Nanoencapsulation mitigates the toxicity of thymol in human cells and zebrafish embryos

Abstract

This study evaluated the cytotoxicity of free thymol (FT) and thymol encapsulated in monolayer (MNCT) and layer-by-layer nanocapsules (LNCT) in normal human (MCF10A, HaCaT, HdFn), and cancer cell lines (MDA-MB-231, A549, HT-29, HCT-116), using the Alamar Blue assay. The study also assessed the acute and organ-specific toxicity of these formulations in zebrafish embryos (ZFEs). FT exhibited concentration-dependent cytotoxicity in the tested cell lines, with IC₅₀ values of 57-194 µg/mL, classifying it as moderately toxic (NCI guidelines). In contrast, MNCT and LNCT displayed IC50 values above 501 µg/mL, indicating nontoxic profiles. In vivo mortality assays in ZFEs corroborated these findings, yielding LC₅₀ values of 47.0 µg/mL (FT), 880.7 µg/mL (MNCT), and > 900 µg/mL (LNCT). NOEC values were 25 µg/mL (FT), 300 µg/mL (MNCT), and > 900 µg/mL (LNCT). Accordingly, classifying FT as slightly toxic and MNCT and LNCT as practically nontoxic (USFWS classification). MNCT and LNCT also rescued the thymol-induced reduction in ZFE hatching rates. In consistence, functional assays in ZFE revealed that FT significantly impaired locomotor activity, heart rate, and blood flow at ≤50 µg/mL, whereas MNCT required > 100 µg/mL to affect locomotion and > 300 µg/mL and > 900 µg/mL to impact cardiac function, respectively. Notably, LNCT remained nontoxic up to 900 µg/mL for all the tested functions. Collectively, nanoencapsulation significantly attenuates thymol toxicity in human cells and ZFEs, with LNCT providing superior protection, possibly through controlled release. These findings highlight thymol nanocapsules as promising, safer, and effective delivery systems for food and other applications.