The Experimental Study Of The Hypocrellin Modified Nano-lipid Microbubbles With LED Light On Human Tongue Carcinoma Tca8113Cells

Objective:1.The modified hypocrellinsB nano lipid microbubbles was made bylaboratory, then determine its Particle size、electric potential、encapsulationefficiency and And other physical properties.2. Conventionally cultured human tongue carcinoma, Observe thelaw of absorption and metabolism of the modified hypocrellinsB nano lipidmicrobubbles in the human tongue carcinoma, choose the best time, thenobserve the cells with the LED light.Methods:Part1:1.production of microbubbles The hypocrellin modifiednano-lipid microbubbles was made by mechanical vibration inlaboratory,then determine its Particle size、electric potential、encapsulationefficiency byMalvin laser particle size analyzer、Malvern surface potentialdetector and UV-2102PCultraviolet and visible spectrophotometer.2.HypocrellinsB was dissolved in DMSO, of which the concentration was1 mg/ml, which color is brown yellow.Then join100ml5%RPMI-1640offetal bovine serum-containing culture medium, diluted to HB concentrationof0.5μm. To produce the HB absorption spectrometry by UV-2102PCultraviolet spectrophotometer in the wavelength range400nm-700nmprogressive scan, then select the highest value as a follow-up experimentconditions.Part2:1. Cell culture: Tca8113cells routinely cultured in RPMI-1640culture medium containing5%fetal bovine serum.2. The Absorption andmetabolism laws of hypocrellin modified nano-lipid microbubbles in thehuman tongue carcinoma Tca8113cells: Take Tca8113cells which were inthe logarithmic growth phase into RPMI1640containing of20μM nanophotosensitizer, cultured2h,4h,6h,8h,10h,12h. The cells were lysed withDMSO centrifuged twice after had been washed twice by PBS.After30minutes, detect its fluorescence values by Spectrophotometer, excitationwavelength is475nm, and emission wavelength is600nm.3. Observe thehypocrellin modified nano-lipid microbubbles with LED light on humantongue carcinoma Tca8113cells: Tca8113cells in logarithmic growth phasewere added to96-well plates,2*104cells per well.There are3complexholes in every section. The cells were added by the hypocrellin modifiednano-lipid microbubbles after cultured overnight, of which theconcentration is0-10μmol/L. The cells were treated by the light after hadbeen incubated for6hours in a dark room.The light energy density is 0-3J/cm2.And its wavelength is475nm. Then cultured the cells for18hours, detected OD value of the absorbance value of each well by MTT.The inhibition rate of the cells in each group were calculate by the formula.The results of all the experiments repeated three times. Cell inhibition rate(%)=(control group OD value-treatment group OD value)/OD value ofthe control group*100%.4. Observe the hypocrellin modified nano-lipidmicrobubbles with LED light on human tongue carcinoma Tca8113cells byFlow cytometry: The cells were divided into five groups,①experimentalcell group: HB-microbubble concentration is10μM, light energy is3J/cm2;②blank control group：HB-microbubble concentration is0μM, light energyis0J/cm2；③pure light group：HB-microbubble concentration is0μM, lightenergy is3J/cm2;④pure HB-microbubble group： HB-microbubbleconcentration is10μM, light energy is0J/cm2；⑤blank microbubblegroup：blank microbubble concentration is10μM, light energy is3J/cm2;After each group had been treated by microbubbles6h accroding to thegrouping requirements, lighted18h, then, doubled cell dye by the byAnnexinV-FITC/PI apoptosis detection kit, detected by Flow cytometry.Results: part1:1.The hypocrellin modified hypocrellinsB nano lipidmicrobubbles is nanoscale,the particle size is (1050.4士323.0)nm, Zetapotential is+(21.3±7.3)mV, encapsulation efficiency is (85.1±4.6)％; Blankmicrobubbles’ particle size is (1029.8士134.0)nm, Zeta potential is+(20.9±8.6)mV;2. There are absorption peaks in475nm,545nm and 585nm of hypocrellinsB,475nm at maximum; part2:1. HypocrellinsBmicrobubbles in Cells’ absorption varies with time and change, six hours ofthe best, then there is a platform downward trend of the hypocrellinsBmicrobubbles in the cells.3.Except the experimental cell group, apoptosisrate and lmortality rate are ower,there is no significant difference （p＞0.05）.Though apoptosis rate and mortality rate of the blank microbubblegroup showed a little higher, there is still no statistically significant.Apoptosis rate and mortality rate of the experimental cell group are higher,showed a significant difference statistically significant (p <0.05).Conclusion:1. The modified hypocrellinsB nano lipid microbubbles made by laboratoryis nanoscale, suitable for laboratory cells use.2. HypocrellinsB exerted the best photodynamic effect because of itsabsorption maxima at a laser wavelength of475nm.3. HypocrellinsB microbubbles in Cells’ absorption varied with time andchange, six hours of the best.4. Hypocrellin modified nano-lipid microbubbles with LED light showed asignificantly inhibition on the cells Tca8113.