Conjugation with L,L-diphenylalanine Self-Assemblies Enhances In Vitro Antitumor Activity of Phthalocyanine Photosensitizer

Abstract

We present the synthesis and characterization of new peptide conjugates obtained by hierarchical co-assembly of L,L-diphenylalanine (FF) and zinc phthalocyanine complexes (ZnPc) in water. Self-assembly capabilities under defined conditions were investigated by scanning electron microscopy, and photophysical properties were evaluated using UV-Vis and fluorescence spectroscopy. AFM observations demonstrated that these ZnPcs form different highly ordered arrays on the crystalline faces of the FF microplates and that surface roughness significantly changes with the presence of differently substituted phthalocyanine units. XRD assays showed that the overall molecular packing of the conjugates is organized according to a hexagonal symmetry, with ZnPcs hosted in the interstices of the peptide phase. In vitro photodynamic studies were conducted on human breast cancer MCF-7 cells to investigate both cellular uptake and cytotoxicity. It was shown that FF self-assemblies are not toxicity and enhance accumulation of ZnPc in MCF-7 cells, improving apoptotic cell death upon irradiation. Our findings demonstrate enhancement of ZnPc antitumor efficiency by FF conjugates and a proof-of-concept for new photosensitizer carriers based on peptide conjugates.

Figure 1

Zinc phthalocyanines used in this study.

Figure 2

SEM images of FF-MNSs conjugated with different zinc(II) phthalocyanines: (A) ZnPc1, (B) ZnPc2, (C) ZnPc3, (D) ZnPc4.

Figure 3

AFM topography images of ZnPc/FF-MNSs hybrids (A) ZnPc1, (B) ZnPc2, (C) ZnPc3, (D) ZnPc4. Left magnified image of the surface.

Figure 4

(left) Rietveld plots from the final refinements. Black open circles represent the observed patterns; red lines indicate the calculated patterns; blue lines at the bottom of each diffractogram display the difference between observed and calculated patterns. The magenta vertical bars represent the Bragg reflections of the FF crystal structure. From top to bottom: FF-MNSs, ZnPc1/FF-MNSs, ZnPc2/FF-MNSs, ZnPc3/FF-MNSs and ZnPc4/FF-MNSs. (right) Variation of unit cell parameters from FF-MNSs to ZnPc4/FF-MNSs.

Figure 5

Representative variation of the emission and absorption (inset) maxima of ZnPc3 (close circles) and ZnPc3/FF-MNSs (open circles) with chromophore concentration. Emission was measured at 693 nm (λ_exc.= 615 nm) and absorption at 680 nm.

Figure 6

Representative decay rates of absorption intensity of DPBF (417 nm) following photosensitization by 0.12 mM ZnPc3 (close circles) and ZnPc3/FF-MNSs (open circles).

Figure 7

Cell death profile investigated by annexin V-FITC and PI double staining flow cytometric analyses. MCF-7 cells (6 × 10⁵ cells/experiment) were incubated with 0.42 µmol/L ZnPc3, 0.2 mg/mL FF-MNSs and 0.2 mg/mL ZnPc3/FF-MNSs for 2 hours and irradiated at 660 nm for 10 minutes. Flow cytometry was done 24 h after irradiation and compared to the same experimental conditions in the dark. (A) Representative dot plot panels of one experiment (An⁻/PI⁻ viable cells An⁻/PI⁺ suggests necrosis; An⁺/PI⁻, early apoptosis; An⁺/PI⁺, late apoptosis or apoptosis followed by necrosis), and (B) quantification of the percentage of cell population in each quadrant obtained from two independent experiments done in duplicate. *One-way ANOVA followed by Tukey’s post hoc test showing statistically different from control (p < 0.05).

Figure 8

ZnPc3 uptake by MCF-7 cells. (A) Representative image of fluorescence emission in MCF-7 cells after 2 h incubation with 0.62 µmol/L ZnPc3 (left panels) and 0.3 mg/mL ZnPc3/FF-MNSs (right panels). Upper and bottom panels refer to images obtained under 200 and 630x magnification, respectively, in a Leica DMI 6000B microscopy system using Y5 filter with excitation at 675 nm. Scale bars: 50 µm (200x) and 20 µm (630x). (B) Fluorescence quantification was performed at 630x magnification using Leica Application Suite software (LAS, v. 3, Leica Microsystems, Germany). *One-way ANOVA followed by Tukey’s post hoc test showing statistically different from control (p < 0.05).