Functionalization of single-walled carbon nanotubes and their binding to cancer cells

Abstract

Background: Single-walled carbon nanotubes (SWCNTs) have novel properties including their nanoscale size and ease of cellular uptake. This makes them useful for drug delivery, and their photo-thermal effects make them potentially useful in a wide range of applications, particularly the treatment of solid tumors. The poor solubility of SWCNTs has, however, been an issue that may potentially limit the utility of SWCNTs for cancer treatment. Functionalization of the surface of the tubes may be an approach to overcome this problem.

Methods: SWCNTs were refluxed in HNO₃/H₂SO₄ (1:3) at 120°C for 120 minutes. Transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), contact angle measurements, and near infrared (NIR) light exposure were used to assess the functionalization process. The attachment of a carbohydrate-binding protein (lectin) labeled with fluorescein isothiocyanate to the functionalized SWCNTs enabled evaluation of the functionalization step via confocal microscopy. The lectin from Helix pomatia, (Helix pomatia agglutinin [HPA]), can detect changes in protein glycosylation associated with aggressive metastatic cancer. The interaction between the lectin HPA alone and HPA conjugated to the functionalized SWCNTs with human breast cancer cells (MCF-7) was measured using a quartz crystal microbalance biosensor.

Results: Following the functionalization process, TEM images showed a layer had formed on the surface of the SWCNTs. In the FTIR experiment, results illustrated the presence of the -COOH group on the functionalized SWCNTs. Contact angle measurements showed that upon functionalization the hydrophilicity of the SWCNTs increased. The temperature increase in the liquid (supernatant) surrounding the functionalized SWCNTs following exposure to light in the NIR (808 nm) was greater than for non-functionalized SWCNTs. The biosensor work showed that HPA binds with high affinity (nanomolar range) to human breast cancer cells; HPA-binding properties to MCF-7 cells were retained following conjugation to the functionalized SWCNTs.

Conclusion: Treating pure SWCNTs with HNO₃/H₂SO₄ (1:3) at 120°C for 120 minutes is an effective method for functionalization of SWCNTs. HPA linked to SWCNTs is a suitable candidate for the delivery of the functionalized SWCNTs to cancer cells.

Keywords: HPA lectin; SWCNT; human breast cancer cells.

Figure 1

Transmission electron microscopy of SWCNTs pre- and post-functionalization. SWCNTs before (A) and after (B) oxidization using HNO₃:H₂SO₄. Treatment with acid resulted in chemical modifications of the SWCNTs and the formation of carboxyl groups on the surface. Inset: tube containing SWCNTs, showing the enhancement of dispersibility of SWCNTs 2 weeks post-functionalization. Abbreviation: SWCNT, single-walled carbon nanotube.

Figure 2

HPA-lectin-FITC-SWCNTs. A sample of functionalized SWCNTs conjugated to HPA-FITC was viewed under the confocal microscope, in transmission mode (A) and in fluorescence mode, following excitation at 488 nm (B). The results show that a proportion of the SWCNTs had been successfully conjugated to the FITC-labeled lectin. Abbreviations: FITC, fluorescein isothiocyanate; HPA, Helix pomatia agglutinin; SWCNT, single-walled carbon nanotube.

Figure 3

The effect of different acid exposure times on attachment of lectin to SWCNTs. The SWCNTs were treated with acid for 0.5, 1, 2, 3, 4, and 5 hours, followed by HPA-lectin-FITC conjugation. Confocal microscopy was used to determine the relative fluorescence for the SWCNTs prepared. An increase in the fluorescence was observed as the exposure time of SWCNTs to HNO₃ and H₂SO₄ increased. The relative fluorescence following 0.5 hours treatment with HNO₃ and H₂SO₄ was compared with longer incubation periods. Notes: Statistical analysis was performed using the Student’s t-test, and the P-values are indicated. Values are mean ± standard deviation. *P < 0.05 (2 hrs); **P < 0.01 (3 hrs, 4 hrs and 5 hrs). Abbreviations: FITC, fluorescein isothiocyanate; HPA, Helix pomatia agglutinin; SWCNT, single-walled carbon nanotube.

Figure 4

Fourier transform infrared spectroscopy analysis of SWCNTs. Before (red) and after (blue) functionalization. Following functionalization, peaks observed at 1750 cm⁻¹ and 3450 cm⁻¹ indicate that −COOH groups had been successfully grafted onto the surface of the SWCNTs. Abbreviation: SWCNT, single-walled carbon nanotube.

Figure 5

Confocal microscopy of MCF-7 breast cancer cells incubated with HPA-FITC and HPA-lectin-FITC linked to SWCNTs. Lectin binding to MCF-7 and SW480 cells as viewed using a Leica TCS SP2 confocal microscope. Cells were incubated with FITC-labeled HPA (green) and lectin-SWCNTs (green), and the nuclei were counterstained with To-Pro^®3 (blue). MCF-7 were observed to interact with the HPA lectin either linked to FITC or to SWCNT; in contrast, the SW480 cells did not bind the lectin. Abbreviations: FITC, fluorescein isothiocyanate; HPA, Helix pomatia agglutinin; SWCNT, single-walled carbon nanotube.

Figure 6

Temperature profile of SWCNTs, pre- and post-functionalization. Following exposure to light at λ 808 nm, functionalized SWCNTs achieved a higher temperature profile compared with non-functionalized CNTs. Note: Mean values ± standard deviation are shown. Abbreviations: CNT, carbon nanotube; SWCNT, single-walled CNT; F-SWCNT, functionalized SWCNT.

Figure 7

Sensorgrams illustrating the binding profile of HPA-FITC and HPA-lectin-SWCNTs to MCF-7 cells grown on a biosensor chip surface. The association (85 second) and dissociation (165 second) phases were monitored giving a change in the resonance frequency (ΔF) as the lectin interacted with the surface of the MCF-7 cells on the chip surface. The lectin interaction with MCF-7 cells was investigated at 12.5 μg/mL and 25 μg/mL as shown, using HPA alone and HPA-lectin-SWCNTs. The flow rate was maintained at 25 μL/min, and the temperature was 22°C. The binding profile for native HPA was comparable to the profile obtained for HPA-lectin-SWCNTs, indicating that the conjugation process did not affect the rate of epitope binding by the lectin. Abbreviations: FITC, fluorescein isothiocyanate; HPA, Helix pomatia agglutinin; SWCNT, single-walled carbon nanotube.