CN111603437A - 一种包载药或活菌的递送复合物及其制备方法 - Google Patents
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Abstract
本发明涉及复合凝胶载体技术领域,具体涉及一种包载药或活菌的递送复合物及其制备方法,所述方法包括如下步骤:将海藻酸钠溶液与目的药物溶液或目的活菌菌液混合,配置成海藻酸钠混合溶液,后将其滴入CaCl2溶液中,获得海藻酸钙凝胶微球,静置,获得形态稳定的海藻酸钙凝胶微球;将微球洗涤后吸干表面水分,将微球浸入不饱和脂肪酸的甘油脂中,取出微球与未凝固的明胶凝胶混合后加入到模具,降温至明胶凝胶完全凝固,静置后,脱去模具,即得递送复合物。本发明适用于蛋白、多肽等易于受到胃肠环境影响的药物以及益生菌活菌制剂,能够有效地保护其装载的药物或活菌,免于胃部环境的影响,而其使药物或活菌到达肠道内目标位置进行释放。
Description
技术领域
本发明涉及复合凝胶载体技术领域,具体涉及一种包载药或活菌的递送复合物及其制备方法。
背景技术
口服给药是最为方便、安全和舒适的给药途径,也是目前许多药物设计与开发的首选途径。但是由于消化道环境的复杂性,蛋白、多肽类药物在消化道中很容易被胃酸与蛋白酶破坏,导致药物吸收利用度低。口服的情况下,由于胃中的强酸性环境,以及胃肠环境的酸碱变化,使得服用的益生菌活菌数量大大降低。
凝胶是介于固体和液体之间的一种特殊的半固体状态。许多聚合物材料可以制备成凝胶载体,这些载体对药物具有一定的保护作用,同时可以根据pH,离子强度,温度,氧化还原条件等因素的变化而改变自身状态,实现对生物大分子物质在特定部位进行的可控装载和释放,同时凝胶的水溶液核心也增强了药物稳定性和生物活性。
海藻酸钠是一种水溶性的天然线性非分枝的多糖,可以与二价金属离子如钙离子等形成水凝胶。由于其具有很好地生物相容性,粘膜吸附性,生物可降解性,以及温和的凝胶化条件,常被作为一种安全的生物材料应用于食品与医药领域。明胶是通过纤维胶原蛋白降解得到的天然高分子多肽化合物,无味、半透明、微带光泽,干燥条件下能够长时间保存,在食品、医药、工业等领域具有广泛的用途。近些年,由于其优良的性能,使其在医药领域的药物控释材料方面有更加广泛的应用。
综上,开发一种适于蛋白、多肽类药物以及益生菌活菌制剂的口服给药载体,通过其包载蛋白、多肽类药物以及益生菌活菌制剂,提高其口服生物利用率仍是口服给药制剂研发的关键问题。
发明内容
为解决上述技术问题,本发明提供了一种包载药或活菌的递送复合物及其制备方法。为实现上述目的,本发明采用如下技术方案:
本发明的第一个目的是提供一种包载药或活菌的递送复合物的制备方法,包括如下步骤:
S1、将海藻酸钠溶液与等体积的目的药物溶液或目的活菌菌液混合,配置成海藻酸钠质量分数为1-3%的海藻酸钠混合溶液;
S2、将所述海藻酸钠混合溶液滴入到浓度为0.1mol/L的CaCl2溶液中,形成海藻酸钙凝胶微球,静置10-15min,获得形态稳定的海藻酸钙凝胶微球;
S3、将所述海藻酸钙凝胶微球洗涤后吸干表面水分,随后将海藻酸钙凝胶微球浸入不饱和脂肪酸的甘油脂中;
S4、将甘油脂处理过的海藻酸钙凝胶微球与未凝固的明胶凝胶混合后加入到模具中,降温至明胶凝胶完全凝固,静置20-30min后,脱去模具,即得到复合凝胶。
进一步的,所述目的药物为为蛋白质或多肽,所述目的活菌为益生菌。
进一步的,上述制备方法的S4中,明胶凝胶由以下步骤制备而成:
(1)将明胶颗粒与水按照10g:15mL的比例混合,加热并搅拌至明胶颗粒完全溶解,获得明胶溶液;
(2)称取果胶1.2g、葡萄糖5.9g、柠檬酸1g、柠檬酸钠0.8g、麦芽糖27g,溶于15mL水中,后与步骤(1)的明胶溶液混合,再加入10mL水,除去气泡,获得明胶凝胶。
进一步的,上述制备方法的的S4中,将明胶溶液加热至70-80℃,后降温降至35℃,并维持温度在35℃,获得未凝固的明胶凝胶,向未凝固的明胶凝胶中加入甘油脂处理过的微球,二者混合均匀,加入到模具中,降温至明胶凝胶完全凝固,静置30-40min后,脱去模具,即得到递送复合物。
进一步的,所述模具形状为立方体。
本发明的第二个目的是提供上述方法制备的包载药或活菌的递送复合物。
与现有技术相比,本发明具有如下有益效果:
1、单独一种凝胶形成的载体往往具有较为明显的缺点,海藻酸钙凝胶微球中的网状结构非常松散,导致在海藻酸钙凝胶微球制备过程中会发生装载物质的渗漏损失及快速释放,这限制了它的应用。明胶的脆性较大,热稳定性差,同时容易在水环境中被降解,在冷水中溶解性较差,很难达到缓释的效果。本发明开发的包载药或活菌的递送复合物,能够弥补单一凝胶的缺陷,并发挥两者的优势,有效地保护装载的药物或活菌,使其能够到达肠道后释放。
2、本发明所述包载药或活菌的递送复合物,结合两种凝胶的优势,蛋白、多肽类药物以及益生菌活菌制剂装载于内层凝胶海藻酸钙微球中,其较高含水量能够起到有效的保护作用,微球外层的不饱和脂肪酸的甘油脂能够防止水分的流失,其性质使其在碱性环境中才会逐步解聚,达到在肠道内缓慢释放的目的。外层明胶凝胶,能够保护内侧的海藻酸钙凝胶微球,维持凝胶的结构,减缓胃部消化作用的影响,避免其内部蛋白、多肽的流失与提前释放。
附图说明
图1为本发明的递送复合物的构建过程示意图。
图2为实施例1制备得到的海藻酸钙凝胶微球实物图。
图3为实施例1制备得到装载BSA的递送复合物。
图4为实施例1体外释放实验结果。
图5为实施例2体外释放实验结果,图中A为实验组,B为对照组。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明,但不应理解为本发明的限制。如未特殊说明,下述实施例中所用的技术手段为本领域技术人员所熟知的常规手段,下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1
一种包载药的递送复合物的制备方法
本实施例的模式药物为牛血请白蛋白(Bovine Serum Albumin,BSA)(浓度为1mg/mL),具体包括如下步骤:
S1、将4g海藻酸钠粉末溶于100mL无菌水中,配置成质量分数为4%的海藻酸钠溶液,量取一定体积的海藻酸钠溶液,加入等体积BSA的溶液混合,配置成质量分数为2%的海藻酸钠混合溶液;
S2、吸取2%的海藻酸钠混合溶液,采用使用恒流泵将液滴滴入到浓度为0.1mol/L的CaCl2溶液中,滴入位置与液面相距30cm,滴注的同时,使用磁力搅拌器以250rpm进行搅拌,液滴进入CaCl2溶液中立即形成海藻酸钙凝胶微球,如图2所示,静置10min,使钙离子与海藻酸钠形成聚合结构,获得形态稳定的海藻酸钙凝胶微球;
S3、将上述海藻酸钙凝胶微球用无菌水洗涤1次,用滤纸吸干表面水分,随后将让微球浸入不饱和脂肪酸的甘油脂中,让微球表面吸附一层油脂;
S4、制备明胶凝胶:称取10g食品级明胶颗粒,加入15mL无菌水,在常温下浸泡15min,使其完全膨胀***。80℃水浴加热,期间不断搅拌直至明胶颗粒完全溶解。分别称取果胶1.2g,葡萄糖5.9g,柠檬酸1g,柠檬酸钠0.8g,麦芽糖27g,溶于15mL无菌水中,搅拌使各组分完全溶解。随后将其加入到溶解后的明胶溶液中,再加入10mL无菌水,将明胶溶液混合均匀,使用超声波法除去溶液中的气泡,获得明胶凝胶;
将明胶凝胶加热至70℃,后降温降至35℃,并维持温度在35℃,获得未凝固的明胶凝胶,向未凝固的明胶凝胶中加入甘油脂处理过的微球,二者混合均匀,加入到立方体的模具中,降温至明胶凝胶完全凝固,静置30min后,脱去模具,即得到包载药的递送复合物,实物如图3所示。
将本实施例制备得到的包载药的递送复合物进行体外释放实验,方法如下:
将包载药的递送复合物置于20mL模拟胃液中(氯化钠2g,7mL浓盐酸,加入超纯水定容至1L,溶液pH为1.2),37℃水浴100rpm恒温孵育,每30min取样一次,进行检测,连续取样6次,直至孵育3h为止。随后除去模拟胃液,加入20mL模拟肠液(磷酸二氢钾6.8g,加入超纯水定容至1L,调节溶液pH至7.4,),37℃水浴100rpm继续孵育,每30min取样一次,进行检测,连续取样10次,孵育5h。使用Bradford法测定样品中的蛋白浓度,绘制蛋白的释放曲线。
结果如图4所示,包载药的递送复合物在模拟胃液中处理3h后,蛋白释放了约10%。当微球移入模拟肠液中,随着pH的升高,外层的凝胶逐渐吸膨胀,孔隙变大,内部的海藻钙微球,氨基之间的静电相互作用减弱,聚合结构也随之解聚,蛋白随着渗漏的水分一起扩散至微球外部。复合凝胶中的蛋白在4h内基本释放完全。
实施例2
一种包载活菌的递送复合物的制备方法
本实施例除目的活菌为乳酸菌模式菌株(浓度为1×1010cfu/mL),其余制备方法同实施例1。
将本实施例制备得到的包载活菌的递送复合物进行体外释放实验,方法如下:
实验组将包载活菌的递送复合物置于20mL模拟胃液中(氯化钠2g,7mL浓盐酸,加入超纯水定容至1L,溶液pH约为1.2),37℃水浴100rpm恒温孵育3h。随后除去模拟胃液,加入20mL模拟肠液(磷酸二氢钾6.8g,加入超纯水定容至1L,调节溶液pH至7.4),37℃水浴100rpm继续孵育,孵育1h。随后取出递送复合物,在温和条件下将其完全裂解,1000rpm离心,除去未溶解的凝胶微粒,取上清液稀释,进行平板涂布,37℃恒温培养24h,检测平板上单克隆数量,计算活菌数。对照组将相同浓度的菌液放入3KD透析袋中采用相同方法进行处理。
结果如图5所示,平板涂布的结果表明,递送复合物对装载的乳酸菌起到了有效的保护作用,经过模拟胃液与肠液的处理,递送复合物的活菌数可达约1.2×109cfu/mL,而无载体保护的对照组活菌数仅为3×107cfu/mL。
需要说明的是,本发明权利要求书中涉及数值范围时,应理解为每个数值范围的两个端点以及两个端点之间任何一个数值均可选用,为了防止赘述,本发明描述了优选的实施例。
尽管已描述了本发明的优选实施例,但本领域内的技术人员一旦得知了基本创造性概念,则可对这些实施例作出另外的变更和修改。所以,所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。
显然,本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样,倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内,则本发明也意图包含这些改动和变型在内。
Claims (6)
1.一种包载药或活菌的递送复合物的制备方法,其特征在于,包括如下步骤:
S1、将海藻酸钠溶液与等体积的目的药物溶液或目的活菌菌液混合,配置成海藻酸钠质量分数为1-3%的海藻酸钠混合溶液;
S2、将所述海藻酸钠混合溶液滴入到浓度为0.1mol/L的CaCl2溶液中,形成海藻酸钙凝胶微球,静置10-15min,获得形态稳定的海藻酸钙凝胶微球;
S3、将所述海藻酸钙凝胶微球洗涤后吸干表面水分,随后将海藻酸钙凝胶微球浸入不饱和脂肪酸的甘油脂中;
S4、将甘油脂处理过的海藻酸钙凝胶微球与未凝固的明胶凝胶混合后加入到模具中,降温至明胶凝胶完全凝固,静置20-30min后,脱去模具,即得到包载药或活菌的递送复合物。
2.根据权利要求1所述一种包载药或活菌的递送复合物的制备方法,其特征在于,S1中,所述目的药物为蛋白质或多肽,所述目的活菌为益生菌。
3.根据权利要求1所述一种包载药或活菌的递送复合物的制备方法,其特征在于,S4中,明胶凝胶由以下步骤制备而成:
(1)将明胶颗粒与水按照10g:15mL的比例混合,加热并搅拌至明胶颗粒完全溶解,获得明胶溶液;
(2)称取果胶1.2g、葡萄糖5.9g、柠檬酸1g、柠檬酸钠0.8g、麦芽糖27g,溶于15mL水中,后与步骤(1)的明胶溶液混合,再加入10mL水,除去气泡,获得明胶凝胶。
4.根据权利要求3所述一种包载药或活菌的递送复合物的制备方法,其特征在于,S4中,将明胶凝胶加热至70-80℃,后降温降至35℃,并维持温度在35℃,获得未凝固的明胶凝胶,向未凝固的明胶凝胶中加入甘油脂处理过的海藻酸钙凝胶微球,二者混合,加入到模具中,降温至明胶凝胶完全凝固,静置30-40min后,脱去模具,即得到递送复合物。
5.根据权利要求4所述一种载药明胶-海藻酸钙复合凝胶的制备方法,其特征在于,所述模具形状为立方体。
6.权利要求1-5任一项所述的方法制备的包载药或活菌的递送复合物。
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