Claims (10)
3.根据权利要求1所述GSH响应型吉西他滨聚合物,其特征在于,其数均分子量为3000~10000;所述亲水性聚合物的数均分子量为500~2000。
4.根据权利要求1所述GSH响应型吉西他滨聚合物,其特征在于,所述亲水性聚合物、含两个羧基的二硫键化合物、吉西他滨的摩尔比为(0.1~0.5):(1~4.5):1。
5.权利要求1~4任一所述GSH响应型吉西他滨聚合物的制备方法,其特征在于,包括以下步骤:
S1.将吉西他滨、含两个羧基的二硫键化合物HOOC-R2-S-S-R2-HOOC、DCC以及DMAP混合,溶于有机溶剂中,反应18~36h,得到含二硫键的吉西他滨前药溶液;所述R2选自CH2、CH2-CH2或CH2-CH2-CH2;
S2.在步骤S1的含二硫键的吉西他滨前药溶液中加入亲水性聚合物、DCC、HOBT,反应24~48h,过滤产物,取滤液,透析,再经过旋蒸、冰乙醚沉淀、抽滤、真空干燥得到GSH响应型吉西他滨聚合物粉末。
6.根据权利要求5述制备方法,其特征在于,步骤S1所述含两个羧基的二硫键化合物、DCC、DMAP的摩尔比为1:(2~2.4):(0.1~0.6)。
7.根据权利要求6述制备方法,其特征在于,步骤S2所述亲水性聚合物、HOBT、DCC的摩尔比为1:(0.74~1.1):(0.45~0.61)。
8.一种GSH响应型吉西他滨纳米粒子,其特征在于,由权利要求1~4任一所述的GSH响应型吉西他滨聚合物、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000组成,所述GSH响应型吉西他滨聚合物和二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000的质量比为1:(0.1~0.8)。
9.权利要求8所述GSH响应型吉西他滨纳米粒子的制备方法,其特征在于,包括以下步骤:将权利要求1~4任一所述的GSH响应型吉西他滨聚合物溶于溶剂中,充分溶解后加入二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000,混匀后滴加到水中,搅拌,过滤,离心,除杂,即得。
10.权利要求9所述制备方法制备得到的GSH响应型吉西他滨纳米粒子在制备抗肿瘤药物中的应用。
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