Folic Acid-Functionalized MWCNT-Conjugated Zirconium Oxide Nanoparticles for Targeted Cancer Cell Delivery of Astaxanthin

Abstract

In this study, zirconium oxide nanoparticles (ZrO₂ NPs) were synthesized using astaxanthin (AST) rich extract (AZ) and subsequently conjugated with multiwalled carbon nanotubes (MWCNTs) (AZM) and functionalized with folic acid (FA) (FAZM) to develop a cancer-targeting nanocomposite with enhanced anticancer efficacy. The physicochemical properties of the synthesized materials were characterized using transmission electron microscopy (TEM), dynamic light scattering (DLS), electrophoretic light scattering (ELS), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FT-IR). FAZM exhibited the highest antioxidant activity, with IC₅₀ values of 822.78 μg/mL against ABTS and 320.70 μg/mL against DPPH free radicals. Biocompatibility assessments revealed that FAZM exhibited little cytotoxicity in normal human skin cells and demonstrated improved hemocompatibility, as confirmed by a hemolysis assay. Furthermore, FAZM significantly inhibited the proliferation of MDA-MB-231 breast cancer cells, inducing apoptosis and exhibiting potent cytotoxic effects (IC₅₀: 115.84 μg/mL). These findings suggest that FA and MWCNTs enhance the cancer-targeting capability of AZ while maximizing its selective cytotoxicity against cancer cells. This study highlights that FA-functionalized MWCNT-conjugated ZrO₂ NPs are a promising nanoplatform as an AST delivery system for targeted cancer therapy.

Keywords: anticancer; astaxanthin; folic acid; multiwalled carbon nanotubes; targeting; zirconium oxide nanoparticles.

Scheme 1

Synthetic process and applications of AZ, AZM, and FAZM.

Figure 1

(a) TEM and SAED and (b) EDS images of AZ, AZM, and FAZM.

Figure 2

Particle size, PDI, and zeta potential of AZ, AZM, and FAZM.

Figure 3

XRD pattern of AZ and AZM. #: Carbon, ∗: ZrO₂.

Figure 4

FT-IR spectra of AST, AZ, AZM, and FAZM.

Figure 5

(a) DPPH, (c) ABTS results, and (b, d) IC₅₀ values of AZ, AZM, and FAZM. Data are expressed as means ± standard deviation (n = 3). ^∗p < 0.1, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.

Figure 6

Cytotoxicity in (a) normal human skin cells HaCaT, (c) breast cancer cells MDA-MB-231, and (b, d) IC₅₀ values of AZ, AZM, and FAZM. Data are expressed as means ± standard deviation (n = 3). ^∗p < 0.1, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.

Figure 7

(a) Visualization of hemolysis properties and (b) hemolysis percentage (%). Data are expressed as means ± standard deviation (n = 3). ^∗p < 0.1, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.

Figure 8

Measurement of ROS expression in MDA-MB-231 using DCFH-DA fluorescence staining analysis (a) and flow cytometry (b), and (c) statistical analysis of (b).

Figure 9

Fluorescence staining analysis results of (a) control, (b) AZ, (c) AZM, and (d) FAZM in MDA-MB-231 cells. The concentration of the samples was 250 μg/mL (scale bar, 50 μm).

Figure 10

Apoptosis assay for (a) control (PBS), (b) AZ, (c) AZM, and (d) FAZM in MDA-MB-231 cells. The concentration of the samples was 250 μg/mL.

Figure 11

(a) Micrograph of cell migration in MDA-MB-231 treated with (A) control (PBS), (B) AZ, (C) AZM, and (D) FAZM. (b) Quantification of wound closure (%). Data are expressed as means ± standard deviation (n = 3). ^∗p < 0.1, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.

Figure 12

Cellular internalization of AZ, AZM, and FAZM formulations in MDA-MB-231 cells. (a) TEM images showing intracellular localization of the formulations. (b) Zr standard calibration curve for quantitative analysis of intracellular zirconium content using ICP-OES. (c) Quantification of intracellular zirconium content in cells treated with AZ, AZM, and FAZM.