CN111214441B - 一种古罗糖醛酸低聚糖脂质体及其制备方法 - Google Patents
一种古罗糖醛酸低聚糖脂质体及其制备方法 Download PDFInfo
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Abstract
本发明属于医药制剂技术领域，具体涉及一种古罗糖醛酸低聚糖脂质体、其制备方法、其促免疫活性的用途。本发明通过将所述质量比的古罗糖醛酸低聚糖、卵磷脂、胆固醇和吐温80制作成脂质体，尤其是制备过程中对形成的脂质体悬液进行超声处理和均质处理时，通过控制超声处理的温度、时间、频率及均质的压强和时间，所制备的古罗糖醛酸低聚糖脂质体形态良好，呈球形，稳定性好，粒径小，包封率较高，古罗糖醛酸低聚糖脂质体的包封率为68.26±2.11％，粒径为45.69±3.6nm。本发明制备的古罗糖醛酸低聚糖脂质体具有促免疫活性的作用。
Description
技术领域
本发明属于医药制剂技术领域，具体涉及一种古罗糖醛酸低聚糖脂质体、其制备方法、其促免疫活性的用途。
背景技术
古罗糖醛酸低聚糖(Polyguluronate，PG)主要是由褐藻胶通过分级处理得到的，它是由多个α-L-古罗糖醛酸(G)通过α-1，4糖苷键连接而成的糖链。褐藻胶具有特殊的生理、生化特性，如无毒、稳定性、抗肿瘤和增强免疫活性等，近年来受到广泛关注，但其高黏性限制了其功能的发挥，将褐藻胶经过分级处理，得到的低聚糖的生物活性显著提高，并可产生新的生物活性。但褐藻胶低聚糖价格较高，因此提高古罗糖醛酸低聚糖的生物利用度尤为重要。
脂质体是由具有双亲性的磷脂分子分散于水中自发形成的闭合囊泡，具有亲水性和疏水性，既能包埋水溶性物质又能包埋脂溶性物质，是一种特殊型载体。为了增加脂质体膜的流动性，在脂质体的制备过程中往往会加入胆固醇，胆固醇能够调节脂质体双分子层膜的流动性，在一定程度上能促进药物包封，并提高其稳定性。脂质体作为一种新型药物载体，能够增强药物的稳定性和靶向性，提高药物的生物利用度，降低药物的毒副作用，延长药物在体内的循环时间，达到药物控释缓释的目的。
发明内容
本发明针对古罗糖醛酸低聚糖的生物利用度有待进一步提高的问题，提供一种稳定性好、粒径小、包封率高的古罗糖醛酸低聚糖脂质体，及该种古罗糖醛酸低聚糖脂质体的制备方法，以及该古罗糖醛酸低聚糖脂质体促免疫活性的用途。
为实现上述目的，本发明采用以下技术方案。
本发明的第一方面，提供一种古罗糖醛酸低聚糖脂质体，由古罗糖醛酸低聚糖、脂质体膜、吐温80组成，古罗糖醛酸低聚糖与脂质体膜的质量比为1∶(9-11)，古罗糖醛酸低聚糖与吐温80的质量比为1∶(3.5-4.5)；所述脂质体膜由卵磷脂和胆固醇组成，脂质体膜中胆固醇的含量为18-22％。
优选的，古罗糖醛酸低聚糖与脂质体膜的质量比为1∶10，古罗糖醛酸低聚糖与吐温80的质量比为1∶4；脂质体膜中胆固醇的含量为20％。
本发明的另一方面，提供以上所述的古罗糖醛酸低聚糖脂质体的制备方法，包括以下步骤：
S1、将卵磷脂和胆固醇溶于有机溶剂中，然后除去有机溶剂，形成脂质体膜。
优选的，所述有机溶剂为无水乙醇。
更优选的，通过减压蒸发除去有机溶剂。
将古罗糖醛酸低聚糖和吐温80溶于去离子水中，形成糖溶液；所述糖溶液中，古罗糖醛酸低聚糖的浓度为4.5-5.5mg/mL，吐温80的浓度为18-22mg/mL。
优选的，所述糖溶液中，古罗糖醛酸低聚糖的浓度为5mg/mL，吐温80的浓度为20mg/mL。
S2、将糖溶液转移至盛装脂质体膜的容器中，使脂质体膜悬浮在糖溶液中，得到脂质体悬液。
S3、对脂质体悬液进行超声处理，得到超声悬液。
优选的，在室温下对脂质体悬液进行水浴超声，超声频率为20-30 kHz，超声时间为13-17min。
更优选的，超声时间为15min。
S4、对超声悬液进行均质处理，得到溶于水中的古罗糖醛酸低聚糖脂质体。
优选的，脂质体悬液用高压均质机均质3min，设置压强为60-80 Mpa。
更优选的，脂质体悬液用高压均质机均质时设置压强为80Mpa。
优选的，上述溶于水中的古罗糖醛酸低聚糖脂质体保存于4℃的环境中。
本发明的再一方面，提供以上所述的古罗糖醛酸低聚糖脂质体促免疫活性的用途。
本发明所述的古罗糖醛酸低聚糖脂质体具有促免疫活性的作用，具有用于促免疫活性的用途，可应用于促免疫活性药物、食品等产品的制备。
与现有技术相比，本发明的有益效果是：
本发明通过将所述质量比的古罗糖醛酸低聚糖、卵磷脂、胆固醇和吐温 80制作成脂质体，尤其是制备过程中对形成的脂质体悬液进行超声处理和均质处理时，通过控制超声处理的温度、时间、频率及均质的压强和时间，所制备的古罗糖醛酸低聚糖脂质体形态良好，呈球形，稳定性好，粒径小，包封率较高，古罗糖醛酸低聚糖脂质体的包封率为68.26±2.11％，粒径为 45.69±3.6um。本发明制备的古罗糖醛酸低聚糖脂质体具有促免疫活性的作用。
附图说明
图1为吸光度对古罗糖醛酸低聚糖含量的标准曲线图；
图2为本发明的古罗糖醛酸低聚糖脂质体的粒径分布图；
图3为本发明的古罗糖醛酸低聚糖脂质体的扫描电镜图；
图4为本发明的古罗糖醛酸低聚糖脂质体储藏稳定性的结果；
图5为本发明的古罗糖醛酸低聚糖脂质体温度稳定性结果；
图6为本发明的古罗糖醛酸低聚糖脂质体pH稳定性结果；
图7为本发明的古罗糖醛酸低聚糖脂质体对小鼠单核巨噬细胞 (RAW264.7)细胞活力的影响；
图8为本发明的古罗糖醛酸低聚糖脂质体对RAW264.7细胞分泌NO的影响。
具体实施方式
为了更充分的理解本发明的技术内容，下面结合具体实施例对本发明的技术方案作进一步介绍和说明。
一、实验材料及试剂
卵磷脂、胆固醇、吐温80购于上海麦克林生化公司；无水乙醇购于广东光华科技股份有限公司；小鼠单核巨噬细胞(RAW264.7)购于美国模式培养保藏所；高糖DMEM培养基、青霉素、链霉素、胎牛血清、脂多糖(LPS) 购于Gibco公司；磺胺、萘基乙烯基二胺购于上海生工生物工程股份有限公司。
二、古罗糖醛酸低聚糖脂质体的制备
本发明所述的古罗糖醛酸低聚糖脂质体及其制备方法，具体如下：称取4g卵磷脂和1g胆固醇溶于40mL无水乙醇中，待完全溶解后转移至 500mL圆底烧瓶中，60℃水浴减压蒸发除去有机溶剂，形成脂质体膜。称取0.5g古罗糖醛酸低聚糖和2g吐温80，加去离子水定容至100mL，形成糖溶液。将糖溶液转移到圆底烧瓶中，继续旋转洗膜至瓶壁上的薄膜完全脱落，形成脂质体悬液。将得到的脂质体悬液在室温下超声15min(超声频率20-30kHz)，得到超声悬液。将超声悬液用高压均质机(80Mpa)处理3 min，得到溶于水中的古罗糖醛酸低聚糖脂质体。制备所得溶液置于4℃冰箱保存备用。
三、标准曲线的制作
称取100mg的古罗糖醛酸低聚糖，加去离子水定容至100mL，作为标准溶液，备用。取8支试管并编号，向1至7号管中加1mL去离子水，第 8号管中加入2mL标准溶液，并从中吸取1mL加入到第7号管，以此类推， 1号管为去离子水。向所有试管中加入0.5mL 5％苯酚溶液，再加入2.5mL 浓硫酸溶液，沸水浴20min，冷却到室温后，490nm波长下测其吸光度。以吸光度为横坐标，糖浓度为纵坐标，作图，标准曲线图如图1所示。
四、古罗糖醛酸低聚糖脂质体包封率测定
将1mL古罗糖醛酸低聚糖脂质体稀释10倍后，取3mL稀释液加入到超滤管中(截留分子量：30000Da)，8000rpm，4℃，离心10min，利用硫酸苯酚法分别测定超滤管内外溶液中的糖含量(W外、W内)。根据下面公式计算古罗糖醛酸低聚糖脂质体包封率：
包封率(％)＝(1-W外/W总)×100％(W总＝W外+W内)
本发明的古罗糖醛酸低聚糖脂质体的包封率为68.26±2.11％。
五、古罗糖醛酸低聚糖脂质体粒径测定
将1mL古罗糖醛酸低聚糖脂质体稀释5倍后，利用纳米激光粒度分析仪测定其粒径和多分散指数(PDI)，本发明制备的古罗糖醛酸低聚糖脂质体的粒径分布图如图2所示。本发明制备的古罗糖醛酸低聚糖脂质体的粒径为45.69±3.6nm。
六、本发明制备的古罗糖醛酸低聚糖脂质体扫描电镜图
将制备好的古罗糖醛酸低聚糖脂质体稀释10倍后，滴加到铜网上，待自然风干后，利用扫描电镜观察脂质体形貌特征，图3为本发明制备的古罗糖醛酸低聚糖脂质体的扫描电镜图。
七、古罗糖醛酸低聚糖脂质体储藏稳定性
将古罗糖醛酸低聚糖脂质体放置于4℃的环境中。取放置不同天数的古罗糖醛酸低聚糖脂质体，稀释5倍后，利用纳米激光粒度分析仪测定其粒径，以粒径作为评价脂质体稳定性指标。图4为本发明制备的古罗糖醛酸低聚糖脂质体储藏稳定性的结果。
储藏时间对本发明制备的古罗糖醛酸低聚糖脂质体稳定性影响结果如图4所示，随着储藏时间延长，古罗糖醛酸低聚糖脂质体的粒径逐渐增大，这是因为脂质体是热力学不稳定体系，容易聚集和降解。由图可知，前12天，古罗糖醛酸低聚糖脂质体粒径增加较快，但12天后，粒径基本稳定，可见本发明制备的古罗糖醛酸低聚糖脂质体稳定性较好。
八、古罗糖醛酸低聚糖脂质体温度稳定性
取3mL本发明制备的古罗糖醛酸低聚糖脂质体，分别置于15mL 4、 20、37、55和80℃的水溶液中，100rpm水浴2h，测定脂质体的粒径，本发明制备的古罗糖醛酸低聚糖脂质体温度稳定性结果如图5所示。
温度对古罗糖醛酸低聚糖脂质体的稳定性结果如图5所示，从图中可以看出，随着温度升高，脂质体粒径越大，4℃时脂质体粒径最小，表明4℃下脂质体最稳定。
九、古罗糖醛酸低聚糖脂质体pH稳定性
取3mL本发明制备的古罗糖醛酸低聚糖脂质体，分别置于15ml pH＝2.5、5.0、7.0和8.5的0.01mol/L的PBS(150mM NaCL)缓冲液中，室温处理2h，测定脂质体的粒径，本发明制备的古罗糖醛酸低聚糖脂质体pH稳定性结果如图6所示。
pH对古罗糖醛酸低聚糖脂质体稳定性的影响如图6所示，酸性条件下对脂体粒径影响较大，在pH＝7.0时，质体粒径最小，表明在中性条件下，古罗糖醛酸低聚糖脂质体稳定性好，因此，古罗糖醛酸低聚糖脂质体应保存在中性环境中。
十、本发明制备的古罗糖醛酸低聚糖脂质体对小鼠单核巨噬细胞 (RAW264.7)细胞活力的影响
将RAW264.7细胞接种于96孔板中，每孔细胞数为2×105个，贴壁培养2-6h后，弃去上清，分别加入0.1、0.5、1.0mg/mL的古罗糖醛酸低聚糖脂质体，培养24h，之后每孔加入10μLCCK-8试剂，孵育1h 后，用酶标仪检测在波长450nm下的吸光值。本发明制备的古罗糖醛酸低聚糖脂质体对小鼠单核巨噬细胞(RAW264.7)细胞活力的影响如图7 所示。
古罗糖醛酸低聚糖脂质体对小鼠单核巨噬细胞(RAW264.7)细胞活力的影响结果如图7所示，与对照组相比，甚至高达1mg/mL的古罗糖醛酸低聚糖脂质体溶液，对RAW264.7细胞活力均无明显影响。
十一、本发明制备的古罗糖醛酸低聚糖脂质体对小鼠单核巨噬细胞 (RAW264.7)分泌NO的影响
将RAW264.7细胞接种于96孔板中，每孔细胞数为2×105个，贴壁培养2-6h后，弃去上清，分别加入0.1、0.5、1.0mg/mL的古罗糖醛酸低聚糖脂质体，培养24h。利用Griess法检测细胞培养液中NO2 -的含量，间接获得NO的生成量。具体操作如下：从每孔培养基上清液中吸取50μL细胞悬液，加入50μL磺胺试剂反应5min，再加入50μL 萘基乙烯基二胺试剂，避光孵育5min，然后用酶标仪检测545nm波长下的吸光值。本发明制备的古罗糖醛酸低聚糖脂质体对RAW264.7细胞分泌NO的影响如图8所示。
古罗糖醛酸低聚糖脂质体对小鼠单核巨噬细胞(RAW264.7)分泌NO 的影响如图8所示，古罗糖醛酸低聚糖脂质体能够促进RAW264.7细胞分泌 NO，呈浓度依赖性，且效果要优于同浓度的古罗糖醛酸低聚糖。
以上所述仅以实施例来进一步说明本发明的技术内容，以便于读者更容易理解，但不代表本发明的实施方式仅限于此，任何依本发明所做的技术延伸或再创造，均受本发明的保护。
Claims (6)
1.一种古罗糖醛酸低聚糖脂质体，其特征在于，由古罗糖醛酸低聚糖、脂质体膜、吐温80组成，古罗糖醛酸低聚糖与脂质体膜的质量比为1：10，古罗糖醛酸低聚糖与吐温80的质量比为1：4；所述脂质体膜由卵磷脂和胆固醇组成，脂质体膜中胆固醇的含量为20％；
所述的古罗糖醛酸低聚糖脂质体的制备方法，包括以下步骤：
S1、将卵磷脂和胆固醇溶于有机溶剂中，然后除去有机溶剂，形成脂质体膜；
将古罗糖醛酸低聚糖和吐温80溶于去离子水中，形成糖溶液；所述糖溶液中，古罗糖醛酸低聚糖的浓度为5mg/mL，吐温80的浓度为20mg/mL；
S2、将糖溶液转移至盛装脂质体膜的容器中，使脂质体膜悬浮在糖溶液中，得到脂质体悬液；
S3、对脂质体悬液进行超声处理，得到超声悬液；
S4、对超声悬液进行均质处理，得到溶于水中的古罗糖醛酸低聚糖脂质体。
2.根据权利要求1所述的古罗糖醛酸低聚糖脂质体的制备方法，其特征在于，步骤S1中，所述有机溶剂为无水乙醇。
3.根据权利要求1所述的古罗糖醛酸低聚糖脂质体的制备方法，其特征在于，步骤S1中，通过减压蒸发除去有机溶剂。
4.根据权利要求1所述的古罗糖醛酸低聚糖脂质体的制备方法，其特征在于，步骤S3中，在室温下对脂质体悬液进行水浴超声，超声频率为20-30kHz，超声时间为13-17min。
5.根据权利要1所述的古罗糖醛酸低聚糖脂质体的制备方法，其特征在于，步骤S3中，超声时间为15min。
6.根据权利要求1所述的古罗糖醛酸低聚糖脂质体的制备方法，其特征在于，步骤S4中，脂质体悬液用高压均质机均质3min，设置压强为60-80Mpa。
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