Preparation Of MTX-loaded Silk Fibroin Nanoparticles And The Mechanism Of Targeted Treatment Of Psoriasis

Psoriasis is an autoimmune-mediated chronic inflammatory skin disease,which is mainly manifested by red,scaly,and plaque bulges caused by inflammatory cell infiltration and angiogenesis which is accompanied by excessive proliferation of keratinocytes.Patients who suffer with psoriasis often experience itching,pain,bleeding on the skin as common symptoms.This occurrence is prone to recurrence and cause systemic damage of immune system.In addition,some other diseases such as cardiovascular disease,metabolic syndrome and diabetes are known associated with psoriasis.Moreover,patients are more likely to develop depression,and are at risk of pain,disfigurement,and disability.Methotrexate(MTX)is one of treatment options for psoriasis and can be administered both as injection and oral intake.Nonetheless,there is a great limit of oral administration in clinical application of MTX due to the low solubility level and toxicity effect.The effectiveness of topical administration is higher compare to injection and oral administration due to the direct application into the blood circulation targeting the infected sites.Hence,topical administration of MTX is preferable for psoriasis treatment in order to increase the bioavailability and reduce the drug toxicity level.Interestingly,the technology of Nanoparticles(NPSs)can be applied to improve the efficacy of local drugs and reduce the toxicity effect.The solubility and stability of the drugs can be achieved by controlling the release of drugs on the infected site.Moreover,silk fibroin(SF)has been widely used in biomedical field due to its biocompatibility,controllable degradability and low immunogenicity level.Besides,SF is easy to be processed and modified under low cost of operation.Since SF has a unique structure which made it as a good local delivery carrier.The unique structure is crystalline region-specific hydrophobic and amorphous regions of hydrophilic domains which create balance between hydrophilicity and hydrophobicity conditions.Hence,in this study,Hyaluronic acid modified methotrexate silk fibroin nano-drug delivery system(Hyaluronic acid)modified(HYaluronic acid)(HA-MTX-SFNPs)was successfully constructed.It was assumed that HA-MTX-SFNPs might function to reduce the toxicity effects of MTX and improve the efficacy rate of MTX by specifically bound on the overexpressed cd44 protein at the site of psoriasis inflammation.The therapeutic effect on psoriasis was evaluated by assessment of HA-MTX-SFNPs efficacy at the cellular level and animal test.Firstly,MTX-fibroin nanopharmaceuticals(MTX-SFNPs)were constructed by optimized decontamination method,and then functional targeted modifications test were performed on the surfaces with HA and physicochemical properties were characterized.At the same time,the anti-inflammatory properties and biosafety properties of multi-load MTX filamentine nanopharmaceuticals were verified via in vitro and in vivo.The result shown that HA-MTX-SFNPs were aggregated in the inflammatory foci of the epidermis without entering the in vivo circulation after injection on the skin.Moreover,the HA-MTX-SFNPs was specifically engulfed by target cells and increased the accumulation of MTX in inflamed skin.However,topical application of HA-MTX-SFNPs showed an improvement of the therapeutic effect on psoriatic-like inflammatory skin in mice which induced by imiquimod ointment(imiquimod,IMQ).Hence the results obtained are explained as follows:Part I: Construction and Characterization of HA-MTX-SFNPsFirstly,the silk protein was extracted from the cocoon of silkworms using alkali degradation method.The silk protein was obtained and MTX-SFNPs were constructed by decontamination method followed by modification using hyaluronic acid to obtain HA-MTX-SFNPs.Some characteristics such as particle size(Size),multi-dispersion index(PDI)and Zeta potential of HA-MTX-SFNPs were measured using dynamic light scattering(DLS)technology.Stability of HA-MTX-SFNPs was evaluated by measuring the particle size for 7 days consecutively.The morphology of MTX nanoparticles was observed under Scanning electron microscopy(SEM)and transmission electron microscopy(TEM).The ability of SFNPs to successfully load MTX and HA was examined using Fourier Infrared(FITR)and the successful modified MTX-SFNPs was obtained.MTX nanoparticles ability to control the release of drug sustained was evaluated.Constructed HA-MTX-SFNPs consists of uniform particle size and a stable surface charge,with a particle size of about 243 nm and a surface charge of-24.5 m V.Stability of HA-MTX-SFNPs was achieved,which will be beneficial to the effective aggregation of HA-MTX-SFNPs in the site of psoriasis lesions.The results of electron microscopy and drug release performance show that HA forms a gel protection network on the surface of HA-MTX-SFNPs.This feature represents a strong sustained release ability of drugs,which is conducive to the delivery of drugs to the lesion site during local administration.Part II: Evaluation of the efficacy and biosafety of HA-MTX-SFNPs in the treatment of psoriasis at cellular and Animal testThe cytotoxicity test was conducted and the number of cells was counted using Cell Counting Kit 8(CCK8)to detect the cytotoxicity of various silk fibroin nanoparticles on human skin keratinocytes(Ha Ca T).The safety of NPs without MTX was tested by topical application on the skin and oral administration in mice that induced by IMQ.Hence,the comparative efficacy level of different administration route was evaluated.In addition,the biosafety of HA-MTX-SFNPs was evaluated by the cytocompatibility of silk fibroin nanocarriers,hemolysis test in vitro and blood routine test after administration in vivo.The results showed that the IC50 of HA-MTX-SFNPs was 8.81 μg/m L,which showed the strongest killing effect on Ha Ca T cells.In animal test,the treatment effect of skin administration group was better than oral administration group.By comparing the histopathological sections of mouse ears,it was found that the dermal administration group had stronger inhibition ability on the proliferation of keratinocytes and subcutaneous tissue cells induced by IMQ than the oral administration group,among which the HA-MTX-SFNPS group had the most obvious inhibition effect and the cuticle recovered best.In individual trials,the treatment effect of skin administration group was better than oral administration group.By comparing the histopathological sections of mouse ears,it was found that the dermal administration group had stronger inhibition ability on the proliferation of keratinocytes and subcutaneous tissue cells induced by IMQ than the oral administration group,among which the HA-MTX-SFNPS group had the most obvious inhibition effect and the cuticle recovered best.CCK8 experiment showed that pure silk fibroin nanoparticles showed good biocompatibility,and even high concentration silk fibroin nanoparticles did not show obvious cytotoxicity.In vitro blood compatibility test results showed that high concentration of HA-MTX-SFNPs did not lead to hemolysis.There was no significant difference between the experimental group and the control group in blood routine test results.Thus,the results indicate that HA-MTX-SFNPS is safe to be used.Part III: Study on the mechanisms of HA-MTX-SFNPs as a targeted therapy for psoriasisThe phagocytosis rate of Ha Cat cells that engulfed various silk protein NPs was assessed under laser confocal microscopy(CLSM).The effect of NPs on apoptosis rate was examined by observation of drugs uptake by the cells using flow cytometry analysis.The permeability and skin retention tests were conducted after administration of MTX nanoparticles.Finally,the effect of each group of NPs on the expression of cellular inflammatory factors was detected by quantitative PCR.Phagocytosis test showed that MTX-SFNPs modified by HA had strong targeting ability.After co-incubation with Ha Ca T cells treated with TNF-α for 4 h,the uptake rate of Ha Ca T cells was 45.88%,which could effectively promote the uptake of Ha Ca T cells.The apoptosis rate of Ha Ca T cells induced by HA-MTX-SFNPs was 21.64%,and the apoptosis rate of Ha Ca T cells treated with TNF-α was 31.16%,indicating that it could effectively inhibit the proliferation of keratinocytes,and the targeting ability of HA further enhanced its inhibition ability,which provided the possibility for the effective treatment of psoriasis.Quantitative PCR results showed that the expression of inflammatory factors(IL-17,IL-22,IL-1β,TNF-α)were significantly inhibited in each treatment group,of which indicates that the topical HA-MTX-SFNPs had the best effect.In short,hyaluronic acid-modified MTX-contained fibrous nanopharmaceutical(HA-MTX-SFNPs)was successfully constructed and the physicochemical properties of it was tested.The results obtained revealed the ability of HA-MTX-SFNPs in enhancing the efficacy of MTX by targeting on the inflammatory foci of osmosis psoriasis.Besides,HA-MTX-SFNPs has the ability in reducing the toxicity effects by significantly inhibiting the expression of psoriasis-related cellular inflammatory factors.Hence,it is suggested that HA-MTX-SFNPs has a broad application prospects in the treatment of psoriasis and deserve further study.