Multimodal Imaging And Photothermal Synergistic Immunotherapy Of Retinoblastoma With Tuftsin-loaded Carbonized MOF Nanoparticles

Part 1 Preparation and characterization,in vitro and in vivo safety and targeting of CMT NPsObjective CM nanoparticles with optical properties and stable magnetic properties of carbon materials were prepared.Tuftsin was successfully encapsulated to obtain multifunctional nanoparticles CMT,which laid a foundation for further research.Methods CMT nanoparticles were prepared by high-temperature carbonization,acidification and hydration,and mechanical agitation.The morphology of CMT nanoparticles was observed by electron microscopy,the particle size potential was analyzed,the FTIR spectrum was measured,magnetic saturation was measured,the tuftsin encapsulation rate and drug loading rate were measured,and the characterization characteristics of CMT nanoparticles were evaluated by photothermal conversion ability.The in vitro and in vivo safety of nanoparticles was evaluated by the survival of cells coincubated with nanoparticles and blood biochemistry after in vivo injection of nanoparticles and sections.The in vivo and in vitro magnetic targeting ability of nanoparticles was evaluated by observing the uptake rate of nanoparticles in Y79 cells under the action of a magnetic field and the distribution of nanoparticles in tumor-bearing nude mice.Results The size of the CMT nanoparticles was 303.1±48.53 nm,and the zeta potential was-12.1±2.88 mV.The magnetic strength of the CMT nanoparticles was 92.84 emu/g.Fourier transform infrared spectroscopy(FTIR)showed that Tuftsin was successfully encapsulated in CMT nanoparticles.The encapsulation rate and drug loading rate of Tufsin were 29±4.09%and 2.64±0.37%,respectively.After laser irradiation,the temperature of the CMT nanoparticle suspension reached 57.5℃.The survival rates of ARPE-19 cells and Y79 cells were greater than 90%after coincubation with nanoparticles.There were no obvious abnormalities in routine blood tests or blood biochemistry in nude mice after injection of CMT nanoparticles.In vivo and in vitro targeting studies found that CMT nanoparticles had good magnetic targeting,and the accumulation of nanoparticles in the tumor reached a maximum at 4 h after injection of CMT in vivo.Conclusion Multifunctional drug-carrying carbonized nanoparticles with uniform size,good dispersibility and stability were successfully prepared.The nanoparticle has good photothermal conversion ability and is an ideal material for photothermal therapy.The nanoparticle has good safety and targeting properties in vivo and in vitro and is a kind of safe,magnetic targeting multifunctional nanoparticle.Part 2 In vitro and in vivo bimodal imaging of CMT NPsObjective To investigate the in vitro and in vivo photoacoustic and NMR dual-mode imaging capabilities of CMT nanoparticles and to provide a basis for the diagnosis and treatment of RB.Methods Six groups of samples(CMT NPs(100,200,400,800μg/ml),CM NPs(800 μg/ml),normal saline group,)were photoacoustic imaged in vitro.Seven groups of samples[CMT NPs(5,10,20,40,80μg/ml),CM NPs(80 vg/ml),normal saline group]were examined by a MRI scanner in vitro.In addition,15 Rb subcutaneous tumor-bearing nude mice were randomly divided into 3 groups:CMT NPs(800 μg/ml),CM NPs(800 μg/ml)and normal saline groups.After tail vein injection of nanoparticles or normal saline in each group,photoacoustic imager and MAGNETIC resonance scanner were used to scan the tumor sites at different time points,and the analysis images and data results were recorded.Results(1)Photoacoustic imaging:In the in vitro study,the photoacoustic signal increased with increasing CMT NPs concentration,and there was no significant difference between the CM NPs group and the CMT NPs group,while the normal saline group had no photoacoustic signal.In in vivo studies,the photoacoustic signals of naked mice were gradually enhanced 1 h after intravenous injection of CMT NPs and CM NPs,peaked at 4 h,and then gradually decreased.The photoacoustic signals were still present 24 h after injection of CMT NPs and CM NPs in the normal saline group.(2)Magnetic resonance imaging:In vitro studies,T2-weighted MRI signals decreased with the increase of CMT NPs concentration.There was no significant difference between the CM NPs group and the CMT NPs group,while the normal saline group showed a high signal.In the in vivo study,the T2-weighted MR signals of nude mice gradually decreased after intravenous injection of CMT NPs and CM NPs for 1 hours and reached the lowest at 4 hours,while the normal saline group continued to have high signals.Conclusion CMT nanoparticles exhibit excellent photoacoustic and magnetic resonance imaging properties,which provides application value for the diagnosis of RB.Part 3 Study of laser combined with CMT nanoparticle photothermal/immunotherapy for retinoblastomaObjective To investigate the tumor killing and immune activation ability of a laser combined with CMT nanoparticles,to evaluate the effectiveness of photothermal/immunotherapy with a modified nanoparticle system and to provide a basis for the treatment of RB.Methods Apoptosis of Y79 cells in 6 groups[control group,Tuftsin,CMT NPs,laser,laser+CM NPs,laser+CMT NPs]was detected.After in vivo treatment of subcutaneously implanted RB tumors,the tumor cell morphology,apoptosis index and proliferation index were observed,and the effectiveness of the laser combined with CMT nanoparticles in killing retinoblastoma cells in vivo and in vitro was evaluated.The expression of CD 16/32 and CD206 in Ana-1 macrophages after treatment in each group was detected,and the expression of CD86 and TNF-α in tumor tissues was detected after treatment in vivo to evaluate the ability of CMT nanoparticles in the laser combined group to activate macrophage immunity in vivo and in vitro.Results In vitro,it was found that the apoptosis rate of Y79 cells was higher after laser irradiation combined with CM nanoparticles and laser irradiation combined with CMT nanoparticles.In the in vivo study,after subcutaneous implantation of RB tumors in each group,the tumor volume of the laser combined with CMT group gradually decreased,and tumor cell swelling with a large amount of nuclear fragmentation and obvious necrosis were observed under section.Compared with the other groups,the tumor cell proliferation index was the lowest,and the apoptosis index was the highest.In addition,after anA-1 macrophages were treated in each group,the CD206-positive cells in all groups were low,and the proportion of CD16/32-positive cells in the laser combined with CMT nanoparticle group was higher than that in the other groups.After in vivo treatment,the expression of CD86 and TNF-α in tumor tissues of the laser combined with CMT nanoparticles group was higher than those of the other groups.Conclusion CMT NPs enhance the therapeutic effect of laser therapy,which can achieve dual-mode guided laser therapy of RB,inhibit tumor growth and achieve good therapeutic effects.