Size-Dependent Photodynamic Anticancer Activity of Biocompatible Multifunctional Magnetic Submicron Particles in Prostate Cancer Cells

Abstract

In this study, newly designed biocompatible multifunctional magnetic submicron particles (CoFe₂O₄-HPs-FAs) of well-defined sizes (60, 133, 245, and 335 nm) were fabricated for application as a photosensitizer delivery agent for photodynamic therapy in cancer cells. To provide selective targeting of cancer cells and destruction of cancer cell functionality, basic cobalt ferrite (CoFe₂O₄) particles were covalently bonded with a photosensitizer (PS), which comprises hematoporphyrin (HP), and folic acid (FA) molecules. The magnetic properties of the CoFe₂O₄ particles were finely adjusted by controlling the size of the primary CoFe₂O₄ nanograins, and secondary superstructured composite particles were formed by aggregation of the nanograins. The prepared CoFe₂O₄-HP-FA exhibited high water solubility, good MR-imaging capacity, and biocompatibility without any in vitro cytotoxicity. In particular, our CoFe₂O₄-HP-FA exhibited remarkable photodynamic anticancer efficiency via induction of apoptotic death in PC-3 prostate cancer cells in a particle size- and concentration-dependent manner. This size-dependent effect was determined by the specific surface area of the particles because the number of HP molecules increased with decreasing size and increasing surface area. These results indicate that our CoFe₂O₄-HP-FA may be applicable for photodynamic therapy (PDT) as a PS delivery material and a therapeutic agent for MR-imaging based PDT owing to their high saturation value for magnetization and superparamagnetism.

Keywords: biocompatible multifunctional submicron particles; cytotoxicity; folic acid; hematoporphyrin; magnetic resonance imaging; photodynamic anticancer therapy.

Figure 1

Morphology and crystal structure of CoFe₂O₄ submicron particles with controlled external size: (A) FE-SEM images of CoFe₂O₄ particles with controlled external size synthesized using different volume ratios of V_EG/V_DEG: (a) 5/15, (b) 10/10, (c) 15/5, and (d) 20/0. Other experimental parameters were kept constant (NaOAc = 1.0 g, Na acrylate = 0.5 g, temperature = 200 °C, and time = 10 h); (B) XRD patterns of CoFe₂O₄ particles with tunable grain sizes obtained using different mass ratios of NaOAc/Na acrylate (w/w): (a) 1.4/0.1, (b) 1.2/0.3, (c) 1.0/0.5, and (d) 0.5/1.0. Other experimental parameters were kept constant (EG = 15 mL, DEG = 5 mL, temperature = 200 °C, and time = 10 h).

Figure 2

Magnetic and optical properties of multifunctional magnetic particles: (A) Temperature dependence of magnetic susceptibility of monodispersed CoFe₂O₄ particles in zero-field cooled (ZFC) and field-cooled (FC) conditions measured using a SQUID magnetometer at an applied field of 5 kOe; (B) PL and PLE spectra of pure HP and CoFe₂O₄-HP-FA in THF. The excitation and detection wavelengths were 500 and 625 nm for PL and PLE spectra, respectively; (C) Irradiation time-dependent UV-Vis spectra of DPBF in THF solution with CoFe₂O₄-HP-FA excited by the Xe lamp. The inset represents the absorption OD of DPBF in THF at 435 nm as a function of irradiation time. In the inset, (a) DPBF only with the light, (b) DPBF with CoFe₂O₄-HP-FA without the light, and (c) DPBF with CoFe₂O₄-HP-FA with the light are shown; (D) MR (T₂-weighted) images of CoFe₂O₄-HP-FA and PC-3 cells treated with CoFe₂O₄-HP-FA. (a) Different concentrations of 60-nm CoFe₂O₄-HP-FA. (b) PC-3 cells treated with 60-nm CoFe₂O₄-HP-FA. Cells treated with different concentrations of 60-nm CoFe₂O₄-HP-FA were incubated for 2 h in the dark before acquisition of MR images.

Figure 3

Cytotoxicity and photodynamic anticancer activity of multifunctional magnetic particles in PC-3 cells. (A) Cytotoxicity in fibroblast (L-929 cells) and (B) prostate cancer (PC-3 cell). Cells were cultured with different concentrations of CoFe₂O₄-HP-FA for 24 h at 37 °C in the dark; (C) Photodynamic anticancer activity of each size of CoFe₂O₄-HP in PC-3 cells; (D) Photodynamic anticancer activity of each size of CoFe₂O₄-HP-FA in PC-3 cells. Cells were incubated with different concentrations of CoFe₂O₄-HP and CoFe₂O₄-HP-FA for 2 h in the dark prior to irradiation for 30 min. Data are expressed as the mean ± standard deviation (n = 6) and were analyzed by Student’s t-test. Statistical significance was defined as p < 0.05 (* p < 0.05, ** p < 0.005, *** p < 0.001 vs. control at the same time).

Figure 4

TEM images of PC-3 cells. (A) Cells only; (B) Cells with 60-nm CoFe₂O₄-HP; (C) Cells with 60-nm CoFe₂O₄-HP-FA. Cells were incubated with 6.25 μg/mL CoFe₂O₄-HP or CoFe₂O₄-HP-FA for 2 h in the dark. Scale bar: 2 μm.

Figure 5

Apoptotic DNA fragmentation, Capase-3/7 activity, cell membrane translocation, and nuclear fragmentation in PC-3 cells. (A) Apoptotic DNA fragmentation in PC-3 cells. Apoptosis was induced by irradiation for 30 min after treatment with each size of CoFe₂O₄-HP-FA at 6.25 µg/mL in PC-3 cells. Lane 1; DNA size marker (1 kb), Lane 2; control, Lane 3; 60-nm MNPs, Lane 4; 133-nm MNPs, Lane 5; 245-nm MNPs, Lane 5; 335-nm MNPs; (B) Caspase-3/7 activity was evaluated after treatment with 60-nm CoFe₂O₄-HP-FA for 2 h without irradiation and at 0, 1, and 2 h post irradiation. Data are expressed as the mean ± standard deviation (n = 6) and were analyzed by Student’s t-test. Statistical significance was defined as p < 0.05 (* p < 0.05, ** p < 0.005 vs. control at the same time); (C) Confocal fluorescence images of Annexin V-FITC (green) staining indicating cell membrane inversion and PI (red) for nuclear staining in PC-3 cells at 12 h post irradiation. Apoptosis was induced by irradiation for 30 min after treatment with 60-nm CoFe₂O₄-HP-FA at 3.13 µg/mL for 2 h. Control cells were incubated without MNPs. Arrows show apoptotic cells (line arrows; early stage of apoptosis, dashed arrows; late stage of apoptosis); (D) Confocal fluorescence images for nuclear fragmentation (line arrows); Hoechst 33342 (blue) shows nuclei and Texas Red (red) shows whole cells in PC-3 cells at 12 h post irradiation. Apoptosis was induced by irradiation for 30 min after treatment with 60-nm CoFe₂O₄-HP-FA at 3.13 µg/mL for 2 h. Control cells were incubated without MNPs. Scale bar represents 25 µm.