In recent years, special attention has been paid to the electrospinning process as a simple method of making nanofibre-based structures. Due to the high porosity and area/volume ratios of nanofibres, different active substances with specific properties and with applications in food packaging [1
], medicine [2
], pharmaceutical drugs [3
], fibres based electronics [4
] or water treatment [5
] can be included.
Electrospinning is a versatile technique for obtaining nano- or micrometric fibre-based mats with variable properties by applying high voltages between the tip of the injector needle (+) and a support fixed to a metal collector (−) on which they are deposited. In general, the electrospinning process involves a polymer that generates the carrier matrix consisting of continuous nanofibres, in which the presence of a network of high molecular weight facilitates the formation of a stable jet during electrospinning. However, in the last decade research has been made on electrospinning of polymer-free materials with functional molecules for other new applications, such as phospholipids [6
], ammonium gemini surfactant [7
], cyclodextrin derivatives [8
] and tannic acid [9
Nanofibre membranes made by electrospinning take precedence over other materials due to their high porosity and interconnectivity [10
]. This structure permits the introduction of active substances giving the possibility for further functionalization [11
Collagen is a biopolymer with regenerative and tissue reconstruction properties used in various treatments in medicine [12
], drugs [13
] or urinary drains [14
]. In addition, collagen is used as food supplements [15
] and antimicrobial emulsion formulations for cosmetic purposes [16
Recently, the use of collagen loaded with bioactive compounds derived from plants for design of natural wound dressings classified as sponges, electrospun nanofibre matrices, films and hydrogels was investigated to overcome the toxicity and reduced antimicrobial activity of existing products [17
]. In the present paper, collagen hydrolysate was used in high concentration due to the associative properties of collagen hydroysate molecules with effect on increased viscosity and improved electrospinnable properties. As compared to gelatin, collagen hydrolysate represents a more versatile, cheaper and available biomaterial. The combination of concentrated collagen hydrolysate with essential oils for new wound dressing manufacture is an original approach for skin wound healing by cell proliferation stimulation and antimicrobial protection. The use of essential oils represents a natural alternative for synthesis antimicrobials with pathogen resistance potential for new non active wound dressings design.
Plant essential oils are gaining a wide interest in medicine, agriculture and food industry due to their antibacterial, antifungal, antioxidant and anti-inflammatory properties [18
]. The beneficial effect of essential oils to promote wound healing was extensively described [19
]. The antimicrobial activity of plant essential oils is attributed to their chemical structure, in particular the presence of hydrophilic functional groups, such as hydroxyl groups of phenolic components and/or lipophilicity of some essential oil components [20
]. To overcome the essential oils volatility, different formulation techniques and methods have been developed, such as casting [21
], emulsion ionic gelation [22
] or encapsulation by electrospinning [23
Although many studies have demonstrated the antimicrobial effect of essential oils and their bioactive compounds against a broad spectrum of pathogenic bacteria in food [24
] and pharmaceutical applications [20
], there are no publications that discuss their incorporation into high concentrated collagen hydrolysate by electrospinning.
Peiwen and Mele [25
] prepared electrospun mats based on clary sage and black pepper essential oil combined with polylactic acid (PLA) dissolved in acetone solvent showing antimicrobial properties for biomedical applications as dressings.
In another paper, cinnamaldehyde essential oil was incorporated inside chitosan/polyethylene oxide (PEO) nanofibre mats, without the use of a surfactant, to obtain new flexible scaffolds with antimicrobial properties against nosocomial infections [26
]. The encapsulation of oregano alcoholic extract in Eudragit E100 (cationic copolymer based on dimethylaminoethyl methacrylate, buthylacrylate and methyl methacrylate) was performed and showed that the active substance delivery from electrospun mats was highly influenced by the polymer concentration [27
The novelty of this paper consists in the successful preparation of collagen hydrolysate-based nanofibres loaded with essential oils, with potential use for natural wound dressings. The investigations on antioxidant and antimicrobial activities and the biocompatibility of electrospun collagen loaded with thyme or oregano essential oils showed the potential of new biomaterials as friendly alternative to similar materials based on more expensive native collagen, synthesis polymers, organic solvents and pathogen resistant antibiotics.
Commercially available antimicrobial dressings used to control the bacterial infections include antimicrobial agents such as antibiotics, quaternary ammonium salts, metal oxide nanoparticles and metal salt solutions [55
]. Bioactive wound dressings have been developed to overcome toxic nature and reduced antimicrobial activity of classical wound dressings due to the antibiotics resistant pathogens.
In this context, nanofibre mats based on thyme or oregano essential oils loaded collagen hydrolysate with low cytotoxicity, antioxidant and antimicrobial properties were prepared by electrospinning. Electrospinning is a process that involves a high voltage supply, a syringe pump, a capillary tube with a needle and a manifold. For this, a polymer solution is pumped through a syringe; at the same time, a high voltage is applied to the spinneret to attract the drop to the collector. The main process parameters that influence the production of nanofibres are the feed rate, the applied voltage, the distance from the collector to the needle tip, the type of spinneret, the polymer and solvent properties [56
]. Typical electrospinning parameters using collagen as a biopolymer were 0.3 mL·h−1
to 1 mL·h−1
for feed flow, voltage 10–25 kV and 7.5–13 cm for needle to collector spacing. However, the most common values were 0.7 mL·h−1
, 19 kV and 13 cm for the feed flow, voltage and distance between the needle tip and the collector, respectively. The mixture of gelatin/PVA/keratin [57
] was electrospun using 20 kV voltage, at 10 cm tip-to-collector distance and with a feed rate of 0.1 mL·h−1
. In our research, we have exploited the molecule association property of collagen hydrolysate and the increase of polydispersion viscosity with concentration. We found that the viscosity of collagen hydrolysate increases 1082 times with increasing concentration 6.5 times. After many trials, we found that the collagen hydrolysate at the concentration of 60% in water, with and without 10% essential oils can be successfully electrospun by using typical parameters: the voltage of 10–25 kV, the distance of 7.5–13 cm from needle to collector spacing and the feed flow of 0.7 mL·h−1
. Concentrated collagen hydrolysate in water represents an alternative to the use of more expensive native collagen or gelatin, usually solved in 1,1,1,3,3,3 hexafluoro-2-propanol (HFP), for tissue engineering applications. The use of acetone, ethylic alcohol as solvents or synthesis tensides for essential oils emulsifying in different polymer mixtures [25
], glutaraldehyde for collagen nanofibres crosslinking [57
] represents also more toxicological approaches as compared to proposed electrospinning process. The combination of concentrated collagen hydrolysate with essential oils for new wound dressing manufacture is an original and efficient approach for skin wound healing by cell proliferation stimulation and antimicrobial protection. Essential oils represent a natural alternative for synthesis antimicrobials with pathogen resistance potential for new non active wound dressings design.
The composite formulations based on natural materials like collagen hydrolysate and essential oils represent sustainable alternatives for more expensive native collagen, synthesis solvents, polymers, surfactants and antimicrobials with pathogen resistance and inflammatory potential [59
Due to the associative properties of collagen hydrolysate, the viscosity of water polydispersion increased from 1.5 cP, the viscosity of 10% concentration polydispersion (resulted after the alkaline-enzymatic hydrolyses) to 1623 cP, the viscosity of 60% concentrated collagen hydrolysate, with improved electrospinnable properties. The essential oil emulsion formation was assisted by collagen hydrolysate tenside properties [28
], which were shown to be superior to native collagen or aminoacids, with improved biocompatility as compared to synthesis surfactants [33
The efficiency of essential oils loading was found to be 29% in the case of collagen nanofibres loaded with thyme essential oil and 39% in the case of collagen nanofibres loaded with oregano essential oil due to electrospinning conditions and essential oils characteristics. Similar encapsulation yields (21–29%) were reported for carvacrol included in starch or PCL matrices [60
] or orange essential oil loaded gelatin (35–69%) [61
], as a function of essential oil concentration. The total phenol content analysis carried out for thyme essential oil, oregano essential oil and nanofibres based on collagen and essential oils proved a higher value in the case of oregano essential oil incorporated into collagen, according to the total phenol content of tested essential oils. It was demonstrated that the high content of polyphenols will ensure the stabilization of collagen and protection against enzyme degradation [62
Antioxidants assessment in terms of total phenolic content, and DPPH radical scavenging showed improved antioxidant activity of nanospun collagen loaded with essential oils due to the synergetic effect of collagen and loaded essential oils. Nanospun collagen loaded with essential oils can act as long lasting delivery support of antioxidant ingredients both from collagen and essential oils, to prolong the antimicrobial properties and storage conditions.
The ATR-FTIR analysis indicated that by incorporation of thyme and oregano essential oils into collagen hydrolysate (P2 and P3 samples) both spectra show the typical pattern of protein molecules, suggesting that the prominent bands characteristic of essential oils (2925 cm−1
with low intensity and absorption band displacement from 2869 cm−1
in oregano essential oil) were overlapped with the characteristic bands of collagen. The same behaviour was reported by other authors [22
] in film-forming emulsion based on clove essential oil and melaleuca essential oil incorporated into chitosan.
SEM images showed that the bead-free and porous morphology of nanofibre mats are preserved but the collagen nanofibre diameter increased from 342 nm, to 471 nm for collagen nanofibres loaded with thyme essential oil and to 580 nm for collagen nanofibres loaded with oregano essential oil, in agreement with phenol content. The influence of essential oil on nanofibre diameter increase was found also for clove essential oil included in electrospun poly(ɛ-caprolactone)/gelatin nanofibres [63
], fish oil encapsulated in poly(vinyl alcohol) nanofibres [64
] and candeia essential oil included in polylactic acid nanofibres [65
] and was attributed to electrical conductivity and viscosity changes. EDX analyses confirmed higher concentration of carbon in essential oil loaded nanofibres, with higher values for oregano oil loaded nanofibres.
Biocompatibility assay performed on NCTC clone 929 fibroblastic cells showed that the electrospun collagen nanofibres with thyme essential oil have slight and moderate cytotoxic effect only at 1000 µg·mL−1
concentration and the electrospun collagen nanofibres with oregano essential oil have slight and moderate cytotoxic effect at 500 μg·mL−1
concentration. As compared to other reported results related to the cytotoxicity concentration of 0.08 to 0.16 μL·mL−1
for thyme oil after 24 h [66
], in our case the cytotoxicity limits of collagen nanofibres loaded with essential oils are very high.
Antimicrobial tests performed against Staphylococcus aureus
ATCC 25923, Escherichia coli
ATCC 25922, Pseudomonas aeruginosa
ATCC 27853 and Candida albicans
ATCC 10231, have shown antimicrobial efficiency, an important characteristic for wound healing process. Minimum inhibitory concentrations values of essential oils loaded collagen nanofibres presented strong antimicrobial activity against Staphylococcus aureus
ATCC 25923 and Pseudomonas aeruginosa
ATCC 27853 (MIC lower than 500 μg·mL−1
) and moderate antimicrobial activity against Escherichia coli
ATCC 25922 and Candida albicans
ATCC 10231 (MIC ranging from 600 to 1500 μg·mL−1
), if we take into consideration a proposed classification for antimicrobial activity of plant extract materials [67
]. We have to mention that the same MIC value of 1 mg·mL−1
, found for collagen nanofibres loaded with essential oils, was recorded for essential oil extracted from Thymus ciliatus Desf
., tested for antifungal properties [68
]. The minimum inhibitory concentrations of essential oils loaded collagen nanofibres against Staphylococcus aureus
ATCC 25923 were lower (0.03125 mg·mL−1
and 0.125 mg·mL−1
) than the value found for thyme oil against Staphylococcus aureus
Rosenbach (0.25–4 mg·mL−1
) reported by other authors [18
Minimal concentration for biofilm eradication results confirmed the potential of essential oils loaded collagen nanofibres to be used for chronic wound treatments or supporting pathogenic resistant antibiotics.
The association of collagen hydrolysate with essential oils cumulates the easily available, low molecular components for skin restructuring with natural antimicrobials in a completely biodegradable product. It is recognized that there are few studies regarding the inclusion of essential oils in electrospun fibres for wound dressings [63
] and it is considered that the technology is recent, moreover the use of only natural materials without organic solvents or surfactants can be considered without correspondent.
Electrospun collagen nanofibres loaded with essential oils with bioactive properties can be used for obtaining bandages for wounds, clothing with specific properties, antimicrobial socks and gloves or in tissue engineering.