Multifunctional Nanoparticles For Multimodal Imaging-guided LIFU/immuno-synergistic Retinoblastoma Therapy

PART 1 THE CONSTRUCTION,CHARACTERIZATION,SAFETY AND TARGETED RESEARCH OFMULTIFUNCTIONAL NANOPARTICLESObject The gold nanocages modified by sodium citrate and Fe₃O₄nanoparticles with amino terminal were connected by carbodiimide method,and the AuNCs-Fe₃O₄ nanoparticles were constructed as carrier materials.A multifunctional nanoparticle,AuNCs-Fe₃O₄/MDP/PFP,containing MDP and PFP,was prepared by phacoemulsification method.The physicochemical properties,safety and targeting ability of AuNCs-Fe₃O₄/MDP/PFP were tested.Method AuNCs-Fe₃O₄/MDP/PFP nanoparticles were prepared by carbodiimide method and phacoemulsification method.The physical and chemical properties of AuNCs-Fe₃O₄/MDP/PFP were evaluated by observing the morphological characteristics,analyzing the particle size potential,infrared and UV spectra.The magnetic properties,encapsulation efficiency and loading capacity of Fe₃O₄ and MDP,capacity of liquid gas phase transformation and drug release in vitro were also detected.The safety of AuNCs-Fe₃O₄/MDP/PFP in vitro and in vivo was evaluated by the cell survival rate of cells after incubation with nanoparticles and the general situation,blood biochemical indicators of mice injected with nanoparticles.The in vitro and in vivo magnetic targeting ability of AuNCs-Fe₃O₄/MDP/PFP was evaluated by analyzing the cell phagocytosis of nanoparticles and the distribution of nanoparticles in Y79 tumor-bearing mice.Results The results of transmission electron microscopy showed that the AuNCs-Fe₃O₄/MDP/PFP had uniform size and good dispersion,with a particle size of?159.3±33.25?nm and zeta potential of?-15.8±4.64?mV.The FTIR spectrum showed that the AuNCs and Fe₃O₄ were bound by amide bond,the encapsulation efficiency of Fe₃O₄ was?27.11±1.20?%and loading capacity was?35.15±1.01?%.And the nanoparticles had superparamagnetism,the saturation magnetization was 29.9 emu/g.The encapsulation efficiency of MDP was?82.60±2.07?%,and the loading capacity was?4.60±0.11?%.The UV spectra further confirmed that AuNCs successfully loaded with Fe₃O₄ and MDP.The liquid-gas phase transition of AuNCs-Fe₃O₄/MDP/PFP after LIFU irradiation showed that PFP was loaded successfully.In vitro study showed that LIFU promoted the release of MDP from AuNCs-Fe₃O₄/MDP/PFP.The cell viability after incubation with nanoparticles was higher than 90%.There was no abnormality in general condition and blood biochemical test in healthy mice after nanoparticles injection.In vitro and in vivo,it was found that AuNCs-Fe₃O₄/MDP/PFP had good targeting ability under the action of magnetic field.At 4 h after injection,the aggregation of nanoparticles in tumor site reached the maximum.Conclusion AuNCs-Fe₃O₄/MDP/PFP with uniform size,good dispersion,high drug loading and good stability were successfully prepared.The nanoparticles have good optical properties and phase transformation ability and good controlled-release performance in vitro.LIFU irradiation promotes drug release.It has good in vitro and in vivo safety and targeting ability,which shows that it is a safe,stable and targeted multifunctional nanoparticles.PART 2 RESEARCH OF THE MULTIMODE IMAGING ABILITY OF MULTIFUNCTIONAL NANOPARTICLESObject To explore the photoacoustic,ultrasonic and magnetic resonance imaging capabilities of AuNCs-Fe₃O₄/MDP/PFP to provide thepremise for application in RB diagnosis and treatment.Method In vitro photoacoustic,ultrasonic and magnetic resonance imaging were performed on 7 groups of samples [AuNCs-Fe₃O₄/MDP/PFP?50,100,200 and 400 ?g/m L?,AuNCs-Fe₃O₄/MDP?400 ?g/m L?,Au NCs/MDP/PFP?400 ?g/m L?and saline group,n=5] and the image data were analyzed.In addition,the nude mice with RB subcutaneous implant tumor were randomly divided into four groups [AuNCs-Fe₃O₄/MDP/PFP?400 ?g/m L?,AuNCs-Fe₃O₄/MDP?400 ?g/m L?,Au NCs/MDP/PFP?400 ?g/m L?and saline group,n=5].After injecting nanoparticles or saline into tail vein at different times,photoacoustic imaging,ultrasound diagnostic instrument and MRI scanner were used to perform photoacoustic,ultrasound and MRI imaging of the tumor in vivo,and the image data were analyzed.Results Photoacoustic imaging: the photoacoustic signal increased with the increase of the concentration of AuNCs-Fe₃O₄/MDP/PFP in vitro.The photoacoustic signal in the AuNCs-Fe₃O₄/MDP/PFP and AuNCs-Fe₃O₄/MDP was higher than that in the Au NCs/MDP/PFP,and there was no photoacoustic signal in the normal saline group.One hour after the injection of AuNCs-Fe₃O₄/MDP/PFP and AuNCs-Fe₃O₄/MDP,the photoacoustic signal in the tumor gradually increased,peaked at 4 hours,and then decreased.In the Au NCs/MDP/PFP,the photoacoustic signal began to increase after 4 hours,and reached the peak value at 12 hours.Ultrasound imaging: the results of in vitro study showed that the echosignal of nanoparticles in each group reached the maximum at 8 min during 0-10 min of LIFU irradiation,and the echo intensity increased with the increase of the concentration of AuNCs-Fe₃O₄/MDP/PFP.After LIFU irradiation,the echo intensity of AuNCs-Fe₃O₄/MDP/PFP and Au NCs/MDP/PFP was higher than that of AuNCs-Fe₃O₄/MDP and saline.MR imaging: the signal intensity of AuNCs-Fe₃O₄/MDP/PFP was higher in T1 weighted MR imaging,but the effect of different concentrations on signal intensity was not obvious.In T2 weighted MR imaging,the signal intensity decreased gradually with the increase of the concentration of AuNCs-Fe₃O₄/MDP/PFP,showing a negative enhancement effect.In vivo study,the signal intensity of tumors in the AuNCs-Fe₃O₄/MDP/PFP and the AuNCs-Fe₃O₄/MDP decreased gradually at 1 h and reached the lowest at 4 h.However,there was no significant change in tumor signal intensity in Au NCs/MDP/PFP and saline.Conclusion AuNCs-Fe₃O₄/MDP/PFP nanoparticles show excellent photoacoustic,ultrasonic and magnetic resonance imaging characteristics,which has a certain value in the diagnosis of RB.PART 3 EFFICACY OF LIFU COMBINED WITH MULTIFUNCTIONAL NANOPARTICLESULTRASOUND/IMMUNOTHERAPY FOR RETINOBLASTOMAObject To explore the antitumor and immune activation ability of LIFU combined with AuNCs-Fe₃O₄/MDP/PFP to evaluate the effectiveness of ultrasound/immunotherapy of RB with the nanosystem.It provides a basis for clinical application and transformation.Method The survival rate and apoptosis of Y79 cells treated with 8 groups of samples?control,MDP,LIFU,AuNCs-Fe₃O₄/MDP/PFP,AuNCs-Fe₃O₄/MDP+LIFU,AuNCs-Fe₃O₄/PFP+LIFU,Au NCs/MDP/PFP+ LIFU and AuNCs-Fe₃O₄/MDP/PFP+LIFU?were measured.After RB subcutaneous implant was treated in vivo,the tumor tissue structure was observed,the apoptosis and proliferative index of tumor cells were measured,and the killing effect of LIFU combined with AuNCs-Fe₃O₄/MDP/PFP on RB in vitro and in vivo was evaluated.The expression of CD11 c and CD83 in bone marrow-derived DCs,the expression of NOD2 and CD83,the content of IFN-? and MCP-1 in tumor tissue were measured after treatment in vivo,and the ability of LIFU combined with AuNCs-Fe₃O₄/MDP/PFP to activate immune system in vitro and in vivo was evaluated.Results In vitro study,LIFU combined with AuNCs-Fe₃O₄/MDP/PFP,AuNCs-Fe₃O₄/PFP and Au NCs/MDP/PFP had lower cell viability and higher apoptosis rate,while Fe₃O₄ and MDP did not affect the ability ofnanosystem to kill tumor cells in vitro.After the treatment of RB subcutaneous implant tumor in vivo,the tumor volume of AuNCs-Fe₃O₄/MDP/PFP+LIFU gradually decreased.There were many swelling tumor cells,nuclear fragmentation and cll necrosis.Compared with other groups,the tumor cell apoptosis index was the highest,and the proliferation index was the lowest.In addition,CD11 c positive cells were more than 70% in each group after different treatment of DCs,and CD83 positive cells in the AuNCs-Fe₃O₄/MDP/PFP+LIFU and MDP were significantly higher than other groups.After in vivo treatment,the expression of NOD2 and CD83,IFN-? and MCP-1 in the AuNCs-Fe₃O₄/MDP/PFP+LIFU were significantly higher than those in other groups.Conclusion AuNCs-Fe₃O₄/MDP/PFP nanoparticles can enhance the therapeutic effect of LIFU,realize ultrasound/immunotherapy of RB under the guidance of multimodal imaging,inhibit the growth,recurrence and metastasis of tumor,and achieve better therapeutic effect.