| BackgroundThe increasingly widespread application of computed tomography(CT)in the screening and follow-up of patients with lung disease has concomitantly increased the detection rate of pulmonary nodules.Currently,Video-assisted thoracic surgery(VATS)has become the preferred method of surgery for patients with pulmonary ground-glass nodules(GGNs)due to its advantages minimal invasiveness and rapid recovery.However,target nodule identification during VATS is sometimes challenging due to the inherent characteristics of these nodules,especially when they are small and distant from the pleura.Intraoperative failure rate of nodule identification is extremely high,requiring extended resection or conversion to open-heart surgery.Currently,there are various ways to localize lung nodules,but all of them have their own drawbacks and shortcomings,so there is an urgent need for a simple,rapid and non-invasive localization method.Intraoperative fluorescence imaging will become a trend in the studies on neoplasms localization because of its non-radiation,non-invasive,simple operation,no need to know the location of nodes in advance,possible detection of imaging-negative nodes,real-time,and intuitive features.Gold nanoparticles(AuNPs)have been studied for neoplasms’ detection,diagnosis and treatment,and they are very stable and non-immunogenic in vivo and have low toxicity.They can easily pass through the neovascularisation of tumor tissue and the lymphatic system through passive ways,enhanced permeability and retention effects(EPR),and active targeting process,which enhance diagnostic imaging sensitivity and therapeutic efficacy.In terms of delivery modes,current contrast agents and drug carriers(including dry powders,aerosols,and sprays)prepared from many nanoparticle and particulate formulations are capable of being delivered via the introtracheal tube to enhance the therapeutic efficacy of the disease.Therefore,targeted fluorescent gold nanoclusters delivered through the airways are expected to improve the uptake of target lesions and become a preferred mode of localization.ObjectiveA novel targeted gold nanocluster for lung adenocarcinoma targeting would be prepared,which can specifically identify lung adenocarcinoma tissues in a non-invasive way,intratracheal delivery.Then the cancer tissues emit bright and persistent fluorescence,which allows surgeons recognize lung adenocarcinomas and provides guidance assistance for surgical identification in order to improve the accuracy of lung adenocarcinoma localization during VATS.MethodsFirstly,Au-GSH-anti Napsin A(Nap A-AuNCs)nanoclusters were synthesized and their physicochemical properties and optical properties were characterized.In the characterization,transmission electron microscopy(TEM)and dynamic light scattering(DLS)were used to observe the morphology and size of Au-GSH-anti Napsin A.Zeta potential test,X-ray photoelectron spectroscopy(XPS),infrared spectroscopy,and UV absorption spectroscopy were used to determine whether Napsin A antibodies were successfully attached to AuNCs,and to measure their surface charge,optical properties,time and solution stability of fluorescence.Artificial broncheal mucus stimulant(BMS)were prepared,and the mucus penetration of Nap A-AuNCs in the BMS was qualitatively and quantitatively examined.The mucus penetration of Nap A-AuNCs in the airways of mice was observed by fluorescence confocal imaging.and penetration ability in BMS.Secondly,in vitro experiments were performed in lung adenocarcinoma cells A549 and normal lung epithelial cells Beas-2b to observe the effect,safety and fluorescence time curve of Nap A-AuNCs,including cell viability assay(CCK-8),cell imaging experiments(laser confocal imaging,High Content System),fluorescence intensity assay(flow cytometry),cytotoxicity experiments(Calcein AM/PI double-staining assay,flow cytometry,hemolysis assay),etc.Subsequently,A549 cell tumorsphere model was constructed,and the penetration ability of Nap A-AuNCs into tumor tissues was tested using the High Content System,and fluorescence time curves were plotted.Finally,a mouse subcutaneous xenograft tumor model was constructed to observe the difference in visualization between Nap A-AuNCs and AuNCs by tail vein administration and to observe the time required for the strongest fluorescence visualization.A mouse lung cancer in situ xenograft tumor model was constructed by intrapulmonary injection to observe the fluorescence visualization of lung adenocarcinomas at different times after Nap A-AuNCs were given by the airway.Another mouse lung cancer in situ xenograft tumor model was constructed by tail vein injection,and the fluorescence localization of Nap A-AuNCs on lung adenocarcinomas of different sizes and locations was investigated by airway administration.The pharmacokinetic changes,in vivo distribution,metabolism and safety testing of Nap A-AuNCs were also observed.Data were analyzed by t-test and ANOVA,and Graphpad Prism 9 was used for statistical analysis,Origin 2022 and Graphpad Prism 9 for plotting software and Image J 1.53 c for image processing.ResultsA nanocluster Nap A-AuNCs targeting lung adenocarcinoma with stable and bright red fluorescence were synthesized,with 5.1 nm in size and 120 nm in hydration radius,-12.1 m V in zeta potential.The optimal excitation light of the complex is 410 nm,and the emission light ranges from 600-650 nm.Nap A-AuNCs can emit bright light,that can be up to 3000 a.u.in fluorescence intensity.Nap A-AuNCs has excellent fluorescence properties and can be stable at extreme p H conditions.Both Nap A-AuNCs and AuNCs penetrate artificial tracheal mucus mimics and rapidly penetrate mouse airways.In vitro experiments have verified that Nap A-AuNCs have the targeting property of A549 cells and have very low toxicity to normal lung epithelial cells.They can enter the cells rapidly to emit bright red fluorescence,which reach peak in 2-8 hours,and gradually decreasing in fluorescence intensity when time passes,but it still can still be seen after 24 hours.Nap A-AuNCs can penetrate into tumor tissues from the periphery to the center,and it was observed that it can penetrate into the center of the tumor within 4 hours with a good penetration rate.In vivo experiments have demonstrated that Nap A-AuNCs can be targeted to lung adenocarcinoma tissues with stronger fluorescence than non-targeted AuNCs.Nap A-AuNCs can be effectively localized in lung adenocarcinoma tissues,and can even fluorescently identify very small,clinically indistinguishable lung adenocarcinoma nodules.Inhaled administration is faster fluorescence visualization of lung adenocarcinoma tissues and less accumulation in non-target organs than intravenous administration.Nap A-AuNCs have a long half-life and are mainly metabolized by the liver and kidney,which excreted through bile and urine.They also have very low hepatotoxicity,nephrotoxicity,and pulmonary effects.ConclusionA nanocluster targeting lung adenocarcinoma,Au-GSH-anti Napsin A,was successfully conjugated,which can increase the accumulation in lung adenocarcinoma.Intratracheal delivery way is a safe,effective and rapid way for intraoperative localization for lung adenocarcinoma.This non-invasive,targeted,rapid and low-toxic method can provide a better strategy for intraoperative localization of lung adenocarcinoma and has a better application prospect. |
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