Studies On Nutrients Loaded Nanoemulsion Transdermal Delivery System

IntroductionMulti-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.Methods1. To prepare nanoemulsion and investigate the physic-chemical properties of nanoemulsion1.1 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 system2.1 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 B1The 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 studyThe 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 systemTo 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 systemSingle 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 system4.1 HPLC analysis of Vitamin A palmitate in blood serumThe 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 studyPharmacokinetic parameters were calculated after the male rats were transdermally administrated single dose of nutrients loaded nanoemulsion transdermal delivery system. Results1.Effect factors for the formation of nanoemulsionSurfactants 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 system2.1 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 systemIt is clear that nanoemulsion is non-irritating carrier for transdermal drug delivery of nutrients, according to the resultant indices of treated skin.4. pharmacokinetics study4.1 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.Conclusion1. 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.