| Objective In this study,we designed a simple but effective nanoparticle system based on phospholipid,Pluronic F68 and pullulan for carrying IR780 and PTX,thus to realize the synergistic effects against HCC by combing PTT/PDT and chemotherapy.Contents The first part is was the preparation and characterization of PDFI and PDFP nanoparticles,including the preparation and characterization of nanoparticles,photostability of PDFI nanoparticles,the carrying capacity of nanocarriers for drugs.The second part was the in vitro studies of PDFI and PDFP nanoparticles,including evaluations of in vitro releases capabilities,PTT/PDT efficiencies of PDFI nanoparticles in MHCC-97 H cells.The third part was the in vivo study of PDFI and PDFP nanoparticles,including in vivo PTT/PDT efficiencies of PDFI nanoparticles.Methods1.The preparation and characterization of PDFI and PDFP nanoparticles:Nanoparticles were prepared by a thin-film hydration method.Pullulan is modified on the surface by incubation.The sizes,size distributions and Zeta potentials of them were measured using Zetasizer Nano ZS90 apparatus.Their morphologies were characterized by TEM.The UV-vis absorption spectra of nanoparticles were recorded to evaluate the photostability of these systems.The ultraviolet-visible spectroscopic and UPLC method were used to determine the loading contents and encapsulation efficiencies of IR780 and PTX.2.The in vitro studies of PDFI and PDFP nanoparticles: Drug release of nanoparticles were analyzed by dynamic dialysis.The in vitro PTT efficacy of PDFI nanoparticles was assessed under NIR laser irradiation.The temperature changes were recorded using a visual IR thermal imaging camera.A fluorescence probe DCFH-DA was used to evaluate the intracellular ROS production.The cellular internalization of PDFI nanoparticles were analyzed by CLSM.Effects of nanoparticles on the apoptosis and cell cycle were analyzed by flow cytometry.3.The in vivo study of PDFI and PDFP nanoparticles: The in vivo PTT efficacy of PDFI nanoparticle4.The in vivo PDT efficiency of PDFI nanoparticles was analyzed by SOSG.The tumor growth in the tumor bearing mice was evaluated after intratumoral injection of PDFI nanoparticles and PDFP nanoparticles.The synergistic effect of PTT/PDT combined with chemotherapy on HCC was analyzed.Results1.Nanoparticles were successfully prepared and DF nanoparticles had exhibited a typical spherical shape.PDFI nanoparticles with the IR780 loading content was4.6%.The loading content of PDFP nanoparticles was 3.8%.IR780 loaded by DFI nanocores and PDFI nanoparticles showed the absorption peak at 802 nm.2.The release profiles of PTX from DFP and PDFP nanoparticles both displayed a biphasic pattern.DFI and PDFI nanoparticles showed much higher PDT/PTT efficiencies.PDFI nanoparticles combined with PDFP nanoparticles had synergistic inhibitory effect on growth of MHCC-97 H cells.3.The tumor temperatures of mice of PDFI nanoparticles increased to higher.It indicated that these treatments triggered the generations of large amounts of 1O2 in the tumors.PDFI nanoparticles were mainly distributed in the tumor sites of HCC bearing mice.PTT/PDT combined with chemotherapy can effectively inhibit the growth of tumor in mice,and can obviously inhibit the angiogenesis of tumor,and there was no obvious organ necrosis.Conclusions In this study,a simple but effective nanoparticle system was designed for carrying drugs,hoping to effectively combine PTT/PDT and chemotherapy to treat HCC.At cellular level,PDFI nanoparticles combined with PDFP nanoparticles significantly inhibited cell growth and induced cell apoptosis and cell cycle arrest at G2/M phase.At animal level,PDFI nanoparticles exhibited an excellent hepatoma-targeting capability and combined treatment effectively inhibited tumor growth and tumor angiogenesis in HCC tumor-bearing mice.Our results suggested this novel nanoparticle system can efficiently combine PTT/PDT and chemotherapy to treat HCC and exhibits considerable potential for clinical applications. |
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