纳米乳营养素透皮传递系统的研究
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摘要
营养素补充剂是指由一种或多种必需的微量营养素组成的产品,如维生素和矿物质。近年来.我国居民食物质量和营养摄入较过去都有明显改善,但营养不良问题仍严重存在,主要表现在营养摄入不足和营养结构失衡.直接后果是与营养相关的慢性退行性疾病的快速增长。适量补充外源性维生素、矿物质,可弥补膳食供给的不足、预防营养缺乏和降低发生某些慢性退行性疾病的风险性,是改善营养不良的有效途径之一
营养素补充剂的剂型目前以口服和注射为主,注射的优点是吸收迅速,主要应用于接受肠外营养支持的病人。缺点是必须考虑使用的安全性,因此应在医院里由护士使用,而不适合个人单独应用；口服营养素补充剂的应用非常广泛,是目前营养素补充剂最主要的补充方式。但是对于胃肠道功能紊乱、某些特殊作业条件下(如消防队员、矿业工人、化工生产、宇航人员等应激或有害的作业环境)以及老人、小孩等,口服补充存在一定的局限性,依从性不高。基于以上,我们认为有必要探讨一种新的营养素补充途径。
透皮给药系统已经在药学上得到了广泛的应用,如在治疗心绞痛、运动病、止痛、高血压以及女性/男性激素替代疗法中都采用了经皮给药系统。而在营养素的透皮给药系统研究中,目前大量研究集中在化妆品领域,如维生素C、维生素A等采用经皮给药系统,达到局部治疗的作用。但是对于要起到全身作用的营养素补充剂来说,目前国内外均无该方面的研究报道。与传统药物经皮给药系统比较,营养素经皮给药系统的研究难点在于如何制备适当的载药载体,以帮助各营养素渗透进深部皮肤,从而进入血液循环达到全身作用。
纳米乳是一种热力学稳定的体系,它的高度分散性使其具备了高度的皮肤或生物膜渗透性能。当纳米乳涂布皮肤后,水被吸收从而出现过饱和现象。过饱和的出现使药物热力学活性增加,药物极易由纳米乳中向皮肤内释放。由于药物扩散通过基底层,进入皮肤更深部位的药物量迅速增加。
本课题以纳米乳作为载体,维生素A棕榈酸酯和维生素B1为模型营养素,研究可以携带不同水溶性、脂溶性的维生素进入皮肤深部,从而发挥其全身作用,达到营养素补充的作用的纳米乳透皮传递系统的制备工艺及影响因素,并对其体内外释药的性质进行评价,以其为开辟新的营养素补充途径打下重要的基础。
研究方法
1.纳米乳的制备及理化性质的考察
1.1在具塞的平底玻璃试管中,将表面活性剂与助表面活性剂按一定比例(如1：1,1：2,2：1等)混合溶解,混合物在室温(25±1℃)下加入油相(含维生素A棕榈酸酯)用恒温磁力搅拌器搅拌,搅拌下用蒸馏水(含维生素B1)滴定三种成分的混合物,直至澄清透明的纳米乳形成。继续滴定,观察可能发生的变化,以确定纳米乳区界限。本研究采用吐温-80 (Tween-80),聚氧乙烯蓖麻油(Cremophor EL-35)及聚氧乙烯氢化蓖麻油(Cremophor RH-40)做为待考察表面活性剂；乙醇,丙二醇,甘油作为助表面活性剂；亚油酸乙酯,十四酸异丙酯,花生油,豆油作为油相；生理盐水,葡萄糖溶液为水相,同时考察表面活性剂与助表面活性剂质量比(Km)和温度的变化对纳米乳制备及制备区域大小的影响。
1.2在待考察的纳米乳组分的拟三元相图纳米乳区域中选择某纳米乳比例测定各组纳米乳的粒径、粘度,并考察不同组分对其粒径、粘度的影响。结合拟三元相图,选择纳米乳区域大、粒径、粘度适宜的纳米乳处方。
2.营养素透皮传递系统体外透皮实验
2.1建立维生素A含量测定方法
建立以高效液相色谱法(high-performance liquid chromatography, HPLC)测定维生素A棕榈酸酯含量的方法。色谱条件如下：色谱柱DiamonsilC18柱(250×4.6mm,粒径5μm)；流动相：甲醇-乙腈-水(V/V/V：66：32：2),使用前超声处理；流速：1.0ml/min；检测波长：275nm；柱温：室温；检测灵敏度1AUFS;进样量：20μl。并进行精密度、重现性、回收率实验。以浓度C (ug/ml)对峰面积A进行线性回归,得到维生素A棕榈酸酯标准曲线。
2.2建立维生素B1测定方法
建立以紫外分光光度法测定维生素B1含量的方法。精密称取维生素B1对照品加盐酸溶液(9-1000)稀释至刻度,摇匀,制成系列浓度的溶液,在246nm的波长处测定吸光度。并进行精密度、重现性、回收率实验。以浓度C (μg/ml)对吸收度A进行线性回归,得到维生素B1标准曲线。
2.3 Franz扩散池体外透皮实验
根据纳米乳的组成特点,利用水平Franz扩散池进行体外透皮实验,通过正交设计,考察纳米乳各组分因素对纳米乳累积释药量的影响,优化透皮系统的处方。选用180～200g左右雄性SD大鼠腹部皮肤,剥离腹部皮肤,小心刮去皮下脂肪组织和粘连物,用生理盐水清洗干净,用滤纸拭干,直接使用。将皮肤平整置于水平Franz扩散池结合部,角质层面向供给池,真皮层面向接受池,在37±0.1℃恒温水浴扩散池中加入纳米乳4ml,接受液为4ml生理盐水。分别于于不同时间点从接受池中取样0.5ml,同时补入37±0.1℃生理盐水0.5ml。按照已经建立的维生素A棕榈酸酯和维生素B1的含量测定方法,由标准曲线计算出各时间点接受池中两种维生素的浓度,绘制体外透皮释放曲线,计算12h累积渗透量(Q)。
2.4营养素透皮传递系统稳定性实验
按照优化处方制备纳米乳透皮传递系统,并于室温和40℃下放置3个月,通过药物浓度的变化考察其稳定性。
3.营养素透皮传递系统皮肤刺激性研究
分别进行单次给药和多次给药的皮肤刺激性实验。
单次给药实验采用SD大鼠同体左右侧自身对比,分完整皮肤组及破损皮肤组。左侧去毛区涂布自制纳米乳1g,右侧涂空白纳米乳作为对照。给药24h后,用温水洗去残留的药物,去除药物后肉眼观察并记录涂抹部位有无红斑和水肿情况,计算各组动物反应平均分值。并对刺激强度作出评价。多次给药实验每天1次,连续涂抹1周。于末次给药24h后,用温水洗去残留的药物,去除药物后用肉眼观察并记录涂抹部位有无红斑和水肿情况,进行评分,计算各组动物反应平均分值,并对刺激强度作出评价。并观察涂抹部位是否有色素沉着、出血点、皮肤粗糙或皮肤菲薄等情况,记录其发生时间及消退时间。
4.营养素透皮传递系统药代动力学研究
4.1建立血清维生素A棕榈酸酯含量测定方法
色谱条件如下：分析柱为DiamonsilC18柱(250×4.6mm,粒径5μm)；检测波长325 nm；流动相：甲醇：水(95：5,V/V)；流速1.2ml/min;进样量20μl。并进行精密度、重现性、回收率实验。以浓度C (ng/ml)对峰面积A进行线性回归,得到维生素A棕榈酸酯标准曲线。
4.2营养素透皮传递系统药代动力学
以SD大鼠为研究对象,给药前禁食12h,自由饮水。在其去毛区涂布自制纳米乳1ml。于给药后0.5、1、2、3、4、8、12、24、48h,从大鼠眼眶取血0.5ml,按建立的维生素A棕榈酸酯含量测定方法进行含量测定。并求算有关药代动力学参数。
实验结果
1.纳米乳制备的影响因素
在所研究的体系中,我们发现表面活性剂的HLB值,助表面活性剂的碳链长度,表面活性剂和助表面活性剂的质量比(Km),油相的结构,水相中添加剂的变化及温度的变化对纳米乳的形成均有影响。
纳米乳黏度与粒径的大小变化与表面活性剂/醇质量比有关,在实验的条件下,该比值越大,纳米乳粒径越小；同时载药纳米乳粒径的大小也随载药量的不同而不同。
2.正交实验设计优化营养素透皮传递系统的处方
2.1 HPLC法测定维生素A棕榈酸酯的出峰时间在9.4min,空白纳米乳各辅料对其测定无干扰,专属性好。标准曲线为A=75704C+45799 (R2=0.999),表明在8～160μg/ml范围内线性关系良好,精密度、回收率等符合实验要求。
2.2紫外分光光度计法测定维生素B1的吸收峰在264nm处,空白纳米乳各辅料对其测定无干扰,专属性好。标准曲线为A=0.043C+0.001 (R2=0.999),表明在7.5～17.5μg/ml范围内线性关系良好,精密度、回收率等符合实验要求。
2.3根据正交实验的结果,考察的各因素对纳米乳营养素透皮传递系统12h累积释药量影响大小为：Km>油相(%)>水相(%),优化的最佳处方为：水相(%)：油相(%)：Km为：40：10：(4：1)。
2.4通过温度加速实验考察了营养素透皮传递系统的稳定性,实验表明,在室温和40℃的环境下放置3个月,该系统保持稳定。
3.营养素透皮传递系统皮肤刺激性研究
3.1通过单次和多次的皮肤给药,均未见皮肤出现红斑、水肿等现象,表明该纳米乳营养素透皮传递系统对皮肤无刺激性。
4.营养素透皮传递系统药代动力学研究
4.1采用HPLC法测定血清中维生素A棕榈酸酯含量,维生素A棕榈酸酯的出峰时间在9.745min,血液中其他组分对其测定无干扰,专属性好。标准曲线为A=646.6C-4351 (R2=0.999),表明在10-400ng/ml范围内线性关系良好,精密度、回收率等符合实验要求。
4.2大鼠给予纳米乳营养素透皮传递系统后,在血清中可以监测到维生素A棕榈酸酯,3 h时血清内维生素A棕榈酸酯浓度达峰值。梯形法计算血药浓度-时间曲线下面积, AUC0-48h为2403.42ng/ml×h。
结论
1.选择聚氧乙烯氢化蓖麻油(Cremophor RH-40)为表面活性剂,乙醇为助表面活性剂,十四酸异丙酯(IPM)为油相,可制备出稳定的纳米乳,其粒径、粘度等理化性质均符合要求。
2.根据体外透皮渗透实验,优化的纳米乳透皮传递系统最佳处方为：水相(%)：油相(IPM,%):Km (Cremophor RH-40:乙醇)为：40：10：(4：1),在室温和40℃的条件下,保存3个月对维生素A棕榈酸酯和维生素B1含量无影响。
3.通过单次和多次给药皮肤刺激性实验,自制的纳米乳透皮传递系统应用后,皮肤无红斑、水肿等现象,表明该制剂对皮肤无刺激性。
4.大鼠给予纳米乳营养素透皮传递系统后3 h,血清内维生素A棕榈酸酯浓度达峰值。48h AUC0-48h为2403.42ng/ml×h。
综上所述,我们以维生素A棕榈酸酯和维生素B1为模型药物,研制的纳米乳营养素透皮传递系统通过体内外实验均表明,其中的营养素可以透过皮肤的屏障作用,达到全身给药的目的,对开辟新的营养素补充剂给药途径具有重要的指导意义。
Introduction
Multi-micronutrient supplements are products which are consist of one or some micronutrients, such as vitamin and mineral. Although food quality and nutrients intake for Chinese people are better than the past years, the problem of nutrition deficiency is very serious. The main problem exists in the nutrients deficiency and imbalance of nutrition structure. These result in the increase of some chronic disease in china. To supply some exogenous vitamin and mineral is a better way to prevent nutrition deficiency and decrease the risk of some chronic disease.
The pharmaceutical dosage forms of multi-micronutrient supplements major in oral and injection forms. The injections can be absorbed quickly, and applied for the patients who receive parenteral nutrition. And orally administration of multi-micronutrients supplements is quite common. But for some people,such as firemen, miner, astronaut or some people who work in some particular environment, oral multi-micronutrients supplements cann't be administrated. Based on these, we believe that a new administration method should be discussed.
Transdermal drug delivery has many advantages over the oral route of administration:it avoids hepatic metabolism, the administration is easier and more convenient for the patient, and there is the possibility of immediate withdrawl of the treatment if necessary. Despite the great potential of transdermal delivery of drugs, only a few drug formulations are available commercially. The main reason is the barrier function of human skin that is considered to be the most impermeable epithelium to exogenous substances.
Nanoemulsion have been subjected to numerous studies during the last decades because of their great potential in many applications. Due to their rather complicated phase behavior and the fascinating microstructures encountered in microemulsion forming systems, many researchers have made significant efforts to obtain a better understanding of these microstructures. Nanoemulsions seem to be ideal liquid vehicles for drug delivery since they provide all the possible requirements of a liquid system including thermodynamic stability(long shelf life),easy formation(zero interfacial tension and almost spontaneous formation).low viscosity with Newtonian behavior, high surface area(high solubilization capacity),and very small droplet size. The small droplets have better chance to adhere to membranes and to transport bioactive molecules in a more controlled fashion.
Methods
1. To prepare nanoemulsion and investigate the physic-chemical properties of nanoemulsion
1.1 The pseudo-ternary phase diagrams were constructed by the dilution method at room temperature. Tween-80, Cremophor EL-35, Cremophor RH-40 as the surfactant, ethanol,1,2-isopropyl alcohol as the cosurfactant, IPM, ethyl linoleate, peanut oil and soy oil as the oil phase. These elements were mixed on a magnetic stirrer. Based on the pseudo-ternary phase diagrams, we investigated the effects of nanoemulsion components and Km(the ratio of surfactant to cosurfactant weight) on the formation and the areas of the nanoemulsion.
1.2 Appropriate quantities of surfactant, cosurfactant, oil and water were constructed in the nanoemulsion region of the pseudo-ternary phase diagrams. The mean diameter and apparent viscosity were measured. Meanwhile, we studdied the influence of nanoemulsion elements on the diameters and apparent viscosities and chose a good nanoemulsion system, which has larger nanoemulsion area and suitable diameter viscosity.
2.1n vitro skin permeation study of nanoemulsion transdermal delivery system
2.1 HPLC analysis of Vitamin A palmitate The amount of vitamin A palmitate in receptor phase was quantitated with the HPLC methods. The HPLC column used was a C18 column (Diamonsil,450X4.6mm,5μm particle size). The mobile phase consisted of methanol, acetonitrile and water(v/v/,66:32:2), at the floww rate aand injection volume of 1.0ml/min and 20μl, respectivelly. The detection waveleength was 275nm. All operations were carried out at room temperature. The calibration curve of vitamin A palmitate was constructed. The precision, reproduction and percent recovery of the method were analyzed.
2.2 Ultraviolet spectrophotometry analysis of Vitamin B1
The amount of vitamin B1 in receptor phase was quantitated with the ultraviolet spectrophotometry methods. The detection waveleength was 246nm. All operations were carried out at room temperature. The calibration curve of vitamin B1 was constructed. The precision, reproduction and percent recovery of the method were analyzed.
2.3 In vitro skin permeation study
The extent and rate of skin permeation of vitamin A palmitate and vitamin B1 from several different nanoemulsion which were prepared according to the orthogonal design were determined using Franz diffusion cells fitted with excised rat skins. Skins were obtained from male sprague-Dawley(SD) rats weighing 180-200g. after hair was removed carefully with a clipper, the subcutaneous fat and other extraneous tissues were trimmed. The excised skins were stored at-20℃and used within one week after the skin harvest. The receptor phase was 0.9% sodium chloride and 4ml was injected to the receptor phase. The effective diffusion area was 0.75cm2. after 4ml of each nanoemulsion was applied on the skin surface,0.5ml of receptor medium were withdrawn at different time. Concentration of vitamin A palmitate and vitamin BI in each sample were quantitated through the calibration curve of themselves, and the cumulative amount of vitamin A palmitae and vitamin B1 over a 12h period(Q) and the skin permeation rate(Jss)were calculated.
2.4 stability of nanoemulsion transdermal delivery system
To prepare the nanoemulsion transdermal delivery system according to the result of the orthogonal design. These nanoemulsion are stored for 3 month in room temperature and 40℃. The stability of nanoemulsion were investigated according to the change of vitamin A palmitate and vitamin B1.
3.in vivo irritating test for nanoemulsion nutrients transdermal delivery system
Single vs. multiple doses are performed in rat to test the irritation fo nutrients loaded nanoemulsion transdermal delivery system.
4. pharmacokinetics study of nutrients loaded nanoemulsion transdermal delivery system
4.1 HPLC analysis of Vitamin A palmitate in blood serum
The amount of vitamin A palmitate in blood serum was quantitated with the HPLC methods. The HPLC column used was a C18 column (Diamonsil,450X4.6mm,5μm particle size). The mobile phase consisted of methanol and water(v/v,95:5), at the floww rate aand injection volume of 1.2ml/min and 20μl, respectively. The detection waveleength was 325nm. All operations were carried out at room temperature. The calibration curve of vitamin A palmitate was constructed. The precision, reproduction and percent recovery of the method were analyzed.
4.2 pharmacokinetics study
Pharmacokinetic parameters were calculated after the male rats were transdermally administrated single dose of nutrients loaded nanoemulsion transdermal delivery system.
Results
1.Effect factors for the formation of nanoemulsion
Surfactants play a key role in many of the novel drug dilivery systems developed,and a wide range of surfactant-containing systems,including emulsions,liposomes,liquid crystalline phase,and microemulsions,are being extensively investigated in relation to drug delivery.Non-iron surfactants are investigated in our research.according to the phase diagram,we investigated the effects of the elements in the formation of the nanoemulsions. the ration of the surfactant to the co-surfactant,hydrocarbon length of co-surfactant,HLB value of surfactant,and the structure of oil,the change of additives in the water,the temperature are important in the microemulsion system.
2. In vitro skin permeation study of nanoemulsion transdermal delivery system
2.1 The retension time for vitamin A palmitate was observed to be 9.4min, and the elements of nanoemulsion did not have any effect on the determination of vitamin A palmitae. The calibration curve of vitamin A palmitae was A=75704C+45799 (R2=0.999) using an HPLC method, A was the peak area and C was the concentration of vitamin A palmitate standard solution. The standard curve was liner in the range of 8～160μg/ml. The RSD, reproducibility RSD and the average recovery confirmed the accuracy of this method as described above.
2.2 The calibration curve of vitamin B, was A=0.043C+0.001(R2=0.999) using an UV spectrophotometry method, A was the absorbance and C was the concentration of vitamin B1 standard solution. The standard curve was liner in the range of 7.5～17.5μg/ml. The RSD, reproducibility RSD and the average recovery confirmed the accuracy of this method as described above.
2.3 According to the orthogonal design, the relation among these tested factors is Km>oil phase (%)>water phase (%), the optimum formula is water phase(%):oil phase(%):Km(40:10:(4:1).
2.4 temperature accelerated test suggested the nutrients loaded nanoemulsion transdermal delivery system has been stability at 40℃for 3 months.
3.in vivo irritating test for nanoemulsion nutrients transdermal delivery system
It is clear that nanoemulsion is non-irritating carrier for transdermal drug delivery of nutrients, according to the resultant indices of treated skin.
4. pharmacokinetics study
4.1 The retension time for vitamin A palmitate in rat blood serum was observed to be 9.745min, and the elements of serum did not have any effect on the determination of vitamin A palmitae. The calibration curve of vitamin A palmitae was A=646.6C-4351(R2=0.999) using an HPLC method, A was the peak area and C was the concentration of vitamin A palmitate standard solution. The standard curve was liner in the range of 10-400ng/ml. The RSD, reproducibility RSD and the average recovery confirmed the accuracy of this method as described above.
4.2 pharmacokinetic parametes were calculated. Area under the drug concentration-time curve (AUC) were calculated using the trapezoidal rule. The maximal blood concentration were also obtained from blood data.AUC0-48h in this study is 2403.42ng/ml X h.
Conclusion
1. The present study clearly demonstrates that nanoemulsion can be well prepared with Cremophor RH-40 as surfactant, ethanol as cosurfactant and IPM as oil phase. The particle size and viscosity of nutrients loaded nanoemulsion and other physic-chemical properties all are desirable.
2. According to the orthogonal design, the relation among these tested factors is Km>oil phase (%)>water phase (%), the optimum formula is water phase(%):oil phase(%):Km(40:10:(4:1). temperature accelerated test suggested the nutrients loaded nanoemulsion transdermal delivery system has been stability at 40℃for 3 months.
3. It is clear that nanoemulsion is non-irritating carrier for transdermal drug delivery of nutrients, according to the single dose and multi-dose administration in the rat skin.
4. pharmacokinetic parametes were calculated. AUC0-48h in this study is 2403.42ng/ml×h. It means that nutrients loaded in the nanoemulsion can reach the systemic circulation.
营养素补充剂的剂型目前以口服和注射为主,注射的优点是吸收迅速,主要应用于接受肠外营养支持的病人。缺点是必须考虑使用的安全性,因此应在医院里由护士使用,而不适合个人单独应用；口服营养素补充剂的应用非常广泛,是目前营养素补充剂最主要的补充方式。但是对于胃肠道功能紊乱、某些特殊作业条件下(如消防队员、矿业工人、化工生产、宇航人员等应激或有害的作业环境)以及老人、小孩等,口服补充存在一定的局限性,依从性不高。基于以上,我们认为有必要探讨一种新的营养素补充途径。
透皮给药系统已经在药学上得到了广泛的应用,如在治疗心绞痛、运动病、止痛、高血压以及女性/男性激素替代疗法中都采用了经皮给药系统。而在营养素的透皮给药系统研究中,目前大量研究集中在化妆品领域,如维生素C、维生素A等采用经皮给药系统,达到局部治疗的作用。但是对于要起到全身作用的营养素补充剂来说,目前国内外均无该方面的研究报道。与传统药物经皮给药系统比较,营养素经皮给药系统的研究难点在于如何制备适当的载药载体,以帮助各营养素渗透进深部皮肤,从而进入血液循环达到全身作用。
纳米乳是一种热力学稳定的体系,它的高度分散性使其具备了高度的皮肤或生物膜渗透性能。当纳米乳涂布皮肤后,水被吸收从而出现过饱和现象。过饱和的出现使药物热力学活性增加,药物极易由纳米乳中向皮肤内释放。由于药物扩散通过基底层,进入皮肤更深部位的药物量迅速增加。
本课题以纳米乳作为载体,维生素A棕榈酸酯和维生素B1为模型营养素,研究可以携带不同水溶性、脂溶性的维生素进入皮肤深部,从而发挥其全身作用,达到营养素补充的作用的纳米乳透皮传递系统的制备工艺及影响因素,并对其体内外释药的性质进行评价,以其为开辟新的营养素补充途径打下重要的基础。
研究方法
1.纳米乳的制备及理化性质的考察
1.1在具塞的平底玻璃试管中,将表面活性剂与助表面活性剂按一定比例(如1：1,1：2,2：1等)混合溶解,混合物在室温(25±1℃)下加入油相(含维生素A棕榈酸酯)用恒温磁力搅拌器搅拌,搅拌下用蒸馏水(含维生素B1)滴定三种成分的混合物,直至澄清透明的纳米乳形成。继续滴定,观察可能发生的变化,以确定纳米乳区界限。本研究采用吐温-80 (Tween-80),聚氧乙烯蓖麻油(Cremophor EL-35)及聚氧乙烯氢化蓖麻油(Cremophor RH-40)做为待考察表面活性剂；乙醇,丙二醇,甘油作为助表面活性剂；亚油酸乙酯,十四酸异丙酯,花生油,豆油作为油相；生理盐水,葡萄糖溶液为水相,同时考察表面活性剂与助表面活性剂质量比(Km)和温度的变化对纳米乳制备及制备区域大小的影响。
1.2在待考察的纳米乳组分的拟三元相图纳米乳区域中选择某纳米乳比例测定各组纳米乳的粒径、粘度,并考察不同组分对其粒径、粘度的影响。结合拟三元相图,选择纳米乳区域大、粒径、粘度适宜的纳米乳处方。
2.营养素透皮传递系统体外透皮实验
2.1建立维生素A含量测定方法
建立以高效液相色谱法(high-performance liquid chromatography, HPLC)测定维生素A棕榈酸酯含量的方法。色谱条件如下：色谱柱DiamonsilC18柱(250×4.6mm,粒径5μm)；流动相：甲醇-乙腈-水(V/V/V：66：32：2),使用前超声处理；流速：1.0ml/min；检测波长：275nm；柱温：室温；检测灵敏度1AUFS;进样量：20μl。并进行精密度、重现性、回收率实验。以浓度C (ug/ml)对峰面积A进行线性回归,得到维生素A棕榈酸酯标准曲线。
2.2建立维生素B1测定方法
建立以紫外分光光度法测定维生素B1含量的方法。精密称取维生素B1对照品加盐酸溶液(9-1000)稀释至刻度,摇匀,制成系列浓度的溶液,在246nm的波长处测定吸光度。并进行精密度、重现性、回收率实验。以浓度C (μg/ml)对吸收度A进行线性回归,得到维生素B1标准曲线。
2.3 Franz扩散池体外透皮实验
根据纳米乳的组成特点,利用水平Franz扩散池进行体外透皮实验,通过正交设计,考察纳米乳各组分因素对纳米乳累积释药量的影响,优化透皮系统的处方。选用180～200g左右雄性SD大鼠腹部皮肤,剥离腹部皮肤,小心刮去皮下脂肪组织和粘连物,用生理盐水清洗干净,用滤纸拭干,直接使用。将皮肤平整置于水平Franz扩散池结合部,角质层面向供给池,真皮层面向接受池,在37±0.1℃恒温水浴扩散池中加入纳米乳4ml,接受液为4ml生理盐水。分别于于不同时间点从接受池中取样0.5ml,同时补入37±0.1℃生理盐水0.5ml。按照已经建立的维生素A棕榈酸酯和维生素B1的含量测定方法,由标准曲线计算出各时间点接受池中两种维生素的浓度,绘制体外透皮释放曲线,计算12h累积渗透量(Q)。
2.4营养素透皮传递系统稳定性实验
按照优化处方制备纳米乳透皮传递系统,并于室温和40℃下放置3个月,通过药物浓度的变化考察其稳定性。
3.营养素透皮传递系统皮肤刺激性研究
分别进行单次给药和多次给药的皮肤刺激性实验。
单次给药实验采用SD大鼠同体左右侧自身对比,分完整皮肤组及破损皮肤组。左侧去毛区涂布自制纳米乳1g,右侧涂空白纳米乳作为对照。给药24h后,用温水洗去残留的药物,去除药物后肉眼观察并记录涂抹部位有无红斑和水肿情况,计算各组动物反应平均分值。并对刺激强度作出评价。多次给药实验每天1次,连续涂抹1周。于末次给药24h后,用温水洗去残留的药物,去除药物后用肉眼观察并记录涂抹部位有无红斑和水肿情况,进行评分,计算各组动物反应平均分值,并对刺激强度作出评价。并观察涂抹部位是否有色素沉着、出血点、皮肤粗糙或皮肤菲薄等情况,记录其发生时间及消退时间。
4.营养素透皮传递系统药代动力学研究
4.1建立血清维生素A棕榈酸酯含量测定方法
色谱条件如下：分析柱为DiamonsilC18柱(250×4.6mm,粒径5μm)；检测波长325 nm；流动相：甲醇：水(95：5,V/V)；流速1.2ml/min;进样量20μl。并进行精密度、重现性、回收率实验。以浓度C (ng/ml)对峰面积A进行线性回归,得到维生素A棕榈酸酯标准曲线。
4.2营养素透皮传递系统药代动力学
以SD大鼠为研究对象,给药前禁食12h,自由饮水。在其去毛区涂布自制纳米乳1ml。于给药后0.5、1、2、3、4、8、12、24、48h,从大鼠眼眶取血0.5ml,按建立的维生素A棕榈酸酯含量测定方法进行含量测定。并求算有关药代动力学参数。
实验结果
1.纳米乳制备的影响因素
在所研究的体系中,我们发现表面活性剂的HLB值,助表面活性剂的碳链长度,表面活性剂和助表面活性剂的质量比(Km),油相的结构,水相中添加剂的变化及温度的变化对纳米乳的形成均有影响。
纳米乳黏度与粒径的大小变化与表面活性剂/醇质量比有关,在实验的条件下,该比值越大,纳米乳粒径越小；同时载药纳米乳粒径的大小也随载药量的不同而不同。
2.正交实验设计优化营养素透皮传递系统的处方
2.1 HPLC法测定维生素A棕榈酸酯的出峰时间在9.4min,空白纳米乳各辅料对其测定无干扰,专属性好。标准曲线为A=75704C+45799 (R2=0.999),表明在8～160μg/ml范围内线性关系良好,精密度、回收率等符合实验要求。
2.2紫外分光光度计法测定维生素B1的吸收峰在264nm处,空白纳米乳各辅料对其测定无干扰,专属性好。标准曲线为A=0.043C+0.001 (R2=0.999),表明在7.5～17.5μg/ml范围内线性关系良好,精密度、回收率等符合实验要求。
2.3根据正交实验的结果,考察的各因素对纳米乳营养素透皮传递系统12h累积释药量影响大小为：Km>油相(%)>水相(%),优化的最佳处方为：水相(%)：油相(%)：Km为：40：10：(4：1)。
2.4通过温度加速实验考察了营养素透皮传递系统的稳定性,实验表明,在室温和40℃的环境下放置3个月,该系统保持稳定。
3.营养素透皮传递系统皮肤刺激性研究
3.1通过单次和多次的皮肤给药,均未见皮肤出现红斑、水肿等现象,表明该纳米乳营养素透皮传递系统对皮肤无刺激性。
4.营养素透皮传递系统药代动力学研究
4.1采用HPLC法测定血清中维生素A棕榈酸酯含量,维生素A棕榈酸酯的出峰时间在9.745min,血液中其他组分对其测定无干扰,专属性好。标准曲线为A=646.6C-4351 (R2=0.999),表明在10-400ng/ml范围内线性关系良好,精密度、回收率等符合实验要求。
4.2大鼠给予纳米乳营养素透皮传递系统后,在血清中可以监测到维生素A棕榈酸酯,3 h时血清内维生素A棕榈酸酯浓度达峰值。梯形法计算血药浓度-时间曲线下面积, AUC0-48h为2403.42ng/ml×h。
结论
1.选择聚氧乙烯氢化蓖麻油(Cremophor RH-40)为表面活性剂,乙醇为助表面活性剂,十四酸异丙酯(IPM)为油相,可制备出稳定的纳米乳,其粒径、粘度等理化性质均符合要求。
2.根据体外透皮渗透实验,优化的纳米乳透皮传递系统最佳处方为：水相(%)：油相(IPM,%):Km (Cremophor RH-40:乙醇)为：40：10：(4：1),在室温和40℃的条件下,保存3个月对维生素A棕榈酸酯和维生素B1含量无影响。
3.通过单次和多次给药皮肤刺激性实验,自制的纳米乳透皮传递系统应用后,皮肤无红斑、水肿等现象,表明该制剂对皮肤无刺激性。
4.大鼠给予纳米乳营养素透皮传递系统后3 h,血清内维生素A棕榈酸酯浓度达峰值。48h AUC0-48h为2403.42ng/ml×h。
综上所述,我们以维生素A棕榈酸酯和维生素B1为模型药物,研制的纳米乳营养素透皮传递系统通过体内外实验均表明,其中的营养素可以透过皮肤的屏障作用,达到全身给药的目的,对开辟新的营养素补充剂给药途径具有重要的指导意义。
Introduction
Multi-micronutrient supplements are products which are consist of one or some micronutrients, such as vitamin and mineral. Although food quality and nutrients intake for Chinese people are better than the past years, the problem of nutrition deficiency is very serious. The main problem exists in the nutrients deficiency and imbalance of nutrition structure. These result in the increase of some chronic disease in china. To supply some exogenous vitamin and mineral is a better way to prevent nutrition deficiency and decrease the risk of some chronic disease.
The pharmaceutical dosage forms of multi-micronutrient supplements major in oral and injection forms. The injections can be absorbed quickly, and applied for the patients who receive parenteral nutrition. And orally administration of multi-micronutrients supplements is quite common. But for some people,such as firemen, miner, astronaut or some people who work in some particular environment, oral multi-micronutrients supplements cann't be administrated. Based on these, we believe that a new administration method should be discussed.
Transdermal drug delivery has many advantages over the oral route of administration:it avoids hepatic metabolism, the administration is easier and more convenient for the patient, and there is the possibility of immediate withdrawl of the treatment if necessary. Despite the great potential of transdermal delivery of drugs, only a few drug formulations are available commercially. The main reason is the barrier function of human skin that is considered to be the most impermeable epithelium to exogenous substances.
Nanoemulsion have been subjected to numerous studies during the last decades because of their great potential in many applications. Due to their rather complicated phase behavior and the fascinating microstructures encountered in microemulsion forming systems, many researchers have made significant efforts to obtain a better understanding of these microstructures. Nanoemulsions seem to be ideal liquid vehicles for drug delivery since they provide all the possible requirements of a liquid system including thermodynamic stability(long shelf life),easy formation(zero interfacial tension and almost spontaneous formation).low viscosity with Newtonian behavior, high surface area(high solubilization capacity),and very small droplet size. The small droplets have better chance to adhere to membranes and to transport bioactive molecules in a more controlled fashion.
Methods
1. To prepare nanoemulsion and investigate the physic-chemical properties of nanoemulsion
1.1 The pseudo-ternary phase diagrams were constructed by the dilution method at room temperature. Tween-80, Cremophor EL-35, Cremophor RH-40 as the surfactant, ethanol,1,2-isopropyl alcohol as the cosurfactant, IPM, ethyl linoleate, peanut oil and soy oil as the oil phase. These elements were mixed on a magnetic stirrer. Based on the pseudo-ternary phase diagrams, we investigated the effects of nanoemulsion components and Km(the ratio of surfactant to cosurfactant weight) on the formation and the areas of the nanoemulsion.
1.2 Appropriate quantities of surfactant, cosurfactant, oil and water were constructed in the nanoemulsion region of the pseudo-ternary phase diagrams. The mean diameter and apparent viscosity were measured. Meanwhile, we studdied the influence of nanoemulsion elements on the diameters and apparent viscosities and chose a good nanoemulsion system, which has larger nanoemulsion area and suitable diameter viscosity.
2.1n vitro skin permeation study of nanoemulsion transdermal delivery system
2.1 HPLC analysis of Vitamin A palmitate The amount of vitamin A palmitate in receptor phase was quantitated with the HPLC methods. The HPLC column used was a C18 column (Diamonsil,450X4.6mm,5μm particle size). The mobile phase consisted of methanol, acetonitrile and water(v/v/,66:32:2), at the floww rate aand injection volume of 1.0ml/min and 20μl, respectivelly. The detection waveleength was 275nm. All operations were carried out at room temperature. The calibration curve of vitamin A palmitate was constructed. The precision, reproduction and percent recovery of the method were analyzed.
2.2 Ultraviolet spectrophotometry analysis of Vitamin B1
The amount of vitamin B1 in receptor phase was quantitated with the ultraviolet spectrophotometry methods. The detection waveleength was 246nm. All operations were carried out at room temperature. The calibration curve of vitamin B1 was constructed. The precision, reproduction and percent recovery of the method were analyzed.
2.3 In vitro skin permeation study
The extent and rate of skin permeation of vitamin A palmitate and vitamin B1 from several different nanoemulsion which were prepared according to the orthogonal design were determined using Franz diffusion cells fitted with excised rat skins. Skins were obtained from male sprague-Dawley(SD) rats weighing 180-200g. after hair was removed carefully with a clipper, the subcutaneous fat and other extraneous tissues were trimmed. The excised skins were stored at-20℃and used within one week after the skin harvest. The receptor phase was 0.9% sodium chloride and 4ml was injected to the receptor phase. The effective diffusion area was 0.75cm2. after 4ml of each nanoemulsion was applied on the skin surface,0.5ml of receptor medium were withdrawn at different time. Concentration of vitamin A palmitate and vitamin BI in each sample were quantitated through the calibration curve of themselves, and the cumulative amount of vitamin A palmitae and vitamin B1 over a 12h period(Q) and the skin permeation rate(Jss)were calculated.
2.4 stability of nanoemulsion transdermal delivery system
To prepare the nanoemulsion transdermal delivery system according to the result of the orthogonal design. These nanoemulsion are stored for 3 month in room temperature and 40℃. The stability of nanoemulsion were investigated according to the change of vitamin A palmitate and vitamin B1.
3.in vivo irritating test for nanoemulsion nutrients transdermal delivery system
Single vs. multiple doses are performed in rat to test the irritation fo nutrients loaded nanoemulsion transdermal delivery system.
4. pharmacokinetics study of nutrients loaded nanoemulsion transdermal delivery system
4.1 HPLC analysis of Vitamin A palmitate in blood serum
The amount of vitamin A palmitate in blood serum was quantitated with the HPLC methods. The HPLC column used was a C18 column (Diamonsil,450X4.6mm,5μm particle size). The mobile phase consisted of methanol and water(v/v,95:5), at the floww rate aand injection volume of 1.2ml/min and 20μl, respectively. The detection waveleength was 325nm. All operations were carried out at room temperature. The calibration curve of vitamin A palmitate was constructed. The precision, reproduction and percent recovery of the method were analyzed.
4.2 pharmacokinetics study
Pharmacokinetic parameters were calculated after the male rats were transdermally administrated single dose of nutrients loaded nanoemulsion transdermal delivery system.
Results
1.Effect factors for the formation of nanoemulsion
Surfactants play a key role in many of the novel drug dilivery systems developed,and a wide range of surfactant-containing systems,including emulsions,liposomes,liquid crystalline phase,and microemulsions,are being extensively investigated in relation to drug delivery.Non-iron surfactants are investigated in our research.according to the phase diagram,we investigated the effects of the elements in the formation of the nanoemulsions. the ration of the surfactant to the co-surfactant,hydrocarbon length of co-surfactant,HLB value of surfactant,and the structure of oil,the change of additives in the water,the temperature are important in the microemulsion system.
2. In vitro skin permeation study of nanoemulsion transdermal delivery system
2.1 The retension time for vitamin A palmitate was observed to be 9.4min, and the elements of nanoemulsion did not have any effect on the determination of vitamin A palmitae. The calibration curve of vitamin A palmitae was A=75704C+45799 (R2=0.999) using an HPLC method, A was the peak area and C was the concentration of vitamin A palmitate standard solution. The standard curve was liner in the range of 8～160μg/ml. The RSD, reproducibility RSD and the average recovery confirmed the accuracy of this method as described above.
2.2 The calibration curve of vitamin B, was A=0.043C+0.001(R2=0.999) using an UV spectrophotometry method, A was the absorbance and C was the concentration of vitamin B1 standard solution. The standard curve was liner in the range of 7.5～17.5μg/ml. The RSD, reproducibility RSD and the average recovery confirmed the accuracy of this method as described above.
2.3 According to the orthogonal design, the relation among these tested factors is Km>oil phase (%)>water phase (%), the optimum formula is water phase(%):oil phase(%):Km(40:10:(4:1).
2.4 temperature accelerated test suggested the nutrients loaded nanoemulsion transdermal delivery system has been stability at 40℃for 3 months.
3.in vivo irritating test for nanoemulsion nutrients transdermal delivery system
It is clear that nanoemulsion is non-irritating carrier for transdermal drug delivery of nutrients, according to the resultant indices of treated skin.
4. pharmacokinetics study
4.1 The retension time for vitamin A palmitate in rat blood serum was observed to be 9.745min, and the elements of serum did not have any effect on the determination of vitamin A palmitae. The calibration curve of vitamin A palmitae was A=646.6C-4351(R2=0.999) using an HPLC method, A was the peak area and C was the concentration of vitamin A palmitate standard solution. The standard curve was liner in the range of 10-400ng/ml. The RSD, reproducibility RSD and the average recovery confirmed the accuracy of this method as described above.
4.2 pharmacokinetic parametes were calculated. Area under the drug concentration-time curve (AUC) were calculated using the trapezoidal rule. The maximal blood concentration were also obtained from blood data.AUC0-48h in this study is 2403.42ng/ml X h.
Conclusion
1. The present study clearly demonstrates that nanoemulsion can be well prepared with Cremophor RH-40 as surfactant, ethanol as cosurfactant and IPM as oil phase. The particle size and viscosity of nutrients loaded nanoemulsion and other physic-chemical properties all are desirable.
2. According to the orthogonal design, the relation among these tested factors is Km>oil phase (%)>water phase (%), the optimum formula is water phase(%):oil phase(%):Km(40:10:(4:1). temperature accelerated test suggested the nutrients loaded nanoemulsion transdermal delivery system has been stability at 40℃for 3 months.
3. It is clear that nanoemulsion is non-irritating carrier for transdermal drug delivery of nutrients, according to the single dose and multi-dose administration in the rat skin.
4. pharmacokinetic parametes were calculated. AUC0-48h in this study is 2403.42ng/ml×h. It means that nutrients loaded in the nanoemulsion can reach the systemic circulation.
引文
[1]中华人民共和国卫生部,中华人民共和国科学技术部,中华人民共和国国家统计局.中国居民营养与健康现状.2004年10月12日
[2]Fairfield KM, Fletcher RH, Vitamins for chronic disease prevention in adults: Scientific review, [J] JAMA 2002,287(23):3116-3126.
[3]Fletcher RH, Fairfield KM, Vitamins for chronic disease prevention in adults: Clinical applications, [J] JAMA 2002,287(23):3127-3129.
[4]刘炳智.美军筹划研制粘贴式营养膳食.军队卫生学环境医学动态,2003,12：3—4
[5]Jenning V,Gysler A, Schafer-Korting M,et al.Vitamin A loaded solid lipid nanoparticles for topical use:Occlusive Properties And drug targeting to th upper skin. Eur J Pharm Biopharm,2000,49:211-218
[6]Guo JX,Ping QN,Sun GQ,et al. Lecithin vesicular carriers for transdermal delivery of Vitamin A. Int J Pharm,2000,194:201-207
[7]Sentjurc M.Liposomes as a topical delivery system the role of size on transport studied by the EPR imaging method. J Contr Rel,1999,59:87-97
[8]Delgado Charro MB, Iglesias Vilas G,Blanco Mendez J,et al.Delivery of a hydrophilic solute through the skin from novel nanoemulsion systems.Eur J Pharm Biopharm,1997,43(1):37-42
[9]Cevc G. Drug delivery across the skin. Expert Opin Invest Drugs,1997,6: 1887-1937
[1]Friberg.S.in Nanoemulsions, Theory and Practice,Academic. Prince.L.M.ed New York:Academic Press,1977.133-148
[2]王晓黎,蒋雪涛.微乳在药剂学上的应用.解放军药学学报.2000,(2)：
[3]俞媛,高申,陈琰,等.盐酸哌甲酯一卵磷脂微乳的制备及其透皮吸收作用[J].中国药学杂志,2005,40(11)：843—845
[4]祝伟伟,翟光喜,于爱华,等.喷昔洛韦微乳经皮渗透的研究.山东大学学报,2008,46(3)：320-323
[5]CHEN H, CHANG X, WENG T, et al. A study of microemulsion systems for transdermal delivery of triptolide [J]. Control Release,2004,98(3):427—436
[6]李宁,张敬一,李川,等.氟比洛芬微乳的制备及其透皮吸收的研究.中国 医院药学杂志,2008,28(10)：819—822
[7]Warisnoicharoen.W, Lansley.A.B, Lawrence.M. Nonionic oil-in-water nanoemulsions:the effect of oil type on phase behaviour. Int J Pharm,2000,198(1):7
[8]郭荣,王秀文,李干佐.微乳液的微观结构与稳定理论.日用化学工业.1989,44：299
[9]Parris.N, Joubran.R.F, Lu.D.P. Triglyceride nanoemulsions:effect of nonionic surfactants and the nature of the oil. J Agric Food Chem.1994,42:1295
[10]李干佐.微乳液分类.特性和结构.见：李干佐,郭荣编微乳液理论及其应用.北京：石油工业出版社,1995.141-142
[11]崔正刚,殷福珊.微乳体系的配方设计.见：崔正刚,殷福珊编微乳化技术及应用1999,205-206
[12]Paschalis Alexandridis,Dali Zhou,Ali Khan Lyotropic Liquid Crystallinity in Amphiphilic Block Copolymers:Temperature Effects on Phase Behavior and Structure for Poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethelene oxide) Copolymers of Different Composition Langmuir 1996,12,2690-2700
[1]Charman SA,Charman WN,Rogge MC et al Self-emulsifying drug delivery systems.formulation and biopharmaceutic evaluation of an investigation lipophilic compound Pharm Res 1992,9,87-93
[2]Kale NJ,Allen LV,Studies on nanoemulsions using Brij 96 as surfactant and glycerin,ethylene glycol and propylene glycol as co-surfactant. Int.J.Pharm 1989,57.87-93
[3]Kyung-Mi Park,Chong-Kook Kim.Preparation and evaluation of flurbiprofen loaded nanoemulsion for parenteral delivery. Int.J.Pharm,181(1999):173-179
[4]park ES, Cui Y, Yun BJ, et al. Transdermal delivery of piroxican using microemulion. Arch Pharm Res.2005,28(2):243-248
[5]姚静, 周建平, 杨宇欣,等。微乳对难溶性药物增溶机理的研究。中国药科大学学报,2004,35(6)：495-498
[1]Varani J, Warner RL, Gharaee KM et al. Vitamin A antagonizes decreased cell growth and elevated collagen-degrading matrix metalloproteinases and stimulates collaen accumulation in naturally aged human skin. J Invest Dematol.2000,114:480-486
[2]Dong GK, Young-II J, Changyng Choi et al. Retinol-encapsulated low molecular water-soluble chitosan nanoparticles.2006,319:130-138
[3]Nasrin Ghouchi Eskandar, Spomenka Simovic, Clive A. Prestidge. Nanoparticle coated submicron emulsions:sustaind in-witro release and improved dermal delivery of all-trans-retinol.2009,26(7):1764-1775
[4]郭青敏赵小萍.复合多维中维生素A紫外分光光度法测定的改进.中国饲料.2001,12：26-26
[5]李雷,陈文利。高效液相色谱法测定脂溶性维生素的含量。浙江化工,2003,4：27-28
[1]高艳玲,杨迪,路福平,杜连祥紫外分光光度法测定LYCD中维生素B1含量 的研究.化学与生物工程,2008,25：73-75
[2]吕惠卿高效液相色谱一荧光检测法测定复合维B片中维生素B1含量.药物鉴定,2008,17(15)：24-25
[1]Lopcz A, Linares F, Cortell C, Herraez M. Int J Pharm 2000,202:133
[2]Fang JY. Yu SY, Wu PC, et al. Int J Pharm 2001,215:91
[3]0bata Y. Takayama K.Maitani Y, Machida Y, Nagai T. Int J Pharm,1993,89:191
[4]Williams AC, Barry BW.Adv Drug Deliv Rev,2004,56:603
[5]Pershing LK, Lambert LD, Knul\tson K. Pharm Res,1991,8:938
[6]Megrab NA, Williams AC, Barry BW, Int J Pharm,1995,116:101
[1]郑俊民 经皮给药新剂型.北京：人民卫生出版社,2006：473-479
[2]G.J. Nohyneka, W.J.A. Meuling, et al.Repeated topical treatment, in contrast to single oral doses, with Vitamin A-containing preparations does not affect plasma concentrations of retinol, retinyl esters or retinoic acids in female subjects of child-bearing age. Toxicology Letters 163 (2006) 65-76
[1]王增寿,陈慧,崔九虎,张慧,赵依娜.HPLC法测定人血清中维生素A浓度中国临床药学杂志,2005,14(6)：382-383
[2]李勇.营养与食品卫生学.北京；北京大学医学出版社,2005：139-141
[3]郑俊民经皮给药新剂型.北京：人民卫生出版社,2006：26-28
[1]郑俊民经皮给药新剂型.北京：人民卫生出版社,2006：480-486
1.T.P. Hoar, J. H. Schulman, Transparent water in oil dispersions:Oleopathic hydromicelle, Nature 152 (1943) 102.
2. Kreilgaard M, Pedemen EJ, Jaroszewski JW. NMR characterization and transdennal drug delivery potential of nanoemulsion systems. J Control Release,2000,69(3): 421
3 赵建忠、宴马成.油酸微乳对利多卡因透皮吸收的影响.医药导报2005年9月第24卷第9期
4.俞媛、高中、陈琰、钟延强、程明和.盐酸哌甲酯-卵磷脂微乳的制备及透皮吸收作用.中国药学杂志2005年6月第40卷第11期
5. Kemben J·Ziegler A, Mfiller BW. Influence of supersaturation on the pharmacodynamic effect of bupranolol after dermal administration using microemulsjOYIS as vehicle Pharm Res.1992; 9(4):554～558
6. Kemken J, Ziegler A, MUller BW. Pharmacodynamic effects of trasdermal bupranolol and timolol in vivo:comparison of nanoemulsions and matrix patches as vehicle. Methods Find Exp Clin Pharmacol,1991,13(5):36
7. Ogiso T, Shiraki T, Okajima K, et al. Transfolicular drug delivery: penetration of drugs through human scalp skin and comparison of penetration between scalp and abdominal skins in vitro. J Drag Target,2002,10:369-378.
8. Lademann, J., Otberg, N., Richter, H., Weigman, H. J., Lindemann, U., Schaefer, H., Sterry, W.,2001. Investigation of follicular penetration of topically applied substances. Skin Pharmacol. Appl. Skin Physiol.14,17-22.
9.陈国坤,龚赛君,周蓉蓉等,月桂氮卓酮对酮对雌二醇皮肤渗透的促进作用及其机理[J].中国药学杂志,1994,29(8)：467：2.
10. Lopez A, Linares F, Cortell C, Herraez M. Int J harm 2000:202:133. Fang JY, Yu SY, Wu PC, Huang YB, Tsai YH. Int J Pharm 2001:215:91.
11.鲁莹,吉小欣,高申,刘毅清.利多卡因—卵磷脂微乳处方的初步研究.药学实践杂志2004年第22卷第3期
12.汪杨,吴伟,阙俐.油吐温- 醇- 水体系伪三元相图在自微乳化制剂研究中的应用中国医药工业杂志 2005,36(6)
13 吴顺芹,李三鸣,赵国斌.微乳及其在药剂学中的应用.沈阳药科大学学报,2003,20(5)：381-385
14.张正全,陆彬. 微乳给药系统研究概况[J].中国医药工业杂志,2001,32(3)：139-142.
15. Jang-Hoon Kweon, Sang-Cheol Chi, Eun-Seok Park. Transdermal Delivery of Diclofenac Using Nanoemulsions. Arch Pharm Res Vol 27, No 3,351-356,2004
16. Eun-Seok Park, YuCui, Bum-JinYun, In-JaKo, and Sang-CheolChi, ransdermal Transdermal Delivery of Piroxicam Using Nanoemulsions, College of Pharmacy, Sungkyunkwan University, Suwon, Kyunggi-Do 440-746, Korea, Arch Pharm Res Vol 28, No 2,243-248,2005
17. Tanojo H, BosvanGeest A, Bouwstra JA, Junginger HE, Bodde HE. Thermochim Acta 1997;293:77.
18. Amnon.C Sintov, Shapiro L. New nanoemulsion vehicle facilitates percutaneous penetration in vitro and cutaneous drug bioavailability in vivo. J Control Release, 2004,95:173
19. Kriwet K, Muller-Goymann CC. Diclofenac release from phospholipids drug systems and permeation through excised human stratum corneum[J]. Int J Pharm,1995,125(2): 231-242.
20. Baroli B, Lopez-Quintela MA, Delgado-Charro MB, et al. Nanoemulsions for topical delivery of 8-methoxsalen. J Control Release,2000,69(1):209-218.
21.廉云飞,李娟,平其能,严菲.吲哚美辛微乳的制备及经皮吸收研究中国医药工业杂志Chinese Journal of Pharmaceuticals 2005,36 (3)
22. Gupta RR, Jain SK, Varshney M. Colloids Surf B Biointerfaces 2005:41:25.
23. Bolzinger MA, Carduner C. Bicontinuous sucrose ester nanoemulsion:a new vehicle for topical delivery of niflumic acid.Int J Pharm,1998,176(1):39-45.
24. Kreilgaard M. Dermal pharmacokinetics of nanoemulsion formulations determined by in vivo microdialysis. Pharm Res,2001,18(3):367-373.
25. Kreilgaard M, Kemme MJ, Burggraaf J, et al. Influence of a nanoemulsion vehicle on cutaneous bioequivalence of a lipophilic model drug assessed by microdialysis and pharmacodynamics. Pha rm Res,2001,18(5):593-599.
26. Jaehwi Lee, Yoonin Lee, Jongseok Kim, Mikyeong Yoon, and Young Wook Choi. Formulation of Nanoemulsion Systems for Transdermal Delivery of Aceclofenac. Arch Pharm Res Vol 28, No 9,1097-1102,2005
27. Arellano, S., Santoyo, S., Martin, C., and Ygartua, P..Enhancing effect of terpenes on the in vitro percutaneous absorption of diclofenac sodium. Int. J. Pharm. 130,141-14 (1996).
28. Williams, A. C. and Barry, B. W., Terpenes and the lipid-proteinpartitioning theory of skin penetration enhancement. Pharm. Res.,8,17-24(1991a).
Williams, A. C. and Barry, B. W., The enhancement index concept applied to terpene penetration enhancers for human skin and model lipophilic(oestradiol) and hydrophilic (5-fluorouracil) drugs. Int. J. Pharm.,74,157-168 (1991b)
[2]Fairfield KM, Fletcher RH, Vitamins for chronic disease prevention in adults: Scientific review, [J] JAMA 2002,287(23):3116-3126.
[3]Fletcher RH, Fairfield KM, Vitamins for chronic disease prevention in adults: Clinical applications, [J] JAMA 2002,287(23):3127-3129.
[4]刘炳智.美军筹划研制粘贴式营养膳食.军队卫生学环境医学动态,2003,12：3—4
[5]Jenning V,Gysler A, Schafer-Korting M,et al.Vitamin A loaded solid lipid nanoparticles for topical use:Occlusive Properties And drug targeting to th upper skin. Eur J Pharm Biopharm,2000,49:211-218
[6]Guo JX,Ping QN,Sun GQ,et al. Lecithin vesicular carriers for transdermal delivery of Vitamin A. Int J Pharm,2000,194:201-207
[7]Sentjurc M.Liposomes as a topical delivery system the role of size on transport studied by the EPR imaging method. J Contr Rel,1999,59:87-97
[8]Delgado Charro MB, Iglesias Vilas G,Blanco Mendez J,et al.Delivery of a hydrophilic solute through the skin from novel nanoemulsion systems.Eur J Pharm Biopharm,1997,43(1):37-42
[9]Cevc G. Drug delivery across the skin. Expert Opin Invest Drugs,1997,6: 1887-1937
[1]Friberg.S.in Nanoemulsions, Theory and Practice,Academic. Prince.L.M.ed New York:Academic Press,1977.133-148
[2]王晓黎,蒋雪涛.微乳在药剂学上的应用.解放军药学学报.2000,(2)：
[3]俞媛,高申,陈琰,等.盐酸哌甲酯一卵磷脂微乳的制备及其透皮吸收作用[J].中国药学杂志,2005,40(11)：843—845
[4]祝伟伟,翟光喜,于爱华,等.喷昔洛韦微乳经皮渗透的研究.山东大学学报,2008,46(3)：320-323
[5]CHEN H, CHANG X, WENG T, et al. A study of microemulsion systems for transdermal delivery of triptolide [J]. Control Release,2004,98(3):427—436
[6]李宁,张敬一,李川,等.氟比洛芬微乳的制备及其透皮吸收的研究.中国 医院药学杂志,2008,28(10)：819—822
[7]Warisnoicharoen.W, Lansley.A.B, Lawrence.M. Nonionic oil-in-water nanoemulsions:the effect of oil type on phase behaviour. Int J Pharm,2000,198(1):7
[8]郭荣,王秀文,李干佐.微乳液的微观结构与稳定理论.日用化学工业.1989,44：299
[9]Parris.N, Joubran.R.F, Lu.D.P. Triglyceride nanoemulsions:effect of nonionic surfactants and the nature of the oil. J Agric Food Chem.1994,42:1295
[10]李干佐.微乳液分类.特性和结构.见：李干佐,郭荣编微乳液理论及其应用.北京：石油工业出版社,1995.141-142
[11]崔正刚,殷福珊.微乳体系的配方设计.见：崔正刚,殷福珊编微乳化技术及应用1999,205-206
[12]Paschalis Alexandridis,Dali Zhou,Ali Khan Lyotropic Liquid Crystallinity in Amphiphilic Block Copolymers:Temperature Effects on Phase Behavior and Structure for Poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethelene oxide) Copolymers of Different Composition Langmuir 1996,12,2690-2700
[1]Charman SA,Charman WN,Rogge MC et al Self-emulsifying drug delivery systems.formulation and biopharmaceutic evaluation of an investigation lipophilic compound Pharm Res 1992,9,87-93
[2]Kale NJ,Allen LV,Studies on nanoemulsions using Brij 96 as surfactant and glycerin,ethylene glycol and propylene glycol as co-surfactant. Int.J.Pharm 1989,57.87-93
[3]Kyung-Mi Park,Chong-Kook Kim.Preparation and evaluation of flurbiprofen loaded nanoemulsion for parenteral delivery. Int.J.Pharm,181(1999):173-179
[4]park ES, Cui Y, Yun BJ, et al. Transdermal delivery of piroxican using microemulion. Arch Pharm Res.2005,28(2):243-248
[5]姚静, 周建平, 杨宇欣,等。微乳对难溶性药物增溶机理的研究。中国药科大学学报,2004,35(6)：495-498
[1]Varani J, Warner RL, Gharaee KM et al. Vitamin A antagonizes decreased cell growth and elevated collagen-degrading matrix metalloproteinases and stimulates collaen accumulation in naturally aged human skin. J Invest Dematol.2000,114:480-486
[2]Dong GK, Young-II J, Changyng Choi et al. Retinol-encapsulated low molecular water-soluble chitosan nanoparticles.2006,319:130-138
[3]Nasrin Ghouchi Eskandar, Spomenka Simovic, Clive A. Prestidge. Nanoparticle coated submicron emulsions:sustaind in-witro release and improved dermal delivery of all-trans-retinol.2009,26(7):1764-1775
[4]郭青敏赵小萍.复合多维中维生素A紫外分光光度法测定的改进.中国饲料.2001,12：26-26
[5]李雷,陈文利。高效液相色谱法测定脂溶性维生素的含量。浙江化工,2003,4：27-28
[1]高艳玲,杨迪,路福平,杜连祥紫外分光光度法测定LYCD中维生素B1含量 的研究.化学与生物工程,2008,25：73-75
[2]吕惠卿高效液相色谱一荧光检测法测定复合维B片中维生素B1含量.药物鉴定,2008,17(15)：24-25
[1]Lopcz A, Linares F, Cortell C, Herraez M. Int J Pharm 2000,202:133
[2]Fang JY. Yu SY, Wu PC, et al. Int J Pharm 2001,215:91
[3]0bata Y. Takayama K.Maitani Y, Machida Y, Nagai T. Int J Pharm,1993,89:191
[4]Williams AC, Barry BW.Adv Drug Deliv Rev,2004,56:603
[5]Pershing LK, Lambert LD, Knul\tson K. Pharm Res,1991,8:938
[6]Megrab NA, Williams AC, Barry BW, Int J Pharm,1995,116:101
[1]郑俊民 经皮给药新剂型.北京：人民卫生出版社,2006：473-479
[2]G.J. Nohyneka, W.J.A. Meuling, et al.Repeated topical treatment, in contrast to single oral doses, with Vitamin A-containing preparations does not affect plasma concentrations of retinol, retinyl esters or retinoic acids in female subjects of child-bearing age. Toxicology Letters 163 (2006) 65-76
[1]王增寿,陈慧,崔九虎,张慧,赵依娜.HPLC法测定人血清中维生素A浓度中国临床药学杂志,2005,14(6)：382-383
[2]李勇.营养与食品卫生学.北京；北京大学医学出版社,2005：139-141
[3]郑俊民经皮给药新剂型.北京：人民卫生出版社,2006：26-28
[1]郑俊民经皮给药新剂型.北京：人民卫生出版社,2006：480-486
1.T.P. Hoar, J. H. Schulman, Transparent water in oil dispersions:Oleopathic hydromicelle, Nature 152 (1943) 102.
2. Kreilgaard M, Pedemen EJ, Jaroszewski JW. NMR characterization and transdennal drug delivery potential of nanoemulsion systems. J Control Release,2000,69(3): 421
3 赵建忠、宴马成.油酸微乳对利多卡因透皮吸收的影响.医药导报2005年9月第24卷第9期
4.俞媛、高中、陈琰、钟延强、程明和.盐酸哌甲酯-卵磷脂微乳的制备及透皮吸收作用.中国药学杂志2005年6月第40卷第11期
5. Kemben J·Ziegler A, Mfiller BW. Influence of supersaturation on the pharmacodynamic effect of bupranolol after dermal administration using microemulsjOYIS as vehicle Pharm Res.1992; 9(4):554～558
6. Kemken J, Ziegler A, MUller BW. Pharmacodynamic effects of trasdermal bupranolol and timolol in vivo:comparison of nanoemulsions and matrix patches as vehicle. Methods Find Exp Clin Pharmacol,1991,13(5):36
7. Ogiso T, Shiraki T, Okajima K, et al. Transfolicular drug delivery: penetration of drugs through human scalp skin and comparison of penetration between scalp and abdominal skins in vitro. J Drag Target,2002,10:369-378.
8. Lademann, J., Otberg, N., Richter, H., Weigman, H. J., Lindemann, U., Schaefer, H., Sterry, W.,2001. Investigation of follicular penetration of topically applied substances. Skin Pharmacol. Appl. Skin Physiol.14,17-22.
9.陈国坤,龚赛君,周蓉蓉等,月桂氮卓酮对酮对雌二醇皮肤渗透的促进作用及其机理[J].中国药学杂志,1994,29(8)：467：2.
10. Lopez A, Linares F, Cortell C, Herraez M. Int J harm 2000:202:133. Fang JY, Yu SY, Wu PC, Huang YB, Tsai YH. Int J Pharm 2001:215:91.
11.鲁莹,吉小欣,高申,刘毅清.利多卡因—卵磷脂微乳处方的初步研究.药学实践杂志2004年第22卷第3期
12.汪杨,吴伟,阙俐.油吐温- 醇- 水体系伪三元相图在自微乳化制剂研究中的应用中国医药工业杂志 2005,36(6)
13 吴顺芹,李三鸣,赵国斌.微乳及其在药剂学中的应用.沈阳药科大学学报,2003,20(5)：381-385
14.张正全,陆彬. 微乳给药系统研究概况[J].中国医药工业杂志,2001,32(3)：139-142.
15. Jang-Hoon Kweon, Sang-Cheol Chi, Eun-Seok Park. Transdermal Delivery of Diclofenac Using Nanoemulsions. Arch Pharm Res Vol 27, No 3,351-356,2004
16. Eun-Seok Park, YuCui, Bum-JinYun, In-JaKo, and Sang-CheolChi, ransdermal Transdermal Delivery of Piroxicam Using Nanoemulsions, College of Pharmacy, Sungkyunkwan University, Suwon, Kyunggi-Do 440-746, Korea, Arch Pharm Res Vol 28, No 2,243-248,2005
17. Tanojo H, BosvanGeest A, Bouwstra JA, Junginger HE, Bodde HE. Thermochim Acta 1997;293:77.
18. Amnon.C Sintov, Shapiro L. New nanoemulsion vehicle facilitates percutaneous penetration in vitro and cutaneous drug bioavailability in vivo. J Control Release, 2004,95:173
19. Kriwet K, Muller-Goymann CC. Diclofenac release from phospholipids drug systems and permeation through excised human stratum corneum[J]. Int J Pharm,1995,125(2): 231-242.
20. Baroli B, Lopez-Quintela MA, Delgado-Charro MB, et al. Nanoemulsions for topical delivery of 8-methoxsalen. J Control Release,2000,69(1):209-218.
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