WO2021244597A1 - 一种脂肪酸基vc脂质体的制备方法 - Google Patents

一种脂肪酸基vc脂质体的制备方法 Download PDF

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WO2021244597A1
WO2021244597A1 PCT/CN2021/098057 CN2021098057W WO2021244597A1 WO 2021244597 A1 WO2021244597 A1 WO 2021244597A1 CN 2021098057 W CN2021098057 W CN 2021098057W WO 2021244597 A1 WO2021244597 A1 WO 2021244597A1
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fatty acid
acid
fatty acids
liposomes
mass
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夏咏梅
柳欢
孟新宇
方云
樊晔
沈洁
刘湘
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Jiangnan University
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    • A—HUMAN NECESSITIES
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    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1277—Preparation processes; Proliposomes
    • A61K9/1278—Post-loading, e.g. by ion or pH gradient
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A61K31/375—Ascorbic acid, i.e. vitamin C; Salts thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/03—Organic compounds
    • A23L29/035—Organic compounds containing oxygen as heteroatom
    • A23L29/04—Fatty acids or derivatives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/03—Organic compounds
    • A23L29/055—Organic compounds containing sulfur as heteroatom
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/115—Fatty acids or derivatives thereof; Fats or oils
    • A23L33/12—Fatty acids or derivatives thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/15—Vitamins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/20—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
    • A61K8/14—Liposomes; Vesicles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/36—Carboxylic acids; Salts or anhydrides thereof
    • A61K8/361—Carboxylic acids having more than seven carbon atoms in an unbroken chain; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/46—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing sulfur
    • A61K8/463—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing sulfur containing sulfuric acid derivatives, e.g. sodium lauryl sulfate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/67—Vitamins
    • A61K8/676—Ascorbic acid, i.e. vitamin C
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/02—Nutrients, e.g. vitamins, minerals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • 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
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the invention relates to a preparation method of fatty acid-based VC liposomes, which belongs to the field of pharmaceutical preparations.
  • Vitamin C also known as L-ascorbic acid
  • VC is the main ingredient of commonly used food additives and cosmetics, and can be used as vitamin supplements and antioxidants in cosmetics.
  • VC is very unstable in neutral or alkaline aqueous solutions.
  • Figure 1 At 25°C, when the initial concentration of VC in deionized water is 0.27mg/mL, after 12h, VC is degraded by 29%; when the temperature is higher than 50°C, VC passes through in deionized water. After 6h, the degradation rate of VC can reach 80%.
  • Liposome is a closed system formed spontaneously by dispersing amphiphilic molecules in an aqueous solution.
  • the most typical one is liposome composed of natural phospholipids and cholesterol. It has good biocompatibility and degradability, and is often used for encapsulation. Biologically active substances.
  • the problem of liposome encapsulation of VC is that the raw materials such as lecithin and cholesterol are expensive; the preparation method is complicated, the steps are long, and organic solvents are required; most of the above liposomes encapsulate VC at pH Performed in a neutral environment.
  • VC is extremely unstable in an environment above neutral pH, and VC can be effectively encapsulated and stabilized only under acidic conditions.
  • Conjugated linoleic acid is a single substance or a mixture of various octadecadienoic acid isomers. It is either naturally occurring or conjugated from plant-derived linoleic acid or linoleic acid.
  • CLA and other fatty acids have good compatibility with food and cosmetics, and are beneficial to human health. Using them to encapsulate VC can do two things with one stone. However, since both CLA and fatty acids can only self-assemble to form a liposome structure in an alkaline environment (pH 8.5 ⁇ 0.5), and the liposomes formed by CLA itself are very small, the encapsulation efficiency will be low. In order to achieve the encapsulation of VC, it is particularly important to make the pH range formed by fatty acid liposomes shift to the acidic direction and increase the liposome size.
  • the technical problem to be solved by the present invention is that vitamin C is stable under acidic conditions, while the existing methods of using liposomes to embed vitamin C need to be carried out under neutral or alkaline conditions, which will destroy the stability of vitamin C , And the encapsulation efficiency is low.
  • the present invention provides a method for preparing fatty acid-based vitamin C liposomes, which uses a complex of industrial conjugated linoleic acid, other fatty acids, and sodium lauryl sulfate as a capsule material.
  • the capsule material is exposed to water under acidic conditions.
  • Phase self-assembly encapsulates vitamin C to form fatty acid-based vitamin C liposomes; the molecular formula of the other fatty acids conforms to the general formula C n H 2n+1 COOH, where n is a positive integer greater than 7;
  • step (2) Under stirring conditions, add VC aqueous solution dropwise to the suspension obtained in step (1), and after the dropwise addition, shake at 20-50 rpm for 10 min-1 h.
  • the fatty acids in the fatty acid group include conjugated linoleic acid (CLA), other fatty acids, and various carboxyl-containing organic acids with 8 or more carbon atoms in the molecule contained in industrial conjugated linoleic acid raw materials.
  • CLA conjugated linoleic acid
  • the other fatty acid is a single or mixed fatty acid whose molecular formula conforms to the general formula C n H 2n+1 COOH or C n H 2n COOH or C n H 2n-1 COOH or C n H 2n-3 COOH, where n is greater than A positive integer of 7.
  • the industrial conjugated linoleic acid is a product obtained by the alkali-catalyzed conjugation of food-grade industrial linoleic acid, and the conjugated linoleic acid is a single substance or a mixture of various octadecadienoic acid isomers And, the total octadecadienoic acid isomer content accounts for 70%-100% of the industrial conjugated linoleic acid dry matter (calculated based on the gas chromatographic peak area percentage).
  • the mass of the other fatty acids is 1%-40% of the mass of the total octadecadienoic acid isomers.
  • the sum of the final concentration of the fatty acid in the self-assembly solution is 10-500 mM.
  • the added amount of the SDS is 5% or 10% of the mass of the total fatty acid.
  • the final concentration of the VC in the suspension system after the dripping is completed is 1-50 mg/mL.
  • the fatty acid self-assembles and encapsulates the VC under the conditions of 5-50°C and 10min-2h.
  • VC is very unstable in neutral or alkaline aqueous solutions.
  • the invention provides a method for preparing fatty acid-based VC liposomes in acidic water.
  • the materials used have good biocompatibility, simple process and easy-to-obtain raw materials.
  • the present invention widens the formation range of fatty acid liposomes by adding sodium dodecyl sulfate (SDS) to fatty acids, which can not only make CLA, but also make the pH of other fatty acids form liposomes move to an acidic environment, and make it It can self-assemble to form liposomes under acidic conditions to encapsulate VC.
  • SDS sodium dodecyl sulfate
  • the present invention can enhance the size of liposomes and improve the encapsulation efficiency. Moreover, the higher the saturation of other fatty acids added, the larger the particle size of the liposomes formed.
  • the fatty acid liposome prepared by the present invention can not only encapsulate VC under acidic conditions, but also can release VC slowly.
  • the carrier fatty acid used in the present invention is one of the essential fatty acids for the human body, and the synergistic effect with VC can further promote the absorption of the two, and can be used in food or cosmetics. Moreover, the preparation process of the invention does not use organic solvents and other harmful substances to the human body, and has the characteristics of safety and health.
  • FIG. 1 TEM image of fatty acid-based VC liposomes, (A): palmitic acid-CLA-VC, (B): stearic acid-CLA-VC.
  • Figure 3 VC release curve of VC aqueous solution and mixed fatty acid-based VC liposomes in water,
  • A 1mg/mL VC aqueous solution
  • B palmitic acid-CLA(10mM)-VC(1mg/mL) liposomes
  • C Palmitic acid-CLA(100mM)-VC(2mg/mL) liposome
  • D Stearic acid-CLA(100mM)-VC(5mg/mL) liposome
  • E Palm Acid-stearic acid-CLA (10mM)-VC (5mg/mL) liposomes.
  • Chromatographic conditions a) Capillary chromatographic column: polydicyanopropylsiloxane strong polar stationary phase, column length 100m, inner diameter 0.25mm, film thickness 0.2 ⁇ m. b) Sampler temperature: 270°C. c) Detector temperature: 280°C.
  • the fatty acid standard measurement solution and the sample to be tested are respectively injected into the gas chromatograph, and the content of each fatty acid is quantitatively calculated based on the chromatographic peak area.
  • Morphological characterization of fatty acid-based VC liposomes characterization by TEM images. Grasp the copper mesh with tweezers and immerse the copper mesh in a 10mM fatty acid-based VC liposome dispersion for 2 seconds, then quickly remove the copper mesh and freeze it in liquid nitrogen, and then transfer the copper mesh to a freeze dryer After drying for 24 hours, observe the morphology of fatty acid-based VC liposomes on copper mesh with TEM (120kV) and take pictures.
  • VC encapsulation rate and sustained release rate respectively prepare VC (L-ascorbic acid) aqueous solutions with a certain concentration interval and the concentration range of 2-30 ⁇ g ⁇ mL -1 , and measure the absorbance of the corresponding solution at 260nm (A 260 ) , Draw the concentration-absorbance standard curve of VC aqueous solution.
  • the absorbance (A 260 ) of the solution outside the dialysis bag of the fatty acid-based VC liposome sample was measured at 260 nm, and the concentration of VC in the solution outside the dialysis bag was calculated using the concentration-absorbance standard curve of the VC aqueous solution.
  • the difference between the amount of VC used to prepare fatty acid-based VC liposomes and the amount of free VC released by dialysis is the amount of encapsulated VC. Therefore, in fatty acid-based VC liposomes, the encapsulation efficiency (EE) of fatty acid liposomes for VC can be calculated using the following formula:
  • m 1 is the amount of VC in the initial dialysis bag where the fatty acid-based VC liposomes are located (that is, the amount of VC used to prepare 5 mL of fatty acid-based liposomes);
  • m 2 is the amount of VC in the solution outside the dialysis bag after 1 hour of dialysis .
  • the amount of VC released from the fatty acid-based VC liposomes can be calculated, thereby calculating the cumulative release rate of VC in the fatty acid-based VC liposomes.
  • the calculation formula is as follows:
  • c n and c i the concentration of VC in the dialysate outside the dialysis bag at the nth sampling, n ⁇ 1;
  • V 0 The volume of dialysate outside the dialysis bag when the VC is released;
  • V s the volume of each sampling
  • m The quality of VC in fatty acid-based VC liposomes.
  • the fatty acid composition (wt%) of 95% industrial CLA is: 93.5% of total stearidienic acid isomers, oleic acid 3.7%, palmitic acid 0.7%, stearic acid 0.4%, linoleic acid 1.7%, and its average molecular weight is 278.73.
  • the fatty acid composition (wt%) of 80% industrial CLA is: 81.1% of total octadecadienoic acid isomers, 9.8% of oleic acid, 5.3% of palmitic acid, 2.5% of stearic acid, and 1.3% of linoleic acid.
  • the molecular weight is 278.71.
  • Example 1 VC liposomes prepared with palmitic acid-CLA-SDS
  • the concentration of fatty acid was 10 mmol/L, and the concentration of VC was 1 mg/mL.
  • the measured VC encapsulation rate is 53.5%.
  • the morphology of the obtained VC liposome is shown in Figure 2A.
  • step (1) If, in step (1), the pH of the mixture system is adjusted to 7.4 or 6.0, liposomes can be formed, and the encapsulation efficiency of VC is 23.6% and 29.7%, respectively.
  • Example 2 VC liposomes prepared with palmitic acid-CLA-SDS
  • the concentration of fatty acid is 100 mmol/L
  • the concentration of VC is 5 mg/mL
  • the measured VC encapsulation efficiency is 64.1%.
  • the morphology of fatty acid-based VC liposomes is shown in Figure 2B.
  • Example 1 Compared with Example 1, it can be seen that the increase in fatty acid concentration can increase the VC encapsulation efficiency.
  • Example 3 VC liposomes prepared with stearic acid-CLA-SDS
  • the concentration of fatty acid is 100 mmol/L
  • the concentration of VC is 5 mg/mL
  • the encapsulation rate of VC is 66.5%.
  • Example 1 Compared with Example 1, it can be seen that the increase in the carbon number of saturated fatty acids will slightly increase the size of the self-assembly, which is beneficial to increase the VC encapsulation efficiency.
  • Example 4 VC liposomes prepared with palmitic acid-stearic acid-CLA
  • 0.1g mixed palmitic acid-stearic acid (mass ratio 1:1) was added to 1.3g 80% industrial CLA (CLA average molecular weight 278.71), and palmitic acid-stearic acid-CLA was shaken at 60°C and 120rpm 60min. Then, 250 mL of the SDS aqueous solution (the mass concentration of the SDS aqueous solution is 0.056%, containing SDS 0.14 g) is added to the above-mentioned mixed fatty acid sample, and simply mixed to obtain a mixture system. Then, adjust the pH of the mixture system to pH 3.8 with dilute hydrochloric acid, and obtain a suspension after homogenization.
  • the concentration of fatty acid is 10 mmol/L, and the concentration of VC is 5 mg/mL.
  • the encapsulation rate of VC is 58.4%.
  • this example uses a common aqueous medium to analyze the sustained release of VC in different fatty acid-based VC liposomes.
  • the fatty acid-based VC liposome prepared by the process of the above-mentioned embodiment was used to analyze the sustained release of VC by dialysis with a general aqueous medium. The results are shown in Figure 3.
  • the cumulative release rate of VC in the control sample (1mg/mL VC aqueous solution) was 95.5%; stearic acid-CLA(100mM)-VC(5mg/mL) prepared in Example 3
  • the cumulative release rate of VC in liposomes was 43.2%, and the cumulative release rate of VC in palmitic acid-CLA(100mM)-VC(2mg/mL) liposomes prepared in the same way as in Example 3 was 36.5%; description Short-chain fatty acids tend to delay the release of VC.
  • the VC release rate in the palmitic acid-CLA(10mM)-VC (1mg/mL) liposomes prepared in Example 1 was 40.9%, indicating that low concentrations of CLA accelerated the release of VC; while the palmitic acid prepared in Example 4-
  • the cumulative release rate of VC in stearic acid-CLA(10mM)-VC(5mg/mL) liposomes is 48.9%, because the concentration of high concentration of VC can accelerate the release of VC.
  • Preparation of the oil phase mix 5 g of monoglycerides, 1 g of mink oil, 15 g of sterols, 2 g of Centella asiatica 50% ethanol extract and 5 g of liquid paraffin at 60°C to obtain the oil phase;
  • the industrial conjugated linoleic acid raw materials used in the above embodiments are commercially available products, which can be purchased from many companies such as Dalian Yinuo Biotechnology Co., Ltd. and Qingdao Haizhiyuan.

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Abstract

一种脂肪酸基VC脂质体的制备方法，以工业共轭亚油酸和其它脂肪酸以及十二烷基硫酸钠的复合物为囊材，囊材在酸性条件(pH＜7)下于水相自组装包埋维生素C形成脂肪酸基维生素C脂质体。所述制备方法不会用到有机溶剂等对人体有害的物质，具有安全、健康的特性，制备得到的脂肪酸脂质体除了能够在酸性条件下包封VC，同时还能对VC起到缓释的作用。

Description

一种脂肪酸基VC脂质体的制备方法 技术领域
本发明涉及一种脂肪酸基VC脂质体的制备方法，属于医药制剂领域。
背景技术
维生素C(Vitamin C，VC)，又称L-抗坏血酸，是常用的食品添加剂和化妆品的主要成分，可以作为维生素补充剂和化妆品中的抗氧化剂。然而，VC在中性或碱性的水溶液中很不稳定。如附图1所示：在25℃下，VC在去离子水中的初始浓度为0.27mg/mL时，12h后，VC降解了29％；当温度高于50℃时，VC在去离子水中经过6h后，VC降解率可达80％。
因此，VC的贮藏稳定性是一个需要解决的问题。为增强VC的贮藏稳定性，用各种壁材，如多糖、蛋白质、聚乙烯或聚乙二醇，以及各种脂质体包封VC的技术应运而生。
脂质体是由双亲性分子分散在水溶液中自发形成的闭合体系，其中最典型的是天然磷脂和胆固醇组成的脂质体，其具有良好的生物相容性和可降解性，常用于包封生物活性物质。脂质体包封VC已经有不少报道。例如，Farhang等人(Farhang,2012)采用微射流技术制备磷脂纳米乳脂质体，对抗坏血酸的包埋效率达到26％。杨水兵等人(杨,2010)以卵磷脂和胆固醇为膜材(总脂质：VC＝25:1)，采用薄膜-超声法制备VC脂质体，制备出的VC脂质体最佳条件下包封率可达42.1％。鲍士宝等人(鲍,2013)以卵磷脂和胆固醇为膜材(总脂质：VC＝5:2)，通过乙醇注入法在pH 6.5、0.05mol/L的Na 2HPO 4-KH 2PO 4的缓冲盐中制备VC纳米脂质体悬浮液，VC包封率达到71.4％。
除了以上的传统脂质体外，还有多糖修饰的脂质体。例如，邹雪坤等人(邹,2017)将壳聚糖和海藻酸钠两种多糖通过静电相互作用，pH 5.5下层层自组装修饰在包封VC的传统脂质体表面，形成新型多聚物运载体系。Zhang等人(Zhang,2019)通过热熔挤出技术制备了麦芽糊精、麦芽糊精阿拉伯胶和麦芽糊精海藻糖基抗坏血酸(分散相)玻璃挤出物，所有挤出物的玻璃化转变温度(Tg)均在40℃以上，抗坏血酸则均匀地分散在玻璃基质中。
以上举例中，脂质体包封VC的问题在于：所用的卵磷脂和胆固醇等原料价格较高；制备方法复杂，步骤较长，需要有机溶剂；以上脂质体对VC的包封大多在pH偏中性环境下进行。然而，VC在pH中性以上环境中极其不稳定，只有在酸性条件下才能有效地包封和稳定VC。
因此，需要提供一种成本低、简单易得又绿色环保的方法来在酸性条件下稳定包封VC的脂质体囊材。
共轭亚油酸(CLA)是各种十八碳二烯酸异构体的单一物质或者混合物，既天然存在也可从植物源亚油酸或亚麻油酸共轭化而来。CLA和其它脂肪酸对食品和化妆品都有良好的相容性，对人类健康有益，用它们包封VC一举两得。然而，由于CLA和脂肪酸都只能在碱性环境(pH 8.5±0.5)自组装形成脂质体结构，而且CLA本身形成的脂质体很小，包封效率就会低。为了实现对VC的包封，使脂肪酸脂质体形成的pH范围向酸性方向迁移并且脂质体尺寸增大显得尤为重要。
发明内容
[技术问题]
本发明要解决的技术问题是，维生素C在酸性条件下稳定，而现有的利用脂质体包埋维生素C的方法需要在中性或碱性条件下进行，这会破坏维生素C的稳定性，且包封效率低。
[技术方案]
本发明提供了一种制备脂肪酸基维生素C脂质体的方法，是以工业共轭亚油酸和其它脂肪酸以及十二烷基硫酸钠的复合物为囊材，囊材在酸性条件下于水相自组装包埋维生素C形成脂肪酸基维生素C脂质体；所述其它脂肪酸的分子简式符合通式C nH 2n+1COOH，其中n为大于7的正整数；
包括以下步骤：
(1)将工业共轭亚油酸和其它脂肪酸混合均匀，向其中添加SDS水溶液，SDS的加入量为总脂肪酸的质量的5-10％，用稀酸调整自组装液的pH至pH 3.5-4.0，然后均质得到悬浊液；
(2)在搅拌条件下，向步骤(1)得到的悬浊液中滴加VC水溶液，滴加结束后，在20-50rpm下振摇10min-1h。
所述脂肪酸基中的脂肪酸是包括共轭亚油酸(CLA)、其它脂肪酸以及工业共轭亚油酸原料中含有的各种含羧基的分子中碳数在8以上的有机酸。所述其它脂肪酸是分子简式符合通式C nH 2n+1COOH或C nH 2nCOOH或C nH 2n-1COOH或C nH 2n-3COOH的单一或混合脂肪酸，其中n为大于7的正整数。
所述工业共轭亚油酸是通过食品级工业亚油酸的碱催化共轭化所得的产品，所述共轭亚油酸是各种十八碳二烯酸异构体的单一物质或者混合物，且，总十八碳二烯酸异构体含量占所述工业共轭亚油酸干物质的质量百分比为70％-100％(以气相色谱峰面积百分比计算)。
所述其它脂肪酸的质量为总十八碳二烯酸异构体质量的1％-40％。
所述脂肪酸在自组装液中的终浓度之和为10-500mM。
所述SDS的加入量为总脂肪酸的质量的5％或10％。
所述VC在滴加完毕后在悬浊液体系中的终浓度为1-50mg/mL。
在本发明的可选的实施方式中，在5-50℃、10min-2h条件下脂肪酸进行自组装并包封VC。
[有益效果]
VC在中性或碱性水溶液中很不稳定。本发明提供了一种在酸性水中制备脂肪酸基VC脂质体的方法，所用材料生物相容性好，过程简单、原料易得。
本发明通过向脂肪酸中加入十二烷基硫酸钠(SDS)，拓宽了脂肪酸脂质体的形成范围，不仅可以使CLA，也可以使其它脂肪酸形成脂质体的pH向酸性环境迁移，使其能够在酸性条件下自组装形成脂质体来包封VC。
把本发明通过加入一定量的其它脂肪酸，特别是饱和脂肪酸，可以增强脂质体的尺寸，提高包封效率。而且所加入的其它脂肪酸的饱和度越高，形成的脂质体粒径越大。
本发明制备的脂肪酸脂质体除了能够在酸性条件下包封VC，同时还能对VC起到缓释的作用。
本发明所用的载体脂肪酸本身就是人体必需的脂肪酸之一，与VC的协同效应更能促进二者的吸收，可用于食品或化妆品中。而且，本发明制备过程中不会用到有机溶剂等对人体有害的物质，具有安全、健康的特性。
附图说明
图1 VC水溶液中在不同温度下VC质量百分数随时间的变化。
图2 脂肪酸基VC脂质体的TEM图，(A)：棕榈酸-CLA-VC，(B)：硬脂酸-CLA-VC。
图3 VC水溶液和混合脂肪酸基VC脂质体在水中的VC释放曲线，(A)：1mg/mLVC水溶液，(B)：棕榈酸-CLA(10mM)-VC(1mg/mL)脂质体，(C)：棕榈酸-CLA(100mM)-VC(2mg/mL)脂质体，(D)：硬脂酸-CLA(100mM)-VC(5mg/mL)脂质体，(E)：棕榈酸-硬脂酸-CLA(10mM)-VC(5mg/mL)脂质体。
具体实施方式
分析方法：
1.CLA中脂肪酸组成的测定：气相色谱法检测其甲酯衍生物。依据GB5009.168—2016食品安全国家标准食品中脂肪酸的测定(气相色谱内标法)：在0.2g CLA中加入2％氢氧化钠甲醇溶液8mL，连接回流冷凝器，80℃±1℃水浴上回流，直至油滴消失；从回流冷凝器上端加入7mL 15％三氟化硼甲醇溶液，在80℃±1℃水浴中继续回流 2min；用少量水冲洗回流冷凝器，停止加热，从水浴上取下烧瓶，迅速冷却至室温；准确加入10mL～30mL正庚烷，振摇2min,再加入饱和氯化钠水溶液，静置分层；吸取上层正庚烷提取溶液大约5mL，移至25mL试管中，加入大约3g～5g无水硫酸钠，振摇1min，静置5min，吸取上层溶液到进样瓶中等待测定。色谱条件：a)毛细管色谱柱：聚二氰丙基硅氧烷强极性固定相，柱长100m，内径0.25mm，膜厚0.2μm。b)进样器温度：270℃。c)检测器温度：280℃。d)程序升温：初始温度100℃，持续13min；以升温速率10℃/min升温至180℃，保持6min；以升温速率1℃/min升温至200℃，保持20min；以升温速率4℃/min升温至230℃，保持10.5min。e)载气：氮气。f)分流比：100：1。g)进样体积：1.0μL。h)检测条件应满足理论塔板数(n)至少2000/m，分离度(R)至少1.25。
在上述色谱条件下将脂肪酸标准测定液及待测样品分别注入气相色谱仪，以色谱峰峰面积定量计算出各脂肪酸含量。
2.脂肪酸基VC脂质体的形貌表征：用TEM影像表征。用镊子夹取铜网并将铜网浸入浓度为10mM的脂肪酸基VC脂质体分散液中2秒，然后迅速将铜网取出并冷冻于液氮中，再将铜网转移至冷冻干燥机中干燥24h，用TEM(120kV)观察铜网上脂肪酸基VC脂质体的形貌并拍照。
3.VC包封率及缓释率：分别配制有一定浓度间隔的、浓度区间为2-30μg·mL -1的VC(L-抗坏血酸)水溶液，测量相应溶液在260nm处的吸光度(A 260)，绘制VC水溶液的浓度-吸光度标准曲线。
将5mL脂肪酸基VC脂质体溶液置于透析袋(MW3500)中，将透析袋放置于装有200mL去离子水的250mL烧杯中，在25℃下搅拌进行透析。同时用与此脂肪酸基VC脂质体溶液中同样终浓度的VC溶液作为对照样。透析1h后，对照样中的VC浓度与透析介质(200mL去离子水)中的VC浓度达到平衡，可以认为脂肪酸基VC脂质体样品中的游离VC也已经被透析出来。此时测量脂肪酸基VC脂质体样品透析袋外溶液在260nm处的吸光度(A 260)，利用VC水溶液的浓度-吸光度标准曲线计算透析袋外溶液中VC的浓度。制备脂肪酸基VC脂质体所使用的VC的量，与透析释放出来的游离VC的量的差，即为被包封VC的量。因此，脂肪酸基VC脂质体中，脂肪酸脂质体对VC的包封率(EE)可采用如下公式进行计算：
Figure PCTCN2021098057-appb-000001
m 1为脂肪酸基VC脂质体所在初始透析袋中VC的量(即为制备5mL脂肪酸基脂质体所 使用的VC的量)；m 2为透析1h后，透析袋外溶液中VC的量。
将上述被透析处理1h后的脂肪酸基VC脂质体置于另一装有新鲜的200mL去离子水或缓冲液的250mL烧杯中进行透析，每隔一定时间，从透析袋外的透析液中取出3mL溶液，立即测量这3mL溶液在紫外分光光度计上260nm处的吸光度，并及时向烧杯中补充等体积新鲜的透析液(去离子水或缓冲液)。通过测定透析袋外的透析液中的VC含量，可以计算从脂肪酸基VC脂质体中释放出的VC的量，从而计算脂肪酸基VC脂质体中VC的累计释放率，计算公式如下：
Figure PCTCN2021098057-appb-000002
c n和c i——第n次取样时，透析袋外的透析液中的VC的浓度，n≥1；
V 0——开始释放VC时透析袋外的透析液的体积；
V s——每次取样的体积；
m——脂肪酸基VC脂质体中VC的质量。
以下实施例中，95％工业CLA的脂肪酸组成(wt％)为：总十八碳二烯酸异构体93.5％、油酸3.7％、棕榈酸0.7％、硬脂酸0.4％、亚油酸1.7％，其平均分子量为278.73。
80％工业CLA的脂肪酸组成(wt％)为：总十八碳二烯酸异构体81.1％、油酸9.8％、棕榈酸5.3％、硬脂酸2.5％、亚油酸1.3％，其平均分子量为278.71。
实施例1用棕榈酸-CLA-SDS制备的VC脂质体
步骤(一)
60℃下，将0.1g棕榈酸和1.3g 95％的工业CLA(CLA平均分子量278.73)混合均匀，然后将混合后的CLA-棕榈酸加到250mL SDS水溶液(SDS水溶液的质量浓度0.056％，含SDS 0.14g)中，简单混匀，得到混合物体系。用稀盐酸调整混合物体系的pH至pH3.5，均质后得到悬浊液。
步骤(二)
在25℃、150rpm下边搅拌边向上述悬浊液中滴加2mg/mL的VC水溶液250mL，VC水溶液在30min内滴加完毕；将滴加了VC后的混合的溶液在50rpm、25℃下振摇30min。
所得的脂肪酸基VC脂质体溶液中，脂肪酸的浓度为10mmol/L，VC浓度为1mg/mL。测得VC的包封率为53.5％。所得VC脂质体形貌如附图2A所示。
对照例1用去离子水替代SDS，其余条件同实施例1
步骤(一)
60℃下，将0.1g棕榈酸加入到1.3g 95％的工业CLA(CLA平均分子量278.73)中，混合均匀。然后将混合后的CLA-棕榈酸加到250mL去离子水溶液中，简单混匀，得到混合物体系。然后，用稀盐酸调整混合物体系的pH至pH3.5，均质后得到悬浊液。
步骤(二)
在25℃、150rpm下边搅拌边向上述悬浊液中滴加2mg/mL的VC水溶液250mL，于30min内完成滴加的动作，使得含VC的混合体系中脂肪酸的最终浓度为10mmol/L，VC浓度为1mg/mL。将含VC的混合体系在50rpm、25℃下振摇30min。
振摇结束后，用显微镜观察，观察结果显示此时脂肪酸未形成脂质体，因而VC没有被包封。
对照例2用非离子表面活性剂Tween 80替代SDS，其余条件同实施例1
步骤(一)
60℃下，将0.1g棕榈酸和1.3g 95％的工业CLA(CLA平均分子量278.73)混合均匀，然后将混合后的CLA-棕榈酸加到250mL Tween 80水溶液(Tween 80水溶液的质量浓度0.255％，含Tween 800.637g)中，简单混匀，得到混合物体系。然后，用稀盐酸调整混合物体系的pH至pH3.5，均质后得到悬浊液。
步骤(二)
在25℃、150rpm下边搅拌边向上述悬浊液中滴加2mg/mL的VC水溶液250mL，于30min内滴加完毕。将含VC的混合体系在50rpm、25℃下振摇30min。
振摇结束后，用显微镜观察，观察结果显示此时脂肪酸未形成脂质体，因而VC没有被包封。
如果，在步骤(一)种，将混合物体系的pH调整为7.4或6.0，则能够形成脂质体，VC的包封率分别为23.6％、29.7％。
对照例3用阴离子表面活性剂CLA-Na替代SDS，其余条件同实施例1
步骤(一)
60℃下，将0.1g棕榈酸和1.3g 95％的工业CLA(CLA平均分子量278.73)混合均匀，然后将混合后的CLA-棕榈酸加到250mL CLA-Na水溶液(CLA-Na水溶液的质量浓度0.056％，含CLA-Na 0.014g)中，简单混匀，得到混合物体系。然后，用稀盐酸调整混合物体系的pH至pH3.5，均质后得到悬浊液。
步骤(二)
在25℃、150rpm下边搅拌边向上述悬浊液中滴加2mg/mL的VC水溶液250mL，于30min 内滴加完毕。将含VC的混合溶液在50rpm、25℃下振摇30min。
振摇结束后，用显微镜观察，观察结果显示此时脂肪酸未形成脂质体，因而VC没有被包封。
实施例2用棕榈酸-CLA-SDS制备的VC脂质体
步骤(一)
60℃下，将1g棕榈酸和13g 95％的工业CLA(CLA平均分子量278.73)混合均匀，然后将250mL SDS水溶液(SDS水溶液的质量浓度0.56％，含SDS 1.4g)加到混合后的CLA-棕榈酸中，简单混匀，得到混合物体系。然后，用稀盐酸调整混合物体系的pH至pH3.5，均质后得到悬浊液。
步骤(二)
在25℃、150rpm下边搅拌边向上述悬浊液中滴加10mg/mL的VC水溶液250mL，30min内滴加完毕。将含VC的混合溶液在30rpm、25℃下振摇90min。
所得的脂肪酸基VC脂质体体系中，脂肪酸的浓度为100mmol/L，VC浓度为5mg/mL，测得VC的包封率为64.1％。脂肪酸基VC脂质体形貌如附图2B所示。
与实施例1相比，可以看出，脂肪酸浓度的增加可提高VC包封率。
实施例3用硬脂酸-CLA-SDS制备的VC脂质体
步骤(一)
60℃下将1g硬脂酸和13g 95％的工业CLA混合均匀，然后将250mL SDS水溶液(SDS水溶液的质量浓度0.56％，含SDS 1.4g)加到混合后的CLA-硬脂酸中，简单混匀，得到混合物体系。然后，用稀盐酸调整混合物体系的pH至pH3.8，均质后得到悬浊液。
步骤(二)
在25℃、150rpm下边搅拌边向上述悬浊液中滴加10mg/mL的VC水溶液250mL，30min内滴加完毕。将混合后的溶液在20rpm、20℃下振摇50min。
所得的脂肪酸基VC脂质体体系中，脂肪酸的浓度为100mmol/L，VC浓度为5mg/mL，VC的包封率为66.5％。
与实施例1相比，可以看出，饱和脂肪酸碳数的增加会稍许增加自组装体的尺寸，有利于增大VC包封率。
实施例4用棕榈酸-硬脂酸-CLA制备的VC脂质体
步骤(一)
将0.1g混合棕榈酸-硬脂酸(质量比1:1)加入到1.3g 80％的工业CLA(CLA平均分子 量278.71)中，将棕榈酸-硬脂酸-CLA在60℃、120rpm下振荡60min。然后，将250mL SDS水溶液(SDS水溶液的质量浓度0.056％，含SDS 0.14g)加到上述混合脂肪酸样品中，简单混匀，得到混合物体系。然后，用稀盐酸调整混合物体系的pH至pH3.8，均质后得到悬浊液。
步骤(二)
在25℃、150rpm下边搅拌边向上述悬浊液中滴加250mL浓度为10mg/mL的VC水溶液，于30min内滴加滴加完毕，在25℃、50rpm下振荡60min。
所得的脂肪酸基VC脂质体体系中，脂肪酸的浓度为10mmol/L，VC浓度为5mg/mL。VC的包封率为58.4％。
可见，CLA中总十八碳二烯酸异构体含量降低会降低VC包封率。
实施例5脂肪酸-CLA-VC脂质体中VC的缓释
脂肪酸基VC脂质体用于药物时，对VC脂质体中VC的缓释有一定时间要求，用于化妆品时则缓释越慢越好。由于无法测定膏霜状态下VC的缓释，本实施例用通用的水介质透析分析不同脂肪酸基VC脂质体中VC的缓释。
将不同脂肪酸与95％CLA复配后，参照上述实施例的工艺制备的脂肪酸基VC脂质体，用通用的水介质透析分析VC的缓释情况。结果如图3所示，透析160min后，对照样(1mg/mL VC水溶液)中VC的累计释放率为95.5％；实施例3制备的硬脂酸-CLA(100mM)-VC(5mg/mL)脂质体中VC的累计释放率为43.2％，按照以实施例3同样方法制备的棕榈酸-CLA(100mM)-VC(2mg/mL)脂质体中VC的累计释放速率为36.5％；说明短碳链脂肪酸倾向于延缓VC的释放。在实施例1制备的棕榈酸-CLA(10mM)-VC(1mg/mL)脂质体中VC释放率为40.9％，说明低浓度CLA加快VC的释放；而在实施例4制备的棕榈酸-硬脂酸-CLA(10mM)-VC(5mg/mL)脂质体中VC的累计释放率为48.9％，这是因为高浓度VC的浓度势能加快VC的释放。
实施例6用脂肪酸基VC脂质体制备VC护肤霜
取用缓释最慢的棕榈酸-CLA(100mM)-VC(2mg/mL)脂质体，按下列配方和工艺制备VC护肤霜：
油相的制备：60℃下将5g的单甘酯、1g貂油、15g的甾醇、2g积雪草50％乙醇提取物以及5g的液体石蜡混合均匀得到油相；
水相的制备：将5g甘油和2g人参皂甙(50％总甙)溶解在50mL去离子水中，最后加入5mL棕榈酸-CLA(100mM)-VC(2mg/mL)脂质体，混合均匀得到水相；
将油相和水相混合后在6000r/min下均质7min，脱气。在50℃下加入质量分数为0.3％ 的香精和0.2％的防腐剂，继续冷却搅拌至室温，脱气后得到VC护肤霜。
用5mLVC溶液(2mg/mL)替代上述配方中的5mL棕榈酸-CLA(100mM)-VC(2mg/mL)脂质体，按照相同的工艺制备得到用VC溶液配制的VC护肤霜对照样。
取利用脂肪酸基VC脂质体制备得到的VC护肤霜和利用VC溶液制备得到的VC护肤霜各100g，分别无菌装入5只30mL样品瓶中(每瓶中20g)，加盖置于37℃培养箱内加速氧化。利用脂肪酸基VC脂质体制备得到的VC护肤霜在放置3个月后未见颜色变化、没有分层。而利用VC溶液制备得到的VC护肤霜放置3个月后样品没有分层，但1个月后颜色就变黄并随时间加深。
上述实施例中所用的工业共轭亚油酸原料为市售商品，可购自大连医诺生物技术有限公司和青岛海之源等多家公司。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (16)

  1. 一种制备脂肪酸基维生素C脂质体的方法，其特征在于，以工业共轭亚油酸和其它脂肪酸以及十二烷基硫酸钠的复合物为囊材，囊材在酸性条件下于水相自组装包埋维生素C形成脂肪酸基维生素C脂质体；所述其它脂肪酸的分子简式符合通式C nH 2n+1COOH，其中n为大于7的正整数；
    包括以下步骤：
    (1)将工业共轭亚油酸和其它脂肪酸混合均匀，向其中添加SDS水溶液，SDS的加入量为总脂肪酸的质量的5-10％，用稀酸调整自组装液的pH至pH 3.5-4.0，然后均质得到悬浊液；
    (2)在搅拌条件下，向步骤(1)得到的悬浊液中滴加VC水溶液，滴加结束后，在20-50rpm下振摇10min-1h。
  2. 根据权利要求1所述的一种制备脂肪酸基维生素C脂质体的方法，其特征在于，所述工业共轭亚油酸是通过食品级工业亚油酸的碱催化共轭化所得的产品，所述共轭亚油酸是各种十八碳二烯酸异构体的单一物质或者混合物，且，总十八碳二烯酸异构体含量占所述工业共轭亚油酸干物质的质量百分比为70％-100％。
  3. 根据权利要求1所述的一种制备脂肪酸基维生素C脂质体的方法，其特征在于，所述脂肪酸基中的脂肪酸是包括共轭亚油酸、其它脂肪酸以及工业共轭亚油酸原料中含有的各种含羧基的分子中碳数在8以上的有机酸；所述其它脂肪酸的分子简式符合通式C nH 2n+1COOH，其中n为大于7的正整数。
  4. 根据权利要求1所述的一种制备脂肪酸基维生素C脂质体的方法，其特征在于，所述脂肪酸基的脂肪酸中，其它脂肪酸的质量为总十八碳二烯酸异构体的质量的1％-40％。
  5. 根据权利要求2所述的一种制备脂肪酸基维生素C脂质体的方法，其特征在于，所述脂肪酸基的脂肪酸中，其它脂肪酸的质量为总十八碳二烯酸异构体的质量的1％-40％。
  6. 根据权利要求3所述的一种制备脂肪酸基维生素C脂质体的方法，其特征在于，所述脂肪酸基的脂肪酸中，其它脂肪酸的质量为总十八碳二烯酸异构体的质量的1％-40％。
  7. 根据权利要求2所述的一种制备脂肪酸基维生素C脂质体的方法，其特征在于，所述脂肪酸在自组装液中的终浓度之和为10-500mM。
  8. 根据权利要求3所述的一种制备脂肪酸基维生素C脂质体的方法，其特征在于，所述脂肪酸在自组装液中的终浓度之和为10-500mM。
  9. 根据权利要求1所述的一种制备脂肪酸基维生素C脂质体的方法，其特征在于，步骤(2)中，滴加结束后，悬浊液体系中的VC的终浓度为1-50mg/mL。
  10. 根据权利要求2所述的一种制备脂肪酸基维生素C脂质体的方法，其特征在于，步骤(2)中，滴加结束后，悬浊液体系中的VC的终浓度为1-50mg/mL。
  11. 根据权利要求1所述的一种制备脂肪酸基维生素C脂质体的方法，其特征在于，步骤(1)中SDS的加入量为总脂肪酸的质量的5％或10％。
  12. 根据权利要求2所述的一种制备脂肪酸基维生素C脂质体的方法，其特征在于，步骤(1)中SDS的加入量为总脂肪酸的质量的5％或10％。
  13. 根据权利要求4所述的一种制备脂肪酸基维生素C脂质体的方法，其特征在于，步骤(1)中SDS的加入量为总脂肪酸的质量的5％或10％。
  14. 根据权利要求7所述的一种制备脂肪酸基维生素C脂质体的方法，其特征在于，步骤(1)中SDS的加入量为总脂肪酸的质量的5％或10％。
  15. 根据权利要求9所述的一种制备脂肪酸基维生素C脂质体的方法，其特征在于，步骤(1)中SDS的加入量为总脂肪酸的质量的5％或10％。
  16. 应用权利要求1-15任一项所述方法制备得到的脂肪酸基维生素C脂质体。
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