CN115381793A - 白蛋白负载阿苯达唑纳米药及其制备方法 - Google Patents
白蛋白负载阿苯达唑纳米药及其制备方法 Download PDFInfo
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Abstract
本发明提供了一种白蛋白负载阿苯达唑纳米药及其制备方法,属于生物纳米合成技术领域,其通过下述步骤得到:第一步将白蛋白、胰凝乳蛋白酶和弹性蛋白酶加入水中得到第一溶液;第二步,将阿苯达唑溶解于有机溶剂得到第二溶液,第三步将第一溶液与第二溶液充分混合;第四步和第五步进行去溶剂化交联,第六步先将第三步所得到的阿苯达唑纳米药进行透析纯化,第七步,将纯化后产物进一步加热固化然后冷却冻干获得纳米级阿苯达唑。本发明显著提高了阿苯达唑药物的溶解度和溶出速度,明显改善阿苯达唑生物利用度,有效地发挥阿苯达唑抗组织器官寄生虫的治疗作用。
Description
技术领域
本发明属于生物纳米合成技术领域,特别涉及一种兽药和医药原料阿苯达唑的制备方法。
背景技术
阿苯达唑(albendazole,ABZ),亦名为丙硫咪唑,为苯并咪唑类衍生物,是应用于临床 的广谱抗寄生虫药。但由于ABZ水溶解性差,导致其吸收率和生物利用度低。寄生虫感染人 体和动物后,主要分布于胃肠道和组织器官。旋毛虫病呈世界性广泛分布,国内旋毛虫病呈 现局部与暴发感染流行的特点。旋毛虫成虫寄生在小肠,幼虫寄生在横纹肌内。旋毛虫幼虫 在肌纤维间形成纵轴与肌纤维平行的梭形囊包,保护幼虫成长。传统的阿苯达唑为粉剂或片 剂,这类普通剂型ABZ对于移行到肌肉的旋毛虫治愈率不到30%。为改善ABZ的生物利用 度,人们对ABZ的制剂进行了大量研究,制备了阿苯达唑的乳剂,粉雾剂,喷雾剂,和凝胶 剂。这些制剂共同缺点是粒子尺寸大,生物利用度低,作用于机体的时间短,因而无法发挥 组织器官内抗寄生虫作用。除了制剂,在药剂学方面,成功开发了ABZ纳米载体系统,如 ABZ脂质体,ABZ聚合物,其中高分子单体聚合制备负载ABZ的聚合物纳米球,能显著提 高ABZ生物利用度,因而增强ABZ在组织器官内的抗寄生虫作用。虽然ABZ的生物利用度得到了积极改善,但是高分子在体内微环境中的稳定性差,ABZ容易泄露或在体内循环未达到靶组织就分解。
发明内容
为了解决阿苯达唑在组织器官的生物利用度和现有技术在体内微环境中的稳定性差问题, 本发明提供了白蛋白负载阿苯达唑纳米药,其是由白蛋白作为载体包裹阿苯达唑、胰凝乳蛋 白酶和弹性蛋白酶形成的纳米颗粒,纳米颗粒的粒径范围为200~350nm;
所述的阿苯达唑为具有式(I)所示结构的分子:
本发明中所述的白蛋白来源于哺乳动物,优选为牛血清白蛋白。
所述白蛋白负载阿苯达唑纳米药的制备方法,具体步骤如下:
第一步,将白蛋白、胰凝乳蛋白酶和弹性蛋白酶加入水中得到第一溶液;在第一溶液中, 白蛋白的含量为20~40mg/mL,胰凝乳蛋白酶的含量为5~10mg/mL,弹性蛋白酶的含量为3~5 mg/mL;
第二步,将阿苯达唑溶解于有机溶剂得到第二溶液;在第二溶液中,阿苯达唑的含量为 1~5mg/mL,所述的有机溶剂为吐温80、DMSO的一种;
第三步,在0℃~40℃的温度下将第一溶液缓慢滴加到第二溶液中,两种溶液等体积充分 混合得到纳米复合体系;
第四步,将第三步所得到的纳米复合体系中滴加乙醇去溶剂化,得到白蛋白负载的阿苯 达唑纳米药。
第五步,所述的纳米颗粒水溶液经过诱导交联、洗涤、纯化、固化、冷却干燥后获得所 述的阿苯达唑纳米药。
其中,诱导交联、洗涤、纯化、固化、冷却、干燥的步骤如下:
A、阿苯达唑纳米药纳米复合体系中加入质量浓度为1-5%戊二醛诱导粒子交联,得到稳 定的纳米粒子。
B、调节体系pH至9进行超滤和透析纯化去除纳米体系中游离小分子。
C、纯化后的产物加热到50-70℃固化2-3h。
D、然后再将固化后产物冷却冻干获得所述阿苯达唑纳米药。
第三步中充分混合的条件为:采用搅拌方式进行充分混合,其搅拌时间为10分钟至30 分钟、搅拌速度为500转/分钟至1000转/分钟;或/和,采用超声方式进行充分混合,其超声 时间为5分钟至30分钟。
本发明的有益效果:
采用本发明方法获得的白蛋白负载阿苯达唑纳米药可作为组织器官内治疗寄生虫感染, 本发明显著提高了阿苯达唑药物的溶解度和溶出速度,明显改善阿苯达唑生物利用度,有效 地发挥阿苯达唑抗组织器官内寄生虫的治疗作用。
附图说明
图1白蛋白负载阿苯达唑纳米药的制备方法的技术路线图;
图2为白蛋白负载阿苯达唑纳米药的透射电镜照片;
图3为白蛋白负载阿苯达唑纳米药的高效液相质谱图;
图4为显微镜10×10放大倍数观察体外药物对旋毛肌幼虫的作用。
图5为体内药物实验小白鼠的膈肌病理组化染色。
图6为体内药物实验小白鼠的膈肌内旋毛虫幼虫扫描电镜照片。
具体实施方式
下面以具体实施例的形式对本发明技术方案做进一步的解释和说明。
如图1所示,为本发明中白蛋白负载阿苯达唑纳米药的制备方法的制备路线图,该方法 具体如下:
1)分别称取白蛋白(BSA)100-200mg、胰凝乳蛋白酶25-50mg和弹性蛋白酶15-25mg, 并溶解于5mLH2O中,轻摇至全溶,恒温加热至30℃,获得第一溶液;
2)称取1-5mg阿苯达唑溶解在5mL的有机溶剂DMSO或吐温-80中,获得第二溶液;
3)将步骤1)获得的第一溶液加入步骤2)得到第二溶液中,充分混合获得纳米复合体 系;充分混合的条件为:采用搅拌方式进行充分混合,其搅拌时间为10分钟至30分钟、搅拌速度为500转/分钟至1000转/分钟;或/和,采用超声方式进行充分混合,其超声时间为5分钟至30分钟。
4)向步骤3)得到的纳米复合体系中加入乙醇去溶剂化,经超声处理后,得到纳米颗粒 水溶液;
5)向步骤4)得到的纳米颗粒水溶液中加醛诱导粒子交联,得到稳定的纳米粒子。
6)步骤5)得到稳定的纳米粒子,加入碳酸氢钠调节体系pH至9,进行超滤和/或透析 纯化去除纳米体系中游离小分子。
7)将步骤6)中纯化后的产物加热到50-70℃固化2-3h。
8)将步骤7)中产物冷却后冻干保存得到所述白蛋白负载阿苯达唑纳米药;
如图2所示,通过上述方法制备的白蛋白复合纳米颗粒粒径范围在200-350nm;图3高 效液相质谱联用定量分析白蛋白复合纳米颗粒中的阿苯达唑成分,确认阿苯达唑的质量百分 比浓度为45%。
分别将阿苯达唑(对照组)和白蛋白负载阿苯达唑纳米药,超声处理30min后加入无血 清RPMI 1640培养基,药物的浓度均为10μg/ml。显微镜下观察细胞培养板中与药物共培养 24h后幼虫的活动情况。图4展示了体外药物对旋毛虫的作用,如图4(a)所示阿苯达唑药 物对照组液体中有大量不溶颗粒悬浮,旋毛虫肌幼虫呈现“蛇形运动”;图4(b)中白蛋白复 合阿苯达唑纳米药组液体清澈,无不溶颗粒,旋毛虫肌幼虫呈现“螺旋状”,且活性低下,不 喜运动。证明本发明显著提高了阿苯达唑药物的溶解度,明显改善阿苯达唑生物利用度。
将感染旋毛虫肌幼虫的小白鼠处死后取膈肌,样本用4%福尔马林固定24小时。该程 序包括制备石蜡块和切片厚度为5μm的切片。在显微镜下检查用苏木精和伊红染色的切片 的组织病理学特征。图5(a)阿苯达唑对照组膈肌组织切片肌幼虫数量多,嗜酸性粒细胞大 量浸润。图5(b)白蛋白负载阿苯达唑纳米药物组肌幼虫包囊数量减少,伴有轻度的嗜酸性 粒细胞浸润。将膈肌粉碎制备组织上清,清洗组织上清。在24孔板中用聚-D-赖氨酸包被8 mm盖玻片。然后用PBS洗涤三次后,将含有旋毛虫肌幼虫的上清固定在聚赖氨酸处理的盖玻片上放置-20℃,静置48h后用扫描电子显微镜上进行SEM成像。图6(a)阿苯达 唑(对照组)中对照组显示完整的旋毛虫肌幼虫。图6(b)白蛋白负载阿苯达唑纳米药物组 肌幼虫虫体被破坏。证明本发明能够有效地发挥阿苯达唑抗组织器官内寄生虫的治疗作用。
Claims (8)
2.根据权利要求1所述的白蛋白负载阿苯达唑纳米药,其特征在于,所述白蛋白为牛血清白蛋白。
3.根据权利要求1所述白蛋白负载阿苯达唑纳米药的制备方法,其特征在于,该方法的具体步骤如下:
第一步,将白蛋白、胰凝乳蛋白酶和弹性蛋白酶加入水中得到第一溶液;在第一溶液中,白蛋白的含量为20~40mg/mL,胰凝乳蛋白酶的含量为5~10mg/mL,弹性蛋白酶的含量为3~5mg/mL;
第二步,将阿苯达唑溶解于有机溶剂得到第二溶液;在第二溶液中,阿苯达唑的含量为1~5mg/mL,所述的有机溶剂为吐温80、DMSO的一种;
第三步,在0℃~40℃的温度下将第一溶液缓慢滴加到第二溶液中,两种溶液等体积充分混合得到纳米复合体系;
第四步,将第三步所得到的纳米复合体系中滴加乙醇去溶剂化,得到白蛋白负载的阿苯达唑所述的纳米颗粒水溶液;
第五步,所述的纳米颗粒水溶液经过诱导交联、洗涤、纯化、固化、冷却干燥后获得所述的白蛋白负载阿苯达唑纳米药。
4.根据权利要求3所述的白蛋白负载阿苯达唑纳米药的制备方法,其特征在于,第五步中诱导交联的步骤如下:向所述的纳米颗粒水溶液中加入质量浓度为1-5%戊二醛诱导粒子交联,得到稳定的纳米粒子。
5.根据权利要求3所述的白蛋白负载阿苯达唑纳米药的制备方法,其特征在于,第五步中洗涤纯化的步骤如下:调节体系pH至9进行超滤或透析纯化去除纳米体系中游离小分子。
6.根据权利要求3所述的白蛋白负载阿苯达唑纳米药的制备方法,其特征在于,第五步中的固化是将纯化后的产物加热到50-70℃固化2-3h。
7.根据权利要求3所述的白蛋白负载阿苯达唑纳米药的制备方法,其特征在于,第五步中的冷却冻干的步骤如下:先将加热固化后产物冷却再冻干获得所述白蛋白负载阿苯达唑纳米药。
8.根据权利要求3所述的白蛋白负载阿苯达唑纳米药的制备方法,其特征在于,第三步中充分混合的条件为:采用搅拌方式进行充分混合,其搅拌时间为10分钟至30分钟、搅拌速度为500转/分钟至1000转/分钟;或/和,采用超声方式进行充分混合,其超声时间为5分钟至30分钟。
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