Fluorecently labeled bionanotransporters of nucleic acid based on carbon nanotubes

Here we propose the approach to design of the new type of hybrids of oligonucleotides with fluorescein-functionalized single-walled carbon nanotubes. The approach is based on stacking interactions of functionalized nanotubes with pyrene residues in conjugates of oligonucleotides. The amino- and fluorescein-modified single-walled carbon nanotubes were obtained, and their physico-chemical properties were investigated. The effect of carbon nanotubes functionalization type on the efficacy of sorption of pyrene conjugates of oligonucleotides was examined. Proposed non-covalent hybrids of fluorescein-labeled carbon nanotubes with oligonucleotides may be used for intracellular transport of functional nucleic acids.

💡 Research Summary

The paper presents a novel strategy for constructing fluorescently labeled carbon‑nanotube‑based bionanotransporters capable of delivering functional nucleic acids into cells. Starting from commercially available single‑walled carbon nanotubes functionalized with carboxyl groups (SWNT‑COOH), the authors first introduce primary amine functionalities using either hexamethylenediamine (HMDA) or a third‑generation polyamidoamine dendrimer (PAMAM G3.0). The presence of amino groups is confirmed by the Kaiser test (0.11–0.206 mmol g⁻¹). In a second step, fluorescein isothiocyanate (FITC) is covalently attached to these amines, yielding two fluorescent nanotube variants: SWNT‑HMDA‑FITC and SWNT‑PAMAM‑FITC. Comprehensive physicochemical characterization—including FTIR (N–H, C=O, C–N vibrations), Raman spectroscopy (increase of the I_D/I_G ratio indicating defect introduction), thermogravimetric analysis, elemental analysis, and electron microscopy (TEM/SEM)—demonstrates successful multifunctionalization and quantifies the amount of organic material grafted onto the nanotube surface.

The core of the delivery system relies on non‑covalent π‑π stacking between the nanotube surface and pyrene‑modified oligonucleotides. The authors synthesize 5′‑pyrene conjugates of both DNA and 2′‑O‑methyl RNA (including versions with a hexa‑ethylene‑glycol spacer) using solid‑phase phosphoramidite chemistry. These conjugates are mixed with the fluorescent nanotubes under ultrasonic agitation (30 min) to form hybrids. Because pyrene fluorescence is efficiently quenched by proximity to the nanotube, the authors exploit this quenching to develop a quantitative assay for the amount of oligonucleotide adsorbed. Isotherm experiments at 25 °C reveal that, when the nanotube concentration reaches ~50 µg mL⁻¹, 90–95 % of the pyrene‑oligonucleotide is adsorbed, corresponding to an adsorption capacity of roughly 100 µmol g⁻¹. The type of primary amine (HMDA vs. PAMAM) and the presence of FITC have only minor effects on adsorption efficiency, whereas the inclusion of a PEG spacer between pyrene and the nucleic acid slightly reduces binding at lower nanotube concentrations.

Microscopic analysis shows individual nanotubes up to 1.5 µm in length and ~1.7 nm in diameter, both as isolated tubes and as aggregates. Fluorescent nanotubes are directly visualized by confocal microscopy, confirming that the FITC label remains functional after the multi‑step synthesis. To verify the non‑covalent nature of the hybrids, the authors displace the pyrene‑oligonucleotides using methylene blue, which precipitates the nanotubes; gel electrophoresis of the supernatant demonstrates a concentration‑dependent release of the oligonucleotides. Stability tests indicate that the ultrasonic processing and interaction with SWNT‑COOH do not introduce apurinic/apyrimidinic sites or cause strand cleavage, as confirmed by piperidine treatment and reversed‑phase HPLC analysis.

In conclusion, the study establishes a straightforward, modular approach to fabricate fluorescent carbon‑nanotube carriers that bind nucleic acids through reversible π‑π interactions. The system combines (i) facile chemical functionalization (amine → FITC), (ii) a robust non‑covalent loading mechanism (pyrene stacking), (iii) quantitative characterization of loading capacity, and (iv) preservation of nucleic‑acid integrity. These attributes make the hybrids promising candidates for intracellular delivery of siRNA, DNAzymes, aptamers, or other therapeutic nucleic acids, with the added benefit of real‑time optical tracking. Future work should focus on cellular uptake studies, endosomal escape mechanisms, and in‑vivo efficacy and toxicity assessments to translate this platform into practical biomedical applications.