Preparation,Characterization And Evaluation Of Minoxidil Transfersomes And Its Gel For The Topical Treatment Of Alopecia

Objective:Androgenetic alopecia（AGA）is a disease with a high incidence at present,and with the improvement of living standards,there is an increasing demand for its treatment products from patients.Minoxidil（MXD）is generally regarded as the first-line drug for the treatment of AGA in China and abroad.Due to its poor water solubility,the current commercially available topical preparations are tinctures and applications made of ethanol and propylene glycol as solvents,which are poorly absorbed by the skin,and with the evaporation of ethanol,a large number of drug crystals will remain on the skin surface,causing adverse reactions including pruritus,rash,dandruff and allergic contact dermatitis,affecting patients’compliance and therapeutic efficacy.Transferosomes is the most effective nanotechnology-based transdermal drug delivery systems,however,low drug loading and entrapment efficiency limit their potential dermal delivery capabilities.The objective of this paper is to prepare MXD transfersomes with high drug loading and entrapment efficiency for more effective topical treatment of alopecia.Methods:A method for the in vitro analysis of MXD was established by high performance liquid chromatography（HPLC）for methodological validation.The solubility of MXD in different solution was investigated to determine the receiving solution for in vitro transdermal assays.The unencapsulated drug in MXD transfersomes was separated by ultrafiltration centrifugation,and the determination methods of entrapment efficiency and drug loading were established,and they were used as evaluation indexes to screen the preparation methods of the transfersomes.Based on the single-factor examination,the Box-Behnken response surface optimization method was used to determine the prescription and preparation process of the transfersomes with the particle size,entrapment efficiency and drug loading as the evaluation indexes.The physicochemical properties of the optimized transfersomes were investigated by the determination of appearance morphology,particle size,polydispersity index（PDI）,zeta potential and p H,and characterized by Transmission Electron Microscopy（TEM）,the determination of entrapment efficiency and drug loading,Differential Scanning Calorimetry（DSC）and Fourier Transform Infrared Spectroscopy（FT-IR）.The preparation method of MXD transfersomal gel was determined by screening the dosage of gel matrix,and the stability was initially evaluated by physical and chemical property examination.The permeation activity of MXD transfersomes and its gel through the skin of isolated guinea pigs was investigated using the steady-state drug permeation rate(J_ss)and drug skin retention（D_R）as indicators.The effect of MXD transfersomes and its gel on hair regeneration in mice with artificially induced alopecia C57BL/6 mice was investigated by assessing the hair growth on the back of mice and histological observation using commercially available MXD applicator and saline as positive and negative controls,respectively.Skin irritation and histopathological studies of MXD transfersomal gel were performed to evaluate its safety using guinea pig skin as a study subject.Results:The chromatographic conditions established for MXD HPLC were as follows:Kromasil 100?C18 column（4.6 mm×250 mm,5μm）;mobile phase:methanol-water-acetic acid（50:50:1,v/v/v）;flow rate:0.8 m L/min;detection wavelength:279 nm;column temperature:35℃;injection volume:10μL.MXD showed good linearity in the range of10～250μg/m L,A=132354 C-144521,R²=0.9998,The established analytical method meets the methodological requirements.The maximum solubility of MXD in 30%ethanol-saline was（4.42±0.84）mg/m L,so 30%ethanol-saline was selected as the accepting medium for in vitro transdermal.MXD transfersomes was prepared by thin film dispersion method,and the drug loading and entrapment efficiency of MXD by the transfersomes could be significantly improved by dissolving MXD and edge activator in the aqueous phase.Based on the single-factor experiment,the optimal mass ratio of phospholipid,cholesterol and drug was5.28:1:1 by Box-Behnken response surface optimization,and the MXD transfersomes was light yellow translucent solution with clear appearance,no precipitation and flocculent.The average particle size was（76.73±0.93）nm,PDI was（0.23±0.01）,zeta potential was（-50.60±0.44）m V,p H was（7.03±0.45）;DSC results showed that MXD was in an amorphous state in the transfersomes,indicating that MXD had been uniformly wrapped in the vesicles of the transfersomes;FT-IR characterization showed no significant difference between the IR spectra of the MXD transfersomes and the blank transfersomes,and only the characteristic peak of-OH stretching vibration was enhanced,probably through the formation of hydrogen bonds by interaction,proving that MXD was encapsulated in the vesicles of the transfersomes.The concentration of carbomer 980 was chosen to be 0.5%and the mass ratio of 1:1with the transfersomes was chosen to prepare the transfersomal gel.The appearance of the resulting transfersomal gel was light yellow gel with no particles or lumps visible to the naked eye,easy to pick up,moderate viscosity,fine and uniform texture;the average particle size was（88.57±0.45）nm,PDI was（0.29±0.03）,zeta potential was（-46.70±0.72）m V,p H was（7.04±0.66）,and viscosity was（66.95±0.31）Pa·s.The subspherical vesicle shape was observed under TEM,with no aggregation and uniform dispersion,and the presented particle size was basically consistent with the results of the particle size measurement.In the centrifugation experiments,no precipitation and delamination were observed for MXD transfersomes and its gel,which indicated that they had good physical stability.The results of stability investigation showed that the transfersomes was stable when stored at 4℃for 3 months,but unstable when stored at 25℃and 40℃for 3months;the transfersomal gel had low and high temperature stability due to the transfersomal vesicles embedded in the gel skeleton,which increased its stability and made the MXD transfersomal gel stable at low and high temperatures,and long-term stability experiments showed that it was still stable at 4℃or even 25℃for 6 months,but only stable at 40℃for 1 month.The results of in vitro transdermal experiments showed that both the J_ssand D_Rof MXD transfersomes and its gel were dose-related and significantly higher than those of commercially available formulations,indicating that transfersomes as a transdermal drug delivery vehicle can significantly improve the transdermal penetration rate of MXD.Among them,the D_Rof MXD transfersomal gel was 1.91 times higher than that of the transfersomes,indicating that it has a stronger drug reservoir effect.Hair growth assessment experiments and histological observations showed that for alopecia-induced C57/BL6 mice,MXD preparations promoted melanin synthesis in the hair follicles and prolonged the anagen phase and shortened the resting phase of the hair follicles.The hair growth-promoting effect of MXD transfersomes and its gel was significantly better than that of commercially available preparations,with MXD transfersomal gel being superior to MXD transfersomes due to their better dermal drug storage potential.The results of both single and multiple administration safety evaluation experiments showed that the MXD transfersomal gel formulation had no irritating effect on the epidermis and dermis of guinea pig dorsal skin and the developed formulation can be considered safe.Conclusion:The MXD transferosomes designed and prepared in this paper has a high drug loading and entrapment efficiency,and has a significantly better hair growth promoting effect than commercially available MXD products.Preparing it into a gel with significantly improved stability and hair growth effect,and with easy administration and no skin irritation,is expected to be a promising strategy for the topical treatment of alopecia.