| objective: IgA nephropathy(IgAN)is a common primary glomerulonephritis,but there is currently no effective treatment.The deposition of abnormal glycosylated IgA1(Gd-IgA1)and its immune complexes in glomeruli is a key link in the pathogenesis of IgAN.Therefore,drugs that eliminate abnormal glycosylated IgA1 and its immune complexes as therapeutic targets have become potential therapeutic agents for IgAN therapy.Our previous research work suggested that bacterial IgA protease can degrade abnormally glycosylated IgA1 and its immune complex in vitro and in vivo through the specific cleavage of IgA1,potentiating its application for IgAN biological therapy.However,the absence of kidney preferential distribution accompanied by immunogenicity and potential toxicity compromises its clinical prospect.In this study,based on previous results,we plan to encapsulate the IgA protease,engineered from the H.influenzae(ATCC 49247),with nanoparticles.we try to validate the hypothesis that,compared with regular IgA protease,nanoparticles formulated IgA protease would demonstrated enhanced performance of both kidney mesangial targeting and pharmaceutic efficiency with eliminated or decreased physical toxicity.Methods : 1.Preparation of recombinant IgA protease with high biological activity.The full length of IgA gene was cloned from H.influenzae(ATCC 49247),and the prokaryotic expression vector of His6-IgA was constructed and expressed in E.coli(BL21).Nickel column affinity chromatography was used.High-purity biologically active IgA protease was prepared by ion exchange chromatography.Then its ability to cleave human IgA1 molecule was detected by SDS-PAGE electrophoresis,silver staining,Elisa method and the protein concentration was determined by BCA method.2.Preparation of nanoparticles and determination of renal targeting.Preparation of two differently charged nanoparticles with a diameter of 60-120 nm by utilizing nanoparticle packaging process.The experimental group is a positively charged nanoparticle that has electrostatic affinity for the mesangial area and the basement membrane with a significantly negative charge.The ddab nanoparticles with negatively-charged incompatible glomerular mesangial regions were used as controls,and albumin(nanoparticle preparation material)was labeled with rhodamine-DHPE fluorescent substance during preparation.Male BALB/c mice were randomly divided into 3 groups,namely PBS group(negative control),positively charged nanoparticles(nanoparticles)and negatively charged granules(ddab nanoparticles).Then,respectively PBS or two different nanoparticles were injected into BALB/c mice through the tail vein.The mice were sacrificed at different time points(5 min/15 min/30 min),and the hearts,liver,spleen,lung and kidney tissues were taken.The frozen sections observation of fluorescence distribution in kidney and other tissues was conducted to determine the kidney-targeted optimal diameter of the nanoparticles.3.Preparation of IgA protease-albumin nanoparticles and determination of renal targeting.After determining the optimal targeting diameter of the nanoparticles,IgA protease nanoparticles were prepared,and the biological activity of the IgA protease nanoparticles was detected by SDS-PAGE electrophoresis followed by silver staining.Normal male BALB/c mice were randomly divided into IgA protease nanoparticles group,blank nanoparticles group,and PBS control groups,with 3 in each group.The mice in each group was injected into through the tail vein with optimal diameter(~ 120 nm)and fluorescently labeled IgA protease-albumin nanoparticle,nanoparticles or PBS,respectively.After 5 min,the mice were sacrificed to obserbe the distribution of IgA protease-albumin nanoparticle in organs(heart,liver,spleen,lung),and especially the location in kidney mesangial area.In addition,the observation of renal targeting of nanoparticles by small animal in vivo fluorescence imaging was conducted.This part was to determine that IgA protease-albumin nanoparticle can reach the kidney with a targeted effect.4.Study on the degradation of IgA1 immune complex deposited in passive IgAN model mice by IgA protease-albumin nanoparticles.A passive IgAN model characterized by deposition of mesangial immune complexes was constructed by injecting a human hypoglycosylated IgA1-Ig G immune complex into the BALB/c mice via the tail vein.Male BALB/c mice were randomly divided into 4 groups,3 in each group,namely PBS group(PBS),model group(IgA1-Ig G),IgA protease treatment group(IgA protease)and IgA protease-albumin nanoparticles treatment group(IgA protease-NPs).After 12 hours of injection of the immune complex,the treatment groups were given the optimal optimal diameter(~ 120 nm)of IgA protease-albumin nanoparticles or conventional IgA protease via the tail vein.After 2 hours,the mice were sacrificed and fresh kidney tissues were taken for frozen section,and immunofluorescence was used to compare the clearance of IgA1-Ig G immune complexes.5.Analysis of physiological toxicity of IgA protease-albumin nanoparticles.Male BALB/c mice were randomly divided into 4 groups,3 in each group,namely PBS control group(negative control),model group(model),IgA protease treatment group(IgA protease)and IgA protease-albumin nanoparticles treatment group(nanoparticles).After 12 hours of modeling,the treatment group were given the targeted optimal diameter IgA protease-albumin nanoparticles or conventional IgA protease through tail vein.After 2h,7days and 30 days,the mice were respectively sacrificed,blood was taken for biochemical tests,liver and kidney function(ALT,AST and CREA,BUN)were measured,and heart,liver,spleen,lung and kidney tissues were taken for hematoxylin-eosin staining(HE staining)to observe the morphology of each tissue.Results: 1.Silver staining results showed the successful purification of IgA protease that originated from H.infuenzae(ATCC 49247),and the IgA protease with nanoparticle packaged possess good biological activity and there were no activity difference compared to the conventional IgA protease;2.Fluorescence results proved that both nanoparticles and IgA protease-albumin nanoparticles can be targeted to reach the renal mesangial area,and the results of small animal in vivo fluorescence imaging showed that after 5 minutes of injection of fluorescently labeled nanoparticles into the tail vein,the nanoparticles showed significant renal accumulation compared with the control group,and the difference was statistically significant(P < 0.05).;3.Immunofluorescence results revealed that a large number of immune complexes were deposited in the mesangial area of the kidney,thus,the successful construction of the passive IgAN model was verified.Compared with the model group,the immune complexes of the mesangial area in IgA protease nanoparticles and the conventional IgA protease treatment group were significantly reduced;At the same time,compared with the conventional IgA protease treatment group,the immune complex of the mesangial area of the IgA protease nanoparticle group was significantly less,and even some complexes were completely eliminated.4.HE staining results showed that there were no pathological changes in the heart,liver,spleen,lung and kidney tissues in the IgA protease nanoparticle group and the IgA protease group,which was not different from the normal PBS control group.The biochemical test results also indicated the liver and renal function of each group is normal.Conclusion: This study has identified that nanoparticle-encapsulated IgA protease presents higher drug accumulation in kidney and lower drug accumulation in off-target organs and tissues than conventional IgA protease. |
PDF Full Text Request