Preparation, Characterization, and Antibiofilm Effect of Free and Nanoencapsulated Tetradenia riparia (Hochst) Codd Leaves Essential Oil

1. Introduction

Staphylococcus aureus is a Gram-positive and aerophilic bacterium responsible for the most common skin infections, including dermatitis. The pathogen colonizes more than 20% of the population asymptomatically but can cause symptomatic infections when the epithelial barrier is compromised [1]. Planktonic S. aureus can adhere to surfaces, proliferate, produce extracellular matrix, and ultimately form biofilms. Once mature, biofilms release individual cells or fragments that can colonize new surfaces. Biofilms account for approximately 80% of microbial infections and exhibit up to 1000-fold greater resistance and tolerance to antibiotics compared with planktonic cells [2,3].

Conventional antibacterial agents present several limitations, including adverse side effects, poor solubility, drug–drug interactions, induction of antimicrobial resistance, and reduced efficacy [4]. Consequently, there is increasing interest in developing safer and more effective antibacterial alternatives. Aromatic plants and their essential oils (EOs) have long been used in traditional medicine and demonstrate broad antimicrobial properties [5]. Essential oils are complex mixtures of volatile compounds, predominantly monoterpenes, sesquiterpenes, and phenylpropanoids, with terpenes primarily responsible for their biological activity [6]. Their antimicrobial effect arises mainly from their high hydrophobicity and abundance of short-chain carbon compounds (particularly terpenes), which interact strongly with membrane lipids, altering membrane fluidity and integrity. This disruption ultimately leads to pathogen death [7].

Tetradenia riparia (Hochst.) Codd, a member of the Lamiaceae family, is an herbaceous shrub 1–3 m tall, widely distributed across Africa. It is commonly known as false myrrh, lemon verbena, lavandula, misty plume, or incense [8,9]. The species has been traditionally used to treat cough, edema, diarrhea, fever, headache, malaria, and toothache. Its essential oil exhibits larvicidal, insecticidal, antimalarial, antimicrobial, and antinociceptive activities. The oil contains a complex mixture of monoterpenes, diterpenes, and sesquiterpenes, with calyculone, caryophyllene, β,13β-epoxy-7-abietene, fenchone, terpineol, 14-hydroxy-9-caryophyllene, and germacrene-D-4-ol identified as major constituents responsible for its biocidal properties [10,11,12].

Despite their strong antimicrobial potential, essential oils are prone to volatilization, degradation, and loss of activity upon exposure to light, heat, or pressure. Nanoencapsulation of EOs into polymeric nanoparticles helps preserve their functional properties, enhance stability, control release, reduce toxicity, and improve water solubility [7,13]. Therefore, the aim of this study was to prepare polymeric nanoparticles containing T. riparia essential oil using nanoprecipitation, characterize their physicochemical properties, and evaluate their cytotoxicity and antibiofilm activity against S. aureus.

2. Materials and Methods

2.1. Plant Material, Botanical Identification, and Extraction of Essential Oils

Fresh Tetradenia riparia leaves were collected in western Paraná State, Brazil, in December 2015. The plant was identified by Professor Ezilda Jacomasi, Department of Pharmacy, Paranaense University (UNIPAR), Paraná. A voucher specimen was deposited in the UNIPAR Herbarium (code 2502).

The essential oil (EO) was extracted by hydrodistillation for 3 h using a modified Clevenger-type apparatus, according to the procedure described in reference [14]. The distilled EO was collected, dried over anhydrous Na₂SO₄, and stored in amber glass flasks at −4 °C until use, following the method described in [15].

2.2. Chemical Identification of Essential Oils

The chemical compositions of T. riparia EO before and after storage were determined by gas chromatography–mass spectrometry (GC–MS). Compounds were identified by comparing their Kovats retention indices with those of known substances and by comparison with mass spectra from reference databases. Relative quantities (%) were calculated directly from GC peak areas.

GC–MS analyses were performed using a Focus GC system (Thermo Electron Corporation) equipped with a DBS-MS column (30 m × 0.25 mm × 0.25 µm). The injector and transfer line temperatures were set at 230 °C. The oven temperature program was as follows: initial temperature 60 °C for 1 min, then increased at 3 °C/min to 220 °C and held for 5 min. Hydrogen was used as the carrier gas. The injection volume was 5 µL (split 1:10), and the ionization energy was 70 eV. Mass spectra were obtained at 230 °C using a DSQ II detector (Thermo Scientific) in total ion current (TIC) acquisition mode, with a mass range of 50–659 m/z.

2.3. Nanoparticle Preparation

Poly(L-lactide) nanoparticles (NP) were prepared by the nanoprecipitation method using the dropping technique. The organic phase, consisting of 50 mg EO and 50 mg PLA (MW 90,000–120,000; Sigma-Aldrich) in 4 mL acetone, was added dropwise into the aqueous phase (10 mL of 1.0% w/v Pluronic F68; Sigma-Aldrich) under stirring at 1200 rpm for 30 min. Acetone was removed using a rotary evaporator. Blank PLA nanoparticles (without EO) were prepared using the same procedure. The final NP suspension was stored at 4 °C until further analysis.

2.4. Nanoparticle Characterization

Particle size and distribution were measured by dynamic light scattering (DLS) using a NanoPlus zeta/nanoparticle analyzer. Samples were diluted in ultrapure water prior to analysis.

Surface morphology was examined using a Shimadzu SS-550 scanning electron microscope (SEM). Samples of the nanosuspension were placed on glass plates, dried under reduced pressure at 25 °C, coated with gold using a sputter coater, and examined by SEM.

Transmission electron microscopy (TEM) was used to further evaluate morphology and size distribution. Samples were deposited onto copper grids, stained with 1% w/v uranyl acetate for 1 min, dried, and analyzed by TEM.

2.5. Thermal Analysis

Thermal properties of blank NP and EO-loaded NP were determined by differential scanning calorimetry (DSC; PerkinElmer DSC4000). Samples (3–5 mg) were placed in aluminum pans and heated from 20 to 200 °C at 20 °C/min under nitrogen flow (50 mL/min).

2.6. Encapsulation Efficiency

The EO content of NP was quantified by UV–visible spectroscopy (Shimadzu UV–VIS). A standard curve was prepared by serial dilution of EO in absolute ethanol and measuring absorbance at 325 nm [14].

For EO quantification in NP, 2 mg of sample was dissolved in 2 mL absolute ethanol, and absorbance at 325 nm was compared to the standard curve. Encapsulation efficiency was calculated as:

%EE = (total loaded EO / initial EO) × 100.

2.7. In Vitro Release Profile

Ten milligrams of NP or EO were placed in individual dialysis bags containing 40 mL PBS and incubated on a shaker at 37 °C. A 2 mL aliquot of the release medium was collected at 0, 15, 30, 60, 90, 120, and 360 min, and replaced with fresh PBS. EO release was quantified by UV–visible spectroscopy at 325 nm [16].

2.8. Stability Assays

A stability assay was conducted over 30–45 days at 25 ± 2 °C according to RDC 45 (August 9, 2012) [17]. The antibacterial activity of T. riparia EO and NP was subsequently analyzed using the broth microdilution assay following CLSI guidelines.

2.9. Strains and Growth Conditions

Staphylococcus aureus ATCC 29213 was used as the test strain. The bacterium was maintained on Mueller–Hinton agar (MHA; Difco) at 4 °C and cultured in Mueller–Hinton broth (MHB; Difco) before assays.

2.10. Microdilution Assay

Antibacterial activity was assessed by broth microdilution following CLSI guidelines [17]. Serial two-fold dilutions of EO and NP were prepared in 96-well plates containing 100 µL sterile MHB. Bacterial inoculum (10⁵ CFU/mL) was added to each well. Plates were incubated at 37 °C for 24 h.

The minimum inhibitory concentration (MIC) was defined as the lowest concentration preventing visible growth. Minimum bactericidal concentration (MBC) was determined by subculturing 10 µL from wells with no visible growth onto MHA plates, followed by incubation at 37 °C for 24 h.

2.11. Antibiofilm Activity

A 100 µL bacterial suspension (1 × 10⁸ CFU/mL) prepared in tryptic soy broth (TSB) supplemented with 1% glucose was added to wells containing EO or NP dilutions and incubated at 37 °C for 24 h. Wells were washed with PBS.

For the MTT assay, 20 µL MTT solution (2 mg/mL in PBS) was added, and plates were incubated for 2 h at 37 °C. After removing the MTT solution, 100 µL DMSO was added to solubilize formazan crystals. Absorbance was measured at 570 nm [18].

The biofilm inhibitory concentration (BIC₅₀) was defined as the minimum concentration that inhibited ≥50% of biofilm viability relative to untreated controls.

2.12. Cytotoxicity Assay

Cytotoxicity of EO and NP was evaluated using an MTT assay according to Tangarife-Castaño et al. [19]. VERO cells (African green monkey kidney; Cercopithecus aethiops) were cultured in DMEM for 72 h. Cell monolayers were trypsinized, washed, and seeded at 2.5 × 10⁵ cells/well in 96-well plates.

After 24 h, EO and NP (1, 10, 100, and 1000 µg/mL) were added to wells, followed by incubation for 72 h at 37 °C in 5% CO₂. Cells were washed with PBS and incubated with 2 mg/mL MTT for 4 h. Formazan crystals were solubilized with 200 µL DMSO, and absorbance was measured at 530 nm using a Biotek PowerWave XS reader.

The IC₅₀ values were obtained by linear regression of dose–response curves generated in R software. Selectivity indices (SI) were calculated as SI = IC₅₀ / MIC.

3. Results and Discussion

3.1. Chemical Composition of Tetradenia riparia Leaf Essential Oil

The antimicrobial activity of essential oils (EOs) depends on their chemical composition. The major compounds present in Tetradenia riparia EO were identified by GC–MS. The EO yield was 0.33% of plant material. The composition and yield of essential oils are known to be affected by seasonal variations [10]. Previous studies reported the highest EO yield from T. riparia leaves in winter (0.265%). In contrast, EO content decreased to 0.168% during spring, likely due to the substantially higher rainfall observed in this season.

Table 1 lists the compounds identified in the EO prior to nanoencapsulation (free EO). The major chemical classes were oxygenated monoterpenes (28.94%), with fenchone (27.19%) as the predominant constituent, and oxygenated sesquiterpenes (34.30%), mainly represented by α-cadinol (16.12%) and 14-hydroxy-9-epi-caryophyllene (13.09%) (Figure S1 supplementary data). Hydrocarbon sesquiterpenes (11.7%) were also noteworthy, particularly due to the presence of caryophyllene (8.40%). These results agree with the chemical characterization reported for T. riparia leaf EO by Gazim et al. [10].

3.2. Nanoparticle Characterization

Incorporating T. riparia EO into PLA nanoparticles (NPs) prevented rapid EO evaporation and degradation, thereby enhancing its stability. DLS measurements indicated that NP size ranged from 221.9 to 396.5 nm. SEM and TEM images showed that the NPs were spherical, had smooth surfaces, and exhibited nanometric diameters (Figure 1).

Nanoencapsulation of aromatic molecules into capsules measuring 10–1000 nm can protect volatile compounds and significantly increase their biological activity. At the nanoscale, delivery systems may enhance passive cellular uptake, reduce mass transfer resistance, and consequently increase biological activity [39].

The surface morphology of the nanoparticles was also relevant. TEM images showed no cracks or pores on the nanocapsule surfaces. Considering that the bioactive constituents of T. riparia EO are highly volatile, the absence of surface fissures indicates effective protection against premature release, degradation, or interactions with environmental factors.

Nanoencapsulation is a promising strategy for controlling EO release. It prolongs antimicrobial effects, improves pharmacological efficacy, enhances water solubility, reduces toxicity, and increases patient compliance and convenience [7].

3.3. Differential Scanning Calorimetry (DSC)

DSC analysis provides insights into thermal stability by quantifying enthalpy changes associated with thermal transitions. Table 2 summarizes the DSC results. The melting temperature and melting enthalpy of blank NPs were 54.29 °C and 429.63 J/g, respectively. For NPs loaded with EO, these values decreased to 52.71 °C and 115.83 J/g. The reduction in melting enthalpy (from 429.63 to 115.83 J/g) suggests that the incorporation of EO favors controlled release.

The DSC thermograms in Figure 2 show a larger endothermic peak for blank NPs. In contrast, EO-loaded NPs exhibited a smaller peak area, indicating that the EO may alter the crystalline organization of the polymer matrix, facilitating melting and consequently promoting EO release. This behavior is advantageous for controlled-release applications.

3.4. Encapsulation Efficiency

The amount of EO encapsulated in the nanoparticles was quantified by UV–Vis spectrophotometry at 325 nm. Encapsulation efficiency was 88.1%, indicating high EO loading and suggesting that the NPs are effective carriers [40].

Zeta potential measurements were used to assess NP stability. Zeta potential reflects surface charge, which influences dispersion stability, flocculation behavior, and interactions with negatively charged cell membranes [41,42]. The NPs exhibited a zeta potential of −23.1 mV, indicating adequate stability [43].

3.5. In Vitro Release

In vitro release profiles of free EO and NP-encapsulated EO were measured at 325 nm. Neither formulation exhibited complete release within the assay period; maximum release reached 90.0% for free EO and 25.5% for NP-encapsulated EO within 360 minutes (Figure 3). Free EO was rapidly released, whereas NP encapsulation markedly slowed the release rate. NPs maintained approximately 25.0% release after 100 minutes. The controlled-release behavior likely results from the compact network formed by poly(L-lactide) crosslinking, consistent with previous studies.

3.6. Antibacterial Activity

The antibacterial activity of EO and NP was evaluated against S. aureus ATCC 29213 using broth microdilution assays (Table 3). Samples were classified as follows: MIC ≤ 0.5 mg/mL indicated strong antibacterial activity; 0.6–1.5 mg/mL indicated moderate activity; and MIC > 1.6 mg/mL indicated inactivity [44].

EO and NP exhibited similar antibacterial activity against both planktonic and biofilm forms of S. aureus, indicating that nanoencapsulation did not diminish EO activity. However, enhanced antibacterial activity after encapsulation would have been desirable.

The antibiofilm effects of EO and NP were compared with vancomycin. BIC₅₀ values were 310 µg/mL for EO and 330 µg/mL for NP.

Biofilm morphology after treatment is shown in Figure 4. SEM analysis revealed that untreated cells (A) appeared spherical, dense, and surrounded by abundant extracellular matrix. EO-treated biofilm (B) showed reduced extracellular matrix, altered cell morphology, and fewer cells. NP-treated biofilm (C) also exhibited decreased cell numbers and notable alterations in the cell wall and membrane, suggesting irreversible membrane damage, as previously described by Kang et al. [45].

Kwieciński et al. [46] reported that treatment with 1% tea tree EO completely eradicated S. aureus biofilm, causing not only bacterial death but also matrix disruption and biofilm detachment from surfaces.

3.7. Cytotoxicity Assay

Although EOs are widely used as antimicrobial agents, their clinical effectiveness depends on the relationship between effective in vitro concentrations and the concentrations achievable at the site of action. Cytotoxicity varies depending on EO constituents, and several attempts have been made to correlate in vitro and in vivo toxicities. Antimicrobial agents must selectively target microorganisms, exhibit minimal effects on host cells, and avoid interfering with normal physiological pathways. According to the U.S. National Cancer Institute (NCI), crude extracts with IC₅₀ ≤ 20 µg/mL are considered cytotoxic [47,48,49].

Table 4 summarizes the cytotoxicity results. IC₅₀ values for NP and EO were 533.96 µg/mL and <125 µg/mL, respectively. NPs were less cytotoxic to VERO cells than free EO, demonstrating that encapsulation reduced EO toxicity. This finding supports nanoencapsulation as an effective approach to modulate release profiles while minimizing toxicity [50].

5. Conclusions

Tetradenia riparia essential oil displayed a consistent chemical profile rich in oxygenated monoterpenes and sesquiterpenes, which supports its biological potential. PLA nanoencapsulation successfully stabilized the EO, provided high encapsulation efficiency, and enabled controlled and sustained release. Although antibacterial activity against Staphylococcus aureus was not enhanced, nanoencapsulation preserved the EO’s efficacy while significantly reducing cytotoxicity. These findings demonstrate that PLA nanoparticles are an effective delivery system for optimizing the stability and safety of T. riparia EO, reinforcing their suitability for future pharmacological development.