Essential Oil Blends or Their Component Blends as Antimicrobial Compounds of Polysaccharide Coatings on Metallic Biomaterials

Highlights

What are the main findings?

• influence of polysaccharide matrix and substrate on EPD process and coatings properties;
• alginate coatings showed superior antibacterial and anti-biofilm activity;
• alginate and chitosan coatings differed in droplet size and additive distribution;
• interactions between oils, active compounds, and polymer matrices were observed;
• alginate-based coatings showed high activity against S. aureus, but also cytotoxicity.

What are the implications of the main findings?

• both polymers enable multi-component loading but require improved coating stability;
• control of droplet size and distribution is critical for coating performance;
• optimization of plant-derived compound type and concentration is required.

Abstract

The work provides novel insight into the development of advanced antibacterial surfaces using the combination of essential oils, cinnamon oil, thyme oil, and tea tree oil, as well as their active compounds, including cinnamaldehyde, thymol, and terpinene-4-ol, embedded in the chitosan and sodium alginate matrix. All coatings obtained in a two-stage electrophoretic deposition process on stainless steel and titanium substrates were characterized by high adhesion strength. The microstructural differences between the coatings were mainly related to the size and location of the additives. Structural investigation showed the impact of individual oil components on intermolecular bonds between polysaccharide chains and the formation of molecular interactions in a specific spatial conformation. The surface of all coatings was minimally rough and had a hydrophilic character. A clear matrix-dependent trade-off between antibacterial efficacy and cytocompatibility was observed: alginate-based coatings achieved strong anti-Staphylococcus aureus activity (2.81 log CFU/mL) at the expense of increased cytotoxicity, while chitosan-based systems provided a more favorable cytocompatibility profile, maintaining cell viability above 70% for selected formulations. This work provides insight into the development of natural antibacterial surfaces by the combination of active compounds and shows the distinctions on many levels between the coatings with various polysaccharide matrices.

1. Introduction

Essential oils (EOs) originating from various parts of plants like cinnamon oil extracted from the bark (CBO), thyme oil (TO) and tea tree oil (TTO) from the leaves, as well as their main individual components incorporating cinnamaldehyde (CA), thymol (TH) or terpinen-4-ol (T4), possess many valuable properties in terms of their impact on the surrounding environment. Their main benefits include the elimination of many strains of bacteria and free radicals and the combat of inflammation [1]. EOs or their constituents (OCs) are being analyzed today as potential microbicidal agents or for wide use in the biomedical industry, especially as a factor in medical devices for hard tissues [2]. Most studies focus on the impact of a single EO or constituent on bacterial strains, cell lines, and other biologically connected factors [3,4]. However, there are several papers concentrating on blended EOs or constituents. For example, Chaiwong et al. [5] prepared blends consisting of five EOs, including vetiver, lavender, lemongrass, eucalyptus, and phlai. They showed that such blends had very promising antioxidant properties due to their ability to act as proton radical scavengers. In terms of anti-inflammatory properties, experiments conducted against nitric oxide showed only minor abilities of such blends to prevent inflammation. Another work worth mentioning is the research conducted by Leigh et al. [6] in which they prepared nanoemulsions consisting of Hyssopus officinalis var. angustifolius and Salvia rosmarinus var. angustifolius EOs as potential therapeutic agents used to cure respiratory tract diseases. They found that such a blend had a very high microbicidal potential against Staphylococcus aureus, Streptococcus pneumoniae, or Klebisella pneumoniae pathogens responsible for such diseases, indicating its potential use in their treatment.

EOs are currently considered potential antimicrobial agents used in implantology [7]. One of the approaches to utilizing the properties of EOs in this area is to obtain antimicrobial coatings on various implants. Although there are several methods that allow the surface of the metallic implant to be coated with a film several micrometers thick consisting only of EO [8,9], most of them require the presence of the host matrix. The most promising and commonly used are polysaccharides, including sodium alginate (Alg) and chitosan (Chit). They are often used as a platform for the release of antimicrobial substances due to their natural origin, biocompatibility, and biodegradability [10]. The appropriate method used to obtain polysaccharide-based coatings on metallic substrates is electrophoretic deposition (EPD) [11]. Sodium alginate and chitosan are charged oppositely, negatively and positively, respectively, which is important in the EPD process and in the interaction with bacteria [12]. The mechanism of antibacterial action of Chit is not yet fully understood. The most well-known proposed model states that positively charged Chit molecules interact with negatively charged bacterial membranes, leading to leakage of internal organelles of cells [13]. Based on that, it can be said that Gram-negative bacteria would be more susceptible to Chit than Gram-positive bacteria, although there are several works showing that Chit acts strongly against both groups of bacteria [13]. The investigation results of the Alg-based multifunctional films described by Riahi et al. [14] indicate bacterial elimination, but it was explained by the presence of sulfur remaining from the prior processing of the polymer. Pure Alg as a raw material is not characterized by antibacterial properties.

The possibilities of blends that can be used to improve the antimicrobial properties of polysaccharides are mixtures of EOs [15,16,17], OCs [18,19], EOs and OCs [20], EOs [21,22], or OCs [23,24] with antibiotics. Combining two or three types of EO or OC together would allow a broadening of the antimicrobial spectrum of action, improving or increasing efficacy, while reducing the content of this additive. Such an approach allows limiting the use of antibiotics in favor of substances of natural origin, which is so important because the trend toward antibiotic use is constantly increasing, leading to antibiotic resistance [25]. This results in higher treatment costs, namely the use of other antibiotics, additional harmful environmental pollutants [26], and, in special cases, it can result in the patient’s death. The variable parameter for observing antimicrobial interactions is primarily the concentration of oils or oils and antibiotics, selected on the basis of previously verified minimal inhibitory concentration (MIC). The fractional inhibitory concentration index (FICI) helps make the final decision about which oil is worth mixing [27]. Such a mixture of antimicrobial additives can significantly change biological properties and produce synergistic, additive, indifferent, and antagonistic effects [28]. An example of the additive effect obtained from blends against the Listeria monocytogenes bacterium was reported by Cava–Roda et al. [20]. The FICI value was obtained as 0.70 and 0.75 for the combination of cinnamon leaf oil and cinnamon bark oil, as well as eugenol and cinnamaldehyde, respectively. The calculated FICI for the same binary combinations of EOs and OCs antimicrobial compounds against Escherichia coli was 1 and 0.65, respectively. In turn, in the work of García–García et al. [18], studies were conducted on Listeria innocua, determining the fractional bactericidal concentration index (FBCI) for blends of two and three components and at various concentrations of each component. They demonstrated that the bactericidal effect, additive or synergistic, can be influenced by controlling both the composition and the concentration.

In our previous works [29,30], we developed Chit and Alg coatings with a single EO or its constituent additive. We proved that it is possible to electrophoretically deposit coatings with relatively high adhesion strength, which was strongly dependent on the oil concentration in emulsions used for the EPD and the substrate used. For Alg-based coatings, unlike Chit-based coatings, it was necessary to use a two-step EPD process. It should be noted that, in the case of Chit-based coatings, they had a high adhesion to ground titanium substrates [29], while for Alg-based coatings, chemical etching of the austenitic stainless steel substrate was necessary [30]. The coatings also revealed a high antimicrobial potential. Both polymers in their chains could interact and form bonds with molecules of different materials. However, there are some differences in the bond formation capabilities. The proposed mechanisms of interaction with EOs or OCs describe the formation of bonds with Chit molecules regardless of the substance used [29,31,32]. It was observed that almost all of the constituents used were able to create hydrogen bonds with nitrogen atoms from the amino groups of Chit chains. Other possible bonds were detected for oxygen atoms from hydroxyl groups of Chit, bonds between rings, and rings themselves. In turn, the Alg chain made of the M units is arranged more linearly in contrast to its G units [33]. Therefore, M units of this polymer are more exposed to the specific functional groups, i.e., hydroxyl and carboxyl, attached to the aromatic rings of the EOs and Ocs, as well as the oxygen atoms in the rings and between them. All of the studies mentioned above confirm that it is possible to fabricate polysaccharide coatings with liquid additives on metallic biomaterial substrates and that there is potential for the development of antibacterial oil blends.

The aim of this work is to investigate the influence of the polysaccharide, anionic Alg or cationic Chit, used as a coating matrix, on the microstructure, surface topography, and selected properties of multicomponent coatings with EOs or OCs blends obtained by the EPD process on metallic biomaterial substrates, titanium and austenitic stainless steel. A key issue was also to verify the antimicrobial properties of Alg- and Chit-based coatings with CBO-TO-TTO, CBO-TO, CA-TH-T4, and CA-TH agents.

2. Materials and Methods

2.1. Blend Materials and Emulsion Preparation Path

Alg powder, no. 9005-38-3, Chit powder, no. 448877, cinnamon bark oil (CBO), no. 8015-91-6, thyme oil (TO), no. 8007-46-3, cinnamaldehyde (CA), no. 104-55-2, thymol (TH), no. 89-83-8, terpinen-4-ol (T4), no. 20126-76-5 and Polysorbate 80 (PS 80), no. 9005-65-6 was obtained from Merck, Poznań, Poland. Tee tree oil (TTO), no. 68647-73-4, was delivered from Pol-Aura, Morąg, Poland.

First, the Alg solution was prepared with a concentration of 4 g/L in an EtOH:H₂O mixture in a 2:3 volume ratio as described in our previous works [30,34]. The Chit solution was prepared with a concentration of 2 g/L in a 0.5% acetic acid aqueous solution obtained as described elsewhere [31,32] and later mixed with ethyl alcohol of 99.8% purity in a 1:1 volume proportion. Next to polymer solutions, two or three phytotherapeutics with a total concentration of 2 mL/L were added. Based on our previous work [30], to minimize the diameter of additive droplets and obtain a high adhesion strength of coatings, the PS 80 surfactant was added to the Alg solution. Binary (with a compound ratio of 1:1) and ternary (1:1:1) plant-derived Alg emulsion blends also contained 2 mL/L of PS 80. All emulsions were homogenized for 6 min with mixing settings at 20,000 rpm, using a Unidrive X1000D homogenizer (CAT, Lehrte, Germany). Additionally, the Alg blend emulsion was subjected to ultrasonic dispersion at room temperature (RT) for 1 min. Figure 1 shows the sequences of adding selected components to polymer solutions for blends containing two or three antibacterial additives.

Figure 1. Binary and ternary emulsion preparation for EPD of coatings. TH was the only ingredient added in the solid state; therefore, the method of feeding into the solution is presented graphically differently from the liquid components.

2.2. Deposition of the Coatings

As substrate materials for depositing the Alg-based coatings, plates (30 × 15 × 2 mm) of AISI 316L austenitic stainless steel (supplied by Saritaş Çelik Sanayi ve Ticaret A.S., Gebze, Turkey) were selected, whereas for Chit-based coatings, Commercially Pure Titanium (CP Ti) Grade 1, according to ASTM F67-13 (2017) [35], (from Shanghai Huaxia Industry Co. Ltd., Shanghai, China) in the form of plates with dimensions of 36 × 15 × 0.5 mm was used.

Both substrates were subjected to the same surface modification. Namely, before the etching process, the samples were cleaned and degreased using distilled water and ethyl alcohol. Then, in order to ensure the highest possible adhesion of the coating to the substrate, a series of steps were carried out, i.e., washing in acetone using ultrasound-emitting equipment, soaking in a 0.06 M Na₃PO₄∙12H₂O solution at 80 °C, rinsing with distilled water and EtOH, air drying, ending with 10 min chemical etching process using a solution with the following composition 5 mL of 40% HF + 35 mL of 70% HNO₃ in 60 mL of H₂O [36,37].

The EPD process ran at direct current and was supplied using an EX752M PSU multimode power supply, Huntingdon (UK) in a standard two-electrode configuration. The EPD parameters were adopted from our previous works [29,30]. The EPD procedure is presented in Table 1.

Table 1. Information on substrate materials, the EPD path, solutions, blends, and emulsions.

substrate		AISI 316L	CP Ti
EPD path		Two-stage EPD
1st stage		Alg solution	Chit solution
2nd stage		Emulsion: blend + Alg solution	Emulsion: blend + Chit solution
Blend marking	EO2	nCBO-TO	CBO-TO
	EO3	nCBO-TO-TTO	CBO-TO-TTO
	OC2	nCA-TH	CA-TH
	OC3	nCA-TH-T4	CA-TH-T4

n—blend with PS 80 addition.

The conditions of the coating deposition system for both matrices were almost identical. For Alg, the working electrode was AISI 316L stainless steel, and for Chit, CP Ti, with interelectrode distances of 15 mm and 10 mm and voltages of 5 V and 10 V, respectively. The first stage of EPD consisted of the deposition of the Alg or Chit interlayer that lasted 1 min and 2 min, respectively. The sample was then allowed to dry at RT. The second layer, composed of Alg or Chit + the chosen phytotherapeutic combination of EO2 or OC2 or EO3 or OC3, was deposited for 2 min and 3 min, accordingly. Finally, all composite coatings were left to dry in a desiccator at RT for 48 h.

2.3. Adhesion Tests

To verify the adhesion strength of the coatings, tape tests were performed according to the ASTM D3359-23 standard (method B) [38]. Cross-cuts in the coatings were made using an MFS 3000 multifunctional plate and an NT cutting knife (Festool, Baden-Württemberg, Germany).

2.4. Microstructural Characterization

The morphology of the coatings was observed with the use of FEI Inspect S50 scanning electron microscope (SEM), Eindhoven, The Netherlands, with operating parameters: 10–15 kV, spot size 4.0–4.5, working distance 9.6–11 mm. The microstructure of the selected coatings in their cross-section was investigated with a JEOL JEM-ARM200F NEOARMex transmission electron microscope (TEM), Tokyo, Japan, acquired using accelerating voltages of 60 kV and 200 kV. Emulsion samples were prepared by placing them onto a carbon-coated ultrathin Cu grid (Ted Pella Inc., Redding, CA, USA) and leaving them to dry at RT. Lamellae from the coatings for TEM investigation were prepared using a Quanta 3D 200i dual-focused ion beam FIB-SEM (Thermo Fisher Scientific, Eindhoven, The Netherlands).

2.5. FT-IR Studies

FT-IR spectroscopy by the specular reflection technique was employed to investigate the structure of the obtained coatings. All measurements were performed using a Vertex 70v spectrometer (Bruker, Billerica, MA, USA) equipped with a Seagull attachment (Harrick Scientific, Mt Kisco, NY, USA). The measurements were carried out in the range of 400–4000 cm⁻¹ at the resolution of 4 cm⁻¹ with 128 scans gathered. The resulting data was processed using OPUS 7.2 software (Bruker, USA).

2.6. Surface Topography

Surface topography and roughness parameters were investigated using Veeco Dimension Icon with ScanAsyst atomic force microscope (Veeco, Plainview, NY, USA). For each sample, a rectangular area of 20 µm × 20 µm with a resolution of 512 × 512 px and 1024 lines was selected for measurements. The roughness parameters: arithmetic mean roughness (Sa), root mean square roughness (Sq), distance between the highest peak and the lowest valley of the measured area (St), and image surface area difference (ISAD), were determined using Gwyddion-2.67 software [39]. The ISAD parameter was determined using the following equation:

$$ISAD={\displaystyle \frac{M_A-I_A}{I_A}}\times 100\%$$

where M_A—real surface area including peaks and valleys, I_A—area of image.

The process of obtaining surface roughness data consisted of two steps. First, the background was subtracted, and basic filters were utilized, and then the surface statistical parameters were acquired.

2.7. Wettability and Surface Free Energy

Kruss DSA25E goniometer (Hamburg, Germany) was used for wettability testing. Distilled water, which is a polar liquid, and diiodomethane, a non-polar liquid, were used as liquids in measurements using the sessile drop technique. The measuring droplets had a volume of 1 µL. Each measurement was repeated nine times. The wettability angles (WA) were determined by using the ellipse-fitting technique. The Owens, Wendt, Rabel, and Kaelble (OWRK) method was applied to obtain surface free energy (SFE) from the acquired data.

2.8. Drug Release Studies

The in vitro drug release study for the nCA-TH-T4/Alg//Alg, nCA-TH/Alg//Alg, CA-TH-T4/Chit//Chit, and CA-TH/Chit//Chit coatings was determined by incubation in phosphate-buffered saline (PBS) at a temperature of 37 °C. PBS was the reference medium for coatings and individual additive compounds, i.e., oil components and surfactant. However, polymers, i.e., Alg and Chit, were analyzed in H₂O:EtOH solution. The collected samples were analyzed at specific time durations, specifically 10 min, 20 min, 1 h, 2 h, 4 h, 5 h, 24 h (additionally for Chit coatings due to the longer degradation time observed during 168 h) and were recorded in the wavelength range of 190–1000 nm by using a UV–vis spectrophotometer UV7 (Mettler Toledo, Greifensee, Switzerland). CA, TH, T4, and PS 80 content was specified at λmax 286 nm, 275 nm, 214 nm, and 282 nm, respectively. Two methods differing in measurement time were used to determine the absorbance of individual wavelengths, one for coatings and one for individual additive compounds. The first measure for 5 s is the absorbance for each sample, namely the reference and the incubated for a certain period. For the second method, each measurement of the reference and individual additive compounds samples lasted 10 s to better illustrate the spectrum. Based on the replicates performed (triplicate measurements), the mean absorbance and standard deviation (SD) for the tested coatings were calculated (Table S1). Figure 2 represents the absorbance results, depending on the wavelength.

Figure 2. Graphical representation of absorption measurement points for given polymer solutions, reference solution, and phytocompounds.

2.9. Antibacterial Adhesion and Biofilm Activity Assessment

The antibacterial performance of the investigated surfaces was evaluated using two well-characterized reference strains: Staphylococcus aureus ATCC^® 25923, representing Gram-positive bacteria, and Escherichia coli ATCC^® 25922 as a Gram-negative model of the cell wall. Bacterial cultures were cultured overnight in Tryptic Soy Broth (TSB; Becton Dickinson, Franklin Lakes, NJ, USA) at 37 °C under aerobic conditions. After 18 h of incubation, cultures were harvested by centrifugation (4000 rpm, 3 min) and rinsed three times with phosphate-buffered saline (PBS; Chempur, Piekary Śląskie, Poland). The final bacterial suspensions were adjusted to a concentration of 1 × 10⁶ CFU/mL in PBS. Individual material tested surfaces were aseptically placed into 24-well plates (Nest Biotechnology, Wuxi, China). Subsequently, standardized bacterial suspensions were introduced to the wells in volumes of 1 mL. The plates were incubated at 37 °C for 4 h to promote early-stage bacterial adhesion. Following incubation, loosely attached cells were removed by careful washing with PBS.

To quantify the viable bacteria remaining attached after the adhesion phase, the tested surface was transferred to individual wells of a sterile 24-well plate containing 1 mL of PBS. Surface-bound microorganisms were detached by shaking, and the resulting suspensions were subjected to serial dilution in 1 mL of PBS. 0.1 mL of aliquots were plated onto Mueller–Hinton agar (Oxoid, Hampshire, UK) and incubated at 37 °C for 24 h. Colony counts were used to calculate the number of colony-forming units (CFU/1 mL). Glass surfaces processed under identical conditions served as reference materials. All experiments were performed in triplicate.

To assess the early biofilm formation, the tested surfaces after 4 h of incubation in PBS with bacteria were transferred to new sterile wells and flooded with fresh 1 mL TSB and incubated at 37 °C for 24 h. After biofilm maturation, the tested surfaces were gently rinsed with PBS to eliminate planktonic bacteria. The metabolic activity of the established biofilms was assessed using the AlamarBlue assay. Fresh 1 mL TSB containing 10% AlamarBlue reagent (Sigma-Aldrich, St. Louis, MO, USA) was applied directly onto the biofilm-covered surfaces. After 20 min of incubation, 100 µL of the supernatant was collected in triplicate and transferred to a black 96-well microplate (Thermo Scientific, Waltham, MA, USA). All experiments were performed using three independent material specimens. Fluorescence measurements were conducted using a SpectraMax^® Mini plate reader (Molecular Devices, San Jose, CA, USA) at excitation/emission wavelengths of 544/590 nm. Biofilm metabolic activity was expressed as the percentage of resazurin reduction, calculated according to the following formula:

$$\% resazurin reduction={\displaystyle \frac{S_x-S_0}{S_{100}-S_0}}\times 100\%$$

where S_x—denotes the fluorescence signal of the tested sample, S₀—corresponds to the fluorescence of medium supplemented with 10% AlamarBlue (0% reduction control), and S₁₀₀—represents the fluorescence obtained after autoclaving the medium containing 10% AlamarBlue (100% reduction reference).

2.10. Evaluation of Cytotoxicity of the Tested Samples

The influence of the investigated materials on mammalian cell metabolic activity was quantified using two human cell lines. MG-63 (ATCC^® CRL-1427) is a human osteosarcoma-derived, osteoblast-like cell line commonly used as an in vitro model of bone-forming cells. FaDu (ATCC^® HTB-43) is a human hypopharyngeal squamous cell carcinoma cell line exhibiting epithelial morphology. Cells were routinely cultured at 37 °C in a humidified atmosphere containing 5% CO₂ in Eagle’s Minimum Essential Medium (EMEM; Gibco, Grand Island, NY, USA), supplemented with 10% fetal bovine serum and ZellShield^® (Minerva Biolabs, Berlin, Germany) as antimicrobial protection.

For biocompatibility testing, the tested surface variants were first aseptically placed into individual wells of 24-well culture plates. Subsequently, cells were seeded onto the surfaces at a density of 1.5 × 10⁵ cells per well and allowed to attach. Wells containing cells cultured in medium alone served as negative controls. Cell cultures were maintained in contact with the materials for 24 h, under standard culture conditions described above.

After the exposure period, the culture medium was removed and replaced with fresh EMEM supplemented with 10% AlamarBlue reagent (Sigma-Aldrich, Merck KGaA, St. Louis, MO, USA). The plates were incubated at 37 °C, 5% CO₂ for 4 h to enable metabolic conversion of resazurin. Subsequently, 100 µL aliquots of the supernatant from each well were collected in triplicate and transferred to black 96-well microplates (Thermo Scientific, Waltham, MA, USA). Fluorescence signals resulting from resazurin reduction were measured using a fluorometric plate reader under the same settings as described previously. Cellular metabolic activity was expressed as the percentage of resazurin reduction and calculated according to the formula outlined in Section 2.9. All experiments were performed using three independent material specimens.

Cell viability was subsequently calculated by normalizing the percentage of resazurin reduction obtained for each tested material to the reference surface, according to the following equation:

$$cell viability \left[\%\right]={\displaystyle \frac{F_{sample}}{F_{reference}}}\times 100\%$$

where F_sample and F_reference denote the fluorescence intensity resulting from resazurin reduction measured for the tested material and the reference surface, respectively. The reference sample was defined as the unmodified metallic surface, (respectively, stainless steel or titanium) without any surface treatment or coating.

Cytotoxicity was defined as a decrease in cell viability relative to the reference surface. In accordance with ISO 10993-5 [40], materials exhibiting cell viability below 70% were classified as cytotoxic, whereas those showing cell viability equal to or greater than 70% were considered non-cytotoxic.

2.11. Statistics Method

All statistical analyses were performed using IBM SPSS Statistics for Windows, Version 27.0 (IBM Corp., Armonk, NY, USA, 2020). The number of adhered bacteria and biofilm activity on the tested surfaces were analyzed using one-way analysis of variance (ANOVA). When the assumption of homogeneity of variances was met, post hoc comparisons were conducted using Hochberg’s GT2 test. In cases where homogeneity of variances was violated, Welch’s ANOVA was applied, followed by the Games–Howell post hoc test. Differences were considered statistically significant at p < 0.05.

3. Results and Discussion

Currently, the mechanism of EPD of both Chit and Alg is known and described elsewhere [41,42,43,44,45,46], respectively. In the case of Chit, its amino groups protonate in an environment of pH below 6.3 and in the presence of H₃O⁺ ions. Under applied voltage, such molecules migrate towards the cathode according to the electric field lines. Near the surface of the cathode, electrolysis of the water, consisting of its disintegration into H⁺ and OH⁻ ions, takes place. The presence of OH⁻ ions results in a local increase in the pH value and interaction of those ions with Chit molecules, which leads to deprotonation of the amino groups and the deposition of coating. On the other hand, Alg in aqueous solutions in the pH range of 3 to 9 and under applied voltage dissociates into a negative Alg ion and a positive Na ion. This also leads to the electrochemical decomposition of water, causing a drop in the pH at the anode. In this way, to neutralize the charge, the H⁺ ion attracts macromolecules with the opposite charge, namely Alg¯. Therefore, these polymers behave oppositely in the electric field, which may result in a different thickness of deposited coatings, different interaction with the antibacterial components, and thus different volume fraction of antibacterial additives in the coatings.

In addition to EPD parameters, such as voltage and deposition time, which were modified and optimized in our previous work [30], electrophoretic mobility strongly influences the deposition kinetics and the macroscopic homogeneity of coatings. This applies to both Alg and Chit solutions, as well as their multicomponent Alg or Chit with EO/OC dispersed systems. The zeta potential values of these systems were determined over a wide range of pH values. Based on the obtained zeta potential values, the stability of Alg-based emulsions in the pH range of 6–9 used for coatings deposition, regardless of the type of additive tested, was high (above −30 mV) [30,33] and within the range considered in the literature [47] for stable emulsions. Chit-based emulsions with a pH lower than 6.3 also met this criterion.

It should also be noted that the co-deposition of EOs and their constituents with Chit [31,32] and Alg polymer [30,33] was investigated in our previous studies. Therefore, in the present work, we did not repeat the investigations of EPD kinetics and mechanisms for multicomponent coatings. Instead, we adapted the process route and parameters and focused on verifying whether, under the same parameters, the coatings would be macroscopically homogeneous. It should be mentioned that, in the case of Alg-based coatings, to ensure high adhesion strength of coatings, it was necessary to develop a two-stage EPD route, i.e., the deposition of the interlayer from an Alg solution and then the outer composite layer from an emulsion with Alg and oil.

In the present work, the focus was on examining the adhesion of coatings to substrates (Section 3.1), their morphology and micro/structure, bond interaction and formation (Section 3.2), surface properties (Section 3.3), release studies of additives, and verification of the antimicrobial response of the coating (Section 3.4).

3.1. Adhesion Strength of Coatings

Firstly, coatings deposited from all Chit-based dispersed systems containing all blends of EOs or OCs, EO2, EO3, OC2, and OC3, were deposited directly on ground titanium substrates. However, the adhesion tape tests showed complete delamination and separation of the coatings from the substrates. Following this result, modification of the deposition procedure or a change in the substrate surface modification was needed. Titanium substrates were chemically etched, as described in the experimental section, and then the deposition of coatings was performed. However, no improvement in the adhesion strength of coatings was achieved. All coatings were separated from the surface of the substrates. Then it was decided to change the way of deposition. The process was divided into two stages. The first Chit interlayer was deposited with a voltage of 10 V for 2 min, and subsequently the composite layer consisting of Chit and EO was obtained at 10 V through 3 min. The coatings deposited in this way on ground titanium also showed a lack of adhesion strength. Only the combination of both, etched titanium and two-stage deposition, allowed for excellent adhesion of coatings to the substrates (class 5B) (Figure 3).

Figure 3. SEM images of the etched titanium with Chit-based coatings after tape tests. Coatings were deposited in a two-stage EPD. The outer layer was obtained from emulsions of concentration of 2 mL/L of the following mixtures: (a) EO2, (b) EO3, (c) OC2, and (d) OC3.

The reasons for such poor performance of coatings deposited directly on ground titanium may be connected to (i) the coating phase composition, (ii) interactions between the EO constituents and the Chit matrix, and (iii) the chemical composition of the surface of substrates and interactions between the coating and the substrate. The chemistry of the titanium surface, used in this work, was previously examined in both the as-delivered state and after etching with the same procedure [43]. Titanium in the delivered state had only one oxidation state (Ti⁴⁺) in the oxide layer covering the surface of the substrate, and after etching had three oxidation states (Ti⁴⁺, Ti²⁺, and Ti⁰). As for ground titanium, it is known that a passive oxide layer is present on its surface. This suggests that the oxide layer on the etched titanium has three possibilities for forming chemical bonds with a coating in addition to physical intermolecular interactions. In the case of ground titanium, probably only intermolecular forces connect the coatings to the surface. This was sufficient for the high adhesion strength of Chit coatings with a single additive developed in our previous works [29,31,32]. In the present study, we work with two or three constituents that may not only act differently with the Chit matrix or titanium oxide, but also exhibit various interactions between themselves. However, there is a lack of information in the available literature on this topic. Only reports on the interactions of individual constituents with Chit are available [44,45,46]. It is known that CA and probably CBO can form imine bonds with Chit [4,48,49,50], which also affect the formation of bonds with metallic substrates, influencing not only physical connections but also chemical ones. Despite this, in our work, we did not find imine bonds in CA-Chit coatings, indicating that CA, similarly to TH [50,51,52,53,54], creates only hydrogen or van der Waals bonds with Chit. This indicates that CA, TH, and T4 molecules may compete with each other to form bonds with Chit. This can lead to saturation of the Chit chain, and the presence of many free molecules occurs in the coatings, which may aggregate and accumulate near the substrate surface, preventing the formation of Chit-substrate bonds. The application of a pure Chit interlayer on the etched substrate surfaces probably leads to the formation of more stable intermolecular interactions and chemical bonds between the polysaccharide and the substrate. Therefore, there are more possibilities to target the functional groups of EOs or their OCs, while these may result in strengthening the polymer adhesion to the oxide layer covering the titanium substrate.

Drawing on previous experience in the development of Alg-based coatings on chemically etched stainless steel substrates [30], the deposition of multicomponent coatings in a single-stage EPD process turned out to be ineffective in obtaining their high adhesion to the substrate; only class 2B was achieved. However, for all coatings with such a composition, high adhesion strength, class 4B, was possible by using an Alg interlayer (Figure 4a–d). Despite constant attributes such as substrate material and polysaccharide type, as well as the EPD route of multicomponent coatings related to the distinctions between deposition time and the use of the surfactant, it was possible to develop a standard operating procedure regarding the high adhesion strength of coatings. This emphasizes the importance of depositing an interlayer, which experimentally increases the adhesion strength of coatings as described by Friedrich [55] and hinders direct contact of oil droplets with the metal substrate during the formation of bonds in the drying stage of the coating. The completion of this stage of research allowed robust coatings to be achieved, which can be subjected to further investigation of properties.

Figure 4. The etched stainless steel with Alg-based coatings after tape tests. Coatings were deposited in a two-stage EPD. The outer composite layer was obtained from dispersed systems of 2 mL/L concentration containing the following mixtures: (a) EO2, (b) EO3, (c) OC2, and (d) OC3.

3.2. Morphology and Micro/Structure of Coatings

Essential oils EO2 and EO3 embedded in Chit were visible as droplet-like deposits scattered homogeneously across the entire coating surface (Figure 5a,b). Their size ranged from 0.5 µm to 4.0 µm.

Figure 5. Surface morphology of Chit-based coatings deposited in a two-stage EPD. The outer layer was obtained from dispersed systems of 2 mL/L concentration containing the following mixtures: (a) EO2, (b) EO3, (c) OC2, and (d) OC3. Enlarged areas of the coating’s surface are also present in each image. SEM.

Regarding the coatings containing OC2 and OC3, the number of visible droplets was very low (Figure 5c,d) due to the better solubility of these compounds in ethyl alcohol [56]. Those that were observed had a size similar to that of EO droplets and were also scattered homogeneously across the surface of coatings. Thus, the main difference in visibility between multi-EOs and multi-OCs on the coating’s surface is related to the chemical composition, as the EOs consist of many compounds of different solubility in water or ethanol.

The morphology of Alg-based coatings shown in Figure 6 indicates the suitability of using PS 80 in emulsions to act as an emulsifier because no large droplets, typical when only EO was used, were observed. The morphology of the observed top layer of these coatings is very similar to each other, and there are no differences depending on whether they are EOs or OCs or whether they are two- or three-component blends. However, it should be noted that these images show only the surface morphology features of the coating. The use of a surfactant that fragments the droplets further enhances this effect. The additive distribution on the cross-section of the coating, described in further part of Section 3.2, is of great importance.

Figure 6. Surface morphology of Alg-based coatings deposited in a two-stage EPD. The outer layer was achieved from dispersed systems of 2 mL/L concentration containing the following mixtures: (a) EO2, (b) EO3, (c) OC2, and (d) OC3. SEM.

Li et al. [57] described that a common method for organic crystal growth can be solvent diffusion. To determine whether crystallization of cinnamic aldehyde molecules occurring in the oil-in-water (O/W) emulsion is possible, a TEM investigation was performed on specimens prepared by inserting a carbon film-coated copper grid into the previously homogenized emulsion and leaving it for a day to dry. Fine CA nanocrystals with flower-like shape up to 50 nm in size were found to be present in the Alg matrix (Figure 7a,b). The Selected Area Electron Diffraction (SAED) patterns (for example, in Figure 7c) confirmed the crystalline structure of the CA nanoprecipitates with a monoclinic crystal system.

Figure 7. TEM images showing distribution of CA nanocrystals in the Alg matrix (a) and the flower-like shape of nanocrystals (b), as well as the SAED pattern taken from the area marked as a circle in Figure 7a (c). The identification of the SAED pattern as CA (monoclinic) is given in Figure S1.

The microstructure of the selected multicomponent coatings was observed using TEM on cross-sectional specimens prepared by the FIB technique. Figure 8 shows a microstructure of the nCA-TH/Alg//Alg coating. Inset A shows images of the coating after primary preparation (inset A1) and additional FIB thinning (inset A2). The additives are not visible in the original lamella, but they are noticeable in the thinner lamellae; during the additional preparation, the coating started to separate from the substrate. The nOC2/Alg//Alg coating thickness was estimated at 1.8 µm. The inset B indicates individual additives and also the presence of agglomerates. The SAED pattern taken from areas with additives revealed an amorphous character (inset C). However, it should be emphasized that the investigated lamella was relatively thick despite additional thinning using the FIB, and the number of additives in the Alg matrix was relatively low. Therefore, the possible crystalline structure of the additives (e.g., CA) may not be detected. Moreover, with regard to the shape of the additives, it can be assumed that the presence of solid particles is highly possible. The inset D strongly points out the presence of additives with a diameter in the range of 30–80 nm.

Figure 8. TEM images of nOC2/Alg//Alg coating after primary preparation and additional FIB thinning (inset (A1) and (A2), respectively), higher magnification of the coating focusing on additives (inset (B)), the amorphous character of the coating visible on the SEAD pattern taken from the area marked as a circle (inset (C)), additives representing different shapes (inset (D)). Arrows in insets (B,D) indicate additives.

The CA-TH/Chit//Chit coating adhered very well to the chemically etched titanium substrate, and its thickness was about 0.7 µm (Figure 9). In the investigated area of the coating, several oil droplets were located within the Chit matrix, especially in areas close to the coating surface. However, observation of the droplets was difficult because the coating contained numerous artifacts resulting from the sample preparation process.

Figure 9. TEM images of OC2/Chit//Chit coating. An inset (A,B) showing oil droplets in the Chit matrix.

The clear difference for dual-component coatings, nOC2/Alg//Alg and OC2/Chit//Chit, is primarily related to their average thickness, and above all, the distribution and quantity of additives in the matrix. Although the deposition of the Chit-based coating at higher voltage and for a longer time, its thickness is 2.6 times smaller than that of the corresponding Alg-based coating. Moreover, this is primarily related to the substrate material, polymer type, electrophoretic mobility of each of the constituent phases of O/W emulsions, and, to a minor extent, the distance between the electrodes in the EPD cell during the deposition process. The electrophoretic mobility of particles in the emulsion and the potential difference during the EPD process may result directly from the use (or not) of a surfactant, as well as the type of polymer used as a matrix.

FT-IR spectra of pure Alg and Chit coatings, as well as all individual components of the obtained coatings, have been described in detail in our previous publications [29,30,33]. Based on those investigations, it is known that the basic mechanism of the interactions between chains of polysaccharides, such as sodium alginate and chitosan, and molecules of additives is the formation of new hydrogen bonds. An additional effect may also be the cleavage of primary hydrogen bonds between the chains. This rearrangement of intermolecular interactions involves hydroxyl groups (-OH) as well as oxygen atoms in C-O-C bridges, methoxy groups (-OCH₃), and aldehyde groups (-CHO) present in the structure of the coatings components.

The main components of the essential oils used as additives are as follows: for CBO—CA and eugenol (EUG), for TO—TH, for TTO—T4, 1,8-cineole, γ-terpinene, and α-terpinene. The structure of CA includes an aldehyde group, while there are hydroxyl and methoxy groups attached to the aromatic ring in the structure of eugenol. TH also has a hydroxyl group bonded to the aromatic ring in the structure. In the case of T4, the structure contains a hydroxyl group attached to an unsaturated six-membered ring, while 1,8-cineole has a C-O-C bridge in the bicyclic structure. Terpinenes, as unsaturated hydrocarbons, do not possess oxygen atoms in the structure and therefore do not participate in the rearrangement of hydrogen bonds. The structure of the analyzed molecules is shown in Figure 10.

Figure 10. The structure of the additives molecules: (a) CA, (b) EUG, (c) TH, (d) T4, and (e) 1,8-cineole.

The FT-IR spectra of the Alg coating and the Alg coatings with different additives are shown in Figure 11, and their characteristics are compiled in Table S2. All spectra reveal similar characteristic bands, but the positions of some have shifted after the introduction of the additives. The most significant change is observed for the band corresponding to the C-O stretching in the C-O-C bonds between the sodium alginate rings. Its position shifted to the lower wavenumbers, from 1055 cm⁻¹ to 1039–1044 cm⁻¹, depending on the composition of the coating. This effect is attributed to the formation of new hydrogen bonds between oxygen atoms in the C-O-C bonds and hydroxyl groups in the structure of the additives. Hydroxyl groups are present in the structure of EUG, TH, and T4, enabling all of them to participate in the formation of new intermolecular interactions. The largest shift (to 1039 cm⁻¹) is observed for the coating with the binary CA-TH composition, which contains the highest content of TH. The hydroxyl group in TH is attached directly to the six-membered aromatic ring, whereas in T4 it is bonded to the tertiary carbon atom, to which a large isopropyl group is also attached. The easier formation of new hydrogen bonds and stronger interaction with TH molecules are therefore associated with the presence of less steric hindrance.

Figure 11. FT-IR spectra of the Alg coating and the Alg coatings with different additives, from the top OC2, OC3, EO2, and EO3.

It could be expected that the T4 molecule, with an unsaturated six-membered ring in the structure, acquires the spatial conformation that should better facilitate the formation of new molecular interactions. In contrast, this is more difficult for the TH molecule, which has a flat aromatic ring structure. However, since the largest shift in the band position related to the formation of the strongest interactions is observed for the coatings with the highest TH content, the main factor appears to be the absence of steric hindrance that could be caused by other substituents on the rings. Differences in the additive content could also explain the shifts in the position of the band associated with the C-O stretching of the C-O-C bonds within the six-membered rings of the polymer chains. For the coatings with binary CBO-TO and CA-TH compositions, the position is changed from 1098 cm⁻¹ to 1096 cm⁻¹ and 1092 cm⁻¹, respectively. Similarly to the band analyzed previously, this effect is related to the higher content of TH, which is also the main component of TO. However, this shift is smaller because the formation of new hydrogen bonds is affected by the steric hindrance associated with the presence of large carboxyl groups attached to the six-membered rings of the polymer chains. Contrary to this, for the ternary CBO-TO-TTO and CA-TH-T4 compositions, a slight shift towards higher wavenumbers is observed, from 1098 cm⁻¹ to 1104 cm⁻¹ and 1100 cm⁻¹, respectively. This indicates that, for these coatings, the changes in the intermolecular interactions are not primarily related to the formation of new hydrogen bonds with TH molecules (which content is lower), but rather to the cleavage of the primary intermolecular interactions between sodium alginate chains associated with the presence of the other additives. It can be assumed that this results from the cleavage of the primary hydrogen bonds involving oxygen atoms in the C-O-C bonds within the six-membered rings, along with the formation of new hydrogen bonds involving CA and EUG (as the component of CBO). This is confirmed by the observed shifts in the bands for the C=O stretching and the C-O stretching in carboxyl groups, from 1745 cm⁻¹ to 1740–1742 cm⁻¹ and from 1618 cm⁻¹ to 1613–1614 cm⁻¹, respectively. In particular, it can be noted that EUG, which has both a hydroxyl group and an oxygen atom in the methoxy group in the structure, can more easily form new hydrogen bonds with carboxyl groups than CA, which has only an oxygen atom in the aldehyde group. Hence, the band at 1098 cm⁻¹ for the C-O stretching is slightly shifted towards higher wavenumbers for the coating with the ternary CBO-TO-TTO composition (to 1104 cm⁻¹) containing both CA and eugenol than for the coating with the ternary CA-TH-T4 composition (to 1100 cm⁻¹) containing only CA. This indicates that the presence of EUG has a more significant impact on the cleavage of the primary intermolecular bonds between the polymer chains.

The FT-IR spectra of the Chit coating and the Chit coatings with different additives are shown in Figure 12, and their characteristics are compiled in Table S3. As with the previously analyzed spectra of the Alg coatings, these spectra also exhibit shifts in the position of certain bands after the introduction of the additives. The most significant shift, from 1085 cm⁻¹ to 1062–1064 cm⁻¹, is observed for the band corresponding to the C-O stretching of the C-O-C bonds within the six-membered rings of the Chit chains. This indicates the formation of new hydrogen bonds with hydroxyl groups in the structure of EUG, TH, and T4. The position of the band at 1032 cm⁻¹ for the C-O stretching in the C-O-C bonds between the rings is changed slightly to 1028–1029 cm⁻¹. Therefore, the formation of new intermolecular interactions is more favorable involving oxygen atoms in the C-O-C bonds within the ring than between the rings. The band at 1569 cm⁻¹, attributed to the N-H stretching in unsubstituted amino groups, shifts towards higher wavenumbers to 1573 cm⁻¹ for the binary CBO-TO and CA-TH compositions with the highest content of TH and T4. Therefore, it can be assumed that the formation of new intermolecular interactions involving hydroxyl groups of the additives results in the cleavage of the primary interactions between the polymer chains, which involve their amino groups. The band at 1420 cm⁻¹ for the C-H stretching in hydroxymethyl groups (-CH₂OH) attached to the rings shifts to 1411–1414 cm⁻¹, while the position of the band at 1152 cm⁻¹ for the C-O stretching in hydroxyl groups attached directly to the rings remains unchanged. The molecules of CA, EUG, and 1,8-cineole, containing oxygen atoms in the structure, thus form new intermolecular interactions more easily with hydroxyl groups more distant from the rings (attached through methylene groups) than with those attached directly, for which a steric hindrance associated with the proximity of other substituents occurs. The formation of new intermolecular interactions involving oxygen atoms in the structure of the additives is further confirmed by the largest shift (to 1411 cm⁻¹) observed for the coating with the binary CBO-TO composition with the highest content of CA and EUG. The spectrum for the chitosan coating reveals the band at 1657 cm⁻¹ attributed to the C=O stretching in N-acetyl substituted amino groups. In the spectra of the chitosan coatings with the additives, its position remains unchanged (observed as the shoulder at 1657–1659 cm⁻¹), but the additional band at 1635–1636 cm⁻¹ could also be noticed. This indicates the formation of new intermolecular interactions involving carboxyl groups in the polymer chains after the introduction of the additives. However, since this characteristic band appears both at its original position and shifted towards lower wavenumbers, it can be assumed that this leads to the formation of two different types of polymer chain ordering.

Figure 12. FT-IR spectra of the Chit coating and the Chit coatings with different additives, from the top OC2, OC3, EO2, and EO3.

3.3. Surface Topography and Surface Properties

The roughness of the surface of the coatings showed minor differences between the coatings with similar EOs or OCs and different polysaccharide matrices (Figure 13a,b). They can be classified as “minimally rough” based on the Sa value, as proposed by Albrektsson and Wennerberg [58]. Alg-based coatings were rougher compared to Chit-based coatings (Figure 13a,b) with slightly more developed surface (Figure 13c), except for coatings containing CA-TH-T4 agents. For comparison, as reported in our previous works, the surface roughness of the same stainless steel substrate etched in the same way was considered smooth [30], while that of titanium substrates after etching, also in the same procedure, was classified as between “minimally rough” and “smooth” [43] according to Albrektsson’s classification [58].

Figure 13. Roughness parameters of Chit-based (a) and Alg-based coatings (b) with OC2, OC3, EO2, EO3 additives and their ISAD (c).

The distinction in roughness parameter values, i.e., Sa, Sq, and St, is not very large and may be directly related to the polymer used, the total deposition time of 5 min vs. 3 min, and the presence of surfactant as an additional component of the Alg coatings. Shao et al. [59] described SiO-ZnO coatings on polyethylene terephthalate surfaces, which, for similar Sa values, have high antibacterial efficacy against E. coli and S. aureus bacteria. Based on our roughness values of Alg and Chit-based coating surfaces, it can be assumed that multicomponent coatings will respond in a similar way.

The major differences between the coatings regarding the polymeric matrix used were visible in the AFM images. In the case of Chit-based coatings, it was observed that on the surface of each coating, many individual additives or their clusters were visible (Figure 14). The highest diameter of single additives was about 2 µm, and the lowest diameter was less than 100 nm. These additives were visible as peaks that differed from the background (chitosan matrix) (Figure 15), which suggests that most of them were located in the upper layer of the coatings. This claim is supported by TEM investigation of coatings performed on the FIB lamella (Figure 9). This great visibility of the additives corresponds well to the surface morphology of the coatings observed by SEM (Figure 5), on which many droplets or clusters occurred.

Figure 14. AFM images of surfaces of Chit-based coatings with EO2 (a), EO3 (b), OC2 (c), and OC3 (d).

Figure 15. Three-dimensional visualization of surfaces of Chit-based coatings containing EO2 (a), EO3 (b), OC2 (c), and OC3 (d). The color scale bar indicating the measured data is the same as in Figure 14.

In the case of Alg-based coatings, a very low number of visible droplets was observed on the surface (Figure 16). It is probably due to the use of PS 80 as a surfactant and a better overall fragmentation of droplets in the dispersed systems used for EPD. The lack of visible peaks on the surface (Figure 17) suggests that most of the additives were embedded deep in the coatings, which could lead to the assumption that the size of droplets or clusters was below 100 nm or that the additives were successfully integrated with the matrix, forming a single-phase layer.

Figure 16. AFM images of surfaces of Alg-based coatings containing EO2 (a), EO3 (b), OC2 (c), and OC3 (d).

Figure 17. Three-dimensional visualization of surfaces of Alg-based coatings with EO2 (a), EO3 (b), OC2 (c), and OC3 (d). The color scale bar indicating the measured data is the same as in Figure 16.

The differences in surface topography between Alg-based and Chit-based coatings are quite significant. This is mainly due to the use of different polymers, different substrates, and the PS 80 surfactant influencing the additive size. Additionally, it should be noted that embedding two or three antibacterial components (while maintaining their concentration constant of 2 mL/L for emulsions) in the coating does not significantly affect the results of surface parameters between coatings with a given base polymer. However, an exception is the CA-TH-T4/Chit//Chit coating, which has a higher roughness compared to all variants and combinations of coatings developed. The possible reason for such behavior is the fact that TH, as the only substance, was added to the dispersed systems in the solid state and may crystallize after deposition. Similarly, CA can also crystallize in the Chit matrix, so the presence of precipitates of both compounds may be the reason for such differences in roughness.

The wettability of the coatings was investigated to evaluate the ability of the coatings to adhere cells. WA values for Alg-coated samples indicate a significant decrease compared to the etched stainless steel substrate control sample (Figure 18a). For the water droplet, the WA value changed from approx. 94° to approx. 35° (average value for all multicomponent Alg-coatings), and for the diiodomethane droplet, from approx. 44° to approx. 20°. Figure 18b shows a multiple increase in the polar component of SFE to a value of more than 20 mN/m and a slight change in the value of the dispersive component of SFE of 10 mN/m. The total SFE value for all Alg-based coatings was almost identical and amounted to approximately 70 mN/m. Chit-based coatings had slightly lower WA for both water and diiodomethane than etched titanium (Figure 18c) and were very similar to each other. The values of contact angles for water were close to 80°, and those for diiodomethane droplets were close to 30°. As for SFE (Figure 18d), all coatings exhibited higher values than the substrate with the most visible polar part. In our previous work [60], we showed that sodium alginate coating deposited on chemically treated titanium substrate also has a hydrophilic character with WA close to 35°. Multi-EOs or OCs/Alg coatings developed in this work have similar results for H₂O and CH₂I₂ droplets, 35° and 20°, appropriately. Karpiński et al. [61] reported that the WA for the water and diiodomethane droplets applied to an etched titanium substrate was greater than 95° and 50°, which is close to our results of 90° and 45°, respectively. The present study showed that polar and nonpolar liquid droplets placed on the surface of Chit-based coatings with all blend additives, i.e., EO2, EO3, OC2, and OC3, only slightly reduced the contact angle. This contrasts with the properties of multi-EOs/Chit or OCs/Chit coatings, for which the WA for both liquids decreased at least twice. As described by Hamad et al. [62], the differences in WA values for Chit or Alg composite coatings may be related to their porosity. Nyström et al. [63] also reported that the less hydrophilic character of Chit is strongly related to free amino or hydroxyl groups present along the main chain of the polymer. Qiang Sun [64] described that -CH₃ bonds that occur in the Chit chain are considered hydrophobic groups. This may be the reason for the discrepancy in WA results between polymers.

Figure 18. WA and SFE of Alg-based coatings (a,b) and of Chit-based coatings (c,d) with EO2, EO3, OC2, and OC3 additives on etched stainless steel and etched titanium substrates, respectively.

Upon comparing the SFE values for both types of coatings and their metallic substrates, regardless of the polymer used for the coating matrix, it can be concluded that a high value of the polar component of SFE is not so crucial in ensuring high adhesion strength of the coatings. The reason for the differences in the results of the polar component for all multicomponent coatings may be the properties of polymers, where Alg is easily soluble in water at a pH close to neutral, in contrast to Chit being soluble in an acidic medium. Therefore, it is easier for Alg (potentially more hydrophilic groups compared to Chit) to form more hydrogen bonds, which will be more resistant to the drying process. Based on WA values for water droplets for all types of coatings, a range of 20–44° for Alg-based coatings and close to 80° for Chit-based coatings, it can be assumed that cells will have positive/optimal hydrophilic environments for adhesion and proliferation, as described by Li et al. [65]. However, more favorable conditions are provided by Alg-based coatings.

3.4. Release of Bactericidal Additives from the Coatings

The intensity of release of microbicidal substances from two distinct Chit and Alg-based coatings containing OCs was investigated using UV-vis spectroscopy (Figure 19). There were minor differences between the coatings and their behavior in PBS medium. In general, the results indicate a less intensive release of antibacterial additives for coatings containing three versus two blend constituents. This is the result of maintaining the total blend concentration in the emulsion used for EPD for all variants at 2 mL/L. Furthermore, for the nCA-TH/Alg//Alg coating after only two hours of incubation under target conditions, the percentage share of the released TH and CA substances was 68.8% and 68.9%, respectively (Figure 19a). For the nCA-TH-T4/Alg//Alg coating, the release was presented in the sequence of 60.9%, 60.8% and for T4 it was 67.9% (Figure 19b). For PS 80, these values were also high, 69.4% and 61.4%, respectively (Table S1). These percentage values mean a very fast release of antibacterial substances. Therefore, in further studies, it would be worth using material modifications related to thickening of the coating, polymer cross-linking, or using alternating deposition of Alg and Alg+EO/OC layers to slow down this process. In the case of a two-constituent coating, it was found that the maximum absorbance for CA occurred for a time of 300 min, and later the absorbance decreased slightly over time (Figure 19c). For T-mol, it was observed that up to 300 min, the increase in absorbance was very fast, and after that, it still increased, but with a lower rate. This behavior may be related to the possible degradation of constituents in PBS over time (CA) or constant release from the deeper layer of coating (TH). For the three-constituent coating, the maximum absorbance was observed for each constituent for a time of 1440 min, and later the level of absorbance became stable with only a minor decrease for TH (Figure 19d). The longer degradation time of the Chit-based coating did not show a clear increase in antibacterial agent concentration, indicating the release of the compounds tested after only 24 h.

Figure 19. Releasing of antimicrobial substances from (a,b) Alg- and (c,d) Chit-based with OC2 (a,c), and OC3 (b,d) coatings.

Such differences between coatings may be related to the chemical composition and interactions between constituents, which means that in the case of CA-TH-T4, some sort of balance state was achieved. It should be noted that the dynamics of antibacterial agent release at the established 4 h incubation time for the Chit-matrix (Table S1c,d) is slower or similar (lower concentration in the collected fluid) than for the Alg-matrix (Table S1a,b). Therefore, by describing Alg and Chit-based coatings, it can be concluded that in both cases the release of the active substance occurs too quickly, and that it is necessary to take steps to extend this time. An effective approach in the future can be the modification of the polymer matrix of the coatings.

3.5. Antimicrobial and Antibiofilm Activity of Materials Developed

Analysis of the number of viable S. aureus cells revealed clear differences between the tested surfaces. The strongest antimicrobial properties among all variants were exhibited by the Alg coating, for which the average number of bacteria was 2.81 ± 0.56 log CFU/mL, compared to the control surface (5.37 ± 0.23 log CFU/mL; p < 0.001). Among coatings containing oil additives, the greatest effectiveness in reducing the number of bacteria was observed for the three-component oil nEO3/Alg//Alg coating, for which the number of bacteria decreased to 3.38 ± 0.86 log CFU/mL (p = 0.04). The other coatings had weaker antimicrobial activity, including nOC3/Alg//Alg (3.98 ± 0.50 log CFU/mL; p = 0.006) and nEO2/Alg//Alg (4.38 ± 0.94 log CFU/mL). However, the result was not significantly different from the control. The smallest effect was observed for the two-component nOC2/Alg//Alg coating, for which the number of bacteria (5.43 ± 0.36 log CFU/mL) was comparable to the control surface (Figure 20a).

Figure 20. Viability of bacterial cells (log CFU/mL; bar charts) and metabolic activity of biofilm (%; line plots) on polysaccharide-based coatings. Results obtained for S. aureus ATCC^® 25,923 on pure Alg and Alg-based coatings (a) as well as pure Chit and Chit-based coatings (b), and for E. coli ATCC^® 25,923 on Alg and Alg-based coatings (c) as well as Chit and Chit-based coatings (d). Data are presented as mean ± SD. *—indicates statistically significant differences in bacterial viability (CFU). #—indicates statistically significant differences in biofilm metabolic activity.

The evaluation of biofilm metabolic activity confirmed the varying effectiveness of the tested surfaces in limiting the growth of the S. aureus biofilm. The strongest anti-biofilm effect was observed for the Alg coating (49.55% ± 8.95%, p = 0.001) and for the nEO3/Alg//Alg coating (31.85% ± 15.09%, p < 0.001), compared to the control surface (68.4% ± 9.21%). The other effects were not significant. However, the three-component nOC3/Alg//Alg coating showed a moderate effect (60.10% ± 10.16%). In contrast, the two-component coating variants, both nOC2/Alg//Alg (76.59% ± 7.92%) and nEO2/Alg//Alg (73.51% ± 9.75%), did not inhibit biofilm formation (Figure 20a).

In contrast to the results obtained for S. aureus, analysis of the number of live E. coli cells showed no differences between the control surface and the tested Alg-based coatings. The number of live E. coli cells showed no significant differences between the control surface and the Alg coatings with oil additives. On the control surface, the number of bacteria was 5.77 ± 0.29 log CFU/mL, while for the Alg coating without additives, the value was 5.36 ± 0.34 log CFU/mL. Coatings containing oil additives did not lead to a reduction in the number of bacteria. For the nOC2/Alg//Alg and nEO2/Alg//Alg variants, the number of E. coli (5.88 ± 0.43 log CFU/mL and 5.82 ± 0.39 log CFU/mL). Subsequent samples also did not show significant differences. For three-component coatings, values of 6.35 ± 0.16 log CFU/mL (nOC3/Alg//Alg) and 5.84 ± 0.17 log CFU/mL (nEO3/Alg//Alg) were obtained. Similar observations were noted for the metabolic activity of the E. coli biofilm. In the case of the anti-biofilm effect of the tested coatings, the following observations were made. The biofilm activity on the control surface was 73.49% ± 7.07%, while on the Alg coating without additives, it was 75.52% ± 7.60%, indicating no biofilm inhibition. In the case of coatings containing oil additives, only a significant decrease in biofilm metabolic activity was observed for the two-component coatings (64.14% ± 4.78% for the nOC2/Alg//Alg coating and 62.62% ± 6.51% for the nEO2/Alg//Alg coating; p = 0.026 and p = 0.005, respectively). The three-component nOC3/Alg//Alg (66.62% ± 5.80%) and nEO3/Alg//Alg (70.23% ± 8.97%) coating, biofilm activity remained similar to the control and was statistically insignificant (Figure 20c).

In the next stage of the study, the antimicrobial activity of Chit coatings deposited on a titanium substrate against S. aureus was evaluated. On the control surface, the number of bacteria was 5.89 ± 0.17 log CFU/mL, while on the Chit coating without additives, it was 5.54 ± 0.25 log CFU/mL (p = 0.04). For the OC2/Chit//Chit (p = 0.023) and EO2/Chit//Chit coatings, the number of S. aureus was 5.48 ± 0.27 log CFU/mL and 5.58 ± 0.91 log CFU/mL, respectively. Similar values were observed for the three-component coatings: OC3/Chit//Chit (5.34 ± 0.66 log CFU/mL) and EO3/Chit//Chit (5.39 ± 0.34 log CFU/mL, p = 0.023). The assessment of the metabolic activity of the S. aureus biofilm showed only limited anti-biofilm activity. However, for the OC2/Chit//Chit coating, the result (58.59% ± 7.54%, p < 0.001) was significant compared to the control, indicating a moderate anti-biofilm effect. The activity of the biofilm on the control surface was 79.29% ± 8.07%, while on the Chit coating without additives, it decreased to 69.85% ± 22.72%. The other coatings did not inhibit biofilm formation. For the EO2/Chit//Chit, biofilm activity increased to 82.94% ± 13.14%, and for the three-component coating systems, it remained like the control, 72.97% ± 15.99% for the OC3/Chit//Chit and 80.71% ± 19.23% for the EO3/Chit//Chit coating (Figure 20b).

Some antimicrobial effects were observed for E. coli. Analysis of the number of live E. coli cells showed that the Chit coatings did not show an antimicrobial effect. On the contrary, a significant increase in the number of cells was demonstrated for all four multi-EOs and multi-OCs variants, Chit-based coatings (p < 0.05). On the control surface, the number of bacteria was 4.33 ± 0.34 log CFU/mL, while on the Chit coating without additives, the value was 4.61 ± 0.30 log CFU/mL. The use of EOs and OCs additives did not result in a reduction in the number of bacteria. For the OC2/Chit//Chit and EO2/Chit//Chit coating variants, the number of E. coli was 4.86 ± 0.31 log CFU/mL and 5.02 ± 0.18 log CFU/mL, respectively. In the case of three-component systems, a further increase in the number of bacteria was observed to 5.26 ± 0.36 log CFU/mL for OC3/Chit//Chit and 4.88 ± 0.39 log CFU/mL for EO3/Chit//Chit. In contrast, the assessment of the metabolic activity of the E. coli biofilm showed only a moderate and non-selective anti-biofilm effect of the tested surfaces. The biofilm activity on the control surface was 38.10% ± 11.52%, while on the Chit coating without additives, it was 36.49% ± 6.72%. Coatings containing EOs and OCs additives were characterized by reduced biofilm metabolic activity, although this effect was not correlated with a reduction in the number of live cells. The lowest values were recorded for OC2/Chit//Chit and EO3/Chit//Chit coatings, 25.64% ± 4.07%, p = 0.012 and 26.88% ± 3.05%, p = 0.023, respectively. For the other variants (statistically insignificant), biofilm activity was 29.31% ± 6.97% for the EO2/Chit//Chit and 30.58 ± 2.32% for the OC3/Chit//Chit coating (Figure 20d).

A comparison of the two matrices showed that Alg coatings had significantly better antimicrobial and anti-biofilm properties than Chit coatings, especially against S. aureus, while in the case of E. coli, none of the tested surfaces showed significant antimicrobial activity. Within Alg coating systems, the best results were obtained for the three-component oil nEO3/Alg//Alg coated stainless steel, while the weakest effect was observed for the two-component OC2/Alg//Alg coated stainless steel. Chit coatings on titanium substrates, regardless of their composition, showed only limited and inconsistent anti-biofilm effects, with no clear reduction in bacterial survival. The observed strain-dependent response may be attributed to fundamental differences in bacterial cell envelope structure. In particular, the outer membrane of Gram-negative bacteria such as E. coli constitutes an additional barrier limiting the penetration and local activity of hydrophobic EO and OC constituents, whereas Gram-positive bacteria such as S. aureus are more susceptible to membrane-targeting agents. The predominant antimicrobial mechanism of EOs involves interactions with the cell membrane, including permeabilization, depolarization, and induction of oxidative stress, which may result in reduced metabolic activity or biofilm inhibition without a proportional decrease in viable cell counts [66,67,68].

To discuss microbiological responses of Alg or Chit-based coatings with single EO and OC reported in our previous publications [29,30,33] and multicomponent coatings developed in the present work, significant differences in the results can be observed. A single antibacterial additive embedded in the coating significantly or almost eliminated G⁺ bacteria. However, the G⁻ bacteria in most of the examined coatings were resistant. Undeniably, for both strains of bacteria, these responses were significantly stronger compared to Alg- and Chit-based coatings with multicomponent EO and OC additives obtained from emulsions with the same (2 mL/L) additive concentration. This may be due to the differences in the coating microstructure, including distribution, morphology, and diameter of EOs and OCs additives, incorrect additives ratio, their coexistence, and interactions between particular additives and with Alg and Chit molecules.

3.6. Cytotoxicity of Materials Developed

The assessment of cell metabolic activity demonstrated a pronounced decrease in the viability of both MG-63 and FaDu cell lines after contact with pure Alg and Alg-based coatings containing EO and OC additives (Figure 21a). Relative to the stainless steel reference surface, Alg coatings induced a substantial reduction in cell metabolic activity, which was further intensified by the incorporation of EO and OC additives. A similar trend was observed for the FaDu cell line. Relative to the stainless steel reference surface, Alg coatings reduced cell metabolic activity, while coatings containing two-component EO or OC systems exhibited a comparable response to that of pure Alg. In contrast, three-component systems resulted in a slightly more pronounced reduction in cell viability.

Figure 21. Metabolic activity and cytotoxicity of MG-63 and FaDu cells after contact with reference metallic surfaces: (a) metabolic activity—stainless steel, (b) cytotoxicity relative to stainless steel, (c) metabolic activity—titanium, (d) cytotoxicity relative to titanium. The red dashed line indicates the cytotoxicity threshold corresponding to 70% cell viability, in accordance with ISO 10993-5. Data are presented as mean ± SD.

Based on the applied cytotoxicity criterion (70% cell viability), Alg-based coatings were classified as cytotoxic for both MG-63 and FaDu cell lines (Figure 21b). Relative to the stainless steel reference surface, pure Alg coatings reduced cell viability below the cytotoxicity threshold, and the incorporation of EO and OC additives led to a further decrease. This effect was more pronounced for coatings containing multicomponent additive systems.

In contrast to alginate-based systems, Chit coatings exhibited a milder cytotoxic effect (Figure 21c). Relative to the titanium reference surface, pure Chit coatings showed a moderate influence on the metabolic activity of MG-63 cells. The incorporation of EO and OC additives resulted in a varied but generally moderate reduction in cell viability, with no clear trend indicating a strong enhancement of cytotoxicity compared to the Chit matrix alone. A comparable response was observed for the FaDu cell line. Relative to the titanium reference surface, Chit coatings improved cell tolerance, and the incorporation of EO and OC additives did not result in a further deterioration of cell metabolic activity (Figure 21c).

Based on the applied cytotoxicity criterion (70% cell viability), the biological response to Chit-based coatings depended on the cell line tested (Figure 21d). For MG-63 cells, the OC2/Chit//Chit surface was classified as cytotoxic, whereas the remaining Chit-based coatings exhibited cell viability values close to the defined threshold, being either slightly above or slightly below 70%. In contrast, for FaDu cells, all tested Chit-based coatings, including those containing EO and OC additives, showed cell viability values above the cytotoxicity threshold and were therefore classified as non-cytotoxic relative to the titanium reference surface.

Overall, the results demonstrate that Alg-based coatings exhibited a markedly higher cytotoxic potential for both MG-63 and FaDu cell lines than Chit-based coatings, particularly in systems containing EOs and COs additives. Among Alg coatings, the presence of additives, especially in three-component formulations, resulted in the strongest reduction in cell viability, leading to cytotoxic responses in both cell models. In contrast, Chit-based coatings showed a milder and more variable cytotoxic profile, with several variants maintaining cell viability above the defined 70% threshold. In some cases, Chit coatings improved cell viability relative to the metallic substrate alone, indicating a more favorable cytocompatibility profile for this polymeric matrix. In the future, studies on more cell lines derived from oral tissues could be considered to optimize the indication of the least cytotoxic response.

4. Conclusions

• For Alg-based coatings deposited on stainless steel and Chit-based coatings obtained on titanium, substrate etching and two-stage EPD processes were necessary to achieve a high adhesion strength. Differences in the surface morphology of the coatings were mainly related to the use of the PS 80 surfactant for EPD of Alg-based coatings and fragmentation of EOs or OCs additives, although they did not have such an impact on the roughness parameters.
• The microstructure of multicomponent coatings with EOs and OCs consisted of antibacterial additives embedded in a polymeric matrix. The microstructure of the dual-component nOC2/Alg//Alg coating indicates the presence of additives differing in size and shape located in the deeper part of the coating. However, the microstructure of the OC2/Chit//Chit coating compared to the nOC2/Alg//Alg coating contains additives of larger size, located mainly in the upper zone of the coating.
• FT–IR investigation of the multicomponent coatings revealed interactions of single essential oil components, both with polymer matrices and between each other, depending on the degree of steric hindrance (correlated with substituents on the aromatic rings), the concentration of additives, and their type.
• The contact angle of water was significantly higher for the Chit-based coatings than for the Alg-based coatings. This difference is primarily due to the type of polymer used as the coating matrix.
• Too rapid release of antibacterial additives from coatings indicates the need for modification, e.g., cross-linking of the polymer chain, which would prolong the degradation time.
• Alg-based coatings exhibited significantly stronger antimicrobial and anti-biofilm activity than Chit-based coatings, particularly against S. aureus, but at the expense of markedly higher cytotoxicity, especially in formulations containing EOs and COs. In contrast, the chitosan coatings showed weaker and inconsistent antimicrobial effects while demonstrating a more favorable cytocompatibility profile, with several variants maintaining cell viability above the 70% threshold.

This work provides insight into the development of an electrophoretic co-deposition route of polymers with essential oil or their component blends to fabricate multicomponent coatings with high adhesion strength to metallic biomaterial substrates and with a broad spectrum of antimicrobial activity. However, the microbiological studies performed in this work show that the selection of individual components of multicomponent blends requires further advanced systematic research to determine the appropriate chemical composition of the blend. Therefore, future development of coatings should focus on screening of blends in terms of type and concentration gradient, in terms of interaction with various bacterial strains.