Cold Physical Plasma-Mediated Fenretinide Prodrug Activation Confers Additive Cytotoxicity in Epithelial Cells

Abstract

Cold physical plasma is a partially ionized gas operated at body temperature and utilized for heat-sensitive technical and medical purposes. Physical plasma is a multi-component system consisting of, e.g., reactive species, ions and electrons, electric fields, and UV light. Therefore, cold plasma technology is an interesting tool for introducing biomolecule oxidative modifications. This concept can be extended to anticancer drugs, including prodrugs, which could be activated in situ to enhance local anticancer effects. To this end, we performed a proof-of-concept study on the oxidative prodrug activation of a tailor-made boronic pinacol ester fenretinide treated with the atmospheric pressure argon plasma jet kINPen operated with either argon, argon-hydrogen, or argon-oxygen feed gas. Fenretinide release from the prodrug was triggered via Baeyer-Villiger-type oxidation of the boron-carbon bond based on hydrogen peroxide and peroxynitrite, which were generated by plasma processes and chemical addition using mass spectrometry. Fenretinide activation led to additive cytotoxic effects in three epithelial cell lines in vitro compared to the effects of cold plasma treatment alone regarding metabolic activity reduction and an increase in terminal cell death, suggesting that cold physical plasma-mediated prodrug activation is a new direction for combination cancer treatment studies.

Keywords: boronic pinacol ester; cancer therapy; cold atmospheric pressure plasma; gas plasma technology; prodrug; reactive oxygen species (ROS).

Figure A1

¹H NMR (top) and ¹³C NMR (bottom) of prodrug (FenP, (2E,4E,6E,8E)-3,7-dimethyl-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nona-2,4,6,8-tetraenamide).

Figure A2

(a) ESI-MS(+), (b) expanded ESI-MS(+), and (c) ESI-tandem MS² spectrum of fenretinide prodrug matching the [M + H]⁺.

Figure A3

Cold-plasma-derived ROS/RNS. Quantification of cold-plasma-induced H₂O₂ (a) and ONOO⁻ (b) under Ar/H₂ and Ar/O₂ discharge regimes. (*) Prodrug-treated solution; Ar = argon; H₂ = hydrogen; O₂ = oxygen.

Figure A4

Plausible mechanism of fenretinide isomerization [56].

Figure A5

Trans-FenD release at t_R = 7.10 min in chromatogram upon plasma treatment incubated for 2 h (a) and 24 h (b); abundance (peak area) of FenH degradation at m/z 420.2 and FenD release at m/z 392.2 upon plasma treatment under the Ar/H₂ discharge regime at 2 h (c,d) and 24 h (e,f) incubation time. Ar = argon; H₂ = hydrogen; O₂ = oxygen; n.d. not determined.

Figure A6

The abundance (peak area) of FenH degradation at m/z 420.2 and FenD release at m/z 392.2 upon cold plasma treatment under Ar and Ar/O₂ discharge regimes with 2 h (a,b) and 24 h (c,d) incubation. (*) Ar/O₂ discharge regime; Ar = argon; H₂ = hydrogen; O₂ = oxygen; n.d. = not determined.

Figure A7

The abundance (peak area) of FenH degradation at m/z 420.2 and FenD release at m/z 392.2 upon addition of 50 µM to 1000 µM H₂O₂ after 2 h (a,b), 24 h (c,d), and 72 h (e,f) of incubation; 3 mM and 5 mM H₂O₂ after 2 h (g,h), and 24 h (i,j) of incubation. n.d. = not determined.

Figure A8

pH of solution (mixture of H₂O–MeOH (1:1, v/v)) after plasma treatment ignited with varying gas admixtures.

Figure A9

(a) ESI-MS(−) of samples before and after cold plasma treatment under Ar (pink color), Ar/H₂ (red color), and Ar/O₂ (green color) regimes; (b) expanded spectrum from m/z 390 to 650.

Figure 1

(A) Activation mechanism of arylboronic acid and esters prodrugs with (a) a phenol-based prodrug, (b) an aromatic nitrogen mustard prodrug, and (c) a belinostat prodrug; (B) synthesis of fenretinide prodrug; (C) schematic view of the cold-plasma-treated prodrug solutions and plausible reactive species production in the gas phase and liquid media. EDC–HCl = 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; DMAP = 4-dimethylaminopyridine; Ar = argon.

Figure 2

Cold-plasma-derived ROS/RNS. (A,B) H₂O₂ deposition and consumption kinetics after argon plasma treatment (A) and consumption of chemically added H₂O₂ (B); (C) deposition of ONOO⁻/^•OH after argon plasma treatment. RFU = relative fluorescence units; Ar = argon; H₂ = hydrogen; O₂ = oxygen; n.d. = not determined.

Figure 3

(A) Proposed activation mechanism of the prodrug upon cold plasma treatment and chemically added H₂O₂; (B) HPLC–MS² of the boronate pinacol ester fenretinide prodrug solution incubated at ambient temperature for 2 h ((a), FenP) and 72 h ((b), hydrolysis step: FenH in solution) and after plasma treatment ((c), FenD release); (C–F) abundance (combined sum of peak areas) of FenH and FenD after cold plasma treatment with varying admixture gases (Ar, Ar/H₂, and Ar/O₂ regimes) at 2 h (FenH (C) and FenD (D)) and 24 h (FenH (E) and FenD (F)); (G,H) abundance of FenH/FenD after incubation with chemically added H₂O₂ at concentrations up to 1000 μM (G) and 5 mM (H) for specific durations. Ar = argon; H₂ = hydrogen; O₂ = oxygen; n.d. = not determined.

Figure 4

Prodrug cleavage and fenritinide release upon H₂O₂ addition. FenH degradation (A) and FenD release (B) upon incubation with different concentrations of H₂O₂ after 24h of incubation. I: 13-cis; II: 11-cis; III: 9-cis; IV: trans. Quantitative FenH degradation and FenD release in solution upon direct addition of H₂O₂ after 24h of incubation (C) and plasma treatment (D) (Ar, Ar/O_2, and Ar/H₂ discharge regime) at a given incubation time points. Ar = argon; H₂ = hydrogen; O₂ = oxygen.

Figure 5

Metabolic activity reduction in three epithelial cell lines with plasma and drug combination treatments. (A) Representative macroscopic image of the resazurin assay; (B–D) metabolic activity of HaCaT (B), A431 (C), and SCaBER (D) cells 72 h after cold plasma treatment of PBS or the addition of non-treated and plasma-treated FenP or FenD; (E–G) metabolic activity of HaCaT (E), A431 (F), and SCaBER (G) cells 72 h after cold plasma treatment of the cells alone or prior (timing 1) or after (timing 2) addition of FenP or FenD. Data are mean and S.E. of three independent experiments. Statistical analysis (E–G) was performed using one-way analysis of variances (ANOVA) with Dunnett’s post hoc test against untreated PBS vehicle (gray symbols) or cells treated with plasma alone (blue symbols). *** = p < 0.001 compared to PBS without plasma exposure (-); ## = p < 0.01 compared against PBS with plasma exposure (+); ns = p > 0.05.

Figure 6

Viability in three epithelial cell lines with plasma and drug combination treatments. (A) Representative brightfield (BF, (top)), digital phase contrast (DPC, (middle)), and DAPI (bottom) microscopy of HaCaT cells 72 h following plasma, drug, and plasma–drug combination treatments; (B–D) cell count (growth) of HaCaT (B), A431 (C), and SCaBER (D) cells up to 72 h after cold plasma treatment of PBS or the addition of non-treated and plasma-treated FenP or FenD; (E–G) number of dead (DAPI⁺) cells in HaCaT (E), A431 (F), and SCaBER (G) cells 72 h after cold plasma treatment of the cells alone or prior (timing 1) or after (timing 2) addition of FenP or FenD. Data are mean and S.E. of three independent experiments. Statistical analysis (E–G) was performed using one-way analysis of variances (ANOVA) with Dunnett’s post hoc test against untreated PBS vehicle (gray symbols). ** = p < 0.01; *** = p < 0.001; ns = p > 0.05.