Nanostructured delivery system for zinc phthalocyanine: preparation, characterization, and phototoxicity study against human lung adenocarcinoma A549 cells

Figures & data

Table 1 Mathematical models used for linearization of the photosensitizer release profile data

Mathematical models	Equation
Zero order	$F=k_{0}t$
First order	$ln(1-F)=-k_{\text{f}}t$
Higuchi	$F=k_{\text{H}}{t}^{1/2}$
Baker–Lonsdale	$3/2[1-{(1-F)}^{2/3}]-F=k_{3/2}t$
Hixon–Crowell	$1-{(1-F)}^{1/3}=k_{1/3}t$
Square root of mass	$1-{(1-F)}^{1/2}=k_{1/2}t$
Three seconds root of mass	$1-{(1-F)}^{2/3}=k_{2/3}t$

Notes: F denotes fraction (%) of drug released up to time t. k₀, k_f, k_H, k_3/2, k_1/3, k_1/2, and k_2/3 are constants of the mathematical models.

Figure 1 Photographs of TEM. A) and C) ZnPc-loaded PCL nanoparticles; B) and D) empty nanoparticles.

Abbreviations: TEM, transmission electron microscopy; ZnPc, zinc phthalocyanine; PCL, poly-ɛ-caprolactone.

Figure 2 A) In vitro releasing profile of the ZnPc from nanostructured system. B) Graphic representation of the Higuchi model obtained after regression. Mean and SD of n = 6 determinations.

Abbreviations: ZnPc, zinc phthalocyanine; SD, standard deviation.

Table 2 Released drug in the acceptor solution and remaining content of photosensitizer in the nanoparticles

Released drug (%)1	Remaining drug in the nanoparticles (%)1	Total (%)2	Loss (%)2
22.1 ± 0.24	70.8 ± 0.48	92.9	7.1

1 Notes: Media ± standard deviation of n = 6 determinations;

2 Media of n = 6 determinations.

Table 3 Determination coefficient (R²) and correlation coefficient (r) obtained after linear regression of the release data

Mathematical models	R^2	r
Zero order	0.9556	0.9775
First order	0.9663	0.9830
Higuchi	0.9861	0.9932
Baker–Lonsdale	0.9782	0.9890
Hixon–Crowell	0.9629	0.9812
Square root of mass	0.9612	0.9824
Three seconds root of mass	0.9594	0.9794

Figure 3 Influence of the incubation time (h) in the phototoxicity of free ZnPc or ZnPc-loaded nanoparticles. The cells were incubated with 5 μg (250 μL) of free ZnPc and ZnPc-loaded nanoparticles at different times, washed, and irradiated at a light dose of 100 J/cm². Dark toxicity was studied for all samples. The MTT assay was performed 24 h after light exposure. Each data point represents the mean (±SD) of n = l0 determinations.

Notes: *Significant difference (P < 0.05), **No significant difference (P > 0.05).

Abbreviations: ZnPc, zinc phthalocyanine; MTT, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-biphenyl tetrazolium bromide; MB, methylene blue; SD, standard deviation.

Figure 4 Influence of the photosensitizer concentration in the photobiological activity of free ZnPc, free MB, or ZnPc-loaded nanoparticles. The cells were incubated with 250 μL of free photosensitizer (ZnPc or MB) and ZnPc-loaded nanoparticles for 4 h, washed, and irradiated at a light dose of 100 J/cm². Dark toxicity was studied for all samples. The MTT assay was performed 24 h after light exposure. Each data point represents the mean (±SD) of n = 10 determinations.

Notes: *Significant difference (P < 0.05), **No significant difference (P > 0.05).

Abbreviations: ZnPc, zinc phthalocyanine; MB, methylene blue; MTT, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-biphenyl tetrazolium bromide; SD, standard deviation.

Figure 5 Influence of light dose on phototoxicity of free ZnPc and ZnPc-loaded nanoparticles. The cells were incubated for 4 h, at an equivalent drug dose of 5-pg ZnPc (250 μL), washed, and irradiated with red light (660 nm). MTT assay was performed 24 h after light exposure. Each data point represents the mean (±SD) of n = 10 determinations.

Notes: *Significant difference (P < 0.05), **No significant difference (P > 0.05).

Abbreviations: ZnPc, zinc phthalocyanine; MTT, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-biphenyl tetrazolium bromide; SD, standard deviation.

Figure 6 Morphological evaluation of the A549 cells using light microscope: A) and B) cells incubated with PBS (control). C) and D) cells incubated with empty nanoparticles suspension. E) and F) cells incubated with ZnPc-loaded nanoparticles. (A), (C), and (E) were observed at magnification of ×50 (scale 200 μm). (B), (I), and (F) were observed at magnification of ×400 (scale 50 μm).

Abbreviations: PBS, phosphate-buffered saline; ZnPc, zinc phthalocyanine.

Figure 7 Photobleaching of ABMDMA by singlet oxygen generated by encapsulated ZnPc and free ZnPc. Empty nanoparticles were used as control. The change in ABMDMA absorption at 400 nm was measured as a function of the irradiation time. Each data point represents the mean (±SD) of n = 3 determinations.

Abbreviations: ABMDMA, 9,10-anthracenediyl-bis(methylene)dimalonic acid; ZnPc, zinc phthalocyanine; SD, standard deviation.