| PART I PREPARATION AND CHARACTERIZATION OFTRANSFERRIN MODIFIED PACLITAXEL LOADED PHASECHANGE NANOPARTICLESObjective1.To prepare a kind of paclitaxel loaded liquid PFP core encapsulated phase change PLGA nanoparticles which was modified with transferrin(Tf-PTX-PFP-PLGA),and to detect the properties.2.To study thermo-and acoustic-induced phase change characteristics of nanoparticles.3.To study the ability of the nanoparticles to target C6 glioma cells and the toxic effects on cells.Methods1.Tf-PTX-PFP-PLGA nanopartles were prepared by double emulsion and carbodiimide technique.Tf-PTX-PFP-PLGA was observed by light microscope(LM)and transmission electron microscope(TEM).The particle size distribution and ZETA potential were detected by dynamic light scattering.The encapsulation efficiency and drug loading ratio of PTX and the drug release characteristics influenced by sonication were detected by High Performance Liquid Chromatography(HPLC);The successful modefication of Tf was detected by immunofluorescence method and flow cytometry method was used to detect the connect ratio.2.The thermo-induced phase change characteristic of the nanoparticles were detected by heating and observed under the inverted microscope.The images of B-model and contrast enhanced ultrasound(CEUS)of nanoparticles before and after sonication with different acoustic pressure(0.5 MPa,1.0 MPa,1.5 MPa)and sonication duration(1min,3min,5min)were captured by ultrasonic diagnostic apparatus,and gray scale and acoustic intensity value were analyzed by DFY software.3.The expression of transferrin receptor(TfR)on C6 glioma cells was detected by flow cytometry method.The in vitro targetting ability to C6 cells of Tf-PTX-PFP-PLGA,PTX-PFP-PLGA and Tf-PTX-PFP-PLGA +free Tf was detected by confocal laser scan microscopy(CLSM).Results1.The microscopic images showed Tf-PTX-PFP-PLGA nanoparticles were uniform,spherical and dispersed.The TEM showed shell core structure and indicated the PFP inner core.The size distribution was(352.3±14.6)nm.The ZETA potentional was(-24.3±0.4)mv.The encapsulation efficiency was(63.3±1.8)% and drug loading ratio was(3.7±0.1)%.Fluorescence of nanoparticles and antibody coincided well under CLSM and the connect ratio was(99.29±0.46)%.2.Microbubbles appeared under invert microscope when the temperature was heated to 48℃.The B-model and CEUS images of suspenssion before and after sonication showd the phase changge of nanoparticles could be induced by FUS.3.The expressions transferrin receptor on C6 was(98.87±0.57)%.CLSM observation of in vitro targeting results showed that the binding of nanoparticles to C6 cells was significantly higher than that of non-targeting and competitive inhibition groups.CCK8 results showed that Tf-PTX-PFP-PLGA had the strongest cytotoxicity to C6,and non drug loaded Tf-PFP-PLGA had little effect on the proliferation of C6 which showd good safety.ConclusionSuccessful preparation of Tf-PTX-PFP-PLGA nanoparticles,with thermo-and acoustic-induced phase change ability.It had good ability to target C6 glioma cells in vitro and could specifically kill the cells.PART II IN VIVO STUDIES OF PHASE CHANGE NANOPARTICLES COMBINED WITH FOCUSED ULTRASOUNDFOR BLOOD BRAIN BARRIER OPENING IN RATObjectiveTo explore the feasibility of using phase change nanoparticles as alternative media agent for focused ultrasound induced(FUB)blood brain barrier(BBB)opening in rat.To find safe and effective parameters of sonication.To test the closing time of BBB opening.Methods1.51 SD were used to perform skull window opening by rat brain stereotaxic apparatus and dental drill.2.24 rats were randomly divided into 3 groups.After intravenously injection of phase change nanoparticles,rat brains were sonicated by Focused ultrasound(FUS,1 MHz)with 0.5 MPa,1.0 MPa and 1.5 MPa acoustic pressure and the sonication duration was 3min.Intravenously injection of Evans’ blue(EB)were administrated,and BBB opening was observed by EB extravasation within the sonicated area.5 rats of each group were selected for quantitative analysis of EB extravasation.The rest 3 rats for HE staining to understand the effect of focused ultrasound on the brain tissue with different acoustic pressure,and to screen out the safe and effective acoustic pressure.3.24 rats were randomly divided into 2 groups,duration of exposure was 3 min and 5 min respectively and acoustic pressure was 1.0 MPa.After sonication,3 from each group were taken at at 0h,1h,2h and 4h for EB extravasation test.4.3 rats were sonicatied with FUS combined with lipied-shelled MBs,comparing the difference of distribution partten of EB extravasation between nanoparticles and MBs.Results1.There were no EB extravasation in the brain tissue of the target area with the acoustic pressure of 0.5 MPa after irradiation,and obvious EB extravasation was observed in the 1.0 MPa group and 1.5 MPa group,and the extent and extent of EB extravasation were more pronounced in the 1.5 MPa group.The amount of EB extravasation in each group were(5.9±0.7)μg/g,(9.9±2.0)μg/g and(31.8±4.9)μg/g,respectively.Compared to the contralateral side,p>0.05 in the 0.5 MPa group,p<0.05 in the1.0 MPa group and p<0.001 in the1.5 MPa group,respectively.2.HE stain: No erythrocyte extravasations or tissue damages were observed in 0.5 MPa group and 1.0 MPa group.Band like erythrocyte extravasations and sporadic intracerebral hemorrhages were observed in 1.5 MPa group,accompanied by obvious brain tissue liquefaction and necrosis.3.Time window: The amount of EB extravasation reached the highest at 0h,and decreased as time gone by with a fast-slow trend.The amount of EB extravasation in 3min group decreased to the level of the contralateral side.The amount of EB extravasation in 5min group was higher than 3min group and the window extended to 4h.4.The distribution partten of EB extravasation showed a relative high focal performance by phase change nanoparticles.ConclusionSuccessful BBB opening in rat was achieved by serving phase change nanoparticles as mediated agent combined with FUS,and the optimum acoustic pressure was 1.0 MPa which met both effectiveness and bio security,and laid foundation for subsequent treatment experiment.PART III IN VIVO STUDIES OF PACLITAXEL-LOADED PHASECHANGE NANOPARTICLES COMBINED WITH FOCUSEDULTRASOUND FOR GLIOMA THERAPY IN RATObjectiveTo investigate the therapeutic effect of Tf-PTX-PFP-PLGA nanoparticles combined with FUS on C6 orthotopic glioma model in SD rats.Methods1.Establishment of orthotopic glioma model: With the help of stereotaxic apparatus,the C6 cells were inoculated into SD rats’ brain tissue by puncture injection.MRI and HE staining were used for verification and the expression of TfR in tumor tissue was detected by immunohistochemistry.2.6 glioma bearing SD rats were randomly divided into 2 groups and DiI labeled PTX-PFP-PLGA or Tf-PTX-PFP-PLGA suspension were injected via tail vein.The distribution of nanoparticles in each group were observed by CLSM.3.40 glioma bearing SD rats were randomly divided into 5 groups:saline,PTX,PTX-PFP-PLGA,Tf-PTX-PFP-PLGA and Tf-PTX-PFPPLGA+FUS(1.0 MPa+5min).The changes of glioma were observed in 5 rat from each group by MRI before and after treatment and the survival time were recorded(n=5).The other 3 rats were HE and TUNEL stained for measure tumor necrosis and apoptosis(n=3).4.10 Healthy adult male SD rats were treated with Tf-PFP-PLGA or saline(n=5).The body mass was recorded and the main organs were HE stained to examine the toxicity in vivo.ResultsImmunohistochemistry showed that TfR was highly expressed in murine C6 glioma and matched with flow cytometry.The distribution of Tf-PTX-PFP-PLGA in tumor tissue was more than that in PTX-PFP-PLGA group under CLSM.MRI showed the obvious suppression of tumor growing in Tf-PTX-PFP-PLGA+FUS group.HE and TUNEL stain showed that the tumor necrosis and apoptosis were the highest in Tf-PTX-PFP-PLGA+FUS group,the differences were statistically significant.The median survival time was 22 days,20 days,22 days,26 days and 33 days respectively in each treatment group.No significant difference were detected between Tf-PFP-PLGA group and saline group about the increase in body mass.HE stain showed no obvious pathological necrosis or injury were revealed in the liver,spleen,lung and kidney.ConclusionThe prepared Tf-PTX-PFP-PLGA nanoparticles had good targeting ability in vivo.Drug loaded phase change nanoparticles combined with FUS could effectively inhibit the proliferation of intracranial orthotopic C6 glioma in SD rats,and promote necrosis and apoptosis of the tumor,and prolong the media survival time.Tf-PFP-PLGA had no negtive effects on healthy rats. |
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