Experimental Study On Multi-modality Targeted Nanoprobes Mediated Diagnosis And Photothermal Therapy In Micro-liver Cancer

Research BackgroundMicro hepatocellular carcinoma refers to a tumor less than 20 mm in diameter.Early diagnosis and treatment are key factors to improve the prognosis of patients.Traditional imaging techniques,such as CT and MRI,can efficiently detect lesions with a diameter of 10?20 mm,but they have low sensitivity for the detection of lesions below 10 mm,especially those with cirrhosis,and the location of them is difficult during operation.While,optical molecular imaging technology has the advantages of high sensitivity,which provides a new opportunity for the detection of microscopic liver cancer.This technology combined with multimodal nanoprobes has been applied to the diagnosis and treatment of tumors.However,the application of multi-modal targeting diagnosis and treatament integration probes in microscopic liver cancer has rarely been reported.Epithelial cell adhesion molecule(EpCAM)is highly expressed in the early stage of liver cancer.Anti-EpCAM antibody jointed with multimodal nanoprobe can be used to target tiny hepatocellular carcinoma,collect multimodality imaging and perform fluorescence navigation photothermal therapy.ObjectivesThrough the synthesis of ICG-Gd-CuS@BSA-EpCAM nanoprobe,preoperative detection of small hepatocellular carcinoma and the photothermal therapy of the near-infrared(NIR)fluorescence imaging navigation were realized by photoacoustic,magnetic resonance,and fluorescence imaging.Materials and Methods1.Indocyanine green(ICG),gadolinium(Gd),copper sulfide(CuS),bovine serum albumin(BSA),epithelial cell adhesion molecule(EpCAM)were selected to synthesize ICG-Gd-CuS@BSA-EpCAM multimodal targeting nanoprobe.2.Ultraviolet-visible(UV)spectrophotometer was used to analyze the absorption spectrum of nanoparticles.Malvern particle size analyzer measured its hydration particle size.Transmission electron microscopy(TEM)detected its morphology and size;In vivo imaging system(IVIS),photoacoustic,magnetic resonance and 808 nm lasers were used to verify the fluorescence,photoacoustic,magnetic resonance signals and photothermal properties of the probes,respectively.3.Laser confocal microscopy validated the expression of EpCAM and targeted uptake of probes in the LO2,Huh7 and HepG2 cells.The microplate reader or live dead cell staining measured cytotoxicity and cell photothermal effects of probes.4.The IVIS system analyzed the metabolic process of the probe in subcutaneous tumor-bearing nude mice and obtained the optimal timing of tumor fluorescence signal to background ratio(TBR);the fiber viable laser confocal microscopy imaging(Cellvizio)analyzed the accumulation of nanoparticles in tumors.Fluorescence,photoacoustic and magnetic resonance imaging of orthotopic liver cancer tumor-bearing nude mice in the best metabolic time points were obtained by IVIS system,photoacoustic and magnetic resonance,respectively.Sham surgery group,PBS group and probe group were treated with photothermal therapy under fluorescence navigation in orthotopic HepG2-tumor-bearing mice using an 808 nm laser irradiation at dose of 1 w/cm2.The survival time and weight of each group were recorded.Tissue sections were stained with hematoxylin and eosin staining(H&E)method.Results1.Characterization of the ICG-Gd-CuS@BSA-EpCAM nanoprobes:The UV absorption peak of the ICG-Gd-CuS@BSA-EpCAM nanoparticle was 800 nm,the hydrated particle size was approximately 38 nm,and the particle size was about 10 um.The probe respectively dissolved in deionized water,PBS,DMEM,and BSA were placed in 4°C environment for a week,and the UV absorption peak was not observed to change significantly.The probe has a maximum temperature of up to 53 ? in the near-infrared light.2.At the cellular level,EpCAM was higher expressed in Huh7 and HepG2 cell,and was lower expressed in L02 cell.The probe had little toxicity to the L02 cell lines at 1 mg/ml.The uptake of nanoparticles was the highest in HepG2 cell and the lowest in L02 cell.After photothermal treatment,the HegG2 cell membranes and its cytoplasm were disintegrated,and the cell viability was less than 10%.3.At the animal experimental level,the uptake of tumors was the highest after intravenous injection of nanoparticles for 24 h.Image of magnetic resonance,photoacoustic,and optical were acquired at 24 h respectively and photothermal therapy of micro-liver cancer under fluorescence navigation was performed.Compared with sham-operated group and PBS group,the survival rate in probe group was the highest and weight loss rate was the slowest.H&E staining showed loose cell arrangement,a large number of cell necrosis and interstitial hemorrhage.ConclusionThe multimodality targeted nanoparticles,ICG-Gd-CuS@BSA-EpCam,has good stability,photothermal characteristics,liver cancer targeting and biocompatibility.As a photoacoustic,magnetic resonance and fluorescence trimodal targeted nanoprobe,it can be used for the detection of in situ micro-hepatocellular carcinoma and the experimental study of intraoperative near-infrared fluorescence navigation photothermal therapy,which has potential application value.