A research team has demonstrated that discrete, oxidized multi-walled carbon nanotubes formulated with a dual-surfactant coating can deliver large plasmid DNA into mammalian cells with high efficiency and lower cytotoxicity than leading commercial lipid nanoparticles. Published in Bioscience Nanotechnology, the study details how a stable dispersion of multi-walled carbon nanotubes, designated do-MWCNT, was engineered using an amphiphilic surfactant alongside a branched cationic polymer to overcome historical barriers in carbon-based gene delivery. Evaluated in cultured Chinese hamster ovary cells, the formulation successfully delivered an 8.78-kilobase plasmid encoding antibody sequences, driving gene expression that exceeded commercial Lipofectamine 3000 by an average of eleven percent at full dosing. Concurrently, the nanotube complexes preserved cell viability without the significant thirty-percent cell loss observed with the lipid reagent. Parallel experiments with single-walled carbon nanotubes demonstrated further gains in cell health, confirming that dispersed carbon nanomaterials can achieve competitive gene transfection while maintaining a remarkably benign toxicity profile.

Transfection technologies serve as an indispensable cornerstone across modern biotechnology and medicine, underpinning advanced therapeutics, biomanufacturing, research tools, and next-generation vaccines. The global market is currently dominated by lipid nanoparticles, with the top two producers commanding exceeding 44.7 billion dollars in market share. Despite widespread adoption driven by affordability, effective protocols, and low immunogenicity, lipid nanoparticles suffer from critical physical limitations. They struggle to deliver large plasmids exceeding five kilobases, decrease host cell viability, require stringent storage to avoid breakdown, and exhibit short shelf lives once formulated. Carbon nanotubes have long presented an alternative due to their high aspect ratios and large surface areas, but earlier biological attempts were hampered by severe cytotoxicity, non-competitive transfection rates, particle aggregation, and catalyst impurities. Unfunctionalized nanotubes aggregate in physiological environments and fail to reversibly bind nucleic acids, demonstrating an urgent need for engineered surface chemistry to unlock their gene-delivery potential.

To overcome these material hurdles, the authors utilized carbon nanotubes produced via chemical vapor deposition and functionalized by Molecular Rebar Design LLC using concentrated nitric acid and high-energy dispersion. This washing process removed toxic residual catalysts, yielding purified multi-walled nanotubes supplied as a wet cake containing approximately six percent solid nanotube content by weight. Structural measurements confirmed physical dimensions of 530 nanometers in length, 13 nanometers in outer diameter, 5 nanometers in inner diameter, and an average of 13 walls, with outer wall surface oxidation of roughly twenty percent. The team also evaluated single-walled carbon nanotubes produced by chemical vapor deposition, supplied as a wet cake of approximately ten percent solids. These single-walled structures measured around 1100 nanometers in length and 4 nanometers in diameter, exhibiting surface oxidation of about ten percent—half that of the multi-walled tubes due to structural stability limitations inherent to single graphene cylinders.

Transforming the raw multi-walled material into a stable, biocompatible transfection vector required developing a refined dual-surfactant protocol in aqueous solution. The six-percent wet cake was processed in water at a solids concentration not exceeding one percent by weight. The primary surfactant, DSPE-PEG-Amine with a molecular weight of 2000, was added to the dilute mixture and vortex mixed. The suspension underwent bath sonication for 30 minutes at room temperature to disperse the oxidized tubes before introducing secondary branched polyethylene-imine with a molecular weight of 270 kilodaltons. Following a second 90-minute bath sonication, the dispersion was filtered through dialysis with a 1,000,000 molecular weight cutoff to eliminate unbound, free surfactants. Filtered dispersions were then autoclaved for sterilization, subjected to another 120 minutes of restorative sonication, and centrifuged at 20,000 gravities for 10 minutes to pellet larger particles, yielding an aggregate-free, stable colloidal suspension ready for nucleic acid complexation.

The dual-surfactant architecture was engineered to balance dispersion stability with electrostatic nucleic acid binding. While non-covalently bound DSPE-PEG-Amine prevented nanotube agglomeration, the positively charged branched polyethylene-imine stimulated DNA binding, cellular uptake, and endosomal escape. To optimize binding, the authors tested three molecular weights of branched polyethylene-imine—10, 25, and 270 kilodaltons—across nanotube-to-DNA mass ratios from 2.5:1 to 20:1 using one-percent agarose gel electrophoresis at 100 volts for 30 minutes. In this assay, tightly bound DNA complexes remained stationary at the well while unassociated plasmid migrated. The 270-kilodalton polymer proved most effective at inhibiting DNA migration, and the 20:1 ratio achieved superior binding across all polymer sizes. Subsequent in vitro testing in Chinese hamster ovary cells confirmed that the 20:1 ratio induced significantly greater target gene expression than lower ratios of 10:1 or higher ratios of 25:1, establishing 20:1 as the definitive benchmark formulation for subsequent biological evaluations.

Investigating complexing conditions revealed that incubation temperature and duration profoundly influence transfection efficiency. When nanotube-plasmid mixtures were prepared at 4 degrees Celsius, 27 degrees Celsius, and 37 degrees Celsius over seven hours, qPCR demonstrated that refrigeration at 4 degrees Celsius maximized gene expression, with a significant drop observed at 37 degrees Celsius. Tracking incubation duration across intervals from 1 hour to 168 hours revealed that transfection efficiency increased progressively over time, reaching a distinct peak at 96 hours with statistically significant advantages over 48-hour and 168-hour timepoints. Cell population monitoring showed that extending incubation to 96 hours maintained high cell viability. However, extending incubation to 168 hours caused a significant drop in cell health, which the authors attributed to the accumulation of nanotube-plasmid flocculants and aggregates that impair cultured cells. Consequently, 96 hours of refrigerated incubation was selected as the optimal protocol for achieving peak transfection efficiency without compromising cell survival.

The optimized multi-walled complex was directly benchmarked against Lipofectamine 3000 in CHO-K1 cells plated at 200,000 cells per milliliter in 12-well plates. Transfection delivered an 8.78-kilobase plasmid, pTRIOZ_hIgG1, encoding human anti-HIV antibodies targeting the VRC01 light chain variable region. Following a five-day incubation, non-adherent dead cells were rinsed away with phosphate-buffered saline before automated trypan blue counting on a Countess II counter. Lipofectamine treatment caused a statistically significant thirty-percent drop in cell population compared to untreated controls, whereas multi-walled nanotube treatment showed no significant difference from untreated cells, demonstrating exceptional cytocompatibility. Quantitative PCR analysis—normalized against Beta-Actin and Aldo-Keto-Reductase controls with PGDM antibody sequences as negative controls—demonstrated that full-dose do-MWCNT generated a statistically significant eleven percent higher VRC01 transcript expression than Lipofectamine 3000. This established that the multi-walled nanotube vehicle drives higher gene expression per cell while fully preserving the cultured cell population, although Lipofectamine maintained an advantage when doses were halved.

To assess the influence of tube geometry, the formulation was translated to single-walled carbon nanotubes using surface-area particle matching calculations. At a particle-matched ratio of 10 parts nanotubes to 27.7 parts DSPE-PEG-Amine to 17 parts 270-kilodalton polyethylene-imine, an aggregate-free dispersion was achieved at an SWCNT-to-DNA mass ratio of 2.1:1, requiring fifteen-fold less carbon mass than the multi-walled system. Testing dose escalation at 1x, 2x, and 4x demonstrated that single-walled nanotubes exhibited remarkable safety, causing no significant increase in cytotoxicity even at elevated doses where multi-walled nanotubes showed significant cell loss at 2x concentrations. However, higher single-walled doses caused a decline in gene expression, suggesting that particle crowding and aggregation limit transfection when nanotube concentrations rise. Because both dispersions contained approximately 9.04 times 10 to the tenth power nanotubes per milliliter, absolute particle count appears to be a critical factor governing cellular delivery mechanics and biological efficacy across distinct tube configurations.

An economic evaluation revealed substantial material cost advantages for carbon nanotubes compared to standard commercial reagents. Lipofectamine 3000 requires specialized Opti-MEM reduced-serum media and a multi-solution preparation protocol, costing roughly 2.725 dollars per 12-well dose containing one microgram of plasmid DNA. In contrast, the multi-walled nanotube formulation forms stable complexes in sterile water and is introduced directly into complete serum-containing growth media as a single-component system, costing just 0.084 dollars per dose, while single-walled nanotubes cost 0.07 dollars per dose. Evaluated by price per unit of relative quantification of gene expression, Lipofectamine scales to 2.3 times 10 to the negative sixth power dollars, whereas do-MWCNT scales to 6.4 times 10 to the negative eighth power dollars. This demonstrates that multi-walled nanotubes are 36 times cheaper per transfection unit while achieving equivalent expression magnitude. Single-walled nanotubes scale to 1.2 times 10 to the negative seventh power dollars, proving 18.5 times cheaper than Lipofectamine.

Optical detection and biological degradation profiles also highlight practical differences between nanotube geometries. Multi-walled nanotubes strongly quench reporter fluorescence such as enhanced green fluorescent protein because dark accumulations absorb emitted light and obscure visualization. In contrast, single-walled nanotubes possess lower optical absorption per unit mass, enabling direct fluorescence detection of green fluorescent protein in CHO-K1 cells after three and five days, which facilitates flow cytometry applications. Furthermore, single-walled nanotubes feature only one graphene wall that can be cleaved by cellular enzymes and peroxides, avoiding the shielded, non-functionalized interior walls of multi-walled tubes that resist degradation and trigger aggregation. The primary operational drawback of the multi-walled platform remains the 96-hour pre-incubation required for plasmid binding, reflecting slow structural coiling between branched polyethylene-imine and plasmid DNA. The authors propose that sonication may accelerate complexation, potentially resolving the incubation barrier and enhancing adoption of carbon nanotube transfection technologies across research and therapeutic biomanufacturing workflows.

Subject of Research: Carbon nanotube-mediated plasmid DNA transfection in mammalian cell cultures

Article Title: Discrete, oxidized multi-walled carbon nanotubes for efficient plasmid DNA transfection

Article References: Quinlan, C., Foote, A., Castillo, K., Bosnyak, C., & Swogger, K. (2026). Discrete, oxidized multi-walled carbon nanotubes for efficient plasmid DNA transfection. Bioscience Nanotechnology, 2(1), Article 6. https://doi.org/10.1186/s44331-026-00010-0

Image Credits: AI Generated

DOI: 10.1186/s44331-026-00010-0

Keywords: carbon nanotubes, gene delivery, transfection, plasmid DNA, nanobiotechnology, Lipofectamine 3000, cytotoxicity, molecular biology, Discrete, oxidized, multi-walled, carbon