A Melanin-based Targeted Nanodrug For Combination Therapy Of Diabetic Nephropathy

Objective:Diabetic nephropathy(DN)is a serious microvascular complication of diabetes.It is the most common form of chronic kidney disease and the leading cause of end-stage renal disease.Traditional therapies are limited in many ways.Currently,there is no effective treatment that could be a cure for DN.The aim of this work is to develop a melanin-based targeted nanodrug(CSMDNPs).The CSMDNPs are based on melanin nanoparticles loaded with dapagliflozin(DA)and coated with polyethylene glycol chitosan(GCS).The aim is to provide a new idea for the treatment of DN through a combination of hypoglycaemic and antioxidant effects guided by melanin photoacoustic imaging(PAI).Methods:1.Synthesis and characterization of CSMDNPsFollowing the previous work,small water-soluble melanin nanoparticles(MNPs)were prepared by ultrasonic fragmentation.The mixture of MNP and DA was centrifuged overnight at low temperature.After removal of the free DA,the solution of the mixture and the GCS solution were stirred overnight.Finally,by water washing and centrifugation,the CSMDNPs were successfully prepared.Ultraviolet-visible spectrophotometer(UV-Vis),transmission electron microscopy(TEM)and Zetasizer nanoparticle size potentiometer(Zetasizer)were used to investigate the physicochemical properties of the CSMDNPs.The High Performance Liquid Chromatography(HPLC)method was used to measure the loading of DA in the nanodrugs.2.In vitro experimentsThe cytotoxicity of CSMDNPs at different concentrations was determined using the CCK-8 assay.The cellular uptake of CSMDNPs by rat renal tubular epithelium was studied using confocal microscopy and flow cytometry.In addition,the scavenging ability of CSMDNPs on reactive oxygen species(ROS)at the cellular level was measured using the DCFH-DA kit.3.In vivo experimentsA model of diabetic nephropathy was established in C57BL/6J male mice by intraperitoneal injection of streptozotocin(STZ)combined with a high-fat,high-sugar diet.The drug was administered by tail vein injection after successful establishment of the model.PAI monitors the metabolism of nanoparticles in mice.Meanwhile,the mice’s body weight and blood glucose levels were under regular monitoring.To comprehensively evaluate the efficacy of the nanodrugs,haematological and pathological analyses were performed by blood biochemistry,urine routine,H&E,Masson’s and PAS staining.The levels of oxidative factors were characterised by enzyme-linked immunosorbent assay.Finally,the therapeutic mechanism of the nanodrugs was investigated by Western blot.Results:1.The results of TEM and Zetasizer nanoparticle size potentiometer showed that the CSMDNPs with a negatively charged surface had a suitable size,good dispersion,and excellent stability.The UV-Vis absorption spectra showed a clear absorption peak of DA,confirming the successful loading of DA.Further,the loading rate of DA calculated by HPLC was 5.48 ± 1.14%.2.The CCK-8 cytotoxicity assay results showed that,after incubation of nano-drugs at concentrations of 12.5-500 μg/mL with NRK-52 E cells for 24 h,no significant decrease in cell survival was observed,indicating the CSMDNPs has excellent biocompatibility.Meanwhile,cell confocal and flow cytometry results showed that the cells after incubation with the chitosan-coated nanoparticles had a higher uptake efficiency at the same period of time.Besides,DCFH-DA results showed that the levels of intracellular ROS decrease gradually with increasing concentrations of CSMDNPs.3.PAI results show longer retention of CSMDNPs in the kidney of DN mice.Compared with the control group,the experimental group mice showed body weight loss,blood glucose levels decreased,and a significant recovery in hematological indexes and pathological examination.In addition,Western Blot results indicated that nano-drugs could inhibit NAGL expression,inhibit Nrf2/keap-1 pathway,and regulate HO-1 at the protein level to play an antioxidant role and protect renal function.In conclusion,the nano-drugs could significantly slow down diabetic nephropathy.Conclusion:This study provides a biocompatible,melanin-based targeted nanomedicine that combines integrated hypoglycemic,antioxidant and photoacoustic imaging for the combined treatment of diabetic nephropathy.This work provides a new idea for the effective treatment of diabetic nephropathy by constructing targeted,sustained-release,and multifunctional nano-drugs.