CN111317715A - 一种高尔基体靶向的粘多糖纳米胶束及其制备方法与应用 - Google Patents
一种高尔基体靶向的粘多糖纳米胶束及其制备方法与应用 Download PDFInfo
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Abstract
本发明涉及一种高尔基体靶向的粘多糖纳米胶束及其制备方法,此纳米胶束以粘多糖作为亲水端,通过酯键的方式连接疏水羧酸类物质。该胶束作为药物载体,可携载高尔基体干扰剂传递到高尔基体。此外,该粘多糖纳米胶束具有生物相容性好、肿瘤细胞特异性靶向、肿瘤微环境内酸敏感释药等优点,具有广泛的应用前景。
Description
技术领域
本发明涉及粘多糖纳米胶束及其制备,具体涉及一种高尔基体靶向的粘多糖纳米胶束及其制备方法与应用。
背景技术
粘多糖(mucopolysaccharide,MPSH)是含氮的不均一多糖,是构成细胞间***的主要成分,也广泛存在于哺乳动物各种细胞内,是生物体的重要结构、能源和功能分子。重要的粘多糖有:硫酸类肝素,硫酸软骨素和透明质酸等。由于具有无毒无免疫原性、较好的生物相容性、生物可降解及易于修饰等特性,常应用于纳米给药***的构建中。
高尔基体(Golgi apparatus,Golgi complex)是真核细胞的细胞器之一,是由一些扁平囊形成的类似于扁盘的堆叠结构。高尔基体的主要功能是将内质网合成的多种蛋白质进行再加工、分类与包装,然后将其运送到细胞特定的部位或分泌到细胞外。因此,高尔基体是细胞内大分子运输的一个重要的交通枢纽。此外,高尔基体还是细胞内糖类合成的工厂,在细胞生命活动中起重要作用。研究发现多种疾病的发生都与高尔基体有关,比如高尔基体修饰并分泌肿瘤转移相关蛋白,促进肿瘤的转移;高尔基体还是真核生物糖基化修饰的重要场所。修饰异常可导致糖尿病、心血管疾病、肿瘤和阿尔茨海默病等多种疾病的发生。因此许多研究将高尔基体作为疾病治疗诊断的靶点。专利文献US5516921A提出了一种具有高尔基体干扰作用的化合物brefeldin A衍生物,该化合物可抑制高尔基体蛋白运输,具有抗炎抑瘤作用。专利文献WO2013151161A1提出了一种八氢萘化合物,该化合物通过抑制高尔基体功能,从而发挥抑瘤作用。专利文献WO200228387A1提出了一种新生血管抑制剂,该制剂由高尔基体干扰剂和醇类等有机溶剂组成,用于肿瘤治疗。这些干扰剂虽然具有一定的抑瘤生长及抗转移效果,但均缺乏高尔基体靶向性,在干扰高尔基体的同时,也会作用到其他细胞器,导致副作用的产生。除此之外,部分高尔基体干扰剂还存在溶解度低、稳定性差等问题,限制了此类药物的临床应用和研究。有文献报道(Xue F, Wen Y, Wei P,et al. A smart drug: a pH-responsive photothermal ablation agent for Golgiapparatus activated cancer therapy[J]. Chemical Communications, 2017, 53(48):6424.)利用非对称菁染料和牛血清白蛋白自主装成纳米粒,靶向高尔基体,作为高尔基体诊断探针。但是非对称菁染料合成操作复杂,产率低,且生物相容性有待进一步验证,所以该载体未能得到广泛的应用。
因此,开发一种生物相容性好,且可以广泛应用到治疗领域的高尔基体靶向载体具有很好的研究价值。
发明内容
本发明人在前期研究中发现,粘多糖与疏水羧酸类物质以酯键的方式连接,在水中自组装形成胶束,该胶束具有较强的高尔基体靶向性。
本发明提供了一种高尔基体靶向的粘多糖纳米胶束及其制备方法,此纳米胶束利用粘多糖良好的水溶性、生物相容性及易修饰性等优点,与疏水羧酸类物质以酯键的方式连接,由于含有亲水性粘多糖部分和疏水羧酸部分,故在水中可自组装形成纳米胶束。
该胶束可作为药物载体,可携载药物传递到高尔基体。进而该胶束可以作为高尔基体靶向载体,广泛应用到治疗领域。
本发明所提供的技术方案为:
一种高尔基体靶向的粘多糖纳米胶束,其特征在于,包括:粘多糖及疏水羧酸类物质。
所述粘多糖,选自透明质酸、软骨素、硫酸软骨素、低分子量肝素、脱硫酸化肝素中的一种或多种混合,其分子量为4kDa~1000kDa,优选为5kDa~300kDa。
进一步,所述的粘多糖优选为透明质酸或硫酸软骨素。透明质酸(hyaluronicacid,HA)和硫酸软骨素是(chondroitin sulfate,CS)是天然的线性粘多糖,广泛分布在哺乳动物骨髓细胞外基质和疏松***中,具有诸多优点,如水溶性和生物相容性良好、无免疫抗原性、无致炎性等。另外,透明质酸和硫酸软骨素的特异性受体CD44,在多种恶性肿瘤细胞表面均过度表达,因此可通过特异性受体介导的细胞内吞作用,选择性地进入肿瘤细胞,提高其在体内的靶向性,减少药物在正常组织的蓄积。
所述疏水羧酸类物质,碳原子数选自10~50,选用的疏水基团物质的碳原子数与所形成的纳米胶束的粒径相关,因此选择适合的碳原子数,可以控制胶束的粒径。进一步优选为胆酸、脱氧胆酸或硬脂酸。
所述疏水羧酸类物质还可选自羧酸类药物,优选为维甲酸、冈田酸。羧酸类药物可以和粘多糖形成前药胶束,具有药理活性,实现高尔基体靶向。
所述的粘多糖纳米胶束,其粒径优选为100 nm~200 nm。
一种高尔基体靶向的粘多糖纳米胶束的制备方法,包括以下步骤:
(1)将疏水羧酸类物质溶解于有机溶剂中,以1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDCl)和4-二甲氨基吡啶(DMAP)为活化剂,0℃活化30 min,得到活化的疏水羧酸类物质。
(2)将粘多糖溶于甲酰胺中,按粘多糖与活化疏水羧酸类物质的摩尔比例为0.05~0.5:1,将粘多糖缓慢滴入活化的疏水羧酸类物质,室温反应至完全。反应结束后,加入丙酮沉淀产物,抽滤得沉淀;加水复溶沉淀,透析,冷冻干燥,即得最终产物粘多糖纳米胶束。
所述合成路线图解如下:
在上述粘多糖纳米胶束的制备方法中:步骤(1)所述的有机溶剂,选自N,N-二甲基甲酰胺、二甲基亚砜或四氢呋喃,优选为N,N-二甲基甲酰胺。
步骤(2)所述的粘多糖与活化的疏水羧酸类物质的摩尔比优选为0.1~0.25:1。
本发明还提供一种包载高尔基体干扰剂的纳米制剂,该纳米制剂是以上述粘多糖纳米胶束作为载体,包载高尔基体干扰剂的纳米制剂。其特征在于,所述纳米制剂包括:粘多糖、疏水羧酸类物质及高尔基体干扰剂。
所述的包载高尔基体干扰剂的纳米制剂,其中所述高尔基体干扰剂,选自维甲酸、紫杉醇、布雷菲尔德菌素A、伊马醌、诺可达唑、冈田酸、巴弗洛霉素。
进一步优选,所述的高尔基体干扰剂选自维甲酸或紫杉醇。
所述包载高尔基体干扰剂的纳米制剂的制备方法,包括下列步骤:
将高尔基体干扰剂溶于有机溶剂,按高尔基体干扰剂与粘多糖纳米胶束质量比为0.1~0.8:1,滴入粘多糖纳米胶束的水溶液中,室温磁力搅拌30~150 min,探头超声(功率120w, 30 min)。透析,冷冻干燥,即得包载高尔基体干扰剂的纳米制剂。
所述的有机溶剂,选自N,N-二甲基甲酰胺、二甲基亚砜、四氢呋喃或乙醇。
所述的高尔基体干扰剂与粘多糖纳米胶束的质量比优选为0.125~0.5:1。
所述的磁力搅拌时间优选为60~120 min。
所述的粘多糖纳米制剂,其载药量为2%~24%,优选为5%~22%。
发明益处
(1)本发明的粘多糖纳米胶束,其特异性强、高尔基体靶向效果好,并有很好的安全性和生物相容性。
(2)本发明的粘多糖纳米胶束,其中疏水羧酸类物质可选自羧酸类药物,从而形成前药胶束,具有药理活性,实现高尔基体靶向。
(3)本发明的粘多糖纳米胶束,具有pH响应性,可以在肿瘤细胞酸性微环境中释药,可用于肿瘤细胞递药。
(4)本发明的粘多糖纳米胶束,可通过特异性受体CD44靶向多种恶性肿瘤细胞,具有靶向治疗载体的潜力。
附图说明
图1 CS-RA氢谱结果
图2 胶束TEM表征
图3 胶束体外释放结果
图4 高尔基体药物含量测定
图5 胶束对高尔基体形态的作用
图6胶束的细胞摄取
图7胶束对肿瘤细胞的迁移抑制作用
图8胶束对肿瘤细胞的侵袭抑制作用
图9胶束对细胞血管生成的抑制作用。
具体实施方式
实施例1
合成硫酸软骨素-维甲酸纳米胶束(CS-RA)。称取0.33 mol RA加入10 mL DMF,避光搅拌至完全溶解。加入2eq EDCl和0.2eq DMAP,0℃活化30 min。称取0.05 mol CS溶于2.5 mL甲酰胺,缓慢滴入RA溶液中,室温搅拌反应两天。反应结束后,将得到的溶液倒入大量的丙酮中沉淀,用 G5 垂熔玻璃滤器抽滤取沉淀。将沉淀用去离子水分散,透析袋中透析两天,冷冻干燥,即得最终产物CS-RA。用激光粒度仪测定CS-RA粒径约为169 nm。
实施例2
合成透明质酸-维甲酸纳米胶束(HA-RA)。称取0.33 mol RA加入10 mL DMF,避光搅拌至完全溶解。加入2eq EDCl和0.2eq DMAP,0℃活化30 min。称取0.08 mol HA溶于2.5 mL甲酰胺,缓慢滴入RA溶液中,室温搅拌反应两天。反应结束后,将得到的溶液倒入大量的丙酮中沉淀,用 G5 垂熔玻璃滤器抽滤取沉淀。将沉淀用去离子水分散,透析袋中透析两天,冷冻干燥,即得最终产物HA-RA。用激光粒度仪测定HA-RA粒径约为183 nm。
实施例3
合成硫酸软骨素-脱氧胆酸纳米胶束(CS-DOCA)。称取0.4 mol DOCA加入10 mL DMF,避光搅拌至完全溶解。加入2eq EDCl和0.2eq DMAP,0℃活化30 min。称取0.05 mol CS溶于2.5 mL甲酰胺,缓慢滴入DOCA溶液中,室温搅拌反应两天。反应结束后,将得到的溶液倒入大量的丙酮中沉淀,用 G5 垂熔玻璃滤器抽滤取沉淀。将沉淀用去离子水分散,透析袋中透析两天,冷冻干燥,即得最终产物CS-DOCA。用激光粒度仪测定CS-DOCA粒径约为181 nm。
实施例4
合成透明质酸-脱氧胆酸纳米胶束(HA-DOCA)。称取0.4 mol DOCA加入10 mL DMF,避光搅拌至完全溶解。加入2eq EDCl和0.2eq DMAP,0℃活化30 min。称取0.09 mol HA溶于2.5mL甲酰胺,缓慢滴入DOCA溶液中,室温搅拌反应两天。反应结束后,将得到的溶液倒入大量的丙酮中沉淀,用 G5 垂熔玻璃滤器抽滤取沉淀。将沉淀用去离子水分散,透析袋中透析两天,冷冻干燥,即得最终产物HA-DOCA。用激光粒度仪测定HA-DOCA粒径约为179 nm。
实施例5
取18 mg 实施例1制备的CS-RA,溶解于5 mL去离子水中,搅拌下缓慢逐滴加入300 μL紫杉醇(PTX)的乙醇溶液(20 mg/mL),室温下搅拌100 min,冰浴探头超声30 min(功率120W,工作3 s,间歇3 s),随后将溶液转移到透析袋内,以去离子水透析12 h,3000 rpm离心10min,取上清液过0.45 μm微孔滤膜,冷冻干燥,得到载紫杉醇的纳米制剂(PTX-CS-RA)。
实施例6
取18 mg 实施例4制备的HA-DOCA,溶解于5 mL去离子水中,搅拌下缓慢逐滴加入300 μL 维甲酸(RA)的二甲亚砜溶液(10 mg/mL),室温下搅拌80 min,冰浴探头超声30 min(功率120 W,工作3 s,间歇3 s),随后将溶液转移到透析袋内,以去离子水透析12 h,3000 rpm离心10 min,取上清液过0.45 μm微孔滤膜,冷冻干燥,得到载维甲酸的纳米制剂(RA-HA-DOCA)。
实施例7
取18 mg 实施例3制备的CS-DOCA,溶解于5 mL去离子水中,搅拌下缓慢逐滴加入200 μL PTX的乙醇溶液(20 mg/mL),室温下搅拌90 min,冰浴探头超声30 min(功率120 W,工作3s,间歇3 s),随后将溶液转移到透析袋内,以去离子水透析12 h,3000 rpm离心10 min,取上清液过0.45 μm微孔滤膜,冷冻干燥,得到载紫杉醇的纳米制剂(PTX-CS-DOCA)。
实验例1 CS-RA的结构确认
将实施例1制备的CS-RA冻干后,溶于氘代水进行核磁共振分析,参见图1。
图1为CS-RA的氢谱结果。可以看出,CS的特征峰出现在了1.80ppm(-COCH3-)。而RA在6.20-6.80ppm出现了特征峰(=CH-),表明RA耦合到了CS上,从而成功制备了CS-RA。
实验例2 胶束TEM表征
将实施例1制备的CS-RA和实施例5制备的PTX-CS-RA 纳米溶液,用 1%磷钨酸负染,滴至专用铜网上,自然挥干后用透射电子显微镜(TEM)观察脂质体形态并拍摄照片。
图2为TEM表征结果,A为CS-RA电镜结果,可观察到纳米胶束呈类球状,粒径约在150 nm左右。B为PTX-CS-RA的电镜结果,粒径约在170 nm左右。
实验例3 胶束的理化性质
将实施例5的PTX-CS-RA、实施例6的RA-HA-DOCA、实施例7的PTX-CS-DOCA胶束混悬液稀释后,用激光粒度仪测定粒径和Zeta电位;精密称取一定量的胶束纳米粒冻干粉,加去离子水配成浓度为3 mg/mL的溶液,30°C下搅拌30 min,过0.45 μm微孔滤膜过滤,用流动相稀释至适宜浓度,HPLC测定RA及PTX浓度。如表1所示。
表1.胶束的理化表征
实验例4 胶束的体外释放
使用透析法来研究药物从胶束中的释放行为。称取实施例5的PTX-CS-RA胶束,制备终浓度为2.5 mg/mL的纳米粒溶液,取1.0 mL至截留分子量为3500 Da的透析袋中。以50 mL磷酸盐缓冲液(pH分别为 7.4、6.0、5.0,含有0.2%吐温80)为释放介质,置于37°C,振荡速度为100 rpm的恒温振荡摇床中。每隔一定时间取样1 mL,并立即补以相同体积的释放介质。测定PTX及RA的累积释放量。释放的药物含量通过HPLC法进行测定,按照公式计算药物的累积释放率,参见图3。
图3(a)和(b)分别为胶束中PTX和RA的累计释放量,随透析介质pH的降低,PTX和RA的释放速度均加快。以上结果说明PTX-CS-RA在酸性环境中更易解聚释放药物,且随酸性加强,药物释放量增多,该胶束具有酸环境响应性。
实验例5高尔基体药物含量测定
发明人通过测定高尔基体的药物含量,验证实施例5的PTX-CS-RA胶束的高尔基体靶向性。4T1细胞以1×106个/孔接种于6孔板中,37℃培养后,分别加入PTX-RA-CS、RA溶液(RA-sol)、PTX溶液(PTX-sol)继续孵育,于0.5、1、2、4、8、12 h取出,冰冷的PBS洗涤三遍,刮刀收集细胞。高尔基体提取试剂盒提取高尔基体,将提取出的高尔基体重悬于高尔基体保存液中,4℃保存备用。向各组细胞混悬液中加入适量乙酸乙酯,漩涡15 min后,3500 rpm离心10min,取有机层氮气吹干,沉淀用乙腈溶解后,利用LC-MS测定RA与PTX的含量。实验结果参见图4。
在不同时间点PTX-CS-RA组在高尔基体中的药物含量均高于游离药物组,且浓度随孵育时间的延长,有增长的趋势。说明PTX-CS-RA可将药物转运至高尔基体。
实验例6 胶束对高尔基体的形态作用
考察实施例1的CS-RA和实施例5的PTX-CS-RA对高尔基体形态的作用,发明人选取GM130作为高尔基体形态标记物,观察高尔基体的形态变化。
将4T1细胞接种到共聚焦皿中,在5%CO2存在下于37℃培养24小时,然后加入CS-RA、PTX-CS-RA与细胞各孵育4 h、8 h、12 h。设置不加入制剂的组别,作为空白对照组。除去含药介质,用冰冷的PBS洗涤细胞3次。10% formalin室温固定20 min,PBS洗涤细胞,0.1%Triton X-100室温孵育5 min,PBS洗涤,5% BSA封闭30 min。加入5μg/mL Anti-GM130一抗(含1% BSA的PBS溶液),4℃孵育过夜。封闭液洗涤细胞,加入FITC标记的IgG二抗,室温孵育1h。PBS洗涤细胞,细胞核用DAPI(10 mM)染色5分钟。然后用PBS洗涤细胞三次,并用激光扫描共聚焦显微镜观察,参见图5。
空白对照组在各时间点,高尔基体蛋白GM130均呈现集中紧凑的状态。与空白对照组相比,CS-RA、PTX-CS-RA组随孵育时间的延长GM130蛋白均有不同程度的解聚和分散。说明胶束对高尔基体的形态具有破坏作用。
实验例7 胶束的细胞摄取
用亲脂性荧光染料香豆素6(C6)标记CS-RA,对胶束进行示踪,制备方法与本发明实施例5相似,将其处方中的300 μL 紫杉醇(PTX)的乙醇溶液(20 mg/mL)替换为330 μL香豆素6(C6)的DMF溶液(0.2 mg/mL)即可,制备得到C6-CS-RA胶束。
采用流式细胞术对胶束的细胞摄取进行定量分析。取对数生长期的 B16F10、4T1以及L929细胞,0.25%胰酶消化得细胞悬液。细胞接种于12孔细胞培养板中,每孔2 mL,37℃条件下孵育 24 h,细胞贴壁生长。吸弃培养基,PBS洗 2遍,加入用不含血清培养基稀释的游离C6和C6-CS-RA(C6 终浓度为 0.5 μg/mL)。37℃条件下孵育 4 h。吸弃含C6的培养基,PBS洗3遍,0.25%胰酶消化,2000 rpm 离心4 min,吸弃上清液,加入PBS溶液重悬细胞,置于流式细胞仪中测定制剂的摄取量,参见图6。
B16F10及4T1细胞表面过表达CD44,L929细胞不表达CD44。图6结果显示提前用CS溶液饱和细胞表面的CD44受体之后,B16F10及4T1细胞对纳米胶束的摄取量明显降低,而游离C6组的摄取没有受到影响。L929细胞对CS纳米粒和游离C6的摄取没有明显差异。该实验说明纳米胶束的摄取可以通过肿瘤细胞表面CD44受体的介导来实现。
实验例8胶束对肿瘤细胞的迁移抑制作用
采用划痕实验,考察实施例1的CS-RA和实施例5的PTX-CS-RA的肿瘤细胞迁移抑制能力。收集生长状态良好的 B16F10、4T1细胞,接种于 12 孔的细胞培养板中培养 24 h,待细胞生长密度达到80%左右后, 用10 µL移液枪头沿培养孔的直径方向划出一条线,并用 PBS洗涤细胞三次,使划痕处没有细胞,然后加入 900 µL的无血无抗培养基。接着分别给药,加入100 µL PBS、CS-RA、 PTX-CS-RA、RA溶液(RA-sol)、RA和PTX的混合溶液(PTX-RA-sol) 给药浓度为1 µg/mL RA,10 µg/mL PTX。在37 ˚C 温度,含5% CO2 的培养箱中培养24 h后,于显微镜下观察划痕的宽度和细胞迁移情况。参见图7。
细胞划痕实验结果显示,PBS组的细胞向中间划痕区域生长, 即向“伤口”内运动迁移。而经制剂处理后的细胞向伤口内运动的现象减弱。PTX-CS-RA和CS-RA组与响应游离药物组对比,细胞抑制作用最明显。结果说明,胶束能够有效抑制 B16F10和4T1细胞的迁移,且包载PTX后,抑制作用更强。
实验例9胶束对肿瘤细胞的侵袭抑制作用
采用小室模型研究实施例1的CS-RA和实施例5的PTX-CS-RA对肿瘤细胞的侵袭抑制作用。将B16F10和4T1细胞接种于12孔板中,置于37 ˚C,5 % CO2 及饱和湿度条件下培养24h。待细胞生长至密度为80%时,弃去培养基,用无血无抗的培养基轻轻清洗细胞3次,然后分别加入100 µL PBS、CS-RA、 PTX-CS-RA、RA-sol、PTX-RA-sol,给药浓度为1 µg/mL RA,10 µg/mL PTX。给药24 h后,弃去给药溶液,并用无血清培养基洗涤3次。然后用胰酶溶液消化细胞, 将细胞重悬于不含FBS的1640完全培养基中,调整细胞浓度为1×106 cell/mL。取100µL细胞悬液加入到膜孔被 Matrigel 基质胶覆盖的 transwell 小室中, 然后在下室中加入600 µL 含 10% FBS 的1640完全培养基。于37 ˚C下继续孵育24 h后,用棉签轻轻擦除transwell 小室筛网上层未穿过基质胶的非侵袭细胞,然后将小室放在1 mL甲醇中,室温固定细胞20 min,之后放入1 mL 0.005%结晶紫溶液中,室温染色10 min,用PBS轻轻洗涤小室后,将小室置于1 mL 33%醋酸溶液中,浸泡10 min,使紫色结晶充分溶解于醋酸溶液,然后在酶标仪上测定各溶液在 570 nm 波长处的吸收值(OD),该OD值间接反映了侵入下层的细胞数即细胞的侵袭能力。结果参见图8。
图8结果显示,制剂组CS-RA和PTX-CS-RA处理后的细胞与相应游离药物组相比,侵入transwell小室下层的细胞量明显减少。说明胶束能够有效抑制肿瘤细胞的侵袭。
实验例10胶束对细胞血管生成的抑制作用
采用小管生成实验,考察实施例1的CS-RA和实施例5的PTX-CS-RA对血管生成的抑制作用。取对数生长期的HUVEC细胞,0.25%胰酶消化得细胞悬液,分别加入用不含血清培养基稀释的PBS、CS-RA、 PTX-CS-RA、RA-sol、PTX-RA-sol (PTX和RA浓度分别为10 ng/mL和1 ng/mL)中。以3×105个/孔接种在含Matrigel的96孔板中,5% CO2,37°C条件下孵育8 h。电镜下观察小管形成情况,记录每个视野中小管形成数目。结果参见图9。
图9结果显示,与PBS对照组相比,含有PTX或RA制剂组的小管数量均有降低,其中CS-RA和PTX-RA-CS组的抑制作用比游离药物组的更明显。说明胶束具有良好的血管生成抑制作用。
Claims (10)
1.一种高尔基体靶向的粘多糖纳米胶束:其特征在于所述胶束包含粘多糖、疏水羧酸类物质;所述粘多糖,通过酯化反应将其侧链羟基与所述疏水羧酸类物质的羧基相连,制备得到两亲性的粘多糖纳米胶束。
2.根据权利要求1所述的纳米胶束,其特征在于,所述粘多糖,选自透明质酸、软骨素、硫酸软骨素、低分子量肝素、脱硫酸化肝素中的一种,其分子量为4kDa~1000kDa,优选为5kDa~300kDa。
3.根据权利要求1所述的纳米胶束,其特征在于,所述疏水羧酸类物质的碳原子数为10~50;优选为维甲酸、胆酸、脱氧胆酸、硬脂酸中的一种或多种。
4.一种权利要求1-3任一项的纳米胶束的制备方法,其特征在于,将粘多糖溶于甲酰胺,疏水羧酸类物质溶于有机溶剂,按粘多糖与疏水羧酸类物质的摩尔比为0.05~0.5:1,室温反应至完全;所述的有机溶剂选自N,N-二甲基甲酰胺、二甲基亚砜或四氢呋喃,优选为N,N-二甲基甲酰胺。
5.根据权利要求4所述的制备方法,其特征在于,所述的粘多糖与疏水羧酸类物质的摩尔比优选为0.1~0.25:1。
6.一种以粘多糖纳米胶束作为载体,包载高尔基体干扰剂的纳米制剂;其特征在于,包括组分:粘多糖、疏水羧酸类物质及高尔基体干扰剂,所述粘多糖,通过酯化反应将其侧链羟基与所述疏水羧酸类物质的羧基相连,制备得到两亲性的粘多糖纳米胶束。
7.根据权利要求6所述的纳米制剂,其特征在于,所述高尔基体干扰剂,选自维甲酸、紫杉醇、布雷菲尔德菌素A、伊马醌、诺可达唑、冈田酸、巴弗洛霉素,优选地,所述的高尔基体干扰剂选自维甲酸或紫杉醇。
8.根据权利要求7所述的纳米制剂的制备方法,其特征在于,按高尔基体干扰剂与粘多糖纳米胶束质量比为0.1~0.8:1,将高尔基体干扰剂滴入粘多糖纳米胶束的水溶液中,室温磁力搅拌30~150 min,探头超声,透析,冷冻干燥,即得包载高尔基体干扰剂的纳米制剂;所述的高尔基体干扰剂与粘多糖纳米胶束的质量比优选为0.125~0.5:1;所述的磁力搅拌时间优选为60~120 min。
9.一种根据权利要求1-3任一项所述的胶束或权利要求4或5所述的制备方法在制备具有高尔基体靶向的纳米胶束的用途。
10.一种根据权利要求6或7所述的胶束或权利要求7或8所述的制备方法在制备具有高尔基体靶向的纳米胶束的用途。
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