Phase-transition Nanodroplets For Photoacoustic/Ultrasound Dual-modality Imaging Of Sentinel Lymph Node And Photothermal Therapy Of Metastatic Lymph Node In Breast Cancer

PART Ⅰ PREPARATION AND CHARACTERIZATION OF CARBON NANOPARTICLES-LOADED PHASE-TRANSITION NANODROPLETS FOR PHOTOACOUSTIC/ULTRASOUND DUAL-MODALITY IMAGINGOBJECTIVE: To synthesize a kind of carbon nanoparticles(CNs)/perfluorocarbon(PFH)-loaded phase-transition PLGA nanodroplets for photoacoustic(PA)/ultrasound(US)dual-modality imaging(CNPs),and investigate their properties of optics,physics,bio-safety,and liquid-gas phase-transition in vitro.METHOD: CNPs were synthesized with dual-emulsion method,and were characterised with the shape,internal structure,size,zeta-potential,absorption spectra,as well as the morphologic change before and after laser irradiation;after preparation of Di I marked CNPs(Di I-CNPs),the Di I-CNPs were incubated with RAW264.7 cells,then,the phagocytosis by the cells and the influence of Di I-CNPs on cell activity were observed.RESULT: CNPs appeared dark black in double distilled water.They showed regular sphere,uniform size,good dispersion,and the obviously increased volume due to liquid-gas phase-transition after laser irradiation.The TEM images exhibit that the internal structure of CNPs was clearly visible,the dark black granular CNs were inserted into PLGA shell,and the gray black PFH was encapsulated in the white PLGA shell.CNPs exhibited a narrow size distribution with an average hydrodynamic diameter of 483.6 ± 42.11 nm,the average zeta potential was determined to be-26.3 ± 5.02 m V.The CNPs had good light absorption in 400-900 nm wavelength range,which was similar to CNs.The results of phagocytosis test demonstrated that CNPs were phagocytosed and resided inside the macrophages.However,the phagocytosed CNPs had no obvious effect on cell activity.CONCLUSION: The CNs/PFH-loaded PLGA polymeric nanodroplets were successfully synthesized with regular sphere,uniform size,good dispersion,good Stability,good biological safety.The introduction of CNs in CNPs could endow the nanodroplets with the property of optical absorption.The absorption spectra of CNPs and pure CNs were similar to each other.Hence,the CNPs have the potential to be a new dual-modality contrast agent for PA/US imaging.PART Ⅱ CARBON NANOPARTICLES-LOADED PHASE-TRANSITION NANODROPLETS FOR ENHANCED PA/US IMAGING AND STAINING OF LYMPH NODESECTION Ⅰ CARBON NANOPARTICLES-LOADED PHASE-TRANSITION NANODROPLETS FOR ENHANCED PA/US IMAGING IN VITROOBJECTIVE: To observe the effect of enhancing PA/US imaging in vitro with CNPs,and explore the feasibility of CNPs as a dual-modalilty contrast agent.METHOD: PA signals of four CNPs concentrations(5,10,15 and 20 mg/ml)in agarose gel phantom were measured to investigate the relationship between concentrations and PA signals using the PA imaging system(700 nm).After exposure to the laser(808 nm,1 W/cm2)for 10 s,four CNPs concentrations(5,10,15,20 mg/ml)in 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.RESULT: The PA signals induced by CNPs were srong after 700 nm laser irradiation,and gradually increased with the increase of CNPs concentrations after laser irradiation.The PA signals originated from various concentrations of CNPs were also quantitatively analyzed,and the results were confirmed with the imagings.After laser(808 nm 1 W/cm2)irradiation,CNPs appeared high echoes,and echo intensity showed a trend of increase with the increase of concentration of CNPs.The ultrasonic images of ROI were quantitatively analyzed with “average gray scale” using DFY ultrasound imaging analysis software,which was confirmed with the imagings.CONCLUSION: CNPs have the ability of enhancing PA and US imaging in vitro,and the effect gradually increased with the increased concentration.Hence,CNPs have the potential to be a PA/US dual-modality contrast agent.SECTION Ⅱ CARBON NANOPARTICLES-LOADED PHASE-TRANSITION NANODROPLETS FOR ENHANCED PA/US IMAGING IN VIVOOBJECTIVE: Observing enhanced effect of PA and US imaging in lymph nodes(LNs)with CNPs,to explore the feasibility of CNPs as a dual-modalilty contrast agent and the rule of enhancing LNs imaging with PA and US at different time points.METHOD: Popliteal fossa lymph node metastasis model with VX2 tumor in rabbits was established,and tumor-bearing rabbits were used for experiments when popliteal fossa lymph nodes could be palpated about 3 weeks after tumor inoculation.New Zealand white rabbits with detectable popliteal fossa lymph nodes were anesthetized.The rabbits were divided into three groups(three in each group)randomly,the CNPs solution(2 ml,20 mg/ml)group,the NPs solution(2 ml,nanodroplets without any CNs,same PLGA concentration as CNPs)group,and the CNs solution(2 ml,same CNs concentration as that incorporated in CNPs)group,the NPs and CNs groups were taken as control.The popliteal fossa lymph nodes of rabbits were observed using the PA imaging system with B-mode and PA-mode at 700 nm wavelength.The average PA signals of CNPs group at each point(pre,15 min,1 h,24 h and 48 h)were recorded and quantitatively analyzed.After the rule was mastered,the average PA signals of control group at each point(pre,15 min,1 h and 24 h)were recorded and quantitatively analyzed to reveal the rule of lymph nodes PA imaging.The rabbits were divided into three groups randomly(three in each group),including the CNPs solution group,the NPs solution group,and the CNs solution group,the NPs and CNs groups were taken as control.The dose and concentration of every group were the same with the PA imaging test in vivo.These rabbits received a percutaneous injection of CNPs solution,NPs solution and CNs solution via foot pad after anesthesia.After the solution injection of CNPs(15 min,30 min,1 h and 2 h)and CNs,NPs(15 min,30 min and 1 h),the lymph nodes area were exposed to the laser(808 nm,1 W/cm2)for 30 s.Then,the lymph nodes were observed with B-mode and contrast-mode.The same laser irradiation was required before each observation.After the images were acquired,the images of ROI were quantitatively analyzed with “average gray scale” using DFY ultrasound imaging analysis software.RESULT:The PA imaging of LNs was obvious at 15 min after CNPs injection,and the whole signal of LNs was gradually enhanced after 1 h,the better PA-signal enhancement was acquired at 24 h,furthermore,the results showed that even at 48 h,the desirable PA imaging in LNs was also observed.However,It was shown that the NPs group had no effect in enhancing PA imaging,and the CNs group had the same or better effect in enhancing PA imaging.PA signal of the images was also quantitatively analyzed,and the results were consistent with that observed in PA images.To US imaging,It was noticed that the signal of LNs began to enhance from 15 min in contrast-mode,and the best enhancement effect was obtained at about 30 min.At 1 h and 2 h after injection,the effect of contrast-enhanced ultrasonography(CEUS)was still good and stable,but a bit weaker compared with that at 30 min.However,It was obviously shown that both of the control groups had no effect in CEUS.ROI of the images was quantitatively analyzed with DFY,and the result was consistent with that observed in the CEUS images.CONCLUSION:CNPs have the ability of enhancing PA and US imaging in lymph nodes,and the enhanced imagings were quite related to the time.Hence,CNPs is potential to be a PA/US dual-modalilty imaging contrast agents for LN.SECTION Ⅲ CARBON NANOPARTICLES-LOADED PHASE-TRANSITION NANODROPLETS IN STAINING LYMPH NODE AND PHAGOCYTOSIS TEST IN VIVOOBJECTIVE:Observing the staining of LN with CNPs,and the CNPs phagocytosed by macrophage in LN,to explore the ability of CNPs in staining LN and the possibility of CNPs phagocytosed by macrophage as a way that the CNPs enter into the LN.METHOD:Popliteal fossa lymph node metastasis model with VX2 tumor in rabbits was established,and tumor-bearing rabbits were used for experiments when popliteal fossa lymph nodes could be palpated about 3 weeks after tumor inoculation.Six VX2 rabbits were randomly divided into two groups(three in each group).After intravenous anesthesia,the CNPs(2 ml,20 mg/ml)and saline(2 ml)were respectively injected via foot pad.One hour after the injection of CNPs,the LNs were removed and examinated with the naked eye.Another 3 VX2 rabbits were also injected with CNPs(2 ml,20 mg/ml)in accordance with the same method.After 48 h,LNs were removed and soaked in formalin,sectioned and stained with hematoxylin and eosin(HE)for pathological examination.RESULT:By gross examination with the naked eye,it was apparent that the popliteal LNs with CNPs injection were stained black,while the LNs with saline injection as control were not stained.After pathologic examination,it was shown that a large number of CNPs were phagocytosed by macrophage,while there were still many CNPs around the tumor cells.It has been found that there is no obvious neutrophil infiltration in both of the two images.CONCLUSION:After subcutaneous injection of CNPs,the LNs can be dyed black,which is benefit to LN detection.It was shown that CNPs can be phagocytosed by macrophage in vivo,which could be an important way of CNPs entering into LNs.However,CNPs did not cause acute inflammation in LNs.PART Ⅲ PHOTOTHERMAL THERAPY OF CARBON NANOPARTICLES-LOADED PHASE-TRANSITION NANODROPLETS IN VITRO AND IN VIVOOBJECTIVE:Observing the effect of CNPs,CNs,NPs on breast cancer cells in vitro after laser irradiation,to explore the therapeutic effect due to the photothermal effect of CNPs;Observing the effect of CNPs,CNs,NPs on metastatic lymph nodes in vivo after laser irradiation,to explore the therapeutic effect of metastatic lymph nodes which was caused by the photothermal effect of CNPs.METHOD: CNPs(20 μl,20 mg/ml),NPs(20 μl,same PLGA concentration as CNPs),CNs(20 μl,same CNs concentration as that incorporated in CNPs)were incubated with MDA-MB-231 cells for 12 h.Then,they were irradiated with laser(808 nm,1 W/cm2)for 30 s,60 s and 120 s.The test was divided into 3 groups in accordance with the added material,then,every group was divided into another 3 groups in accordance with irradiation time.The cell apoptosis rate of the 9 groups were tested with flow cytometry.Popliteal fossa lymph node metastasis model with VX2 tumor in rabbits was established,and tumor-bearing rabbits were used for experiments when popliteal fossa lymph nodes could be palpated about 3 weeks after tumor inoculation.New Zealand white rabbits with detectable popliteal fossa lymph nodes were anesthetized.The rabbits were subcutaneously injected with CNPs(2 ml,20 mg/ml),NPs(2 ml,same PLGA concentration as CNPs),and CNs(2 ml,same CNs concentration as that incorporated in CNPs),the NPs and CNs groups were taken as control.Then,the LN areas were irradiated with laser(808 nm,1 W/cm2)for 2 min,4min and 8 min.The test was divided into 3 groups in accordance with the added material,then,every group was divided into another 3 groups in accordance with irradiation time.All of the lymph nodes were excised,sectioned and stained with HE for pathological examination.One week after irradiation,colour,necrosis,ulceration of the skin were observed.RESULT:With the increase of irradiation time,MDA-MB-231 cells apoptosis rate increased in CNPs group.The cell apoptosis rate of CNPs group was significantly higher than that of NPs group in the case of different irradiation time(P < 0.05).However,The cell apoptosis rate of CNPs group was significantly higher than that of CNs group in the case of different irradiation time(P < 0.05).Lymphatic metastasis was found in all groups under HE examination.Metastatic foci was expressed in good condition in group with NPs.However,massive areas of cell apoptosis were found in groups with CNPs and CNs.With the extension of irradiation time,the area of cell apoptosis was also enlarged.In addition,the rabbit skin was found without redness,necrosis and ulceration till to one week after laser irradiation.CONCLUSION : CNPs generated photothermal effect after laser irradiation,which had obvious inhibitory effect on breast cancer cells in vitro,and with the extension of irradiation time,the inhibition enhanced.CNPs generated obvious photothermal effect in vivo,which had obvious inhibitory effect against the lymph nodes metastasis,and with the increase of laser irradiation time,the inhibitory effect increased.The energy of the laser did not cause redness,necrosis and ulceration of the skin,which confirmed the safety of the laser energy.