CN109485748A - 一类苄泽修饰壳聚糖嫁接物及其制备方法、应用 - Google Patents
一类苄泽修饰壳聚糖嫁接物及其制备方法、应用 Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
- C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
- C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- 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
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
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Abstract
本发明提供苄泽修饰壳聚糖嫁接物,通过两步反应以丁二酸将苄泽末端羟基与壳聚糖中氨基发生化学键合,生成苄泽修饰壳聚糖嫁接物。苄泽修饰壳聚糖嫁接物通过自聚集方法和静电吸引与负电性多聚磷酸钠复合形成纳米递释系统。苄泽的修饰使得此嫁接物具有P糖蛋白外排的抑制作用,提高小肠上皮细胞的摄取,以增加药物的吸收,从而提高药物生物利用度提高疗效。该纳米递送系统具有显著的抑制P糖蛋白底物外排能力,可在口服药物中应用。
Description
技术领域
本发明属于制药领域,涉及苄泽与壳聚糖嫁接物的化学键合,以及所合成的苄泽修饰壳聚糖嫁接物在口服药物治疗中的应用。
背景技术
药物通过胃肠道上皮细胞进入血循环的跨膜过程主要通过以下三种机制:被动转运,载体介导转运和膜动转运,近年来研究胃肠道上皮细胞存在的P糖蛋白(P-glycoprotein,Pgp,MDR1)具有将药物从上皮细胞基底侧转运到顶侧的外排作用。这种药物泵出机制大大减少药物吸收。P糖蛋白成为大部分药物吸收的屏障,大大降低了药物的口服生物利用度。P糖蛋白其实是一种肿瘤多药耐药蛋白,一种能量依赖转运体,属于ATP结合盒式膜转运体(ATP-bindingcassettetransporter)超家族,由ATP供能将细胞内的药物泵出细胞外。
壳聚糖(Chitosan,CS)是自然界唯一大量存在的高分子碱性氨基多糖,与合成高分子材料相比,具有安全无毒,价廉易得,以及良好的生物相容性和生物可降解性等优点,广泛用于纳米给药系统的研究中。由于壳聚糖在生理pH的条件下难溶于水,限制了其作为在医药领域的应用。如果在其主链上引入合适的疏水功能分子,不但可以弥补其分子链中亲脂性的不足,还可增加特异的新功能,使其有望成为一种特殊的药物载体材料。壳聚糖的主链上的-OH和-NH2反应基团,为其化学修饰提供了可能。
苄泽是FDA批准的药物辅料,广泛应用于医药行业。研究表明苄泽可短期抑制P糖蛋白外排功能,不影响其长期的蛋白表达。此特点可作为非药理活性P糖蛋白抑制剂联合药物一同服用,从而抑制药物被P糖蛋白外排,提高药物吸收,最终提高药物口服生物利用度。因此,将苄泽引入壳聚糖分子主链不但可改善壳聚糖的疏水性,得到一种两亲性的苄泽壳聚糖接枝物。还可使其具有抑制P糖蛋白外排活性,提要药物口服生物利用度。
发明内容
本发明提供苄泽修饰壳聚糖嫁接物,其具有代表性的结构通式为:
式中m为苄泽的化学修饰比例,范围为0%~55%,n为壳聚糖乙酰基的比例,范围为20%~30%,一般药用级别的壳聚糖脱乙酰度为70~80,即n=20~30%。苄泽修饰位点在壳聚糖C2位置的氨基,其范围在0~55%。剩余部分是未被修饰的氨基,三者相加为100%。
本发明提供苄泽修饰壳聚糖嫁接物的制备方法:取苄泽1mmol溶于无水二氯甲烷中,滴加到丁二酸酐1mmol,4-二甲基氨基吡啶1mmol的无水二氯甲烷溶液中,无水氮气保护下反应24h。所得反应物与10%盐酸通过液液萃取除杂后减压蒸馏干燥备用。干燥后产物与N-(3-二甲基氨基丙基)-N′-乙基碳二亚胺盐酸盐,1-羟基苯并三唑按照摩尔比1:1.2:1.2在无水二氯甲烷中反应24h,加入到1%壳聚糖醋酸溶液中(pH 4)继续反应24h。将反应液置于透析袋中透析72小时,透析液冷冻干燥,得苄泽修饰壳聚糖嫁接物。合成路线如下:
所述的苄泽修饰壳聚糖嫁接物的制备方法,其特征在于,苄泽和壳聚糖化学反应的投料的摩尔比1:1-1:10。
所述的苄泽修饰壳聚糖嫁接物在制备P糖蛋白底物药物中的应用,进一步来说具体针对P糖蛋白底物药物。苄泽修饰壳聚糖嫁接物通过自聚集与静电吸引和负电性多聚磷酸钠复合形成纳米递释系统,包载P糖蛋白底物药物,提高其在胃肠道吸收从而提到口服生物利用度。
所述的应用,其特征在于:所述的药物选自疏水性P糖蛋白底物。
所述的苄泽修饰壳聚糖嫁接物在制备纳米混悬剂中的应用。
所述的应用,其特征在于:所述的纳米混悬剂通过如下方法制备:将苄泽修饰壳聚糖嫁接物的醋酸水溶液与药物粉末或该药物的有机溶液混合,冰浴探头超声处理,随后滴加三聚磷酸钠溶液搅拌得到粒径为50-500nm的纳米混悬剂。
所述的应用,其特征在于:苄泽修饰壳聚糖嫁接物的醋酸水溶液浓度为0.1-10mg/mL,,醋酸浓度0.1-2%,三聚磷酸钠溶液浓度为0.1-5mg/mL。
所述的应用,其特征在于:所述的有机溶剂为为甲醇、乙醇、丙酮;有机溶液中药物浓度为1-200mg/mL;所述的纳米混悬剂为液体状混悬剂或干纳米混悬剂;干纳米混悬剂为喷雾干燥或冷冻干燥的液体混悬剂,可加入或不加入冻干保护剂;所述的冻干保护剂为蔗糖、果糖、乳糖、葡萄糖、海藻糖、甘露醇中的一种或任意几种的混合物。
有益效果:本发明首次合成了一系列不同嫁接度的苄泽修饰壳聚糖嫁接物,并对其进行了表征和报道。结果表明,苄泽对壳聚糖进行功能改性后,其理化性能显著提高。苄泽修饰壳聚糖嫁接物在水溶液中具有良好的水溶性。此外,苄泽修饰壳聚糖嫁接物在抑制P糖蛋白高表达的MDCK-MDR1细胞中的药物外排方面显示出巨大的潜力。总之,苄泽修饰壳聚糖嫁接物可以进一步发展为有前途的多功能聚合物纳米载体,用于递送水溶性差和P糖蛋白介导的外排的药物。
附图说明
图1.苄泽修饰壳聚糖嫁接物的谱图,其中A核磁共振氢谱图为B为傅里叶红外光谱图;
图2.苄泽修饰壳聚糖嫁接物对P糖蛋白外排抑制作用。*p<0.05,**p<0.01,相对于模型对照组有极显著性差异;
图3.苄泽修饰壳聚糖嫁接物纳米混悬液投射电镜图;
图4.苄泽修饰壳聚糖嫁接物溶解度图
图5苄泽修饰壳聚糖嫁接物X射线衍射图;
上述图中BS20/Brij-S20为苄泽S20;CS为壳聚糖,BC3-BC55为苄泽取代度为3-55的壳聚糖嫁接物,PM为物理混合物。
具体实施方式
苄泽S20购自SigmaAldrich。
实施例1:本发明苄泽修饰壳聚糖嫁接物的制备
取苄泽S20(BS20)(1mmol)溶于10mL无水二氯甲烷中,滴加到丁二酸酐(1mmol),4-二甲基氨基吡啶(1mmol)的10mL无水二氯甲烷溶液中,无水氮气保护下反应24h。所得反应物与10%盐酸通过液液萃取除杂后减压蒸馏干燥备用。干燥后产物与N-(3-二甲基氨基丙基)-N′-乙基碳二亚胺盐酸盐,1-羟基苯并三唑按照摩尔比1:1.2:1.2在无水二氯甲烷中反应24h,加入到1%壳聚糖(Mw 200,000,DD 75%)醋酸溶液中(pH 4)继续反应24h。将反应液置于透析袋中透析72小时,透析液冷冻干燥,得苄泽修饰壳聚糖嫁接物;经测定,其接枝率(氨基取代度)为2%-55%(氨基取代度测定参考Guo Y,Chu M,Tan S,Zhao S,Liu H,Otieno BO,Yang X,Xu C,Zhang Z.Chitosan-g-TPGS nanoparticles for anticancerdrug delivery and overcoming multidrug resistance.Mol Pharm.2014Jan 6;11(1):59-70)。
实施例2:本发明苄泽修饰壳聚糖嫁接物的结构表征
(1)核磁共振氢谱(见图1A)
苄泽-S20在3.64ppm处的特征峰在苄泽-S20和苄泽-S20SA的光谱中都能观察到。新出现的信号在2.65pp归属于琥珀酰基亚甲基的BS20-SA,这证实了成功的酰化BS20-SA。第二步合成了Brij-S20接枝率不同苄泽修饰壳聚糖嫁接物。所获得的BC55共聚物在1.05ppm的光谱上显示了BS20的长链烷基的甲基质子和4.65个ppm的壳聚糖(C1)的异位碳质子的信号的组合。
(2)傅里叶红外光谱(见图1B)
在2850,1100和1060cm-1处出现的Brij-S20的特征吸收峰分别归属于-CH2、C-O-C和C-O的伸缩振动。壳聚糖的峰在1661和1590cm-1处,对应于酰胺I(νC=O)和酰胺II(δ-N-H)。在BC55,观察到在1090和1045cm-1处的新吸收带,这可以归属于Brij-S20的C-O-C和C-O的拉伸振动。此外,与壳聚糖相比,在BC55中,在1565cm-1处N-H的剪切振动更强的吸收和-CH2在1410cm-1处的弯曲振动,证实了酰胺化的发生;物理混合物的光谱表现Brij-S20和壳聚糖的简单重叠。
实施例3:本发明苄泽修饰壳聚糖嫁接物纳米混悬液制备
以苄泽修饰壳聚糖嫁接物为载体材料,制备药物的纳米混悬剂:将苄泽修饰壳聚糖嫁接物以0.1-5mg/mL的浓度范围分散于0.1-2%醋酸水中,与药物的有机溶液混合,选自甲醇、乙醇、丙酮,有机溶液的药物浓度为1-200mg/mL,冰浴下探头超声处理,滴加0.1-5mg/mL的三聚磷酸钠溶液搅拌得到粒径为50-500nm的纳米混悬剂,见图3;
冷冻干燥:加入或不加入冻干保护剂,将纳米混悬剂冷冻干燥,制得质量稳定的纳米混悬剂冻干品。所得的冻干品经适当稀释后可迅速重建,粒径略有增加。其中冻干保护剂优选蔗糖、果糖、乳糖、葡萄糖、海藻糖、甘露醇中的一种或任意几种的混合物;纳米混悬剂在4℃下,一周内粒径保持稳定,冻干产物水复溶后粒径略微增加。所示所制得的冻干品具有良好的长期稳定性,且有利于药物长期储存和运输。
实施例4抑制P糖蛋白外排能力
罗丹明-123(Rho123)作为P糖蛋白的底物,由于自身荧光而广泛应用于P糖蛋白外排研究中。维拉帕米(Verapamil,Ver)是FDA推荐的P糖蛋白抑制剂,增加P糖蛋白底物药物的摄取。在图2中,维拉帕米作为阳性对照,将MDCK-MDR1细胞内Rho123摄取增加了2.7倍。此外,Brij-S20也增加了细胞摄取1.8倍,而壳聚糖和低接枝度BC3嫁接物对Rho123的细胞摄取没有任何增加。随着接枝度的增加,BC嫁接物显著抑制了P糖蛋白外排,提高了Rho123的细胞摄取量,尤其是BC55嫁接物(3.0倍)效果最强。这些结果表明苄泽修饰壳聚糖嫁接物具有抑制P糖蛋白外排能力。同时用BC55制备的罗丹明-123纳米混悬液同样具有抑制P糖蛋白外排,提高罗丹明-123摄取作用。
实施例5化学修饰后壳聚糖溶解度提高
如图4所示,壳聚糖在水中溶解度差,因其结构中具有大量氨基和羟基基团,容易形成分子内和分子间氢键,具有很强的结晶性。化学修饰过程中破坏了壳聚糖的氢键,使得晶型变弱或形成无定型晶型,因此修饰后的壳聚糖在水中溶解度大大增加。
Claims (10)
1.苄泽修饰壳聚糖嫁接物,其结构通式为:
式中m为苄泽的化学修饰比例,范围为0%~55%,n为壳聚糖乙酰基的比例,范围为20%~30%。
2.根据权利要求1所述的苄泽修饰壳聚糖嫁接物,其特征在于,所述的的苄泽修饰壳聚糖嫁接物重均分子量在于200,000-600,000。
3.一种如权利要求1或2所述的苄泽修饰壳聚糖嫁接物的制备方法,其特征在于,按以下步骤实现:
取苄泽1mmol溶于无水二氯甲烷中,滴加到丁二酸酐1mmol,4-二甲基氨基吡啶1mmol的无水二氯甲烷溶液中,无水氮气保护下反应24h;所得反应物与体积百分比为10%的盐酸通过液液萃取除杂后减压蒸馏干燥备用;干燥后产物与N-(3-二甲基氨基丙基)-N′-乙基碳二亚胺盐酸盐,1-羟基苯并三唑按照摩尔比1:1.2:1.2在无水二氯甲烷中反应24h,加入到1%壳聚糖醋酸溶液中继续反应24h,所述的1%壳聚糖醋酸溶液pH4;将反应液置于透析袋中透析72小时,透析液冷冻干燥,得苄泽修饰壳聚糖嫁接物。
4.根据权利要求3所述的苄泽修饰壳聚糖嫁接物的制备方法,其特征在于,苄泽和壳聚糖化学反应的投料的摩尔比1:1-1:10。
5.根据权利要求1所述的苄泽修饰壳聚糖嫁接物在制备P糖蛋白底物药物中的应用。
6.根据权利要求5所述的应用,其特征在于:所述的的药物选自疏水性P糖蛋白底物。
7.权利要求1所述的苄泽修饰壳聚糖嫁接物在制备纳米混悬剂中的应用。
8.根据权利要求7所述的应用,其特征在于:所述的纳米混悬剂通过如下方法制备:将苄泽修饰壳聚糖嫁接物的醋酸水溶液与药物粉末或该药物的有机溶液混合,冰浴探头超声处理,随后滴加三聚磷酸钠溶液搅拌得到粒径为50-500nm的纳米混悬剂。
9.根据权利要求7所述的应用,其特征在于:苄泽修饰壳聚糖嫁接物的醋酸水溶液浓度为0.1-10mg/mL,,醋酸浓度0.1-2%,三聚磷酸钠溶液浓度为0.1-5mg/mL。
10.根据权利要求7所述的应用,其特征在于:所述的有机溶剂为为甲醇、乙醇、丙酮;药物的有机溶液的药物浓度为1-200mg/mL;所述的纳米混悬剂为液体状混悬剂或干纳米混悬剂;干纳米混悬剂为液体纳米混悬剂经喷雾干燥或冷冻干燥得到,可加入或不加入冻干保护剂;所述的冻干保护剂为蔗糖、果糖、乳糖、葡萄糖、海藻糖、甘露醇中的一种或任意几种的混合物。
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