| Background and ObjectiveThe last few decades have witnessed a great revolution in Nanoscience in every way and have become an important part of our today life;in which drug delivery,diagnostics and cosmetics are the major part of it.With the continuous improvement of medical research and the progress of molecular biology techniques,combined with chemical and physical materials and other disciplines,and genomics,proteomics technology,people have known more about prevention,diagnosis and treatment of diseases.Nano has the characteristics of tiny,versatility,low cost and high performance,which contributes to develop as a targeting drug delivery,to infiltrate the biological barrier,the immune system barrier,etc,that the conventional carrier encountered.Therefore,it has become a new and efficient choice of disease diagnosis and treatment.In the field of medicine,including dental,tumor,immune antibody preparation and other disciplines,are involved in the introduction and application of nano materials.The nano therapeutics or nanoparticles(NPs)are the main focus of many research groups as it offers several benefits over conventional dosage forms such as;improved bioavailability,targeted delivery.A nano-drug carrier with better application value can transport the drug to the target area,and should meet the following requirements:appropriate and uniform size,biocompatibility,degradability,low toxicity,sufficient drug package and circulation in the body,in order to ensure that the drug from blood circulation can be reached and accumulate in the lesion tissue through the capillaries,release and reach the effective therapeutic concentration,meanwhile,mediate endocytosis and cause no damage to normal cells.Chitosan(CS);a polymer derived from the deacetylation of chitin,is easy to obtain and low cost.CS-NPs can be prepared between CS and tripolyphosphate sodium(TPP)with a simple ionic gelation method.CS has various positive factors such as it is biocompatible,biodegradable and nontoxic and also the absorption drug encapsulated into CNPs can be improved and can also effectively protect them from enzyme degradation in vivo,so it is widely used as a drug delivery carrier.Phosphatidylserine,also named compound nerve acid,referred to as PS,is a kind of widespread phospholipid,usually located in the inner layer of the cell membrane,is one of the essential components of the cell membrane,have to do with a series of membrane function.Phosphatidylserine(PS)is a glycerol derived phospholipid,which plays a key role in cell cycle signaling.PS has been used as targeting legend due to its ability to be recognized by the receptors including CD68,CD 14,annexins,?2 glycoprotein I and GAS6 and scavenger receptors.These receptors can play a vital role in recognition of PS coated nanoparticles and can effectively target tumar cells of kidney.Curcumin(CU),highly pleiotropic molecule,secluded from Curcuma longa(turmeric)rhizome,.has been proved to possess a wide spectrum of pharmacological actions,such as antitumor,antdiabetes,antioxidant,neutralizing free radicals,anti-inflammatory,lipid-lowering,anti-atherosclerosis,anticoagulation,anti-aging and etc.It has demonstrated anticarcinogenic potential,either alone or in combination with other agents,against colorectal cancer,prostate cancer,multiple myeloma,breast cancer,oral cancer and pancreatic cancer with a low intrinsic toxicity and also having positive effect on patients with renal conditions.It has been reported that curcumin may improve the function of pancreatic islet beta cells and reduce insulin resistance,thus prevent diabetes progression.In spite of these merits,CU suffers from its poor aqueous solubility,which in turn results in low bioavailability,thus makes a hurdle in the utility of it as a therapeutic.CU complexed with phospholipids and its liposomes has also been evaluated to improve its oral bioavailability.The nano carrier itself has very small particle size,takes advantage of the circulation in the body,prolongs the action time,enhances the drug concentration at targeted sites and increases drug aqueous solubility and stability to some extent.Therefore,a new goal is to combine curcumin and nanotechnology,which provides the possibility of curcumin delivery system for achieving better efficacy,so that curcumin in the clinical applications can be more effective.But there are also some problems,including a high concentrations of curcumin due to large dosage or via inappropriate nano-carriers,can result in increased ROS and oxidative stress,then impair DNA and reduce the activity of tumor suppressor gene p53,is also one of the factors that lead to cancer.Therefore,an appropriate nanotechnology of preparation of curcumin nanoparticles is so expected.Diabetic nephropathy(DN)is one of the most common severe complications of diabetes mellitus.The incidence of it in our country is also on the rise.Now,DN is the second cause of end-stage renal diseases(ESRD),only less than all kinds of glomerulonephritis.Because of the complex pathogenesis and metabolic disorders,once a patient with DN developes to ESRD,treatment is usually more difficult and intractable than other kidney diseases.Therefore,it is very important to delay the progression of diabetic nephropathy by prevention and treatment in time.The etiology and pathogenesis of diabetic nephropathy is not fully understood.Tthe main pathological feature of DN is that extracellular matrix(ECM)accumulates in the mesangial region,and the glomerular mesangial cell(GMCs)is the target cell of diabetic nephropathy.Oxidative stress induced by hyperglycemia is a common pathophysiological basis of complications in patients with diabetes mellitus.It is known that high glucose can lead to the increase of ROS production in the glomerular mesangial cells through the mitochondrial pathway and activation of NADPH oxidase aftet it comes into the glomerular mesangial cells.ROS is a signaling molecule of high glucose,activates a serial of of transcription factors in the glomerular mesangial cells,such as nuclear factor kappa B(NF-?B)and active protein 1(AP-1),which in turn increases the expression of cytokines,monocyte chemotactic protein 1(MCP-1),transforming growth factor-beta 1(TGF-?1)and PA-1,etc,eventually participates in the occurrence of renal tissue fibrosis.Therefore,inhibition of the accumulation of ROS within the glomerular mesangial cells in high glucose can potentially block the pathological effect of high glucose,which is of great significance.In this study we have prepared chitosan(CS)nanoparticles(NPs)loaded with CU(CNPs-CU)and PS coated CNPs loaded with CU(PS-CNPs-CU),analyzed characteristics and performance of CNPs-CU and PS-CNPs-CU(that is to evaluate their size,poly dispersity index,amount of drug entrapped,and in vitro CU release study),evaluated cytotoxicity of the prepared PS-CNPs-CU against human embryonic kidney cells and which has been compared with pure CU,the CNPs-CU.The further investigation for its genotoxicity was carried out in rats in order to prove the safety of prepared PS-CNPs-CU.Glomerular mesangial cells were cultured in glucose environment.Inhibition effect of CU,CNPs-CU and PS-CNPs-CU against the proliferation of GMCs,and related mechanisms were also analyzed.MethodsThe ionic gelation method allowed us to prepare uniform blank CNPs and CNPs-CU successfully,which is procedured between free amino group of CS and anion of tripolyphosphate sodium(TPP).The CNPs-CU and CNPs were coated with PS by hydrating the phospholipid film with CNPs-CU and CNPs dispersion.The average particle size,size distribution(PDI)and zeta potential of CNPs-CU,PS-CNPs-CU,blank CNPs and PS-CNPs have been determined by zetasizer 2000(Malvern Instruments)and the zeta potential was determined by laser Doppler anemometry using a Malvern Zetasizer.All the NPs were diluted with tri-distilled water(TDW)to an appropriate concentration.The measurements were carried out in the fully automatic mode.%encapsulation efficiency(%EE)is a function of total amount of CU loaded in the CNPs-CU and PS-CNPs-CU.The morphology of PS-CNPs-CU was observed using high-resolution transmission electron microscopy(HR-TEM).CNPs-CU and PS-CNPs-CU(equivalent of 10 mg CU)release was carried out using dialysis membrane(cut off mol.wt.12 KD).The amount of released CU was estimated using high performance liquid chromatography(HPLC).The in vitro cellular cytotoxicity of the prepared NPs were analyzed and compared with plain CU.The assessment was carried out by the MTT assay(calorimetric assay)on human embryonic kidney cell lines(HEK 293).The genotoxic potential of formulated CNPs-CU and PS-CNPs-CU were evaluated by short-term assays measuring aneugenicity and clastogenicity.Experiments have been carried out according to the reported procedures.The genotoxicity study was carried out by dividing the animals in four test groups;positive control(cyclophosphamide,40 mg/kg BW),CNPs-CU(having 100 mg/kg of CU equivalent dose),PS-CNPs-CU(having 100 mg/kg of Cu equivalent dose),control(having vehicle and distilled water).At least 1,000 polychromic erythrocytes(PCEs)were observed for the assessment of micronuclei for assessing the genotoxicity along with normochromic erythrocytes with or without micronucleus.One hundred well-spread metaphase cells were observed for chromosome aberration(CA)determination as a marker of genotoxicity.Glomerular mesangial cells(GMCs)were randomly divided in six groups:respectively high glucose control group(containing 30 mmol/L glucose in cell culture fluid and establishing a model of GMCs exposed to high glucose environment),curcumin group(Cu),CNPs group,PS-CNPs group,CNPs-CU group,and PS-CNPs-CU group.The other groups were equally cultured in high glucose conditions along with corresponding above-mentioned drugs(the concentration of curcumin is 10?g/ml,24h action).The changes of ROS content in the cells of each group were analyzed.Changes of glutathione activity and superoxide dismutase activity in the cells of each group were detected.NF-?B protein was detected in the cells of each group by Western Blot.Real time PCR assay was used to detect the changes of NF-?B mRNA,TNF-a mRNA,IL-1? mRNA and TGF-?1 mRNA expression in the cells of each group.The expression of NF-?B,TNF-a,IL-1? and TGF-?1 in the supernatant of each group was detected by ELISA.The proliferation of GMCs was analyzed by MTT.Results1.The mean diameter of CNPs and CNPs-CU were 99.6±2.15 nm and 167.6±3.53 nm,respectively with narrow size distributions.The polydispersity index(PDI)of the both was 0.072±0.02 and 0.115±0.014.It was showed the positive zeta potential of the both,19.9± 1.27 mV and 21.9±1.31 mV.2.The mean diameter of PS-CNPs and PS-CNPs-CU were 176.7±2.21 nm and 220.5±3.67 nm,also respectively with narrow size distributions.PDI of the both was 0.124±0.016 and 0.14810.019.Whereas it was showed the negative zeta potential of the both,-19.9±1.33 mV and-25.4±2.13 mV.3.%EE in PS-CNPs-CU was found to be 50.4±3.7%,which was less when compared CNPs-CU(59.4±3.1%).But the difference was not statistically significant.4.The morphological characteristics of PS-CNPs-CU was observed using high-resolution transmission electron microscopy(HR-TEM).PS-CNPs-CU were spherical and have a more regular shape.lipidic layering was observed as a continuous layer over CNPs.5.The amount of CU released at pH 7.4 was 31.82%and 22.64%for CU-CNPs and PS-CNPs after 2h at 37?.CU released in the next 12 h was 81.82%and 73.77%from CNPs-CU and PS-CNPs-CU respectively.After 24 h the release was found to be 94.17%and 83.94%for CNPs-CU and PS-CNPs-CU respectively.This data indicates that formulation retard CU release in the medium.6.The cell cytotoxicity remained<5%for both CNPs and PS-CNPs against HEK 293 cell lines at all equivalent concentrations.The cell cytotoxicity at higher equivalent concentrations(1?g/ml,5?g/ml and 10?gg/ml)of PS-CNPs-CU was more than CNPs-CU,but it was not significantly different.7.The administration of CNPs-CU and PS-CNPs-CU did not result in any significant increase in the rate of micronucleated cells,in the animals,when compared with the cyclophosphamide treated mice.However a significant increase in(P<0.001)of micronucleated cells were observable in the animals treated with cyclophosphamide.CNPs-CU and PS-CNPs-CU were comparable with the control,which were non-clastogenic..8.The administration of CNPs-CU and PS-CNPs-CU did not induce aberrations and were comparable to control.Cyclophosphamide treated mice,however produced a significant number(P<0.001)of aberrations.9.All the CU-containing group could reduce the production of ROS in GMCs under high glucose,the difference was statistically significant(P<0.05)while compared with the control group.And CNPs-CU group and PS-CNPs-CU group could further reduce the production of ROS,the difference was statistically significant(P<0.05)while compared with the CU group,which PS-CNPs-CU group could reduce more significantly(P<0.01).10.All the CU-containing group could increase the activity of GSH and SOD in GMCs under high glucose,the difference was statistically significant(P<0.05)while compared with the control group.And CNPs-CU group and PS-CNPs-CU group could further increase the activity of SOD and GSH,the difference was statistically significant(P<0.05)while compared with the CU group,which PS-CNPs-CU group could increase more significantly(P<0.01).11.All the CU-containing group could inhibit the expression of NF-?B mRNA and protein in GMCs under high glucose,the difference was statistically significant(P<0.05)while compared with the control group.And CNPs-CU group and PS-CNPs-CU group could further inhibit the expression of NF-?B mRNA and protein,the difference was statistically significant(P<0.05)while compared with the CU group,which PS-CNPs-CU group could inhibit more significantly(P<0.01).12.All the CU-containing group could inhibit the expression of TNF-a mRNA,IL-1?mRNA,and TGF-?1 mRNA in GMCs under high glucose,the difference was statistically significant(P<0.05)while compared with the control group.And CNPs-CU group and PS-CNPs-CU group could further inhibit the expression of TNF-a mRNA,IL-1? mRNA,and TGF-?1 mRNA,the difference was statistically significant(P<0.05)while compared with the CU group,which PS-CNPs-CU group could inhibit more significantly(P<0.01).13.All the CU-containing group could inhibit the secretion of TNF-?,IL-1?,and TGF-?1 in the supernatant of GMCs under high glucose,the difference was statistically significant(P<0.05)while compared with the control group.And CNPs-CU group and PS-CNPs-CU group could further inhibit the secretion of TNF-a,IL-1?,and TGF-?1,the difference was statistically significant(P<0.05)while compared with the CU group,which PS-CNPs-CU group could inhibit more significantly(P<0.01).14.All the CU-containine group could inhibit the proliferation of GMCs under high glucose,the difference was statistically significant(P<0.05)while compared with the control group.And CNPs-CU group and PS-CNPs-CU group could further inhibit the proliferation of GMCs,the difference was statistically significant(P<0.05)while compared with the CU group,which PS-CNPs-CU group could inhibit more significantly(P<0.01).Conclusions1.CNPs-CU and PS-CNPs-CU were successfully prepared by ionic gelation method,CNPs-CU were coated with PS by hydrating the phospholipid film.2.CNPs-CU and PS-CNPs-CU achieved sustained or controlled release,and then provided a new method and reference for developing new nanoparticles for disease treatment.3.PS-CNPs-CU or CNPs-CU has low cytotoxicity and genotoxicity,which may be safe and reliable as a drug delivery carrier.4 CU could inhibit the proliferation of GMCs in high glucose environment by regulating oxidative stress reaction and inhibiting the inflammatory factors,which is involved in the regulation of the occurrence and development of diabetic nephropathy.5 PS-CNPs-CU could intensify the inhibitory effect of CU on the proliferation of GMCs,is expected to be an ideal targeting drug carrier for diabetic nephropathy. |
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