**1. Introduction**

Essential oils are liquids with concentrated molecules resulting from the metabolism of a plant. They are very popular in the cosmetics, perfumery, food preservation, and pharmaceutical sectors [1]. The oxidative stress that harms biological molecules can be diminished by essential oils, and many degenerative diseases, including, diabetes, cardiovascular diseases, and neurological disorders, can also be successfully treated [2]. Despite the great biological potential that essential oils currently possess, they are under-utilized when their applications become difficult due to certain physicochemical characteristics provided by the compounds present in their compositions [3]. It is difficult to use essential oils in aqueous environments because they are generally highly volatile at room temperature, easily oxidized when exposed to oxygen and light, and above all have a high lipophilicity [4]. Aqueous solubilization and preservation of bioactive substances present in

**Citation:** Berraaouan, D.; Essifi, K.; Addi, M.; Hano, C.; Fauconnier, M.-L.; Tahani, A. Hybrid Microcapsules for Encapsulation and Controlled Release of Rosemary Essential Oil. *Polymers* **2023**, *15*, 823. https://doi.org/10.3390/ polym15040823

Academic Editors: Lorenzo Antonio Picos Corrales, Angel Licea-Claverie and Grégorio Crini

Received: 16 January 2023 Revised: 2 February 2023 Accepted: 4 February 2023 Published: 7 February 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

essential oils are crucial for their application, which requires the use of a system to promote their dispersion in aqueous media and regulate their release. These issues can be resolved through the incorporation of essential oils into various systems. Included in these systems are emulsions, beads, bioactive films, capsules, liposomes, nanocarriers, and inclusion complexes [5]. Encapsulation systems have attracted great interest in recent decades with a view to achieving an efficient drug delivery process [6]. Particular attention has been paid to finding an inert carrier where the drug is dispersed or incorporated. Recently, the preparation, characterization and application of controlled drug delivery systems prepared from biopolymer compounds has become popular owing to their distinctive properties such as a high encapsulation efficiency, biodegradability, nontoxicity, and controlled release characteristics [7]. The encapsulation of essential oils or their isolated components has been investigated by some researchers where they worked on the possibilities of using essential oils as a drug vehicle in delivery systems for lipophile drugs. Similarly, Rodriguez et al. [8] reviewed the possible strategies for essential oils encapsulation and showed the benefits of polymeric nanoencapsulation [9].

Out of the many biopolymers used for encapsulation purposes, alginate is widely used due to its biosafety, mild gelation properties, environmentally friendly [10], hydrophilicity, as well as its ability to be modified or combined with other biomaterials [11]. It is a hydrophilic polysaccharide, composed of two monomeric structures: β-D mannuronic acid (M) and α-L-guluronic acid (G) which are arranged in MM or GG blocks interspersed with MG blocks [12]. This structure has the unique property of forming water-insoluble calcium–alginate gel through ionotropic gelation with divalent cross-linking salt such as calcium chloride [13]. But the low efficiency of encapsulating water-soluble substances in alginate is one of the problems in developing a controlled delivery system [14]. In addition to the many advantages of polymeric encapsulation, it suffers from low strength. This disadvantage can be overcome by using natural clays as a filler; these systems are called hybrid capsules. Clay minerals have been proposed as fundamental constituents of various modified carriers and have different purposes. Among the promising clay materials, montmorillonite (MTN), which belongs to the smectites family, is of particular interest. The smectite clays are a layered aluminosilicate derived from the assembly of tetrahedral [SiO4] <sup>4</sup><sup>−</sup> and octahedral [AlO3(OH)3] <sup>6</sup><sup>−</sup> sheets. Hybrid systems on the basis of an organic and inorganic materials amalgam have been developed to solve the problem of low chemical stability, low mechanical strength and the erratic release of the encapsulated substances. They are very attractive for diverse applications due to the features afforded by the combination of the organic and the inorganic materials. Among its popular applications is active packaging. In this technology, materials loaded with active agents as essential oils are incorporated to the packaging to inhibit food degradation by oxidation, contamination and many other factors [15,16]. Research work has dealt with nanohybrids valued as possible containers for the encapsulation of rosemary essential oil using pectins and halloysite nanotubes HNTs (a rare type of kaolinite) [17]. It was observed that the release of rosemary essential oil was much slower in the nanohybrid material than the release of the same essential oil blended into the pectin. In addition, the release from the composite filled with the clay shows multiple steps, a rapid diffusion in the first stage, followed by a decreased rate of release in the remaining stages. The antimicrobial activity showed a mold formation in the pectin composites after two weeks of storage, but was not detected in the hybrid composites even after three months. This shows that to elaborate efficient active packaging, it is necessary to entrap the active agent for as long as possible instead of enabling its fast release and that this characteristic can be provided by clay filling.

The preparation of a polymeric layered silicate composite offers the possibility of enhancing the properties of individual components. Different biocompatible and biodegradable polymers are suitable drug carriers that can release components at a constant rate [18]. To improve the active substance entrapment efficiency and thereby modulate the release of the active substance, it is desirable to incorporate materials such as clays. Thus, the alginate–clay composite formed would decrease the active substance release by increasing

its loading capacity in the composite matrix. However, one of the main challenges facing the scientific community is the development of appropriate matrices for essential oils encapsulation and protection, as well as their controlled release under specific stimuli, which we call "smart carriers" [19,20].

The main objective of the present study is the elaboration of composites prepared in situ by ionotropic gelation of sodium alginate and the combination of the latter with natural sodium bentonite from the eastern region of Morocco for the encapsulation of rosemary essential oil (REO) and the pathway release as a function of microparticles formulation and REO concentration. The microcapsules were characterized by Fourier transform infrared spectroscopy (ATR-FTIR), thermogravimetric analysis (TGA), and scanning electron microscopy (SEM). In addition, the release profile of the composites in an aqueous medium was studied in vitro at an ambient temperature of 20 ◦C and the ability of the microcapsules to scavenge the DPPH free radicals was also examined.

#### **2. Materials and Methods**

#### *2.1. Materials*

Sodium alginate (SA) (15% loss during drying at 105 ◦C, 30% ignition residue and 0.004% heavy metal content) was purchased from Panreac Quimica (Barcelona, Spain). The polymer presented intrinsic viscosities of 1.03 <sup>×</sup> <sup>10</sup><sup>3</sup> mL/g and 5.39 <sup>×</sup> <sup>10</sup><sup>3</sup> mL/g in 0.1 M sodium chloride and distillate water, respectively, measured using an Ubbelohde capillary viscometer at 25 ◦C. The values of the typical molecular weight are 5.48 <sup>×</sup> <sup>10</sup><sup>4</sup> g/mol and 3.38 <sup>×</sup> <sup>10</sup><sup>5</sup> g/mol for 0.1 M sodium chloride and distillate water, respectively. In addition, the M/G ratio was determined using infrared spectra and was equal to 1.37. The raw bentonite was taken from the Azzouzet deposit (Nador, Morocco), previously characterized [21], purified and exchanged before the experiment to obtain a sodic clay. The main parameters of the raw and purified/exchanged clay are shown in Table 1.

**Table 1.** The main parameters determined for the raw and homosodic clay. CEC: is the cationic exchange capacity using the copper ethylene diamine complex [Cu (EDA)2]2<sup>+</sup> and BET: specific surface of clays.


*Rosmarinus officinalis* L. essential oil (REO) was obtained from a distillation unit in Jerrada (eastern region of Morocco) and was stored at −1 ◦C until examination. Tween 80 (density of 1.06 and viscosity of 300–500 mPa·s at 25 ◦C) was provided by Panreac Quimica (Barcelona, Spain) and calcium chloride (powder, 97% with a molar mass of 110.99 g/mole) was acquired from Riedel-de-Haën (Seelze, Germany).

#### *2.2. Preparation of Rosemary Essential Oil Loaded Microcapsules*

The preparation of the loaded microcapsules followed two steps as in Figure 1. Firstly, the preparation of emulsions containing alginate/montmorillonite/REO and alginate/REO was carried out, followed by the preparation of the microcapsules through the addition of those emulsions into a gelling bath.

**Figure 1.** Illustration of the CA and hybrid CA-MTN microcapsules elaboration process*.* **Figure 1.** Illustration of the CA and hybrid CA-MTN microcapsules elaboration process.

For calcium alginate CA microcapsules, a sodium alginate solution (1% *w*/*v*) was dissolved in distillate water while being stirred magnetically (350 rpm) at room temperature (20 °C). An oil/water emulsion was formed by mixing REO with sodium alginate solution and left to stir overnight to reach a final concentration of 1, 2 and 3% of rosemary essential oil. For the CA-MTN hybrid microcapsules, a dispersion of sodium bentonite MTN (4%) in distillate water was left to stir overnight. Different concentrations of rosemary essential oil (1, 2 and 3%) were added to the sodium bentonite dispersion and left to stir overnight for the maximum adsorption of essential oil on the bentonite. The sodium MTN/REO emulsion was then blended with sodium alginate solution with a 1:2 ratio and left to stir for 5 h. The amount of alginate solution and bentonite dispersion used in hybrid CA-MTN microcapsules were calculated to obtain the same final essential oil concentration as in CA microcapsules. The CA and CA-MTN hybrid microcapsules were generated by adding the emulsions dropwise to calcium chloride solution (0.1 M) for 60 min under magnetic For calcium alginate CA microcapsules, a sodium alginate solution (1% *w*/*v*) was dissolved in distillate water while being stirred magnetically (350 rpm) at room temperature (20 ◦C). An oil/water emulsion was formed by mixing REO with sodium alginate solution and left to stir overnight to reach a final concentration of 1, 2 and 3% of rosemary essential oil. For the CA-MTN hybrid microcapsules, a dispersion of sodium bentonite MTN (4%) in distillate water was left to stir overnight. Different concentrations of rosemary essential oil (1, 2 and 3%) were added to the sodium bentonite dispersion and left to stir overnight for the maximum adsorption of essential oil on the bentonite. The sodium MTN/REO emulsion was then blended with sodium alginate solution with a 1:2 ratio and left to stir for 5 h. The amount of alginate solution and bentonite dispersion used in hybrid CA-MTN microcapsules were calculated to obtain the same final essential oil concentration as in CA microcapsules. The CA and CA-MTN hybrid microcapsules were generated by adding the emulsions dropwise to calcium chloride solution (0.1 M) for 60 min under magnetic stirring.

stirring. The microcapsules were retrieved through filtration, repeatedly rinsed with dis-

tilled water and then put into storage at 4 °C.

The microcapsules were retrieved through filtration, repeatedly rinsed with distilled water and then put into storage at 4 ◦C.

#### *2.3. Identification of Rosemary Essential Oil Chemotype*

The gas chromatographic-MS analysis of *Rosmarinus officinalis* L. essential oil was performed via a Hewlett-Packard 6890 gas chromatograph interfaced with a Hewlett-Packard mass selective detector (Agilent Technologies, Santa Clara, CA, USA). An HP1 fused silica column (Phynel-methyl Siloxane 30 m × 0.25 mm i.d., film thickness 0.25 µm) was used. An interface temperature of 280 ◦C was used for the gas chromatography parameters, while for the mass spectrometry parameters an interface temperature of 250 ◦C. The MS source temperature was of 200 ◦C, the ionization energy and the ionization current were of 70 eV and 2A, respectively [22]. The carrier gas was helium and the flow rate along the column was 1.4 mL/min.

#### *2.4. Characterization of Microcapsules*

#### 2.4.1. Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR)

The attenuated total reflectance-Fourier transform infrared (ATR-FTIR) analysis was carried out using a Shimadzu Jasco 4700-ATR spectrophotometer (Kyoto, Japan) to determine the functional groups in the rosemary essential oil, as well as unloaded and loaded CA and CA-MTN hybrid microcapsules in the wavelength region between 400 and 4000 cm−<sup>1</sup> . The obtained spectra for each sample were affected by averaging 32 scans at a resolution of 4 cm−<sup>1</sup> .

#### 2.4.2. Thermogravimetric Analysis (TGA)

The thermogravimetric analysis was carried out via a SHIMADZU TA-60WS thermal analyzer (Kyoto, Japan) with an initial sample mass of 22 and 31.8 mg for CA and CA-MTN hybrid microcapsules, respectively, in an alumina sample holder at a heating rate of 20 ◦C/min in an N<sup>2</sup> atmosphere.

#### 2.4.3. Particle Size and Morphology

The microcapsules sizes and shapes are related to their circularity, which indicates how closely the shape of the microparticle resembles a circle. Its value ranges from 0 to 1; the latter indicates the perfect sphere. The size was estimated using an Olympus SZ Stereomicroscope (Japan), equipped with a digital camera (D5000 Color Video Camera, Nikon (Tokyo, Japan), and the shape was characterized by aspect ratio and shape factor using an Image J Software program (NIH). The morphology analysis of the microcapsules was conducted by scanning electronic microscopy (SEM), realized in JNCASR (Bangalore, India), using a ZEISS Gemini model. Samples were dried using a critical point dryer and then fixed into a carbon tape for SEM imaging.

#### 2.4.4. Loading Capacity and Encapsulation Efficiency

The amount of encapsulated rosemary essential oil was determined using a Rayleigh UV–VISIBLE 1800 spectrophotometer (Model Jasco 560, Pekin, China), following the method described by El Hosseini [23] with some modifications; 200 mg of the microcapsules were poured into a beaker containing distilled water and Tween 80 (1% *w*/*v*) then left to stir. The absorbance of the obtained solution was measured at 257 nm. The loading capacity and encapsulation efficiency were determined at the final concentration measured at the end of the release study and calculated using the rosemary essential oil calibration curve in the aqueous solution (1% Tween 80). The values were calculated following Equations (1) and (2) [24,25] for loading capacity and encapsulation efficiency, respectively.

$$\text{L\textbf{C} (\%)} = \frac{\text{Weight of essential oil in microcapsules}}{\text{Weight of microcapsules}} \times 100\tag{1}$$
