Preparation And Cosmetic Efficacy Evaluation Of Transfersome Gel Containing 6 Functional Components

Objective:Antioxidation,anti-aging,and whitening are the three most important functions pursued by luxury skincare market.However,skincare containing functional ingredients cannot effectively exert their effects well,because ingredients with clear skincare effects are difficult to be absorbed by the skin.With lipid bilayer nano-dispersed structure and high deformability,transfersomes can smoothly through pores that are dozens of times smaller than their own volume to promote the transdermal absorption of functional ingredients.In this paper,6 kinds of functional ingredients commonly used in luxury skincare were combined in a certain proportion and designed into a complex transfersomes gel to improve the skincare efficacy of the product.Methods:Human skin fibroblasts（AT3）were stimulated with hydrogen peroxide（H₂O₂）to screen modeling conditions to build cell models of oxidative stress and senescence.Arbutin,tranexamic acid,hyaluronic acid,Vitamin B₁₂(VB₁₂),allantoin,and hydroxyethyl urea were mixed in a certain proportion then the mixture was used as active pharmaceutical composition（APC）.The effect of APC on the change of AT3 cell viability was detected by the Cell Counting Kit-8;the effect of APC on reactive oxygen species（ROS）in oxidative stress cell model was analyzed by the Reactive Oxygen Species Assay Kit and the effect of APC on positively blue-stained senescent cells in a H₂O₂-induced senescent cell model was detected using the Senescenceβ-Galactosidase Staining Kit.The in vitro analysis method of VB₁₂was established by high performance liquid chromatography,and its methodology was validated.VB₁₂ with high molecular weight and strong hydrophilicity,which is not easy to penetrate the skin,was selected as the model drug and the VB₁₂ transfersomes were prepared by the traditional film hydration method.Through the VB₁₂ transfersomes formulation and process investigation,the particle size,polydispersity index（PDI）,Zeta potential and encapsulation efficiency were used as evaluation indicators to screen out the preparation process and formulation of VB₁₂transfersome with high encapsulation efficiency and stable properties.The VB₁₂transfersomes were prepared by the optimum formulation and process,and they pharmacological characterization were evaluated.In vitro transdermal studies of VB₁₂transfersomes were carried out in guinea pig skin to evalute the effect of promoting the transdermal penetration of the transfersomes.The VB₁₂ transfersomes with arbutin,tranexamic acid,hyaluronic acid,allantoin,hydroxyethyl urea and other preparation excipients were loaded into the gel matrix carbomer 980 in a certain proportion to prepare a composite transfersomes gel（CTG）.By observing the appearance and microscopic morphology of CTG,the particle size,PDI,Zeta potential,viscosity,p H value and other indicators were measured to investigate its physical and chemical properties.The stability of CTG was preliminarily investigated under different temperature conditions.Taking the zebrafish as an in vivo activity evaluation model,the safe concentration of CTG to zebrafish larvae under various experimental conditions was firstly investigated.At a safe concentration,the inhibitory effect of different concentrations of CTG on zebrafish embryo melanin production was compared and observed;the neutrophil-red fluorescent zebrafish Tg（lyz:Ds RED2）was used to construct an inflammatory model to evaluate the anti-inflammatory activity of CTG;the AB zebrafish was used to evaluate the antioxidant activity of CTG active.Using guinea pigs as experimental animals,the skin irritation of CTG and its negative control was investigated,and the histopathological changes of guinea pig skin were observed to evaluate the safety of CTG transdermal drug delivery system.Results:Through the investigation of the experimental conditions of the in vitro cell model,a single stimulation of 20μmol/L H₂O₂ for 1 h was selected as the test condition for cell viability.After H₂O₂ induction,the DMEM medium was used for further incubation for 4 h as the experimental time to detect intracellular ROS,and H₂O₂ stimulation was selected for1 h then incubate for another 3 d for staining as an experimental condition for AT3 cell senescence.The results of cell viability showed that APC could alleviate H₂O₂-induced AT3cell viability decrease,oxidative stress injury and cell senescence,and also had a concentration-dependent trend in alleviating H₂O₂-induced AT3 cell viability decrease and ROS increase.A VB₁₂ high performance liquid chromatography content analysis method was established.The result of the linear relationship was in the concentration range of 5～200μg/m L.The VB₁₂ chromatographic peak area A had a good linear relationship with the concentration C.The experimental results of all methods were in line with the methodology and the established analytical methods were scientific and reasonable.Through the screening of the preparation method,formulation and process of VB₁₂ transfersomes,it was determined that the film dispersion method was used to prepare the VB₁₂ transfersomes,and its optimal formulation and process were determined as:0.1 g soy lecithin and 0.04 g cholesterol were placed in a round-bottomed flask,dissolved in a ratio of methanol:chloroform of 5:3,then under the condition of a water bath at 45°C.After the organic solvent was removed by rotary evaporation under reduced pressure,0.01 g VB₁₂was added to dissolve Tween 80 in 10 m L of deionized water,then hydrated at 50°C for 60min,and left at room temperature for 120 min.Finally,the ice bath probe was sonicated for20 min to obtain the VB₁₂ transfersomes.The VB₁₂ transfersomes suspension prepared in this study was clear and transparent,and no bad odor.The particle size was about 114nm,the PDI value was about 0.2,the Zeta potential was about-38 m V,the p H value was 6.55approximately,the encapsulation efficiency was about 65%,and the drug loading was about10.26%.The results of in vitro transdermal experiments showed that the VB₁₂transferosomes prepared in this study had significant transdermal permeability and skin retention（P<0.05）,compared with the directly dissolved VB₁₂ aqueous solution and traditional VB₁₂ liposomes.Using carbomer 980 as the gel matrix,the screening results showed that the dosage of0.5%of carbomer 980 was the most suitable,and the obtained CTG was in the form of a fine gel,with suitable viscosity,easy to stir up,and could spread evenly on the skin.Determine the final prescription composition of CTG:with contain 2%glycerin,1%arbutin,0.8%tranexamic acid,0.2%hyaluronic acid,0.2%allantoin,0.1%hydroxyethyl urea,0.1%phenoxyethanol,0.5%carbomer 980,the hydrogel was mixed with VB₁₂ transfersomes 1:1（w/w）to prepare the complex transfersomes gel.The quality evaluation results showed that the CTG obtained was pink gel-like in appearance,fine and uniform in texture;the average particle size of the CTG was（131.6±0.28）nm,and the particles were round and uniformly distributed under the transmission electron microscope;the PDI was（0.21±0.08）;the average Zeta potential was（-48.38±0.55）m V;the average viscosity was（43.85±0.25）Pa·s,suitable for smearing;the average p H was（6.82±0.01）.There was no obvious change in the appearance of CTG after centrifugation,indicating that it had good physical stability.The CTG stability test results showed that compared with the control group,the appearance,particle size,PDI value,viscosity and p H value of the samples did not change significantly,indicating that CTG had low temperature,high temperature and long-term stability.The zebrafish tolerance experiment showed that under the conditions of the whitening experiment,the safe concentration range of CTG to zebrafish was 20 mg/m L and below;in the zebrafish anti-inflammatory experiment,the safe concentration range of CTG was 100mg/m L and above;The safe concentration range of CTG in anti-oxidant experiments is 200mg/m L and below.The results of the zebrafish activity experiment showed that CTG had a significant inhibitory effect on melanin in zebrafish;the treatment of zebrafish with Cu SO₄significantly caused zebrafish neutrophils to migrate to the lateral line neuromast,proving that the zebrafish model of inflammation was build successfully.Using zebrafish model,it was found that different concentrations of CTG could inhibit the migration of neutrophils to the lateral line neuromast caused by Cu SO₄,and there was a statistical difference,which suggested an anti-inflammatory effect.Under the experimental conditions,different concentrations of CTG had inhibitory effects on 5 mmol/L H₂O₂-induced apoptosis and ROS generation in zebrafish.The results of the CTG safety evaluation showed that neither CTG nor negative control caused serious irritation when applied to the skin of guinea pigs.The pathological tissue sections of the drug-treated guinea pig skin showed that the overall structure of the skin was intact,and no obvious inflammatory histopathological changes were caused,indicating that CTG was relatively safe as a transdermal drug delivery system.Conclusion:The composition of 6 cosmetic functional ingredients had clear cellular anti-oxidant and anti-aging activities.The prepared transfersomes can promote the transdermal absorption of functional ingredients.The composition transfersomes gel had significant anti-melanin,anti-inflammatory and anti-oxidant effects on zebrafish,and had good stability and no obvious skin irritation.It will have strong market competitiveness when developed as a functional cosmetic.