Construction Of Black Phosphorus Nanosheet Drug Delivery System Based On Targeted Peptide Modification And Its Research On Improving Acute Kidney Injur

As one of the common critical illnesses in clinical practice,acute kidney injury(AKI)refers to a clinical syndrome caused by various etiologies that lead to rapid decline in renal function within a short period of time.Despite the high incidence and mortality rates of AKI,the current mainstay of clinical treatment is supportive therapy,including correction of electrolyte disturbances,maintenance of acid-base balance,and provision of nutritional support,with no more proactive and effective targeted therapies available.Due to the complexity of AKI pathogenesis and the special structure of the kidney,targeting drug delivery systems have always been a research challenge,and there is an urgent need to develop more effective drugs and more targeted delivery strategies to prevent and treat AKI.The mechanisms of AKI injury usually include oxidative stress,inflammation,cell apoptosis,cell membrane damage,and intracellular acid-base imbalance.Therefore,it is possible to improve AKI by reducing reactive oxygen species(ROS)level,inhibiting inflammation,and reducing cell apoptosis.In order to screen compounds for the treatment of AKI,by searching bibliographic databases such as PUBMED and SciFinder,full-text databases such as CNKI and Wanfang,citation databases such as Web of Science and Chinese Science Citation Database,and new drug research and development databases such as Yaodu and Yaoyiyun,a library of AKI candidate compounds containing 341 compounds was established.After the screening of cytotoxicity model,cell injury model,cell inflammation model,and rat AKI model,it was found that the small molecule compound ruxolitinib phosphate(RP)could improve AKI through anti-inflammatory and cell-protective effects,and had good potential for treating kidney diseases.However,RP had certain hematological toxicity,which may lead to a decrease in the levels of circulating leukocytes,lymphocytes,platelets,and neutrophils,which limited its clinical application to a certain extent.Therefore,this study envisaged improving the renal targeting of RP and reducing its toxic side effects on non-target tissues through pharmaceutical formulation,and enhancing the therapeutic effect of RP on AKI.In recent years,black phosphorus nanosheets(BPNSs),a novel two-dimensional material,have received extensive attention and application in biomedical field.BPNSs have several advantages in kidney-targeted drug delivery:firstly,their high surface area to volume ratio is conducive to increasing drug loading;secondly,their layered structure allows for shape-dependent passive targeting to the kidneys;in addition,BPNSs could accelerate drug release in a lower pH environment,and the diseased kidney in AKI has an acidic microenvironment under inflammation,so the pH-responsive release of BPNSs drug loading is beneficial to renal drug delivery;finally.BPNSs can quickly clear ROS accumulated in diseased areas,effectively protecting the kidneys.Therefore,in this study,BPNSs were chosen as drug delivery carriers to efficiently load RP for kidney-targeted delivery.Renal tubular injury is one of the main manifestations of AKI,so renal tubular epithelial cells are an important target for AKI therapy.The positively charged renal tubular targeting peptide G3-C12 can be reabsorbed in the proximal tubule through megalin-mediated endocytosis.which efficiently improves the accumulation of the conjugated drug in the kidney.To improve the targeting ability of BPNSs,G3-C12 peptide is used to modify BPNSs by electrostatic interaction to construct the RP@BPNSs@G3C12 functionalized drug delivery system.The system enters the blood circulation through intravenous injection,actively targets renal tubular epithelial cells,releases RP after endocytosis by renal tubular epithelial cells,and relieves renal damage.In this study,BPNSs were prepared using liquid phase exfoliation and RP@BPNSs@G3-C12 was prepared through electrostatic interaction.An orthogonal experiment was conducted to optimize the preparation method,and the influence of RP to BPNSs mass ratio,stirring time,centrifugation speed and centrifugation time on the encapsulation efficiency of RP@BPNSs@G3-C 12 was investigated.The optimized preparation method was verified and the encapsulation efficiency of RP@BPNSs@G3C12 was found to be 87.4%,indicating that the method had good stability and feasibility.Next,the physicochemical properties of the nanocarrier system were characterized.DLS results showed that the particle size of non-targeted BPNSs was 106.0 nm with a Zeta potential of-9.6±6.81 mV.The particle size of targeted BPNSs@G3-C12 was 122.4 nm with a Zeta potential of-13.3±4.59 mV.While,the particle size of RP@BPNSs@G3C12 was 132.8 nm with a Zeta potential of-5.31±4.83 mV.The change in Zeta potential indicated successful adsorption of RP and G3-C12 on the surface of BPNSs through electrostatic interaction.Transmission electron microscopy observed that BPNSs had a flake structure.In vitro release results showed that the release rate of RP increased significantly at pH 5.0 compared to pH 7.2,indicating that the delivery vehicle accelerated drug release in acidic environments,which was beneficial for kidney drug delivery under diseased conditions.Stability evaluation results showed that the delivery system had good stability during storage and circulation in vivo.Meanwhile,the cytotoxicity and cellular uptake mechanism of the delivery system were studied,and it was found that BPNSs and other preparations had no significant cytotoxicity in the concentration range of 0～200μg/mL.Moreover,the study found that the cellular uptake of the G3-C12 modified delivery system was increased,and the endocytosis process was mainly mediated by energydependent micropinocytosis.The in vitro anti-inflammatory effects of RP@BPNSs@G3-C12 were evaluated using a lipopolysaccharide(LPS)-induced NRK-52E cell model.After co-incubation with the delivery system,the levels of tumor necrosis factor α(TNF-α)and interleukin 6(IL-6)in the LPS-induced cell culture supernatant were found to be reduced.The mRNA levels of TNF-α and IL-6,as well as the protein phosphorylation levels of JNK and NF-κB,also decreased,indicating that the delivery system could effectively inhibit LPS-induced inflammation in NRK-52E cells.The cell protective effect of RP@BPNSs@G3-C12 was evaluated using the NRK-52E cell injury model induced by doxorubicin and hydrogen peroxide(H2O2).It was found that the delivery system protected the cells from doxorubicin-induced cell death in a concentration-dependent manner,as well as inhibited H2O2-induced cell oxidative stress and reduced ROS levels.In the study of anti-cell apoptosis in vitro,it was found that the delivery system inhibited H2O2-induced cell apoptosis.After the delivery system pretreatment,the protein level of cleaved Caspase-3 and the activity of Caspase-3 in cells were significantly reduced,indicating that the delivery system had a good protective effect on doxorubicin-induced cell damage,H2O2-induced oxidative stress and cell apoptosis in vitro.The in vivo pharmacodynamics of RP@BPNSs@G3-C12 were evaluated using a doxorubicin-induced AKI rat model,and the mechanism of action was investigated.Firstly,the biocompatibility of the delivery system was evaluated,and it was found to have good biosafety and improved the hematological toxicity of RP.Moreover,after intravenous injection,it quickly targeted and specifically accumulated in the kidneys of AKI rats.Next,the renal function indicators were assessed,and histological staining with H&E,TUNEL,and CD68 immunostaining were performed on the kidneys of the rats.The results showed that the delivery system significantly improved kidney function in AKI rats.The kidney mRNA was extracted and RNA-Seq was performed to study the action mechanism of the delivery system.The therapeutic effect was found to be mediated through apoptosis,inflammation,and oxidative phosphorylation pathways.These pathways were further validated,and it was found that delivery system treatment reduced the levels of TNF-α,IL-6,and interleukin 1 β(IL-1 β)in the blood of rats,indicating an improvement in systemic inflammation.The levels of serum kidney injury molecule-1(KIM-1),neutrophil gelatinase-associated lipocalin(NGAL),and the activity of Caspase-3 in AKI rats were reduced,indicating a reparative effect on kidney damage.Finally,the activity of ROS and superoxide dismutase(SOD),and the content of malondialdehyde(MDA)in the kidneys of rats were measured.It was found that the delivery system significantly increased SOD activity,reduced ROS activity and MDA content in the kidneys,indicating that delivery system treatment inhibited oxidative stress in rat kidneys.In summary,this study discovered the great potential of RP as a therapeutic drug for AKI,and constructed the RP@BPNSs@G3-C12 drug delivery system,which exhibited excellent AKI kidney targeting ability and pH-responsive drug release characteristics.In vivo and in vitro studies showed that RP@BPNSs@G3-C12 had significant inhibitory effects on inflammation,oxidative stress and apoptosis,and had a significant therapeutic effect on doxorubicin-induced AKI injury,indicating that RP@BPNSs@G3-C12,as a new renal drug delivery system,had great advantages and potential clinical application value in the treatment of AKI.