**1. Introduction**

Essential oils (EOs) have been widely used in folk medicine throughout the history of humankind. The application of EOs covers a wide range from therapeutic, hygienic, and spiritual to ritualistic purposes. EOs are aromatic, volatile, lipophilic liquids extracted from different parts of plant materials such as barks, buds, flowers, fruits, seeds, and roots [1]. EOs are mixtures of complex compounds with variable individual chemical composition and concentrations that includes primarily terpenoids, like monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), acids, alcohols, aldehydes, aliphatic hydrocarbons, acyclic esters or lactones, rare nitrogen- and sulfur-containing compounds, coumarin, and homologues of phenylpropanoids [1,2]. The biological effects of EOs cover a wide range of effects, including antioxidant, antimicrobial, antitumor, anti-inflammatory, and antiviral activity [3].

The increase in demand for the use of aromatherapy as complementary and alternative medicine has led people to believe in the myth that EOs are harmless because they are natural and have been used for a long time [4]. However, there might be several side effects of EOs even if topical administration is applied and, among these, allergic reactions are the most frequent (many EOs can cause, e.g., rashes on the skin). Some of them can be poisonous if absorbed through the skin, breathed, or swallowed. Previous studies also report the interaction of EOs with other drugs [5]. The continuous production of new aroma chemicals and their widespread and uncontrolled usage as alternative therapies together with many carrier diluents have brought serious problems, especially among children. In this regard, it is of utmost importance to study the mode of action of essential oils and to find a proper, unharmful formulation. Another serious problem is the highly lipophilic nature of EOs, which makes it impossible to measure their biological effects in an aqueous environment [1,6,7].

One major characteristic and application of EOs are their strong antimicrobial activity, including antibacterial and antifungal effects without the development of microbial resistance. Numerous studies are found in the literature describing the antimicrobial activities of a large variety of EOs [8–13]. Most of these assays include conventional broth dilution method, disk diffusion method, and bioautography assay to measure the antimicrobial activity of EOs. Efforts have been made to overcome the lipophilic nature of the oils usually by application of EOs diluted in seemingly suitable solvents/detergents. In the case of natural lipophilic volatile compounds like EOs, solvents of varying polarity, e.g., DMSO, ethanol, and methanol, are most commonly used. However, previous studies have reported the antimicrobial effects of the solvents themselves (DMSO, ethanol, and other solvents in various microbial assays) or their influence on the true antimicrobial effects of EOs [14]. The usage of solubilizing agents limits the precise determination of the antimicrobial activities of EOs. Also, a major problem might arise in the classical assays due to the evaporation of EOs during the assay or the inability of the test microbes to reach the lipophilic range of the tested EOs (in bioautography, as an example) [15,16].

Therefore, new formulations have been determined to increase the solubility or to emulsify the EOs in an aqueous environment. These efforts help to stabilize the oils, to produce an even release of the active components into the required environment, and to maintain their antioxidant and antimicrobial activities [6,11,17,18]. Detergents and organic solvents are not welcome in this regard. Attempts have been made to entrap EOs by modified cyclodextrins for the exact determination of their antimicrobial characteristics [19,20].

The application of Pickering nanoemulsion is a quite novel approach to stabilize oil-in-water (O/W) and water-in-oil (W/O) emulsions by solid particles instead of surfactants. The mechanism involves the adsorption of solid particles on the oil–water interface, causing a significant decrease in the interfacial surface tension that results in high emulsion stability [18]. Previous studies have reported decreased evaporation of EOs from O/W emulsion of nanoparticle-stabilized formulations versus EO–surfactant systems to be a beneficial factor [21,22].

Despite the numerous existing studies on EO–Pickering emulsion, we could not find any literature data on chamomile volatile oil–nanoparticle formulation [7,23]. The main aim of the present work is to use Pickering emulsion of chamomile EO stabilized with modified Stöber silica nanoparticles and characterize its antimicrobial effect using Gram-positive and Gram-negative bacteria as well as *Candida* fungal species. We could demonstrate the strong antimicrobial effects of the chamomile EO–Pickering emulsion and suggest a plausible mode of action of this formulation. Experimental efforts were made to support the suggested mode of action.

#### **2. Results**

#### *2.1. Characteristics of Stöber Silica Nanoparticles*

The mean diameter, PDI value (polydispersity index), and zeta potential of modified Stöber silica nanoparticles (SNPs) were determined by dynamic light scattering (DLS), and these values were 20 nm, 0.01, and −21.3 mV, respectively. The size and morphology of SNPs were examined by TEM (see Figure 1). The size distribution obtained by DLS was confirmed by TEM, which showed that the mean diameter of silica samples was 20 nm; they are highly monodisperse and have a spherical shape.

**Figure 1.** TEM images of silica nanoparticles (SNPs) with different resolutions: 100,000× magnification (**A**) and 500,000× magnification (**B**), accelerating voltage: 80 kV; dTEM = 20 nm. PDI = 0.015.
