Near-infrared Light-triggered Nanoparticles For Multiple Imaging And Photothermal Therapy In Murine Mammary Carcinoma Models

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PREPARATION AND CHARACTERIZATION OF NEAR-INFRARED LIGHT-TRIGGERED NANOPARTICLES(NP-IR780)ObjectiveTo prepare near-infrared light-triggered nanoparticles(NP-IR780)loaded with PFOB and IR780 and to characterize the features.MethodsThe NP-IR780 was developed through thin-film dispersion and ultrasound emulsification,and then the features was characterized for morphology,dispersion,microstructure,particle size,zeta potential,and drug-loading efficiency.NP-IR780 was diluted to different concentrations for near-infrared fluorescence(NIR FL)imaging,photoacoustic(PA)imaging,and CT imaging respectively in vitro.To detect the photothermal properties,the NP-IR780 was irradiated by laser and the temperatures were monitored by an infrared thermal imaging camera.The toxicity of NP-IR780 to 4T1/Luc cells was determined using the CCK-8 assay.The tumor targeting ability of NP-IR780 to 4T1/Luc cells was detected by confocal laser scanning microscopy(CLSM)and flow cytometry(FCM).ResultsThe NP-IR780 was spherical,uniform in size and dispersed well in aqueous solution.The size,zeta potential,and IR780-loading were 266.00±50.20 nm,-23.97±4.40 mV,and 4.37±0.50-%respectively.The NP-IR780 with excellent photothermal properties could be used as a contrast agent for NIR FL,CT and PA imaging in vitro.The 4T1/Luc cells viability exhibited negligible toxicity from 5 ?g/mL to 30 ?g/mL after co-incubation with NP-IR780.Through CLSM and FCM,the NP-IR780 was demonstrated unique targeting ability to 4T1/Luc cells.ConclusionWe have successfully developed a multifunctional nanoplatform(NP-IR780)with good general properties through thin-film dispersion and ultrasound emulsification.The NP-IR780 with excellent photothermal properties and tumor targeting ability could be used as a contrast agent for NIR FL/CT/PA multimode imaging in vitro.PART ? EXPERIMENTAL STUDY OF MULTI-MODE IMAGING OF NEAR-INFRARED LIGHT-TRIGGERED NANOPARTICLES(NP-IR780)ObjectiveTo evaluate the biosafety and tumor targeting ability of NP-IR780 in vivo,and to explore whether it could be used as a contrast agent for NIR FL/CT/PA multiple imaging in vivo.MethodsHealthy female BALB/c mice were divided into 3 groups(5 in each group):Control group(PBS),NP-IR780 low dose group(25 mg/kg),and NP-IR780 high dose group(50 mg/kg).After 14 days of tail vein administration,circulation blood count,biochemical indicators(ALT,AST,TPBL,CK,LDH,CR,and BUN)and organs pathology were analyzed.The multiple imaging studies were performed on a 4T1/Luc mouse orthotopic breast cancer model.Tumor-bearing mice were randomly divided into two groups(n=3 per group):free IR780 group(1.1 mg/kg)and NP-IR780(25 mg/kg,the corresponding IR780 dose was 1.1 mg/kg).After intravenous administration,the bioluminescence images of the tumor and the near-infrared fluorescence images before and after the injection(1 h,3 h,24 h,48 h)were acquired using an in vivo IVIS bioluminescence and fluorescence imaging system to evaluate the NIR FL imaging ability and tumor targeting of NP-IR780 in vivo.To further study the factors of tumor-targeting ability,25 mg/kg NP-IR780 or NP labeled with DiI was injected into 4T1/Luc tumor-bearing mice via the tail vein.At 48 h post administration,the mice were sacrificed,and tumor tissues were removed for frozen sectioning and observed.After confirming the tumor targeting and safety of NP-IR780 in vivo,images were acquired by a Vevo LAZR PA imaging system and a Philips Ingenuity CT system before and after injection(3 h,24 h)to evaluate PA and CT imaging ability.ResultsCompared with the control group,the blood cells(RBC,WBC,and PLT),biochemical indicators(ALT,AST,TPBL,CK,LDH,CR,and BUN)and organs(heart,liver,spleen,lung,and kidney)histopathology of the mice injected with NP-IR780(25mg/kg and 50mg/kg)showed no significant changes,which confirmed the biosafety of NP-IR780 in vivo.The FL images showed both free IR780 and NP-IR780 were targeted to tumor tissues,while the FL signal of NP-IR780 in tumor was not only higher but remained higher for a longer time.Further comparison of the tumor accumulation between NP and NP-IR780 in tumor tissues revealed that the FL signal of the NP-IR780 group was approximately 5 times higher than that of the NP group.PA and CT images confirmed that NP-IR780 gradually aggregated in the tumor area with time,and the PA intensity and CT signal were increased by approximately 3 times and 1.3 times respectively at 24 h compared with pre injection.ConclusionNP-IR780 has biosafety and excellent tumor targeting ability in vivo,and can be used as a contrast agent for NIR FL/CT/PA multimode imaging in vivo.PART ? EXPERIMENTAL STUDY OF NEAR-INFRARED LIGHT-TRIGGERED NANOPARTICLES(NP-IR780)FOR IMAGE-GUIDES CARCINOMA SURGERY AND PHOTOTHERMAL ABLATION OF RESIDUAL TUMOR TISSUESObjectiveTo evaluate the NIR fluorescence imaging ability of NP-IR780 on delineating the tumor regions during surgery and the therapeutic effect of intraoperative photothermal ablation residual lesions.MethodsAfter intravenous administration of NP-IR780 into the mice with orthotopic breast cancer,the tumor area was exposed to a stereofluorescence microscope at different time points(n=3 per group).Under the guidance of white and FL images,the tumors were removed for pathology.To establish a cervical lymph metastasis model by implanting tumor cells on the mouse ear pinna and observed the process of metastasis by an in vivo IVIS imaging system.After successfully establishing a model of cervical lymph metastasis model,the mice were intravenously injected with NP-IR780.The lymph metastasis areas were exposed to a stereofluorescence microscope at 2 d postinjection(n=3 per group).Under the guidance of images,the lymph metastasizes were removed for pathology.The mice with orthotopic breast cancer were randomly divided into 6 groups(n=5 per group):Control,NP-IR780,Laser-only,PTT(NP-IR780-mediated photothermal therapy),Surgery,and Surgery+PTT(surgery with adjuvant PTT).After different treatments,the tumor volumes,body weight and survival times were monitored.At 24 hours after treatment,3 mice were randomly sacrificed in each group,and tumor tissues were taken for histological analysis(H&E;TUNEL;PCNA).ResultsAfter injection of NP-IR780,the boundary of tumor tissues(less than 1 cm)was clearly displayed in real time by near-infrared fluorescence imaging,which was consistent with the white light.Higher TBR and tumor mean fluorescence intensity(MFI)were obtained simultaneously after 2 days of injection of NP-IR780.The pathological analysis showed that the aggregation of NP-IR780 in tumor tissues was significantly higher than that of surrounding normal tissues.Through the mouse lymph node metastasis model,it was also confirmed that two days after the injection of NP-IR780,the location,boundary and size of the metastatic lymph nodes were clearly revealed by near-infrared fluorescence imaging.Compared with the control group,there was no significant change in tumor volume and survival time in the Laser only and the NP-IR780 groups;while surgery and photothermal therapy had different therapeutic effects on the tumor including the tumor volume was reduced and the survival time was prolonged.The Surgery+PTT group had the best tumor treatment effect and the complete cure rate reached 80%and the median overall survival(OS)was greater than 90 d.Only one mouse developed tumor recurrence in the Surgery+PTT group.There was no significant difference in body weight between the groups in the 2 weeks after treatments.The pathological section analysis 24 hours after treatment showed that the proliferating PCNA-positive cells were dominated in the control group,the laser group,the IR780 group,and the surgery group,while the TUNEL-positive cells were dominated in the PTT and the Surgery+PTT groups.In the PTT group,there were a few PCNA-positive tumor cells were also observed.H&E staining showed a large number of necrotic cells in the PTT and the Surgery+PTT groups.Conclusion:NP-IR780 can be used not only as a near-infrared fluorescence imaging probe to real-time guide precise resection of tumor and metastatic lymph node but also as photosensitizer mediated PTT for the ablation of unresectable residual tumor tissues during surgery to prevent the tumor recurrence.