Effect Of Apatite Nanoparticle-mediated Intracellular Delivery Of Trehalose On The Survival Rate Of Frozen Cells

BackgroundCryopreservation of tissue cells is an important method to maintain cell viability and cellular function.However,cell viability and function are less than ideal by conventional cell cryopreservation methods,which may result in apoptosis and necrosis of cells in cryopreservation.Trehalose plays a role in maintaining cell structure and protecting cells from stress.However,owing to the difficulty in transport of trehalose across the cell membrane,its antifreeze effect is limited.In recent years,many methods have been explored for introducing trehalose into mammalian cells.Among them,apatite nanoparticles are an ideal mediator,which has little effect on cell activity and function,and can provide a sufficient amount of trehalose to cells.Objective?1?Explore the biological evaluation of apatite nanoparticles,including cytotoxicity evaluation based on apatite nanoparticles,effects of apatite nanoparticles and trehalose on cell viability.?2?Explore the process by which apatite nanoparticles mediate the entry of trehalose into cells and the amount and loading efficiency of trehalose into tissue cells.?3?Explore the effects of intracellular trehalose on cell viability and cell function after cryopreservation.Methods and results?1?The study used human aortic vascular smooth muscle cells?HA-VSMC?and human coronary artery endothelial cells?HCAEC?as experimental materials,and the cytotoxicity of NP to both cells was evaluated by CCK-8 test.Five different concentrations of NP in the test medium were tested:cell viability at 0,0.5,1,2,4 and 10 mg/mL and cell viability at different contact times?1 to 7 days?between the nanoparticles and the cells.The results showed that the cell survival rate was close to 100%for the NP concentration in the medium between 0 and 1 mg/mL.During the same incubation time,as the NP concentration increased,the cell viability decreased,and the results showed a significant concentration dependence.In the experiments herein,the optimal concentration was 1 mg/ml.?2?The process of NP-mediated trehalose entry into cells was demonstrated by fluorescent dyes?CFSE and PI?.We used trehalose?0-200 mM?and apatite?1 mg/mL?in a medium for 6 hours to calculate the loading efficiency of trehalose.The results showed that the NP+PI group showed significant red fluorescence in 1 hour or 6 hours.The intracellular trehalose concentration is greater than the extracellular trehalose concentration?up to 237±8.5 mM?,and trehalose is efficiently delivered intracellularly with the help of nanoparticles?NP?with a loading efficiency of up to 137.3±34.5%.?3?The cell viability of the frozen cells was measured by fluorescence microscopy imaging and flow cytometry,and the cell function of the frozen cells was measured using the CCK-8 method.The results showed that the addition of apatite NP to the medium significantly increased the aortic smooth muscle cell cryopreservation rate,up to 83.6%?30%improvement compared to the NP-free control?,at levels comparable to the traditional Me₂SO cryoprotection regimen.In the detection of cell function after cryopreservation,the cryopreservation adhesion efficiency of the NP+Tre group was higher?69.6±2.9%?,and the results were satisfactory.ConclusionBased on these result we conclude that:?1?Apatite nanoparticles have high biocompatibility and low cytotoxicity and are suitable as mediators for mediating trehalose into tissue cells.?2?The apatite nanoparticles can mediate a sufficient amount of trehalose into the tissue cells in a relatively short period of time.?3?Apatite nanoparticles mediate intracellular delivery of trehalose can increase the survival of cryopreserved cells.This method provides a new option to enhance the activity of valvular cells for cryopreservation.