Synthesis Of Water-soluble Cyanine Molecule PEG-808-NM2 And Its Effect On Tumor Targeting And Radiosensitization

Objective:To synthesize a mitochondria-targeting water-soluble indocyanine fluorescent small molecule（designated as PEG-808-NM2）via PEG modification and explore its radiotherapy sensitization effect in renal cell carcinoma as well as underlying mechanism.Methods:PEG-808-NM2 was chemically designed using 2,3,3-trimethylindole,2-nitroimidazole,and polyethylene glycol monomethyl ether as the starting materials（molar ratio 1:1:1.5）and synthesized via a six-step reaction.The successful synthesis of PEG-808-NM2 and other chemical intermediates were characterized using 1H nuclear magnetic resonance（NMR）and high-resolution mass spectrometry.UV-vis-NIR spectra and fluorescence spectra analysis also used to confirm the photophysical properties of PEG-808-NM2.The biosafety assessment of PEG-808-NM2 was evaluated using hemolysis assay and H＆E staining of histological slices from vital organs.The cancer-targeting ability were validated by comparing the cell uptake of PEG-808-NM2 by normal renal tubular epithelial cells（HK-2）and renal cell carcinoma cells（RENCA）.Subcellular localization of PEG-808-NM2 in mitochondrial were observed using fluorescence confocal microscopy after staining with a mitochondrial tracker（Mito Tracker）.The synergistic effects of PEG-808-NM2 with radiotherapy were verified through evaluating cell viability,colony formation ability,intracellular ROS level,DNA damage,and tumour growth.Results:（1）The polyethylene glycol modified indocyanine molecule was synthesized by chemical reaction,and its structure was characterized by ~1H NMR and mass spectrometry,indicating that nitroimidazole and polyethylene glycol were introduced into the indocyanine molecule.（2）The UV-vis spectra absorption results demonstrated that 808-NM2 in PBS exhibits obvious double absorption peaks（680 nm and c）.In contrast,PEG-808-NM2 only present the same absorption peak at 780 nm,indicating PEG modification could effectively increase the solubility and stability of 808-NM2.（3）Fluorescence confocal microscopy images revealed preferential PEG-808-NM2accumulation renal cancer cells RENCA than those in normal HK-2 cells（P<0.01）,indicating the excellent tumor-targeting capability.Moreover,the fluorescence signals of PEG-808-NM2 in RENCA cells were obviously higher than 808-NM2 at same condition（P<0.01）.（4）Subcellular localization analysis of PEG-808-NM2 in RENCA cells revealed the high degree（near 100%）overlap of green fluorescence signal（mitochondria-tracker）and red fluorescence signal（PEG-808-NM2）（5）In contrast to irradiated cells with 90%cell viability,>40%of irradiated cells were killed when treated with PEG-808-NM2（P<0.01）.（6）Flow cytometry analysis demonstrated cell apoptosis in combined group was 26.1%,while 8.5%in irradiated cells.（7）Comet assay results showed the tail DNA content in combination treatment group was 80%,which is 2 times higher than in irradiation group（40%）（P<0.05）.（8）Single-click multi-target model analysis demonstrated that the sensitizer enhancement ratio（SER）value was nearly 1.40,indicating the excellent radio-sensitization effect of PEG-808-NM2.（9）Hemolysis analysis also evidenced PEG-808-NM2 exhibited excellent hemocompatibility as no detectable hemolysis was found even at a relative high concentration（2μg/m L）.（10）H＆E staining of the main organs（heart,liver,spleen,lung,and kidney）did not reveal any abnormalities compared with control.In the treatment experimental group,the drug plus irradiation group had a significant inhibitory effect on the tumor compared with the simple irradiation group（P<0.05）.Conclusion:（1）PEG modification of 808-NM2 could effectively improve the 808-NM2 and stability.（2）PEG-808-NM2 could preferentially accumulate in cancer cells and exhibit excellent mitochondria targeting capability.（3）PEG-808-NM2 exhibits excellent biocompatibility without obvious systemic toxicity.（4）PEG-808-NM2 could effectively suppress tumor growth synergistically by radiotherapy in vitro and vivo.（5）NIR fluorescent imaging property also can be used to precisely distinguish the tumors from their boundaries.