Construction Of Ceria Nanozymes-loaded Dissolvable Microneedles For Androgenetic Alopecia Therapy

Objective:The study aims to design and construct ceria nanozymes(CeNZ s)-loaded dissolvable microneedles(Ce-MNs)for the treatment of androgenetic alopecia,assess the therapeutic effect and explore the underlying mechanism.Methods:1.The ceria nanoparticles were synthesized by a previously reported reverse micelle method.Moreover,the ceria nanoparticles were modified with DSPEPEG2000 on the surface to obtain CeNZs,which could disperse in water phase with uniform size.The ceria nanoparticles were characterized by transmission electron microscopy(TEM),X-ray diffraction(XRD),and X-ray photoelectron spectroscopy(XPS).Meanwhile,dissolved oxygen analyzer,superoxide dismutase detection kit-WST and hydroxyl radical test kit were used to investigate the anti-oxidant ability of CeNZs in vitro;2.Ce-MNs were fabricated through a two-step casting method,with oligomeric hyaluronic acid as the matrix material of needles and polyvinylpyrrolidone-K90(PVPK90)as the matrix material of backing layer.The morphological characterization of CeMNs was achieved by scanning electron microscopy(SEM)and confocal laser microscopy,and the mechanical strength of Ce-MNs was evaluated by compression test and skin insertion test,as well as the ability of Ce-MNs to deliver CeNZs transdermally was assessed through an in vitro transdermal penetration test;3.The hair regeneration effects of Ce-MNs was evaluated on testosterone-induced androgenetic alopecia mice,with the regenerated hair coverage,diameter and density were used as indicators to evaluate hair regrowth quality.Moreover,the transition of hair follicle cycle was investigated through immunofluorescence staining and hematoxylin-eosin(HE)staining;4.The effect of Ce-MNs on the regulation of oxidative stress and angiogenesis in the perifollicular microenvironment was investigated by immunofluorescence staining,HE staining,enzyme-linked immunosorbent assay(ELISA)kits and malondialdehyde(MDA)kits,to explore the underlying mechanism.5.Preliminary evaluation of the biosafety of the Ce-MNs system through cell experiments and HE staining.Results:Ultra-small ceria nanoparticles(～3 nm)were successfully synthesized,and DSPE-PEG2000 was successfully modified on its surface to obtain CeNZs,with a particle size of about 10 nm and uniformly dispersed in the water phase.Moreover,CeNZs owned excellent in vitro antioxidant capacity,which can effectively catalyze the decomposition of hydrogen peroxide,scavenge superoxide anions and hydroxyl free radicals.2.Pyramid-shaped Ce-MNs were successfully prepared,with a height of 550 μm and a base diameter of 200 μm.Ce-MNs possessed sufficient mechanical strength to penetrate the excised porcine skin,and delivered CeNZs to 200～300 μm deep in the skin,thus significantly enhancing the transdermal penetration of CeNZs.3.Testosterone-induced androgenetic alopecia mice model was successfully established.Compared with the positive drug,minoxidil,Ce-MNs achieved comparable effect on the coverage,diameter and density of regenerated hair with a lower administration frequency.Moreover,CeMNs could accelerate the telogen-to-anagen transition of hair follicles more efficiently.4.Ce-MNs delivered CeNZs to the skin to scavenge excessive reactive oxygen species(ROS)in the tissues,thereby remodeling the oxidative microenvironment around hair follicles;at the same time,the mechanical stimulation induced by the administration of microneedles was able to up-regulate the expression of vascular endothelial growth factor(VEGF),thereby inducing angiogenesis around hair follicles.The dual regulation both on oxidative stress and microvasculature restored the perifollicular microenvironment to homeostasis,thereby promoting hair regeneration.Conclusion:The resulted Ce-MNs can efficiently deliver CeNZs to the hair follicles in a minimally invasive manner.Ce-MNs are able to remodel the perifollicular microenvironment through scavenging excessive ROS and inducing angiogenesis on androgenetic alopecia mice model,thereby promoting hair regeneration.The CeNZsloaded microneedle system is simple to prepare and possesses satisfactory therapeutic effect and high safety for the treatment of androgenetic alopecia,which presents a potential in clinic practice.Moreover,the concept of dual regulation of the perifollicular microenvironment provides a new idea for alopecia therapy.