Potential Application Of Hyaluronic Acid Microneedles Loaded With β-Elemene And Silicate Materials In The Treatment Of Psoriasis

Psoriasis(Ps)is a chronic inflammatory skin disease.Pain,itching,disfigurement,and other symptoms strongly damage the physical and mental health of patients.The formation of Ps involves many pathological processes,such as the excessive proliferation of keratinocytes,excessive secretion of inflammatory factors,and inflammatory infiltration of macrophages.At present,the therapeutic outcomes of treatments targeting a single pathological process are not ideal,and many side effects have been observed after their application in clinic.Natural products bring a potential solution to address this issue as they usually hold multi-bioactivities which enable the use of one single molecule to regulate different pathological processes simultaneously.In addition,silicate biomaterials are a type of multi-functional material.The release of various bioactive ions from silicate enables the materials to perform multiple bioactivities.Furthermore,the microneedle(MN)technique has been demonstrated to greatly increase the delivery efficiency of drugs and improve the utilization of drugs.It also has the advantages of painless and convenient use,and can effectively target the epidermal thickening caused by psoriasis.Objective : The aim is to explore the therapeutic effect and potential mechanism of natural product β-elemene(ELE)and silicate materials on Ps,and to increase its transdermal effect by using microneedle system,which provides a new idea for the treatment of psoriasis.Methods:1.Human keratinocytes(HaCaT)and skin fibroblasts(HFF-1)were co-cultured with ELE solution in 96-well plates for 72 h,and the cytotoxicity of ELE to HaCaT and HFF-1 was detected by CCK-8 kit.HaCaT was stimulated by M5 inflammatory factor aggregates and ELE,and the cell viability of HaCaT stimulated by M5 was detected by CCK-8 kit.TUNEL apoptosis detection kit(chromogenic method)was used to detect ELE-induced M5-stimulated HaCaT apoptosis.2.HaCaT was co-cultured with M5 and ELE in 48-well plates for 48 h,and the expression levels of inflammation and psoriasis-related factors were detected by q PCR.3.Mouse M(?)(RAW264.7)cells were co-cultured with ELE solution,100 ng / m L LPS and 20 ng / m L IFN-γ in 48-well plates for 48 h.4.The steps were the same as 3.The macrophages were co-cultured for 24 h and then replaced with complete medium for 24 h.The complete medium of each group of macrophages was collected for culturing HaCaT cells for 48 h.The expression level of inflammatory factors was detected by q PCR.5.Hyaluronic acid(HA)and different volumes of ELE solutions were mixed in deionized water to prepare microneedle matrix solutions with different concentrations of ELE,which were poured into polydimethylsiloxane(PDMS)microneedle molds,and dried at room temperature to prepare ELE-loaded HA microneedles(HA-ELE-MN).6.The morphology of drug-loaded microneedles was observed by field emission scanning electron microscopy(SEM),and the mechanical strength of drug-loaded microneedles loaded with different concentrations of ELE was detected by electronic universal material testing machine.7.The HA-ELE-MN was inserted into the normal skin or Ps-like skin of mice for 1minute,and the tissues were collected for histological analysis.The skin penetration ability of drug-loaded microneedles was detected by tissue sections stained with hematoxylin and eosin.8.HA-ELE-MN was inserted into the skin of mice at different time points,and the dissolution rate of drug-loaded microneedles in vivo was observed by Olympus stereomicroscope.9.Ps-like mouse model was established by imiquimod(IMQ).During the experiment,70 mg 5 % imiquimod cream was applied to the exposed part of the back of the mice for 7 days.Three days later,the treatment of microneedle and smear was given at night.The experiment lasted for 7 days,photographed and recorded every day,and collected animal specimens for histological analysis after the end.10.Different concentrations of cuprorivaite were prepared and co-cultured with HaCaT for 72 h.The cytotoxicity of cuprorivaite on HaCaT and HFF-1 was detected by CCK-8 kit.ELE and forsythin(PHI)were co-cultured with the extract of cuprorivaite and HaCaT for 72 h,respectively.The cytotoxicity of cuprorivaite to HaCaT and HFF-1was detected by CCK-8 kit.Results:1.ELE can specifically inhibit the proliferation of HaCaT stimulated by M5 and induce apoptosis,reduce the expression of IL1,IL6 and other inflammation-related genes in HaCaT,reduce the expression of KRT6 and promote the expression of FLG.2.ELE can not only inhibit the secretion of pro-inflammatory factors by macrophages stimulated by inflammatory factors,but also indirectly inhibit the inflammation and proliferation of HaCaT by regulating M1 macrophages.3.The results of microneedle characterization showed that the drug-loaded microneedles we prepared had complete morphology,sufficient mechanical strength to successfully penetrate Ps thickened skin and could be dissolved quickly.4.The results of animal experiments showed that the drug-loaded microneedles made the appearance of Ps skin closer to normal skin,and effectively reduced the epidermal thickness,increased the number of apoptotic cells,and had a good control effect on Ps.5.The cuprorivaite extract can specifically inhibit the viability of HaCaT cells at a concentration of 1/ 64-1 /16.Conclusion:In this study,we demonstrated that ELE could inhibit M5-stimulated HaCaT proliferation and induce apoptosis.It is proved that ELE can reduce the expression level of inflammatory factors,and it is proved that ELE can reduce the expression level of inflammatory factors in macrophages;it was also proved that ELE had an indirect regulatory effect on macrophage-HaCaT.Furthermore,through our designed microneedle-loaded ELE strategy,we demonstrated that HA-ELE-MN can effectively prevent and treat Ps in vivo,and the effect is better than that of drug application.