New Indocyanine Green Functionalized Melanin Nanoparticles For Bimodal Imaging And Photothermal Therapy

Objective:Diagnosis and treatment integration is a new technology combining disease diagnosis and treatment,which brings a new direction for the cure of cancer.Melanin as endogenous biomolecule is widely distributed in living organisms and has been widely used especially bioimaging and therapy for cancer.Herein,we present a simple method to prepare IR820functionalized MNP nanoparticles for dual-modal Imaging and cancer therapy medicated by laser irradiation in the NIR windows.Methods:1.Synthesis and characterization of IR820-PEG-MNP and determination of solution properties:Firstly,IR820-PEG-MNP nanoparticles were prepared by PEG linking IR820 and MNP,and then the successful preparation of R820-PEG-MNP nanoparticles was proved by some their characterizations such as particle size and potential,transmission electron microscopy,and UV absorption,Fourier transform infrared spectrum,etc.Next,we studied the signal of photoacoustic and fluorescent imaging and photothermal effects of this nanoparticle aqueous solution.2.Cell experiments:in vitro cytotoxicity of IR820-PEG-MNP toward Hep2 cells was evaluated using Cell Counting Kit-8(CCK-8)assay.Cellular uptake of IR820-PEG-MNP was investigated by confocal laser scanning microscopy(CLSM)and flow cytometry.Subsequently,apoptosis and necrosis analysis of cells were detected by flow cytometry,To verify the fluorescence and photoacoustic imaging effects at the cellular level.Cell viability was evaluated by live/dead assay after the photothermal action.3.In vivo experiments:To evaluate the capability of IR820-PEG-MNP for cancer imaging,the dual-modal imaging in vivo was investigated in Hep2 tumor-bearing mice model by tail vein injection of IR820-PEG-MNP.We monitor the NIR fluorescence signal at various time points and observe the enrichment and metabolism of the nanoparticles in the tumor site.To observe the photothermal treatment capacity of IR820-PEG-MNP in the vivo,four groups nude mice employ 808 nm laser(0.4 W/cm~2)for 5 min irradiation after48 h postinjection.Varity about body weight and tumor volume of nude mice were recorded within 16 days.Results:1.Transmission electron microscopy(TEM)image revealed the size of IR820-PEG-MNP ranged from 16.3 to 53.6 nm,which was smaller than the result obtained from the dynamic light scatter(DLS)with an average diameter of 50.7 nm.This phenomenon may be due to the swollen of PEG chains and surface hydration of NPs.Additionally,the zeta potential was determined to be-29.0 for IR820-PEG and-45.6 mV for IR820-PEG-MNP,verifying the successful conjugation of MNP.The Fourier-transform infrared(FT-IR)analysis and the UV–vis spectroscopy was confirmed the successful fabrication of IR820-PEG-MNP.In light of outstanding optical property of IR820-PEG-MNP the NIR-I fluorescence was investigated in vitro,there are two stage of the FL signal with variation of concentration.Foremost,when the concentration of IR820-PEG-MNP increase from0.01563 to 0.0625 mg/mL,the FL signal intensity enhanced rapidly,then the FL signal intensity decrease gradually with increasing concentration ranging from 0.0625 to 1 mg/mL.This phenomenon may be ascribed to the fluorescence self-quenching effect due to self-aggregation of the IR820 on the surface of IR820-PEG-MNP at high concentrations.The intensity of PA signal and brightness of PA images are proportional to the IR820-PEG-MNP concentration.showing a linear correlation with R~2=0.98.2.The results of cck8 illustrated IR820-PEG-MNP have no evident toxic effects on hep2 cells even at concentration up to 100?g/mL within 24 h,implying the outstanding biocompatibility of IR820-PEG-MNP.Flow cytometry analysis proved that the internalization of IR820-PEG-MNP achieved nearly saturation at 4h.According to CLSM images,bright green fluorescence was clearly observed in hep2 cells after 4 h incubation,which was in accordance with flow cytometry analysis.The results of flow cytometry analysis about apoptosis were consistent with live/dead cell staining assay,Without NIR irradiation,cells show high survival ratio and negligible apoptosis or dead behavior.Once treated with NIR irradiation,apparent dead cells appeared in IR820-PEG-MNP group.Subsequently,the fluorescence imaging and the PA imaging at the cellular level was discussed.The intensity of hep2 cells incubated with IR820-PEG-MNP was found to increase with increasing concentration.3.In the group treated with IPM,the temperature of tumor region rapidly reached up67.9?within 5 min,while minimal temperature elevation of tumor was observed under irradiation for PBS group.In contrast,the control groups of IPM and PBS without NIR irradiation exhibited negligible temperature change.These results indicated that IPM could effectively absorb NIR energy to produce hyperthermia for photothermal therapy of cancer.Conclusion:We have successfully prepared IR820-PEG-MNP nanoparticles with good dispersibility,uniform particle size,and excellent biocompatibility.It can realize fluorescence and photoacoustic dual-modality imaging,and the photothermal treatment effect is obvious.It has potential application value as an integrated nanoprobe for diagnosis and treatment.