CN114652849A - 可同时递送多种药物并精确调控药物比例的杯芳烃修饰白蛋白的制备方法和应用 - Google Patents
可同时递送多种药物并精确调控药物比例的杯芳烃修饰白蛋白的制备方法和应用 Download PDFInfo
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Abstract
可同时递送多种药物并精确调控药物比例的杯芳烃修饰白蛋白的制备方法,将磺酸盐偶氮杯[4]芳烃(SAC4A)共价修饰到牛血清白蛋白(BSA)的表面制备而成的,该白蛋白(CaMA)能够精确负载至少两种药物分子,并灵活调控所负载药物分子的比例。本发明通过将多个具有缺氧响应性的杯芳烃结合到一个白蛋白上,CaMA实现了精确的多药负载和多种药物在肿瘤微环境中的同步释放。这种独特的载药和释药机制确保了CaMA可以维持药物比例从初始载药到释放部位,为多药联合治疗在体内达到预期的治疗效果提供了坚实的基础。这种比例递送特性使CaMA成为开发用于癌症治疗的联合化疗和个性化药物的强大工具。
Description
技术领域
本发明属于生物医药领域,涉及一种可同时递送多种药物并精确调控药物比例的杯芳烃修饰白蛋白的制备方法和应用。
背景技术
联合药物治疗是目前癌症治疗中的常见做法,可利用多种药物的不同毒性来改善治疗效果。在该疗法中,联合药物的摩尔比是确定协同效应有效性的关键参数。然而,体外鉴定的组合药物的最优摩尔比由于药物分子不同的生物分布和药代动力学而难以按比例递送到靶标,导致临床转化中的协同效应受限。迄今为止,已经开发了多种纳米药物递送系统,例如脂质体和聚合物胶束,用于多药联合递送。这些策略确保装载药物的药代动力学和生物分布一致,但由于制备和递送过程中潜在的泄漏,精确控制装载比例和剂量仍然是一个挑战。这种不精确装载引起的不确定性可能导致“批次间”的变化,导致在癌症治疗中不可预测的治疗效果。为了解决这个问题,已经开发了通过刺激响应性共价键偶联的策略(包括药物-载体或药物-药物的偶联)。然而,化学偶联所需的特定基团并不总是适用于许多化疗药物,复杂的合成和纯化过程也限制了药物配比的调控。因此,对于有效的组合药物治疗,迫切需要能够满足以下需求的共递送载体:i)精确装载多种药物,ii)轻松调控装载药物的摩尔比和iii)比例共递送组合药物到靶标。
在过去的几十年里,大环分子(柱芳烃、葫芦脲、杯芳烃和环糊精)被广泛用于药物递送,以增强药物稳定性、提高药物溶解度和减少毒副作用。这些大环受体通过具有确定的化学计量(主要是1:1)和特征结合亲和力的主客体相互作用与药物分子复合。这种独特的载药机制可以根据初始浓度预测载药量,为精确控制载药量提供了坚实的基础。然而,大环和药物分子之间1:1的化学计量限制了大环同时装载和共同递送多种药物。此外,作为没有固有靶向能力的小分子,大多数大环不能为装载在内部的药物带来有利的生物分布,这限制了它们作为药物递送载体的效率。因此,为了实现高效的基于大环分子的联合化疗,开发创新策略,使它可以(i)克服1:1的化学计量限制并实现多种药物的精确加载和(ii)有效靶向肿瘤组织以优化药物组合的生物利用度至关重要。
发明内容
本发明目的是解决传统纳米药物载体无法精确调控药物比例并将多药按比例递送至肿瘤组织的问题,提供一种可同时递送多种药物并精确调控药物比例的杯芳烃修饰白蛋白的制备方法和应用。
采用的技术方案是:
一种可同时递送多种药物并精确调控药物比例的杯芳烃修饰白蛋白的制备方法,该杯芳烃修饰白蛋白(CaMA)是通过将磺酸盐偶氮杯[4]芳烃(SAC4A)化学修饰到牛血清白蛋白(BSA)的表面制备得到。
进一步的,BSA表面修饰的SAC4A数目确定为:BSA-(SAC4A)n,其中,n=1,2,3,4,5,6,7。
本发明中,构筑单元BSA具有纳米级粒径,分子量为66.4KDa;构筑单元SAC4A的化学式为C52H36N8S4O16Na4,分子量为1248,SAC4A的结构式如下:
进一步的,本发明制备方法步骤如下:
1)将SAC4A溶解在DMF中,然后向溶液中加入Na2CO3,室温搅拌10分钟后加入环氧溴丙烷并在室温下搅拌24小时,离心去除不溶性Na2CO3,反应溶液在大量冷乙醚中沉淀分离产物,离心收集,真空干燥,获得SAC4A-epoxy;
2)将BSA和SAC4A-epoxy添加到Na2CO3缓冲液(100mM)中,并在室温下搅拌24小时,用水透析(MWCO 10000),超滤(MWCO 30000)和脱盐除去游离SAC4A。
进一步的,本发明提供了可同时递送多种药物并精确调控药物比例的杯芳烃修饰白蛋白作为组合药物递送平台的应用。其中所递送的药物是选自治疗以下一种或多种疾病的药物:癌症、心肌梗塞、中风、动脉粥样硬化、类风湿性关节炎、炎症性肠病、慢性缺氧性肺病和慢性肾病。
由于BSA具有纳米级粒径、稳定的理化结构、高生物相容性和广泛的材料来源,被用作CaMA的结构基础。SAC4A是一种刺激响应性杯芳烃,可在缺氧肿瘤微环境中被降解,用作CaMA加载和控制药物分子释放的功能单元。如图1所示,由于表面有多个SAC4A,CaMA可以在一个纳米结构中加载多个药物分子。更重要的是,CaMA内装载的药物分子可以是多种类型,通过调整根据其特征结合亲和力计算的初始药物浓度,可以精确控制药物配比。在血液循环或正常组织中,CaMA与负载药物之间的强结合力可防止药物发生意外泄漏。同时,得益于其纳米级粒径,CaMA及其负载的药物可以通过增强渗透和保留效应在肿瘤组织中有效积累。到达肿瘤微环境后,结合亲和力随着SAC4A的降解而降低,导致所有药物快速释放。所有这些特性,包括可预测的载药率、运输过程中无药物泄漏、增强的肿瘤积累和所有药物响应肿瘤微环境的快速释放,确保了CaMA对多种药物的成比例递送。
本发明选择临床常用的化疗药物阿霉素和丝裂霉素C作为模型药物组合,并在体外筛选了这些药物达到最佳抗癌疗效的比例。通过以预筛选比例装载DOX和MMC,CaMA实现了将药物组合向肿瘤的比例共递送,与传统的鸡尾酒疗法相比,显著增强了肿瘤抑制并降低了全身毒性。这种比率输送能力使CaMA能够将体外确定的最佳药物组合快速转化为体内治疗效果,为开发联合癌症疗法和个性化药物提供了强大的工具。
附图说明
图1a为CaMA用于药物组合的比率共同递送的图示。图1b为SAC4A在缺氧条件下降解的机制。
图2为CaMA的合成示意图。
图3a显示在25℃下,PBS缓冲溶液中(10mM,pH=7.4),罗丹明B与SAC4A的荧光滴定曲线(λex=554nm);图3b显示罗丹明B与SAC4A的键合常数拟合曲线,根据主客体1:1键合模型进行拟合(λem=575nm)。
图4a显在示25℃下,PBS缓冲溶液中(10mM,pH=7.4),罗丹明B与CaMA的荧光滴定曲线(λex=554nm);图4b显示罗丹明B与CaMA的键合常数拟合曲线,根据主客体1:1键合模型进行拟合(λem=575nm)。
图5a显示在25℃下,PBS缓冲溶液中(10mM,pH=7.4),DOX与CaMA的荧光滴定曲线(λex=497nm);图5b显示DOX与CaMA的键合常数拟合曲线,根据主客体1:1键合模型进行拟合(λem=555nm)。
图6a显示在25℃下,PBS缓冲溶液中(10mM,pH=7.4),MMC与CaMA的荧光滴定曲线(λex=210nm);图6b显示MMC与CaMA的键合常数拟合曲线,根据主客体1:1键合模型进行拟合(λem=423nm)。
图7a显示在25℃下,PBS缓冲溶液中(10mM,pH=7.4),CPT与CaMA的荧光滴定曲线(λex=365nm);图7b显示CPT与CaMA的键合常数拟合曲线,根据主客体1:1键合模型进行拟合(λem=420nm)。
图8显示25℃下,PBS缓冲溶液中(10mM,pH=7.4),DOX与CaMA在干扰物存在下的荧光回复;
图9a显示加入SDT前后,CaMA的紫外吸收曲线;图9b显示加入不同浓度SDT后,CaMA-DOX的荧光曲线。
图10显示CCK-8法细胞毒性实验中,不同浓度的CaMA对于4T1细胞存活率的影响。
图11显示CCK-8法细胞毒性实验中,不同浓度的CaMA-DOX在常氧和乏氧条件下对于细胞存活率的影响。
图12显示CaMA对DOX和CPT的包载比例图。
图13a显示SiPcN2和CaMA-SiPcN2两组小鼠肿瘤和主要脏器的离体荧光成像照片,图13b和13c显示48小时和72小时离体荧光成像对应的定量分析。
图14显示DOX和MMC在4T1细胞中的组合指数和相应的颜色条。
图15a显示PBS、CaMA、DM、CaMA-DOX、CaMA-MMC和CaMA-DM给药后各组小鼠的瘤子体积变化;图15b显示PBS、CaMA、DM、CaMA-DOX、CaMA-MMC和CaMA-DM给药后各组小鼠的瘤子重量;图15c显示PBS、CaMA、DM、CaMA-DOX、CaMA-MMC和CaMA-DM给药后各组小鼠的体重变化。
图16显示各组小鼠肿瘤的TUNLE、H&E和Ki67染色的共聚焦显微镜照片。
具体实施方式
一种可同时递送多种药物并精确调控药物比例的杯芳烃修饰白蛋白的制备方法,参见附图2的合成示意图,包括如下步骤:
将SAC4A用环氧溴丙烷处理以获得SAC4A-epoxy。具体而言,将35mg SAC4A(28μmol)溶解在4mL DMF中,然后向溶液中加入400mg Na2CO3室温搅拌10分钟后加入1g(7.3mmol)环氧溴丙烷继续在室温下搅拌24小时。离心去除不溶性Na2CO3,反应溶液在大量冷乙醚中沉淀分离产物,离心收集,真空干燥。
接下来通过BSA氨基和环氧基之间的反应将SAC4A修饰到BSA上来制备CaMA。将20mg BSA(0.3μmol)和3.75mg SAC4A-环氧树脂(3μmol)添加到5mLNa2CO3缓冲液(100mM)中,并在室温下搅拌24小时。用水透析(MWCO 10000),超滤(MWCO 30000)和脱盐除去游离SA4A。得到产物的质谱数据如下:
MS(MALDI-TOF):CaMA:73689.8,(BSA:66367.0)。
测试实施例
实施例1:CaMA与药物分子键合常数的测定
测试方法:荧光滴定法。
测试工具:仪器型号为Hitachi F4600,石英比色皿,测试光路10mm,配有比色皿控温装置。
试剂及其来源:
罗丹明B(RhB)购置于上海西格玛奥德里奇贸易有限公司;
SiPcN2购置于北京安诺轮生物科技有限公司;
阿霉素(DOX),丝裂霉素(MMC)购置于上海阿拉丁生化科技股份有限公司;
喜树碱(CPT)购置于天津希恩思生化科技有限公司。
CaMA与光致发光分子的荧光滴定实验均在室温下(25℃)进行。首先配制CaMA,RhB,DOX,MMC,CPT,SiPcN2的母液,将其分别溶于PBS(10mM,pH=7.4)缓冲溶液中。测试时先将荧光分子(0.5μM)配置于荧光池内,PBS定容到体积2mL。将CaMA配置在PBS缓冲液中(10mM,pH=7.4),配置浓度为100μM,并向其加入荧光分子,使荧光分子浓度与荧光池内保持一致。随后将CaMA溶液以预定体积加入到荧光池内,记录荧光强度变化。荧光滴定数据根据主客体1:1键合模型进行拟合,测定主客体包结的键合常数Ka。结果参见图3-图7。
药物分子与CaMA的键合常数测试结果如下表1所示。
表1.活性药物分子与CaMA的键合常数
实施例2:CaMA的干扰物竞争测定
测试方法:荧光滴定法。
测试工具:仪器型号为Hitachi F4600,石英比色皿,测试光路10mm。
试剂及其来源:
ATP购置于美国默克公司;
丙氨酸,甘氨酸,精氨酸,缬氨酸,赖氨酸购置于天津希恩思生化科技有限公司;
葡萄糖购置于北京百灵威科技有限公司;
烟草酰胺腺嘌呤二核苷酸(NAD),腺苷二磷酸(ADP),腺苷一磷酸盐(AMP)和肌酐购置于毕得医药;
BSA购置于北京迈瑞达科技有限公司。
实验步骤:
CaMA和荧光分子的干扰物非特异性竞争实验均在室温(25℃)进行。首先配CaMA和DOX的母液,将其分别溶于磷酸盐酸缓冲溶液(PBS,10mM,pH=7.4)中,配置浓度均为100μM。测试时先将CaMA-DOX(5/5μM)配置于荧光池内,PBS定容到体积2mL。将血液中存在的各种竞争物溶于PBS缓冲液中(10mM,pH=7.4)并加入至荧光池子中,搅拌30分钟后监测DOX的荧光强度。血液中竞争物及其浓度为:三磷酸腺苷(ATP)0.2μM或100μM,二磷酸腺苷(ADP)0.1μM,一磷酸腺苷(AMP)10nM,烟酰胺腺嘌呤二核苷酸(NAD)24μM,谷氨酰胺0.5mM,丙氨酸0.4mM,缬氨酸0.2mM,甘氨酸0.3mM和赖氨酸0.2mM;钾盐04.5mM,钙盐2.5mM,牛血清白蛋白(BSA)10μg/mL,谷胱甘肽8.0μM,肌酐80μM和葡萄糖5.0mM。
测试结果如图8所示,添加血液中存在的这些干扰物质后,从CaMA-DOX中没有检测到明显的荧光回复,这表明CaMA在生理环境中具有很高的稳定性。
实施例3:连二亚硫酸钠(SDT)还原实验
测试方法:紫外-可见分光光谱法、荧光分光光谱法。
测试工具:日本岛津UV-1800紫外-可见分光光度计,石英比色皿,光程10mm。荧光仪器型号为Hitachi F4600,石英比色皿,测试光路10mm。
实验步骤:首先配制CaMA,DOX的母液,将其分别溶于PBS缓冲溶液(10mM,pH=7.4)中,配制浓度均为100μM。取CaMA母液稀释到5μM,测试420nm处紫外吸收随时间变化,3.5分钟时加入2.0mM SDT,测试结果如图9a所示。图9a显示CaMA在420nm处紫外吸收随时间变化曲线。从图9a中看出,加入SDT后,CaMA中偶氮键对应的紫外吸收随时间逐渐下降,说明CaMA能被SDT还原,具有乏氧响应性。
配制CaMA-DOX(5/5μM)的溶液,测试荧光光谱。随后加入0-600μM SDT,再次测试荧光光谱。如9b所示:随着SDT浓度的提高,DOX的荧光恢复,说明DOX可以从CaMA中释放出来。
实施例4:CaMA的毒性实验
测试方法:CCK-8法。
测试工具:Tecan Spark多功能酶标仪
试剂及其来源:
胎牛血清(FBS)、DMEM培养基和青霉素链霉素购置于美国赛默飞世尔科技公司。
CCK-8购置于日本同仁公司。
实验步骤:
1、使用含有10%FBS和1%青霉素链霉素的DMEM培养基。将小鼠乳腺癌细胞4T1置于37℃、5%CO2细胞培养箱中孵育,每次在实验之前,将细胞以10000个/孔的细胞密度接种至96孔板中,孔板边缘孔用无菌PBS填充。
2、在5%CO2,37℃下孵育,至细胞单层铺满孔底(96孔平底板),加入浓度梯度的CaMA(0.25μM-64μM)。
3、将96孔板置于5%CO2,37℃培养箱中培养24小时。
4、小心吸去孔内培养液,每孔加入100uL新鲜配置的CCK-8工作液(1/9,v/v),继续培养1.5小时。
5、终止培养。在酶标仪检测OD 450nm处各孔的吸光值。细胞存活率根据以下公式计算得到:细胞存活率=(OD450(samples)/(OD450(control)×100%。
从图10的CCK-8法细胞毒性结果看出,CaMA载体无明显细胞毒性。
实施例5:CaMA-DOX和游离DOX在常氧和乏氧条件下的毒性实验
按照与实施例4相同的方式进行实验,结果参见图10。
图11中,浓度表示DOX的浓度(1μM、2μM、4μM、8μM、16μM、32μM),CaMA-DOX包合物组中CaMA与DOX的浓度比为1:1。从图11的细胞毒性结果可以看出,常氧条件下,与游离DOX相比,包合物CaMA-DOX细胞毒性降低。乏氧条件下,包合物CaMA-DOX细胞毒性比常氧条件的下细胞毒性大,说明包合物CaMA-DOX具有乏氧响应释放的特性。
实施例6:CaMA按预定比例精确包载DOX和CPT实验
测试方法:荧光分光光谱法
测试工具:Hitachi F4600荧光光谱仪,石英比色皿,测试光路10mm。
试剂及其来源:
DOX,MMC购置于上海阿拉丁生化科技股份有限公司
实验步骤:
1、我们给定CaMA的浓度(其中SAC4A的浓度)为180μM,假设DOX和CPT的包载总浓度为150μM,根据预期的摩尔比和Ka计算DOX和CPT的投料浓度。对于不同DOX/CPT比例,计算结果如下0.2=21.5μM/125μM,0.5=42.5μM/100μM,1.0=75μM/88.24μM,2.0=100μM/58.82μM和5.0/=125μM/39.41μM。
2、按计算投料,DOX、CPT和CaMA的混合物在室温下震荡混合30分钟,然后通过超滤离心(MWCO=3kDa)除去未包载的药物。
3、用荧光光谱仪分别在λex=497nm和λex=365nm处测定滤液中未包载的DOX和CPT的浓度,计算CaMA包载的DOX和CPT的浓度及比例。
如图12所示,CaMA包载的DOX和CPT的比例与预期基本一致(0.2与0.20,0.5与0.51,1.0与1.12,2.0与1.97,5.0与5.03),说明CaMA可以实现精确的多药包载,并通过控制投料比调节载药比例。
实施例8:CaMA-SiPcN2活体成像实验
测试方法:离体成像
测试工具:IVIS Lumina成像系统
动物及其来源:雌性6-8周BALB/c小鼠购买于北京维通利华公司
实验步骤:
将1×1064T1癌细胞皮下注射到BALB/c小鼠的左乳脂垫中。待肿瘤生长至400mm3,将荷瘤小鼠随机分为两组,尾静脉分别注射200μL SiPcN2和CaMA-SiPcN2,SiPcN2浓度为200μM。在注射48和72小时后处死小鼠,收集肿瘤以及主要器官(心,肝,脾,肺和肾),然后进行离体成像。
如图13a所示,注射CaMA-SiPcN2的小鼠肿瘤中可以观察到较强的荧光信号。相比之下,注射游离SiPcN2的小鼠的肿瘤部位的荧光较弱。图13b和13c所示的定量分析中也观察到了相似的结果。说明CaMA可以有效地将负载的药物递送至肿瘤组织。
实施例9:DOX和MMC联合指数的测定
测试方法:CCK-8法
测试步骤:
CCK-8法测定不同浓度(0.125μM-16μM)的DOX、MMC及其不同比例(4:1、2:1、1:1、1:2、1:4)的组合药物处理4T1癌细胞后的细胞活力。使用基于Chou-Talalay分析方法的CompuSyn软件对药物组合进行联合指数(CI)分析。药物组合的CI值被绘制为Fa的函数。CI值<1、=1和>1分别表示药物组合的协同作用、相加作用和拮抗作用。值得注意的是,Fa 0.2到0.8之间的CI值被认为是有效的。IC50的最佳拟合CI值显示和比较不同药物比例的药物组合的协同作用。
如图14所示,DOX和MMC的比例为1:1时具有最佳的协同效果
实施例10:CaMA优化组合药物的抗肿瘤功效
测试工具:游标卡尺,天平
实验步骤:
将1x106个4T1细胞原位注射到6-8周的雌性Balb/C小鼠左乳脂垫中,等到瘤子体积大小长到约100mm3时,将小鼠随机分为PBS,DOX+MMC(DM),CaMA,CaMA-DOX,CaMA-MMC和CaMA-DM六组,每组6只小鼠。尾静脉注射200μL各组药物,每次注射前测量小鼠肿瘤长短轴直径。给药量以300μM计。每两天给一次药,共给药3剂。从给药第一天开始测量小鼠瘤子体积及小鼠体重。
如图15a和15c所示,与对照组相比,CaMA-DM组小鼠的肿瘤生长速度明显降低,停止给药后仍有短期抑制作用,证明CaMA-DM具有良好的抑瘤效果。监测小鼠体重可知,六组均无显著毒性。
给药后第18天,将小鼠杀死并取出肿瘤用于拍照和瘤子重量检测,结果如图15b。根据差异显著性分析,CaMA-DM组肿瘤重量与对照组相比具有显著差异性,证明CaMA-DM具有良好的抑瘤效果。
肿瘤大小通过游标卡尺测量,肿瘤体积根据以下公式计算:V=W2×L/2,其中W和L分别是肿瘤的最短和最长直径。
实施例11:H&E,TUNEL,Ki67染色
H&E染色步骤:将小鼠肿瘤细胞置于4%多聚甲醛中固定24小时后,送天津易生源生物技术有限公司进行石蜡切片及H&E染色实验。随后进行显微镜分析。
TUNLE染色步骤:将小鼠肿瘤进行冰冻切片,PBS湿润15min后遵循Roach公司提供的实验手册进行染色。
Ki67染色步骤:将冰冻切片从-80℃取出,恢复至室温,PBS湿润15min后用0.1%TritonX-100处理15分钟后,PBS洗掉Triton,5%BSA封闭1小时后加入一抗(Ki67一抗,小鼠来源,sigma),4℃避光过夜。随后PBS洗掉一抗,然后加入荧光标记的二抗(山羊抗小鼠)。室温孵育1小时后,PBS洗涤。DAPI染色10min后PBS洗涤,封片剂封片,共聚焦显微镜拍照分析。
如图16所示,H&E染色切片在显微镜下观察到CaMA-DM组有明显的细胞核固缩,核质分离和细胞核外流,说明CaMA-DM组的细胞坏死程度明显高于其他几组,证明CaMA组具有良好的杀伤肿瘤的效果。从TUNEL的凋亡指标及Ki67的增殖指标中也可以得到类似的结果,即CaMA-DM具有良好的肿瘤杀伤效果。
工业应用性:
本发明提供了一种可同时递送多种药物并精确调控药物比例的杯芳烃修饰白蛋白的制备方法和应用。本发明的化合物可以与适当的活性药物分子一起制成相应的药物组合物,适于工业应用。
Claims (8)
1.可同时递送多种药物并精确调控药物比例的杯芳烃修饰白蛋白的制备方法,其特征在于:通过将磺酸盐偶氮杯[4]芳烃(SAC4A)共价修饰到牛血清白蛋白(BSA)的表面制备而成的,该白蛋白(CaMA)能够精确负载至少两种药物分子,并灵活调控所负载药物分子的比例。
2.如权利要求1所述的可同时递送多种药物并精确调控药物比例的杯芳烃修饰白蛋白的制备方法,其特征在于,BSA表面修饰的SAC4A数目确定为:BSA-(SAC4A)n,其中,n=1,2,3,4,5,6,7。
3.根据权利要求2所述的可同时递送多种药物并精确调控药物比例的杯芳烃修饰白蛋白的制备方法,其特征在于,n=6。
5.一种权利要求1-4任一项所述的可同时递送多种药物并精确调控药物比例的杯芳烃修饰白蛋白的制备方法,其特征在于步骤如下:
1)将SAC4A溶解在DMF中,然后向溶液中加入Na2CO3,室温搅拌10分钟后加入环氧溴丙烷并在室温下搅拌24小时,离心去除不溶性Na2CO3,反应溶液在大量冷乙醚中沉淀分离产物,离心收集,真空干燥,获得SAC4A-epoxy;
2)将BSA和SAC4A-epoxy添加到Na2CO3缓冲液(100mM)中,并在室温下搅拌24小时,用水透析(MWCO 10000),超滤(MWCO 30000)和脱盐除去游离SAC4A。
6.一种可同时递送多种药物并精确调控药物比例的杯芳烃修饰白蛋白,其特征在于:通过权利要求1-5任一项制备方法制备得到。
7.一种权利要求6所述的可同时递送多种药物并精确调控药物比例的杯芳烃修饰白蛋白作为组合药物递送平台的应用。
8.如权利要求7所述的应用,其特征在于,所递送的药物是选自治疗以下一种或多种疾病的药物:癌症、心肌梗塞、中风、动脉粥样硬化、类风湿性关节炎、炎症性肠病、慢性缺氧性肺病和慢性肾病。
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Citations (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101312986A (zh) * | 2005-09-28 | 2008-11-26 | 日内瓦大学 | 生产修饰(多)肽的方法 |
US20080300164A1 (en) * | 2004-10-04 | 2008-12-04 | Regents Of The University Of Minnesota | Calixarene-Based Peptide Conformation Mimetics, Methods of Use, and Methods of Making |
CN105566129A (zh) * | 2015-12-22 | 2016-05-11 | 南开大学 | 一种两亲杯芳烃AmC5A的纳米超分子组装体及其制备方法和应用 |
CN106974897A (zh) * | 2017-02-20 | 2017-07-25 | 西北农林科技大学 | 一种靶向刺激响应性多功能二氧化铈纳米载药体系 |
CN108314690A (zh) * | 2018-03-15 | 2018-07-24 | 中国科学院苏州生物医学工程技术研究所 | 双杯[4]芳烃衍生物的金属配合物及其合成方法和应用 |
CN108671238A (zh) * | 2018-05-14 | 2018-10-19 | 江苏医药职业学院 | 一种联合治疗高肿瘤渗透性白蛋白纳米系统的制备方法 |
CN109432436A (zh) * | 2018-09-10 | 2019-03-08 | 上海大学 | 基于柱芳烃的多肽缀合物、其制备方法及其应用 |
CN111789963A (zh) * | 2019-04-04 | 2020-10-20 | 苏州健雄职业技术学院 | 一种肿瘤靶向的药物载体及其制备方法和应用 |
CN112274656A (zh) * | 2020-11-19 | 2021-01-29 | 南开大学 | 可将组合药物按比例递送至肿瘤组织的大环两亲自组装纳米颗粒的制备方法和应用 |
WO2021160654A1 (en) * | 2020-02-11 | 2021-08-19 | Universite Libre De Bruxelles | Nanomaterials coated with calixarenes |
CN113876760A (zh) * | 2021-10-26 | 2022-01-04 | 南开大学 | 杯芳烃和/或杯芳烃的衍生物在制备治疗创伤性脑损伤的药物中的应用 |
-
2022
- 2022-03-22 CN CN202210280736.3A patent/CN114652849B/zh active Active
Patent Citations (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20080300164A1 (en) * | 2004-10-04 | 2008-12-04 | Regents Of The University Of Minnesota | Calixarene-Based Peptide Conformation Mimetics, Methods of Use, and Methods of Making |
CN101312986A (zh) * | 2005-09-28 | 2008-11-26 | 日内瓦大学 | 生产修饰(多)肽的方法 |
CN105566129A (zh) * | 2015-12-22 | 2016-05-11 | 南开大学 | 一种两亲杯芳烃AmC5A的纳米超分子组装体及其制备方法和应用 |
CN106974897A (zh) * | 2017-02-20 | 2017-07-25 | 西北农林科技大学 | 一种靶向刺激响应性多功能二氧化铈纳米载药体系 |
CN108314690A (zh) * | 2018-03-15 | 2018-07-24 | 中国科学院苏州生物医学工程技术研究所 | 双杯[4]芳烃衍生物的金属配合物及其合成方法和应用 |
CN108671238A (zh) * | 2018-05-14 | 2018-10-19 | 江苏医药职业学院 | 一种联合治疗高肿瘤渗透性白蛋白纳米系统的制备方法 |
CN109432436A (zh) * | 2018-09-10 | 2019-03-08 | 上海大学 | 基于柱芳烃的多肽缀合物、其制备方法及其应用 |
CN111789963A (zh) * | 2019-04-04 | 2020-10-20 | 苏州健雄职业技术学院 | 一种肿瘤靶向的药物载体及其制备方法和应用 |
WO2021160654A1 (en) * | 2020-02-11 | 2021-08-19 | Universite Libre De Bruxelles | Nanomaterials coated with calixarenes |
CN112274656A (zh) * | 2020-11-19 | 2021-01-29 | 南开大学 | 可将组合药物按比例递送至肿瘤组织的大环两亲自组装纳米颗粒的制备方法和应用 |
CN113876760A (zh) * | 2021-10-26 | 2022-01-04 | 南开大学 | 杯芳烃和/或杯芳烃的衍生物在制备治疗创伤性脑损伤的药物中的应用 |
Non-Patent Citations (2)
Title |
---|
TIAN-XING ZHANG ET.AL.: "A hypoxia-responsive supramolecular formulation for imaging-guided photothermal therapy", 《TIAN-XING ZHANG ET.AL.》, vol. 12, no. 1, pages 1 * |
梁清;官冰;江明;: "两亲性杯芳烃的超分子自组装", 化学进展, no. 1 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115463138A (zh) * | 2022-09-29 | 2022-12-13 | 南开大学 | 包含可辅助化疗药物高效激活免疫原性细胞死亡的大环化合物的药物组合物 |
CN115463138B (zh) * | 2022-09-29 | 2023-08-08 | 南开大学 | 包含可辅助化疗药物高效激活免疫原性细胞死亡的大环化合物的药物组合物 |
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