Development and evaluation of chitosan nanocapsules co-loaded with tea tree oil and thymol: Physicochemical characterization, pH-responsive release, and in vitro antibacterial activity against mastitis-associated pathogens

Bovine mastitis remains one of the most economically significant diseases in dairy production and is a major driver of antimicrobial usage. The increasing prevalence of antimicrobial resistance has necessitated the development of alternative therapeutic strategies. Essential oils (EOs) and nanotechnology-based delivery systems have emerged as promising non-antibiotic approaches. This study aimed to develop chitosan nanocapsules (NC) co-loaded with tea tree oil (TTO) and thymol, and to evaluate their physicochemical characteristics, cytocompatibility, pH-responsive release behavior, and in vitro antibacterial activity against mastitis-associated pathogens.

Chitosan NC containing TTO and thymol were prepared using the ionic gelation method. Physicochemical properties were assessed by dynamic light scattering, scanning electron microscopy, and zeta potential analysis. Cytotoxicity was evaluated using MARC-145 cells. In vitro release studies were performed under simulated bovine mammary pH conditions (pH 6.6 and 7.2). Antibacterial activity was tested against Escherichia coli and Staphylococcus aureus using growth kinetics assays.

The NC exhibited a mean particle size of 383.1 ± 10.2 nm, a polydispersity index of 0.20, and a positive zeta potential of 24.7 ± 1.3 mV, indicating good stability. Encapsulation efficiency reached 77.5 ± 2.3%. Cytotoxicity analysis showed improved cell viability for nanoformulations compared with free oils, particularly for nanoemulsions. Release studies demonstrated a clear pH-dependent behavior, with limited release at pH 6.6 (18.4%) and enhanced release at pH 7.2 (61.7%) over 70 h. Antibacterial assays revealed that chitosan NC achieved the highest inhibitory effect, reducing S. aureus and E. coli growth by 92% and 72%, respectively. The antibacterial activity against S. aureus was comparable to erythromycin under the tested conditions.

Chitosan NC co-loaded with TTO and thymol demonstrated enhanced antibacterial activity, controlled-release, and improved cytocompatibility under in vitro conditions. The pH-responsive release profile highlights their potential as a targeted intramammary delivery system for mastitis management. These findings support further in vivo investigations to validate their applicability as a non-antibiotic alternative in dairy production systems.