Hyaluronate Functionalized Multi-Wall Carbon Nanotubes Loaded with Carboplatin Enhance Cytotoxicity on Human Cancer Cell Lines

Abstract

Cancer is a major global public health problem and conventional chemotherapy has several adverse effects and deficiencies. As a valuable option for chemotherapy, nanomedicine requires novel agents to increase the effects of antineoplastic drugs in multiple cancer models. Since its discovery, carbon nanotubes (CNTs) are intensively investigated for their use as carriers in drug delivery applications. This study shows the development of a nanovector generated with commercial carbon nanotubes (cCNTs) that were oxidized (oxCNTs) and chemically functionalized with hyaluronic acid (HA) and loaded with carboplatin (CPT). The nanovector, oxCNTs-HA-CPT, was used as a treatment against HeLa and MDA-MB-231 human tumor cell lines. The potential antineoplastic impact of the fabricated nanovector was evaluated in human cervical adenocarcinoma (HeLa) and mammary adenocarcinoma (MDA-MB-231). The oxCNTs-HA-CPT nanovector demonstrate to have a specific antitumor effect in vitro. The functionalization with HA allows that nanovector bio-directed towards tumor cells, while the toxicity effect is attributed mainly to CPT in a dose-dependent manner.

Keywords: carboplatin; cytotoxicity; human cancer cells; hyaluronic acid; multiple-walled carbon nanotubes.

Figure 1

(A). Transmission electron micrographs of commercial-grade carbon nanotubes (cCNTs). Notice the disorganization and agglomeration of the nanotubes. In the oxidized and functionalized CNTs loaded with CPT (oxCNTs–HA-CPT), observe the greater scattering, increase in the electrodensity of the walls, and the electrodense spots inside that correspond to the Carboplatin (CPT) (white arrows). Scale = 1.5 µm. (B). Determination of the CD44 receptor in NIH-3T3 (non-tumor cells), HeLa (cervical cancer cells), and MDA-MB-231 (breast cancer cells) using the indirect immunofluorescence technique. Note the increased positivity for the CD44 receptor in tumor cells. Scale = 50 µm.

Figure 2

Micrographs of NIH-3T3 exposed for 24 h to 0, 5, 10, 30, 50 and 100 µg/mL. Note that the higher the concentration of the commercial-grade carbon nanotubes (cCNT), the greater the agglomeration of the nanoparticles, and the lower of cell confluence. In the oxidized CNTs (oxCNTs), oxidized and functionalized CNTs (oxCNT–HA), and the oxidized and functionalized CNTs loaded with CPT (oxCNTs–HA-CPT) treatments, the higher the concentration of the treatment the fewer agglomerates, and the cell confluence does not decrease significantly. Interestingly, in the Carboplatin (CPT) treatments, note that the higher the concentration, the lower the cell confluence. The cells detach from the well due to the cytotoxicity of the treatment and change their morphology. Scale = 200 µm.

Figure 3

Micrographs of HeLa cells in culture and exposed for 24 h at concentrations of 0, 5, 10, 30, 50, and 100 µg/mL. Note that the higher the concentration of cCNTs, the greater the agglomeration of the nanoparticles, and the lower the cell confluence. On the contrary, in the oxCNTs and oxCNT–HA treatments, it is observed that the higher the concentration, the nanoparticle agglomerates are less, and the cell confluence does not decrease significantly. Interestingly, in the treatments with oxCNT–HA–CPT and CPT, it was observed that the higher the concentration of the treatment, the lower the cell confluence. The cells detach from the well due to the cytotoxicity of the treatment and change their morphology. However, the toxicity of CPT is lower compared to that of oxCNT–HA–CPT. Scale = 200 µm.

Figure 4

Graphs of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric, and 4′,6-diamidino-2-phenylindole (DAPI) assays representing viability in NIH-3T3, HeLa, and MDA-MB-231 cell lines after administration of cCNT (blue bar), oxCNT (red bar), and oxCNT-HA (green bar) at doses of 0, 5, 10, 30, 50 and 100 µg/mL and 12, 24, 48, and 72 h. Note that oxidation and functionalization of cCNTs decrease their cytotoxicity. (n = 5). Tukey’s statistical test. P < 0.05. The asterisk (*) indicates statistical significance.

Figure 5

Graphs of the MTT and DAPI assays that represent the viability of the NIH-3T3, HeLa, and MDA-MB-231 cell lines after administration of CPT (blue bar) and oxCNT-HA-CPT (red bar) at doses of 0, 5, 10, 30, 50, and 100 µg/mL and 12, 24, 48 and 72 h of exposure. Note that, after 24 h of exposure, CPT has a greater cytotoxic effect on non-tumor cells, compared to oxCNT-HA-CPT. On the contrary, the oxCNT–HA–CPT nanovector has a greater cytotoxic effect on tumor cells compared to CPT. (n = 5). Statistical test of Student’s T. P < 0.05. The * indicates statistical significance.