Nanoparticle Nucleic Acid Delivery Function And Safety Application Evaluation Study

Introduction and Objective:RNA interference(RNAi)is emerging as one of promising strategies for treatment of human gene related diseases,especially for anti-tumor therapy.Up to now,the mechanism of RNAi is very clear and large numbers of nucleic acid drugs(siRNA,shRNA,miRNA and dsiRNA drugs)and delivery systems(virus,liposome,cationized polymer and metal nanoparticles)have attracted much attention from the public and scientists.Nucleic acid carriers are generally divided into two major types,viral vectors and non-viral vectors.Viral vectors have been proven to be an effective gene delivery system.However,some challenges have restricted their application,such as,their limited gene packing capacity,intrinsic immunogenicity and their potential side effects.Non-viral vector is safe and stable,but the transfection efficiency is lower than that of virus.Thus,safe and efficient non-virus delivery systems are still lack,which has been one of the major challenges for RNAi application.Gelatin derived from animal skin and bone has been an important biomaterial for drug delivery due to the features of biodegradation,biocompatibility and easy chemical modification.The main line of our study is synthesizing cationized gelatin and preparing homogeneously cationized gelatin nanoparticles(CGN),aiming to protect nucleic acid from degradation by nuclease and prolong drugs action time by the controlled release manner of CGN.Meanwhile,we also evaluated the potential effect factors that were associated with the interfering efficiency of CXCR4 siRNA carried by CGN,which includes crosslinking density,serum concentration and tumor cell types.Methods:We synthesized cationized gelatin by introducing ethylenediamine molecular into gelatin and prepared the cationized gelatin nanoparticles with different crosslinking concentration of glutaraldehyde by an improved two-step desolvation method.The uncationzied gelatin nanoparticles(GN)are used as control.The morphology and size of CGN were characterized by scanning electron microscope(SEM)and atomic force microscopy(AFM).Besides,the hydrodynamic size and Zeta potential were measured by dynamic light scattering(DLS).1%agarose gel electrophoresis assay was used to estimate the binding of CGN with siRNA and DLS was used to evaluate the hydrodynamic size and Zeta potential of CGN and siRNA complex(CGN@siRNA).CCK8 assay was used to detected the toxicity of CG,CGN and CGN@siRNA in mouse breast cancer cells 4T1.The cell uptake of CGN@siRNA was determined through flow cytometry and the location of CGN@siRNA in cells was observed by the laser confocal scanning microscopy.Real Time PCR assay was used to detect the level of CXCR4 expression in different tumor cells induced by CGN@siCXCR4 and we estimated the influence rule of crosslinking density and serum concentration on interfering efficiency.Results:(1)We prapared five kinds of CGN with different crosslinking concentrations.SEM micrographs showed that CGNs were homogeneously spherical.With increasing of crosslinking density.the particle size was 488 ± 103.17,412 ± 67.93.328 ± 68.34.297 ±64.80 and 280 ± 72.08 nm.The hydrodynamic diameter was 652± 10.61.533 ± 2.83,375 ±7.07,367 ± 7.08 and 358 ± 3.54 nm.CGN was positively charged with Zeta potential zabout+40 mV.(2)Results obtained from agarose gel electrophoresis assay showed that CGN could bind to siRNA completely when the mass ratio was four to one.(3)The CCK8 assay stated that the cytotoxicity of these particles in 4T1 cells had concentration and size effect.GN and CGN@siRNA complex were less harmful to cells than CGN.In addition,low serum concentration in medium increased cytotoxicity.(4)Flow cytometry analysis showed that CGN could deliver siRNA into 4T1 cells efficiently.High crosslinking density and low serum concentration were beneficial to cellular uptake of CGN@siRNA.The cellular uptake was different in various cells.(5)The laser confocal fluorescence microscopy observed that the CGN@siRNA was located in the endosome and lysosome.(6)The CGN@siCXCR4 could down regulate the level of CXCR4 expression in 4T1 cells and the efficiency was closer to commercial carrier lipofectamine 3000.The CXCR4 silencing effect could be prolonged to 96 h and was related to crosslinking density.Decreasing serum concentration in medium could increase the interfering efficiency.In addition,CGN@siCXCR4 was also effective in other tumor cells.Conclusion:CGN could induce the RNAi of CXCR4 in various tumor cells and prolong the action time by controlled release of siRNA,suggesting a promising siRNA carrier for anti-tumor therapy.Objective:Silver nanoparticles(AgNPs)have become increasingly prevalent as antibacterial agent in many fields including food,agriculture,household appliances,textiles,biomedical devices and environmental protection due to their more potent antimicrobial performance than that of conventional silver compounds.With the increased exposure to AgNPs-containing products,the risk of health,safety and environment has attracted much attention from the public and scientists.AgNPs have been found to inhibit cell growth or even kill cells in vitro by interaction with biology membrane,inducing intercellular oxidative stress reaction,causing DNA damage or releasing Ag ions.At present,a large number of studies report that silver nanoparticles could enter organisms through exposure methods such as skin exposure,oral,inhalation or intravenous exposure and induce toxicity with distributing to most major targets organs,especially to liver,lung,kidney and spleen.However,the studies about the interaction between AgNPs and vascular endothelial cells are still absent and the precise differences in the mechanisms between AgNPs and silver ions also remain elusive.In this study,we evaluated the effects of AgNPs and AgNO3 on vascular endothelial cell junction and compared their mechanisms in vitro cytotoxicity in HUVEC.In addition,we confirm their in vivo toxicity to the blood vessel walls of the liver,kidneys,and lungs of Balb/c mice following intravenous administration.Methods:Three different diameters of AgNPs(10.75、110nm)used in our study were coated with 2mM citrate and silver nitrate(AgNO3)used as control.The physicochemical property of AgNPs was characterized by SEM and DLS.In the in vitro study,we investigated the cytotoxicity of either AgNPs or AgNO3 following 24 h incubations in the presence of primary human umbilical vein endothelial cells(HUVEC)by CCK8 assay and evaluated the apoptosis or necrosis.of cells through Hoechst/PI staining.The cellular uptake and location of AgNPs were determined by ICP-MS analysis and TEM observation.Meanwhile,we detected the effects of AgNPs on intercellular conjunction and intracellular ROS by VE-cadherin staining and 2’,7’-dichlorodihydrofluorescein diacetate(DCFH-DA)assay.In the in vivo study,we used multiple intravenous injections to expose AgNPs or AgNO3 to mice and evaluated the toxicity of AgNPs or AgNO3 and estimated the location of AgNPs by histological analysis of tissue section and TEM observation.Results:(1)TEM micrographs showed that AgNPs are homogeneously spherical and the particle size for AgNP-10,AgNP-75 and AgNP-110 was detected as 11±1.76±6 and 107±8 nm.Three kinds of AgNPs were equally well dispersed in water,cell culture medium containing 10%serum and 5%isotonic glucose solution.The average hydrodynamic diameter for AgNP-10,AgNP-75 and AgNP-110 was 6.2±1.6,72.0±0.9,99.3±1.7 nm in water,10.7±3.3,78.9±0.9,116.8±2.6 nm in 5%isotonic glucose solution and 12.6 ± 3.9,104.6 ± 0.2,147.0 ± 1.7 nm in cell culture medium.Their surface were negatively charged with Zeta potential about-42,3 ± 1.4,-45.5 ± 0.7,-44,5 ± 0.4 mV in water,about 35.7 ± 0.1,38.2 ± 1.1.37.7 ± 0.6 mV in 5%isotonic glucose solution and about-7.9 ± 0.5,-8.0 ± 1.1,-7.2 ± 1.0 mV in cell culture madium.(2)The cytotoxicity of AgNPs was concentration dependent and the larger size was more harmful to HUVEC cells.AgNPs induced apoptosis while AgNO3 caused direct cell death.(4)The results obtained from ICP-MS analysis and TEM micrographs showed that AgNPs were taken up by endothelial cells and mainly located in endosome while AgNO3 was taken up little.(5)AgNPs incubation induced the elevation of intracellular ROS.They also could induce down-regulation of VE-cadherin between the endothelial cells and affect the cytoskeleton actin reorganization,which could be rescued by antioxidant N-acetylcysteine.(2)After muti-dose administration of AgNPs,inflammatory infiltrates were observed in the vessel walls of liver,kidney,and lung.TEM observation showed that AgNPs were located nearby microvascular structures of liver.Conclusion:AgNPs could be taken up by vascular endothelial cells while AgNO3 was taken up little.AgNPs could induce the elevation of intracellular ROS in HUVEC,which attributed to disruption the integrity of endothelial layer.The AgNps-induced leakiness of endothelial cells could attract immune cells in liver,lung and kidney and mediate the common peripheral inflammation.In contrary,AgNO3 caused direct cell death.