Carbon nanotubes as gene delivery vectors: Navigating endosomal escape and intracellular fate

OBJECTIVE: Carbon nanotubes (CNTs) have emerged as non-viral gene delivery vectors, due to their physicochemical properties, high surface area, anisotropic needle-like geometry, tunable surface chemistry, and unique optical and thermal characteristics. This review synthesizes mechanistic understanding of CNT-mediated gene delivery: cellular uptake, receptor-mediated targeting through surface functionalization, and the challenge of endosomal escape. SIGNIFICANCE OF REVIEW: Understanding how CNTs enter cells, behave intracellularly, and what determines functional therapeutic outcomes is essential for advancing their clinical development. KEY FINDINGS: We detail four primary endosomal escape mechanisms: direct cell membrane translocation through intrinsic needle-like property or with cell-penetrating peptide (CPP) facilitation, the proton sponge effect from polyamine coatings, and photothermal/photochemical internalization. We also compare their efficiency against lipid nanoparticle (LNP) and polymeric vector. We further examine CNT's intracellular fate, covering protein corona formation, complement pathway activation and its immune consequences, macrophage recognition, and biodegradation. Intracellular fate trajectories are linked to measurable functional outcomes. Lysosomal sequestration governs their silencing potency, tumor tissue pharmacokinetics determine knockdown duration, and CNS biopersistence coincides with sustained microglial activation. An immunotoxicological evaluation framework adapted from established NCL and regulatory guidelines (ISO/TS 10993-20, ISO/TR 10993-22, ICH S8) is outlined, encompassing complement split product quantification, PBMC cytokine profiling, inflammasome activation assays, and in vivo immunophenotyping. CONCLUSIONS: Translational obstacles remain. Including biopersistence, unpredictable protein corona composition, cytotoxicity, and the absence of standardized manufacturing and characterization protocols. Emerging stimuli-responsive hybrid nanostructures and AI-assisted design offer promising pathways forward, contingent on coordinated progress in reproducible manufacturing, longitudinal fate monitoring, and rigorous immunotoxicological assessment.The graphical abstract is supplied as a separate file.