Application Of Highly Stable Carbon Dot Nanocomposites For The Labeling And Treatment Of Tongue Squamous Cell Carcinoma

Surgical treatment of tongue squamous cell carcinoma(TSCC)has the disadvantages such as high trauma,poorly defined tumor scope,and a propensity for recurrence.In recent years,in order to achieve precise tumor treatment,many scholars have devoted to the development of nanoparticles(NPs)with tumor labeling and killing properties,that taken up by tumor tissue cells through enhanced penetration and retention effects.In terms of tumor fluorescent imaging,many fluorescent labeling materials such as quantum dots and upconversion NPs have been developed for the localisation of TSCC,however,they have drawbacks including complex synthesis,photobleaching,low water solubility and low biocompatibility.Thus,it is still crucial to develop low toxicity and high stability fluorescent materials with outstanding performance for tumor labeling.Carbon dots(CDs)have become ideal fluorescent imaging NPs due to their ideal precursor sources,simple synthesis methods,photobleaching resistance,high water solubility and high biocompatibility.Unfortunately,UV excitation,low thermal stability,and subpar tumor targeting and killing efficacy plague the majority of CDs.We proposed to develop highly stable CDs for labeling TSCC and further develop fluorescent targeted NPs integrating chemotherapy and photothermo therapy with the help of fluorescence characteristics and abundant functional groups of CDs.First,nitrogen-doped CDs(N-CDs)were synthesized via hydrothermal method using ascorbic acid and p-phenylenediamine.The results confirmed that the particle size of N-CDs was 1.9 nm and rich with nitrogen and oxygen-containing groups on their surface,giving them excellent water solubility.The photoluminescence(PL)excitation spectrum demonstrated a peak centered at 400 nm under 544 nm emission with a quantum yield of 10.1%.CCK8 results showed that N-CDs had good biocompatibility and that even at 400μg/m L,cellular viability was still greater than95%.Within 4 hours,the N-CDs were efficiently taken up by TSCC,showing vivid green fluorescence in the cytoplasm.However,the fluorescence intensity decreased by 8%by increasing the solution temperature to 80 ~oC,and the fluorescence intensity decreased with increasing p H,which showed that the stability needs to be further improved.Second,to address the defects of high-temperature decay of hydrothermal synthesized N-CDs,a solvent-free method was chosen to synthesize more stable luminous N-CDs using citrazinic acid and urea as precursors.The size of the N-CDs was found to be 2.05 nm with a 7.9%quantum yield.The p H stability was tested and the results confirmed that the fluorescence intensity was basically constant in the p H range of 4-10.Further analysis of its high thermal stability confirmed that it had excellent stability with no detectable fluorescence decay even at 90 ~oC heating.Long-term toxicity and labeling results confirmed its low toxicity and biocompatibility to exhibit bright fluorescence in TSCC for at least 6 days.Finally,based on the high stability N-CDs with its abundant surface functional groups,polydopamine was modified and anticancer drug doxorubicin(DOX)was loaded.After that,CDs-PDA@DOX-FA/TAT(CPDFT)was synthesized by covalently linking cell membrane targeting chemical folic acid(FA)and nucleus targeting peptide TAT.Characterization was applied to evaluate the process of the synthesis,the UV-visible absorption spectrum confirmed that the DOX entrapment efficiency was90.7%.Under 808 nm laser irradiation and acidic p H,the dual-stimuli release of DOX and the photothermal conversion of the NPs were further confirmed.Long-term toxicity studies later validated the DOX-free nanocarrier CDs-PDA-FA/TAT(CPFT)’s biocompatibility under the concentration of 200μg/m L.The targeted uptake efficiency and mechanism were evaluated using confocal microscopy,which confirmed that cascade-targeted CPFT had the highest cellular uptake efficiency and TSCC mainly relied on clathrin-mediated or caveolae-mediated endocytosis to take up NPs.The killing effiency of the CPDFT fluorescent NPs,which combines cascade targeting,chemotherapy,and photothermal therapy,was further demonstrated during TSCC therapy under 808 nm irradiation.The results of CCK8 demonstrated that only18.6%of cells survived after CPDFT treatment,dead-live staining,flow apoptosis,and the multicellular tumor sphere inhibition assay all supported this conclusion.In this study,a novel fluorescent nanoparticle with simple synsthesis,high biocompatibility,and good degradation properties was developed for the integrated composite cascade targeting,chemotherapy,and photothermal therapy of TSCC.