Protective Effect Of Succinylated Gelatin Injectim Against CRGD-siRNA Nephrotoxicity In Mice

BackgroundSmall interfering RNA(siRNA)is an important part of RNA interference(RNAi),which leads to specific gene silencing as well as reducing target gene expression through inhibiting mRNA translation.For the categoric effect,siRNA has caused extensive attention.However,series obstacles came before clinical application,including its poor serum stability,low cell uptake and the lack of targeting.These questions may be solved by chemical modification or assemble siRNAs into nanoparticles with cationic lipids and polymers,and different kinds of carriers that can deliver siRNAs to targeted positions.Arg-Gly-Asp(RGD)peptide and structurally related compounds are now widely researched and it belongs to the integrin ligand group.Integrin is a kind of cell adhesion molecules which establishes the connection between nucleus and external environment.Among them,integrin-?v?3 receptor plays an important role in the angiogenesis and tumor metastasis,which is over expressed in the endothelial cells of tumor neovasculature.Researchers found that Arg-Gly-Asp(RGD)peptide can specifically bind to the integrin-?v?3 and annular RGD has higher affinity.When applied in vivo,siRNA conjugated with cRGD gathers at the tumor and resulting in targeting efficacy.However,siRNA conjugated with cRGD show kidney toxicity after long-term administration which limits its clinical application.Low-molecular-weight proteins and peptides are filtered and subsequently reabsorbed in the proximal tubules of kidney:this reabsorption leads to the peptides' accumulation in the kidney,thus might contribute to renal toxicity.Gelofusine is gelatin-based plasma expander and usually applicable to blood dilution,extracorporeal circulation,insulin input carrier,as well as used to prevent the hypotension caused by pinal or epidural anesthesia.Researchers found that Gelofusine significantly induced the reduction of the renal reabsorption of A700-RAFT-RGD and 111In-DOTA-RAFT-RGD and at the same time doesn't affect tumor uptake.In this study,mice are co-injected Gelofusine and cRGD-siRNA,then pathological examination,TdT-mediated dUTP nick-end labeling,fluorescence imaging were done.From the results,we could conclude that co-injection Gelofusine and cRGD-siRNA reduced renal toxicity caused by cRGD-siRNA effectively,which clears the obstacles and provides support of cRGD-siRNA clinical treatment.Methods1.Synthesis and identification of cRGD-siRNACyclic RGD was covalently conjugated to the 5'-end of the sense strand of siRNA.The purified molecules are detected by High performance liquid chromatography(HPLC)and Mass spectrometry(MS).2.Determination of serum creatinine and blood urea nitrogenGrouping the mice into three parts:Group 1,injection of saline(100 ?l);Group 2,injection of cRGD-siRNA(5 nmol);group 3,co-injection of cRGD-siRNA(5 nmol)and Gelofusine(4 mg).The mice were dosed continuously 5 times via tail vein,with the interval of 48h.On the second day after the last dosed,cut the tail to gather blood and then the mice were killed.The serum was separated and the creatinine and blood urea nitrogen was determined by the automatic blood instrument.3.Renal index and histological observationThe kidneys were took out and weighed and the renal index(RI)is calculated using the formula:RI=weight of kidney(g)/body weight(g)×100%.After then,the kidneys were cut open.Half of them were put into-80 ? and the others were immediately put in 4%paraformaldehyde for 8 to 12 hours,and then soaked in different concentration of ethanol.The organizations were soaked in the chloroform,and then paraffin embedded and cut into 4?m slices.HE staining was done and tissue pathological changes are observed under microscope.4.TUNEL analysisThe paraffin sections are immersed in xylene twice at at room temperature,5 minutes each time,and then are dipped into gradient ethanol.After 20 minutes'incubation with proteinase k(20?g/ml)at room temperature,50?l mixture with FITC-12-dUTP markup was added.Glass slides were dipped into DAPI for five minutes and then were analyzed under fluorescence microscope.Under the fluorescence of 520 ± 20 nm,the cell nucleus of TUNEL(+)cells were green and under the fluorescence of 460nm,cell nucleus became blue.We took photos and count the cell number.Ten views were chosen for each section.5.Distribution of cRGD-siRNA in vivoMale mice were injected intravenously with both Cy-5 labeled cRGD-siRNA(1 nmol/20 g)and Gelofusine(4 mg)or only cRGD-siRNA-Cy5.The mice were killed at 24h,48h after administration and major organs were excised and imaged using an IVIS Spectrum imaging system(Cy5:? ex = 640 nm,? em = 680 nm).6.Statistical analysisAll the date of this study is analyzed using SPSS 19.0 statistical software.The results are obtained with the X±SD.Use One-way ANOVA to compare multiple groups.When the variance is together,the method of least significant difference(LSD)is used.Dunnett's T3 method is used when the variance is missing.P<0.05 is considered statistically significant.Results1.Synthesis and identification ofcRGD-siRNAThe siRNA sequence was compounded and identified successfully(sense strand:5'-(mG)(mA)(mG)AACCUCACAUGGU(mA)(mC)(mA)dTdT-3';antisense strand:5'-(mU)(mG)(mU)ACCAUGUGAGGUU(mC)(mU)(mC)dTdT-3').HPLC result showed that the purity of cRGD-siRNA exceeded 90%.And MS result showed the molecular mass was homologous with theoretical molecular mass.2.Determination of serum creatinine and blood urea nitrogenAfter two days of administration for 5 times,the serum was separated and the creatinine and blood urea nitrogen was detected by clinical laboratory of Zhujiang Hospital of Southern Medical University.The results both showed that there were statistical significances(P<0.05)among the groups.3.Renal index and histological observationComparing with the group of co-injection of cRGD-siRNA(5 nmol)and Gelofusine(4 mg),the renal index of the group of injection of cRGD-siRNA(5 nmol)significant increased(P<0.05).HE staining results showed that the group of injection of cRGD-siRNA(5 nmol)was observed edema and apoptosis of a large number of renal tubular epithelial cells,as well as glomerular atrophy.In contrast,the renal damages of the group of co-injection of cRGD-siRNA(5 nmol)and Gelofusine(4 mg)were lighter.The results above indicated that Gelofusine can reduce the damage caused by cRGD-siRNA.4.TUNEL analysisThe results of TUNEL analysis showed apoptotic cells were found in every group,but the group of co-injection of cRGD-siRNA(5 nmol)and Gelofusine(4 mg)showed more damaged cells and The difference was statistically significant(P<0.05).5.Distribution of cRGD-siRNA in vivo24 hours and 48 hours after injection,abundant Cy5 fluorescence was detected in the kidney of the group of injection of cRGD-siRNA(5 nmol)and the livers was also observed some fluorescence.In contrast,the Cy5 fluorescence was much more weaker in the group of co-injection of cRGD-siRNA(5 nmol)and Gelofusine(4 mg),and there was little fluorescence in livers.The results showed that Gelofusine expedited the elimination of cRGD-siRNA and alleviated the renal retention of cRGD-siRNA.ConclusionThe cRGD-siRNA molecules were successfully prepared by conjugating cyclic RGD to the 5'-end of the sense strand of siRNA.The results of a variety of experiments showed that Gelofusine can reduce the tubular injuries and glomerular damage caused by cRGD-siRNA.This is an indication that Gelofusine may have promising potential in the clinical application of cRGD-siRNA therefore has a good clinical research prospect.