The application of hyaluronic acid-derivatized carbon nanotubes in hematoporphyrin monomethyl ether-based photodynamic therapy for in vivo and in vitro cancer treatment

Abstract

Carbon nanotubes (CNTs) have shown great potential in both photothermal therapy and drug delivery. In this study, a CNT derivative, hyaluronic acid-derivatized CNTs (HA-CNTs) with high aqueous solubility, neutral pH, and tumor-targeting activity, were synthesized and characterized, and then a new photodynamic therapy agent, hematoporphyrin monomethyl ether (HMME), was adsorbed onto the functionalized CNTs to develop HMME-HA-CNTs. Tumor growth inhibition was investigated both in vivo and in vitro by a combination of photothermal therapy and photodynamic therapy using HMME-HA-CNTs. The ability of HMME-HA-CNT nanoparticles to combine local specific photodynamic therapy with external near-infrared photothermal therapy significantly improved the therapeutic efficacy of cancer treatment. Compared with photodynamic therapy or photothermal therapy alone, the combined treatment demonstrated a synergistic effect, resulting in higher therapeutic efficacy without obvious toxic effects to normal organs. Overall, it was demonstrated that HMME-HA-CNTs could be successfully applied to photodynamic therapy and photothermal therapy simultaneously in future tumor therapy.

Keywords: HA-derivatized carbon nanotubes; hematoporphyrin monomethyl ether; photodynamic therapy; photothermal therapy; synergistic effect; tumor targeting.

Figure 1

Scheme of preparation of hematoporphyrin monomethyl ether hyaluronic acid-derivatized carbon nanotubes (HMME-HA-CNTs). Abbreviations: CNT, carbon nanotubes; HMME, hematoporphyrin monomethyl ether.

Figure 2

(A–C) Characterization of hyaluronic acid-derivatized carbon nanotubes (HA-CNTs). (A) Fourier transform infrared spectra of pristine CNTs (a), COOH-CNTs (b), NH₂-CNTs (c), and HA-CNTs (d); (B) thermal gravimetric analysis curves of pristine CNTs (a), NH₂-CNTs (b), HA-CNTs (c), and HA (d); (C) CNTs (a), COOH-CNTs (b), NH₂-CNTs (c), and HA-CNTs (d) in water.

Figure 3

(A–D) Characterization of hematoporphyrin monomethyl ether hyaluronic acid-derivatized carbon nanotubes (HMME-HA-CNTs). (A) Ultraviolet spectra of HA-CNTs (1) and HMME-HA-CNTs (2) in water; (B) HMME loading at different feeding amounts of HMME; (C) photos of HMME-HA-CNTs in water (1), phosphate-buffered saline (2), plasma of mice (3) and cell culture (4) for 2 weeks; (D) Release profiles of HMME from HMME solution (1) and HMME-HA-CNTs (2).

Figure 4

(A–D) Characterization of hematoporphyrin monomethyl ether hyaluronic acid-derivatized carbon nanotubes (HMME-HA-CNTs). (A and B) Transmission electron microscopy images of HMME-HA-CNTs; (C) scanning electron microscopy image of HMME-HA-CNTs; (D) zeta potential of HMME-HA-CNTs.

Figure 5

Temperature evolution of high (a), middle (b), and low (c) concentrations of hematoporphyrin monomethyl ether hyaluronic acid-derivatized carbon nanotubes (HMME-HA-CNTs) (HA-CNTs approximately 8.7, 6.5, and 4.3 μg/mL; HMME approximately 20, 15, and 10 μg/mL) and phosphate-buffered saline (d), during continuous radiation by 808 nm laser at 1.4 W/cm².

Figure 6

B16F10 cell uptake of (A) fluorescein isothiocyanate (FITC) alone and (B) hematoporphyrin monomethyl ether hyaluronic acid-derivatized carbon nanotubes (HMME-HA-CNTs)/FITC.

Figure 7

(A) Cell viability of B16F10 cells with different concentrations of hyaluronic acid-derivatized carbon nanotubes (HA-CNTs). (B) Relative cell viability of B16F10 cells for 24 hours after treatment with free hematoporphyrin monomethyl ether (HMME), HA-CNTs, and HMME-HA-CNTs under 100 mW/cm² of 532 nm laser and 1.4 W/cm² of near-infrared 808 nm irradiation at different concentrations. Note: Data are presented as means ± standard deviation (n = 6).

Figure 8

(A–F) Fluorescence photomicrographs of B16F10 cells stained with Hoechst 33258. (A) Untreated cells; (B) hematoporphyrin monomethyl ether (HMME) alone (10 μg/mL); (C) HMME, 532 nm laser (10 μg/mL); (D) hyaluronic acid-derivatized carbon nanotubes (HA-CNTs), 808 nm laser (4.35 μg/mL); (E) HMME-HA-CNTs, 532 nm laser (HMME dose, 10 μg/mL; HA-CNT dose, 4.35 μg/mL); and (F) HMME-HA-CNTs, 532/808 nm laser (HMME dose, 10 μg/mL; HA-CNTs dose, 4.35 μg/mL).

Figure 9

(A) Tumor-growth curves of different groups after treatment. The tumor volumes were normalized to their initial sizes. (B) Mean body weights of mice in different groups after treatment. Note: Data are presented as means ± standard deviation (n = 5). Abbreviation: HMME-HA-CNTs, hematoporphyrin monomethyl ether hyaluronic acid-derivatized carbon nanotubes.

Figure 10

Tumor-bearing mice treated with (A) 808 nm near-infrared laser and (B) 532 nm laser. (C) Photo of tumors taken out of the saline group (1), the hyaluronic acid-derivatized carbon nanotubes (HA-CNTs; 808 nm) laser group (2), the hematoporphyrin monomethyl ether (HMME; 532 nm) laser group (3), the HMME-HA-CNT (532 nm) laser group (4), and the HMME-HA-CNT (532/808 nm) laser group after treatment for 8 days (5).

Figure 11

(A–E) Histological assessments of major organs and tumor tissues with hematoxylin and eosin staining in mice (200×). (A) Saline; (B) hyaluronic acid-derivatized carbon nanotubes (HA-CNTs), 808 nm laser; (C) hematoporphyrin monomethyl ether, 532 nm laser; (D) HA-CNTs, 532 nm laser; and (E) HA-CNTs, 532/808 nm laser.