CN109662954B - 石杉碱甲分子印迹水凝胶微球的制备方法 - Google Patents
石杉碱甲分子印迹水凝胶微球的制备方法 Download PDFInfo
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Abstract
本发明公开了一种石杉碱甲分子印迹水凝胶微球的制备方法,其是以石杉碱甲作为模板分子,HPMC为功能单体,TEMED为催化剂,DVS为交联剂,采用反向悬浮聚合法制得所述石杉碱甲分子印迹水凝胶微球。该石杉碱甲分子印迹水凝胶微球具有较高的特异性识别能力,并具有缓释功能且适于口服,是一种新型的药物制剂。
Description
技术领域
本发明属于药物剂型制备领域,主要涉及石杉碱甲的载体制剂,更具体地说,涉及一种石杉碱甲分子印迹水凝胶微球的制备方法。
背景技术
石杉碱甲(Huperzine A,HupA)是从民间草药蛇足石杉中分离得到的中枢乙酰胆碱酯酶(acetycholinesterase,AChE)抑制剂,其表现出广泛的神经保护及抗氧化应激,具有改善脑缺血损伤、修复记忆损伤等作用,已广泛应用于阿尔茨海默病(AD)、血管性痴呆(VD)及智力低下等神经退行性疾病的治疗。临床数据表明,石杉碱甲的总体临床安全性良好,不存在剂量限制的肝脏毒性,心血管临床数据也不存在异常,但有少数患者服用石杉碱甲后会出现轻中度的胃肠道反应。目前上市的剂型主要是口服或注射的普通制剂,因此为了提高口服给药的安全性、依从性和便利性,应对普通剂型进行改良。
石杉碱甲剂型的改良研究主要是制备缓控释载体,使其具有一定的缓控释作用,减少服药次数,避免服药患者因频繁给药所造成的血药浓度波动幅度大,避免产生不良反应。目前较为成熟的缓控释制剂是以水凝胶为原料所制备的微球,而分子印迹聚合物微球(Molecularly Imprinted Polymeric Microspheres,MIPMs)由于选择性高、专属性强等优点已成为国内外的研究热点,主要涉及富集萃取、传感器、色谱分离、手性拆分等方面,也有一部分文献将分子印迹技术应用于药物剂型研究,有较好的发展前景。目前国内外仅有一篇对石杉碱甲分子印迹聚合物制备进行研究的文献(李志平,“石杉碱甲印迹聚合物的制备、表征及其吸附性能”,2013年),该文献中所制备的石杉碱甲分子印迹水凝胶微球是以甲基丙烯酸作为原料,硅胶作为牺牲载体,开发出的一种高效分离富集的新型吸附材料,但若将其应用于药物剂型方面可行性较低。
发明内容
本发明的目的在于提供一种具有缓释功能且适于口服的石杉碱甲分子印迹水凝胶微球的制备方法。
为实现上述目的,本发明采用如下技术方案:
一种石杉碱甲分子印迹水凝胶微球的制备方法,是以石杉碱甲作为模板分子,羟丙基甲基纤维素(HPMC)为功能单体,四甲基乙二胺(TEMED)为催化剂,二乙烯基砜(DVS)为交联剂,采用反向悬浮聚合法制得具有缓释作用的石杉碱甲分子印迹水凝胶微球。其具体包括如下步骤:
1)HPMC-石杉碱甲水溶液的制备:取0.4 g HPMC于30 mL 50 ℃超纯水中搅拌均匀,放冷,加入24 mg石杉碱甲,超声30 min进行预聚合后得HPMC-石杉碱甲水溶液,备用;
2)复配分散剂的制备:取0.04 g吐温-80与0.4 g司盘-80混合,加入180 mL环己烷中,分散均匀得复配分散剂;
3)石杉碱甲分子印迹聚合物的制备:取180 mL复配分散剂,在460~720 r/min的搅拌速度下缓慢加入30 mL HPMC-石杉碱甲水溶液,搅拌3h使其均匀分散后加入40mgTEMED及6 mL体积浓度为1%的DVS溶液,继续搅拌至充分交联后,洗涤产物,冷冻干燥后采用超声辅助抽提法进行模板的洗脱(将聚合物置于锥形瓶中,加入50 mL甲醇超声提取30 min后,弃去提取溶剂,多次重复,直至提取溶剂中检测不到石杉碱甲为止),洗涤产物,所得产物经冷冻干燥后即为所述石杉碱甲分子印迹水凝胶微球。
目前报道的关于石杉碱甲分子印迹聚合物的制备方法不适用于制备载体药物,其主要有两个原因:一是所用甲基丙烯酸具有较大毒性,不适用于口服剂型的制备;二是制备过程中加入硅胶作为牺牲载体,洗脱过程较为繁琐;若洗脱不彻底,可能影响药效,降低生物相容性等。
本发明通过对其原料及实验方法的改进,有效提高了生物相容性,更加适合作为载体药物。与现有技术制备的石杉碱甲药物载体相比,利用本发明方法制备的石杉碱甲分子印迹水凝胶微球具有高的特异识别性,并比一般的水凝胶具有更高的载药量。并且,与市售石杉碱甲片剂的体外释放度相比,本发明制备的石杉碱甲分子印迹水凝胶微球对石杉碱甲具有明显的药物缓释作用。
本发明的显著优点在于;
1、本发明采用羟丙基甲基纤维素(HPMC)作为制备分子印迹水凝胶微球的材料,使产品具有较好的缓释性和生物相容性;同时,HPMC是非离子型性化合物,不与金属盐、离子化合物等发生反应,影响药效的可能性较低;且HPMC良好的抗酶和抗代谢性能可提高药品的稳定性。
2、本发明采用反向悬浮聚合法制备水凝胶微球。相比其他聚合方法,其具有产物后处理简单、易于控制温度、产物的杂质含量较少等优点,更适合应用于医药、生物技术方面;且其制备过程中不需加入牺牲载体,经超声辅助抽提便可去除模板,可有效降低模板洗脱难度。
附图说明
图1为本发明所得石杉碱甲分子印迹水凝胶微球的TEM图。
图2为石杉碱甲(HupA)的红外光谱图。
图3为洗脱前的石杉碱甲分子印迹水凝胶微球的红外光谱图。
图4为非分子印迹水凝胶微球的红外光谱图。
图5为模板分子洗脱后的石杉碱甲分子印迹水凝胶微球的红外光谱图。
图6为石杉碱甲片的体外释放曲线。
图7为石杉碱甲分子印迹水凝胶微球的体外释放曲线。
图8为石杉碱甲片的血药浓度时间曲线图。
图9为石杉碱甲分子印迹水凝胶微球的血药浓度时间曲线图。
具体实施方式
为了使本发明所述的内容更加便于理解,下面结合具体实施方式对本发明所述的技术方案做进一步的说明,但是本发明不仅限于此。
实施例
1)HPMC-石杉碱甲水溶液的制备:取0.4 g HPMC于30 mL 50 ℃超纯水中搅拌均匀,放冷,加入24 mg石杉碱甲,超声30 min进行预聚合后得HPMC-石杉碱甲水溶液,备用;
2)复配分散剂的制备:取0.04 g吐温-80与0.4 g司盘-80混合,加入180 mL环己烷中,分散均匀得复配分散剂;
3)石杉碱甲分子印迹聚合物的制备:取180 mL复配分散剂,在460~720 r/min的搅拌速度下缓慢加入30 mL HPMC-石杉碱甲水溶液,搅拌3h使其均匀分散后加入40mgTEMED及6 mL体积浓度为1%的DVS溶液,继续搅拌至充分交联后,洗涤产物,冷冻干燥后将所得聚合物置于锥形瓶中,加入50 mL甲醇超声提取30 min后,弃去提取溶剂,多次重复,直至提取溶剂中检测不到石杉碱甲为止,洗涤产物,经冷冻干燥后即为石杉碱甲分子印迹水凝胶微球。
用透射电子显微镜、红外光谱分析鉴定分子印迹聚合物的结构特征,并考察石杉碱甲分子印迹水凝胶微球载药量、体外释放度及大鼠药代动力学研究,具体内容如下:
1. 取所得石杉碱甲分子印迹水凝胶微球水溶液,负染色法处理后,采用透射电子显微镜放大2.0×105倍观察微球形貌,结果如图1所示。由图1可见,制备的分子印迹水凝胶微球具有良好的类球形外观,且分散性较好,粒径约为50 nm。
2 分子印迹水凝胶微球的红外光谱分析
采用压片法,分别取石杉碱甲(HupA)、洗脱前的石杉碱甲分子印迹水凝胶微球、非分子印迹水凝胶微球、模板分子洗脱后的石杉碱甲分子印迹水凝胶微球压片后进行红外光谱分析,结果如图2-5所示。
其中,非分子印迹水凝胶微球的制备方法是取80 mL 2 %的氢氧化钠水溶液,水浴加热至50 ℃左右,加入0.2 g HPMC搅拌均匀后放冷备用,得HPMC水溶液;取适量吐温-80与司盘-80混合,加入环己烷中,分散均匀得复配分散剂;然后取复配分散剂,在460~720 r/min的搅拌速度下缓慢加入HPMC水溶液,搅拌3h使其均匀分散后加入适量体积浓度为1%的DVS溶液,继续搅拌至充分交联后,洗涤产物,冷冻干燥得到HPMC水凝胶微球,即非分子印迹水凝胶微球。
图2为石杉碱甲的红外光谱图。由图2所可见,石杉碱甲红外谱线中,3477 cm-1和2963 cm-1处分别为N-H和C-H的伸缩振动吸收峰,1646 cm-1处为C=O的伸缩振动吸收峰,为谱图最强峰,1612 cm-1处为C=C的振动吸收峰,1455 cm-1处为吡啶酮环的伸缩振动吸收峰。
图3为洗脱前的石杉碱甲分子印迹水凝胶微球的红外光谱图,即石杉碱甲与羟丙基甲基纤维素(HPMC)两者形成聚合物的红外光谱图。由图3所可见,两者通过氢键结合,在3455 cm-1处的吸收峰对应于羟基-OH的吸收峰,C=O基、C=C双键和吡啶酮环的吸收峰分别移至1654 cm-1、1601 cm-1和1451 cm-1。
图4为非分子印迹水凝胶微球的红外光谱图。由图中可见,3481 cm-1为羟基-OH的吸收峰,2919 cm-1为甲基-CH3的伸缩振动峰,1472 cm-1与1457 cm-1是甲基-CH3的弯曲振动峰,1058 cm-1处是C-O键的伸缩振动峰。
当将石杉碱甲分子印迹水凝胶微球中的模板分子HupA洗脱后,其红外光谱图如图5所示。由图中可见,与图4非分子印迹水凝胶微球的红外谱线相比,羟基-OH的吸收峰红移至3447 cm-1处,且强度更大,说明洗脱后的分子印迹水凝胶微球结构中有较强的氢键形成;甲基-CH3和C-O键的吸收峰也都发生了一定程度的移动;其次,若聚合物中存在C=O键,则在谱图中1630 cm-1-1700 cm-1处存在极强峰,一般为谱图最强峰,但在图5的谱图中并未出现该强峰,因此说明聚合物中无C=O,即石杉碱甲模板已彻底洗脱。
3. 载药量考察
采用浸泡法进行载药,精密称取未加入石杉碱甲制备的空白非分子印迹水凝胶微球及空白分子印迹水凝胶微球,分别浸泡于3 mL浓度为1 mg/mL的HupA标准溶液中,振摇至吸附饱和后,用超滤离心法分离载药微球与滤液,HPLC法测定滤液中HupA浓度,计算载药量。
实验表明,非分子印迹水凝胶微球的平均载药量为3.66 %,分子印迹水凝胶微球的载药量为8.19 %,分子印迹水凝胶微球的载药量明显高于非分子印迹水凝胶微球的载药量,这可能是由于分子印迹的特异性识别使石杉碱甲在药物载体中的载药量增加。
4. 体外释放考察
分别精密称取市售石杉碱甲片(双益平,上海复旦复华药业有限公司)及本发明制备的石杉碱甲分子印迹水凝胶微球,采用透析袋法进行体外释放实验,其具体是在50 mL溶出介质、恒温37 ℃、固定转速的条件下,每隔一段时间取样1 mL后补加等量恒温的溶出介质,取出的样品溶液经微孔滤膜过滤后,HPLC法测定HupA浓度,计算HupA的累积释放度(%)。实验结果如图6、7所示。
图6、图7分别为石杉碱甲片及石杉碱甲分子印迹水凝胶微球的体外释放曲线。如图6所示,石杉碱甲片在3 h处释放基本达到平衡。而图7的石杉碱甲分子印迹水凝胶微球体外释放曲线中,在0-5 h的阶段,累积释放度持续上升,5-8 h阶段累积释放度曲线较平缓,在8 h处基本达到释放平衡。由此可见,石杉碱甲片与石杉碱甲分子印迹水凝胶微球两者的体外释放曲线存在较大区别,石杉碱甲分子印迹水凝胶微球的体外释放达到平衡的时间明显长于石杉碱甲片,说明分子印迹水凝胶微球对石杉碱甲具有较好的缓释作用,可在体外条件下缓慢释放HupA约8 h。
5. 药代动力学研究
SD大鼠,雄性,分为石杉碱甲片剂组(阳性药组)及石杉碱甲分子印迹水凝胶微球组(对照药组),每组各6只,给药剂量为0.36 mg/kg,分别于灌胃给药后的15 min、45 min、90 min、2 h、3 h、4 h、6 h、8 h、12 h、24 h进行眼眶采血,离心取200 μL血浆样品,前处理后进UPLC-MS测定血中HupA药物浓度。图8、图9分别为石杉碱甲片及石杉碱甲分子印迹水凝胶微球的血药浓度时间曲线图。
如图8所示,石杉碱甲片的血药浓度时间曲线在0-1.5 h阶段,血药浓度不断增大,到1.5 h时达到峰值,血药浓度约为35 μg/mL;达到峰值后,血药浓度迅速下降,6 h后血药浓度为零。
如图9所示,石杉碱甲分子印迹水凝胶微球与石杉碱甲片的血药浓度时间曲线基本呈现同样的变化趋势,但分子印迹水凝胶微球达到血药浓度峰值的时间为6 h,比石杉碱甲片达到血药浓度峰值的时间长,且达到峰值时的血药浓度约为40 μg/mL。灌胃后12 h血药浓度降低到2.9 μg/mL,24 h后血药浓度为零。
在体外释放研究及大鼠药代动力学的研究中,通过对石杉碱甲片及石杉碱甲分子印迹水凝胶微球两种剂型的对比,证实了石杉碱甲分子印迹水凝胶微球相对于石杉碱甲片均明显具有缓释作用。
以上所述仅为本发明的较佳实施例,凡依本发明申请专利范围所做的均等变化与修饰,皆应属本发明的涵盖范围。
Claims (1)
1.一种石杉碱甲分子印迹水凝胶微球的制备方法,其特征在于:以石杉碱甲作为模板分子,HPMC为功能单体,TEMED为催化剂,DVS为交联剂,采用反向悬浮聚合法,制得具有缓释作用且适于口服的石杉碱甲分子印迹水凝胶微球;其具体包括如下步骤:
1)HPMC-石杉碱甲水溶液的制备:取0.4 g HPMC于30 mL 50 ℃超纯水中搅拌均匀,放冷,加入24 mg石杉碱甲,超声30 min进行预聚合后得HPMC-石杉碱甲水溶液;
2)复配分散剂的制备:取0.04 g吐温-80与0.4 g司盘-80混合,加入180 mL环己烷中,分散均匀得复配分散剂;
3)石杉碱甲分子印迹聚合物的制备:取180 mL复配分散剂,在460~720 r/min的搅拌速度下缓慢加入30 mL HPMC-石杉碱甲水溶液,搅拌3h使其均匀分散后加入40mg TEMED及6mL体积浓度为1%的DVS溶液,继续搅拌至充分交联后,洗涤产物,冷冻干燥后采用超声辅助抽提法进行模板的洗脱,所得产物经冷冻干燥后即为所述石杉碱甲分子印迹水凝胶微球。
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