Application Research Of Colorectal Cancer Surgery Guided By The Second Near-infrared Window Fluorescence Imaging

Objective:Colorectal cancer(CRC)commonly spreads through lymph nodes(LNs).Additionally,peritoneal metastasis(PM)is a prevalent distant metastatic route for CRC patients.The detection of occult metastases is still a challenge,significantly impacting patient prognosis.Traditional imaging techniques,such as X-ray,computed tomography(CT),magnetic resonance imaging(MRI),and positron emission tomography(PET),have risks of ionizing radiation,cumbersome equipment,and lack of real-time imaging feedback.Moreover,traditional imaging methods rely on morphological information(size and shape)for assessing metastatic status,limiting sensitivity and specificity.In recent years,second near-infrared(NIR-Ⅱ)fluorescence imaging has been rapidly developed.The red-shifted wavelengths in the NIR-Ⅱ window(1000-3000 nm)exhibit photon attenuation,reduced tissue autofluorescence and light scattering.NIR-Ⅱ fluorescence imaging offers a non-invasive,real-time,high-resolution,and deep imaging diagnostic mode.Indocyanine green,an FDA-approved probe,facilitates NIR-Ⅱ fluorescence imaging with tail fluorescence.There are still some drawbacks of ICG,including short blood circulation time,susceptibility to fluorescence quenching,and poor tumor enrichment.Basing on key issues in CRC surgery,this study design and synthesize a novel NIR-Ⅱ probe with enhanced stability,high resolution,and excellent biocompatibility.This study aims to explore the clinical value of NIR-Ⅱ fluorescent guided surgery in detecting micro-metastases in CRC.Methods:Part I: Firstly,novel donor-acceptor-donor(D-A-D)type NIR-Ⅱ fluorescent probe was designed and synthesized.The absorption and emission spectral characteristics were evaluated,and NMR hydrogen and carbon spectra were characterized.Subsequently,systematic evaluations of the biosafety of these probes will be conducted,including cytotoxicity,long-term biological metabolism,organ toxicity,blood toxicity,hepatic and renal toxicity,and weight monitoring.The fluorescence performance of the probe was evaluated,including photostability,blood circulation,and imaging penetration.Finally,in vivo NIR-Ⅱ fluorescence imaging assessed the lymphatic and vascular systems using the D-A-D type NIR-Ⅱ probe.Part Ⅱ: Subcutaneous tumors,orthotopic CRC,orthotopic CRC with LNs metastasis,and PM-CRC were established.The probe’s tumor targeting efficacy was evaluated in the subcutaneous CRC model.The probe’s performance in guiding intraoperative diagnosis and complete resection of residual tumors was assessed.Additionally,the probe’s ability to detect millimeter-sized mesenteric LNs metastases during surgery was evaluated.Furthermore,in the PM-CRC model,NIR-Ⅱ fluorescence-guided surgery and diagnostic tests were conducted to assess its precise detection capabilities of occult metastatic nodules.The study also evaluated the probe’s performance in achieving rapid intraoperative detection using NIR-Ⅱ microscopy.Finally,the study assessed the application of NIR-Ⅱ dual-color fluorescence imaging strategy in improving surgical accuracy and reducing intraoperative bleeding complications.Results:Part I: A novel D-A-D organic NIR-Ⅱ probe,FE-2PEG,was rationally designed and synthesized.Characterization of NMR hydrogen and carbon spectra confirmed successful synthesis of the probe.The probe exhibited good water solubility,with a fluorescence emission peak in the NIR-Ⅱ region(～1090 nm)in PBS.Cell toxicity assays demonstrated the probe’s safety even at a high concentration of 20 μM.Metabolic monitoring for 30 days showed that FE-2PEG was primarily metabolized through the hepatic-biliary pathway.Blood routine tests and liver-kidney function evaluations after 30 days indicated no significant toxicity.Staining of major organs after 30 days revealed no significant toxicity.Weight monitoring for 2 weeks showed no significant weight loss.FE-2PEG exhibited good photostability,with only a 9.23%decrease in fluorescence intensity after continuous irradiation with an 808 nm laser for 3 hours.The probe demonstrated long blood circulation performance,with 21.7%fluorescence intensity still present in the blood 24 hours after intravenous injection.FE-2PEG showed good deep tissue penetration,providing clear imaging beneath a 2mm intralipid.NIR-Ⅱ fluorescence imaging using FE-2PEG successfully visualized the microstructure of mesenteric lymph nodes,including lymphatic follicles,sinuses,afferent,and efferent lymphatic vessels,allowing for prolonged observation for up to2 hours.Moreover,FE-2PEG enabled long-term observation(4 hours)of blood vessels in the hind limbs and brain of mice.The probe also achieved excellent spatiotemporal resolution in the vascular system,with a signal-to-background ratio(SBR)of 1.95 after 2 hours.Part Ⅱ: Four mouse models were successfully established,including subcutaneous tumors,orthotopic CRC tumors,orthotopic CRC tumors with mesenteric LN metastasis,and PM-CRC models.FE-2PEG effectively targeted tumor sites through high penetration and retention effects,with a tumor-to-normal tissue ratio(TNR)of 7.11 observed in vivo after 24 hours in the subcutaneous tumor.In the subcutaneous CRC,precise intraoperative diagnosis and complete tumor resection of residual lesions were performed,leading to prolonged survival in mice without recurrence within 2 weeks postoperatively.The TNR of orthotopic CRC tumors during laparotomy reached 3.4,successfully guiding NIR-Ⅱ fluorescence surgical navigation.In the orthotopic CRC with mesenteric LNs metastases,the probe accurately identified primary tumors,14 mesenteric LNs metastases,and 3 abdominal metastatic nodules.Among the 14 mesenteric LNs metastases,the smallest diameter was 1.5 mm,with a maximum TNR of 8.22.NIR-Ⅱ fluorescence-guided surgery in PM-CRC mouse models showed a significant decrease in postoperative fluorescence intensity in the abdominal regions of interest(p<0.05).A total of 321 nodules were excised using NIR-Ⅱ fluorescence-guided surgery,with an additional 89 suspicious nodules obtained through supplementary biopsies.The SBR of nodules excised using NIR-Ⅱ fluorescence-guided surgery was significantly higher than that of nodules obtained through supplementary biopsies(48.09 vs.13.56,p<0.001).The sensitivity of NIR-Ⅱ fluorescence-guided surgery in removing PM lesions was 94.51%(92.03%-96.99%),with a specificity of 86.59%(79.05%-94.12%),a positive predictive value(PPV)of 96.57%(94.57%-98.57%),and a negative predictive value of 79.78%(71.27%-88.28%).Subgroup analysis revealed a sensitivity of 96.07%(93.53%-98.61%)for detecting occult PM lesions smaller than 3 mm,with a specificity of82.35%(71.52%-93.18%),a PPV of 96.07%(93.53%-98.61%),and a negative predictive value of 82.35%(71.52%-93.18%).NIR-Ⅱ microscopy facilitated rapid intraoperative detection,and the NIR-Ⅱ dual-color fluorescence imaging strategy using FE-2PEG(1100-1300 nm)for tumor and LNs imaging in the NIR-Ⅱa window and Pb S@Cd S quantum dots(>1500 nm)for imaging surrounding blood vessels in the NIR-Ⅱb window was successfully implemented.Conclusion:We have developed and synthesized a novel D-A-D type organic NIR-Ⅱ probe,FE-2PEG,with good water solubility and a fluorescence emission peak in the NIR-Ⅱ region(～1090 nm)in PBS.A series of biosafety evaluations confirmed the probe’s excellent biocompatibility.FE-2PEG demonstrated reliable photostability,long blood circulation performance,and good deep tissue penetration among other outstanding optical properties.Furthermore,FE-2PEG enabled stable,clear,and long-term NIR-Ⅱ fluorescence imaging of mesenteric LNs,allowing observation of internal LNs structures.The probe also achieved clear long-term NIR-Ⅱ fluorescence imaging of blood vessels in the legs and brain.Through its high penetration and retention effects,FE-2PEG efficiently targeted tumor sites.In the subcutaneous CRC,FE-2PEG facilitated precise intraoperative diagnosis and complete tumor resection of residual lesions,leading to improved survival.FE-2PEG exhibited excellent spatiotemporal resolution in deep-seated orthotopic CRC and successfully guided surgery.In the orthotopic CRC with mesenteric LN metastases,FE-2PEG accurately identified primary tumors and 14 metastatic LNs.NIR-Ⅱ fluorescence-guided surgery in PM-CRC achieved thorough clearance,with a significant decrease in postoperative fluorescence intensity in the abdominal ROIs.The SBR of nodules excised using NIR-Ⅱ fluorescence-guided surgery was significantly higher than that of nodules obtained through supplementary biopsies,enabling precise resection of occult PM lesions smaller than 3 mm.Additionally,NIR-Ⅱ microscopy facilitated rapid intraoperative detection,and the NIR-Ⅱ dual-color fluorescence imaging strategy effectively identified tumors,LNs,and blood vessels during curative surgery,enhancing surgical precision.In summary,the novel D-A-D type organic NIR-Ⅱ probe FE-2PEG shows significant importance in the diagnosis and surgical navigation of micro-metastases in CRC,with potential for clinical translation.