CN116966316A - 一种双功能超分子纳米组装体及应用 - Google Patents
一种双功能超分子纳米组装体及应用 Download PDFInfo
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- CN116966316A CN116966316A CN202310764745.4A CN202310764745A CN116966316A CN 116966316 A CN116966316 A CN 116966316A CN 202310764745 A CN202310764745 A CN 202310764745A CN 116966316 A CN116966316 A CN 116966316A
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- cyclodextrin
- supramolecular
- bifunctional
- nanoessembly
- organic solvent
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- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 claims abstract description 29
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 28
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- KHBQMWCZKVMBLN-UHFFFAOYSA-N Benzenesulfonamide Chemical compound NS(=O)(=O)C1=CC=CC=C1 KHBQMWCZKVMBLN-UHFFFAOYSA-N 0.000 claims abstract description 10
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 10
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Classifications
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
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- A61K47/6951—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes using cyclodextrin
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
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- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
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- C08B37/0012—Cyclodextrin [CD], e.g. cycle with 6 units (alpha), with 7 units (beta) and with 8 units (gamma), large-ring cyclodextrin or cycloamylose with 9 units or more; Derivatives thereof
- C08B37/0015—Inclusion compounds, i.e. host-guest compounds, e.g. polyrotaxanes
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Abstract
本发明公开了一种双功能超分子纳米组装体,其是将苯磺酰胺偶联的环糊精、金刚烷胺/二硫键共同修饰的抗肿瘤药物置于在水中进行自组装,利用环糊精和金刚烷之间的主客体作用形成两亲性包合物;所述双功能超分子纳米组装体可实现药物的靶向递送和特异性释放,增强对肿瘤组织的治疗效果,避免对正常细胞的额外毒副作用,解决了目前化疗药物利用率不高,缺乏靶向性的问题,对于靶向抗肿瘤药物新型制剂的开发具有潜在的应用价值。
Description
技术领域
本发明属于生物医药材料技术领域,具体涉及一种肿瘤靶向的双功能超分子纳米组装体和应用。
背景技术
超分子化学是近年来迅速发展的一门交叉学科。主-客体的组装与识别是超分子化学中一个重要分支。其中,大环化合物是主要的超分子主体,这些主体分子大多具有疏水性空腔(例如环糊精),能够与尺寸匹配的药物小分子进行自组装识别,形成的稳定主-客体系统可用于递送药物。主-客体两亲物是由亲水大环分子与疏水分子通过自组装形成的包合物,该包合物兼具了非共价键驱动的动态特性和类似于共价键的稳定两亲性,可在水中进一步自组装成为两亲性纳米颗粒。它们通过EPR效应优先在肿瘤组织中蓄积,通过胞吞途径被细胞内化,在特定刺激下触发客体的逃逸或断裂和自组装纳米粒的解体,从而进一步导致被封装在内部疏水核心或双层膜中的药物释放
将抗癌药物输送到肿瘤组织并减少对正常组织的选择性仍然是一个亟待解决的问题。碳酸酐酶IX(CAIX)是一种在肿瘤细胞膜上过量表达的跨膜蛋白,负责肿瘤细胞的pH调节,在正常细胞中几乎不表达。已有大量文献证明,CAIX活性的抑制能够显著降低肿瘤的生长和转移,因此CAIX可作为靶标被合理利用,从而设计出具有靶向作用的分子。
环糊精的特殊圆锥形空腔以及对客体的识别组装作用使得其在药物递送中有广泛的应用,其结构骨架本质上由葡萄糖组成,具有生物安全性高、分子识别能力优良、易修饰等特点,已被FDA批准作为商用药用辅料。对其丰富的羟基进行修饰可衍生出功能化的环糊精,打破空腔内部氢键提高其水溶性,赋予其靶向性、刺激响应等功能。目前尚未见到将具有CAIX靶向功能的结构修饰于环糊精载体的相关报道。
发明内容
本发明提供了一种双功能超分子纳米组装体,其是将苯磺酰胺偶联的环糊精、金刚烷胺/二硫键共同修饰的抗肿瘤药物置于在水中进行自组装获得,该双功能超分子纳米组装体能实现药物的靶向递送和特异性释放,增强对肿瘤组织的治疗效果,避免对正常细胞的额外毒副作用,解决了目前化疗药物利用率不高,缺乏靶向性的问题。
本发明苯磺酰胺偶联的环糊精(L-CD)具有特异性靶向于肿瘤细胞的能力,作为抗肿瘤药物载体,与匹配客体形成多分子体系,从而赋予药物靶向性,增强肿瘤治疗效果
所述苯磺酰胺偶联的环糊精(L-CD)化学结构式为:
其中,m为1、2或3;n为1、2或3。
苯磺酰胺偶联的环糊精主体(L-CD)是将羧酸化磺胺溶于有机溶剂,然后加入缩合剂在20~40℃下搅拌反应,待反应完全后将反应液缓慢滴入冷水中产生白色沉淀,分离沉淀物,经干燥后再次溶于碱性有机溶剂,加入氨基环糊精在20~40℃下搅拌反应,然后将混合物滴入丙酮中重结晶,收集晶体粗产物用水溶解后,用去离子水透析纯化制得;
其中羧酸化磺胺与氨基环糊精的摩尔比为1:0.5~1;碱性有机溶剂是添加了碳酸钾、三乙胺或乙醇钠的有机溶剂,有机溶剂包括但不限于丙酮、四氢呋喃、醋酸酐、乙醇、甲醇、N,N-二甲基甲酰胺、二甲基亚砜中,碳酸钾、三乙胺或乙醇钠添加量为羧酸化磺胺摩尔量的1~5倍;缩合剂选自EDCI/NHS、EDCI/DMAP、DCC/NHS、DCC/DMAP中的一种。
所述氨基环糊精参考文献Journal of Molecular Structure,2019,1193:207-214.中的方法合成;羧酸化磺胺参照Tetrahedron,2005,61(40):9478–9483.中的方法制得。
所述金刚烷胺/二硫键共同修饰的抗癌药物(A-SS-Ad)的化学结构式为:
A为疏水性抗癌药物。
抗癌药物的修饰方法均为常规方法,例如参照Nanoscale,2016,8(45):18876-18881.中、Chemical Communications,2020,56(30):4192-4195.中、Angewandte Chemie,2020,132(35):15067-15074.中的方法。
本发明另一目的是将双功能超分子纳米组装体应用在制备治疗肿瘤药物中。
与现有技术相比,本发明具有以下有益效果:
本发明以苯磺酰胺偶联的环糊精为主体,以金刚烷胺/二硫键共同修饰的疏水性抗癌药物为客体,利用环糊精和金刚烷之间的作用形成两亲性包合物,该包合物进一步在水中发生自组装,形成亲水性环糊精为壳层、疏水性抗肿瘤药物为内核的超分子纳米组装体,利用分子之间的互相作用力,进行自组装,制备方法简单易行。
本发明双功能超分子纳米组装体可实现药物的靶向递送和特异性释放,增强对肿瘤组织的治疗效果,避免对正常细胞的额外毒副作用,解决了目前化疗药物利用率不高,缺乏靶向性的问题,对于靶向抗肿瘤药物新型制剂的开发具有潜在的应用价值。
附图说明
图1是实施例1中L-CD(m=2,n=1)的高分辨率质谱(HR-MS)图;
图2是实施例1中L-CD(m=2,n=1)的核磁共振氢谱(1H NMR)谱图;
图3是实施例1中L-CD(m=2,n=1)与CAIX的分子对接,使用临床上使用的CAIX抑制剂乙酰唑胺作为对照;
图4是实施例1中MTX-SS-Ad(MTX为疏水性抗肿瘤药物甲氨蝶呤)的高分辨率质谱(HR-MS)图;
图5是实施例1中MTX-SS-Ad的核磁共振氢谱(1H NMR)谱图;
图6是实施例1中SNPs的动态光散射图;
图7是实施例1中SNPs的透射电镜图像;
图8是实施例1中L-CD(m=2,n=1)与MTX-SS-Ad的2D-ROESY NMR谱图;
图9是实施例2中SNPs(A)、β-CD/MTX-SS-Ad形成的纳米粒(B)、MTX-SS-Ad(C)在不同浓度下的细胞存活率柱形图;
图10是实施例3中负载罗丹明B的SNPs(A)、负载罗丹明B的β-CD/MTX-SS-Ad形成的纳米粒(B)、罗丹明B(C)在三阴性乳腺癌细胞(MDA-MB-231)上的激光共聚焦显微镜下的细胞摄取图像;
图11是实施例3中负载罗丹明B的SNPs(A)、负载罗丹明B的β-CD/MTX-SS-Ad形成的纳米粒(B)、罗丹明B(C)在宫颈癌细胞(Hela)上的激光共聚焦显微镜下的细胞摄取图像;
图12是实施例3中负载罗丹明B的SNPs(A)、负载罗丹明B的β-CD/MTX-SS-Ad形成的纳米粒(B)、罗丹明B(C)在正常肝细胞(LO2)上的激光共聚焦显微镜下的细胞摄取图像;
图13是经过MTX-SS-Ad(A)、β-CD/MTX-SS-Ad形成的纳米粒(B)、SNPs处理后的Hela细胞(C)中CAIX表达的蛋白免疫印迹条带图。
具体实施方式
下面结合实施例对本发明所述方法作进一步的详细描述。
本领域技术人员将会理解,下列实施例仅用于说明本发明,而不应视为限定本发明的范围。实施例中未注明具体技术或条件者,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用材料或设备未注明生产厂商者,均为可以通过购买获得的常规产品,使用的方法如无特殊说明均为常规方法;
实施例1:
1、将磺胺(1.73g,10mmol)溶于DMF/MeCN(v/v,1:4,10mL)的混合溶剂中,加入丁二酸酐(1.12g,11mmol),混合物在70℃下回流搅拌至有大量白色固体产生时停止反应,过滤收集粗产物,粗产物用MeCN洗涤3次,干燥即得2.01g的白色固体羧酸化磺胺;
2、将羧酸化磺胺(2mmol)溶于N,N-二甲基甲酰胺(20mL)中,然后加入EDCI(2.5mmol)和NHS(2.5mmol),混合物在40℃下搅拌24h,待反应完全后将反应液缓慢滴入冷水中产生白色沉淀,分离沉淀物,经干燥后再次溶于N,N-二甲基甲酰胺(15mL),并加入三乙胺(4mmol)、乙二胺环糊精(1mmol),于40℃反应24h,然后将混合物滴入丙酮中重结晶,收集晶体粗产物,粗产物用少量水溶解,用去离子水透析纯化48h(1000Da)得到苯磺酰胺偶联的环糊精L-CD(m=2,n=1);L-CD产物的高分辨率质谱(HR-MS)如图1所示,产物的核磁共振氢谱(1H NMR)如图2所示,从图中可以看出,4.98ppm的质子峰为环糊精骨架上的1号位上的7个氢,3.99-3.64ppm范围内的质子峰归属为环糊精骨架上的H-3,6,5的28个氢,3.62-3.43ppm的质子峰对应环糊精上H-2和H-4的16个氢;2.96-2.79ppm可归属为乙二胺单元上的两个亚甲基;7.90-7.49和2.65-2.33ppm分别对应L-ligand上苯环的4个氢和丁二酸酐单元上的两个亚甲基,综上数据可知L-CD已被成功制备。
通过PDB数据库获得碳酸酐酶IX(CAIX)与乙酰唑胺的共晶结构(PDB:3IAI),在殷赋云平台对所有模型进行预处理及分子对接计算,结果如图3所示,L-CD(m=2,n=1)的苯磺酰胺部分正如预期的那样进入CAIX的大口袋空腔,磺酰胺上的N原子与蛋白质中的Zn2+连接。此外,环糊精上丰富的-OH基团通过氢键与ARG58、GLN67、VAL131和TRP5等残基结合,导致其与CAIX之间的结合能力相对较强,计算的结合能为-94.227kcal/mol(图3a),大于阳性对照乙酰唑胺(-41.897kcal/mol,图3b),表明L-CD(m=2,n=1)对CAIX具有较强的结合能力和显著的抑制作用,从而为靶向性提供了前提条件;
3、将甲氨蝶呤(MTX,0.6mmol)和胱胺二盐酸盐(0.8mmol)溶于DMSO/吡啶(1:1,v/v,8mL),加入DCC(0.7mmol)和DMAP(0.06mmol),室温下避光搅拌18h后,使用丙酮析出粗产物,粗产物经丙酮洗涤(40mL×2)、离心、50℃真空干燥后得到黄色固体MTX-SS;将产物MTX-SS(0.6mmol)溶于吡啶(5mL),加入丁二酸酐(1.0mmol),室温下避光搅拌18h后使用丙酮析出粗产物,粗产物经丙酮洗涤(40mL×2)、离心、50℃真空干燥后得到黄色固体MTX-SS-COOH;将该产物MTX-SS-COOH(0.2mmol)溶于DMSO/吡啶(1:1,v/v,3mL),加入EDCI(50mg,0.24mmol)、NHS(30mg,0.24mmol)和金刚烷胺(0.21mmol),在室温下下避光搅拌24h后,使用丙酮析出粗产物,粗产物经依次经过丙酮、水、乙醇洗涤,离心后得到黄色固体MTX-SS-Ad,产物MTX-SS-Ad的高分辨率质谱(HR-MS)如图4所示,产物的核磁共振氢谱(1H NMR)如图5所示,从图中可以看出3.34-3.21ppm的4个氢和2.74-2.64ppm的四个氢对应二硫键周围的质子信号,2.05-1.35ppm的19个氢包含了金刚烷部分的质子峰,综上数据可知MTX-SS-Ad已被成功制备。
4、将MTX-SS-Ad溶于DMSO,记为G液,将L-CD溶于超纯水,记为H液,在超声条件下将G液缓慢加入到H液得到悬浊液,悬浊液中MTX-SS-Ad和L-CD的终浓度均为50μmol/L,超声30min后静置过夜使其充分组装,反应产物置于透析袋中在去离子水中透析(10000Da)4h以除去DMSO,然后经0.22μm微孔滤膜过滤即得双功能超分子纳米组装体SNPs;图6是SNPs的动态光散射图,从图中可以看出SNPs的平均粒径为77.93±0.62nm;图7是SNPs的透射电镜图像,TEM图像显示了直径为100nm左右的球形中空结构,这与DLS的结果一致。这些球形纳米颗粒为分布均匀的双层囊泡结构,其双层膜间距离为9.66nm,与计算所得的包合物分子尺寸一致(4.32nm×2=8.64nm)。这些图像进一步直观地证明了SNPs的形成及其核壳结构;图8是L-CD(m=2,n=1)与MTX-SS-Ad的2D-ROESY NMR谱图;从图中可以观察到环糊精腔内氢(H-3,5)与金刚烷部分之间存在明显的NOE交叉峰,直观地表明二者间存在主-客体相互作用。
同时采用β-CD和MTX-SS-Ad制备纳米粒,作为对照,方法同制备SNPs。
实施例2:通过MTT法分别评估L-CD(m=2,n=1)和MTX-SS-Ad自组装形成的SNPs(A)、β-CD/MTX-SS-Ad形成的纳米粒(B)、MTX-SS-Ad(C)对宫颈癌细胞(Hela)细胞毒性
将Hela细胞以每孔104个细胞的密度接种在96孔板中培养24h,然后将细胞分别与上述样品孵育44h,孵育结束后加入MTT溶液,并再孵育4h,随后加入DMSO以溶解甲瓒,通过酶标仪在492nm处测定吸光度;
图9为SNPs(A)、β-CD/MTX-SS-Ad形成的纳米粒(B)、单独MTX-SS-Ad(C)在不同浓度下的细胞存活率柱形图,从图中可以观察到SNPs对Hela细胞的毒性明显高于β-CD/MTX-SS-Ad形成的纳米粒、MTX-SS-Ad,表明SNPs增加了药物对癌细胞的杀伤作用。
实施例3:将荧光染料罗丹明B负载到实施例1制得的SNPs、β-CD/MTX-SS-Ad形成的纳米粒中,利用CLSM研究SNPs在三阴性乳腺癌细胞(MDA-MB-231)、宫颈癌细胞(Hela)、正常肝细胞(LO2)中的细胞摄取行为1、将SNPs与过量罗丹明B混合超声30min,然后置于300Da透析袋中,在去离子水中透析,期间多次换水,通过紫外光谱对袋外溶液监测,直至检测不到罗丹明B时停止透析,此时透析袋内即为负载罗丹明B的SNPs;
β-CD/MTX-SS-Ad形成的纳米粒负载罗丹明B方法同上,区别在于将SNPs中的L-CD换成等量的β-CD即可;
2、将上述细胞在35mm培养皿中孵育24h,然后分别加入负载罗丹明B的SNPs、负载罗丹明B的β-CD/MTX-SS-Ad形成的纳米粒、罗丹明B,再培养0.5h,然后用PBS洗涤细胞三次,用共聚焦显微镜60×放大倍率观察样品;
结果见图10-12,从图中可以看出在两种癌细胞中,负载罗丹明B的SNPs(A)在两种癌细胞中的摄取效果良好,体现为荧光较强的细胞成像,而负载罗丹明B的β-CD/MTX-SS-Ad形成的纳米粒(B)和游离罗丹明B(C)效果次之,体现为荧光较弱的细胞成像;而在正常细胞中不存在这种明显差异,这说明SNPs对肿瘤细胞具有较好的靶向性和选择性,可以优先被肿瘤细胞内化并蓄积。
实施例4:SNPs在分子水平上对CAIX靶向作用实验
1、将冻存的Hela细胞接种在25T培养瓶中,6-7h细胞贴壁后换液,传代培养至第三代细胞活力旺盛时,在培养细胞中加入40μM的MTX-SS-Ad(样品A)、β-CD/MTX-SS-Ad形成的纳米粒(样品B)、SNPs(样品C),再孵育24h;同时以等浓度的顺铂、乙酰唑胺作为对照;
2、细胞的蛋白提取
孵育后的细胞弃上层培养基,用冰的PBS清洗细胞两次,胰酶消化至细胞变圆后加DMEM培养基(含10%胎牛血清)终止消化,离心,弃上清液,并用冰PBS吹洗细胞一遍,离心,弃上清液,加入150μL的RIPA细胞裂解液于冰上裂解细胞30min,后离心,收集上清液于0.5mL冰上的EP管中备用。
BCA蛋白浓度检测:使用BCA蛋白定量试剂盒配制0.5mg/mL的蛋白标准品及BCA工作液,后将蛋白标准品按照不同浓度加入到96孔板中,再用蛋白标准品补足至20μL,再吸取每个样品20μL于96孔板中,然后每孔各加BCA工作液200μL,于60℃水浴锅中放置30min,用酶标仪测定562nm处的吸光度,根据蛋白标准品测出的标准曲线计算样品蛋白浓度及样品体积,最终用上样缓冲液将蛋白稀释至同一浓度;
3、将玻璃板对齐后放入卡槽中夹紧,准备灌胶,分别配制上下层分离胶与浓缩胶,将蛋白样品及marker加入各个凝胶泳道,设置电泳条件为80V,60mA,30min;后电泳条件为120V,80mA电泳至底部,转膜液提前配制好放-4℃冰箱冻存,PVDF膜先置于甲醇中激活几秒,转膜时按照“三明治”方法进行,黑胶白膜,按照转膜夹黑面-网格垫-滤纸-胶-PVDF膜(0.22μm)-滤纸-网格垫-转膜夹白面顺序进行,冰浴转膜条件为110V,200mA,2h,转膜结束后,将膜置于丽春红染色液中染色,观察是否有蛋白条带,后用TBST将膜洗至接近无色,再将膜用TBST配制的5%脱脂牛奶封闭2h,后加入一抗CAIX稀释液于4℃冰箱中过夜(12h),TBST清洗膜3次,每次5min,加入二抗稀释液于冰上振摇1h,TBST清洗膜3次,每次5min,配制ECL化学发光显影仪(现配现用)显影;后TBST清洗膜3次,每次5min,加入内参β-actin稀释液于冰上振摇1h,TBST清洗膜3次,每次5min,加入二抗稀释液于冰上振摇1h,TBST清洗膜3次,每次5min,显影,得到条带。
结果见图13,从图中可以看到,经阳性对照(乙酰唑胺)处理后的细胞中CAIX表达量明显低于空白对照组、顺铂对照组,经SNPs(样品C)处理后的细胞中CAIX表达水平也存在降低,而经β-CD/MTX-SS-Ad、MTX-SS-Ad处理后细胞中CAIX表达并无显著降低,以上结果证明了SNPs在分子水平上对CAIX具有明显的靶向作用。
Claims (7)
1.一种双功能超分子纳米组装体,其特征在于:将苯磺酰胺偶联的环糊精、金刚烷胺/二硫键共同修饰的抗肿瘤药物置于在水中,进行自组装获得双功能超分子纳米组装体。
2.根据权利要求1所述的双功能超分子纳米组装体,其特征在于,苯磺酰胺偶联的环糊精的化学结构式为:
其中,m为1、2或3;n为1、2或3。
3.根据权利要求1所述的双功能超分子纳米组装体,其特征在于:苯磺酰胺偶联的环糊精是将羧酸化磺胺溶于有机溶剂,然后加入缩合剂在20~40℃下搅拌反应,待反应完全后将反应液缓慢滴入冷水中产生白色沉淀,分离沉淀物,经干燥后再次溶于碱性有机溶剂中,加入氨基环糊精在20~40℃下搅拌反应,然后将混合物滴入丙酮中重结晶,收集晶体粗产物用水溶解后,用去离子水透析纯化制得;
其中,m为1、2或3;n为1、2或3。
4.根据权利要求3所述的双功能超分子纳米组装体,其特征在于:羧酸化磺胺与氨基环糊精的摩尔比为1:0.5~1。
5.根据权利要求3所述的双功能超分子纳米组装体,其特征在于:碱性有机溶剂是添加了碳酸钾、三乙胺或乙醇钠的有机溶剂,有机溶剂包括但不限于丙酮、四氢呋喃、醋酸酐、乙醇、甲醇、N,N-二甲基甲酰胺、二甲基亚砜中,碳酸钾、三乙胺或乙醇钠添加量为羧酸化磺胺摩尔量的1~5倍。
6.根据权利要求1所述的双功能超分子纳米组装体,其特征在于,金刚烷胺/二硫键共同修饰的抗癌药物的化学结构式为:
其中A为疏水性抗癌药物。
7.权利要求1-6中任一项所述的双功能超分子纳米组装体在制备治疗肿瘤药物中的应用。
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