Toxicological Assessment of 2-Hydroxychalcone-Mediated Photodynamic Therapy: Comparative In Vitro and In Vivo Approaches

Abstract

Background: Photodynamic therapy (PDT) is a treatment modality that uses light to activate a photosensitizing agent, destroying target cells. The growing awareness of the necessity to reduce or eliminate the use of mammals in research has prompted the search for safer toxicity testing models aligned with the new global guidelines and compliant with the relevant regulations.

Objective: The objective of this study was to assess the impact of PDT on alternative models to mammals, including in vitro three-dimensional (3D) cultures and in vivo, in invertebrate animals, utilizing a potent photosensitizer, 2-hydroxychalcone.

Methods: Cytotoxicity was assessed in two cellular models: monolayer (2D) and 3D. For this purpose, spheroids of two cell lines, primary dermal fibroblasts (HDFa) and adult human epidermal cell keratinocytes (HaCat), were developed and characterized following criteria on cell viability, shape, diameter, and number of cells. The survival percentages of Caenorhabditis elegans and Galleria mellonella were evaluated at 1 and 7 days, respectively.

Results: The findings indicated that all the assessed platforms are appropriate for investigating PDT toxicity. Furthermore, 2-hydroxychalcone demonstrated low toxicity in the absence of light and when mediated by PDT across a range of in vitro (2D and 3D cultures) and in vivo (invertebrate animal models, including G. mellonella and C. elegans) models.

Conclusion: There was a strong correlation between the in vitro and in vivo tests, with similar toxicity results, particularly in the 3D models and C. elegans, where the concentration for 50% viability was approximately 100 µg/mL.

Keywords: 2-hydroxychalcone; Caenorhabditis elegans; Galleria mellonella; monolayers; photodynamic therapy; three-dimensional tissue model; toxicological evaluation of compounds.

Figure 1

Chemical structure of 2-hydroxychalcone (A) and absorption spectrum (B). Adapted from [20], Elsevier, 2017.

Figure 2

Images illustrate the characterization of the diameter and viability of three-dimensional spheroids. (A) The mean diameters of spheroids derived from primary dermal fibroblast (HDFa) and adult human epidermal keratinocyte (HaCat) lines demonstrate their suitability for compound testing. The HDFa cell line formed spheroids with an average diameter of 542.07 ± 18.15 μm, while the HaCat cell line produced spheroids with an average diameter of 399.49 ± 45.53 μm. (B) The resazurin method demonstrated high cell viability in both dermal cell lines, with spheroids from the HDFa cell line showing 94.54% viability (standard deviation ± 5.35%) and the HaCat cell line exhibiting 99.25% viability (standard deviation ± 1.14%). The data are presented as mean ± standard deviation.

Figure 3

Representative image of a rounded spheroid from the HDFa line, measuring approximately 591.02 µm (A). The spheroid from the HaCat line measures 432.36 µm (B).

Figure 4

The viability of HDFa cells was evaluated using the resazurin reduction assay following exposure to 2-hydroxychalcone and blue light irradiation at a dose of 150 J/cm² and 2-hydroxychalcone kept in the dark. Results are shown for both the monolayer model (A) and the spheroid model (B). All values are expressed in micrograms per milliliter (μg/mL). The results of the three independent experiments are expressed as the mean ± standard deviation (SD). The statistical significance of the results was determined using one-way ANOVA, with a p-value of less than 0.001 indicated by asterisks (***).

Figure 5

The viability of HaCat cells was evaluated using the resazurin reduction method after exposure to 2-hydroxychalcone and blue light irradiation at a dose of 150 J/cm² and 2-hydroxychalcone kept in the dark. The results are presented for both the monolayer model (A) and the spheroid model (B). Values are expressed in micrograms per milliliter (μg/mL). The results of the three independent experiments are expressed as the mean ± standard deviation (SD). The statistical significance of the results was determined using one-way ANOVA, with an asterisk (*) indicating a p-value of less than 0.05; (***) indicating a p-value of less than 0.001.

Figure 6

The percentage survival of Caenorhabditis elegans at larval stage 4 (L4). Mutant strain AU37 (A) and wild-type N2 (B) after 24 h of treatment with different concentrations of 2-hydroxychalcone in the dark and 2-hydroxychalcone-mediated PDT. The results of the three independent experiments are expressed as the mean ± standard deviation (SD). The statistical significance of the results was determined using one-way ANOVA, with *** p < 0.001.

Figure 7

Representative image of Caenorhabditis elegans (wild-type strain N2) larvae at the L4 larval stage treated with different concentrations of 2-hydroxychalcone and kept in the dark: 250 µg/mL (a); 125 µg/mL (b); 62.5 µg/mL (c); 31.25 µg/mL (d); 15.6 µg/mL (e); 7.8 µg/mL (f); 3.9 µg/mL (g); 1.95 µg/mL (h); 0.98 µg/mL (i); and untreated control (j) (magnification 10×). Rod-shaped larvae were considered dead (blue arrows), while sinusoidal larvae were considered alive (orange arrows).

Figure 8

Representative image of C. elegans N2 larvae at the L4 larval stage treated with different concentrations of 2-hydroxychalcone mediated by PDT: 250 µg/mL (a); 125 µg/mL (b); 62.5 µg/mL (c); 31.25 µg/mL (d); 15.6 µg/mL (e); 7.8 µg/mL (f); 3.9 µg/mL (g); 1.95 µg/mL (h); 0.98 µg/mL (i); and untreated control (j) (magnification 10×). Rod-shaped larvae were considered dead (blue arrows), while sinusoidal larvae were considered alive (orange arrows).

Figure 9

Survival curve of G. mellonella larvae treated with different concentrations of 2-hydroxychalcone (10, 50, 100, and 200 mg/kg) in the dark (A) and mediated by PDT (B). The survival curves were plotted of three independent assays as the mean ± standard deviation (SD).