Study On Low Frequency Focused Ultrasound Combined With GA/PLGA-CMBs Targeting Therapy For Glioma

BackgroundGlioma is the most common intracranial tumor.Most of the patients are treated by comprehensive treatment,mainly by operation,and postoperative chemotherapy is one of the important methods,but the toxic and side effects of tissues and organs are obvious.Gamboxylic acid（GA）is a new broad-spectrum antineoplastic drug,which has some disadvantages such as poor water solubility and low bioavailability.Focused ultrasound（FUS）combined with drugloaded microbubbles is a targeted drug delivery technique,which can target the opening of bloodbrain barrier（BBB）and blood-brain tumor barrier（BBTB）,and further target drug release to brain tumor areas.ObjectiveThe GA/PLGA-CMBs microbubble complex containing GA was constructed,and the BBB was opened by ultrasound combined with GA/PLGA-CMBs with certain parameters.Second ultrasound combined with GA/PLGA blasting cavitation targeted release of GA,glioma cell membrane "sound pore effect",improve the permeability and effect of GA.To achieve the purpose of in vivo and targeted treatment of gliomas and reduce the toxic and side effects of GA,and provide new ideas for targeted therapy of gliomas.Methods1.GA/PLGA nanoparticles containing GA were prepared by double emulsification method,cationic microbubbles（CMBs）with positive charge were prepared by thin film hydration method,and GA/PLGA-CMBs microbubble composites were prepared by positive and negative potential attraction.Malvern particle size analyzer,transmission electron microscope（TEM）,scanning electron microscope（SEM）and small animal ultrasound system were used to detect the potential,particle size,morp Hology and in vivo imaging of GA/PLGA-CMBs.2.The optimal ultrasonic time and intensity parameters for promoting drug release were determined.The viability and apoptosis rate of tumor cells were detected by CCK-8 reagent and flow cytometry,and the survival status of tumor cells was observed.3.BALB/c mice aged 6-8 weeks were selected.GA/PLGA-CMBs suspension was injected into the caudal vein and EB solution was injected into the caudal vein to evaluate the effect of ultrasound combined with GA/PLGA-CMBs on opening the blood-brain barrier.The biosafety was evaluated by hue staining and PI fluorescence apoptosis detection.4.GA/PLGA,CMBs and GA/PLGA-CMBs suspensions were injected into the tail vein of 7-week-old BALB/c mice,and the imaging effect was detected by B-mode ultrasound.The distribution of drugs in tissues was analyzed by high performance liquid chromatograp Hy（HPLC）.U87-GFP-Luc-Puro cells were constructed by double fluorescein labeling,and the models were made in 6-7-week-old BALB/c nude mice.Small animal in vivo imaging（IVIS）was used to determine whether the model was successful or not.The anti-tumor effect was evaluated by immunohistochemical technique,IVIS and statistical survival rate.Results1.The surface of GA/PLGA has negative charge and has the ability of in vivo imaging,while the surface of CMBs and GA/PLGA-CMBs has positive charge and has better imaging effect in vivo.2.Under the action of ultrasound,the optimum ultrasonic parameters for GA/PLGA-CMBs to produce cavitation effect are Freq: 949 k Hz,p Hase 0.00,cycle: 10.000 k,offset 0 mv,interal:1.000 s,ampl: 100 mvpp,time: 1 min.The 50% inhibitory concentration of GA was 1.5 μ mol/L.Ultrasound combined with GA/PLGA-CMBs can improve the effect of GA on glioma cells.3.When the ultrasonic intensity is 0.5-1.01 MPa,FUS alone or FUS combined with GA/PLGA cannot open BBB.When the ultrasonic intensity is 0.63-1.01 MPa,FUS combined with GA/PLGACMBs can open BBB,and the best ultrasonic intensity is 0.63 MPa.4.The mouse model of brain glioma labeled with double fluorescein was successfully constructed.The results of immunohistochemistry,ultrasound imaging and IVIS showed that FUS+GA/PLGA-CMBs group had the best anti-glioma effect.ConclusionPLGA nanoparticles can improve the solubility and bioavailability of GA.Under the action of ultrasound,GA/PLGA-CMBs microbubble complex can open BBB to kill tumor effectively,and can monitor tumor growth by contrast-enhanced ultrasound in vivo.This kind of drug is loaded by PLGA-CMBs and released by targeted blasting under the action of FUS,which provides a promising platform for drug delivery in the treatment of tumor.