CN110354076A - 一种唾液酸修饰复合纳米给药系统及制备与应用 - Google Patents
一种唾液酸修饰复合纳米给药系统及制备与应用 Download PDFInfo
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- CN110354076A CN110354076A CN201910550667.1A CN201910550667A CN110354076A CN 110354076 A CN110354076 A CN 110354076A CN 201910550667 A CN201910550667 A CN 201910550667A CN 110354076 A CN110354076 A CN 110354076A
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- Prior art keywords
- sialic acid
- chitosan oligosaccharide
- delivery system
- drug delivery
- hydroxyapatite
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Abstract
本发明提供一种唾液酸修饰复合纳米给药系统及制备与应用,在唾液酸修饰的壳寡糖‑脱氧胆酸嫁接物胶束的稳定作用下,通过水热法制备羟基磷灰石纳米粒,提高羟基磷灰石的稳定性。该纳米给药系统通过其表面的唾液酸与关节炎症部位血管内皮细胞表面高表达的E选择素的特异性结合,实现药物的关节炎部位靶向递送和高度累积,在关节炎部位以及细胞内较低pH条件下,通过羟基磷灰石的降解,实现药物的快速释放,显著提高药物对炎症反应的抑制作用和耐受性,降低药物的毒副作用,通过促进软骨细胞合成分泌糖胺多糖,实现软骨修复,促进成骨细胞矿化,协同唾液酸自身的骨质修复能力,强化成骨修复与再生功能,可在制备类风湿关节炎药物中应用。
Description
技术领域
本发明属纳米给药系统的制备,涉及一种唾液酸修饰复合纳米给药系统,具体涉及唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统和制备方法,以及唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统在制备类风湿关节炎药物中的应用。
背景技术
类风湿关节炎是一种以多关节滑膜炎症为主要特征的系统性自身免疫疾病,常伴关节外器官受累及血清类风湿因子阳性,最终导致关节畸形及功能丧失,高发于30-50岁的女性,发病率呈逐年上升的趋势。目前临床上类风湿关节炎的治疗以药物治疗和手术治疗为主,但手术治疗易造成关节腔二次损伤,滑膜的切除使关节失去了合成滑液的基本功能,进一步加剧了关节功能的受损。甲氨蝶呤是临床一线的抗风湿药,具有免疫抑制、抗炎作用,能够减轻疼痛、缓解症状、控制病情发展。但因其在体内的非特异性分布,使得进入炎症关节部位的药物分子数量有限,而在正常组织中大量分布,服用高剂量甲氨蝶呤常产生溃疡性口腔炎、腹泻甚至致命的肠穿孔等毒副作用,严重影响了患者的生活质量,而低剂量的治疗又严重制约类风湿关节炎疗效,且长期小剂量使用仅能延缓骨质侵蚀的进展,而不能阻止长期炎症反应导致的骨质疏松的发生,更不能促进已受损骨质的修复与再生。
纳米给药系统的出现为药物递送方式带来了新的技术革命,它通过尺寸效应和提高生物膜的通透性改变药物的体内分布,可显著提高药物疗效及用药安全系数,在类风湿关节炎治疗中已得到广泛研究。纳米羟基磷灰石作为骨组织工程中广泛应用的生物材料,具有比表面积大、生物粘附性强、可吸附药量大等多重优点。此外,纳米羟基磷灰石能够在酸性微环境中降解,释放钙离子和磷酸根离子,发挥良好的骨诱导性能。然而类风湿关节炎为多关节、小关节起病,因此基于纳米羟基磷灰石的支架不仅难以覆盖整个病变区域,而且在支架植入期间可能进一步损伤关节。此外,长期炎症浸润会造成关节周围成骨骨质的丢失和软骨的侵蚀,单纯的纳米羟基磷灰石对关节功能的修复能力不足。
唾液酸是一类位于细胞膜最外层的九碳糖,可与E选择素特异性结合,且唾液酸化的糖复合物可通过抑制破骨细胞成熟,促进成骨细胞分化,进而促进骨的修复和再生。壳寡糖是以氨基葡萄糖为单体通过β-1,4-糖苷键连接的聚合物,可通过刺激软骨细胞合成具有正常多聚体结构的糖蛋白,抑制胶原酶活性,多途径多通路发挥修复关节软骨,改善关节功能的作用。
发明内容
本发明的第一个目的是提供一种唾液酸修饰复合纳米给药系统,该纳米给药系统由唾液酸-壳寡糖-脱氧胆酸嫁接物胶束、羟基磷灰石和甲氨蝶呤所组成,其质量百分比为:唾液酸-壳寡糖-脱氧胆酸嫁接物14.85%-34.85%,羟基磷灰石64.06%-84.06%,甲氨蝶呤1.07%-3.08%,唾液酸-壳寡糖-脱氧胆酸嫁接物的分子量范围为5700~31000Da,结构式如下:
其中壳寡糖的聚合度n=30~180。
本发明中的甲氨蝶呤释放呈现明显的pH值依赖性,在pH 7.4中性介质中释放缓慢,而在pH 5.5酸性条件下释放加快。
本发明的第二个目的是提供唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统的制备方法,具体通过以下途径实现:
1.唾液酸修饰壳寡糖嫁接物胶束的合成
取脱氧胆酸50-80mg,1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐77mg于圆底烧瓶,加入乙醇50mL超声溶解,在60℃下搅拌0.5h,然后加入到50mL浓度为5mg/mL已60℃预热的壳寡糖(MW 5000-30000Da)水溶液中,于400rpm下继续反应24h。反应后的溶液转移至透析袋(MWCO 7.0kDa),纯水透析48h,收集混悬液,离心(6000rpm,15min),取上清经冷冻干燥得壳寡糖-脱氧胆酸嫁接物混合物。将得到的粉末用乙醇多次洗涤,除去体系中未反应的脱氧胆酸,复溶后经冷冻干燥得壳寡糖-脱氧胆酸粉末。取唾液酸10mg,1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐46mg于圆底烧瓶,加入乙醇40mL超声溶解,在60℃下搅拌0.5h,然后加入到20mL浓度为5mg/mL已60℃预热的壳寡糖-脱氧胆酸水溶液中,于400rpm下继续反应24h。反应后的溶液转移至透析袋(MWCO 7.0kDa)中,纯水透析48h,收集透析袋中液体经冷冻干燥得唾液酸-壳寡糖-脱氧胆酸嫁接物。
2.唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统的制备
取CaCl2溶液(50mM)1mL、唾液酸-壳寡糖-脱氧胆酸嫁接物(5mg/mL)0.5-1mL、MTX溶液(320μg/mL)1-4mL,混合均匀,缓慢滴入磷酸盐水溶液(30mM)1mL、柠檬酸钠水溶液(100mg/mL)100μL。100℃搅拌4h后,转移至7.0kDa透析袋中,纯水透析24h,冻干得唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统。
本发明的第三个目的是提供一种唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统在制备类风湿关节炎药物中的应用。本发明药物显著抑制炎症反应和促进关节成骨及软骨的修复。本发明利用唾液酸修饰壳寡糖/羟基磷灰石的电荷吸附作用,有效负载抗类风湿药物甲氨蝶呤。该纳米给药系统可通过其表面的唾液酸与关节炎症部位血管内皮细胞表面高表达的E选择素的特异性结合,实现药物的关节炎部位靶向递送和高度累积;高效靶向至关节炎部位的纳米给药系统能够在关节炎部位以及细胞内较低pH条件下,通过羟基磷灰石的降解,实现药物的快速释放,从而显著提高药物对炎症反应的抑制作用,降低药物的毒副作用;同时残留于炎症关节部位的载体材料中的壳寡糖可通过促进软骨细胞合成分泌糖胺多糖,实现软骨修复,纳米羟基磷灰石可通过促进成骨细胞矿化,协同唾液酸自身的骨质修复能力,强化成骨修复与再生功能。
本发明通过使用唾液酸修饰壳寡糖嫁接物胶束作为稳定剂,制备唾液酸修饰壳寡糖/羟基磷灰石纳米粒,并通过纳米羟基磷灰石有效吸附抗类风湿药物甲氨蝶呤,制备唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统;该纳米给药系统在唾液酸修饰壳寡糖嫁接物胶束的作用下,可更安全高效地治疗类风湿关节炎,显著降低甲氨蝶呤的毒副作用,提高耐受性,并对受损关节部位的软骨和成骨进行修复,恢复关节功能。
本发明提供的一种唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统,首先在唾液酸修饰的壳寡糖-脱氧胆酸嫁接物胶束的稳定作用下,通过水热法制备羟基磷灰石纳米粒,提高羟基磷灰石的稳定性。利用唾液酸修饰壳寡糖/羟基磷灰石的电荷吸附作用,有效负载抗类风湿药物甲氨蝶呤。该纳米给药系统可通过其表面的唾液酸与关节炎症部位血管内皮细胞表面高表达的E选择素的特异性结合,实现药物的关节炎部位靶向递送和高度累积;高效靶向至关节炎部位的纳米给药系统能够在关节炎部位以及细胞内较低pH条件下,通过羟基磷灰石的降解,实现药物的快速释放,从而显著提高药物对炎症反应的抑制作用,降低药物的毒副作用,提高药物的耐受性;同时残留于关节炎部位的载体材料中的壳寡糖可通过促进软骨细胞合成分泌糖胺多糖,实现软骨修复,纳米羟基磷灰石可通过促进成骨细胞矿化,协同唾液酸自身的骨质修复能力,强化成骨修复与再生功能。
附图说明
图1是唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统的透射电镜观察结果。
图2是唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统的体外药物释放曲线。
图3是唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统的体外抑制软骨细胞凋亡结果。
图4是唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统的体外促成骨细胞酶活力结果。
具体实施方式
本发明结合附图和实施例作进一步的说明。
实施例一唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统的制备
唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统的制备分两步进行,第一步合成唾液酸修饰壳寡糖嫁接物。取脱氧胆酸50mg,1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐77mg于圆底烧瓶,加入乙醇50mL超声溶解,在60℃下搅拌0.5h,然后加入到50mL浓度为5mg/mL已60℃预热的壳寡糖(MW 5000Da)水溶液中,于400rpm下继续反应24h。反应后的溶液转移至透析袋(MWCO 7.0kDa),纯水透析48h,收集混悬液,离心(6000rpm,15min),取上清经冷冻干燥得壳寡糖-脱氧胆酸嫁接物混合物。将得到的粉末用乙醇多次洗涤,除去体系中未反应的脱氧胆酸,复溶后经冷冻干燥得壳寡糖-脱氧胆酸粉末。
取唾液酸10mg,1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐46mg于圆底烧瓶,加入乙醇40mL超声溶解,在60℃下搅拌0.5h,然后加入到20mL浓度为5mg/mL已60℃预热的壳寡糖-脱氧胆酸水溶液中,于400rpm下继续反应24h。反应后的溶液转移至透析袋(MWCO7.0kDa)中,纯水透析48h,收集透析袋中液体经冷冻干燥得唾液酸-壳寡糖-脱氧胆酸嫁接物。
采用核磁共振氢谱对唾液酸-壳寡糖-脱氧胆酸嫁接物的结构进行确证。经计算,唾液酸-壳寡糖-脱氧胆酸嫁接物中唾液酸:壳寡糖:脱氧胆酸的摩尔比为1:1:1。
以芘为荧光探针,采用荧光分光光度法对唾液酸-壳寡糖-脱氧胆酸嫁接物嫁接物在水性介质中的临界胶束浓度进行测定。取唾液酸-壳寡糖-脱氧胆酸嫁接物嫁接物10mg,精密称定,将其分散于10mL蒸馏水中,探头超声20次,功率400w,工作2s停3s,制得1mg/mL的嫁接物水溶液,并将其稀释成不同浓度的嫁接物溶液(0.005-1mg/mL)。然后分别加入一定量的芘,使芘的终浓度为6×10-7mol/L,水浴超声30min后,采用荧光分光光度计扫描芘的激发光谱和发射光谱,分别记录374nm及385nm波长下的荧光强度,以I374/I385比值计算得嫁接物的临界胶束浓度为38.45μg/mL。
第二步制备唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统。取CaCl2溶液(50mM)1mL、唾液酸-壳寡糖-脱氧胆酸嫁接物(5mg/mL)0.5mL、MTX溶液(320μg/mL)1mL,混合均匀,缓慢滴入磷酸盐水溶液(30mM)1mL、柠檬酸钠水溶液(100mg/mL)100μL。100℃搅拌4h后,转移至7.0kDa透析袋中,纯水透析24h,冻干得唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统。
采用高效液相色谱法测定甲氨蝶呤的含量。经测定,唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统中的甲氨蝶呤含量为1.09%。
采用透射电子显微镜对唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统进行表观形貌观察。透射电子显微镜结果显示,纳米给药系统形态呈棒状,且粒径大小约为50nm。用微粒粒度与表面电位测定仪测定纳米给药系统的粒径。经测定,唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统的粒径46.08±7.88nm。
采用强酸降解羟基磷灰石,结合药物含量测定,以重量法测定唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统中唾液酸修饰壳寡糖嫁接物、羟基磷灰石和甲氨蝶呤的含量。经测定,唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统中唾液酸修饰壳寡糖嫁接物、羟基磷灰石和甲氨蝶呤的含量分别为14.85%、84.06%和1.09%。
实施例二唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统的制备
唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统的制备分两步进行,第一步合成唾液酸修饰壳寡糖嫁接物。取脱氧胆酸50mg,1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐77mg于圆底烧瓶,加入乙醇50mL超声溶解,在60℃下搅拌0.5h,然后加入到50mL浓度为5mg/mL已60℃预热的壳寡糖(MW 5000Da)水溶液中,于400rpm下继续反应24h。反应后的溶液转移至透析袋(MWCO 7.0kDa),纯水透析48h,收集混悬液,离心(6000rpm,15min),取上清经冷冻干燥得壳寡糖-脱氧胆酸嫁接物混合物。将得到的粉末用乙醇多次洗涤,除去体系中未反应的脱氧胆酸,复溶后经冷冻干燥得壳寡糖-脱氧胆酸粉末。
取唾液酸10mg,1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐46mg于圆底烧瓶,加入乙醇40mL超声溶解,在60℃下搅拌0.5h,然后加入到20mL浓度为5mg/mL已60℃预热的壳寡糖-脱氧胆酸水溶液中,于400rpm下继续反应24h。反应后的溶液转移至透析袋(MWCO7.0kDa)中,纯水透析48h,收集透析袋中液体经冷冻干燥得唾液酸-壳寡糖-脱氧胆酸嫁接物。
采用核磁共振氢谱对唾液酸-壳寡糖-脱氧胆酸嫁接物的结构进行确证。经计算,唾液酸-壳寡糖-脱氧胆酸嫁接物中唾液酸:壳寡糖:脱氧胆酸的摩尔比为1:1:1。
以芘为荧光探针,采用荧光分光光度法对唾液酸-壳寡糖-脱氧胆酸嫁接物嫁接物在水性介质中的临界胶束浓度进行测定。取唾液酸-壳寡糖-脱氧胆酸嫁接物嫁接物10mg,精密称定,将其分散于10mL蒸馏水中,探头超声20次,功率400w,工作2s停3s,制得1mg/mL的嫁接物水溶液,并将其稀释成不同浓度的嫁接物溶液(0.005-1mg/mL)。然后分别加入一定量的芘,使芘的终浓度为6×10-7mol/L,水浴超声30min后,采用荧光分光光度计扫描芘的激发光谱和发射光谱,分别记录374nm及385nm波长下的荧光强度,以I374/I385比值计算得嫁接物的临界胶束浓度为38.45μg/mL。
第二步制备唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统。取CaCl2溶液(50mM)1mL、唾液酸-壳寡糖-脱氧胆酸嫁接物(5mg/mL)0.5mL、MTX溶液(320μg/mL)4.0mL,混合均匀,缓慢滴入磷酸盐水溶液(30mM)1mL、柠檬酸钠水溶液(100mg/mL)100μL。100℃搅拌4h后,转移至7.0kDa透析袋中,纯水透析24h,冻干得唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统。
采用高效液相色谱法测定甲氨蝶呤的含量。经测定,唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统中的甲氨蝶呤含量为3.08%。
采用透射电子显微镜对唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统进行表观形貌观察。透射电子显微镜结果显示,纳米给药系统形态呈棒状,且粒径大小约为50nm。用微粒粒度与表面电位测定仪测定纳米给药系统的粒径。经测定,唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统的粒径43.48±6.28nm。
采用强酸降解羟基磷灰石,结合药物含量测定,以重量法测定唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统中唾液酸修饰壳寡糖嫁接物、羟基磷灰石和甲氨蝶呤的含量。经测定,唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统中唾液酸修饰壳寡糖嫁接物、羟基磷灰石和甲氨蝶呤的含量分别为14.52%、82.40%和3.08%。
实施例三唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统的制备
唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统的制备分两步进行,第一步合成唾液酸修饰壳寡糖嫁接物。取脱氧胆酸80mg,1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐77mg于圆底烧瓶,加入乙醇50mL超声溶解,在60℃下搅拌0.5h,然后加入到50mL浓度为5mg/mL已60℃预热的壳寡糖(MW 30000Da)水溶液中,于400rpm下继续反应24h。反应后的溶液转移至透析袋(MWCO 7.0kDa),纯水透析48h,收集混悬液,离心(6000rpm,15min),取上清经冷冻干燥得壳寡糖-脱氧胆酸嫁接物混合物。将得到的粉末用乙醇多次洗涤,除去体系中未反应的脱氧胆酸,复溶后经冷冻干燥得壳寡糖-脱氧胆酸粉末。
取唾液酸10mg,1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐46mg于圆底烧瓶,加入乙醇40mL超声溶解,在60℃下搅拌0.5h,然后加入到20mL浓度为5mg/mL已60℃预热的壳寡糖-脱氧胆酸水溶液中,于400rpm下继续反应24h。反应后的溶液转移至透析袋(MWCO7.0kDa)中,纯水透析48h,收集透析袋中液体经冷冻干燥得唾液酸-壳寡糖-脱氧胆酸嫁接物。
采用核磁共振氢谱对唾液酸-壳寡糖-脱氧胆酸嫁接物的结构进行确证。经计算,唾液酸-壳寡糖-脱氧胆酸嫁接物中唾液酸:壳寡糖:脱氧胆酸的摩尔比为1:1:1。
以芘为荧光探针,采用荧光分光光度法对唾液酸-壳寡糖-脱氧胆酸嫁接物嫁接物在水性介质中的临界胶束浓度进行测定。取唾液酸-壳寡糖-脱氧胆酸嫁接物嫁接物10mg,精密称定,将其分散于10mL蒸馏水中,探头超声20次,功率400w,工作2s停3s,制得1mg/mL的嫁接物水溶液,并将其稀释成不同浓度的嫁接物溶液(0.005-1mg/mL)。然后分别加入一定量的芘,使芘的终浓度为6×10-7mol/L,水浴超声30min后,采用荧光分光光度计扫描芘的激发光谱和发射光谱,分别记录374nm及385nm波长下的荧光强度,以I374/I385比值计算得嫁接物的临界胶束浓度为45.12μg/mL。
第二步制备唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统。取CaCl2溶液(50mM)1mL、唾液酸-壳寡糖-脱氧胆酸嫁接物(5mg/mL)1mL、MTX溶液(320μg/mL)1.0mL,混合均匀,缓慢滴入磷酸盐水溶液(30mM)1mL、柠檬酸钠水溶液(100mg/mL)100μL。100℃搅拌4h后,转移至7.0kDa透析袋中,纯水透析24h,冻干得唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统。
采用高效液相色谱法测定甲氨蝶呤的含量。经测定,唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统中的甲氨蝶呤含量为1.07%。
采用透射电子显微镜对唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统进行表观形貌观察。透射电子显微镜结果见图1,结果表明纳米给药系统形态呈棒状,且粒径大小约为50nm。用微粒粒度与表面电位测定仪测定纳米给药系统的粒径。经测定,唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统的粒径48.18±5.21nm。
采用强酸降解羟基磷灰石,结合药物含量测定,以重量法测定唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统中唾液酸修饰壳寡糖嫁接物、羟基磷灰石和甲氨蝶呤的含量。经测定,唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统中唾液酸修饰壳寡糖嫁接物、羟基磷灰石和甲氨蝶呤的含量分别为34.85%、64.08%和1.07%。
实施例四唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统在类风湿关节炎中的应用。
1.唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统的体外药物释放行为
配制pH 7.4和pH 5.5的磷酸缓冲盐溶液分别模拟体内正常部位和炎症关节部位的生理环境条件。取唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统10mg(实施例三),分散于10mL蒸馏水中,各取溶液2mL,密封置于透析袋中(MWCO:7.0kDa),置于含有适量体积释放介质的释放管中,使药物的释放满足漏槽条件,在37℃下60rpm振荡。在预设的时间点(0.5、1、2、4、8、12、24、36、48和72h)取样,并将释放管中释放介质全部更换为新鲜介质。采用高效液相色谱法测定样品中的药物含量,并计算药物的累积释放百分率。
经测定,唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统的体外药物释放行为如图2所示,该纳米给药系统呈现出明显的pH值依赖性,经过48h,在pH 7.4缓冲液中的累积释放百分率为30.65±1.16%,而在pH 5.5缓冲液中的累积释放百分率为82.85±4.75%。
2.唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统的体外抑制软骨细胞凋亡研究
按照实施例三的制备方法制备了唾液酸修饰低分子量壳寡糖(MW 2500Da)/羟基磷灰石纳米给药系统,将其作为对照组,采用流式细胞仪考察考察实施例三对白介素1β诱导的受损软骨细胞的保护作用。以每孔2×105个软骨细胞的密度接种6孔培养板,待细胞贴壁后,先分别与磷酸盐缓冲液、唾液酸修饰低分子量壳寡糖(MW 2500Da)/羟基磷灰石纳米给药系统和唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统(实施例三)共孵育30min,再与一定浓度的白介素1β共孵育48h后,收集各孔的培养液置于离心管中,板内的细胞用磷酸盐缓冲液洗三次,加入胰酶消化1min,用收集的培养液终止消化,收集细胞后1000rpm离心5min,弃去上清,磷酸盐缓冲液再洗三次,弃去上清,取0.5mL结合缓冲液加入EP管中,重悬细胞,依次加入Annexin V-FITC(5μL)及PI(10μL),混匀,避光,于室温下染色20min,用流式细胞仪检测。
经测定,唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统的体外抑制软骨细胞凋亡作用如图3所示,白介素1β刺激软骨细胞后,28.25%的细胞发生凋亡,经唾液酸修饰低分子量壳寡糖(MW 2500Da)/羟基磷灰石纳米给药系统处理后,凋亡细胞比例降低至15.33%,而经实施例三处理后,凋亡细胞的比例减小至9.15%,证明了唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统的软骨保护作用。
3.唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统的体外促成骨细胞的酶活力研究
按照实施例三的制备方法制备了唾液酸修饰低分子量壳寡糖(MW 2500Da)/羟基磷灰石纳米给药系统,将其作为对照组,采用磷酸苯二钠法考察考察实施例三对成骨细胞的碱性磷酸酶活力。以每孔2×104个细胞的密度接种24孔板,待其贴壁后,加入磷酸盐缓冲液、唾液酸修饰低分子量壳寡糖(MW 2500Da)/羟基磷灰石纳米给药系统和唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统(实施例三)进行干预,于培养5天后,弃培养液,每孔加入细胞裂解液,收集上清,按碱性磷酸酶活力检测试剂盒步骤检测样本中碱性磷酸酶活性,并以BCA试剂盒测得对应孔内的总蛋白含量进行归一化处理。
经测定,唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统的对成骨细胞的碱性磷酸酶活力的作用如图4所示,磷酸缓冲液处理的成骨细胞内碱性磷酸酶活力较低,为2.20±0.42DEA/mgprog,经唾液酸修饰低分子量壳寡糖(MW 2500Da)/羟基磷灰石纳米给药系统处理后,成骨细胞内碱性磷酸酶活力为8.77±0.57DEA/mgprog,而经实施例三处理后,成骨细胞内碱性磷酸酶的活力显著增强,提高至13.11±0.59DEA/mgprog。
Claims (4)
1.一种唾液酸修饰复合纳米给药系统,其特征在于,该纳米给药系统由唾液酸-壳寡糖-脱氧胆酸嫁接物胶束、羟基磷灰石和甲氨蝶呤所组成,其质量百分比为:唾液酸-壳寡糖-脱氧胆酸嫁接物14.85%-34.85%,羟基磷灰石64.06%-84.06%,甲氨蝶呤1.07%-3.08%,唾液酸-壳寡糖-脱氧胆酸嫁接物的分子量范围为5700~31000Da,结构式如下:
其中壳寡糖的聚合度n=30~180。
2.根据权利要求1所述的一种唾液酸修饰复合纳米给药系统,其特征在于,其中的甲氨蝶呤释放呈现明显的pH值依赖性,在pH 7.4中性介质中释放缓慢,而在pH 5.5酸性条件下释放加快。
3.权利要求1所述的一种唾液酸修饰复合纳米给药系统的制备方法,其特征在于,通过以下步骤获得:
(1)唾液酸修饰壳寡糖嫁接物胶束的合成
取脱氧胆酸50-80mg,1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐77mg于圆底烧瓶,加入乙醇50mL超声溶解,在60℃下搅拌0.5h,然后加入到50mL浓度为5mg/mL已60℃预热的壳寡糖水溶液中,于400rpm下继续反应24h,反应后的溶液转移至透析袋,纯水透析48h,收集混悬液,离心,取上清经冷冻干燥得壳寡糖-脱氧胆酸嫁接物混合物,将得到的粉末用乙醇多次洗涤,除去体系中未反应的脱氧胆酸,复溶后经冷冻干燥得壳寡糖-脱氧胆酸粉末,取唾液酸10mg,1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐46mg于圆底烧瓶,加入乙醇40mL超声溶解,在60℃下搅拌0.5h,然后加入到20mL浓度为5mg/mL已60℃预热的壳寡糖-脱氧胆酸水溶液中,于400rpm下继续反应24h,反应后的溶液转移至透析袋中,纯水透析48h,收集透析袋中液体经冷冻干燥得唾液酸-壳寡糖-脱氧胆酸嫁接物;
(2)唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统的制备
取CaCl2溶液1mL、唾液酸-壳寡糖-脱氧胆酸嫁接物0.5-1mL、MTX溶液1-4mL,混合均匀,缓慢滴入磷酸盐水溶液1mL、柠檬酸钠水溶液100μL。100℃搅拌4h后,转移至7.0kDa透析袋中,纯水透析24h,冻干得唾液酸修饰壳寡糖/羟基磷灰石纳米给药系统。
4.权利要求1所述的一种唾液酸修饰复合纳米给药系统在制备类风湿关节炎药物中的应用。
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CN111821470A (zh) * | 2020-09-01 | 2020-10-27 | 中南大学 | 包载甲氨蝶呤的铁-鞣酸配合物及其制备方法与应用 |
CN114199844A (zh) * | 2021-12-09 | 2022-03-18 | 吉林大学 | 一种金纳米簇及其在制备检测碱性磷酸酶荧光探针中的应用 |
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CN111821470A (zh) * | 2020-09-01 | 2020-10-27 | 中南大学 | 包载甲氨蝶呤的铁-鞣酸配合物及其制备方法与应用 |
CN111821470B (zh) * | 2020-09-01 | 2022-08-12 | 中南大学 | 包载甲氨蝶呤的铁-鞣酸配合物及其制备方法与应用 |
CN114199844A (zh) * | 2021-12-09 | 2022-03-18 | 吉林大学 | 一种金纳米簇及其在制备检测碱性磷酸酶荧光探针中的应用 |
CN114199844B (zh) * | 2021-12-09 | 2024-02-09 | 吉林大学 | 一种金纳米簇及其在制备检测碱性磷酸酶荧光探针中的应用 |
CN114891057A (zh) * | 2022-05-26 | 2022-08-12 | 沈阳药科大学 | 唾液酸衍生物修饰的化合物及其合成方法和应用 |
CN114891057B (zh) * | 2022-05-26 | 2023-10-17 | 沈阳药科大学 | 唾液酸衍生物修饰的化合物及其合成方法和应用 |
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