| Objective:Androgenic alopecia(AGA)is the most common hair loss disease,and the demand for its treatment products is increasing among patients.Transfersome(TF)is a highly deformable nano transdermal drug delivery system.Encapsulating the minoxidil(MXD)in a transfersome can improve the percutaneous absorption of MXD,but the treatment effect is poor for patients with hair follicle inflammation.Ginsenoside Rg3(Rg3)has the effects of inhibiting 5α-reductase and anti-inflammation.In this paper,a carrier(MXD-Rg3 @ TFS)co-loaded with Rg3 and MXD was designed and prepared in order to treat AGA better by inhibiting the androgen transformation and inflammation level of AGA.Methods:A high-performance liquid chromatography(HPLC)method was used to establish an analytical method for simultaneous determination of Rg3 and MXD content,and was validated through specificity experiments,precision experiments,recovery experiments,and repeatability experiments.MXD-Rg3 @TFs was prepared using thin film dispersion method.By measuring particle size,polydispersity index(PDI),Zeta potential,and drug encapsulation efficiency,the formulation and process of delivery systems with good stability and high encapsulation efficiency were screened and determined.The optimized preparation prescription and process were used to prepare the transfersomes.The particle size,PDI,Zeta potential,encapsulation rate and drug loading were measured,and the p H was also determined.MXDRg3 @TFs and MXD-Ch @TFs were prepared with the same prescription and process to investigate the storage stability.Human dermal papilla cells(DPCs)were stimulated by Dihydrotestosterone(DHT),and the cell viability was determined by Cell Counting Kit-8(CCK-8)kit,so as to determine the modeling conditions of AGA cell model.Then CCK-8 kit was used to detect the effects of Rg3,MXD and their combined use(1:1)on the viability of DPCs cells stimulated by DHT.The cell senescenceβ-galactosidase staining kit was used to detect the senescence of DPCs cells after DHT stimulation by drugs.The sexual oxygen detection kit was used to analyze the effect of drugs on reactive oxygen species(ROS)in DPCs cells after DHT stimulation.Finally,the effects of the drugs on the levels of inflammatory factors TNF-αand IL-6 in the DHT-stimulated DPCs cell culture supernatant were detected by enzyme-labeled kit.The AGA model of C57BL/6 mice was established by applying testosterone propionate solution continuously for 21 days.Local application of normal saline and commercial minoxidil tincture were used as negative control and positive control,respectively.The therapeutic effect of MXD-Rg3 @TFs on AGA was evaluated by examining the hair growth and tissue sections on the back of the mouse,and detecting the levels of DHT,TNF-α and IL-6 in the skin tissues of the subjects.The guinea pig was used as an animal model to investigate the skin irritation of the blank transfersome and MXD-Rg3 @TFs,and C57BL/6 mice were used as an animal model to investigate the histopathological changes of the skin after continuous use of the blank transfersome and MXD-Rg3 @TFs for 21 days.Results:HPLC method was developed for the simultaneous determination of Rg3 and MXD using GL Sciences ODS-3 column(4.6×150mm,5μm)with acetonitrile-0.1% phosphoric acid water(60:40)as the mobile phase at the flow rate of 1m L/min,the column temperature of 35℃,and the injection volume of 5 μL.The detection was performed at the wavelength of 203 nm.The peak areas of MXD and Rg3 had a good linear relationship with the concentration in the concentration range of 5–150 μg/m L.Solvent,carrier and excipients did not interfere with the determination of MXD and Rg3.The results of methodology validation met the requirements of analytical methodology,indicating that the established analytical method was scientific and could be used for the simultaneous determination of Rg3 and MXD.MXD-Rg3 @TFs was prepared by film dispersion method.The preparation prescription and process of the transfersome are determined as follows: 0.15 g soybean lecithin and 0.015 g Rg3 were weighed and placed in a round bottom flask,a mixed solution of trichloromethane and methanol with the volume ratio of 10:1 is added for dissolution,and an organic solvent is removed by reduced pressure rotary evaporation under the condition of a constant temperature water bath at 25℃,Then,they were added into 5 m L deionized water in which 0.02 g MXD and 0.01 g Tween 80 were dissolved,hydrated for 60 min under the constant temperature of 50℃,placed at room temperature for 120 min,and then subjected to ultrasonic treatment for 12 min using the probe of an ultrasonic cell breaker to obtain MXD-Rg3 @TFs.The prepared MXD-Rg3 @TFs was a clear and transparent solution.The morphology of MXD-RG3 @TFs was spherical under the transmission electron microscope,and there was no aggregation phenomenon.The mean particle size was(84.17±0.60)nm,PDI was(0.25 ± 0.01),Zeta potential was(--47.8 ± 0.65)m V,encapsulation efficiency was(89.51 ± 0.56)%,drug loading was(15.99 ± 0.06)%,and p H was(6.70 ± 0.15).The storage stability of the transporters was investigated and it was found that MXD-Rg3 @TFs was more stable than Ch-MXD-TFs and it was more stable when stored at 4℃.The DPCs were incubated with MXD,Rg3 and their mixtures for 48 h,and then stimulated with 40 ng/m L DHT for 24 h.The results showed that MXD,Rg3 and their mixtures could alleviate the DHT-induced decline in cell viability,promote cell migration,inhibit the DHT-induced senescence of DPCs cells,and reduce intracellular ROS level.Moreover,the mixture of the two had a better effect than the single use of MXD and Rg3.In addition,Rg3 can also inhibit the levels of TNF-α and IL-6 secreted by DPCs after DHT stimulation,and has a therapeutic prospect for causing inflammation of AGA.After continuous topical application of testosterone propionate solution for 21 days,the C57BL/6 mouse androgenic alopecia model was successfully established.Local application of commercial minoxidil tincture(MT),MXD-Ch @TFs,and MXD-Rg3 @TFs promoted the transformation of mouse hair follicles from the resting phase to the growth phase,extended the growth phase,and increased the length of new hair.The results of skin histopathological sections showed that MT,MXD-Ch @TFs and MXD-Rg3 @TFs could inhibit androgen-induced hair follicle miniaturization,increase hair follicle length,and maintain the normal morphology of hair follicles.MXD-Rg3 @TFs can also inhibit 5α-reductase activity and reduce DHT level in mouse skin,and reduce TNF-α and IL-6 levels in skin tissue to exert anti-inflammatory effect and promote hair regeneration,with better effects than MT and MXD-Ch @TFs.The safety evaluation results of MXD-Rg3 @TFs showed that MXD-Rg3 @TFs was non-irritating to the skin of guinea pigs after single or multiple dosing.The pathological sections of the skin of C57BL/6 mice after 21 days of continuous administration were observed,and the skin was structurally intact and showed no significant inflammatory pathological changes,indicating that MXD-Rg3 @TFs was a safe transdermal drug delivery system.Conclusion:The MXD-Rg3 @TFs prepared in this paper had good stability,could inhibit the inflammatory reaction in AGA,and had better effects than MT and MXD-Ch @TFs in the treatment of AGA.It had no irritation to the skin,and would be a promising transdermal drug delivery system for the treatment of AGA. |
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