Study On Targeted Anti-renal Fibrosis Of Self-assembled Polypeptide Drug-loaded Gold Nanoparticles

Background:Renal fibrosis is a common pathway for chronic kidney disease to progress to end-stage renal disease.Recent studies have found that hypoxic preconditioning has a certain effect in improving acute and chronic kidney injury.Among them,cobalt chloride(CoCl2)is used as a stabilizer for HIF and is now used to study the effects of hypoxic preconditioning on the disease.However,high concentration of cobalt ions has obvious toxicity to cells and genes,thus greatly limiting its application in disease treatment.Nanomaterials have obvious advantages in biomedical applications due to their features such as controllability of size and morphology,easy modification,high surface activity,and good biocompatibility.Gold nanoparticles(AuNPs)are the most in-depth and extensive nanoparticles.Functionalized AuNPs can not only load more drugs,but also have the effect of maintaining drug stability and targeting transport and release of drugs.Thus,it can be seen that functionalized AuNPs may be helpful in targeting drug delivery to the kidney,thus providing a new direction for the targeted treatment of chronic kidney disease.Methods:Transmission electron microscopy(TEM),Dynamic light scattering(DLS)and thermal gravimetric analysis(TGA)was verified the preparation of functional gold nanoparticles(GLAuNPs).Infrared spectroscopy and fluorescence spectrum were used to investigate the hydration medium particle size and stability in a variety of medium,respectively.Cell proliferation assay(CCK-8),cytokines and pathological staining were used to detect the cell toxicity,immune toxicity and systemic toxicity,respectively.Cell fluorescence,in vivo imaging and fluorescence imaging respectively used to observe the cell uptake mechanism,in vivo distribution and organ targeting capability.Inductively coupled plasma mass spectrometry(ICP-MS)was used to study the efficiency of drug loading and release.After establishing unilateral ureteral obstruction(UUO)model,pathology staining,Western Blot and Real-time PCR were used to detect the effects of renal-targeting AuNPs(GLAuNPs-Co)against renal fibrosis,inflammation,apoptosis and oxidative stress.Finally,the mechanism of anti-fibrosis of GLAuNPs-Co was studied by Real-time PCR.Results:The results of TEM and infrared spectra showed that GLAuNPs performed homogeneous spherical particles of monodisperse and its hydrated particle size was 9.9±2.2nm,higher than LAuNPs 3.8±1.0nm,suggesting that glutathione was successfully modified on the surface of the LAuNPs and TGA results also confirmed this result.Fluorescence spectra showed that the fluorescence intensity had no obvious change before and after modification.The results showed that GLAuNPs medium stability can keep stable in a variety of common medium.CCK-8 test demonstrated that at the concentration below 4000 nM,the cell activity still remained at around 100%.Cell fluorescence showed that the cell uptake of GLAuNPs was specific,and caveolin,megalin as well as the energy were involved in the endocytosis process.In vivo imaging and fluorescence imaging of organs showed that GLAuNPs can rapidly accumulate to the kidneys(1 hour),keep on 12 hours and then decrease gradually,which is consistent with semi quantitative fluorescence results of various organs.The intrarenal distribution showed that GLAuNPs mainly located in the proximal tubule cytoplasm.ICP-MS results displayed that when the volume ratio of GLAuNPs and Co2+ was 5:1,the load rate reached the highest.With the decrease of pH and the release amount of Co2+increased gradually,and in less than the pH?6.0,the release efficiency can reach above 80%.Comparative toxicity test results revealed that the modification of GLAuNPs with Co2+ could be significantly reduce cytotoxicity of Co2+.The results of HE staining showed that there was no obvious organ toxicity in the treatment of GLAuNPs-Co.In UUO model,pathological staining results showed that GLAuNPs-Co significantly decreased nephropathy mice renal tubule damage and inflammatory infiltration and interstitial fibrosis in nephropathy mice compared to the group treated with the free equal amounts of CoCl2:Western blot showed downregulated levels of fibronectin,Nox2,Nox4,Collagen-I,Bax,p-ERK,cleaved caspase-3,MCP-1,IL-6 and ICAM-1 protein in GLAuNPs-Co group(P<0.05),upregulated levels of bcl-2,Catalase and SOD1.PCR results displayed that GLAuNPs-Co significantly reduced gene expression levels of fibronectin,Collagen-I,MCP-1,IL-6,ICAM-1,and TNF-?(P<0.05).The mechanism study revealed that GLAuNPs-Co significantly upregulated the expression of HIF and its downstream genes(EPO,Tie-2 and HO-1),and restored the downregulation of miR-29c expression and downregulated its downstream genes expression(TPM-1 and COL3A1).TUNEL results showed that GLAuNPs-Co significantly reduced the number of apoptotic cells in the kidney(P<0.05).Conclusion:GLAuNPs had no obvious toxicity to the cells,with good biocompatibility and specific target capability.Its main enrichment site was proximal renal tubules.In UUO model,GLAuNPs-Co can significantly relieve renal fibrosis,inflammation,apoptosis and oxidative stress and the possible mechanism was induced by activation of HIF-1? through restoring the expression of miR-29c.