LIFU-activated PLGA Phase Change Nanoparticles Loaded With God/Fe₃O₄/PFH For The Treatment Of Retinoblastoma

PART I PREPARATION AND CHARACTERIZATION OF PLGA PHASE CHANGE NANOPARTICLES LOADED WITH PFH/FE3O4/GODObjective To use poly?lactic-co-glycolic acid??PLGA?as the shell,liquid Perfluorohexane?PFH?as the core,oleic acid modified ferroferric oxide nanoparticles?Fe₃O₄?distributed in the shell.Glucose oxidase was distributed in the inner core,and a phase change PLGA nanoparticle?PFH/Fe₃O₄/GOD/PLGA?was prepared and its related characterization was detected.Methods PLGA phase change nanoparticles?PFH/Fe₃O₄/GOD/PLGA?loaded with GOD and Fe₃O₄ and coated with PFH were prepared by traditional double emulsification method.PLGA nanoparticles?PFH/PLGA?,PLGA nanoparticles?PFH/Fe₃O₄/PLGA?loaded with PFH and Fe₃O₄,PLGA nanoparticles?PFH/GOD/PLGA?loaded with PFH and GOD were prepared as experimental control groups respectively.The particle size,potential size,surface morphology,internal structure and other related characteristics were detected.The concentration of Fe in PFH/Fe₃O₄/GOD/PLGA was measured by inductively coupled plasma optical emission spectroscopy?ICP-OES?.The concentration of GOD in PFH/Fe₃O₄/GOD/PLGA was measured by high performance liquid chromatography?HPLC?and the encapsulation efficiency was calculated.Results The prepared PFH/Fe₃O₄/GOD/PLGA was brown yellow emulsion after dissolved in deionized water.The average particle size was255.6nm,the Polymer dispersity index?PDI?was 0.035,and the Zeta potential was-27.3mV as measured by Malvern particle size meter.Ultraviolet absorption spectrum is used to detect the absorption characteristics of nanoparticles.PFH/Fe₃O₄/GOD/PLGA was observed to bepunctiformunderordinaryopticalmicroscope,and PFH/Fe₃O₄/GOD/PLGA was observed to be cubic spherical under scanning electron microscope,with regular morphology,uniform size,good dispersion and smooth surface.Transmission electron microscope observation shows that PFH/Fe₃O₄/GOD/PLGA has a shell-core structure,PLGA polymer shell is outside,PFH has a core structure,and black spots uniformly distributed around the shell-core are Fe₃O₄ nanoparticles.The entrapment efficiency of Fe in PFH/Fe₃O₄/GOD/PLGA was 43.07 1.47%as measured by ICP-OES,while the entrapment efficiency of GOD in PFH/Fe₃O₄/GOD/PLGA was 33.99 4.19%as measured by HPLC.Conclusion PFH/Fe₃O₄/GOD/PLGA phase change nanoparticles have been successfully prepared.The nanoparticles are uniform in size,regular in morphology,cubic in spherical structure,stable in properties,and have good iron and drug loading rates,which can be used for subsequent related research.PART II THE EXPERIMENTAL STUDY ON THE PHASE CHANGE CHARACTERISTICS AND DRUG RELEASE OF PLGA PHASE CHANGE NANOPARTICLES LOADED WITH PFH/FE3O4/GODObjective PFH/Fe₃O₄/GOD/PLGA nanoparticles were excited by low intensity focused ultrasound,and their in vitro phase change characteristics were observed by light microscopy,ultrasound imaging and contrastenhanced ultrasound imaging,and the in vitro drug release behavior of glucose oxidase GOD under LIFU excitation was detected,so as to provide basis for LIFU parameters for subsequent treatment of retinoblastoma.Methods Agar gel model and PFH/Fe₃O₄/GOD/PLGA nanoparticles with different concentrations were prepared.PFH/Fe₃O₄/GOD/PLGA nanoparticles were excited by low intensity focused ultrasound with different powers?1W,2W,3W?and different times?1min,2min,3min?,respectively.The phase change characteristics were observed by small animal photoacoustic imager in ultrasound mode and contrast enhanced ultrasound development mode.Before,during and after the phase change of nanoparticles were observed under light microscope.The release of glucose oxidase GOD was detected.Results PFH/Fe₃O₄/GOD/PLGA nanoparticles can undergo phase transformation under low intensity focused ultrasound.The particle size of the nano-particles after phase change is continuously increasing. Ultrasound imaging?B-Mode?and contrast ultrasound imaging?CEUS?showed that the echo intensity after phase change was significantly enhanced compared with that before phase change,with P values of 0.0108 and 0.0034 respectively,and the difference was statistically significant,it shown that nanoparticles have the function of enhancing ultrasound development after phase change.Under the low intensity focused ultrasound excitation with power of 3W,the cumulative release of drug GOD at 240 s and 270 s reached 79.7967±1.5080% and 81.79±1.4305%,respectively.The basic curve was nearly stable and the release basically reached saturation.Under the excitation of 1W,the cumulative release of drug GOD at 240 s and 270 s was only 16.2667±1.2453% and 17.2567±1.3333%,respectively.Under 2W excitation,the cumulative release of GOD at 240 s and 270 s was 41.2667±1.2684% and 45.26±1.6810%,respectively.The cumulative release of drug GOD under 3W was significantly higher than that under 1 W and 2W.Conclusion The prepared PFH/Fe₃O₄/GOD/PLGA nanoparticles have phase change characteristics.The nanoparticles can undergo phase change when excited by low intensity focused ultrasound at appropriate power.The echo intensity of the nanoparticles after phase change is significantly increased in ultrasound imaging?B-Mode?and contrast enhanced ultrasound imaging?CEUS?modes,which has certain imaging effect.In addition,the nanoparticles after phase change can make GOD have a higher cumulative drug release,which can be used for in vivo and in vitro treatment research of tumors.PART III THE EXPERIMENTIAL STUDY OF PLGA PHASE CHANGE NANOPARTICLES LOADED WITHPFH/Fe₃O₄/GOD IN THE TREATMENT OF RETINOBLASTOMAObjective To observe the therapeutic effect of PFH/Fe₃O₄/GOD/PLGA phase change nanoparticles on retinoblastoma in vitro and in vivo under LIFU excitation,and to explore its therapeutic mechanism,so as to provide a new research idea and method for the treatment of retinoblastoma.Methods In vitro therapeutic experiment: Firstly,different nanoparticles were prepared,then human retinoblastoma Y79 cells were cultured in vitro,and the phagocytosis of Y79 cells on nanoparticles under EPR effect was observed,and compared with normal umbilical vein endothelial cells?HUVEC cells?.The cytotoxicity of nanoparticles under LIFU and nonLIFU excitation was studied.The following different groups were established: PBS group?Control group?,PFH/ PLGA group?PP group?,PFH/Fe₃O₄/ PLGA group?PPF group?,PFH/GOD/PLGA group?PPG group?,PFH/Fe₃O₄/GOD/PLGA group?PPFG group,PH=7.2?,PFH/Fe₃O₄/GOD/PLGA group?PPFG group,PH=6.0?.In vitro therapeutic experiments include CCK-8 cytotoxicity experiment,fluorescence staining of living/dead cells,apoptosis detection,Cell cycle detection,reactive oxygen species detection,etc.In vivo therapeutic experiment: First,35 nude mice were subcutaneously transplanted with Y79 cells.They were randomly divided into the following 7 groups: PBS group,PBS LIFU group,PFH/PLGA+LIFU group?PP+LIFU group?,PFH/Fe₃O₄/PLGA+LIFU group?PPF+LIFU group?,PFH/GOD/PLGA+LIFU group?PPG+LIFU group?,PFH/Fe₃O₄/GOD/PLGA group?PPFG group?,PFH/Fe₃O₄/GOD/PLGA+LIFU group?PPFG+LIFU group?.Tumor growth was observed after being treated according to the above groups.The weight and tumor size of nude mice in each group were recorded,and the nude mice were executed after 3 weeks of observation.HE tests were performed on all important tissues?heart,liver,spleen,lung,kidney and brain?,and TUNEL and PCNA were performed on tumor tissues to detect proliferation and apoptosis of tumor cells.Results In vitro treatment experiments showed that tumor cells phagocytized more than normal cells due to EPR effect.CCK-8 results showed that the cell survival rate of PFH/Fe₃O₄/GOD/PLGA group?PPFG group,PH=6.0?stimulated by LIFU was significantly lower than that of other groups.After adding different concentrations of L-ascorbic acid,the cell survival rates in PFH/Fe₃O₄/GOD/PLGA+LIFU group?PPFG+LIFU group,PH=7.2?and PFH/Fe₃O₄/GOD/PLGA+LIFU group?PPFG+LIFU group,PH=6.0?all increased with the increase of L-ascorbic acid concentration.Under the laser confocal microscope,the Y79 cells in PFH/Fe₃O₄/GOD/PLGA+LIFU group?PPFG+LIFU group,p H = 6.0?can be directly observed to be basically all dead,and more or less living cells still exist in the other groups.Flow cytometry showed that 90.96% of the cells in PFH/Fe₃O₄/GOD/PLGA+ LIFU group?PPFG+LIFU group,PH=6.0?were apoptotic,which was significantly higher than that in other groups.The results of reactive oxygen species detection showed that a large amount of reactive oxygen species were produced in the cytoplasm of PFH/Fe₃O₄/GOD/PLGA+LIFU group?PPFG+LIFU group,PH=6.0?.In vivo treatment experiments showed that the tumor volume of PFH/Fe₃O₄/GOD/PLGA+LIFU group was significantly smaller than that of other groups,and even the tumor volumebasically disappears.HE and other results showed that there was no damage to important organs such as heart,liver,spleen,lung,kidney and brain after treatment.TUNEL and PCNA immunohistochemical of tumor or tumor surrounding tissues showed that tumor cells in PFH/Fe₃O₄/GOD/PLGA+LIFU group?PPFG+ LIFU group?were significantly apoptotic,while tumor cell proliferation in PFH/Fe₃O₄/GOD/PLGA+LIFU group?PPFG+LIFU group?was reduced.Conclusion PFH/Fe₃O₄/GOD/PLGA phase change nanoparticles can accumulate in a large amount in tumors due to enhanced permeability and retention effect?EPR effect?of tumor cells.The nanoparticles excited by low-intensity focused ultrasound can undergo phase change,and the nanoparticles after phase change release the drug glucose oxidase GOD,which reacts with glucose in tumor cells or tissues,and performs enzymelinked catalytic reaction with Fe₃O₄ to generate active oxygen components and kill tumor cells,thereby achieving therapeutic effect in vivo treatment and destroying tumor tissues.Compared with methods such as chemotherapy,radiotherapy,photothermal and immunotherapy,the nanoparticles release drugs and generate active oxygen by using lowintensity focused ultrasound,phase change and enzymatic catalytic reaction,have high biocompatibility and no obvious toxic and side effects,provide a new idea for the treatment of retinoblastoma,and have good development prospects.