Molecular Imaging-guided Therapy For Acute Kidney Injury Based On Melanin Nanoparticles

Part One Application of GPR97-MNP-PJ34（GMP）in acute renal injury induced by rhabdomyolysisObjective:GPR97-MNP-PJ34（GMP）Nanoprobes based on MNP were constructed and exploringtheir therapeutic effect and related mechanism in acute kidney injury induced by rhabdomyolysis through in vivo and in vitro experiments,and the therapeutic process was visualized through photoacoustic imaging,which aiming to provide a new idea for improving the therapeutic effect of acute kidney injury.Methods:1.Probe preparation:PJ34 were loaded on melanin nanoparticles（MNP）throughπ–πinteraction to form MNP-PJ34（MP）,and then coupled with Anti-GRP97 by chemical bonding to prepare GPR97-MNP-PJ34（GMP）nanoprobes successfully.The prepared nanoprobes were systematically characterized.2.In vitro experiments:The cytotoxicity of nanoprobes were detected by CCK-8methods.The uptake of nanomedicines by renal tubular epithelial cells was observed by fluorescence microscopy and flow cytometry.The concentration and time of Co Cl₂-induced hypoxia in renal tubular epithelial cells were determined by flow cytometry.The content of ROS in renal tubular epithelial cells in different treatment groups was detected by DCFH-DA probe.Changes of mitochondrial membrane potential before and after treatment of renal tubular epithelial cells were detected by JC-1 probe.The distribution of apoptosis of renal tubular epithelial cells stimulated by Co Cl₂ under different treatments was analyzed by flow cytometry.The contents of IL-6 and TNF-αin the cell culture supernatant were determined by ELISA kit.3.In vivo experiment:To construct the model of AKI in mice induced by rhabdomyolysis,the metabolism of GMP in kidney,therapeutic effect and safety in vivo were evaluated.The mechanism of GMP anti-oxidation and anti-apoptosis was studied by Western Blot.Results:1.GMP were synthesized successfully,and their size was about 20 nm observed by transmission electron microscope and DLS was about 40 nm.In addition,UV-Vis-NIR absorption and Zeta potential further suggested that MNP were successfully loaded with PJ34 and targeted for modification.2.In vitro experiments showed that GMP nanoparticles had high biocompatibility,and a strong ability to scavenge a variety of toxic ROS,anti-apoptosis and anti-inflammatory.3.In vivo experiments showed that the aggregation and metabolism of GMP in both kidneys of mouse AKI model were monitored in real time by photoacoustic imaging.GMP nanoparticles could effectively reduce oxidative stress,apoptosis and inflammatory responses in rhabdomyolysis-induced AKI mice.Western Blot results showed that GMP reduced the expression of AIF by inhibiting PARP-1;the antioxidant capacity of GMP was mainly achieved by activating the Keap-1/Nrf2/HO-1 pathway.Conclusion:In this study,we successfully constructed a p J34-loaded and anti-GRR97 coupled melanoma nanoparticles targeted nano delivery platform,realizing photoacoustic self-monitoring and triple-collaborative therapy（antioxidant,anti-apoptotic and anti-inflammatory）in rhabdomyolysis AKI mice.Therefore,using melanin nanoparticles as nanocarriers to achieve targeted delivery and efficacy monitoring under the guidance of PA imaging will be a promising method for the treatment of rhabdomyolysis AKI.Part Two Application of HA-MNP-DXM（HMD）in renal injury induced by ischemia reperfusionPurpose:HA-MNP-DXM nanoprobes based on MNP were constructed and exploring their therapeutic effect and related mechanism in acute kidney injury induced by ischemia reperfusion through in vivo and in vitro experiments,and the therapeutic process was visualized through fluorescence imaging,which aiming to provide a new idea for improving therapeutic effect of acute kidney injury.Methods:1.Probe preparation:The melanin nanoparticles were modified by NH₂-PEG-COOH and then combined with-OH on dexamethasone（DXM）through DCC/DMAP reaction to form MNP-DXM（MD）.HA-MNP-DXM（HMD）nanoprobes were successfully obtained after coating the hyaluronic acid（HA）layer on its surface to increase the targeting ability and then prepared nanoprobes were systematically characterized.2.In vitro experiments:The cytotoxicity of nanoprobes was detected by CCK-8 method.The uptake of nanoprobes by renal tubular epithelial cells was observed by fluorescence microscopy and flow cytometry.The content of ROS in renal tubular epithelial cells in different treatment groups was detected by DCFH-DA probe.Changes of mitochondrial membrane potential before and after treatment of renal tubular epithelial cells were detected by JC-1 probe.The distribution of apoptosis of renal tubular epithelial cells stimulated by Co Cl₂ under different treatments was analyzed by flow cytometry.The contents of IL-6 and TNF-αin cell culture supernatant were determined by ELISA kit.3.In vivo experiment:A mouse model of AKI induced by ischemia reperfusion was constructed to systematically evaluate the metabolism of HMD in the kidneys,the therapeutic effect and safety in vivo.The antioxidant mechanism of HMD was investigated by Western Blot.Results:1.The HMD nanoprobes were successfully prepared,and their particle size was about40 nm observed by transmission electron microscope and 60 nm in hydrated particle size analyzed by DLS.In addition,UV-Vis-NIR absorption and Zeta potential suggested that MNP were successfully loaded with DXM and modified with HA.2.In vitro experiments showed that HMD nanoparticles had good biocompatibility,and a strong ability to scavenge a variety of toxic ROS,anti-apoptosis and anti-inflammatory.3.In vivo experiments showed that the aggregation and metabolism of HMD in both kidneys in the mouse AKI model were monitored in real time by fluorescence imaging.HMD nanoparticles could effectively reduce oxidative stress,apoptosis and inflammatory responses in ischemia reperfusion AKI mice.Western Blot results showed that the antioxidant capacity of HMD was mainly achieved by activating the Nrf2/HO-1 pathway.Conclusion:In this study,a targeted nano-delivery platform（HMD）was successfully developed by loading DXM with MNP and targeted modification with HA,realizing the synergistic treatment of antioxidant,anti-apoptotic and anti-inflammatory in ischemia-reperfusion AKI mice.The process of AKI treatment can be visualized by labeling near-infrared second-region imaging small molecule H2.Therefore,using melanin nanoparticles as nanocarriers to achieve targeted delivery and efficacy monitoring under the guidance of fluorescence imaging will be a promising method for the treatment of ischemia-reperfusion AKI.Based on the above two parts,we confirmed that:1.Melanin is an endogenous substance in the human body with good biocompatibility and stability.As a high-efficiency drug-carrying platform,it is beneficial to clinical transformation;2.Melanin nanoparticles can effectively reduce oxidative stress injury in AKI induced either rhabdomyolytic or ischemia reperfusion,and the nuclear Nrf2 plays an important role in reducing oxidative stress;3.Utilizing the unique photoacoustic imaging properties of melanin and its ability to couple with the near-infrared second-region imaging small molecule H2,it can guide the selection of treatment plans for renal injury and monitor the treatment effect.