Folate Receptor-mediated Drug Delivery Systems To Enhance Treatment Of Acute Kidney Injury By Improving Oxidative Stress And Inhibiting Fibrosis

Acute Kidney Injury(Acute Kidney Injury,AKI)is a clinically critical and severe disease characterized by rapid renal failure.There is still no effective treatment.Necrosis of renal tubular epithelial cells(HK-2)is the most common phenomenon of AKI.Excessive oxidative stress and the intense inflammatory response caused by it in HK-2cells lead to rapid degeneration,apoptosis,and shedding,and aggravate the process of AKI.In addition,strong oxidative stress and inflammation can induce tissue fibrosis,and severe fibrotic tissue completely loses its function.Therefore,starting from repairing HK-2 cell damage,taking advantage of the precise targeting,solubilization,and toxicity reduction of the nano-transport system,designing a drug system that can effectively improve oxidative stress and relieve fibrosis is expected to solve the problem of AKI treatment.In this article,folic acid(FA)was used as the targeting group,and curcumin(Cur)and resveratrol(Res),which are polyphenolic Chinese medicine monomers with anti-inflammatory and antioxidant effects,were used as model drugs.Vitamin E polyethylene glycol succinate(TPGS)and Pluronic F127(F127)as carriers,using their high loading rate and good biocompatibility to construct FA receptor-mediated nanosystems(Cur/Res@FAF127/TPGS)to achieve AKI safe and efficient treatment.First,to achieve targeted drug delivery,the F127/TPGS carrier was modified by FA functionalization;then,nanosystems were prepared by thin-film hydration;finally,the targeted and pharmacodynamic studies of AKI induced by cisplatin(CDDP)in vivo and in vitro models were conducted to evaluate the comprehensive therapeutic effect of the nanosystem.The main research contents are as follows:1.Preparation and characterization of Cur/Res@FA-F127/TPGSFirst,FA-COOH and F127-OH were esterified to synthesize FA-F127 under the action of dehydrating agent dicyclohexylcarbodiimide(DCC)and catalyst 4-dimethyl aminopyridine(DMAP).Then,Cur and Res were loaded into the hydrophobic core of FAF127/TPGS by film hydration to obtain Cur/Res@FA-F127/TPGS drug-loaded micelles.Finally,the particle size and morphology of the nanosystem were measured by laser nanoparticle sizer and transmission electron microscope.The results showed that the particle size of the Cur/Res@FA-F127/TPGS nanosystem was 101.5±2.3 nm,with uniform size and good dispersion.2.In vitro specific targeting studiesFluorescein isothiocyanate(FITC)was used as a fluorescent probe to label mixed micelles.The uptake capacity of HK-2 cells to the nanosystem was by fluorescence microscope and flow cytometry.The results showed when HK-2 cells were incubated for4 hours,the fluorescence intensity was the strongest,and the cell uptake rate was as high as 81.2%.The co-incubation of free FA blockers with HK-2 cells proved that the nanosystem quickly entered HK-2 through FA receptor-mediated endocytosis.3.In vitro pharmacodynamic study of nanosystems to improve oxidative stressWhen AKI occurs,the explosive growth of reactive oxygen species(ROS)in HK-2causes the mitochondrial permeability transition pore to open abnormally,which causes a large amount of water and ions to enter the mitochondrial matrix,mitochondrial swelling,mitochondrial outer membrane rupture,mitochondrial membrane potential(MMP)decrease,and Caspase-3 apoptosis signaling pathway is activated.In this paper,HK-2 cells stimulated by CDDP were used as an AKI model in vitro to study the improvement of oxidative stress by nanosystems in vitro.DCFH-DA and JC-1 fluorescent probes were used to investigate the effects of nanosystem on ROS and MMP.The intensity of DCFH-DA green fluorescence can reflect the level of ROS,the relative ratio of JC-1 red-green fluorescence can reflect the change of MMP,and the changes of ROS and MMP can reflect the degree of cell damage.The results showed that the nanosystem could effectively remove ROS,stabilize MMP and improve oxidative stress.Caspase-3 and Bcl-2 proteins are closely related to the process of apoptosis.Western blot results showed that the nanosystem could significantly reduce the pro-apoptotic protein expression of Caspase-3and promote the anti-apoptotic protein expression of Bcl-2,which improved oxidative stress by exerting an excellent anti-apoptotic effect.4.Research on in vivo targeting and anti-inflammatory and anti-fibrosis properties of nanosystemsOxidative stress can also cause lipid peroxidation of the cell membrane,resulting in peroxidation products such as malonaldehyde(MDA),which further activates the nuclear factor-κB(NF-κB)signaling pathway,and promotes the high expression of downstream inflammatory factors such as tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),and cyclooxygenase-2(COX-2),resulting in a severe inflammatory response.In addition,untimely treatment of AKI can induce severe uncontrollable tissue fibrosis and complete loss of tissue function.In this study,BALB/c male mice stimulated by CDDP were used as AKI models to study the targeting,anti-inflammatory,and anti-fibrosis properties of the nanosystem-based system in vivo.In vivo imaging results of small animals showed that the constructed nanosystem could specifically target the kidney,and accumulated to the maximum concentration in the kidney 12 h after caudal vein injection.The contents of serum urea nitrogen(BUN)and serum creatinine(Scr)were determined by the biochemical method,and the levels of superoxide dismutase(SOD)and MDA in renal tissues were determined by the xanthine oxidase method and thiobarbituric acid method,respectively.The results showed that the nanosystem could significantly reduce the contents of metabolites Scr and BUN and improve renal function.It could reduce the content of MDA,increase the activity of SOD,and improved oxidative stress in tissues.The results of SIRT1 immunohistochemistry showed that the nanosystem could effectively activate the SIRT1 signaling pathway.Western Blot and ELISA experiments showed that the nanosystem could significantly inhibit the activation of the NF-κB signaling pathway and reduce the levels of inflammatory factors COX-2,IL-6,and TNF-α.Finally,hematoxylin-eosin(H&E)staining and TUNEL assay of renal histopathology showed that the nanosystem could effectively protect HK-2 cells from CDDP-induced apoptosis and necrosis.The results of Masson,PAS,and collagenous Ⅰ protein staining showed that nanosystems could significantly reduce glycogen deposition,overexpression of collagenous fiber and collagenous protein in renal tissue.Western Blot results showed that the nanosystem significantly reduced the expression of α-smooth muscle actin(α-SMA)and delayed the progression of fibrosis.In combination with the above experiments,it had been proved that nanosystem could effectively reduce the expression of inflammatory factors by inhibiting the activation of the NF-κB signaling pathway,and it could also delay the process of fibrosis by reducing the expression of fibrosis-related proteins,thus playing an excellent comprehensive therapeutic effect in vivo.In summary,this article successfully constructed a drug delivery system that c specifically recognizes the surface target of HK-2.In vivo and in vitro studies have proved that the nanosystem could promote the effective accumulation of Cur and Res in the kidney,and improve oxidative stress,inhibit inflammation and tissue fibrosis,effectively repair the damaged HK-2 cells.It provided a new way of thinking for the treatment of AKI.