| Cell therapy is an emerging technology for disease treatment in recent years,it can enhance body immunity,kill pathogens and tumor cells,promote tissue regeneration and body rehabilitation.Cell therapy usually requires a large number of cells,and in vitro expansion culture is one of the commonly used methods to obtain cells in a larger quantity.However,the senescence of cells in the in vitro expansion process greatly hinders the clinical application of the cell therapy.Therefore,inhibition of cell senescence during the in vitro expansion is extremely important.Resveratrol is a polyphenol compound with good antioxidant and free radical scavenging capacity,which is the basis for its biological activity.Besides,resveratrol is an activator of protein deacetylase sirtuin1(Sirt1),which regulates aging through metabolism peroxisome proliferator-activated receptor ?coactivator-1(Pgc-1).However,it has poor chemical stability,thus being easily oxidized and lose its bioavailability.To improve its bioavailability,a suitable nano-carrier is of critical importance.Metal-organic frameworks(MOFs)are a new type of hybrid materials composed of metal ions and organic ligands.They have a large surface area,adjustable pore size,and biodegradability,opening the possibility for efficient loading of drugs.ZIF-8 is a class of metal-organic framework materials with a zeolite framework structure.It not only has the characteristics of MOFs(such as adjustable composition and structure),but also has high water stability due to its zeolite-like structure.So,ZIF-8 is a potential drug delivery material.In this study,ZIF-8 nanoparticles were used as a carrier of resveratrol to construct a nano-drug delivery system to inhibit the cell senescence in the in vitro expansion process.The main work and conclusions are as follows:1.Zinc nitrate hexahydrate and 2-methylimidazole were used as raw materials,water or methanol were used as media for the ZIF-8 synthesis.In the case of waterbased reaction system,the surfactant of cetyltrimethylammonium bromide(CTAB)and the capping agent((trimethylol)amino methane(TRIS))were used to control the size and morphology of the ZIF-8.The morphology and particle size of the as-prepared ZIF-8 nanoparticles were characterized by X-ray diffractometer(XRD),scanning electron microscope(SEM),transmission electron microscope(TEM).The results show that ZIF-8 prepared in a methanol system presents a uniform polyhedron shape with a size of about 30 nm.When water is used as a medium,ZIF-8 nanoparticles with a petal shape,Luban-lock shape,spherical shape,polyhedron shape,needle rod shape,and tetrahedron shape can be produced by modulation of the pH of the reaction solution and the concentration of surfactants(CTAB)and TRIS.The size of the nanoparticles are as small as 90 nm and as large as 700 nm.2.A solvent method was used to self-assemble Zn ions and 2-methylimidazole to generate ZIF-8 nanoparticles.An adsorption method was used to load resveratrol in the ZIF nanoparticle to build resveratrol/ZIF drug delivery system(Resveratrol@ZIF-8).The effects of resveratrol concentration and loading time on the drug loading efficiency were studied.The Resveratrol@ZIF-8 system was systematically characterized by Xray diffractometer(XRD),scanning electron microscope(SEM),transmission electron microscope(TEM),Fourier infrared spectrometer(FTIR),ultraviolet-visible spectrometer(UV-Vis),Zeta potentiometer,and nitrogen adsorption meter.It was demonstrated that the geometrical and crystalline structures were not compromised during the drug-loading process.It was also revealed that the resveratrol was successfully loaded in ZIF-8 nanoparticles with a loading rate of around 15.3%.The drug release studies proved that the drug-loaded Resveratrol@ZIF-8 system has better stability in neutral and alkaline conditions,and thus showing a low drug release efficiency.By contrast,ZIF-8 undergoes faster degradation in an acidic environment and thus presenting a relatively faster and sustainable release profile.3.To obtain senescent cells,mouse bone marrow mesenchymal stem cells(BMSC)were cultured under the simulation of hydrogen peroxide.The effects of experimental conditions such as hydrogen peroxide concentration and stimulation time on cell senescence were explored.Cell viability test,b-gal staining staining,and reactive oxygen species(ROS)quantitative detection were used to characterize cell senescence behavior.The optimal stimulation time and concentration established for the building of the in vitro cell senescence model were 3 h and 200 ?M,respectively.4.The effect of ZIF-8,Resveratrol,and Resveratrol@ZIF-8 on the proliferation of senescent bone marrow mesenchymal stem cells(S-BMSCs)was studied using the CCK8 method.The results show that the cell co-cultured with the Resveratrol@ZIF-8 has the highest proliferation rate,indicating that our drug delivery system can promote the growth of S-BMSC.BMSCs in the logarithmic growth phase were co-cultured with hydrogen peroxide,hydrogen peroxide + resveratrol,hydrogen peroxide + resveratrol@ZIF-8,and the cell proliferation and osteogenic activity(ALP and osteogenic genes expression)were evaluated.The results show that the resveratrolloaded ZIF-8 has a certain effect on the rejuvenation of the senescent BMSC,which will be furthered validated in our future studies.In summary,we successfully prepared ZIF-8 nanoparticles with which a drugloading system for resveratrol delivery(Resveratrol@ZIF-8)was constructed.The drug-loading efficiency was around 15.3%.It was proved that ZIF-8 not only protects the biological activity of resveratrol,but also can release the drug in a pH-dependent manner.The results of the in vitro cell experiments confirmed that the Resveratrol@ZIF-8 system inhibits,to some degree,the cell senescence in the cell expansion culture process and has a certain effect on the rejuvenation of the senescent BMSC.Our work provides new ideas for resolving the shortcomings of resveratrol,such as poor chemical stability,proneness to oxidation,and low bioavailability. |
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