Construction Of Dissolving Microneedles Loaded With Hypoxia-pretreated Mesenchymal Stem Cells-derived Conditioned Media And Its Application In The Treatment Of Androgenic Alopecia

Androgenetic alopecia（AGA）is the most familiar type of hair loss in clinic,however,widely used therapeutic drugs often need long-term use,having a risk of side reactions and low efficacy.Therefore,designing a safe and efficient strategy is an urgent scientific problem.As insufficient vascularization is assumed to be one of the major causes of AGA,the conditioned media（CM）derived from stem cells have attracted wide concern for their potential in inducing hair regrowth.There are many proangiogenic bioactive factors in CM derived from mesenchymal stem cells（MSCs）,which can induce hair regeneration,and hypoxic preconditioning can further enhance the proangiogenic activity of CM.Based on this background,this study aims to construct a dissolving microneedles（MNs）patch encapsulated with CM derived from hypoxia-pretreated MSCs for the treatment of AGA.The dissolving microneedles patch could deliver the pro-angiogenic factors of CM directly into the skin in an efficient and minimally invasive manner.Through in vitro and in vivo studies,the therapeutic efficacy,mechanism and safety of CM-MNs was evaluated.Firstly,hypoxia CM（Hypo-CM）and normoxia CM（Nor-CM）were collected from MSCs cultured under hypoxic（1%O₂）or normoxic（21%O₂）conditions for 24 h,respectively.Results through enzyme-linked immunosorbent assay（Elisa）kit,scratch assay,and tube formation in vitro demonstrated that the hypoxic pretreatment on MSCs elevated the derived CM’s HIF-1αcontent and promoted angiogenesis comparing to the normoxic pretreatment on MSCs in vitro.In addition,CM-MNs was prepared via a two-step centrifugation casting method using Hypo-CM and oligomeric sodium hyaluronate（Oligo-HA）as needle materials and polyvinylpyrrolidone-K90（PVP-K90）as backing materials.After that,through the observation by scanning electron microscopy,confocal laser microscopy,and texture analyzer,CM-MNs was demonstrated that had a neatly arranged and highly consistent pyramid shape with 550μm in height and 200μm in width.And CM-MNs had enough mechanical strength to penetrate into the rat and porcine skin,delivering the encapsulated Hypo-CM into the skin with a depth of 200-300μm.Based on the successful preparation of CM-MNs,the testosterone-induced AGA mouse model was further established for therapeutic analysis.CM-MNs’ability to promote hair regeneration was investigated through regenerated hair coverage,diameter,and density.Moreover,the hair cycle transition rate he activation of hair follicle stem cells were assessed by Hematoxylin-eosin（H&E）staining and immunofluorescence staining.The outcomes indicated that CM-MNs induced faster hair regeneration and achieved comparable quality in hair coverage and hair density with a lower administration frequency compared with Minoxidil.In addition,CM-MNs successfully activated hair follicle stem cells.Furthermore,the mechanism of hair regeneration induced by CM-MNS was investigated by immunofluorescence staining,H&E staining,and Elisa kit.In vivo experiments found that CM-MNs achieved more pronounced efficacy at boosting perifollicular angiogenesis due to the synergistic pro-angiogenic effects of CM and the mechanical stimulation of MNs.To further investigate the pivotal role of perifollicular neovascularization in CM-MNs-induced hair regeneration,angiogenesis inhibitor TNP-470 was intradermally injected along with the administration of CM-MNs.Hair regeneration and neovascularization were notably affected by angiogenesis inhibitor TNP-470,suggesting CM-MNs induced hair regeneration by promoting perifollicular angiogenesis.Moreover,the inhibition of perifollicular angiogenesis restrained the activation of hair follicle stem cells.This result elucidated the tight relation between perifollicular angiogenesis and hair follicle stem cells activation.Finally,considering the safety of CM-MNs application,the safety of CM-MNs was evaluated by H&E staining,body weight monitoring,blood biochemistry,and the trans-epidermal water loss（TEWL）value.The observation showed that CM-MNs treatment would not cause pathological changes in major organs,blood systems,and skin barriers,having good safety on both AGA model mice and healthy mice.In this study,we designed a dissolving MNs patch integrated CM derived from hypoxia-pretreated MSCs.The synergism of Hypo-CM and MNs patch successfully boosted the perifollicular vasculature and activated the hair follicle stem cells,thereby accelerating hair regeneration with a lower administration frequency in the AGA mouse model.The CM-MNs was simple to prepare and had high biocompatibility as well as therapeutic effect to treat AGA,which presented a promising strategy.Moreover,the tight correlation between perifollicular neovascularization and activation of hair follicle stem cells pointed out an appealing direction for the clinical treatment of AGA.