Magnetite Nanoparticles and Essential Oils Systems for Advanced Antibacterial Therapies

Essential oils (EOs) have attracted considerable interest in the past few years, with increasing evidence of their antibacterial, antiviral, antifungal, and insecticidal effects. However, as they are highly volatile, the administration of EOs to achieve the desired effects is challenging. Therefore, nanotechnology-based strategies for developing nanoscaled carriers for their efficient delivery might offer potential solutions. Owing to their biocompatibility, biodegradability, low toxicity, ability to target a tissue specifically, and primary structures that allow for the attachment of various therapeutics, magnetite nanoparticles (MNPs) are an example of such nanocarriers that could be used for the efficient delivery of EOs for antimicrobial therapies. The aim of this paper is to provide an overview of the use of EOs as antibacterial agents when coupled with magnetite nanoparticles (NPs), emphasizing the synthesis, properties and functionalization of such NPs to enhance their efficiency. In this manner, systems comprising EOs and MNPs could offer potential solutions that could overcome the challenges associated with biofilm formation on prosthetic devices and antibiotic-resistant bacteria by ensuring a controlled and sustained release of the antibacterial agents.


Introduction
Although most species of microorganisms are beneficial to nature and support life on Earth, a small group of infectious agents can cause severe diseases, sometimes leading to emerging outbreaks and difficult-to-handle pandemic situations. Pathogenic microorganisms hold the capacity to cause infections in the host by escaping the defense mechanisms through various strategies and virulence factors. According to The World Health Organization (WHO), bacteria, viruses, toxins, parasites, and harmful chemicals are listed as potential deadly biohazards [1][2][3]. Pathogenic bacteria may produce extracellular polymeric matrices, also known as glycocalyx, consisting of polysaccharides, lipids, and nucleic acids [1,4,5]. By these structures, bacteria adhere to biotic and abiotic substrates where they form biofilms, which are attached to the multicellular consortia of cells with particular architectures and modified phenotypes [1,5,6]. The formation of the biofilm is a primary protection strategy of microorganisms against environmental and antimicrobial agents, e.g., dehydration, starvation, antibiotics or biocides, but also host immune system [4]. Biofilm development is a fundamental factor in infection persistence or recurrence, with 65-80% associated being with chronic infections, according to The National Institutes of Health (NIH) [1,5,7].

Essential Oils as Antimicrobial Agents
As defined by the European Pharmacopeia 7th edition, EOs are "odorant products, with complex composition, obtained from raw plant extract, either extracted by a steam of water, dry distillation or a suitable mechanical method without heating" [35]. EOs comprise complex mixtures of more than 300 different volatile organic compounds, which are found in a partial vapor state at atmospheric pressure and room temperature [36]. Additionally, EOs are characterized by a lipophilic and hydrophobic nature and a density lower than that of water, which makes them highly soluble in organic solvents [37]. Among the large plethora of about 17,000 aromatic plant species, the most common EOs producing families are Lamiaceae, Lauraceae, Poaceae, Asteraceae, Verbenaceae, Myrtaceae, Zingiberaceae, Rutaceae, Piperaceae, Cuppressaceae and Umbelliferae [27,37].
There are various extraction techniques for extracting EOs that use multiple plant segments, including seeds, buds, flowers, leaves, barks, and peels, with extraction yields depending on the species and segment type, solute and solvent properties, such as temperature, polarity, concentration, pH and acidity [37,43]. These techniques usually imply a physical method for avoiding any potentially significant change in the chemical composition of EOs [37]. Conventional EOs extraction methods include cold pressing, solvent extraction, enfleurage, and steam or water distillation. However, there are several disadvantages associated with these methods, such as high energy consumption and carbon dioxide emission [44]. In order to overcome these challenges and to enhance extraction yield and EOs quality, a novel promising "green" techniques that are non-thermal, cost-efficient, sustainable, and capable of producing EOs with similar or improved characteristics have been developed [42][43][44]. These techniques include supercritical fluid extraction, microwave-assisted extraction, ultrasound-assisted extraction, pressurized liquid extraction, accelerated solvent extraction, microwave steam distillation, and steam diffusion, pulse electric field extraction, enzyme-assisted extraction, molecular distillation, and instantaneous controlled pressure drop processes [42,43,45]. The advantages and disadvantages of the main extraction techniques are summarized in Table 1. Table 1. The advantages and disadvantages of the main EOs extraction techniques [44,45]. EOs are complex mixtures of volatile compounds, comprising two or three major components at relatively high concentrations, i.e., 20-70%, and 20-60 components (or up to 100) in trace amounts of different concentrations. Based on their hydrocarbon skeleton, the major components of EOs are classified into two structural families, namely terpenoids, such as carvacrol and thymol, and phenylpropanoids, such as cinnamaldehyde and eugenol. Based on the number of isoprene units, which are 5-carbon building blocks, terpenoids can be further sub-divided into monoterpenes, sesquiterpenes, and diterpenes, produced by the combination of two, three, or four units, respectively. Moreover, depending on the existence of ring structures, double bonds, oxygen addition, and stereochemistry, there are more than 1000 monoterpenes and 3000 sesquiterpenes. While both classes comprise phenolic compounds, their primary metabolic precursors and biosynthesis pathways are different. Specifically, terpenoids are synthesized via the mevalonate and mevalonate-independent pathways, while phenylpropanoids involve the shikimate pathways [28,38,46]. Furthermore, the volatile compounds found in EOs can be categorized based on their chemical classes, namely phenols, aldehydes, ketones, amines, amides, alcohols, and esters [25].

Extraction Method Advantages Disadvantages
The therapeutic effects of EOs are manifested through the composition and concentration of these compounds. Therefore, they can exhibit a plethora of bioactive properties, including cardioprotective, hepatoprotective, neuroprotective, anxiolytic, antioxidant, anti-inflammatory, anticancer, anti-diabetic, anti-hyperpigmentation, antiviral, antifungal, antibacterial, antibiofilm, and acaricidal ( Figure 1) [26,38,46,47]. activity of EOs are illustrated in Figure 2 [25,42]. Additionally, EO antibacterial character is mostly provided by the presence of phenols, aldehydes, and alcohols within their composition [48].  Moreover, more than 60% of EO derivatives have exhibited varied antifungal effects, which could be of great importance in the food preservation industry [49]. The underlying mechanisms of EO antifungal activity are similar to the previously described antibacterial actions, namely membrane depolarization, proton pumps and ion channels disruptions, ATP pool depletion, and subsequent cell death by apoptotic and necrotic processes [50]. The antifungal bioactivity is generally given by the presence of phenolic compounds, including carvacrol, thymol, α-terpinyl acetate and cymene, terpenes, such as pinene and linalool, and the CHO group of unsaturated aldehydes, when conjugated with carbon in the form of C = C [49,50].
Generally, the antiviral activity of EOs is associated with virion envelopment interfering processes, which avert the entrance within the host cell. However, the underlying mechanisms are not completely understood, and further intensive research is required in the field [50].
Recent years have witnessed an incredible interest in the use of nanometric systems as carriers for EO delivery in antimicrobial applications [39,42]. In addition to offering protection against volatilization, nanomaterials have the advantage of increasing EO bioefficiency, as they are absorbed Studies have shown the highest antibacterial activity for EOs containing carvacrol, eugenol, thymol, cinnamaldehyde, citral and linanaldehyde, and weaker activity for ketones or esters, such as β-myrcene, α-thujone or geranyl acetate. EOs containing terpene hydrocarbons are generally inactive [25,26]. The interactions between these compounds result in either enhanced or reduced antimicrobial efficiency, with four possible effect types, namely indifferent, additive, antagonistic, or synergistic. EOs' antibacterial efficiency also depends on the bacteria type and their biochemical profile and ratio in crude [26,42].
Exhibiting both single and multiple target effects, EOs, owing to their hydrophobic nature, are capable of crossing the lipids constituting bacteria cell membranes and subsequently disrupting cell wall structures. In this manner, the increased membrane permeability causes electron flow, proton driving forces, active transport alterations, and cell content coagulation, and, consequently, ions and cellular materials leakage and cell death. The precise mechanisms involved in the antibacterial activity of EOs are illustrated in Figure 2 [25,42]. Additionally, EO antibacterial character is mostly provided by the presence of phenols, aldehydes, and alcohols within their composition [48]. activity of EOs are illustrated in Figure 2 [25,42]. Additionally, EO antibacterial character is mostly provided by the presence of phenols, aldehydes, and alcohols within their composition [48].  Moreover, more than 60% of EO derivatives have exhibited varied antifungal effects, which could be of great importance in the food preservation industry [49]. The underlying mechanisms of EO antifungal activity are similar to the previously described antibacterial actions, namely membrane depolarization, proton pumps and ion channels disruptions, ATP pool depletion, and subsequent cell Moreover, more than 60% of EO derivatives have exhibited varied antifungal effects, which could be of great importance in the food preservation industry [49]. The underlying mechanisms of EO antifungal activity are similar to the previously described antibacterial actions, namely membrane depolarization, proton pumps and ion channels disruptions, ATP pool depletion, and subsequent cell death by apoptotic and necrotic processes [50]. The antifungal bioactivity is generally given by the presence of phenolic compounds, including carvacrol, thymol, α-terpinyl acetate and cymene, terpenes, such as pinene and linalool, and the CHO group of unsaturated aldehydes, when conjugated with carbon in the form of C = C [49,50].
Generally, the antiviral activity of EOs is associated with virion envelopment interfering processes, which avert the entrance within the host cell. However, the underlying mechanisms are not completely understood, and further intensive research is required in the field [50].
Recent years have witnessed an incredible interest in the use of nanometric systems as carriers for EO delivery in antimicrobial applications [39,42]. In addition to offering protection against volatilization, nanomaterials have the advantage of increasing EO bioefficiency, as they are absorbed by cells and can penetrate membranes and biological barriers. However, as the synthesis of nanomaterials usually requires a heating or solvent evaporation step, the development of EOs and nanomaterials-based systems is still challenging [39]. MNPs have received considerable interest in the production of such systems, with many studies in recent years investigating the antimicrobial activity of MNPs and EOs systems.

Magnetite Nanoparticles-Synthesis, Properties, and Functionalization
Nanoparticles offer remarkable possibilities in terms of their electronic, optical, and magnetic properties, which have the potential to produce improved therapeutics and medical devices [51]. In this regard, inorganic nanoparticles have been widely considered as a platform for the development of tools for targeted drug delivery, clinical diagnostics, and medical imaging. The most common inorganic nanoparticles used for these purposes are iron oxide, silver, gold, zinc oxide, and titanium [52]. Among them, magnetic nanoparticles, the first generation of nanomaterials approved for clinical use, are particularly used owing to their superparamagnetic properties, which have increased the possibilities for developing novel and efficient biomedical applications [52][53][54], such as targeted drug and gene delivery, magnetic resonance imaging, biosensors, cancer detection and treatment, diagnosis and magnetic field-assisted radiotherapy, and tissue engineering [55]. The common types of iron oxide nanoparticles, which belong to the ferrimagnetic class of magnetic nanomaterials, are magnetite (Fe 3 O 4 ), maghemite (γ-Fe 2 O 3 ), hematite (α-Fe 2 O 3 ), and mixed ferrites (MFe 2 O 4 , where M = Co, Mn, Ni or Zn) [52,53,56,57].

Synthesis Methods
As their behavior strongly depends on their size, shape, and surface chemistry, MNP synthesis methods have been a hot research topic ever since their discovery. Moreover, any variation in the preparation process could subsequently lead to considerable changes in the final product. Therefore, considering the desired features and the field of application, there are three main routes for MNPs synthesis, namely physical, chemical, and biological methods ( Figure 3) [55,[58][59][60][61].
the preparation of MNPs [69,71]. Through this method, MNPs are formed by the decomposition of iron precursors, such as acetylacetonates, acetates, or oleates, in high-boiling organic solvents containing surfactants and stabilizing agents, such as oleic acid and oleylamine. Moreover, the thermally unstable metal complexes are injected into the hot solution in order to generate a nucleation event followed by the controlled growth process. This allows for modifications of the synthesis parameters for tuning the size and shape of the nanoparticles [69,72].

Physical Methods
Physical methods for the preparation of MNPs follow a top-down approach in which bulk materials or large particles are broken into nanoscale particles. While such methods are preferred for the large-scale production of nanoparticles, there are major setbacks regarding uncontrolled particle size, non-uniform size distribution, and time-consuming and expensive technologies [31,62]. There is a variety of physical methods for synthesizing MNPs, including ball milling and electron beam lithography as the most commonly used, and others, such as laser ablation, sputtering, gas-phase deposition and aerosol spray pyrolysis [31,62,63]. On the one hand, the principle of ball milling, a solid-state synthesis method, is based on the generation of frictional force through the collision between the surfaces of the reactants, which causes an increase in temperature, pressure, and internal energy [64]. While this method is categorized into low energy and high energy milling, the latter is mostly preferred for the synthesis of nanoparticles [65]. On the other hand, electron beam lithography represents the conversion of high-purity bulk iron materials or films, which are deposited onto a substrate, to MNPs by emitting an electron beam across the material. Specifically, the electrons are focused and accelerated toward the materials, generating electron-electron collisions in the interaction volume of the incident electrons and modifying the chemical properties of the resistant layer. The initial iron precursors evaporate, and MNPs are subsequently formed [62,66].

Chemical Methods
Chemical methods are the most commonly used, as they follow the bottom-up approaches, which are more efficient, easier to employ, and time and cost-efficient, and provide a narrower size particle distribution [59,67,68]. There are multiple chemical synthesis routes, and the most common are co-precipitation, thermal decomposition, sol-gel, and microemulsion [31,67,68].
The co-precipitation method is one of the most popular methods for MNPs synthesis, owing to its non-toxic nature, cost-efficiency, and mild reaction conditions [59,69]. It is based on the phenomenon through which a mixture of iron Fe 2+ and Fe 3+ salts in the form of nitrates, perchlorates, sulfates, and chlorides will precipitate at the addition of a base, such as ammonia, sodium, or potassium hydroxide, to form nanoparticles by nucleation and grain growth [59,68,70]. The use of polymers or surfactants is necessary to control particle size [70]. Furthermore, the size, shape, and magnetic properties of MNPs are influenced by the types and concentration of the salts, the pH and temperature of the medium, ionic strength, and mixing rate [59,70]. However, as MNPs obtained through co-precipitation have irregular shapes, wide size distribution, and low crystallinity, high-temperature synthesis methods are necessary to prepare high-quality MNPs [69,70].
In this manner, thermal decomposition is rapidly becoming one of the most common routes for the preparation of MNPs [69,71]. Through this method, MNPs are formed by the decomposition of iron precursors, such as acetylacetonates, acetates, or oleates, in high-boiling organic solvents containing surfactants and stabilizing agents, such as oleic acid and oleylamine. Moreover, the thermally unstable metal complexes are injected into the hot solution in order to generate a nucleation event followed by the controlled growth process. This allows for modifications of the synthesis parameters for tuning the size and shape of the nanoparticles [69,72].
The sol-gel method involves an initial solution of precursors, which are usually inorganic metal salts or metal-organic compounds in an adequate solvent. The addition of a surfactant will generate a polymerization reaction that will form the "sol", which is a colloidal suspension of dispersed particles. Subsequently, a chemical treatment that will disable the surfactant will lead to the development of an extended network of linked particles throughout the solution, which will form the gel. The evaporation of the solvent will lead to the obtaining of MNPs [73]. While the sol-gel method is advantageous owing to its low cost, and the good homogeneity and high purity of the so-obtained nanoparticles, further heat treatments are required to reach a final crystalline state [69].
The microemulsion method implies the preparation of a microemulsion in an aqueous phase with ferric acid and/or ferrous ions, which will be precipitated through the injection of organic precipitating agents, such as cyclohexylamine or oleylamine [74]. While it offers the advantages of controllable size and shape, it also includes disadvantages related to aggregation and stability issues of the resulted MNPs [74,75].

Biological Methods
Biological methods involve green biosynthesis processes through bottom-up approaches. Specifically, metal precursors generate metal atoms that will assemble into nanoparticles. As the biological compounds present within the green substrates act as both reducing and capping agents, the final size, shape, and dispersibility can be controlled through a series of parameters, such as temperature, pressure, pH, and time of incubation [62]. The process involves the formation of small nucleation centers that grow through the sequestration of more metal ions around the nucleation site. Since they have high surface energies, the capping of the nanoparticles is necessary to prevent agglomeration and stabilize them [76]. These methods involve the use of bacteria, algae, fungi, and higher plants [31,62]. While biological methods are the most cost-effective and eco-friendly and generate the most biocompatible nanoparticles, publications within the field are still lacking [31].
Bacteria-based methods involve the use of magnetotactic and iron, reducing bacteria to produce MNPs intracellularly or extracellularly under anaerobic conditions. However, such methods usually necessitate further treatments, including calcination, sonication, and detergent usage, in order to eliminate the excess of capping biomolecules. By contrast, plant-based methods involve the use of various substrates, including callus, leaf, fruit, or seed extracts [62]. Plant-mediated MNPs synthesis represents the most efficient method for large-scale production in a short time. Additionally, waste products are easy to dispose of in the environment as they mostly comprise plant biomaterials [76].

Properties
Magnetite possesses unique physicochemical properties (Table 2), which further determine their interactions with biological tissues and, subsequently, their applicability in the biomedical field. The size and shape of MNPs are the key characteristics that influence their pharmacokinetics, biodistribution, body clearance, and toxicity [78]. Based on their size, MNPs are classified into superparamagnetic iron oxide nanoparticles, ultrasmall superparamagnetic iron oxide nanoparticles, and a subset size of monocrystalline iron oxide nanoparticles [79,80]. Thus, size plays a fundamental role in the magnetic properties of MNPs and their subsequent response to magnetic fields. Specifically, as size decreases, the saturation magnetization decreases, and the nanoparticles exhibit supermagnetism [78]. Moreover, when applied in biomedicine, the size of MNPs should be <200 nm to avoid rapid spleen and liver filtration and to prolong blood circulation time, but >10 nm to avoid rapid kidney filtration [81]. Additionally, their size should be designed based on the type of biological entities they are intended for to promote cellular uptake, namely viruses (20-450 nm), proteins (5-50 nm), and cells (10-100 µm) [82]. MNPs have been synthesized in a variety of shapes, including nanospheres, nanocubes, nanowires, nanotubes, nanoplates, nanohexagons, nanooctahedrons, nanorods, nanorings, nanocapsules, and nanoflowers [81]. Their shape is influenced by the synthesis parameters, such as reactant concentration, reaction temperature, aging time, and interactions between the surface of the nanoparticles and the surfactants and/or capping agents [78].
Magnetite is a common iron oxide with a cubic inverse spinel structure and an Fd3m space group with a cell parameter of 8.394 Å. The 32 O 2− ions form ab fcc (face-centered cubic) closed packing structure, with Fe 2+ ions disposed of in the octahedral sites, and Fe 3+ ions disposed of both in the octahedral and tetrahedral sites [83][84][85]. The presence of the four unpaired electrons in the three-dimensional orbital of iron ions determines MNPs' magnetic properties, which are measured through magnetization saturation, coercivity, and the magnetocrystalline anisotropy constant. Additionally, MNPs exhibit various individualities, such as superparamagnetism, saturation field, additional anisotropy contributions, and shifted loops subsequent to field cooling. Notably, superparamagnetism is the most important characteristic of MNPs, which is associated with particles of less than 20 nm in size. Such small and finite size, and surface effects, dominate the magnetic properties of MNPs, allowing for the free rotation of the particle in response to the magnetic field applied [70,86]. The interaction between MNPs and an external magnetic field will determine two processes, namely the orientation of the particle's magnetic moment in a parallel direction to the magnetic field applied in order to minimize energy and bipolar interactions, and the transition of the particle in the gradient direction, as in magnetophoresis [77]. After removing the magnetic field, MNPs act with single-domain ferromagnetic properties and do not retain their magnetism, which consequently prevents physical aggregation. Size, shape, crystal lattice structure and size distribution directly influence the magnetic properties and behavior of MNPs [70].
When introduced into the organism, MNPs follow a series of steps, namely magnetic guidance to the target tissue or organ, immobilization and drug release through the external magnetic field, and standard clearance [78]. MNPs are a unique class of nanomaterials with well-proven superior biocompatibility in their native form that forms the basis for their widespread applicability in the biomedical field. By contrast to other biocompatible nanomaterials that are chemically and biologically inert, MNPs are catalytically active, exhibiting important physiological effects [87]. The cellular uptake of MNPs occurs through endocytosis, where they tend to accumulate in lysosomes and subsequently be degraded to iron ions. The toxicity of MNPs has been shown to be influenced by a series of factors, such as size, shape, configuration, and surface charge. Specifically, nanosized rod-shaped particles proved to be more toxic than microsized sphere-shaped particles. Furthermore, the toxicity of positively charged MNPs is increased as they undergo non-specific interactions and adsorptive endocytosis with negatively charged cell membranes [88]. An important aspect of the biological interactions of MNPs is microbial toxicity, which is due to a series of mechanisms, such as membrane depolarization and consequent cell integrity impairments, the release of iron ions that reach the nucleus and mitochondria and affect cellular homeostasis and protein coordination, the production of reactive oxygen species with lipid peroxidation, and DNA damage [88,89].

Functionalization
Simple MNPs are associated with issues related to their stability in colloidal solutions and biological fluids. First, as they are usually hydrophobic, positively charged, and have high surface energy, there is a high tendency to form large agglomerates through van der Waals and magnetic forces. Moreover, these agglomerates induce plasma protein adsorption, forming a protein corona on the surface that will favor rapid opsonization by the reticuloendothelial system or retention by the macrophages, and will reduce blood circulation half-life [70,77,78]. Second, their administration is associated with an increased risk of causing thrombosis and clots due to the occlusive effect of these agglomerates, which will hinder normal blood flow through the vessels [70].
In this regard, the surface modification of MNPs is a fundamental process for avoiding agglomeration and oxidation phenomena, such as Ostwald ripening, and improving stability and compatibility by providing interfacial electrostatic or steric repulsion forces between particles [77,78]. Additionally, surface functionalization enables the specific responses of nanoparticles to biological species and eliminates non-specific interactions with components within the system [90].

Antimicrobial Range of Essential Oils-Magnetite Nanosystems
Bacteria are widespread microorganisms generally grouped into genera and species depending on their morphological, biochemical, physiological, and genetic features. Specifically, they are categorized based on their shape, color, presence of flagella, growth ability onto certain media, and Gram staining [92]. Among them, Gram staining provides one of the most important classification systems, developed by Christian Gram in 1884. The approach relied on the cell envelope phenotypic characteristics, as some bacteria retain a crystal violet dye after ethanol discoloring, while others do not [93,94]. Thus, according to the Gram staining test, the bacteria which remain blue are called Gram-positive, while those that appear red are called Gram-negative. Such differences reflect variations in the cell wall structure [94]. With limited exceptions, Gram-positive bacteria possess a relatively simple cell wall of about 20-80 nm, comprising a single membrane surrounded by a thick layer of 30-200 molecules of peptidoglycans [93][94][95]. Additionally, the cell wall of Gram-positive bacteria contains water-soluble polymers of polyol phosphates, namely teichoic acids and lipoteichoic acids [94]. By contrast, Gram-negative bacteria have a more complex cell wall comprising a thin inner layer of less than 10 nm surrounded by a layer of one or two molecules of peptidoglycans, and an outer membrane containing the endotoxin lipopolysaccharide and a pore-containing structure [93][94][95]. Moreover, the space between the two membranes, known as the periplasm, contains a series of degradative enzymes and transport proteins [94]. The Gram staining classification is critically important as the characteristics of the cell envelope are associated with several cell properties, especially related to its response to outer stresses, such as ultraviolet radiation, heat or antibiotics [93].
Gram-positive bacteria are the most common causes of clinical infections, with approximately 60% arising from respiratory, intra-abdominal and urinary tract sources [96,97]. Including the Staphylococcus, Streptococcus and Enterococcus genera, Gram-positive bacteria are associated with diverse pathology spectra, varying from mild soft tissue infections to life-threatening systemic sepsis and meningitis [97,98].
Staphylococcus aureus is a Gram-positive bacterium that colonizes a considerable part of the human population, with 30% as a persistent carrier and 30% as an intermittent carrier, due to the abundant secreted and surface proteins that promote both colonization and immune response evasion. Specifically, tissue adhesion and host cell invasion occur through surface proteins, while immune response evasion and tissue damage are possible through the action of extracellular enzymes and zymogen activators. As most strains carry pathogenicity islands, prophages, transposons and insertion sequences, Staphylococcus aureus has the potential to cause various complications, from benign skin infections to osteomyelitis, endocarditis and septicemia [99,100]. Anghel  The uniform thin coating led to an increased surface biocompatibility, which allowed for a normal endothelial cell development up to 5 days and a significantly reduced Staphylococcus aureus colonization, with four-fold inhibition for incipient biofilms and three-fold for mature biofilms [104]. Furthermore, Negut et al. deposited Nigella sativa EO-functionalized MNPs and Nigella sativa EO-functionalized MNPs mixed with a polymeric matrix of poly(lactic-co-glycolic acid) on glass and silicone surfaces via the MAPLE technique. The results showed enhanced antimicrobial colonization and antibiofilm formation activities, and biocompatibility towards MG-63 cells, proving the potential use of such systems in the development of multifunctional antibiofilm medical surfaces [105]. Nutmeg EO was used by Mousavi et al. for improving MNPs saturation magnetization and antibacterial properties. Compared to the nutmeg ethanolic and methanolic essence, nutmeg EO-functionalized MNPs were shown to have superior antibacterial efficiency. Additionally, nutmeg EO-functionalized MNPs synthesized through a green method enhanced antimicrobial activity when compared to chemically synthesized MNPs [106]. Additionally, Grumezescu et al. developed eugenol-functionalized MNPs embedded into poly(3-hidroxybutyric acid-co-3-hidroxyvaleric acid)-polyvinyl alcohol microspheres through an oil-in-water emulsion technique. The microspheres were transferred through the MAPLE technique for obtaining thin antibacterial coatings. The microbiology tests demonstrated the inhibition of microbial adherence and biofilm development for Staphylococcus aureus at three time-points [107]. Similarly, Anghel et al. compared the microbicidal and anti-adherent properties of MNPs functionalized with eugenol and limonene. The results showed no significant differences when tested on Staphylococcus aureus strains, with the most important decrease in viable biofilm cells observed at 72 h, thus proving the ability of the nanosystem to combat the volatility of EOs when used for antimicrobial applications [108].
Enterococci are one of the main causes of nosocomial infections, which are responsible for both endemic and epidemic healthcare-associated infections [109,110]. Enterococcus faecalis is a Gram-positive coccus and facultative anaerobe, commonly found as a human intestinal commensal. Similar to other enterococci, Enterococcus faecalis is capable of producing oxygen-free radicals within the host, consequently causing chronic inflammation and colorectal cancer. Additionally, it has a high potential for spreading virulence and antibiotic-resistant genes through horizontal gene transfer [110,111]. The previously mentioned nutmeg EO-functionalized MNPs systems developed by Mousavi et al. exhibited a significant antibacterial character against Enterococcus faecalis strains [106].
Considering that Gram-negative bacteria exhibit increased levels of resistance towards most antibiotic classes, the infections caused by these microorganisms imply continuously increasing costs, morbidity, and mortality. Among them, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae are the most common causes of health problems in hospitalized patients [112].
Escherichia coli is a remarkably diverse bacterial species, which is nearly ubiquitously found in the human gastrointestinal tract, despite accounting for a reduced portion of the gut microbiota. Although it does not generally compromise host health, virulence traits expressing strains can cause various diseases, such as urinary tract infections, through multiple mechanisms [113,114]. The thin coatings based on Cinnamomum verum EO-functionalized MNPs developed by Anghel et al. resulted in a slightly reduced Escherichia coli biofilm inhibition, ranging from 2.5-fold for initial biofilms and 2-fold for mature biofilms, which could demonstrate enhanced antimicrobial effects against Gram-positive bacteria [104]. Additionally, the Nigella sativa EO-functionalized MNPs and Nigella sativa EO-functionalized MNPs mixed with a polymeric matrix of poly(lactic-co-glycolic acid) exhibited similar results towards Escherichia coli biofilm, proving the potential of these systems for the prevention and/or treatment of Gram-negative bacteria infections [105]. The growth of Escherichia coli bacteria was also effectively inhibited in the presence of nutmeg EO-functionalized MNPs [106].
Pseudomonas aeruginosa, a Gram-negative bacillus, is an opportunistic pathogen ubiquitously found in nature, particularly in soil and water, characterized by a versatility that allows for its persistent survival under severe conditions [115,116]. While it does not usually cause diseases within healthy hosts, it is a major concern for patients with underlying immunocompromising conditions as it causes serious infections in the respiratory tract [115,117]. As its genome has provided knowledge of the mechanisms underlying the metabolism and pathology of this species, it has become the model for understanding microbial variations at the genome level and chronic disease evolution [115]. Tests on nutmeg EO-functionalized MNPs showed an efficiently inhibited growth of Pseudomonas aeruginosa bacterial strains [106]. The functionalization of MNPs with eugenol [107,108] and limonene [108] led to increased inhibiting effects on Pseudomonas aeruginosa biofilm formation and development. In the case of eugenol functionalization performed by Grumezescu et al., although the biofilm formation was significantly impaired in the first stages of development, the effect of the nanosystem decreased over time. This behavior could be associated with the greater antimicrobial effects against Gram-positive than Gram-negative strains of eugenol [107]. However, the results published by Anghel et al. stated no significant differences between Gram-positive and Gram-negative bacteria but demonstrated a higher antibiofilm inhibition when compared to limonene [108].
Klebsiella pneumoniae, an encapsulated Gram-negative bacterial strain, is widely found in the mouth, skin, and intestinal microbiota, but also in natural environments [118]. As it can cause both nosocomial and community-acquired pneumonia, Klebsiella pneumoniae is associated with high mortality rates in the bacteremia setting, mostly in the neonatal and infant populations [119]. The nanosystems developed by the authors of [103] proved no significant differences between patchouli, vanilla, and ylang-ylang EOs, exhibiting bioactivity mostly against Klebsiella pneumoniae adherence.
Saviuc et al. synthesized a core-shell system comprising MNPs and Anethum graveolens EOs. This method aids in the stabilization of EOs, further enhancing their antibiofilm activity against fungal adhesion on an inert substratum of the Candida albicans, Candida tropicalis, Candida famata, Candida glabrata, and Candida krusei. Results showed a decreased fungal adherence after 24 h of incubation, which increased after 48 h when compared to the untreated substratum [123]. Moreover, Grumezescu et al. used MNPs/oleic acid core-shell nanostructures for the stabilization of Eugenia carryophyllata EO on catheter surfaces. The use of the hybrid system based on eugenol and cariophylene-rich EOs significantly decreased biofilm development when compared to surfaces only treated with MNPs or EOs for Candida albicans, Candida glabrata, and Candida krusei. In some cases, biofilm development was totally inhibited after 24 h incubation [124]. Nanocoated wound dressings were obtained by Anghel et al. by submerging dressing pieces into nanofluids containing MNPs and Satureja hortensis EO. Scanning electron microscopy revealed the strong inhibition of Candida albicans adherence and consequent biofilm formation when compared to uncoated materials. Additionally, biofilm development was impaired both in early and mature phases, as quantified at 24, 48, and 72 h, which could be explained by the presence of phenolic compounds within the EO [125]. Similar results were also obtained in the case of wound dressings coated with MNPs functionalized with Anethum graveolens and Salvia officinalis EOs. Additionally, it was observed that the maximum intensity of inhibition was reached on the 48 h and 72 h biofilms, which could be explained by the different release rates [126]. Furthermore, Chifiriuc et al. investigated the effects of Rosmarinus officinalis EO-functionalized MNPs against the Candida albicans and Candida tropicalis clinical strains. The results showed considerably higher inhibitory effects for nanosystem-treated catheters, with yeast cells practically absent at 48 and 72 h for both strains [127]. Antifungal adherence and antibiofilm properties were also observed when using the Cinnamomum verum EO-functionalized MNPs developed by Anghel et al. [104], and the Nigella sativa EO-functionalized MNPs and Nigella sativa EO-functionalized MNPs mixed with a polymeric matrix of poly(lactic-co-glycolic acid) systems developed by Negut et al. [105]. Table 3 summarizes all the nanosystems based on MNPs and EOs that have been described above for antimicrobial therapies.   Candida parapsilosis MNPs Functionalized with Cinnamomum Verum EO n.r. 9-10 nm n.r. Gastrostomy Tube Coating [104] Where, n.r.-not related; "-" not applicable.

Conclusions and Future Perspectives
Recent studies have focused on the discovery of non-antibiotic antimicrobial agents that could attack multiple molecular targets, such as by blocking DNA replication, altering gene expression, denaturing enzymes, inducing reactive oxygen species, or damaging microbial membranes. Owing to the increasing evidence of their antibacterial, antiviral, antifungal, and insecticidal effects, EOs have attracted tremendous interest in recent years for a variety of applications in the fields of medicine, pharmaceutics, and cosmetics. Additionally, the hydrophobic nature of EOs allows for crossing the lipids constituting bacteria cell membranes and subsequently disrupting cell wall structures, thus inducing promising antibacterial effects. However, as environmental factors might cause EO degradation and/or loss of bioactivity due to volatilization, there has been increasing interest in the use of nanometric systems as carriers for EOs delivery. As they are highly advantageous, owing to their biocompatibility, biodegradability, non-toxicity, ability to specifically target tissue, and primary structures that allow for the attachment of various therapeutics, MNPs represent a potential candidate for the development of EO-based nanosystems for antibacterial therapies. In this manner, promising solutions that could overcome the challenges associated with biofilm formation on prosthetic devices and antibiotic-resistant bacteria could be developed. While the current state of research focuses mainly on the antibacterial and antifungal purposes of such nanosystems, future works should also focus more on developing antiviral and antiparasitical applications, as each individual component has proven to be efficient.

Conflicts of Interest:
The authors declare no conflict of interest.