| Part I Formulation and Characterization of TOI_HNPs,OI_PNPs and TOI_LNPsObjective: To synthesize folate-targeted Lipid–polymer hybrid nanoparticles-loaded indocyanine green/PFP-carrying oxygen(TOI_HNPs)and to explore the essential properties of developed NPs.Methods: TOI_HNPs were prepared by using a combination of two-step method and solution evaporation technique for the frst time.PLGA NPs-encapsulated ICG and PFP-carrying oxygen(OI_PNPs)and folic-targeted lipid NPs-encapsulated ICG and PFP-carrying oxygen(TOI_LNPs)were synthesized by emulsion evaporation process.The morphology of TOI_HNPs was observed by CLSM and the structure was characterized by using TEM.The particle size,zeta potential,encapsulation efficiency and drug loading of the fabricated nanoparticles were determined.The changes of TOI_HNPs diameter and surface potential afternear-infrared excitation were measured.The absorption spectra,fluorescence spectra,stability and oxygen release capacity of the synthesized nanoparticles were compared.Results: TOI_HNPs with shell-core structure were successfully prepared,which showed a uniform spherical shape and could be phase-transition by near-infrared(NIR)laser excitation.The average diameter of the TOI_HNPs,OI_PNPs and TOI_LNPs were 166.83±5.54 nm,214.50±10.87 nm and 340.47±16.06 nm,respectively.The zeta potential of TOI_HNPs,OI_PNPs and TOI_LNPs were-(30.57±1.36)mV,-(24.04±1.17)mV and-(17.50±1.65)mV,respectively.The ICG encapsulation efficiency of TOI_HNPs was 75.29±2.92%,while the OI_PNPs and TOI_LNPs was 62.48±3.39% and 82.50±2.84%,respectively.The absorption and fluorescence spectra were almost similar to the free ICG solution without significant shift in all of the nanoagent.After 15 days of observation,the TOI_HNPs absorbance was decreased to 75.4% of its initial intensity,while the OI_PNPs,TOI_LNPs and free ICG decreased to56.2%,35.4% and 12.5%,respectively.Moreover,the fluorescence intensity of TOI_HNPs remained at 62.7%,while the fluorescence of OI_PNPs,TOI_LNPs and free ICG were reduced to 35.6%,14.8% and4.0%,respectively.Besides,the particle sizes of TOI_HNPs within 30 days was increased to 239.40±15.75 nm and OI_PNPs was increased to386.27±11.29 nm.Particularly,the TOI_LNPs increased significantly to523.97±58.08 nm within 5 days and the monodisperse state almost disappeared on the tenth day.In addition,the total ICG accumulative release of TOI_HNPs,OI_PNPs and TOI_LNPs in PBS was 25.95%,30.51% and 66.68%,respectively;And in 10% BSA was 29.05%,34.97%and 80.59%,respectively.Furthermore,the oxygen concentration in the TOI_HNPs solution increased from 3.36 mg/L to 6.85 mg/L after laser irradiation plus ultrasound exposure.Conclusion: We have successfully constructed core–shell hybrid nanosystem as TOI_HNPs,which have good drug loading ability,phase change potential and oxygen carrying capacity.And TOI_HNPs are more stable than TOI_LNPs and have higher encapsulation efficiency than OI_PNPs.It is a excellent multifunctional nano-delivery system.Part II Dual-mode imaging potential of TOI_HNPsObjective: To investigate the ultrasound and photoacoustic(PA)imaging capabilities of TOI_HNPs in vitro and the photoacoustic imaging in vivo.Methods: In vitro ultrasound and photoacoustic imaging were performed using agar-gel model.The TOI_HNPs were dissolved in degassed water and diluted to 1,2,4,6,8,16?g/ml according to the ICG concentration.The ultrasound and PA imaging after laser irradiation was observed by MyLab 90 ultrasonic diagnostic instrument and VEVO LASRPA imaging system.In addition,the imaging ability of 8 ?g/ml of TOI_HNPs and the same concentration of free ICG and TO_HNPs was compared.In vivo photoacoustic imaging were performed on SKOV3-subcutaneous tumor-bearing nude mice.And the mice were randomly divided into four groups: normal saline,free ICG,OI_PNPs and TOI_HNPs(the ICG concentration were 8?g/ml)and the tumor images obtained at different time points(0,0.5,1,2,4,6,8,12h).Results: In vitro ultrasound experiment,the echo intensity(EI)of B-mode and CEUS in TOI_HNPs were linearly enchaned,following ICG concentration from 0?g/ml to 8?g/ml,after the NIR irradiation.However,the increaseing tendency was not for 16 ?g/ml.In addition,the imaging of TOI_HNPs at 8?g/ml after laser irradiation was significantly enhanced compared with TO_HNPs,and the EI ??in B-mode and CEUS of TOI_HNPs increased from 8.19±0.91 to 81.88±10.73 and from 4.87±0.51 to 49.13±0.91,respectively.In vitro photoacoustic imaging,the PA average values of TOI_HNPs were linearly correlated with ICG concentration.Furthermore,the PA images of TOI_HNPs at 8?g/ml after NIR irradiation was significantly enhanced compared with TO_HNPs.The PA signal intensities increased from 0.145±0.016 to 1.526±0.090;while the ICG at the same concentration decreased from 0.274±0.015 to 0.060 ± 0.003.In vivo photoacoustic imaging experiments,the PA intensities in tumor region were significantly enhanced after injection contrast agent(ICG,OI_PNPsand TOI_HNPs).The TOI_HNPs group reached peak enhancement at 2h post injiection and the PA value was 0.615±0.033.Besides,the ICG and OI_PNPs group reached peak enhancement after 6h injiection and the PA values were 0.380±0.014 and 0.529±0.017,respectively.Conclusion: TOI_HNPs can be used as a dual-mode contrast agent for ultrasound/photoacoustic imaging to enhance the EI and PA value.Moreover,TOI_HNPs have higher sensitivity than OI_PNPs through active targeting.Part III Therapeutic effect and mechanism of TOI_HNPsinduced PSDT in SKOV3 cellsObjective: To investigate the therapeutic effect and underlying mechanism of TOI_HNPs mediated photo–sonodynamic therapy(PSDT)on SKOV3 cancer cells.Methods: The biosafety of TOI_HNPs were determined by MTT assay,and the targeting ability of TOI_HNPs was observed by confocal microscopy and flow cytometry.The experiment was divided into six groups: control group,ICG + PSDT,TO_HNPs + PSDT,TI_HNPs + PSDT,OI_HNPs + PSDT and TOI_HNPs + PSDT.The cytotoxicity of TOI_HNPs induced PSDT on SKOV3 cell were evaluated by MTT assay and the cell apoptosis rate were measured by flow cytometry.The fluorescence increase of intracellular of ROS and cell-free system of SOSG was determined byfluorescence microplate reader,and the expression of hypoxia-inducible factor 1-?(HIF-1?)and IL-6 was detected by Western blot.Results: There were no signifcant cytotoxicity at 24 and 48 hours incubation of SKOV3 cells with TOI_HNPs in 0–8 ?g/mL of ICG concentration.Therefore,8 ?g/mL was used for the experiment.TOI_HNPs can actively target SKOV3 cells by folate and folate receptor linkage.The cell viability of TOI_HNPs+PSDT,OI_PNPs+PSDT and TI_HNPs+PSDT group was 16.39±2.58%,41.58±2.10% and 53.85±4.58%,respectively.The apoptotic rates of TOI_HNPs +PSDT,OI_PNPs+PSDT and TI_HNPs+PSDT group were 81.58±7.68%,52.04±14.66% and43.26±11.10%,respectively.Besides,ICG+PSDT and TO_HNPs+PSDT had no obvious cytotoxicity on SKOV3 cells.Furthermore,the increase of intracellular ROS in the TOI_HNPs+PSDT group was 244.27±29.11%,which was significantly higher than other groups(P<0.05).Moreover,the increase of SOSG fluorescence in the TOI_HNPs+PSDT and OI_PNPs+PSDT group were 203.87±9.74% and 189.23±14.05%,respectively.There was no statistical significance between this two groups,but it was statistically significant compared with the control group(P<0.05).In addition,compared with the other group,the TOI_HNPs+PSDT group significantly down-regulated the expression of HIF-1? and IL-6 proteins(P<0.05).Conclusion: TOI_HNPs can significant actively target SKOV3 cell.TOI_HNPs induced-PSDT caused more serious cell damage and obvious cell apoptosis,increasing intracellular ROS production and down-regulating the expression of HIF-1? and IL-6 proteins. |
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