CN114246336B - 一种高刚性的阳离子纳米脂质体及其制备方法 - Google Patents
一种高刚性的阳离子纳米脂质体及其制备方法 Download PDFInfo
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- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/30—Physical treatment, e.g. electrical or magnetic means, wave energy or irradiation
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
- A23P10/00—Shaping or working of foodstuffs characterised by the products
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
- A23P10/00—Shaping or working of foodstuffs characterised by the products
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- A23P10/35—Encapsulation of particles, e.g. foodstuff additives with oils, lipids, monoglycerides or diglycerides
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
- A23P30/00—Shaping or working of foodstuffs characterised by the process or apparatus
- A23P30/20—Extruding
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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Abstract
本发明公开了一种高刚性的阳离子纳米脂质体及其制备方法,所述纳米脂质体包括甾醇类物质、磷脂类物质、阳离子脂质和功能活性物质;其中甾醇类物质包括胆固醇、游离甾醇、甾醇酯或甾醇糖苷中的一种,磷脂类物质为二棕榈酰磷脂酰胆碱(DPPC)、二油酰基卵磷脂(DOPC)其中之一,阳离子脂质为(2,3‑二油酰基‑丙基)‑三甲胺(DOTAP);功能活性物质为原花青素。本发明不仅采用薄膜分散法制备阳离子纳米脂质体,利用不同C3支链植物甾醇代替胆固醇后,获得了相同理化性质和包封率但刚性更大的阳离子纳米脂质体,从而提高了原花青素的生物利用度,更好地发挥了原花青素的美白功效。
Description
技术领域
本发明涉及纳米脂质体制备技术领域,尤其涉及一种高刚性的阳离子纳米脂质体的制备方法。
背景技术
纳米脂质体是一种特殊的营养物质传递系统,它含有一个或多个同心磷脂双层包围的内部水相,可同时包封亲水性、亲脂性和两亲性活性化合物。纳米脂质体热力学的不稳定导致的其物理化学稳定性较差,容易产生不良的膜融合,以及磷脂降解可导致自组装双分子层的解体和囊泡中生物活性化合物的泄漏。此外,在制造、运输和储存过程中不利的环境条件(温度波动、紫外光照射)以及胃分泌期间的高电解质浓度,都会加剧不稳定。然而脂质体除了容易被消化酶水解外,也容易融合聚集,酰基链易暴露,因而其物理稳定性差,在消化吸收过程中容易破裂,其作为口服载体的应用受到很大的限制。
目前解决方法通常是在纳米脂质体膜中添加胆固醇以增加其机械强度。然而过量摄入胆固醇会提高心血管等疾病的患病几率。植物甾醇与胆固醇有相似的类固醇结构,能通过降低肠道胆固醇的吸收来改善心血管疾病。研究人员已证实植物甾醇可以稳定磷脂双分子层,但大多针选取了游离甾醇(谷甾醇、豆甾醇等),对其他C3支链不同的甾醇衍生物如甾醇酯和甾醇糖苷研究较少。并且目前对植物甾醇脂质体的研究大多停留在形态稳定性、粒径分布等方面,并没有通过脂质体的力学性能来比较几者之间的差别。原花青素是一种有效的天然多酚类抗氧化剂,具有多种生理活性,如抗癌、抗菌、抗炎和抑制黑色素形成的作用。然而由于其特殊的化学结构,原花青素具有不稳定性和易降解性。通常选择脂质体技术对原花青素进行封装来解决这些问题。
发明内容
本发明的目的旨在提供了一种高刚性的阳离子纳米脂质体的制备方法,该方法制备过程耗时短、效率高,获得的阳离子纳米脂质体粒径小,稳定性好。添加不同C3支链植物甾醇可以提高阳离子纳米脂质体的刚性,从而提高其生物利用度,为扩展纳米脂质体在食品领域的应用提供重要理论依据。
具体技术方案如下:
一种高刚性的阳离子纳米脂质体,包括甾醇类物质、磷脂类物质、阳离子脂质和功能活性物质,所述甾醇类物质包括胆固醇、游离甾醇、甾醇酯或甾醇糖苷中的一种。
进一步地,所述的磷脂类物质为二棕榈酰磷脂酰胆碱(DPPC)、二油酰基卵磷脂(DOPC)其中之一,阳离子脂质为(2,3-二油酰基-丙基)-三甲胺(DOTAP)。
进一步地,所述的功能活性物质为原花青素。
一种高刚性的阳离子纳米脂质体的制备方法,包括以下步骤:
(1)将甾醇与DPPC或DOPC混合溶于氯仿,添加DOTAP,置于旋转蒸发器上,减压蒸发形成均匀的脂质薄膜。
(2)在40~50℃的温度下,含功能活性物质的葡萄糖水溶液和适量吐温80将干燥的脂质膜再水合,置于旋转蒸发器上,减压蒸发,然后在浴式超声波处理器中超声处理,获得脂质体悬浮液;
(3)将脂质体悬浮液通过装有100nm孔径滤膜的聚碳酸酯过滤器的微型挤出机,在40~50℃的温度下进行挤出,从而获得阳离子纳米脂质体。
进一步地,步骤(1)中的DPPC/DOPC、DOTAP、甾醇类物质的摩尔比为0.5-0.65:0.1:0.25-0.4,甾醇、磷脂和阳离子脂质在氯仿中的总浓度为1mmol/L。
进一步地,步骤(2)中所述的葡萄糖水溶液中,葡萄糖的质量分数为5%,溶液pH值为5.4,吐温80的含量为0.2mg/mL,葡萄糖水溶液的添加量相当于步骤(1)中氯仿体积的1.5倍。
进一步地,步骤(1)和(2)中旋转蒸发的条件为:温度40~50℃,转速40~50rpm。
进一步地,步骤(2)中的功能活性物质为原花青素,其在葡萄糖水溶液中的浓度为0.1-0.4mg/mL。
进一步地,步骤(2)中超声处理的条件为:采用浴式超声波处理器进行持续超声,超声频率为50Hz,功率为240W,超声时间为10min。
进一步地,步骤(3)中的挤出方法为:通过100nm孔径滤膜的聚碳酸酯过滤器的微型挤出机10-15次。
经试验发现,不同C3支链的植物甾醇会影响阳离子纳米脂质体的刚性。其中,步骤(1)中添加甾醇糖苷的阳离子纳米脂质体的刚性最大。
游离甾醇和甾醇糖苷在C3位上的羟基和糖苷均能与磷脂形成氢键,再加上深锚定的甾醇环减少了烃类运动,它们可能会填充脂质双层的极性顶部空间,从而增加膜的稳定性。甾醇酯在C3位上的羰基也可以和磷脂发生氢键结合,从而稳定脂质体膜。
本发明通过薄膜分散法结合聚碳酸酯过滤器制备阳离子纳米脂质体,游离甾醇、甾醇酯和甾醇糖苷都能通过氢键与磷脂双分子层结合形成稳定的结构,因此C3支链植物甾醇可以代替胆固醇作为阳离子纳米脂质体的稳定剂。
与现有技术相比,本发明具有以下有益效果:
(1)本发明采用薄膜分散法结合聚碳酸酯过滤器制备阳离子纳米脂质体,不仅制备过程耗时短、效率高,获得的分散体系粒径较小,具有重力学稳定的性质,而且增加了纳米脂质体的刚性,提高了纳米脂质体的力学性能。
(2)本发明制备一种新的代替胆固醇的植物甾醇纳米脂质体,既克服了传统胆固醇纳米脂质体中的不良作用,又赋予脂质体植物甾醇的有益生理活性。并且通过提高纳米脂质体的刚性,从而提高了包封在内的原花青素的生物利用度及美白效果。
附图说明
图1为实施例1、实施例2、实施例3、实施例4和对比例3制得的阳离子纳米脂质体的透射电镜图(A)和原子力电镜图(B);
图2为实施例1、实施例2、实施例3、实施例4和对比例3制得的阳离子纳米脂质体的杨氏模量图。
图3为实施例1、实施例2、实施例3、实施例4和对比例3制得的阳离子纳米脂质体的傅里叶红外光谱图。
图4为实施例1、实施例2、实施例3、实施例4和对比例3制得的阳离子纳米脂质体在Caco-2细胞的荧光图像(A)和平均荧光强度(B)。
图5为实施例1、实施例2、对比例1和对比例2制得的阳离子纳米脂质体处理后对斑马鱼头部黑色素信号强度表型图(A)和黑色素形成抑制率(B)。
具体实施方式
下面结合具体实施例对本发明作进一步阐释,本发明根据发明技术方案进行实施,给出了详细的实施方式和操作步骤,但本发明的保护范围并不限于下述的实施例。下列实施例中未注明具体条件的实验方法,通常按照常规条件。
实施例1
首先制备一种可调节刚性的阳离子纳米脂质体,具体步骤如下:
(1)将10μmol的总脂溶解在10mL的氯仿中,总脂的成分为DPPC,DOTAP与甾醇糖苷,三者的摩尔比为0.65∶0.1∶0.25,在50℃,40rpm条件下减压蒸发直到在旋蒸瓶的壁上形成薄薄的脂质膜。
(2)在50℃下,用15mL含0.2mg/mL原花青素和0.2mg/mL吐温80的葡萄糖水溶液(葡萄糖质量分数5%,pH=5.3)将干燥的脂质膜再水合,置于旋转蒸发器上,在50℃,40rpm条件下减压蒸发,然后在浴式超声波处理器中超声10min,获得脂质体悬浮液10mL,浓度为6.5mg/mL。
超声条件为:SB-5200DT浴式超声波处理器,超声频率为40kHz,功率为240W,超声时间为10min。
(3)将脂质体悬浮液通过装有100nm孔径滤膜的聚碳酸酯过滤器的微型挤出机11次,在50℃进行挤出,从而获得阳离子纳米脂质体。
取10mL上述阳离子纳米脂质体放入样品瓶中,获取电位、粒径、包封率、透射扫描电镜图、原子力图;纳米分散系冷冻干燥(BTP-3XL真空冷冻干燥机)后的样品,获取傅里叶红外光谱和示差量热扫描图;通过Caco-2细胞摄取实验评价阳离子纳米脂质体的细胞内化率。结果如表1和图1~5所示。
实施例2
本实例除添加的甾醇为游离甾醇,其余内容与实施例1完全相同,结果如表1和图1~5所示。
对比例1
本对比例除添加的甾醇为游离甾醇,在步骤(2)中添加的葡萄糖水溶液中不含有原花青素,其余内容与实施例1完全相同,用来评价阳离子纳米脂质体对斑马鱼黑色素的抑制作用,结果如图5所示。
对比例2
本对比例仅使用阳性对照药3000μg/mL熊果苷对Caco-2细胞进行处理,用来评价阳离子纳米脂质体对斑马鱼黑色素的抑制作用,结果如图5所示。
实施例3
本实例除添加的甾醇为甾醇酯,其余内容与实施例1完全相同,结果如表1和图1~4所示。
实施例4
本实例使用DOPC代替实施例1中的DPPC,其余内容与实施例1完全相同,结果如表1和图1~4所示。
对比例3
本实例除添加的甾醇为胆固醇,其余内容与实施例1完全相同,结果如表1和图1~4所示。
表1不同阳离子纳米脂质体的电位和粒径
由表1知,不同组的纳米脂质体的粒径和电位没有明显区别,这可能是由于脂质体通过聚碳酸酯过滤器后减少了尺寸异质性,粒径均在129-137nm的范围内。Zeta电位测试显示,脂质混合物中电荷脂质的影响很大,导致含DOTAP的纳米脂质体的Zeta电位较高。实验中所制备的所有纳米脂质体的Zeta电位都带正电,Zeta电位在30.4-36.5mV,表明了所有纳米脂质体都处于稳定阶段。不同组的纳米脂质体对原花青素的包封率没有明显差异,包封率在80.02%-84.97%。原花青素具有一定的亲脂性,能与磷脂的疏水性尾部结合,使它们位于卵磷脂双层内,这可能就是原花青素具有相对较高的保留率的原因。
如图1A所示,通过透射电镜可以观察到一些球形或椭圆形的纳米脂质体囊泡,直径在100nm-200nm之间,并且不同组之间的纳米脂质体没有明显区别。在图B的原子力图中观察到同样的形貌特征。
如图2所示,实施例1的杨氏模量达到126.27±2.06MPa,高于实施例2的56.76±4.46MPa,说明实施例1的刚性大于实施例2。实施例3和实施例4的杨氏模量高于对比例3,说明了使用植物甾醇代替胆固醇可以增加纳米脂质体的刚性。
如图3所示,通过傅里叶红外光谱图发现,CH、FS的羟基和SG的糖苷均能与磷脂形成氢键,降低P=O键的极性,导致PO2-红移。实施例1中的PO2-红移(1243cm-1)比实施例2的PO2-红移(1248cm-1)程度大,说明了SG对PO2-的振动影响强。这可能是由于SG糖苷上含有丰富的羟基(FS只有一个羟基),因此SG对PO2-的红移影响强。由于SG与磷脂极性头部的氢键作用最强,因此在通过AFM得到各组纳米脂质体刚性时,发现实施例1的杨氏模量最大。原花青素在1550到1700cm-1之间的光谱区域存在C=C的红外吸收,在1616cm-1处的峰对应于C=C伸缩振动,并且可以与花青素中芳香族C=C的伸缩相关联。在1520cm-1处的峰对应芳香族C=C拉伸。在各组包封原花青素的阳离子纳米脂质体中,在1645cm-1、1647cm-1、1643cm-1、1649cm-1和1645cm-1处出现吸收峰,这可能是通过脂质的极性头部和原花青素的酚羟基团之间发生氢键结合,使原花青素结合至磷脂双分子层中间。这些信息表明了原花青素被包封在纳米脂质体中,并且会与磷脂双分子层相互作用。
如图4A所示,通过Caco-2细胞摄取刚性不同的纳米脂质体发现,与实施例2相比,刚性大的实施例1更容易被细胞摄取内化,定量结果与定性分析一致(图4B)。实例3和实例4的细胞内化也高于对比例3,说明了使用植物甾醇代替胆固醇可以增加纳米脂质体的细胞内化率。
如图5A所示,通过观察斑马鱼头部黑色素,相比于实施例2,发现用实施例1喂养的斑马鱼头部黑色素斑点数量和面积减少。在图5B中可以看到实施例1处理后,斑马鱼黑色素的抑制率可以达到42.08±13.50%,高于实施例2的29.71±8.37%。这说明了刚性大的实施例1有较高的生物利用度,能使原花青素发挥最佳的美白效果。对比例1的黑色素抑制率最低,是因为没有包封原花青素,从而排除了植物甾醇存在美白活性的可能性。对比例2对斑马鱼黑色素的抑制率也很高,达到46.03±9.77%,与实施例1没有显著性差异。表明了经过纳米脂质体的包封,低浓度的原花青素可以达到与阳性对照药熊果苷相同的美白效果。
Claims (9)
1.一种高刚性的阳离子纳米脂质体,其特征在于,包括甾醇类物质、磷脂类物质、阳离子脂质和功能活性物质,所述甾醇类物质包括游离甾醇、甾醇酯或甾醇糖苷中的一种;所述磷脂类物质为二棕榈酰磷脂酰胆碱DPPC或二油酰基卵磷脂DOPC其中之一,所述阳离子脂质为(2,3-二油酰基-丙基)-三甲胺DOTAP, DPPC/DOPC、DOTAP、甾醇类物质的摩尔比为0.5-0.65:0.1:0.25-0.4。
2.如权利要求1所述的高刚性的阳离子纳米脂质体,其特征在于,所述的功能活性物质包括原花青素。
3.如权利要求1所述的高刚性的阳离子纳米脂质体的制备方法,其特征在于,包括以下步骤:
(1)将甾醇类物质、磷脂类物质与阳离子脂质混合溶于氯仿,置于旋转蒸发器上,减压蒸发,形成均匀的脂质薄膜;
(2)在40~50 ℃下,将含功能活性物质和吐温80的葡萄糖水溶液加入到所述脂质薄膜中,将脂质薄膜再水合,置于旋转蒸发器上,减压蒸发,然后超声处理,获得脂质体悬浮液;
(3)在40~50 ℃的温度条件下,将脂质体悬浮液通过聚碳酸酯过滤器进行挤出,从而获得阳离子纳米脂质体。
4.如权利要求3所述的高刚性的阳离子纳米脂质体的制备方法,其特征在于,步骤(1)中的甾醇类物质、磷脂类物质与阳离子脂质在氯仿中的总浓度为1 mmol/L。
5.如权利要求4所述的高刚性的阳离子纳米脂质体的制备方法,其特征在于,步骤(2)中所述的葡萄糖水溶液中,葡萄糖的质量分数为5%,溶液pH值为5.4,吐温80的含量为0.2mg/mL,葡萄糖水溶液的添加量相当于步骤(1)中氯仿体积的1.5倍。
6.如权利要求3所述的高刚性的阳离子纳米脂质体的制备方法,其特征在于,步骤(1)和(2)中减压蒸发的条件为:温度40~50 ℃,转速40~50 rpm。
7.如权利要求3所述的高刚性的阳离子纳米脂质体的制备方法,其特征在于,步骤(2)中的功能活性物质为原花青素,其在葡萄糖水溶液中的浓度为0.1-0.4 mg/mL。
8.如权利要求3所述的高刚性的阳离子纳米脂质体的制备方法,其特征在于,步骤(2)中超声处理的条件为:采用浴式超声波处理器进行持续超声,超声频率为50 Hz,功率为240W,超声时间为10 min。
9.如权利要求3所述的高刚性的阳离子纳米脂质体的制备方法,其特征在于,步骤(3)中的挤出方法为:通过100 nm孔径滤膜的聚碳酸酯过滤器的微型挤出机10-15次。
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