EFFECT OF NANOENCAPSULATED THYMOL, CARVACROL, AND TRANS-CINNAMALDEHYDE IN HUMAN CELLS AND ZEBRAFISH EMBRYOS

Abstract

Thymol (THY; a monoterpenoid), carvacrol (CARV; a monoterpenoid), and trans-cinnamaldehyde (TC; a phenylpropanoid) are key bioactive constituents of essential oils with diverse pharmacological activities, including anticancer, antimicrobial, preservative, and insecticidal properties. However, their practical applications are limited by poor solubility, high volatility, rapid degradation, low bioavailability, and potential toxicity. Nanoencapsulation offers a promising strategy to overcome these limitations by improving stability and enabling controlled release. This study evaluated the toxicity of free (FT, FC, FTC) and nanoencapsulated [monolayer (ML) and layer-by-layer (LBL)] forms of THY, CARV, and TC in vitro using human cell lines (A549, HT-29, HCT-116) and zebrafish embryos (ZFEs) in vivo. In vitro assays demonstrated that ML and LBL nanoencapsulation significantly reduced the concentration-dependent cytotoxicity of the free compounds in human cells. In vivo, nanoencapsulation enhanced survival and hatching rates of ZFEs, and mitigated the teratogenic and neurotoxic effects observed with the free compounds. The LC₅₀ values of the free compounds were 46.9 μg/mL, 6.78 μg/mL, and 0.91 μg/mL for FT, FC, and FTC, respectively, whereas nanoencapsulation increased LC₅₀ by approximately 10–20-fold, with LBL outperforming ML. Based on U.S. Fish and Wildlife Service (USFWS) guidelines, toxicity classification shifted from moderately–highly toxic for the free forms to slightly–practically non-toxic for the nanocapsules, indicating reduced ecotoxicity. Except for FT and ML-FT, which induced cardiotoxicity at 25 and 900 μg/mL, respectively, none of the tested forms caused cardiotoxicity. Neurotoxicity was induced by the free compounds at concentrations starting at 25 μg/mL for FT, 1.56 μg/mL for FC and 0.195 μg/mL for FTC, yet around 4- to17-fold higher concentrations of the nanoencapsulated forms were required to induce similar effects. Overall, nanoencapsulation markedly reduced the cytotoxicity and developmental toxicity of THY, CARV, and TC, enhancing their safety and environmental compatibility for pharmaceutical, food, and biotechnological applications.