As a form of adaptability and evolution, bacteria managed to establish a well-organized behavior into a very efficient assembly, called biofilm. Bacterial biofilm formation is the prevailing microbial lifestyle in natural and man-made environments and occurs on all surface types, including biological surfaces; it can be defined as a community of microorganisms irreversibly attached to a surface, producing extracellular polymeric substances, exhibiting an altered phenotype compared with corresponding planktonic cells, and interacting with each other [4,5]. One of the most significant clinical aspects is the fact that bacterial biofilms cause chronic infections because they disclose increased tolerance to antibiotics and disinfectants, as well as resisting phagocytosis and other components of the body’s defense system [6]. Approximately, 80% of all human infections are associated with biofilms, and evidence for their role in an ever-growing number of cutaneous disorders is constantly unfolding [7].

In the recent years, researchers aimed to find alternative methods of dealing with infections with biofilm-embedded bacteria, knowing that adherent microbial cells exhibit high antibiotic resistance. One of the most efficient strategies is to interfere with bacterial adherence, the first step in the biofilm formation, by direct blockage of surface receptors [8] or using a non-specific strategy, usually involving compounds with anti-adherence properties [9-11]. Another efficient strategy seems to be the one involving the manipulation of communication processes between bacteria into the biofilm, using different natural or artificially synthetized compounds [12-14]. Bearing in mind that chemically synthetized compounds may be toxic and have usually unpredictable long-term effect on the mammalian host cell, natural compounds exhibiting anti-microbial activity are considered as a more preferred alternative [15,16]. Essential vegetal oils are natural compounds that have proved to be highly efficient as antimicrobial agents, demonstrating significant anti-adherence and anti-biofilm properties [17,18]. However, the use of essential oils can be limited by their high volatility and low stability [19].

Magnetic iron oxide nanoparticles have appeared as a well-established technology and an important research field, mainly because of their superparamagnetism properties that allow to be guided with an external magnetic field, [20,21]. Potential applications in the field of biotechnology and nanomedicine such as biomagnetic separations [22], biosensors [23], carriers for targeted drug delivery [24-28], hyperthermia-producing systems [29], inhibition of biofilm development [30,31], stabilization of essential oils [32], and contrast agents in magnetic resonance imaging [33,34] have been proposed. The material surface chemistry and the electronic configuration of the surface complexes have major influences on the reactivity and properties [35].

In our previous papers, we have demonstrated by scanning electron microscopy the ability of Fe₃O₄@C₁₈ to prevent the fungal adherence of Candida albicans on optimized textile dressing samples coated with functionalized magnetite nanoparticles, as compared to uncoated materials [36]. These functionalized Fe₃O₄@C₁₈ nanoparticles exhibited also the ability to stabilize, limit the volatilization, and potentiate the fungicidal effect of Salvia officinalis essential oil [43]. On the other hand, limonene and eugenol, the major compounds of essential oils extracted from Anethum graveolens (56.53%) and Eugenia caryophyllata (92.45%) proved, to exhibit very good antimicrobial properties [28,44]. In this paper, we report the successful fabrication of two phyto-nanofluids for coating textile wound dressings, based on limonene and eugenol loaded in magnetic nanoparticles, in order to increase their microbicidal and anti-biofilm properties and, thus, combat the cutaneous opportunistic infections.

The obtained nanostructure was characterized by XRD as illustrated in Figure ​Figure2,2, and the results showed that the diffraction patterns and the relative intensities of all diffraction peaks match well with magnetite (based on ICDD 82–1533). Also, the sample has the characteristics of bulk magnetite crystallite phase, and the broad peaks suggest the nanocrystallite nature of magnetite particles [45,46], the average crystallite size being 10.58 nm (based on Scherrer formula). FT-IR spectrum of the nanostructure exhibits a characteristic broad peak of magnetite at about 533 cm⁻¹ (Fe-O stretching) [47]. The FT-IR analysis also identified the organic coating on the surface of the magnetite nanoparticles (Figure ​(Figure3).3). The peaks recorded at about 1,572 and 1,701 cm⁻¹ at FT-IR spectrum of the nanostructure can be assigned to structures of the type COO⁻M⁺. The peaks at 2,915 and 2,848 cm⁻¹ were assigned to stretching vibration of C-H (Figure ​(Figure3).3). The nanostructure diameter was approximated from the TEM images (as presented in Figure ​Figure4),4), showing that the particles are spherical with an average size of 10 nm which, corroborated with the XRD data, means that the obtained nanoparticles are formed by only one crystallite. The presence of essential oils induces a strong modification of the thermal behavior of the two nanostructured materials (Figure ​(Figure5).5). In the case of phyto-E-nanostructurated material, the weight loss increases with about 4.6%, which can be mainly attributed to the eugenol adsorption onto the nanomaterial. The weight loss was surprisingly affected in the phyto-L-nanostructurated material, where the weight loss became even lower than that corresponding to Fe₃O₄@C₁₆. We explain this anomaly by the fact that limonene and C₁₆ interact by special hydrophobic interactions, and the complex may be partially lost during the drying step.

Due to their widespread, easy manipulation, and low side effects, direct contact wound absorptive natural-based plasters are preferred for wound dressing. Specialized literature reports few studies aimed to improve the quality and antibacterial properties of natural or artificial materials used for wound dressing and covering, but the proposed techniques are mainly based on using artificial, new chemically synthetized compounds [16,17].

Essential oils represent an alternative for treating microbial infections because they are natural vegetal compounds with lower or no side effects for the host compared with artificially synthetized antimicrobial compounds, representing one of the ecological anti-infectious strategies. However, their effects can be impaired by their great volatility, highlighting the necessity of novel vectoring stabilizing systems. In the recent years, the usage of nanosystems for clinical issues has emerged, mainly because of their reduced structures and their proved characteristics, as antimicrobial activity. Even though nanosystems are considered a novel challenge for medicine, their usage is largely restricted because of their unknown long term effects and sometimes because of their toxicity on eukaryotic cells. During this study, we have investigated the possibility of improving the antimicrobial activity of wound dressings by modifying their surface using a nanofluid to assure the stability and controlled release of some volatile organic compounds isolated from essential oils. Our results obtained on two in vitro monospecific bacterial biofilm models involving cotton-based wound dressers layered with a phyto-nanostructured coating demonstrated that the functionalized textile materials exhibited antimicrobial effects on wound-related pathogens.

VCCs assessed from mechanically detached biofilm bacteria revealed a slightly different ability of the two modified wound dressings. The results revealed that the nanofluid coating containing L affected both the initial stage of biofilm formation and the development of a mature biofilm, as demonstrated by the lower VCCs obtained at the three harvesting time intervals (i.e., 24 h, 48 h, and 72 h), as comparing with control, uncoated textile materials (P<0.0001). Even though P. aeruginosa ATCC 27853 grew better, the differences between S. aureus and P. aeruginosa VCC values were not significantly different. The nanofluid exhibiting comparative antibiofilm effects in both models (Figure ​(Figure5)5) induced a significantly reduced biofilm development expressed as viable cells in time (P<0.05). The phyto-E-nano-modified wound dressing model has proved to have also a significant antibiofilm activity, determining a pronounced biofilm inhibition on both S. aureus (Figure ​(Figure6)6) and P. aeruginosa (Figure ​(Figure7)7) models at all three tested time points (P<0.0001). The effect of this system seems to be more pronounced on adherence and initial biofilm formation compared to the L-based one, in case of P. aeruginosa.