Objective To explore the feasibility of the application of yak pericardium as anti-calcified heart valve materials in transcatheter aortic valve replacement (TAVR). Cattle pericardium and commercial Sino products were used as controls. Methods Yak and cattle pericardium was decellularized, and then the acellular extent was qualitatively and quantitatively analyzed with hematoxylin-eosin (HE) staining and DNA detection kit respectively. Two cross-linking agents, i.e. glutaraldehyde and genipin, were used to cross-link the acellular pericardium, respectively. After testing the mechanical property, thermodynamic stability and biocompatibility of the pericardium, the cross-linked pericardium was subcutaneously implanted in juvenile Wistar rats. After 2～4 weeks of the implantation, histological staining and tissue calcification analysis were conducted. Results The nuclei of the yak pericardium were almost invisible after decellularized treatment, and the DNA content decreased from (0.90±0.13)μg/mg to (0.09± 0.02)μg/mg that was significantly lower than that of Sino product (P<0.001). The mechanical strength of the acellular yak pericardium, glutaraldehyde or genipin cross-linked yak pericardium was higher than that of the cattle pericardium (all P<0.05). The maximum stress value, obtained in the glutaraldehyde cross-linked yak pericardium, was (8.44±2.61) MPa, which was higher than (7.92±1.81) MPa of the Sino product (P<0.05). The shrinkage temperature of non- and cross-linked yak pericardium was slightly higher than that of cattle pericardium, but difference was not statistically significant (all P>0.05). For the two cross-linked yak pericardium, the hemolysis rate and cell proliferation ratio was similar with that of cattle pericardium (all P>0.05), the tissue regeneration ability of subcutaneous implantations was inferior, and the calcification level was higher than that of cattle pericardium with an no statistically significant difference (all P>0.05). After 4 weeks of the subcutaneously implantation, the calcium content of the glutaraldehyde cross-linked pericardium was 32.62～65.49μg/mg. Conclusions Yak pericardium has better mechanical properties and thermodynamic stability than cattle pericardium, and the biocompatibility can meet the requirements of transcatheter aortic valve replacement.

Objective To explore a new method for synthesizing arginine modified chitosan ( AC ) with mono-arginine substitution and high degree of substitution, and to evaluate the biological effect of AC as gene carriers. Methods The single arginine modified chitosan (sAC) was synthesized by means of protecting and de-protecting the arginine amino group before and after chitosan modified arginine reaction. Liu's arginine-modified chitosan ( LAC ) was prepared according to the methods reported in the literature . The conjunction of arginine to chitosan was detected by infrared spectroscopy and nuclear magnetic resonance spectroscopy. Three kinds of chitosan gene nanoparticles were respectively prepared by complex coacervation and characterized, including sAC gene nanoparticles (sACGNs), the LAC gene nanoparticles (LACGNs) and the chitosan gene nanoparticles (CGNs). A10 rat vascular smooth cells transfected with sACGNs were used to estimate the in vitro cellular uptake, internalization mechanisms and transfection efficiency. Thiazolyl blue tetrazolium blue (MTT) assay was used to measure the cytotoxicity. Results The infrared spectrum analysis confirmed that sAC was obtained via the conjunction of arginine to chitosan. Nuclear magnetic resonance spectrum analysis showed that the degree of substitution of arginine in sAC and LAC was 21.3％and 6.4％, respectively. When the ratio of nitrogen to phosphorus (N/P ratio) was 2:1, the particle sizes of CGN, LACGN, and sACGN were (94.81±2.93) nm, (124.53±2.55) nm, and (128.53±2.04) nm, respectively, and the Zeta potentials were (3.50±1.61) mV, (5.74±0.41) mV, and (6.04±1.39) mV, respectively. For the cellular uptake, CGNs were mainly through the clathrin-mediated endocytic pathway, and LACGNs and sACGN were mainly through the caveolin-mediated endocytic pathway. Compared with CGNs, LACGNs and sACGNs showed higher cellular uptake and transfection efficiency , and the differences were statistically significant ( all P<0 . 05 ) . sACGNs achieved the highest transfection efficiency in the near-neutral pH environment. MTT results showed that when the mass concentration of sACGNs reached 100μg/ml, the survival rate of A10 cells was still higher than 90％, indicating the non-cytotoxicity of sACGNs. Conclusion The new method successfully synthesized single arginine-modified chitosan. As a gene carrier, sACGNs show higher gene transfection efficiency and lower cytotoxicity than CGNs and LACGNs in near neutral pH environment.

Most of the anticancer drugs have some limitations in clinical application,such as poor solubility,low targeting and cytotoxicity to normal tissue and organ.The application of drug carriers offers a solution of these problems to a certain extent.In recent years,some materials such as polymers,liposomes,carbon nanotubes (CNTs) were used as carriers of anticancer drugs.The utilization of these carriers improved drug targeting and reduced adverse reactions by targeted modification of carriers which ensured the slow release of the drugs and maintained the plasma concentration.In these carriers,CNTs,as a novel nano-material,have attracted more attention in nanomedical applications.CNTs not only possess nanoscaled diameter,hollow structure and large aspect ratio,resulting in large drug capacity,but also can selectively absorb near infrared lights and transform them into thermal energy,according to the research finding.The functionalized drug-loaded CNTs in combination with thermotherapy shows potential,which is expected to become a new targeting therapy of cancer.In this paper,the basic structure of CNTs,the application of CNTs as drug carriers,and the recent development of functionalized CNTs as drug carriers combined with thermotherapy in tumor therapy were summarized.

Objective To explore the effects of surface functionalized multi-walled carbon nanotubes (FMWCNTs) on the cytotoxicity of human peripheral blood mononuclear cell (PBMC).Methods Five different types of MWCNTs (hydroxylated,carboxylated,aminated,nickel-plated and pristine MWCNTs (P-MWCNTs)) with the same diameter and length were evaluated the dispersion and characterizations in physiological salt solution by transmission electron microscopy.PBMC were isolated by density gradient centrifugation from human peripheral blood,and 5 types of MWCNTs were ultrasonically dispersed in serum-containing medium respectively.After incubation with PBMC for 12,24,48 or 72 h,cytotoxicity was detected by CCK-8 kits.Results All the MWCNTs had well dispersion,especially the F-MWCNTs.Cytotoxicity results showed that all types of MWCNTs could induced PBMC death,and presented dose-dependence manner and a certain degree of time-dependence manner.Compared with the P-MWCNTs,F-MWCNTs changed cytotoxicity statistically,with the hydroxylated,carboxylated,aminated MWCNTs weakened,aminated MWCNTs significant (P＜0.05),nevertheless the nickel-plated MWCNTs increased.Compared with the P-MWCNTs (25 μg/ml),cell viability of PBMC after 24 and 48 h incubation with the same dose of nickelplated MWCNTs both decreased,and the differences was statistically significant (P＜0.01,P＜0.05).Conclusions The functional group modification affects not only the MWCNTs dispersion in medium,but also the cytotoxicity of the MWCNTs on PBMC.

Objective To prepare stable aqueous dispersions of chitosan/multi-walled carbon nanotubes (CS/MWCNTs) composites,and observe the effects of CS/MWCNTs on the growth of human umbilical vein endothelial cells (HUVEC).Methods CS/MWCNTs composites were prepared by electrostatic interactions between negatively charged MWCNTs and positively charged low-molecular-weight CS.The prepared CS/MWCNTs were characterized by transmission electron microscopy and Zetasizer nano-analyser.The cellular uptake of the fluorescently labeled CS/MWCNTs was observed by laser confocal microscopy after incubating with HUVEC for 24 h at different concentrations.In vitro cytotoxicity and cellular reactive oxygen were also detected.Results When the mass ratio of low-molecular-weight CS to MWCNTs was equal or greater than 10∶1,the CS/MWCNTs can be stabilized in solution.Cellular uptake experiments showed that the CS/MWCNTs could enter into the cells and locate mainly in the cytoplasm.Cytotoxicity study showed that the CS/MWCNTs composites was less toxic than MWCNTs alone at high concentration (10 and 20 μg/ml).However,there was no significant differencein the level of cellular reactive oxygen between the two groups (P＜0.05).Conclusions CS/MWCNTs composites showed low cytotoxicity and high stability,which would be a promising carrier for drug delivery.

Objective To detect the size distribution and Zeta potential of LHRH-MPG△NLS/CDK-siRNA nanoparticles,to observe the effect of different solvents on the nanoparticle size,and to investigate the inhibitory effect of nanoparticles on HepG2 cell growth.Methods LHRH-MPG △NLS and CDK2-siRNA were mixed by continuous stirring to form nanoparticles at different N/P ratios (10/1,20/1 and 40/1).The size distribution and Zeta potential of LHRH-MPG△NLS/CDK2-siRNA nanoparticles were detected by dynamic light scattering,and the stability of the nanoparticles in normal saline,10％ glucose and pure water was discussed.Finally,the inhibitory effect of the nanoparticles on HepG2 cells was determined by CCK8 kit.Results The mean size of the nanoparticles was within 200 nm,and the Zeta potentials were (70±5) mV (N/P=10/1),(120±5) mV (N/P=20/1) and (130±5) mV (N/P=40/1),respectively.The size of the nanoparticles in normal saline was significantly increased,which demonstrated that strong electrolytes had a great impact on the nanoparticles size.When nanoparticle concentration was 200 nmol/L,LHRH-MPG△NLS/CDK2-siRNA nanoparticles (N/P=10/1) showed significantly inhibitory effect on HepG2 cell growth.Conclusions The mean size of the LHRH-MPG△NLS/CDK2-siRNA nanoparticles was within 200 nm,which was ideal for cellular uptake.The Zeta potential of nanoparticles revealed that nanoparticles could be stable in aqueous solution,while strong electrolytes would affect nanoparticle size.When nanoparticle concentration was 200 nmol/L,LHRH-MPG△NLS/CDK2-siRNA nanoparticles (N/P=10/1) showed significantly inhibitory effect on HepG2 cell growth.

ObjectiveTo isolate and identify endothelial progenitor cells (EPCs) from human umbilical cord,and to study the cell proliferation and gene transfection of green fluorescent protein plasmid in vitro.MethodsEPCs were isolated from human umbilical cord in enzyme digestion method.The biological characteristics of EPCs were identified by flow cytometry and laser confocal microscope.The enhanced green fluorescent protein (EGFP) gene transfection mediated by EPCs was investigated using Lipofectamine 2000 as transfection reagent.ResultsEndothelial progenitor cells isolated from umbilical cord formed typical endothelial cell colony 9 days later.These cellsdisplayed an improved positive expression of CD133 and kinase insert domain receptor (KDR).The endotheliallineage characteristics of expanded cells were confirmed by fluorescein isothiocyanate (FITC)-UEA-1 binding and DiI-ac-LDL uptake assay with the aid of laser confocal microscope.The transfection results demonstrated high expression of EGFP taking EPCs as host cell.ConclusionEndothelial progenitor cells isolated from umbilical cord can be propagated and induced to differentiate into endothelial cells in the appropriate culture conditions.EPCs demonstrated to be an ideal carrier for gene and cell therapy.

ObjectiveConjugation of fluorescent dye onto plasmid DNA was investigated in order to monitor delivery process of plasmid DNA.MethodsPlasmid was activated with bromine,stored for different timeintervals at 4 ℃ or room temperature,and subsequently coupled with 1,10-diaminodecane to prepare aminemodified plasmid DNA.Amine-modified plasmid was then reacted with isothiocyanate (FITC) for fluorescent labeling,and the labeling ratio was calculated after purification.The effect of storage conditions (time/temperature) of bromine-actived plasmid (BP) on fluorescent labeling efficacy was estimated,and the cell transfection efficiency of fluorescent plasmid-lipofectamine complex was observed.The fluorescent plasmid delivered by lipofectamine 2000 in A10 cells was observed by laser scanning confocal microscope (LSCM) and flow cytometry.ResultsThe experimental data showed that prolonged storage time of bromine-activated DNA had a negative effect on the labeling ratio,and lower storage temperature had a positive effect on the labeling ratio.It also demonstrated that FITC modification had no effect on the transfection efficiency of plasmid-lipofectamine complex as compared with that of unlabeled plasmid-lipofectamine complex,and FITC modified plasmid had enough fluorescent intensity to monitor cell uptake with flow cytometer and sub-cellular distribution with LSCM.ConclusionA facile method for conjugating fluorescent dye onto plasmid was established in the study,and could be utilized to trace the plasmid delivery for investigating the transfection mechanism.

ObjectiveTo explore the endocytic pathway of TAT-LHRH modified chitosan/DNA nanoparticle (TLCDN) that exhibits high transfection efficiency and targeting to HepG2.MethodsPlasmid DNA was labeled with fluorescein,and the resulting fluorescent DNA was complexed with chitosan or TAT-LHRH modified chitosan to form chitosan/DNA nanoparticle (CDN) and TLCDN by the complex coacervation method.Internalization of TLCDN or CDN by HepG2 cells were measured in the presence of three kinds of inhibitors of endocytic pathway,Chlorpromazine,Filipin or Dynasore,using High-Content Analyzer to collect and analyze the data.ResultsChlorpromazine led to more decreased uptake of CDN than that of TLCDN,although not statistically significant.Filipin demonstrated significant inhibitory effect on the uptake of TLCDN while promoted the uptake of CDN.Dynasore resulted in a similar decrease in the uptake of both nanoprticles.ConclusionIt was demonstrated that CDN was taken up by HepG2 cells mainly through the clathrin-dependent endocytic pathway and TLCDN was more likely to be internalized by HepG2 cells through the caveolin-mediated endocytic pathway although the clathrin-dependent endocytic pathway was also involved.

ObjectiveTo investigate the cytotoxicity and gene transfection mediated by NMPCS/DNA nanoparticles.MethodsN-methylene phosphonic chitosan (NMPCS) was synthesized using one-step reaction under homogeneous conditions.The NMPCS/DNA nanoparticles were prepared using complex coacervation method.The cytotoxicity of NMPCS alone and its complexes with plasmid DNA were determined by MTT assay on HeLa cells.The gene transfection mediated by NMPCS/DNA nanoparticles were investigated using pGL3control vector as reporter gene.ResultsThe MTT results suggested that the NMPCS and NMPCS/DNA complexes showed significantly lower cytotoxicity than PEI and PEI/DNA complexes,respectively.The gene transfection mediated by NMPCS/DNA nanoparticles were greatly improved compared with unmodified chitosan.ConclusionNMPCS would demonstrate great potential as a novel,safe,efficient non-viral vector for gene delivery.