PD-L1~+ Exosomes With Pristimerin Through PD-1/PD-L1 Interaction For Psoriasis-targeted Treatment

Background:Psoriasis is a chronic systemic skin disease characterized by well-defined,varying-sized red plaques with inflammatory rims,slight infiltration,and thick,white scales.The hallmark of psoriasis is the dysfunction of the epidermal keratinocyte and excessive infiltration of immune cells,which are closely related to skin immune dysregulation.Therefore,how to regulate the skin immune microenvironment to restore homeostasis remains an important challenge.Exosomes are one kind of extracellular vesicles,which are cell-derived vesicles with a phospholipid bilayer structure and a diameter of approximately 30-150 nm.They are important mediators of intercellular communication and signaling regulation,which can transmit signaling molecules over long distances,and regulate the functional state of target cells.Because of their good biocompatibility and low immunogenicity,exosomes are often considered as natural drug delivery carriers.Inspired by the role of tumor-derived exosomes in immune escape,this study explored the feasibility of melanoma cell-derived exosomes as a targeted drug delivery platform for treating inflammatory skin diseases.Due to the up-regulated expression of PD-1 on keratinocytes and immune cells in the psoriatic pathological environment,melanoma cell-derived exosomes with high expression of PD-L1 were used to load the potent anti-inflammatory agent,pristimerin(Pri@exo),and target drug delivery for the treatment of psoriasis.Methods:Melanoma cell-derived exosomes were extracted using differential and ultracentrifugation methods,and exosomes loaded with pristimerin was constructed using ultrasonic technology.The Pri@exo was characterized by transmission electron microscopy,atomic force microscopy,particle size analysis,and immunoblotting.A psoriasis model was constructed in vitro by stimulating keratinocytes(Ha Ca T)with tumor necrosis factor-α(TNF-α).PD-1 expression on Ha Ca T cells was detected using flow cytometry.In addition,an Imiquimod(IMQ)-induced psoriasis model was constructed in BALB/C mice.The expression of PD-1 on immune cells from spleens were detected using flow cytometry and immunofluorescence.Primary CD4~+T cells from mice were stimulated in vitro with TNF-α,interleukin-23(IL-23),and transforming growth factor-β(TGF-β)to establish an in vitro psoriasis inflammation model.Cy5.5-labeled Pri@exo was added into CD4~+T cells,and the cell uptake efficiency was detected via flow cytometry.The uptake efficiency of Ha Ca T cells was also monitored.The in vitro anti-proliferative activity of Pri@exo was explored using carboxyfluorescein succinimidyl ester(CFSE)through flow cytometry.Pri@exo was encapsulated in a thermosensitive hydrogel to achieve sustained release and subcutaneously injected into IMQ-induced psoriasis-like mice.The overall therapeutic effect of Pri@exo was evaluated by observing the skin lesions,epidermal thickness,and tissue sections.To evaluate the in vivo anti-inflammatory effects of Pri@exo,changes in immune cell populations in the spleen and lymph nodes were detected using flow cytometry and immunohistochemistry.Enzyme linked immunosorbent assay(ELISA)were used to monitor the levels of pro-inflammatory factors in the serum.Proliferation-related indicators such as proliferating cell nuclear antigen(PCNA)and Ki67 were used to evaluate the in vivo anti-proliferative activity of Pri@exo.The expression levels of 4-hydroxynonenal(4-HNE),long-chain family member 4 of acyl-Co A synthetase(ACSL4),and glutathione peroxidase 4(GPX4)were explored using immunofluorescence and immunoblotting to determine the relationship between Pri@exo and ferroptosis.The levels of reactive oxygen species(ROS)and lipid-ROS in Ha Ca T cells were investigated via flow cytometry after stimulating with RSL3 to establish an in vitro ferroptosis model,and the inhibitory capacity of Pri@exo against ferroptosis was evaluated.Safety evaluations were conducted by performing histopathological examination of the heart,liver,spleen,lung and kidney,as well as hematological analysis.Results:Pri@exo exhibited a uniform size and a cup-shaped morphology under electron microscopy.The particle size and immunoblotting results were consistent with previous literature reports,indicating the successful construction of Pri@exo in this study.PD-1 expression was significantly up-regulated on keratinocytes and T cells in psoriasis.In addition,it is noteworthy that the uptake efficiency of Pri@exo was increased in psoriasis,indicating the potential of Pri@exo to target keratinocytes and T cells.Pri@exo effectively inhibited the proliferation of keratinocytes in vitro.After the application of Pri@exo,the expression level of IL-17 and TNF-αby CD4~+T cells decreased,but the proportion of regulatory T cells(Treg)was augmented,implying the good anti-inflammatory ability of Pri@exo.Pri@exo effectively alleviated psoriasis-like manifestations,including erythema,scales,and epidermal thickening,in the IMQ-induced psoriasis-like mouse model.Moreover,Pri@exo not only improved the elevated ratio of T cells and macrophages in psoriasis but also promoted the polarization of macrophages towards M2 type.The proliferation-related indicators such as PCNA and Ki67 were significantly downregulated under the intervention of Pri@exo.ACSL4,4HNE,and GPX4 showed activated ferroptosis in the psoriasis-like mouse model,while the application of Pri@exo helped inhibit ferroptosis.Additionally,the enhancement of ROS and lipid-ROS in Ha Ca T cells in vitro was attenuated by Pri@exo.The histological staining showed no obvious morphological changes happen in heart,liver,spleen,lung and kidney after the subcutaneous injection of Pri@exo.Besides,the hematological profile also remained within the normal range or comparable to the control mice after administration of Pri@exo.These results indicated the safety of Pri@exo at the current dose.Conclusions:This study characterized melanoma cell-derived exosomes loaded with pristimerin(Pri@exo)and evaluated its PD-1 targeting ability through cell uptake experiments.The anti-inflammatory effects of Pri@exo were observed in both in vitro and in vivo models of psoriasis.This study found that Pri@exo had the potential to target CD4~+T cells and keratinocytes in psoriasis,inhibit the secretion of IL-17 and TNF-α,and promote the differentiation of regulatory T cells.Pri@exo significantly and safely alleviated the psoriasis-like manifestations in IMQ-induced psoriasis-like inflammation mice,with reduced epidermal thickness and improved immune cell populations.Besides,Pri@exo treatment alleviated ferroptosis-related changes in psoriatic skin,thereby dampening excessive inflammation and,in turn,decreasing the abnormal proliferation of keratinocytes in psoriatic lesions.This study demonstrates that our engineered exosomes can not only act as a treat-to-target strategy for psoriasis treatment but also provide insight in clinical application of inflammatory disorders.