(189a) Design of Carbon Nanotube-Enabled Photothermal Therapeutics for Cancer Treatment

CNTs are categorized into single-walled carbon nanotubes (SWNTs) and multi-walled carbon nanotubes (MWNTs) based on their structure. SWNTs consist of a single sheet of graphene and typically have diameters ranging from 1 to 2 nanometers. Their electronic properties depend on their chirality and can be metallic or semiconducting. Because of these properties, SWNTs are being actively studied in nanotransistors, sensors, semiconductor materials, and more. MWNTs, on the other hand, are composed of multiple concentric layers of graphene and are structurally stronger and easier to synthesize, making them suitable for reinforcing composites. However, SWNTs are better suited for biomedical applications due to their distinct electronic structure, large surface area, and ease of surface modification.

One of the most promising biomedical applications of SWNTs is photothermal therapy (PTT). SWNTs exhibit strong light absorption properties in the near-infrared (NIR) region, which makes them ideal for converting light energy into heat energy. PTT is a minimally invasive treatment technique that uses NIR-absorbing nanomaterials to selectively kill tumor cells or inflammatory cells by generating localized high temperatures. The high temperatures generated disrupt cell membranes, denature intracellular proteins, and damage mitochondria, eventually inducing apoptosis or necrosis. This mechanism presents an attractive strategy for cancer treatment because it enables precise targeting of abnormal cells while minimizing damage to surrounding healthy tissue.

However, despite these advantages, CNTs suffer from poor in vivo water dispersibility, which limits their application potential. CNTs are inherently hydrophobic and easily aggregate in aqueous solutions, significantly reducing their in vivo availability and therapeutic efficiency.

To address these issues, effective surface modification and dispersion strategies are essential. In this study, phenoxylated amino dextran (p-dextran) was used as a dispersant for the dispersion of single-walled CNTs. Dextran provides biocompatibility and water solubility, while the phenoxyl groups promote stable binding via π-π interactions with the CNT surface. These dual interactions allow the CNTs to be stably dispersed in aqueous solutions, enabling biological applications.

For the synthesis of p-dextran, amino dextran with a molecular weight of approximately 70 kDa was reacted with 1,2-epoxy-3-phenoxypropane (EPP). The reaction was carried out under stirring at 50 °C, 300 rpm for 6 hours and 45 minutes. After the reaction, the absorbance at 269 nm was measured using UV-Vis spectroscopy, and the amount of phenoxyl introduced was quantified using the Lambert-Beer law. The final phenoxyl content was determined to be 13.6 wt%, selected based on previous studies that showed this concentration achieved optimal dispersibility. At lower concentrations, such as 7.8 wt%, the dispersion was unstable due to a lack of π-π interactions, while at higher concentrations, such as 17.0 wt%, the polymer shrank due to excessive hydrophobicity and the CNTs tended to re-aggregate.

SWNTs prepared by the HiPco method were used for CNT dispersion at a concentration of 1 mg/mL. The SWNTs were mixed in distilled water with 1 wt% p-dextran and subjected to ultrasonic dispersion by running a sonicator equipped with a 1/4-inch tip at 30% power for 2 hours. This process helps to disassemble the CNT bundles and ensure that the polymer is uniformly adsorbed on the CNT surface. After sonication, centrifugation was performed at 16,250 g for 30 minutes to remove free polymer and large aggregates. The supernatant contained well-dispersed CNTs, which were used in subsequent experiments. Dispersion stability was assessed by observing the sharpness and shape of the UV-Vis absorbance peak. A sharp and narrow peak indicates that the CNTs were effectively dispersed and agglomerates were removed. For photothermal characterization, the samples were irradiated with an 808 nm NIR laser at different power and time conditions, and the temperature change was measured in real time using an infrared thermal imaging camera. The relation of temperature increase with CNT concentration and laser power was systematically analyzed.

To evaluate the intracellular uptake properties, the pD-SWNTs were labeled with the fluorophore FITC. FITC-NHS was reacted with the amine group of p-dextran for 2 hours and purified to remove the residue left after the reaction. The fluorescently labeled CNTs were incubated with different cell types to evaluate their intracellular uptake.

The cells used in the experiment were skin cancer cells, normal skin cells, and macrophages. Each cell was inoculated into a 24-well plate, treated with various concentrations of FITC-pD-SWNTs, and incubated for 24 hours. The cells were then washed with PBS, fixed with 4% paraformaldehyde, and the nuclei were stained with DAPI to quantitatively analyze the intracellular uptake by fluorescence microscopy and flow cytometer. The results showed that skin cancer cells and macrophages exhibited significantly higher fluorescence signals compared to normal cells, indicating selective cellular internalization. This selectivity is interpreted as a result of the interaction of dextran on the CNT surface with scavenger receptors expressed on the surface of certain cells. Receptors such as MARCO and CD36 are known to bind dextran and are highly expressed on tumor-associated macrophages and some cancer cells. Therefore, this system can be utilized as a receptor-mediated cell-specific delivery platform.

To evaluate the photothermal therapy effect, skin cancer cells and macrophages were inoculated into 48-well plates at a concentration of 1 × 10⁵ cells/well and incubated for 24 hours. After treatment with various concentrations of pD-SWNTs, the cells were washed with PBS and incubated for an additional 24 hours. The cells were then treated with various concentrations of pD-SWNTs. After washing the cells with PBS, 200 µL of PBS was added to each well and irradiated with an 808 nm NIR laser at 8.84 W/cm² for 10 minutes. Cell viability was measured by Live/Dead staining and CCK-8 assay. The results showed that both skin cancer cells and macrophages showed a concentration-dependent decrease in viability upon laser irradiation, and localized cell death occurred only in the laser-irradiated area, confirming that the photothermal effect was spatially controllable. In particular, at a CNT concentration of 0.05 mg/mL, the local temperature exceeded 50 °C, which proved to be sufficient heat for cell death. Conversely, in the absence of NIR irradiation, cell viability remained above 80% at all concentrations, confirming the system’s low toxicity and high biocompatibility.

This study demonstrates that phenoxylated amino dextran acts as an excellent dispersant to simultaneously improve the water dispersibility and photothermal efficiency of CNTs. The phenoxyl groups induce strong π-π interactions with the CNT surface, while the dextran provides water solubility and biocompatibility. These dual properties enable the construction of stable CNT-based platforms suitable for biomedical applications. In this study, we demonstrated the selective uptake of pD-SWNTs by cancer cells and macrophages, confirming the potential for cell-specific targeted therapy via scavenger receptor-mediated mechanisms, which offers the possibility of precise drug delivery or photothermal therapy without off-target effects. The pD-SWNTs developed in this study exhibited excellent stability, low toxicity, and efficient photothermal conversion, highlighting their potential as a practical and biocompatible photothermal therapy platform.

In conclusion, we developed a CNT-based photothermal therapy platform utilizing phenoxylated amino dextran as a dispersant. The system exhibits excellent water dispersibility, selective cellular uptake, and high efficiency photothermal conversion properties, and is a promising approach for precision therapy for cancer and inflammatory diseases. Future work will include in vivo studies using animal models, along with extended in vitro assays to further optimize therapeutic efficacy and validate clinical applicability.