# Nanoencapsulation of Eucalyptus Essential Oils via Box–Behnken Design: Phytochemical Profiling and Enhanced Antibacterial and Antibiofilm Efficacy

## TL;DR

This study improves the stability and effectiveness of Eucalyptus essential oils by encapsulating them into nanoemulsions, enhancing their antibacterial and antibiofilm properties.

## Contribution

A design-driven approach using Box–Behnken optimization to develop stable and effective Eucalyptus essential oil nanoemulsions.

## Key findings

- Nanoemulsions achieved droplet sizes below 200 nm and showed robust physical stability.
- Antimicrobial activity was up to 4-fold higher, with 80% biofilm inhibition observed.
- Enhanced dispersion and stabilization of volatile compounds likely contributed to improved efficacy.

## Abstract

The increasing burden of infectious diseases and antimicrobial
resistance underscores the urgent need for alternatives to conventional
therapeutics. Although Eucalyptus essential oils
(EOs) are well recognized for their broad-spectrum antimicrobial and
antibiofilm properties, their relatively high volatility and limited
physicochemical stability restrict their practical applications. In
this study, a systematic, design-driven comparative approach was employed
to develop nanoemulsions containing Eucalyptus globulus, E. citriodora, and E. radiata EOs, with the aim of improving their stability
and biological efficacy. Initially, gas chromatography–mass
spectrometry was performed to identify species-specific chemotypes,
which guided rational formulation design. A Box–Behnken design
enabled the precise optimization of critical colloidal parameters,
resulting in nanoemulsions with droplet sizes below 200 nm, polydispersity
indices below 0.3, and ζ-potentials of approximately −22
mV. Comprehensive structural characterization by Fourier transform
infrared spectroscopy and scanning electron microscopy, together with
stress testing, confirmed the robust physical stability of the formulations. In-vitro antimicrobial evaluations against Staphylococcus aureus, Enterococcus
faecalis, and Klebsiella pneumoniae revealed up to a 4-fold enhancement in antimicrobial activity and
up to 80% inhibition of biofilm compared to the corresponding unformulated
essential oils. The enhancements are most likely attributable to enhanced
dispersion, increased interaction with greater microbial interfaces,
and nanoscale stabilization of volatile bioactive constituents. Collectively,
the optimized Eucalyptus nanoemulsions constitute
reproducible, chemically well-defined nanocarrier systems with markedly
improved antibacterial and antibiofilm performance, highlighting their
promise as next-generation delivery systems for phytopharmaceutical
applications. Further comprehensive biological and toxicological investigations
are required to support the safe and effective translation of essential
oil-containing nanocapsulations into practical use.