Drug Delivery Systems For Acute Kidney Injury Based On Antioxidant Peptide SS31

Acute kidney injury(AKI)manifests as a rapid decline in renal function with increased morbidity and mortality.The main causes of AKI include sepsis,kidney ischemia-reperfusion injury,and so on.No effective therapeutic efficiency exists nowadays.Mitochondrial oxidative stress is considered the common pathological mechanism of various AKI.Therefore,anti-oxidative stress has become an important treatment strategy.While the treatments of traditional antioxidants have limited clinical therapeutic efficiency.Poor renal bioavailability and insufficient mitochondrial distribution are the main causes.SS31 is a cationic mitochondrial-targeted peptide with strong antioxidative ability,which is a drug candidate for AKI therapy.However,for SS31,some factors are limiting the bioavailability of peptide therapeutics including systemic proteases,rapid metabolism,and insufficient target tissues distribution.This study prepared two kinds of SS31 delivery systems for different AKI types(sepsisinduced AKI and ischemia-reperfusion induced AKI),protecting SS31 in the blood circulation and targeted delivering SS31 to AKI injured area,and decreasing oxidative stress assisted by mitochondrial-targeted ability.The oxidative stress of microvascular endothelial cells is an important pathological mechanism of sepsis-induced AKI.CD44 receptor is overexpressed in AKI injured vascular endothelial cells.Hyaluronic acid is a biocompatible and biodegradable natural anionic material and has the inherent property to specifically target CD44 receptors.Based on the positive charge of SS31,nanopolyplexes(HA/CS/SS31)consisting of SS31,anionic sodium hyaluronate(HA),and cationic chitosan(CS)are designed and prepared facilitated by electrostatic interaction.We investigated the influences of CS input and its molecular weight on the particle size of HA/CS/SS31.The results showed that the less the CS input was,the smaller the particle size of HA/CS/SS31 was,and the CS with a molecular weight of 20 k Da was easier to form homogeneous nanopolyplexes than that of 2.5 k Da.Therefore,in this study,we chose20 k Da CS as the carrier material,set CS input as 0.33%,and prepared HA/CS/SS31 nanopolyplexes with a diameter of 53 ± 0.17 nm,a PDI of 0.207 ± 0.06 and a surface potential of –19.6 ± 0.7 m V.HA/CS/SS31 has a good SS31 loading capacity,with a drug loading of 10.5 ± 0.2% and an entrapment efficiency of 94.0 ± 2.0%.We measured the release of SS31 from HA/CS/SS31 as a function of p H via the HPLC method.The drug release behaviors of HA/CS/SS31 were highly dependent on the change in p H values.HA/CS/SS31 showed a slight SS31 release of 6% when exposed to the medium with p H 7.4 for 5 min.In comparison,drug release was considerably faster at acidic conditions,with SS31 releasing above 70% at p H 4.5 within 5 min.We also determined the size distribution of HA/CS/SS31 in the different p H conditions by Zetasizer NanoZS and transmission electron microscope.The data showed that,with the decrease of p H,the size of HA/CS/SS31 increased,and the shape became irregular,which further confirmed its p H responsibility.Hemolysis test showed that HA/CS/SS31 had good blood compatibility.Taken human umbilical vein endothelial cell(HUVEC)as model cell,use lipopolysaccharide(LPS)to stimulate HUVEC to model sepsis-induced AKI cells.The cytotoxicity,cell uptake,and intracellular drug release behaviors of HA/CS/SS31 were investigated.HA/CS/SS31 showed low cytotoxicity.Compared with normal cells,LPS stimulated cells with overexpressed CD44 receptors had better HA/CS/SS31 uptake ability.With the extension of incubation time,SS31 was gradually released from HA/CS/SS31.The released SS31 was targeted to mitochondria and played an antioxidant function.Use H2O2 to stimulate HUVECs to prepare the oxidative stress cell model,and investigate the antioxidant stress and anti-apoptotic effects of HA/CS/SS31.The results showed that HA/CS/SS31 could more effectively improve the survival rate of H2O2 stimulated cells,reduce the level of intracellular reactive oxygen species(ROS),stabilize mitochondrial membrane potential,and reduce apoptotic cells,compared with free SS31,HA/SS31,and HA/CS.Intrarenally inject ICR mice with LPS to establish the sepsis-induced AKI animal model.The AKI mice were injected intravenously with HA/CS/SS31,and the reprehensive organs were harvested and visualized by in vivo imaging system.The results showed that HA/CS/SS31 could be effectively distributed in AKI kidneys,and the strong kidney-targeting ability of HA/CS/SS31 was related to the overexpression of CD44 receptors in AKI mice.We further investigated the therapeutic effects of HA/CS/SS31 on sepsis-induced AKI after intravenous administration,with free SS31,HA/SS31,and HA/CS as control groups.The results showed that HA/CS/SS31 treatment significantly reduced the levels of blood creatinine and urea nitrogen in AKI mice;reduced renal tubular necrosis,transparent tubular type and cell abscission in kidney;alleviated the membrane rupture,the loss of ridge and swelling of mitochondria;reduced oxidative stress factor malondialdehyde(MDA),inflammatory factors interleukin-6(IL-6)and tumor necrosis factor(TNF-α),the infiltration of macrophage,and apoptosis of renal cells.The plasma drug concentration-time curve showed that HA/CS/SS31 could effectively prolong the half-life of SS31 and protect SS31.The effective protection to SS31 and targeted delivery are the important reasons for the good therapeutic effect of HA/CS/SS31 on AKI.Renal ischemia-reperfusion(IR)injury is the most common cause of AKI in the clinic.Mitochondrial oxidative stress injury of renal tubular epithelial cells is one of the key pathogenesis of ischemia-reperfusion-induced acute kidney injury(IR-AKI).Therefore,SS31 is also a potential therapeutic drug for IR-AKI.Drugs first reach the renal tubular lumen and then enter the cells from the top side of the renal tubular epithelial cells,which is the main drug delivery approach of the renal tubular epithelial cell-targeted drug delivery system.This process needs to overcome many physiological barriers such as glomerular filtration.Generally,the drug delivery systems with small particle size(less than 5 nm),small molecular weight(less than 70 k Da),and the positive surface charge are easier to be filtered by the glomerular to reach the renal tubules.Conjugate prodrug is a kind of new drug delivery system,which usually uses polymer materials with good biocompatibility as carriers and connects the carrier with the drug through a covalent bond,to deliver the drug.The low molecular weight prodrugs with rational carrier design are expected to protect SS31 and actively target renal tubular epithelial cells,realize the controlled drug release,so as to improve the IR-AKI therapeutic effect of SS31.KIM-1 is a transmembrane protein markedly up-regulated in injured kidneys,particularly renal proximal tubules,which induces the internalization of the clearance of dead cells.KIM-1 is expected to be a target for renal tubular drug delivery.Serine is the substantial structure for interaction with KIM-1 in the process of endocytosis.On this basis,we synthesized the L-serine modified chitosan(Ser-CS)carrier by amide reaction with cationic carrier material CS(molecular weight 2.5 k Da)as the basic skeleton.Taking ICR mice as model animals,the IR-AKI mouse model was constructed by bilateral renal artery occlusion,and the biodistribution of Ser-CS in AKI kidneys was investigated.The results indicated that Ser-CS had good renal aggregation and retention effects in AKI mice,and could quickly and effectively distribute in AKI kidneys,mainly accumulating in renal tubules.Taking human renal tubular epithelial cell(HK-2 cell)as the model cell,the oxidative stress cell model was constructed by stimulating HK-2 cells with H2O2.The cytotoxicity and cell uptake behavior of Ser-CS were investigated.The results showed that Ser CS had low cytotoxicity and could be rapidly taken by injured cells.Western blotting showed that KIM-1 was overexpressed in H2O2 stimulated HK-2 cells.Use confocal microscopy to investigate the immunofluorescence co-localization of Ser-CS and KIM-1 at the cellular and tissue levels.The results showed that the fluorescence of Ser-CS and KIM-1 highly coincided,indicating that the internalization of Ser-CS by H2O2 stimulated cells and the accumulation and retention of Ser-CS in AKI renal tubules were related to KIM-1mediated endocytosis.Kim-1 received much attention in early diagnosis and pathological mechanisms of kidney diseases.However,it is a new field for Kim-1 as a drug target.Based on the high ROS microenvironment in AKI injured renal tubular epithelial cells,we synthesized the ROS responsive conjugate prodrug Ser-CS-TK-SS31 by connecting Ser-CS and antioxidant peptide SS31 through amide reaction with ROS responsive thioketal(TK)as linker.In addition,non-ROS responsive prodrug Ser-CSnonane diacid(NO)-SS31 and non-KIM-1 targeted prodrug CS-TK-SS31 were synthesized as control groups.Confirm the structure of the prodrugs using nuclear magnetic resonance(NMR)and gel permeation chromatography(GPC),and estimate the SS31 graft ratios by peak area and molecular weight tested by NMR and GPC,respectively.The results showed that the graft ratios of SS31 on the 3 conjugated prodrugs were 1/1.The drug loadings of Ser-CS-TK-SS31 and CS-TK-SS31 were 16.8%and 19.6%,respectively,which were investigated by high performance liquid chromatography(HPLC).The grafting ratios of SS31 calculated according to the drug loadings were also 1/1,which was consistent with the results of NMR and GPC.H2O2 is a typical and relatively stable ROS.Ser-CS-TK-SS31 was incubated in a 10 m M H2O2 medium and its time-dependent drug release behavior was detected by HPLC.The results showed that Ser-CS-TK-SS31 had obvious ROS responsive drug release characteristics.Under the condition of H2O2,Ser-CS-TK-SS31 released 60% of SS31 within 8 hours,while there was no drug release after the non-responsive SS31 prodrug Ser-CS-NO-SS31 incubating with H2O2 for 48 hours.In addition,Ser-CS-TK-SS31 had good plasma stability,which was conducive to effective drug delivery.Construct the oxidative stress cell model by stimulating HK-2 cells with H2O2.Use laser confocal microscope to observe the intracellular drug release and mitochondrial targeting of Ser-CS-TK-SS31.The results showed that with the extension of incubation time,the fluorescence signals of carrier and drug of Ser-CSTK-SS31 were significantly separated in oxidative stress model cells,while in normal cells,the fluorescence signals still had a high degree of coincidence,indicating that SerCS-TK-SS31 had ROS responsive release characteristics.The released drug SS31 could be targeted to mitochondria and played an antioxidant stress role.Taking SS31,CSTK-SS31,and Ser-CS-NO-SS31 as control groups,we studied the antioxidant stress and anti-apoptotic effects of Ser-CS-TK-SS31.The results showed that Ser-CS-TKSS31 could more effectively reduce the level of mitochondrial ROS,reduce the expression of antioxidant enzyme HO-1,stabilize mitochondrial membrane potential and reduce apoptosis.With ICR mice as model animals,establish the IR-AKI mouse model by bilateral renal artery occlusion.We evaluated the therapeutic effects of Ser-CS-TK-SS31 on IRAKI after intravenous injection.The results showed that,compared with SS31,CS-TKSS31 and SER-CS-NO-SS31 control groups,Ser-CS-TK-SS31 had better therapeutic effects,significantly reduced the levels of serum creatinine and urea nitrogen in AKI mice;effectively improved the renal tissue structure,reduced the transparent tubular type and cell shedding of renal tubules,retained the brush edge;effectively reduced the membrane rupture,ridge loss and swelling of mitochondria;reduced the expression of nitrotyrosine(a marker of oxidative stress)and the level of MDA;increased the level of reducing agent glutathione,reduced inflammatory factors(IL-6 and TNF-α),the infiltration of macrophage,and apoptosis of renal cells.Investigate the content of SS31 in the main organs of mice after intravenous administration of Ser-CS-TK-SS31 by HPLC.The results showed that SS31 was mainly distributed in the kidneys of mice,indicating that Ser-CS-TK-SS31 had a good renal aggregation effect,which was the important reason for the good therapeutic effect of Ser-CS-TK-SS31 on AKI.In conclusion,based on the oxidative stress injury of vascular endothelium in sepsis-induced AKI and that of renal tubular epithelium in IR-AKI,we constructed two kinds of AKI renal targeted drug delivery systems with mitochondrial-targeted antioxidant peptide SS31 as the drug: HA/CS/SS31 nanopolyplex and Ser-CS-TKSS31 conjugate prodrug.The two drug delivery systems could protect SS31 during delivery,recognize different targets(CD44 and KIM-1)by receptor ligand-specific interaction and deliver SS31 to renal injured cells in an active target way,and controlled release SS31,aggregate SS31 in mitochondria,alleviate oxidative stress and finally realize the efficient AKI treatment of SS31.