Targeted Methotrexate-loaded Plga Nanobubbles For Ultrasound Imaging And Synergistic Therapy Of Trophoblast Cells During HIFU Ablation

PARTⅠ PREPARATIONAND CHARACTERIZATION OFmAbHLA-G/MTX/PLGANANOBUBBLESObjective To Prepare a kind of targeted methotrexate (MTX)-loadedPLGA nanobubbles (mAbHLA-G/MTX/PLGA NBs), and to detect theirphysical and acoustic properties.Methods mAbHLA-G/MTX/PLGA nanobubbles were prepared by a doubleemulsion (water/oil/water) evaporation process and a carbodiimidecovalent conjugation method. The morphological characterization ofmAbHLA-G/MTX/PLGA nanoparticles was analyzed by light microscopeand Scanning Electron Microscope (SEM). Transmission ElectronMicroscopy (TEM) after negative staining with sodium phosphotungstatesolution (2%, w/w) was used to estimate the shape of nanoparticles. ALaser Particle Size Analyzer System was used to obtain the mean diameter, size distribution and Zeta potential. High performance liquidchromatography (HPLC) method was established for the determination ofdrug-loading and encapsulation efficiency. Sonication experiment in vitrowas performed to assess the MTX release behavior in the sound field,including HIFU and low-frequency ultrasound from gene transfectioninstrument (Model UTG1025, Institute of Ultrasound ImagingofChongqing Medical Sciences, Chongqing, China). A gel mold which hadseveral holes with a depth of2cm on the edge was used to assessultrasound imaging of mAbHLA-G/MTX/PLGA NBs in vitro with differentconcentrations (200mg/ml，100mg/ml，50mg/ml，25mg/ml，10mg/ml) anddifferent time points (0h，2h，4h，8h，16h，24h，48h).Results The high dispersity and well-defined spherical morphology ofmAbHLA-G/MTX/PLGA NBs can be directly observed in SEM image. Thepresence of hydrophilic methotrexate in the cores of nanobubbles wasobserved by Transmission electron microscope (TEM). The mean diameterof mAbHLA-G/MTX/PLGA nanobubbles was in a range of477.6±119.7nm(standard deviation) with a polydispersity index of0.171, and zeta potentialof mAbHLA-G/MTX/PLGA NBs was-5.62±5.36mV. MTX loading mountand encapsulation effi ciency were measured by HPLC. The encapsulationeffi ciency of MTX was44.11±1.27%and the loading efficiency was4.41±0.13%(w/w) in the mAbHLA-G/MTX/PLGA NBs. Comparatively, theencapsulation and loading efficiency of MTX/PLGA NBs were46.52± 1.66%,4.65±0.17%(w/w). The amount of MTX released frommAbHLA-G/MTX/PLGA nanoparticles (de-gassed) after low-frequency UStreatment is not different from that of mAbHLA-G/MTX/PLGA NBs ormAbHLA-G/MTX/PLGA nanoparticles (de-gassed). Although HIFU canslightly increase the release of MTX from mAbHLA-G/MTX/PLGAnanoparticles (de-gassed), there is significant difference between the HIFUplus mAbHLA-G/MTX/PLGA NBs group and the HIFU plusmAbHLA-G/MTX/PLGA nanoparticles (de-gassed) group (P <0.001). Thelow-frequency ultrasound irritation can accelerate the release of MTX frommAbHLA-G/MTX/PLGA NBs, while the effect that HIFU promotedmAbHLA-G/MTX/PLGA NBs to unload MTX was significantly differentcompared with that of mAbHLA-G/MTX/PLGA NBs irritated bylow-frequency US and mAbHLA-G/MTX/PLGA NBs control group. It justtook3h that50%of MTX was released from the NBs after sonication inHIFU group and approximately80%of MTX was unloaded after72hpost-irritation. However, only about47.8%of the encapsulated MTX wasreleased from mAbHLA-G/MTX/PLGA NBs by low-frequency US at thetime point of72h after sonication. Compared with de-gas and de-ionizedwater, the mean DB of pure PLGA NBs, MTX/PLGA NBs andmAbHLA-G/MTX/PLGA NBs of concentration gradient had significantdifference (p <0.05). However, the same concentration of pure PLGA NBs,MTX/PLGA NBs and mAbHLA-G/MTX/PLGA NBs did not exhibit the significantly different mean echo intensities (p>0.05). With change ofconcentration of mAbHLA-G/MTX/PLGA NBs, the mean echo intensitieshad significant difference (p <0.05). Compared with de-gas and de-ionizedwater, the prepared mAbHLA-G/MTX/PLGA NBs exhibited strong echoesfrom0h to48h. And there is no significant difference in the echo intensityat the different time points in24h (p>0.05), while there is significantdifference between24h and48h (p<0.05).Conclusion The mAbHLA-G/MTX/PLGA nanobubbles were successfullyprepared. The mAbHLA-G/MTX/PLGA NBs have the properties of nano size,uniform shape, and well dispersion. Compared with the ordinary ultrasound,HIFU more effectively promoted the mAbHLA-G/MTX/PLGA nanobubblesto release MTX. The mAbHLA-G/MTX/PLGA ultrasound contrast agent canalso enhance ultrasonic imaging in vitro. Because of the properties of highstability and well acoustic performance, the mAbHLA-G/MTX/PLGAnanobubbles are a kind of multifunctional contrast agent with goodapplication prospect. PART Ⅱ THE EFFECT OF mAbHLA-G/MTX/PLGANANOBUBBLES COMBINATION WITH HIFU ONTROPHOBLAST CELLS IN VITROObjective To detect the expression and localization of HLA-G protein inthe JEG-3cell line of human chariocarcinoma, to evaluate the targetingability of mAbHLA-G/MTX/PLGA NBs in vitro, and to explore the effect ofmAbHLA-G/MTX/PLGA nanobubbles combination with high intensityfocused ultrasound on the proliferation and invasion of JEG-3cell line.Methods JEG-3cells were cultured in DMEM/F12media with10%fetalbovine serum under5%CO2atmosphere at37℃. Immunofluorescenceexamination was used to detect the the expression and localization ofHLA-G protein in the JEG-3cells. DiI-mAbHLA-G/MTX/PLGAnanobubbles were prepared. The experiment of targeting efficiency wasdivided to three groups:(1) experimental group: DiI-mAbHLA-G/MTX/PLGANBs;(2) control group I: the cells were blocked with excess mAbHLA-G(1mg/mL) for2h before addition of DiI-mAbHLA-G/MTX/PLGA NBs;(3)control group II: the cells were added with DiI-MTX/PLGA NBs. JEG-3cells at a density of5×104cells/mL were seeded into wells of a6-wellplatecontaining a coverslip in each well. After24h of culture, the cells werewashed with PBS three times, and divided into the three groups toco-incubate for targeting experiment and cell phagocytosis experiment. Confocal laser scanning microscopy (CLSM) was used to observe thedistribution of mAbHLA-G/MTX/PLGAnanobubbles.The experiment of the treatment of mAbHLA-G/MTX/PLGA NBs plus HIFUon JEG-3cells in vitro was divided into12groups:(1) PBS,(2) pure PLGANBs,(3) MTX/PLGA NBs,(4) methotraxate,(5) mAbHLA-G/PLGA NBs,(6)mAbHLA-G/MTX/PLGA NBs,(7) PBS plus HIFU,(8) pure PLGA NBs plusHIFU,(9) MTX/PLGA NBs plus HIFU,(10) methotraxate plus HIFU,(11)mAbHLA-G/PLGA NBs plus HIFU,(12) mAbHLA-G/MTX/PLGA NBs plusHIFU. After treatment in each group, cell apoptosis and cell cycle weretested by flow cytometry. The protein expression of Bax, Bcl-2, Caspase3,MMP2and TIMP-2were detected by Western blotting. And the level ofgene expression of Caspase3and MMP2were tested by fluorescencequantitative PCR techonology.Results The HLA-G overexpressed on the membrane and inside thecytoplasm of JEG-3cells. From CLSM images, it could be directlyobserved that large amounts of DiI-labeled mAbHLA-G/MTX/PLGA NBs(red fluorescent dots) were present in the cytoplasm of JEG-3cells. Thethree-dimensional fluorescence reconstruction further demonstrated that thetargeting NBs were located intracellularly, which indicated thatmAbHLA-G/MTX/PLGA NBs can target the HLA-G on the membranes ofJEG-3cells and are uptaken by cells subsequently.In vitro treatment experiment, compared with other groups, HIFU+ mAbHLA-G/MTX/PLGA NBs group presented the strongest early apoptosisand late apoptosis of JEG-3cells. Cells cycle was obviously blocked in Sphase. The expression of Bax and Caspase3in the HIFU+mAbHLA-G/MTX/PLGA NBs group was significantly higher than othergroups, and the expression of Bcl-2was lower than other groups. The cellinvasion ability decreased significantly in the HIFU+mAbHLA-G/MTX/PLGA NBs group. The expression of MMP2in the HIFU+mAbHLA-G/MTX/PLGA NBs group was lowest, and the expression ofTIMP-2was significantly higher than the rest of the groups.Conclusion The mAbHLA-G/MTX/PLGA nanobubbles had well targetingproperty, and they could enhance the ultrasonic cavitation effect andmechanical effect by improving the surrounding environment. Meanwhile,ultrasound effects could promote the release of chemotherapy drugs fromnanobubbles. HIFU combined mAbHL-G/MTX/PLGA NBs significantlyinhibited the proliferation and invasion of JEC-3cells, and the mechanismmay be through the influence on the expression of apoptosis and invasionrelated proteins. PART Ⅲ mAbHLA-G/MTX/PLGA NANOBUBBLES FORSYNERGISTIC TARGETED THERAPY OFTROPHOBLASTCELLS DURING HIFUABLATION IN VIVOObjective To investigate the expression and localization of HLA-Gprotein in JEG-3cells subcutaneous transplant tumor model. To evaluatethe ultrasound imaging and targeting ability of mAbHLA-G/MTX/PLGA NBsin vivo, and to explore the effect of mAbHLA-G/MTX/PLGA nanobubblesfor synergistic targeted therapy of subcutaneous transplant tumor duringHIFU ablation in nude mice.Methods We purchased specific-pathogen-free level BALB/c (strain nu/nu)nude mice (n=143, female,4-6weeks old, weight15-20g) for the animalexperiment. Cultured JEG-3cells (1～2×106) were harvested inlogarithmic growth phase and resuspended in0.1m L PBS. The suspensionwas immediately injected subcutaneously into the flank of nude mice todevelop choriocarcinoma subcutaneous transplant sarcoma model. Twoweeks post-injection, the maximal diameter of the tumor reached0.8-1.0cm. The harvested subcutaneous transplant tumor tissues of nude micewere fixed in10%neutral buffered formalin, routinely processed, paraffinembedded and sreptavidin peroxidase (SP) immunohistochemical stainingfor HLA-G. The ultrasound imaging before and after injection ofmAbHLA-G/MTX/PLGA nannobubbles (post-0h and post-24h), respectively, were observed by the diagnostic US unit of HIFU system. The In VivoImage System (IVIS) was used to detect the targeting distribution ofDiI-labeled mAbHLA-G/MTX/PLGA nanobubbles at different time points(3h,24h,48h) after injection via tail intravenous. The experiment of thetreatment of mAbHLA-G/MTX/PLGA NBs plus HIFU on subcutaneoustransplant sarcoma model in vivo was divided into12groups (n=10):(1)Saline,(2) pure PLGA NBs,(3) MTX/PLGA NBs,(4) Methotraxate,(5)mAbHLA-G/PLGA NBs,(6) mAbHLA-G/MTX/PLGA NBs,(7)Saline plusHIFU,(8) pure PLGA NBs plus HIFU,(9) MTX/PLGA NBs plus HIFU,(10) Methotraxate plus HIFU,(11) mAbHLA-G/PLGA NBs plus HIFU,(12)mAbHLA-G/MTX/PLGA NBs plus HIFU. During HIFU treatment, tissue ofablation appeared strong echo in US image. So pre-and post-ablation,gray-scale of the targeted zone was automatically compared by Gray Val1.0software affiliated to HIFU device. The groups from1st to6th groupwent through this same process, however never received ultrasound. AfterHIFU irritation immediately, three mice in each group from7th to12thgroup were randomly euthanized. The tumor tissues were removedimmediately, and the volume of coagulated tissues in the tumors wascalculated. After HIFU ablation, the remaining mice in the whole twelvegroups were fed for another24h to evaluate the efficiency of HIFUcombination with mAbHLA-G/MTX/PLGA NBs for killing tumor cells. The5mice in each group were remained to observe the growth of tumor and the survival analysis of tumor-burdened mice. Then, all the remaining micewere euthanized and the tumor tissues were removed immediately andfixed in10%neutral buffered formalin, routinely processed, paraffinembedded and sectioned. Hematoxylin and eosin stain on tissues sectionswas used of histopathological analysis. Immunohistochemical staining withantibodies against proliferating cell nuclear antigen (PCNA) was done toassess tumor cell proliferation. To evaluate apoptosis, TUNEL assaymethod was performed on these tissue sections. The proliferating index (PI)and apoptotic index (AI) were calculated as the ratio of positively stainedcells to all cells, which were obtained from at least5randomly selectedhigh power fields (×400magnification) by blind observers.Results The HLA-G overexpressed on the membrane and inside thecytoplasm of tumor cells. The mAbHLA-G/MTX/PLGA nanobubbles ascontrast agent produced stable ulstrasound iamge in vivo. From the IVISimage, they could specifically arrive at the target tissue, improve theresidence time in the target area and increase the release quantity ofchemotherapy drugs in the target area. The mAbHLA-G/MTX/PLGAnanobubbles can significantly enhance HIFU ablation. The mean graydifference after ablation was significantly higher in HIFU+mAbHLAG/MTX/PLGA NBs group. The ablation volume was significantly greaterin HIFU+mAbHLA G/MTX/PLGA NBs group than that in the othergroups. HIFU combined mAbHLA-G/MTX/PLGA NBs effectively inhibited the proliferation of tumor cells and promoted cell apoptosis toextend the survival cycle of tumor-burdened mice (median survival=58days).Conclusion The mAbHLA-G/MTX/PLGA nanobubbles can enhance HIFUablation effect by changing acoustic environment in the target area, andHIFU thermal effect can promote the release of MTX frommAbHLA-G/MTX/PLGA nanobubbles， which can synergistic targetedtherapy residual tumor cells to inhibit tumor growth and metastasis. ThemAbHLA-G/MTX/PLGA ultrasound contrast agents in combination with highintensity focused ultrasound has opened up a novel strategy for thetreatment of trophoblast cell related diseases.