Toxicity and Therapeutic Potential of Oxidized Carbon Nanotubes Functionalized with Doxorubicin and Fluorescein: In Vitro and In Vivo Findings

Background

Carbon nanotubes (CNTs) possess a high surface area, excellent cellular uptake, and the ability to traverse biological membranes, making them attractive vectors for drug delivery, gene therapy, and imaging applications. Functionalization with hydrophilic groups improves solubility and biocompatibility, while covalent attachment of therapeutic molecules can provide targeted, controlled release. Doxorubicin (Dox) is a widely used chemotherapeutic with significant systemic toxicity; linking it to CNTs may reduce off‑target effects and enhance tumor specificity. Previous work has shown that CNTs can be safely administered at low doses, but higher concentrations or inadequate surface chemistry can provoke oxidative stress, inflammation, and organ accumulation. This study builds on these findings to evaluate the safety profile of CNT‑Dox and CNT‑FITC in vitro and in vivo.

Methods

HT29 colon adenocarcinoma cells were cultured in 2‑D monolayers and 3‑D spheroids. CNTs were synthesized by chemical vapor deposition, oxidized with 70 % HNO₃, and functionalized with doxorubicin or fluorescein via carbodiimide chemistry. Suspensions were sonicated, sterilized, and characterized by SEM, zeta potential, and FTIR. Cytotoxicity was assessed using MTT and colony‑area assays. For controlled release, trypsin (0.05–70 %) was added to evaluate doxorubicin liberation. In vivo, Balb/c mice received intraperitoneal injections of CNTox (1.5 mg kg⁻¹), CNT‑Dox (1.5 mg kg⁻¹), or free Dox (20 mg kg⁻¹) every three days for four weeks. Serum biochemical markers (ALT, AST, ALP, total protein, albumin) and hematological parameters (RBC, WBC, platelets, hemoglobin) were measured after the treatment period.

Results and Discussion

Nanoparticle Characterization

SEM images confirmed multi‑walled CNTs with diameters 10–60 nm and lengths 20–500 µm. Functionalization did not alter morphology. Zeta potential measurements showed CNT‑Dox (−28 mV), CNT‑FITC (−32 mV), and CNTox (−36 mV), indicating good colloidal stability. FTIR spectra displayed characteristic amide I and II bands, confirming covalent bonding of doxorubicin and fluorescein to CNTs.

In Vitro Cytotoxicity

MTT assays revealed dose‑dependent cytotoxicity for CNTox (IC₅₀ ≈ 100 µg mL⁻¹) and CNT‑FITC (IC₅₀ ≈ 50 µg mL⁻¹). CNT‑Dox exhibited markedly lower cytotoxicity in the absence of protease (IC₅₀ > 200 µg mL⁻¹), reflecting doxorubicin sequestration. When 0.05 % trypsin was added, CNT‑Dox released free doxorubicin, producing an IC₅₀ of ≈ 5 µg mL⁻¹ and a synergistic decrease in cell viability (≤ 10 % of control). Colony‑area assays confirmed that CNT‑FITC markedly inhibited colony formation, whereas CNT‑Dox had minimal effect unless trypsin was present. In 3‑D spheroid cultures, CNTox increased spheroid volume dose‑dependently, while CNT‑FITC reduced spheroid size; CNT‑Dox showed no significant effect unless protease‑mediated release occurred.

In Vivo Toxicity

Serum analyses showed that CNTox alone did not alter ALT, AST, or ALP levels. CNT‑Dox and free Dox both increased AST (up to 126 %) and ALP (up to 148 %) relative to control, but the elevations were less pronounced for CNT‑Dox. Total protein rose slightly in all treatment groups. Hematology revealed that free Dox caused anemia, thrombocytopenia, and neutropenia, whereas CNT‑Dox produced comparable but milder changes. CNTox had negligible impact on blood cell counts. These findings suggest that CNT‑Dox mitigates systemic toxicity while preserving antitumor potency.

Conclusion

Functionalization of oxidized CNTs with doxorubicin via peptide bonds creates a safe, controlled‑release nanocarrier that reduces systemic toxicity compared with free drug. In vitro, CNT‑Dox remains inert until proteolytic cleavage, after which it achieves potent cytotoxicity comparable to free doxorubicin. In vivo, CNT‑Dox exhibits lower hepatic enzyme elevations and hematologic toxicity than free Dox, while CNTox alone is essentially non‑toxic. These results support further development of CNT‑based delivery systems for highly potent anticancer agents, particularly in gastrointestinal malignancies where protease activity can trigger targeted release.

Abbreviations

Al:

Albumin

ALP:

Alkaline phosphatase

ALT:

Alanine aminotransferase

AST:

Aspartate aminotransferase

CNT-Dox:

Carbon nanotubes functionalized with doxorubicin

CNT-FITC:

Carbon nanotubes functionalized with fluorescein

CNTox:

Oxidized carbon nanotubes

CNTs:

Carbon nanotubes

Dox:

Doxorubicin

FTIR:

Fourier‑transform infrared spectroscopy

MTS:

Multicellular tumor spheroid

MWCNTs:

Multi‑walled carbon nanotubes

PTX:

Paclitaxel

Tp:

Total protein