| Part ?: Preparation and characterization research of FCNPI nanoagentsObjectives: To prepare the folic acid(FA)-modified phase-changeable cationic nanoparticle encapsulating liquid perfluoropentane(PFP)and indocyanine green(ICG)(FA-CN-PFP-ICG,FCNPI)nanoparticles and investigate their basic characteristics,phase-transition property,plasmid loading capacity,biosafety and cell targeted property in vitro and in vivo.Methods: This studying part was divided into two sections.In the first section,the lipid cationic nanoparticles were fabricated with a modified twostep emulsion process.DSPE-PEG(2000)-folate and DC-cholesterol were added to make the nanoparticles have the target ability and be charged positively,respectively.The morphological structure,the zeta and size potential,the encapsulation efficiency(EE)of ICG were detected using several analytical methods.A near-infrared light(NIR)laser(808 nm,1W/cm2,2min)was applied for FCNPI nanoparticles phase transition triggering.Plasmid loading capacity and cell cytotoxicity were explored as well.The second section was focus on the targeting efficiency of FCNPI by cell and mice experiment.Untargeted cationic nanoparticle(CN-PFP-ICG,CNPI)and neutral nanoparticle(NN-PFP-ICG,NNPI)were also made to make comparison with FCNPI.Results: The prepared FCNPI nanoemulsion was green.Nanoparticles showed spherical or dotted morphology under transmission electron microscope(TEM).The mean diameter of prepared nanodroplets was 328.4±5.5nm and the average surface zeta potential was 35.3±1.9 m V.The EE of ICG was above 92.1 ± 1.3% in FCNPI by virtue of ultraviolet absorption spectrometry.It can be seen that the volume of some FCNPI nanoparticles significantly increased after laser irradiation(808nm,1W/cm2,2min)as a result of the PFP liquid-gas phase transition.Successful plasmid binding was exhibited with red fluorescence,as only PI was able to stain the plasmid.The red fluorescence could only been seem in CNPI and FCNPI,which suggests that only cationic nanoparticles were able to attach plasmid DNA successfully by virtue of charge coupling.Gel retardation assay further confirmed the results of confocal laser scanning microscopy(CLMS).When 40?g plasmid was added,the DNA loading capacity for 5×108 NNPI,CNPI and FCNPI were 1.4±0.4?g,22.4±0.1?g and 22.7±0.1?g,respectively.The differences in DNA loading capacity between CNPI and FCNPI were not statistically significant(P>0.05).Results of cytotoxicity revealed that the cell viability of NNPI,CNPI,FCNPI with or without plasmid retained above 85%,and meanwhile these six kinds of nanoparticles had a great stability and they could be stored for 48 h without aggregation and precipitation in water.In vitro targeting experiment,plenty of red dots representing the nanoparticles in the cytoplasm and around the cell membrane of Y79 cells were detected in the FCNPI group,followed by those in the CNPI group.Nonetheless,rare red dots could be detected in the cells of NNPI and antagonized groups.In vivo targeting experiment,as for the NNPI group,no fluorescence was observed in the tumor area throughout the observation time.From 30 min after injection on,the FCNPI group displayed a gradual accumulation of fluorescence,which achieved a peak 2h postinjection.Then,the fluorescence faded out bit by bit till 24 h after injection.Similarly,the CNPI group demonstrated a corresponding tendency of performance change with clearly less fluorescence accumulation.In addition,the measurement results of excised tumor tissue 24 h post-injection demonstrated that the FCNPI group took on the supreme fluorescence intensity,followed by CNPI group and virtually no fluorescence was found in the tumor sections from the NNPI group.Conclusion: The successfully fabricated FCNPI nanoparticles were normal in shape,uniform and stable,with good plasmid capability,favorable biosafety and stability.The nanoagents had good phase-transition and targeted abilities.These results laid the foundation for the future researches.Part ?: FCNPI nanoagents for enhanced ultrasound and PA imagingObjectives: Study on the effect of FCNPI for enhanced US and PA imaging in vitro and in vivo,to investigate the feasibility of the nanoparticles as a multi-modality imaging agents.Methods: This studying part was divided into two sections.In vitro experiment,after exposure to the laser(808 nm,1W/cm2)for 2min,Saline,NNPI(0.5mg/ml),CNPI(0.5mg/ml)and FCNPI(0.5,1.0,1.5,2.0,2.5mg/ml)in 2% agarose gel phantom were observed with B-mode and contrast-mode.After ultrasonic images acquisition,the images of ROI(region of interest)were quantitatively analyzed with ‘average gray scale' using DFY ultrasound imaging analysis software.PA signals of Saline,NNPI(0.1mg/ml),CNPI(0.1mg/ml)and FCNPI(0.1,0.2,0.3,0.4,0.5mg/ml)in agarose gel phantom were measured to investigate the relationship between concentrations and PA signals using the PA imaging system.In vivo imaging experiment,the nude mice with xenograft RB tumor were injected intravenously with saline,NNPI,CNPI and FCNPI(n=5 in each group).With regard to US imaging,images were observed before and after laser irradiation(808nm,2W/cm2,2min)using My Lab 90 in B-mode and CEUS.Then,the intensity of ROI was quantitatively analyzed.Similarly,the tumor mass of xenograft was observed by means of the VEVO LASR PA imaging system continuously for 12 h in order to get the PA images.The average PA value at each time point(0h,1 h,2h,6h and 12h)were recorded and then quantitatively analyzed.Results: As for the US imaging in vitro,no ultrasonic contrast enhancement was observed in the saline group after laser irradiation,whereas there were some enhancements in the NNPI and CNPI groups.Nonetheless,perfect ultrasonic contrast enhancement was obtained in the FCNPI group with various concentrations after laser irradiation.The results of quantitative analysis indicated that echo intensity rose with the increase of FCNPI concentrations.Likewise,no PA signal was observed in the images of the saline group after laser irradiation.The NNPI and CNPI groups exhibited some PA signals.As for FCNPI,PA signals steadily went up in terms of the rise in its concentrations upon laser irradiation and high concentration led to higher signal intensity.In vivo study,both the CNPI and FCNPI groups demonstrated higher echo intensities than those gained before laser irradiation in the B-mode,no obvious change was detected in the control and NNPI groups.While all the NNPI,CNPI and FCNPI groups after laser exposure showed significantly superior echo intensities compared with those before laser exposure in the CEUS.The echo intensities obtained from FCNPI group were the highest,followed by those from the CNPI group and the NNPI group in order.Meanwhile,no enhancing effect of PA imaging was observed in the saline group without optical absorption material,and rare signal was observed for NNPI around 2h after injection.By contrast,with the increase in the accumulation of CNPI and FCNPI nanoparticles,the signals for both groups were gradually enhanced 1h after injection,and a better PA-signal enhancement was acquired 2h after injection.Subsequently,a relatively weaker PA-signal enhancement was detected.At 12 h after injection,only the FCNPI group could still exhibit desirable PA imaging.Conclusion: The FCNPI nanoparticles were successfully acted as dualmodality biological imaging contrast agents for US and PA imaging in vitro and vivo,which had a potential application value in the detection of retinoblastoma.Part ?: The therapy effect of retinoblastoma in nude mice by laser-actived gene transfection with FCNPI/p DNAObjectives: To evaluate the killing effect of targeted cationic molecular probe(FCNPI/p DNA)loading with plasmid against Y79 cells after transferred by laser,and to observe the therapeutic effect in the nude mice with xenograft RB tumor in vivo.Methods: This studying part was divided into two sections.In the first section,different treatments were applied to Y79 cells,including control(no treatment),p DNA,p DNA+Laser,NNPI/p DNA+Laser,CNPI/p DNA+Laser and FCNPI/p DNA+Laser.Then laser(808nm,1W/cm2)for 2min was employed alternately.Y79 cells were collected from different groups after a transfection for 24 h or 48 h for the measurement of the expression level of the plasmid-encoded GFP gene,cell cycle,proliferation,apoptosis,m RNA and protein level of TK,PCNA and caspase-3.In the second section,the sample mice were treated with control,NNPI/p DNA,CNPI/p DNA,and FCNPI/p DNA with/without laser irradiation(808 nm,2W/cm2,2min)at an equal GCV dose of 100 ml/mg every other day through tail intravenous injection.The size and body weight of tumor xenografts were recorded every other day.Fourteen days later,the tumor tissues were excised for q PCR and WB.In addition,they were also sacrificed for H&E,TUNEL and PCNA staining for the effect evaluation of gene therapy.Then the histological analysis of major organs(e.g.spleen,kidneys,liver,lung and heart)as well as the functions of mice liver and kidney after all kinds of treatment would be examined to evaluate the in vivo biosafety of this theranostic system.Finally,the surface temperature of the tumors from different groups was measured with an infrared thermometer during laser exposure.Results: The transfection efficiency of FCNPI/p DNA+Laser group was the highest 24 hours post-transfection,with the biggest proportion of cells inhibited in the G1 phase,and the highest early apoptotic rate.The q PCR and WB analyses of TK,caspase-3 and PCNA gene expression indicated that the FCNPI/p DNA+Laser group presented the highest TK RNA and protein expression,the greatest caspase-3 expression and the least PCNA expression,followed by CNPI/p DNA+Laser group and NNPI/p DNA+Laser group.The FCNPI/p DNA+Laser system also exhibited the best tumor inhibition effect in vivo.The q PCR and WB analyses of TK,caspase-3 and PCNA gene expression also demonstrated that the FCNPI/p DNA+Laser group presented the highest TK RNA and protein expression,the greatest caspase-3 expression and the least PCNA expression,followed by CNPI/p DNA+Laser group and NNPI/p DNA+Laser group.Results of H&E staining for the tumor tissues in the FCNPI/p DNA+Laser group showed distinct and widespread coagulative necrosis,and the tumor cells were arranged in nests.The apoptosis index of TUNER was the highest while the proliferation index of PCNA was the lowest in the FCNPI/p DNA+Laser group compared with those in other groups.During the therapy process,the temperature of FCNPI/p DNA+Laser group was the highest at the end of laser irradiation,raising from nearly 34°C to 40°C,which could not cause any damage to tissue and the results of H&E for major relevant tissues(lung,heart,liver,spleen and kidneys)of all groups with or without laser irradiation showed similar histological structures in comparison with those of control group.Mice's liver and kidney function after different treatment were within the normal range.All of these results suggested that current gene transfection model had good biosafety and biocompatibility in vivo.Conclusion: The transfection efficiency for the FCNPI/p DNA+Laser group was the highest,led to the best therapy effect,better than that of untargeted groups.Meanwhile,it could inhibit the tumor growth successfully,which was expected to establish a new path for the therapy of retinoblastoma. |
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