Biocompatible fluorescent carbon dot-based nanoprobes for G-quadruplex targeting in cancer cells

Abstract

Carbon-based nanomaterials with intrinsic luminescent properties have emerged as promising tools in biological and biomedical research, however tuning their photoluminescent properties for bespoke applications remains challenging. Herein, we report the design and synthesis of a new class of pH-responsive, red-emissive carbon dot-based nanoprobes (average core size 2–5 nm) functionalised with a naphthalene diimide (NDI)-based G-quadruplex (G4) ligand. These hybrid nanoprobes combine the favourable chemical and optical properties of CDs with the high selectivity of classical molecular ligands for G4 DNA. The resulting NDI-CDs exhibit excitation-dependent emission with distinct maxima at ~450, ~490, and ~590 nm upon excitation at 350, 460, and 520 nm, respectively, in contrast to the excitation-independent emission of the parent CDs (λem ≈ 450 nm). Mechanistic investigations indicate that changes in the observed emission arise primarily from covalent surface conjugation with the NDI ligand, that modifies the electronic structure of the CDs surface and introduces additional emissive states. Control experiments rule out significant contributions from electron transfer or aggregation effects, and instead support a mechanism involving surface-state modulation and pH-dependent protonation equilibria of the NDI moiety. Importantly, the NDI-CDs retain strong G4-binding affinity (Kd = 1.38 ± 0.25 µg/mL) and simultaneously enhance ligand bioavailability and reduce cytotoxicity. Confocal microscopy studies of live cells treated with these nanoprobes show efficient cellular uptake, preferential localisation in mitochondria and nuclei, and significantly reduced cytotoxicity relative to the free ligand, confirming their suitability for biological applications, suggesting possible engagement with intracellular G4-associated structures under physiological conditions. Overall, this work establishes a versatile and biocompatible platform for the generation of carbon-based nanomaterials for multifunctional bioimaging and nucleic acid-targeting applications.