| Background and Purpose:The lack of early,effective,and non-invasive diagnostic and treatment methods for lung squamous cell carcinoma results in limited treatment opportunities and a high mortality rate.Therefore,targeted identification and precise treatment of lung squamous cell carcinoma are of great significance.Early and accurate diagnosis is crucial for improving treatment outcomes and survival rates.However,the commonly used contrast agent iodine has certain physiological side effects,and traditional diagnostic methods have limitations.Biological fluorescence imaging(FI)offers the advantages of being intuitive and easily capturing deep tissue information.When combined with CT,it can achieve dual-mode imaging and provide more comprehensive diagnostic information.Meanwhile,photothermal therapy,as a highly controllable,safe,and effective treatment method,can achieve precise treatment.Gold nanomaterials can integrate the advantages of FI and CT and have high photothermal conversion efficiency,making them widely applicable in the biomedical field.The high expression of Desmoglein 3(Dsg3)antigen on the membrane of lung squamous cell carcinoma cells is a precise targeted biomarker that can optimize the nanosystem for precise diagnosis and treatment.Therefore,in this study,the construction of the responsive targeting probe Au-Dsg-3 integrating early diagnosis and effective treatment strategies on a multifunctional nanoplatform holds significant importance for the diagnosis and treatment of lung squamous cell carcinoma.Furthermore,we have explored its photothermal therapy’s pathway in promoting apoptosis.Methods:This study is mainly divided into four parts.In the first part,Au-GSH was synthesized using the template etching method,and then the Au-Dsg-3 nanomaterial was successfully constructed through a coupling reaction.The stability,fluorescence imaging performance,CT imaging properties,and photothermal performance of the gold nanomaterial were studied using methods such as transmission electron microscopy(TEM),dynamic light scattering(DLS),Zeta potential,infrared spectroscopy(FTIR),X-ray photoelectron spectroscopy(XPS),and ultraviolet-visible-near-infrared(UV-Vis-NIR)spectroscopy.In the second part,the study focused on the imaging of Au-Dsg-3 in lung squamous cell carcinoma.Firstly,the cytotoxicity of Au-Dsg-3 on normal lung epithelial cells(Beas-2B)and lung squamous carcinoma cells(NCI-H520)was studied at different concentrations and time points using the CCK8 assay in vitro.Subsequently,confocal microscopy fluorescence localization and flow cytometry were used to analyze the targeting of Au-Dsg-3 to cells.In vivo,Au-Dsg-3 was injected into tumor-bearing nude mice via the tail vein,and its imaging performance was studied at different time points using small animal live optical imaging and CT scanners.The biodistribution and clearance process were also investigated.Finally,serum biochemical indicators and histological examinations were conducted to study the in vivo biosafety.In the third part,the study explored the therapeutic potential of Au-Dsg-3 in lung squamous cell carcinoma.Firstly,NCI-H520 cells and Beas-2B cells were incubated with 300 μg/m L Au-Dsg-3 for 6 hours in vitro,followed by irradiation with a near-infrared laser at a power of 2 Wcm-2 for different durations.Cell apoptosis rates were then measured by flow cytometry.In vivo,NIR laser was precisely applied to the tumor site 6 hours after the administration of Au-Dsg-3,and the temperature changes in the tumor and surrounding tissues were monitored at different time points.Subsequently,the mice were treated with NIR light irradiation at the tumor site for 14 days,with a duration of 600 seconds each time.Changes in body weight,tumor weight,and volume were monitored,and tumor tissue sections were stained with H&E,TUNEL,and PCNA to assess microscopic structural changes,apoptosis,and proliferation.The serum TNF-α level was measured using an enzyme-linked immunosorbent assay to evaluate the antitumor effect in vivo.Finally,H&E staining and serological tests were conducted to assess the biocompatibility after photothermal therapy.In the fourth part,the study investigated the mechanism of Au-Dsg-3photothermal therapy.NCI-H520 cells were divided into four groups: Control,NIR-only,Au-Dsg-3-only,and Au-Dsg-3+NIR.Cells were pretreated with Au-Dsg-3(300 μg/m L)for 6hours and exposed to NIR for 600 seconds.ROS levels were measured using a ROS detection kit,and Western blot was used to detect the protein levels of Hsp70,Bax,Bcl-2,caspase-3,and caspase-9.Additionally,in vitro experiments were conducted using ROS antagonist NAC and Hsp70 agonist ML346.Cells were divided into six groups: control,Au-Dsg-3+NIR,NAC,Au-Dsg-3+NIR+NAC,ML346,and Au-Dsg-3+NIR+ML346.NCI-H520 cells were treated with Au-Dsg-3(300 μg/m L)for 6 hours,followed by pretreatment with NAC(5 m M)for 2 hours or ML346(15 μM)for 1 hour before NIR exposure for 600 seconds.Cell apoptosis rates,ROS levels,and related apoptotic protein levels were measured again.Results:In the first part of the study,the size of Au-Dsg-3 increased from 2.2 nm to 5.1 nm,indicating the successful synthesis of Au-Dsg-3 nanomaterials.The DLS showed the same trend in particle size variation,the Zeta potential indicated charge neutralization,and the infrared spectrum also showed the characteristic absorption peak of the amide bond at the wave number of 1470 cm-1,all proving the occurrence of coupling reaction and the successful construction of Au-Dsg-3 material.In addition,XPS characterization showed that GSH ligands and gold cores formed an effective Au-S bond protective layer,indicating that the nanoprobe has fluorescence stability,which is conducive to long-term targeted imaging and tumor tracing.The characteristic absorption peak at 275 nm in the UV-Vis-NIR spectrum proved the formation of metal-sulfur complex,and the appearance of a broad near-infrared absorption peak at around 780 nm can enhance photothermal absorption,which is beneficial to the improvement of photothermal performance.Moreover,the fluorescence excitation wavelength of Au-Dsg-3 is 503 nm,and its emission wavelength is 670 nm,which can exhibit red fluorescence and contribute to achieving clear visual imaging detection.Under different time conditions,UV light irradiation,and dispersed in various simulated in vivo environments,the results showed that Au-Dsg-3 has excellent fluorescence imaging stability.In addition,the CT signal also increased proportionally with concentration.Finally,according to the temperature changes under different wattages and time conditions and the temperature monitoring during thermal cycles,it was shown that Au-Dsg-3 can serve as a good photothermal converter for photothermal performance.In the second part of the study,Au-Dsg-3 exhibited no significant toxic effects on NCI-H520 and Beas-2B cells when co-cultured at a concentration of 300 μg/m L for 6 h,12 h,and 24 h in vitro,indicating its good biocompatibility.Confocal microscopy localization revealed a high intensity of red fluorescence on the NCI-H520 cell membrane with clear boundaries,while it was relatively weak on the Beas-2B cell membrane,suggesting the excellent targeting ability of Au-Dsg-3.Quantitative fluorescence ratio analysis by flow cytometry showed that NCI-H520 cells exhibited a more pronounced red fluorescence ratio of 99.8% compared to untreated cells,whereas Beas-2B cells only showed 0.032%,further confirming the strong targeting recognition of Au-Dsg-3 towards NCI-H520 cells.In vivo experiments,both small animal live imaging and CT imaging showed that the fluorescence signal and CT signal intensity reached their peak at 6 hours,and the tumor tissue site was fully enriched,which was consistent with the results of in vitro experiments.Moreover,the high-intensity imaging signal could last for at least 2 hours,indicating that Au-Dsg-3 is a potentially ideal non-invasive and rapid FI and CT contrast agent for detecting lung squamous cell carcinoma,and once again proving its good stability.ICP-MS analysis further revealed that Au-Dsg-3 could be effectively and rapidly cleared through kidney filtration.Post-imaging assessments showed no significant differences in aspartate aminotransferase(AST),alanine aminotransferase(ALT),blood urea nitrogen(BUN),creatinine(CR),and creatine kinase(CK)levels between the Au-Dsg-3 group and the control group.Hematoxylin and eosin(H&E)staining of major organs in mice also showed no obvious pathological changes,further confirming the good biocompatibility of Au-Dsg-3 in vivo.In the third part of the study,NCI-H520 and Beas-2B cells were incubated with 300μg/m L of Au-Dsg-3 for 6 hours,followed by irradiation with a 2 W cm-2 laser for 120 s,180 s,300 s,and 600 s.In the absence of near-infrared(NIR)laser irradiation,the number of apoptotic cells in both groups treated with Au-Dsg-3 alone was negligible,further confirming the good biocompatibility of Au-Dsg-3 with both cell types.After 600 seconds of NIR laser irradiation,the apoptosis rate of NCI-H520 cells was 24.7%,while there was no significant difference in Beas-2B cells(P>0.05).The results showed that Au-Dsg-3 had good near-infrared photothermal performance,which could lead to the apoptosis of tumor cells.In vivo,NIR laser was accurately applied to the tumor site,and the temperature of the tumor area in the Au-Dsg-3+NIR group could rapidly reach 43.5 ℃ after 120 seconds and up to53.3 ℃ after 600 seconds,a significant increase of 16.8 ℃,while the temperature in the control group only increased by 2.5 ℃,consistent with the in vitro results.During the process,the temperature of the tissue near the tumor was monitored not to exceed 42 ℃ to avoid photothermal damage to normal cells nearby.These results indicate that Au-Dsg-3 has good stability and strong photothermal ability in vivo.Subsequently,continuous monitoring of tumor volume and weight indicators for 14 days showed significant differences between the Au-Dsg-3+NIR group and the PBS group as well as the PBS+NIR group(P<0.05),while there was no significant difference in body weight changes among the three groups(P>0.05).In addition,pathological changes in the tumor site of the Au-Dsg-3+NIR group were observed through H&E staining,TUNEL,and PCNA detection.The proliferation and apoptosis coefficients were 12.3% and 81.6%,respectively,while there were no significant abnormalities in the other two groups.Furthermore,increased serum TNF-α levels may be due to photothermal-induced or exacerbated inflammatory responses,leading to apoptosis.The results once again confirm that photothermal therapy has local,noninvasive,and mild tumor-killing ability and can inhibit the development of lung squamous cell carcinoma.After photothermal therapy,there were no significant differences in AST,ALT,BUN,CR,and CK indicators compared to the control group,and H&E staining showed no obvious pathological changes.These results suggest that photothermal therapy has no effect on the function of major organs in mice,has no obvious side effects,and has good biocompatibility.In the fourth part of the study,there was no significant difference in ROS levels between NCI-H520 cells treated with NIR alone or Au-Dsg-3 alone compared to the control group(P>0.05).However,when cells were treated with Au-Dsg-3+NIR,there was a significant increase in ROS levels(P<0.05).These results suggest that the nanoprobe exhibits excellent photothermal performance,effectively promoting a significant increase in intracellular ROS.Compared to the other three groups,the Hsp70 protein level was significantly reduced in the Au-Dsg-3+NIR treated group(P<0.05).The results indicate that the high-temperature effect generated by the nanoprobe under near-infrared laser irradiation may directly affect the stability of Hsp70 protein,leading to its inability to perform normal cellular protective functions and thus weakening the cell’s defense against high temperatures.Through Western blot experiments,it was found that the level of Bax protein significantly increased,while the level of Bcl-2 protein was suppressed in lung squamous cell carcinoma cells after Au-Dsg-3+NIR treatment.Additionally,there was an increase in the expression levels of activated caspase-3 and caspase-9 proteins.These results suggest the activation of the mitochondria-mediated endogenous apoptosis pathway,further confirming the photothermal therapeutic ability of Au-Dsg-3.The number of apoptotic cells was significantly reduced in the groups treated with NAC or ML346 compared to the Au-Dsg-3+NIR group(P<0.05).NAC could reduce the intracellular ROS level in cells treated with Au-Dsg-3+NIR(P<0.05),further confirming the efficient performance of the nanoprobe under photothermal action and its ability to induce an increase in intracellular oxidative stress.Similarly,ML346 also reduced the intracellular ROS level in cells treated with Au-Dsg-3+NIR(P<0.05).However,the Hsp70 expression level did not increase in the NAC-only group(P>0.05),and there was no increase in Hsp70 expression level with the addition of NAC to the irradiated cells(P>0.05).In contrast,the Hsp70 expression level was significantly increased in cells irradiated with ML346(P<0.05).These results suggest that the nanoprobe Au-Dsg-3 reduces the protective effect of Hsp70 protein against thermal stress after near-infrared irradiation,and increasing Hsp70 levels can help enhance the cell’s antioxidant capacity.However,Hsp70 protein is not affected by ROS levels.The addition of NAC or ML346 resulted in decreased expression levels of Bax protein,increased expression levels of Bcl-2 protein,and reduced expression levels of cleaved-caspase-3 and cleaved-caspase-9 proteins(P<0.05).These results indicate that inhibiting ROS or activating Hsp70 conditions can prevent tumor cell apoptosis by interfering with caspase activation and the mitochondrial endogenous apoptosis pathway.Conclusion:In summary,we have successfully constructed a responsive targeting probe Au-Dsg-3with excellent multi-functional performance.Both in vitro and in vivo experimental results have demonstrated its ability to perform fluorescence imaging,CT imaging,and near-infrared photothermal therapy for lung squamous cell carcinoma over extended periods,exhibiting good stability,controllability,and biocompatibility.The process of photothermal therapy can cause oxidative stress reaction in cells,and also reduce the level of Hsp70 protein,thus losing the protective effect on lung squamous cell carcinoma.Simultaneously,it can affect and upregulate the levels of apoptosis-related proteins such as Bax,caspase-3,and caspase-9,while downregulating the level of Bcl-2 protein,thereby promoting cell apoptosis. |
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