Evaluation of Antioxidant and Anti-Inflammatory Effects of a Nanoformulation Derived from Annurca Apple Callus Extract in an In Vitro Model of Iron Overload-Induced Inflammation

Abstract

Ferroptosis, a regulated form of cell death driven by iron accumulation and lipid peroxidation, contributes to oxidative stress-related skin damage. This study evaluates the antioxidant and anti-inflammatory effects of a nanoformulation derived from an Annurca apple callus extract in an in vitro model of ferroptosis using human keratinocytes (HaCaT cells). A hydroalcoholic extract from light Annurca apple callus (LCE) was nanoformulated with Pluronic^® F127 and Soluplus^® to enhance stability and bioavailability. The resulting nanoformulation (NF-LCE) exhibited optimal particle size (103.17 ± 0.87 nm), polydispersity index (0.21 ± 0.00), and zeta potential (-1.88 ± 0.64 mV). Iron overload (100 µM) was employed to induce oxidative stress and inflammation in HaCaT cells, resulting in elevated levels of inflammatory markers (COX2, IL-6, TNF-α) and a diminished antioxidant response, as indicated by decreased expression of GPX4 and Nrf2. NF-LCE treatment restored GPX4 and Nrf2 levels (~0.8-fold increase, p < 0.05) while significantly reducing COX2 (36.6%, p < 0.01), IL-6 (79.6%, p < 0.0001), and TNF-α (30.9%, p < 0.1). These results suggest NF-LCE as a promising therapeutic strategy for mitigating ferroptosis-induced skin damage, warranting further investigation in advanced skin models and clinical applications.

Keywords: antioxidant; ferroptosis; nanoformulation; natural compounds; skin protection.

Figure 1

Transmission electron micrographs of nanoparticles of the three nanoformulations: (A) blank nanoformulation (magnification: 65 k); (B) NF-TA (magnification: 135 k); (C) NF-LCE (magnification: 93 k).

Figure 2

HaCaT cell viability. HaCaT cells were treated with different concentrations of LCE (13–0.203125 µM TA concentration) for 24 h (a) and 48 h (b), and cell viability was measured by MTT assay. MEM represents the control condition with cells incubated with treatment-free media. The results are expressed as% of CTRL ± SEM (n = 6; p ≤ 0.05). ANOVA followed by Dunnett’s multiple comparison test was performed (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Figure 3

HaCaT cell viability after exposure to unformulated and nanoformulated compounds. HaCaT cells were treated with different concentrations of encapsulated and non-encapsulated LCE and TA for 24 h (a) and 48 h (b), and cell viability was measured by MTT assay. MEM represents the control condition with cells incubated with treatment-free media. The results are expressed as% of CTRL ± SEM (n = 6; p ≤ 0.05). ANOVA followed by Dunnett’s multiple comparison test was performed (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 4

Quantification of non-bound and ferritin-bound iron levels. Intracellular iron accumulation was quantified using FerroZine^™-based colourimetric assay (a) and Ferritin assay (b). Total non-ferritin-bound iron was obtained subtracting the total iron content obtained from the ferrozine assay from the corresponding mean ferritin concentrations (c). Data are expressed as mean ± SD (n = 6; p ≤ 0.05). ANOVA followed by Dunnett’s multiple comparison test was performed comparing each condition against the treatment condition with normal iron levels (20 µM) (* p < 0.05, **** p < 0.0001).

Figure 5

Cell viability. HaCaT cells were treated with different iron concentrations (20 µM, 50 µM, 100 µM, and 200 µM) for 24 h (a) and 48 h (b), and cell viability was measured by MTT assay. MEM represents the control condition with cells incubated with treatment-free media. Data are expressed as % of CTRL ± SD (n = 6; p ≤ 0.05). ANOVA followed by Dunnett’s multiple comparison test was performed (* p < 0.05, *** p < 0.001, **** p < 0.0001).

Figure 6

Anti-inflammatory activity of formulated and non-formulated LCE and TA on iron-treated HaCaT cells. HaCaT cells were pre-treated for 3 h with formulated and non-formulated LCE and TA (3.25 µM) followed by 24 h incubation with 100 µM Iron. MEM represents the control condition with cells incubated with treatment-free media. Data are expressed as% of CTRL ± SEM (n = 3; p ≤ 0.05). ANOVA followed by Dunnett’s multiple comparison test was performed against the prooxidant condition alone (100 µM Iron) (* p < 0.1, ** p < 0.01, *** p < 0.001, **** p < 0.0001).