NIR-I imaging of indocyanine green-functionalized carbon nanodots in pancreatic adenocarcinoma: Biodistribution, protein corona, and prospects for theranostics

In this work, we report the design and synthesis of zero-dimensional carbon nanodots (CDs) PEGylated and functionalized with indocyanine green (ICG) moieties, yielding stealth near-infrared I (NIR-I) emitting conjugates (CDs–ICG-PEG) with an average diameter of ∼7 nm. Controlled surface engineering was achieved via click chemistry between azido-functionalized CDs and a custom monofunctionalized alkyne–ICG derivative, enabling precise conjugation and stable optical performance. The resulting CDs–ICG-PEG exhibited robust and long-term NIR fluorescence within the biologically transparent window, positioning them as promising candidates for in vivo theranostic applications. Comprehensive in vitro assays demonstrated excellent biocompatibility (IC50 > 500 μg mL−1) across multiple cancer cell lines. Mass spectrometry revealed that the protein corona (PC) formed upon incubation with human serum comprised a limited yet functionally diverse set of proteins, varying in length, molecular weight, and isoelectric point. Gene Ontology enrichment analysis indicated PC enrichment in pathways associated with the complement cascade, innate immune response, and blood coagulation. Functionally, the presence of the PC significantly reduced macrophage uptake of CDs–ICG-PEG, suggesting immune evasion potential. In vivo biodistribution studies using a xenograft mouse model of pancreatic ductal adenocarcinoma (PDAC) revealed time-dependent accumulation of CDs–ICG-PEG within tumor tissue, liver, and kidneys, with pronounced retention in the tumor mass after 4 h, confirmed by ex vivo imaging. Furthermore, immunohistochemical analyses of liver and kidney sections indicated negligible toxicity and minimal inflammatory response, even at prolonged exposure times. Overall, these findings demonstrate that CDs–ICG-PEG are biocompatible, bioeliminable, photostable, and capable of passive tumor targeting, making them strong candidates for next-generation theranostic platforms.