Preparation Of Dermal Papilla Cell Membrane Camouflage Nano Drug Loading System And Its Treatment For Androgenic Alopecia

Background:Androgenic alopecia(AGA)is a common dermatological disease characterized by the miniaturisation of scalp hair follicles.The disease not only seriously affects the mental health and quality of life of patients,but also faces the possible side effects of related drugs.The pathogenesis of AGA may be related to androgen metabolism,with testosterone(TS)converted to dihydrotestosterone(DHT)under the action of 5αreductase,and DHT binds with androgen receptor(AR)in hair follicle cells,resulting in hair loss.At present,the treatment methods for AGA include oral finasteride(FNS),dutasteride(DUT),and external minoxidil(MXD).The inhibitory effect of DUT on 5α-reductase is better than that of FNS,but oral DUT may lead to side effects such as male breast development,testicular pain and impaired sexual function.However,external use of MXD may cause such adverse reactions as desquamation and itching.Therefore,we try a new and controllable way of administration,such as changing oral DUT into local administration to reduce adverse reactions caused by systemic administration.In addition,drug targeting AR is also a research focus.As a specific gene silencing tool,siRNA can target and knock down target genes,but its application is limited due to the poor stability of system circulation and the low permeability of biological membranes.Therefore,a suitable nano-drug delivery system is needed to transport drugs and siRNA simultaneously to achieve the goal of dual targeting.Poly(lactic-co-glycolic acid)(PLGA)is a biodegradable polymer with good biocompatibility and no toxicity.Cell membrane camouflage technology can be used to encapsulate nanoparticles(NPs),so that various surface molecules carried by cell membrane can be transferred to NPs completely,and the biocompatibility and targeting can be improved.Objective:This study aims to deliver DUT and siRNA(siAR)targeting AR through a biomimetic nano drug delivery system and to treat AGA with double targeting by local drug delivery.To achieve the effect of DPC proliferation,promote hair growth,and reduce the side effects of oral DUT.Methods:1.The dermal papilla cells were isolated and extracted by microdissection combined with one-step enzyme digestion,and the dermal papilla cells were identified by the following methods:cell morphology,osteogenesis and adipogenic differentiation,specific alkaline phosphatase staining,flow cytometry to identify DPC surface markers,Western blot,RT-PCR and immunofluorescence techniques to identify DPC marker proteins.2.Nano-polymer PLGA-DUT/siAR NPs were synthesized by the W/O/W method.PLGA-DUT/siAR@DPCM were synthesized by membrane extrusion and assembled to form target nanoparticles.The knockout effect of three alternative siAR on the target gene was evaluated by the RT-qPCR method,which was used to screen the siAR with the highest knockout rate.The stability,size,potential and morphology of PLGA-DUT/siAR NPs and PLGA-DUT/siAR@DPCM NPs were verified by dynamic light scattering(DLS)and transmission electron microscopy(TEM).DUT’s drug loading and entrapment efficiency were determined by detecting DUT content in nanoparticles,and the best input ratio was fixed;similarly,the drug loading and entrapment efficiency of siRNA were detected.The stability of PLGADUT/siAR@DPCM NPs was evaluated by measuring the nano-size of NPs in PBS and FBS at different time points.SDS-PAGE gel electrophoresis combined with Coomassie brilliant blue staining was used to identify whether the cell membrane lysates and the prepared nanoparticles had similar protein distribution.The immunofluorescence technique was used to detect the co-localization of fluorescence-labeled nanoparticles.3.In vitro experiment:the effect of PLGA-DUT/siAR@DPCM NPs on the proliferation and cytotoxicity of DPC was detected by the CCK8 method.Flow cytometry and immunofluorescence technique observed the uptake of PLGA-FITC NPs and PLGA-FITC@DPCM NPs by DPC.In the in vitro release experiment,the content of DUT was detected at different time points to determine the release of PLGA-DUT NPs和PLGA-DUT@DPCM NPs in vitro.The expression of AR was detected by Western blot and RT-qPCR to detect the efficiency of gene silencing.In the skin penetration and retention experiment,the contents of DUT in the receiving chamber,stratum corneum,hair follicles and skin tissue of PLGA-DUT NPs and PLGADUT@DPCM NPs groups were examined at 6 hours and 12 hours.4.In vivo experiment:the mouse model of androgenic alopecia was established by subcutaneous injection of testosterone,and different experimental groups,model groups and positive control groups were found.Hair coverage,density and diameter of other groups were distinguished by intuitive observation and histopathological examination;hair growth was evaluated by hair growth index;the silencing effect of target gene in vivo was determined by immunofluorescence detection of AR;the permeability of nanoparticles in vivo was observed by in vivo uptake experiment.The histopathological examination of the whole body organs proves whether the nanoparticles are safe or not.In addition,the apoptosis of hair follicle cells in different groups was observed by immunofluorescence detection of Bcl-2 and TGF-β2,and the related mechanism of hair follicle growth was explored by immunofluorescence detection of Wnt3a,β-catenin and MMP3 in tissue.Results:1.The DPC was extracted successfully,the adhesion rate of DPC 72h was more than 50%,and the survival rate was high,which reached the demand of the experiment.The extracted DPC could be cultured and passaged normally,and the lower generation DPC grew in vortex shape;calcified nodules deposited by mineral salts and large round lipid droplets appeared in osteogenesis and adipogenic differentiation experiments,respectively,and the corresponding staining tests were all positive;with the increase of generations,the DPC activity decreased,the related alkaline phosphatase staining became lighter and the staining range became smaller.DPC surface markers CD73,CD90 and CD 105 were positive,while DPC markers SOX2,ALP,Versican,α-SMA and Nestin were all positive.2.Preparation and characterization of nanoparticles:PLGA-DUT/siAR NPs and PLGA-DUT/siAR@DPCM NPs were successfully prepared and the core-shell structure of PLGA-DUT/siAR@DPCM NPs was observed to be uniformly distributed,and the average diameters of PLGA-DUT/siAR and PLGA-DUT/siAR@DPCM were 157.23±1.16nm and 194.83±3.18nm,respectively.In detecting Zeta potential,the potential of PLGA-DUT/siAR@DPCM NPs was similar to that of cell membrane vesicles.PLGA-DUT/siAR@DPCM NPs can maintain their stability in both PBS and FBS.When the dosage of DUT was 0.8mg,the entrapment efficiency was 53.31±0.64%,the drug loading was 6.45±0.12%,the entrapment efficiency of siRNA in nanoparticles was 47.1±0.3%,and the drug loading was 21.2±0.2 μ g/10mg.SiAR2843 was screened out to have the highest knockout rate of the target gene.SDS-PAGE gel electrophoresis showed that the protein distribution of DPC cell membrane vesicles was similar to that of PLGA-DUT/siAR@DPCM NPs.In the co-localization experiment,DPCM,FITC(instead of DUT)and siRNA could be observed,and the fluorescence regions coincided.3.In vitro experiment:in vitro release test,the cumulative release of PLGA-DUT NPs was about 57.35±1.84%,higher than that of PLGA-DUT@DPCM NPs(40.54±1.77%).In the permeation and retention experiments,the DUT content in the stratum corneum was similar in PLGA-DUT NPs and PLGA-DUT@DPCM NPs groups at different time points.Compared with PLGA-DUT NPs,PLGA-DUT@DPCM NPs detected higher DUT content in hair follicles at 6 and 12 hours.Flow cytometry and immunofluorescence showed that the uptake efficiency of PLGA-FITC@DPCM NPs was higher than that of PLGA-FITC NPs at two-time points.In the CCK8 test,the PLGA-DUT/siAR@DPCM NPs group showed the most increased cell activity,similar to that of the positive control group.In addition,the results showed that PLGADUT/siAR@DPCM NPs had good gene silencing efficiency.4.In vivo experiment:in the in vivo experiment of AGA model mice,both PLGA-DUT/siAR@DPCM NPs group and the positive control group(MXD)showed ideal hair coverage,density and diameter in visual observation and pathological examination,as well as higher hair growth index.Immunofluorescence staining showed that the experimental group had an obvious knock-down effect on the androgen receptor.In vivo uptake experiment showed that the fluorescence had reached the dermis and the fluorescence-labeled NPs were distributed in and around the hair follicles.No organ injury was observed in the histopathological examination of whole-body organs.In addition,immunofluorescence showed that the expression of Bcl-2 in PLGADUT/siAR@DPCM NPs and the positive control group was higher than that in the model group,but the expression of TGF-β2 was minimal.The fluorescence intensity of Wnt3a,β-catenin and MMP3 in PLGA-DUT/siAR@DPCM NPs and the positive control group was higher than that in the model group.Conclusion:1.DPC was successfully isolated,cultured,identified and amplified and used to prepare DPCM,and DPCM camouflaged co-carrier drugs and gene silencing tools of PLGA-DUT/siAR@DPCMNPs nanoparticles were synthesized.2.PLGA-DUT/siAR@DPCM NPs can be absorbed more efficiently than NPs without DPCM camouflage at the cellular level,can promote the proliferation of DPC after sustained release,and has no cytotoxicity,can effectively reduce the expression of AR in DPC and has good hair follicle targeting ability.3.The in vivo experiments of this study show that PLGA-DUT/siAR@DPCM NPs can be used as an external drug applied in vitro,can reach the hair follicles and surrounding tissues,can be used to guide the treatment of AGA,has good safety,and has a good hair promotion effect on mouse AGA model.At the same time,it is verified that the use of PLGA-DUT/siAR@DPCM NPs can inhibit hair follicle cell apoptosis,make hair more in the growth stage,and may play a role by up-regulating Wnt/βcatenin pathway to promote hair growth.