Mage-targeted Gold Nanoparticles For Ptotoacoustic/Ultrasound Imaging Guided Phototherapy In Melanoma

PART 1 THE PREPARATION AND CHARACTERIZATION OF MAGE-TARGETED GOLD NANOPARTICLESObject: to prepare a novel targeting high biocompatible nanoparticles encapsulating gold nanorod and perfluorohexane liquid fluorocarbon(Melanoma-associated antigens targeting gold nanorod and perfluorohexane encapsulated PLGA NPs: MAGE-Au-PFH-NPs),and investigate it's general physical properties,optical properties,phase transformation capability and targeting capability Methods: Multifunctional targeted PLGA nanoparticles encapsured gold nanorod(GNR/Au-NRs)and liquid fluorohexane(PFH)were prepared by double emulsion method.Using Carbon imide method to connect monoclonal antibody targeting malignant melanoma(MAGE-Antibody).Then,the MAGE-Au-PFH-NPs have been synthesused.The morphology size and dispersion of nanoparticles were observed by transmission electron microscopy and scanning electron microscopy.The size and potential of particles were tested by Malvern laser particle size analyser.The encapsulation rate of nanoparticles was determined by UV-Vis-NIR.The maximum absorption peaks of nanoparticles were detected by photoacoustic imaging.MAGE-Au-PFH-NPs were co-incubated with B16 cells of melanoma to observe the targeting capacity of NPs in vitro.The cells viability under different concentrations of nanoparticles on the activity of B16 cells were tested.Results:The dual-mode multifunctional nanomolecular probe was successfully prepared.The morphology and the dispersion was uniform under microscopy.Under scanning electron and transmission electron microscopy,these NPs showed a spherical shape with a uniform size distribution and had a nanosize of less than 500 nm.The diameters of prepared MAGE-Au-PFH-NPs were determined to be 354.27 ± 23.31 nm,and the Zeta potential was(-4.76±3.7)mV as measured by the Malvern laser particle size analyser;The encapsulation efficiency of Au-NRs was 70.61 ± 7.27%.The antibody was successfully connected to the surface of the nanoparticles by indirect immunofluorescence.A large number of targeting nanoparticles(MAGEAu-PFH-NPs)were found surrounding melanoma B16 cell lines under laser confocal microscopy,demonstrated that MAGE-Au-PFH-NPs could specifically target B16 cells.The maximum absorption wavelength for PAI was approximately 780 nm,which corresponded to the gold absorption peak.And under infrared thermal-imaging camera,the MAGE-Au-PFH-NPs suspension temperature increase could reach as high as 71 °C at the MAGEAu-PFH-NPs concentration of 25 mg/mL within 5 min of NIR irradiation,and under laser irradiation,the MAGE-Au-PFH-NPs were induced to form microbubbles,while no enhancement was found in the MAGE-Au-NPs group.After 24 hours of co-incubation with B16 cells of different concentrations of nanoparticles,the NPs at tested concentrations did not affect cell viability in each group as shown in CCK-8 test.Conclusion:In this study,the Au-NRs and PFH were encapsulated in a PLGA shell through the double emulsion method with a high encapsulation efficiency and successfully connected the MAGE-antibody.The NPs have uniform shape,high biocompatibility and photothermal conversion efficiency,and could specifically target B16 cells.Therefore,MAGE-Au-PFH-NPs is a new kind of multi-functional target nanometer probe,laying an experimental foundation for subsequent enhancement of photoacoustic and ultrasonic imaging-guided photothermal therapy of tumorsPART 2 STUDY ON THE MAGE-AU-PFH-NPS FOR ENHANCEMENT OF PHOTOACOUSTIC AND ULTRASONIC IMAGINGObject:To study the MAGE-Au-PFH-NPs for enhancement of photoacoustic/ultrasonic imaging in vitro and in vivoMethods:Agarose gel model were used in vitro study,to evaluate the photoacoustic(PA)performance of MAGE-Au PFH-NPs,a Vevo LAZR Photoacoustic Imaging System(Visual Sonics Inc.,Toronto,Canada)equipped with an LZ250(fibre-optic bundles: 25.4 1.25 mm;focal depth: 10 mm;centre frequency: 21 MHz;axial resolution: 75 mm)probe was used to acquire PA and ultrasound images.The quantified PA signal intensities within a region of interest(ROI)in each image were then analysed by Vevo LAZR software.For different concentrations of MAGE-Au-PFH-NPs dispersed in PBS ranging from 0 to 25 mg/m L,the 780-nm wavelength was used to acquire the PA images.The PA intensities of each image were measured.B-Mode ultrasound and contrast-enhanced ultrasound(CEUS)-mode images of the NPs were recorded 10 min after NP administration using a Parkson Ultrasound System(Esaote My Lab 90,Genoa,Italy).The NPs were then exposed to an 808-nm laser for 10 min at a power density of 1.00 W/cm2,and B-mode ultrasound and CEUS-mode images were recorded again.In vivo,The photoacoustic signal of tumour-bearing mice was acquired using the Vevo LAZR Photoacoustic Imaging System as demonstrated above.B-Mode ultrasound and CEUS-mode images of the tumour were recorded 30 min after NP administration using the Parkson Ultrasound System(Esaote My Lab 90,Genoa,Italy).The tumours were then exposed to an 808-nm laser for 10 min at a power density of 1.00 W/cm2,and B-mode ultrasound and CEUS mode images were recorded again.Results:In vitro,the photoacoustic signal of NPs was gradually enhanced as the nanoparticle concentration increased.The NPs showed a concentrationdependent increase in absorbance intensity,which followed a linear relationship,as described by the following linear correlation equation: Y =0.0181 X + 0.1383(R2 = 0.8836).Under laser irradiation,the NPs were induced to form microbubbles,remarkably increasing the echogenicity and thus leading to a significant enhancement of ultrasound imaging in the MAGE-Au-PFH-NPs group,while no enhancement was found in the MAGE-Au-NPs group.In vivo,The PA signal increased gradually with time(25 mg/m L,100 ?L)and reached the peak at 2 h after the tail vein injection of MAGE-AuPFH-NPs,indicating the gradual NPs accumulation within the tumor region.While no PA signal appeared at 1 h after injection with Au-PFH-NPs(25 mg/m L,100 ?L),only a weak signal was shown at 2 h post-injection and disappeared after 24 h post injection.The PA signal intensities in the ROI were further quantitatively analyzed.The PA intensity value at 2 h in the MAGE-Au-PFH-NPs group was higher than that in the Au-PFH-NPs group(P < 0.05).The in vivo PAI experiment showed that the targeted NPs accumulated at the tumor site earlier and for a longer time.The ultrasound signals in B-mode and CEUS imaging were significantly enhanced at the tumor site in the MAGE-Au-PFH-NPs group 10 min after laser irradiation.No enhancement of the ultrasound signal was found in the MAGE-Au-NPs groupConclusion:In this part of study,It is proved that the targeted MAGE-Au-PFH-NPs has obvious photoacoustic imaging effect,can enhance ultrasound imaging and specifically target melanoma,and can be used as photoacoustic/ultrasound dual-mode imaging contrast agent for the diagnosis and treatment of tumorsPART 3 STUDY ON MAGE-AU-PFH-NPS FOR IMAGING-GUIDED PHOTOTHERMAL ABLATION OF MELANOMAObject:To study in vitro photothermal ablation against B16 cells,to investigate the therapeutic effect of photothermal ablation combined with phase transition on melanoma in mice,and to explore the prospect of MAGE-AuPFH-NPs as an integrated contrast agent for photoacoustic/ultrasonic imaging and treatmentMethods:The study was divided into two parts: the in vitro study was as follows as below:(1)Photothermal capability of NPs was evaluated by laser irradiation using 808 nm laser in vitro.Besides,different concentration of NPs and different power densities of the 808 nm laser were adopted for irradiation in 5 min.The infrared radiation(IR)thermal images and temperature changes were recorded by an infrared thermal-imaging camera(2)To test the photothermal ablation effects in vitro,the mouse melanoma B16 cells were inoculated to the confocal-specific cell-culture dishes,and were divided into 4 groups: normal saline group(NS),Laser group(Laser only),MAGE-Au-PFH-NPs plus Laser group(NPs + Laser),and nanoparticles(MAGE-Au-PFH-NPs only)group.Both Laser group(Laser only)and MAGE-Au-PFH-NPs plus Laser group(MAGE-Au-PFH-NPs + Laser)receive 808 nm laser irradiation.The cells of four groups were scanned by confocal microscopy after co-staining with Calcein AM and propidium iodide.The in vivo study as followed:(1)To evaluate the in vivo photothermal efficiency of MAGE-Au-PFH-NPs,tumor-bearing mice were randomly divided into three groups: MAGE-Au-PFH-NPs,Au-PFH-NPs and NS group.MAGE-Au-PFH-NPs and Au-PFH-NPs groups which were intravenously injected with 100 ?L MAGE-Au-PFH-NPs and Au-PFH-NPs respectively,NS groups were intravenously injected with 100 ?L NS.The tumours were exposed to an 808-nm laser for 10 min at power density of 1.0 W/cm2.The temperature changes in tumors and infrared radiation(IR)thermal images were recorded by an infrared thermal-imaging camera.(2)For evaluating the in vivo photothermal efficiency and ODV efficiency of MAGE-Au-PFH-NPs,the tumor-bearing mice were divided into four groups randomly including Laser only,Au-NPs +Laser,Au-PFH-NPs+Laser,and MAGE-Au-PFH-NPs + Laser group.The 4 groups were injected with saline solution,Au-NPs suspension,Au-PFH-NPs suspension and MAGE-AuPFH-NPs suspension respectively,then all the mice were anesthetized for PTT evaluation.The tumor-volume change of four groups was monitored with a digital camera and the weight of each mouse was recorded every other day after PTT.One mouse of each group was sacrificed 24 h post treatment,the tumor issues were collected and were stained with H&E,Td T-mediated d UTP Nick-End Labeling(TUNEL)and Proliferating Cell Nuclear Antigen(PCNA)for histopathology analysis.CEUS was used to monitoring the imaging of the tumor ablation effect.Results:In vitro study:Under infrared thermal-imaging camera,the MAGE-Au-PFH-NPs suspension temperature increase could reach as high as 71 °C at the MAGEAu-PFH-NPs group.In contrast,the temperature of normal saline(NS)showed almost no temperature increase.The temperature elevated significantly with the increase of NIR irradiation power and the increase of concentration.All the cells showed a strong red fluorescence in the MAGE-Au-PFH-NPs + Laser group.In contrast,no dead cells were found in the control group,.Thimbleful dead cells were found in the Laser only and MAGE-Au-PFHNPs only groups,as demonstrated by the strong green fluorescence and very weak red fluorescence of PI stainingIn vivo study:The surface temperature of tumors in the MAGE-Au-PFH-NPs+ Laser group increased from 31.6 ± 1.09 °C to 56.1 ± 2.6 °C under irradiation,The Au-PFH-NPs+ Laser group increased from 30.1 ± 1.3 °C to 52.0 ± 2.1 °C under irradiation.Comparatively,the temperature at the tumor region of mice in the Laser only group showed only a slight temperature change.Tumor tissues in the MAGE-Au-PFH-NPs + Laser group were necrotic,leaving black scars in the initial tumor regions.And the black scar disappeared,leaving the complete eradication of tumor after 15 days.While the tumors of the other three groups grew significantly.H&E and TUNEL staining on tumor sections further confirmed serious necrosis of tumor in the MAGE-Au-PFH-NPs + Laser group as compared to the other three groups,From the result of PCNA assay on tumor sections,The MAGEAu-PFH-NPs + Laser group exhibited a significant suppression effecton tumor cell proliferation.The ultrasound signals in CEUS imaging were significantly enhanced at the tumor site in the MAGE-Au-PFH-NPs group during the first laser irradiation.And the enhanced ultrasound signals gradually decreased into a non-detecting state as the treatment progresses till the 15 th day.Conclusion:MAGE-Au-PFH-NPs have high Photothermal conversion efficiency in vitro and in vivo.Combined with PTT/ODV,the tumor can be removed completely and the therapeutic effect can be monitored by ultrasound.This MAGE-Au-PFH-NPs contrast agent could be used as a multifunctional theranostic nanoparticles for highly efficient imaging-guided photothermal ablation of tumor.