Apitherapeutics in electrospun fibers

Different by-products of beekeeping have been used for the design and electrospinning of fibers for wound healing.

Honey

It is said to be the oldest wound dressing. It is an aromatic hydrocarbon with a prominent organic composition, used as a medical treatment since ancient times due to its high biocompatibility ². It is also a complex mixture made up of macrocomponents and microcomponents, the main ones being water, lipids and sugars such as glucose, fructose and sucrose; Within the minority components are amino acids, vitamins, mineral salts and enzymes ^11,12, and chemical species have been reported that have specific functions and are responsible for the biological functionality of honey..

Flavonoids: phenolic compounds that act directly against bacteria, by inhibiting their energy metabolism, the function of the cytoplasmic membrane and the action of DNA gyrase ⁹.

Tetracycline antibiotics: they contribute to the aseptic state of the wound ¹³.

Hydrogen peroxide: antimicrobial produced by the enzyme glucose oxidase, which provides an antioxidant environment by inhibiting the production of inflammation causing free radicals ¹⁴.

Defensin-1: cysteine-rich peptide with antibiotic function against bacteria, fungi and viruses ¹⁵.

For electrospinning of fibers with apitherapeutics, Manuka honey is often used, which is produced in New Zealand from the nectar of the Leptospermum scoparium bush, ^16-18. It differs from other types of honey by the presence of methylglyoxal (MGO), component that reinforces antibacterial activity, with respect to hydrogen peroxide commonly present in honey types ¹⁹. It has a broad spectrum of antibacterial action fighting aerobic, anaerobic, gram-positive and gram-negative bacteria ²⁰. As well as honey, other bee products have also been used to treat different human diseases. This practice is called apitherapy ²¹.

Propolis

It is a resinous and highly viscous product, made by bees from the secretion that they collect from different types of trees and herbaceous plants. Similar to honey, propolis is used medicinally against infections generally caused by microorganisms, due to the properties conferred by phenolic compounds such as those that are antibacterial / bactericidal, antioxidant, antifungal and anti-inflammatory ²². It is also cytostatic for the treatment of tumors, it is astringent, spasmolytic, anesthetic, antiulcer and immunomodulatory, which makes it a special agent for healing wounds ^23,24. The effectiveness of propolis and the polyphenolic content depends on the geographical area and the plant environment where the bees develop ²². In practice, the research by Ullah et al., A ⁹. demonstrated the utility of propolis when impregnated in cellulose acetate nanofibers and CA / PCL polycaprolactone. These bee fibers showed high antioxidant activity and effective antibacterial activity against gram-negative and gram-positive bacteria.

Bee venom

Also called apitoxin, it has a great therapeutic potential in relieving pathological symptoms such as pain, severe inflammation, rheumatic arthritis, skin diseases and tumors ¹⁹. It acts as an analgesic, antiviral, antioxidant and antimicrobial, which makes it a favorable agent for wound healing ²¹. the antibacterial capacity of bee venom was demonstrated in the work of Abou et al.S. and collaborators ⁽¹⁹. Who Iincorporated PVA scaffolds were manufactured with different combinations between pomegranate extract, honey and bee venom. Scaffolds loaded with bee venom were effective against gram-positive bacteria, but showed no activity against gram-negative bacteria. In contrast, propolis has been shown to be fully effective against gram-positive and gram-negative bacteria; with the scaffolds composed of honey and bee venom, better tissue repair was obtained in rats wounds ¹⁹. Thus, despite the fact that apitoxin does not present a complete antibacterial activity against all types of bacteria, its other therapeutic properties provide an improved microenvironment for wound healing. Not only must the antibacterial activity of a substance be taken into account, but its properties as a whole can contribute synergistically to the treatment of wounds.

Honey as a therapeutic agent in wound healing

True wound healing requires that the entire physiological process of normal healing be accomplished in all its stages. In this way, with the presence of a wound, the immune system triggers a healing cascade composed of phases such as: inflammation, production of granulation tissue, re-epithelialization or deposition of collagen fibers ¹³. It has been reported that honey as a therapeutic agent contributes to the development of healing stages by fighting inflammation, causing autolytic debridement, that is, moderate separation between necrotic and healthy tissue, by enzymatic action. In addition, honey, due to its acidic nature, provides an environment with reduced pH, which favors the growth and activity of cells involved in the repair of tissues such as: fibroblasts, keratinocytes, lymphocytes, neutrophils and monocytes ^25-27. Skin tissues they regenerate and the size of the collagen fibers and the epidermis increases, reducing inflammation and the risk of a recurrent opening of the wound ¹. In oxygen-hemoglobin dissociation, honey as an apitherapeutic intervenes by increasing the release of oxygen. It allows the elimination of abnormal collagen and the synthesis of normal collagen with its maturation ⁽²⁸ and promotes the emergence of blood vessels ^29-32.

Dressings need to provide a moist environment to the wound while not allowing the growth of bacteria. Honey gives these characteristics to the polymeric matrices, in addition to constituting an interface between the wound and the external environment, which added to the protection barrier allows the non-adherence of the material to the skin, so that when the the scaffold is removed, does not damage the new tissue ¹⁰. Honey stimulates the activity of macrophages, leukocytes to produce cytosines that fight against infection and decreases the toxicity of substances produced by bacteria such as ammonia ¹⁹. It also counteracts resistant bacteria that put health at risk with a possible systemic infection ²¹.

Fibers electrospun with honey: properties and advantages

Nanofibers are considered fibers with diameters of less than 100 nm ⁽³³ which, due to their morphology and spatial arrangement, offer different advantages as materials for dressings. If the polymeric fibers are hydrophilic and even so their hydrophilicity is increased with the incorporation of honey, they will provide good exudate management ¹⁷. Furthermore, nanofibers with a small pore size will hinder the development of bacteria and infection, they will help the flow of nutrients, waste elimination and gas exchange, fundamental characteristics to providing an optimal environment for tissue renewal ²⁵. The decrease in size of the materials provides differential properties with respect to conventional ones, such as a high surface / volume ratio that allows the excessive liquid to be filtered and retained through the small fissures of the fibers, thereby maintaining a humid environment ^10,25. Honey nanofibers have great therapeutic and clinical potential to be applied in biotechnology, biomedicine, controlled release of active ingredients, local regeneration of skin affected by wounds, design of cell scaffolds / dressings ⁽³⁴⁾ and for biosensor devices ¹⁶. The fibers with honey have been developed to be used mainly in medical treatments and at the same time to replace the common use of raw honey, while innovating how the apitherapy is administered ¹³. A great advantage of nanofibers is that due to their 3D morphology and their small-scale size, they can mimic the structure of the extracellular matrix, favoring the development and proliferation of cells, as happens in their natural environment ³².

Electrospinning of biomaterials from honey and plant extracts

The effectiveness of honey-loaded polymeric fibers for wound healing has been reported; however, it has also been shown that enriching the fibers with natural extracts improves their therapeutic effect and increases their potential as biomaterials for tissue repair. In this way, the wound healing process is dynamized, with fibers that act as vehicles that allow the free or controlled release of medicinal agents on the affected area ⁹⁾ (Table 4).

In the study by Naeimi, A. et al. ¹Nepeta dschuparensis extract was added to chitosan fibers, PVA and honey. This biocomposite facilitated cell attachment and proliferation, characteristics of a material applicable as a dressing for tissue engineering. Fibrous meshes loaded with Nepeta dschuparensis presented an improved capacity to induce a higher percentage of wound closure, compared to clean polymeric fibers loaded with honey or with medicated cream, silver sulfadiazine. The work of Naeimi, A. et al. ¹demonstrates that the addition of the plant extract promotes great advantages for the renewal of skin tissues, due to the total strengthening of the antibiotic and antibacterial properties with natural agents. In a similar investigation by Arunpandian, B. et al. ¹⁰, extract of Carica papaya was incorporated into the electrospinning of polyurethane (PU) fibers and honey, demonstrating that the addition of fruit extract influenced a smaller fiber diameter, due to its effect on the physical properties of the solution being electrospun. The thin nanofibers facilitated the proliferation and spread of skin cells, such as fibroblasts and keratinocytes ¹⁰. As such, the trend of designing functional materials based on synthetic polymers and natural agents has spread ⁶, in order to maximize their wound healing power.

In the study by Abou, S. et al. ³³, a new fibrous wound dressing was developed based on honey and powdered pomegranate peel extract. Pomegranate peel residues were used for electrospinning because they are a major source of polyphenols compared to the fruit juice. The abundance of polyphenols is responsible for the therapeutic effect of the materials designed with these extracts. Additionally, the use of residual biomass is interesting to reduce waste at the environmental level and its use in scalable research processes.

For the design of electrospun dressings for the treatment of wounds, the high potential of plants is being exploited as alternatives for the treatment of diseases. As part of the materials research, an analysis of the phytochemical profile of each species is carried out and the raw materials with the highest content of therapeutically active biomolecules are used. Likewise, the medicinal properties of garlic (Allium sativum) and the plant species Cleome droserifolia have been used for the design of nanofibrous dressings. Garlic is known for its potential for treating infections due to its broad antibacterial activity against gram-positive, gram-negative and resistant bacteria. Garlic's antiseptic activity is attributed to the content of sulfur compounds soluble in both water and oil ⁵⁶. Cleome droserifolia on the other hand, contains compounds such as phenols, flavonoids, terpenoids and alkaloids, used as anti-inflammatories, antioxidants and antibacterials; therefore, they are good wound healing agents ²⁰. Fibers were made with natural extracts of garlic and Cleome in order to enhance the antibacterial activity of the PVA honey and Chitosan HPCS nanofibers. In general, the addition of plant extracts to the honey fibers increased their antibacterial activity, resulting in better results compared to the commercial dressing AquacelAg®.

Polymers used for electrospinning with honey

Different polymers of both synthetic and natural origin have been used for electrospinning of fibers with honey (Table 5 and 6). The polymers mostly mentioned by the authors are described for their versatility for electrospinning with honey.

Polyvinyl alcohol (PVA). It is a polymer of synthetic origin, biocompatible, biodegradable, soluble in water; by Naeimi, A. et al. ¹⁾ its hydrophilic nature makes it suitable for electrospinning of fibers in aqueous solution and with hydrophilic agents such as honey, while promoting a humid environment in the wound ¹⁰. It is a polymer approved by the FDA for the development of medical materials such as dressings, due to its swelling capacity, its null toxicity and high elasticity, which allow the obtaining of uniform fibers without defects.

Chitosan: It is a natural polymer obtained from chitin present in the exoskeleton of crustaceans ^{4 _,8)} . Chitosan has been used for electrospinning of fibers with honey due to its high biocompatibility, biodegradability, its antimicrobial and healing properties and the ease with which it promotes cell growth and regulation ^1,17.

The polycationic nature of chitosan contributes to the antibacterial effect, by affecting the surface membranes of bacteria, causing leakage and cell death ¹⁹. Electrospinning this polymer with honey has been shown to lead to increased fiber diameter and hinders its conversion to fibers. Therefore, to improve fiber morphology, it is necessary to add surfactants and combine chitosan with other polymers such as PVA ^13,17,58.

Sodium alginate: it is a natural polymer present in the cell walls of brown marine algae and to a lesser extent, produced by biotechnology. It is a biocompatible and biodegradable anionic polysaccharide. Electrospinning of alginate with honey has been possible, as long as it is accompanied with another polymer, given the deficiency in chain entanglements which hinder its mechanical resistance, in addition to its high surface tension and high conductivity. Like PVA, it is a hydrophilic compound that facilitates humid conditions to absorb exudates from wounds ⁶⁰.

Techniques reported for production of nanofibres with honey

Honey fibers have been obtained by different electrospinning techniques, among them electrospinning by needle, by centrifuge and coaxial. In Table 7 the parameters used by the various authors for different electrospinning techniques are presented. The system basically consists of subjecting a solution of synthetic and / or natural polymers to high voltage so it can be molded and collected in the form of fibrous strands. Thus fibrous nonwoven matrices are obtained with large ratio area / volume, significant permeability and high porosity ²⁰. The technique offers the ability to predict and design in advance the morphology, composition and applicability of the fibers, to fit the needs of the investigation, while also allowing the control of fiber size to nanometric or micrometric scale by via the elecrospinning parameters ⁶¹. In most studies, the polymer fibers with honey were obtained by electrospinning needle. Table 8 showsare the parameters used in this technique by different investigators to obtain fibers with honey.

Other researchers have manufactured core-shell fibers by coaxial electrospinning, where honey has become part of the core and polymers as the shell or covering of the fibers ³³. In this technique, a dual nozzle is used to connect two tubular conduits, one inside another, so that the honey flows internally while the crust of the nanofiber is formed towards the outside. The fibers give the possibility of releasing their content by diffusion through small pores or holes in the polymeric wall. Therefore, fibers with honey have been obtained by different electrospinning techniques such as: horizontal and vertical needle electrospinning, centrifugal electrospinning and coaxial electrospinning.

Core-shell fibers have a wide application in biomedicine for the controlled release of different therapeutic agents such as, but not limited to, drugs, antibiotics, growth factors, among others ^15,27.

It is interesting to highlight the wide application of honey in another nanomaterial manufacturing technology, which was related to the electrospinning technique. In the work of Mancuso, E.,2019 ¹⁵ they reported the design of a multilayer nanocoating with honey, by layer-by-layer (LbL) electrostatic assembly, as a nanofabrication technique that functionalizes molecules on surfaces.

Mancuso E. et al ⁽¹⁵ exposed poly (ε-caprolactone) meshes to alternating electrostatic interaction between honey as polyanion and poly (allylamine hydrochloride) as polycation to form the layered structure. The result was functionalized biomimetic meshes for the administration of natural antibiotics derived from honey, in the treatment of soft tissue infections ¹⁵. Poly (ε-caprolactone) fibers were manufactured by centrifuge electrospinning (Table 7), a technique that allows the manufacture of nanofibers at large volumes in a short time for possible industrial application. This technique depends on process components such as the electric field and the centrifugal force ⁶². Different results and interesting applications are recorded as antecedents in the manufacture of polymeric fibers with honey. These advances are presented in Table 9; however, reserchers have voiced certain precautions on the subject, and these must be taken into account in order to successfully obtain fibers with honey by means of electrospinning.

Precautions to consider when obtaining electrospun fibers with honey

Honey fibers are highly soluble in water and much more so when the concentration of honey increments ⁵⁸. As such, upon contact with aqueous media, they tend to degrade and lose their fibrous structure and stability. It is therefore very important that the scaffold is resistant, remains for a certain time without degradation in the wound, and in general that mechanical and therapeutic properties are retained. Reticulation or crisscrossing of fibers immediately after electrospinning not only prevents degradation of the honey fibers upon contact with aqueous media or ambient moisture, but also preserves the potential clinical scaffolding, facilitating the implementation of biological testing and analysis necessary to assess the applicability of the fibers. To prevent such degradation, researchers subjected the fibrous matrices to contact with agents that cause crosslinking of fibers and reduce its solubility. For example, in the work by Kadakia P. et al. ²⁰, the scaffolds were completely wetted in methanol for 30 min, which allowed the randomly arranged primary silk fibroin chains to regain their secondary structure β, and the material lost its solubility in water. Besides methanol, it glutaraldehyde (GAH) has also been employed as another crosslinking agent. Sarkar R. et al. ²⁸ subjected their electrospun fibers to contact with GHA vapors for 24 h.

In another study ¹⁹, fibers with vapors of the same compound led to meshes that were highly resistant to instant dissolution when in contact with saline aqueous media or with the wound itself. There was also a moderated release of the loaded active ingredient and the scaffold maintained its functionality until the completion of the healing process. This result is very

important for further research because it also highlighted that the non-crosslinked scaffolds dissolved very quickly in the medium and did not contribute optimally to wound closure and total healing. Furthermore, crosslinking of fibers has been applied in more studies ^19,60) all in order to improve the scaffold's ability to handle exudate, facilitate the constant release of active principles and provide a compact fibrous structure. However, this procedure must be practiced with caution and in a balanced way, since evidence shows that an increased degree of polymer crosslinking interferes with the movement of the bioactive molecules, which are trapped in the entangled chains within the fibers, limiting its functionality ¹⁹. Moreover, care must be taken in establishing the concentration of honey in fibers for its application as dressings in tissue regeneration.

In the study carried out by Kadakia P. et al. ²⁰ it was indicated that by means of in vitro tests, Manuka honey at 5% v/v or more caused cell death, possibly due to the acidity that honey gives to the medium.

obtained for wound healing using lower concentrations of honey such as 0.1 at 40%, but fundamentally due attention must be paid to the needs of the skin at the cellular level, so as not to incur honey saturation without it being necessary ⁶³.

Regarding the handling of solutions to be electrospun, the increase in honey content caused a complex handling of the

solution due to the high viscosity and low fluidity that it produces ⁶¹, and therefore, did not allow satisfactory expulsion from the syringe. Thus, Sarhan & Azzazy ⁽⁶¹ reported a simple experimental step to achieve electrospinning of solutions with high honey content, between 25 and 30%. The mixtures of honey, PVA and chitosan were kept at rest for a week and then their electrospinning was easier. Therefore, the balance between the concentration of honey in solution and its controlled release from the fibers must be promoted, since if excesses are present, the risk of cytotoxicity increases.

Thus, in the investigations of Sarkar, R. et al. ²⁸ and Mancuso, E. et al. ¹⁵, concentrations of honey were moderate, which not only did not result in cytotoxicity, but also supplied better antioxidant and anti-inflammatory needs for wounds. In these studies an experimental design was done under the premise that honey in high concentrations can negatively influence the cellular environment causing toxicity to smooth muscle cells, such as myofibroblasts or mesenchymal stem cells ³⁴.

Techniques used for the characterization of fibers with honey

For the characterization of fibers with honey, different techniques have been used that allow to understand their composition, morphology, crystalline structure, mechanical, thermal properties, wettability, degree of swelling, water absorption capacity and weight loss. The characterization techniques and the results from obtaining electrospun fibers with honey are presented below.

Analysis of composition and chemical structure of fibers with honey

Fourier Transform Infrared Spectroscopy is a characterization technique that has the ability to provide the interactions of functional groups chemical structure of functional groups of materials such as those with polymers in their composition. For fibers electrospun with honey it is important to recognize the characteristic FTIR bands of honey, since these signals will be necessary to observe the behavior of honey when it is incorporated lo cualinto fibers by electrospinning.

The bands mentioned are: 3384 cm⁻¹ (-OH), 2936 cm⁻¹ (CH), 1637 cm⁻¹ (CO) and 1057 cm⁻¹ (CO) ³². The chemical composition of the electrospun honey fibers has been confirmed by means of this technique and on a smaller scale by X-ray photoelectron spectroscopy or XPS. As per the study carried out by Naeimi, A. et al. ¹, the FTIR spectra of chitosan and PVA fibers were analyzed in contrast to electrospun fibers with natural compounds such as honey and a phytoextract. For the fibers loaded with honey, CH bands belonging to the honey carbohydrates and polysaccharides of the Nepeta extract were observed, while in the FTIR spectrum of the polymeric fibers, only bands of functional groups of polymers were observed, such as CO and C=C of PVA, NH₂ and OH chitosan.

The intensity of the C-H bands increased when incorporating honey and Nepeta;, hence,confirming the incorporation of the natural compounds in the fibers was confirmed; there was chemical interaction between the hydroxyl (-OH) and amine groups (-NH₂) of chitosan and PVA with functional groups of honey and Nepeta, forming hydrogen bonds between them. Likewise, the study carried out by Khan, M. et al. ^6,64 comparing the spectra of the PICT fibers Poly (1.4 cyclohexane dimethylene isosorbide terephthalate) with and without honey, confirms the incorporation of the apitherapeutic in the fibers. In the FTIR analysis, for the clean poly(1,4-cyclohexane dimethylene isosorbide trephthalate) (PICT) fibers, a characteristic band of aromatic compounds was obtained due to the presence of terephthalate and a C=C double bond stretching. In contrast, in the spectrum of the fibers with honey, these bands disappear and instead the interaction between O, C and H is evidenced, with the appearance of bands of single bonds for CO at 1270 cm^-1 and at 1850 cm^-1 for CH.

In turn, the PICT and honey fibers were analyzed by XPS spectroscopy. An N1s peak was observed whichthat affirms the interaction between honey and the polymer; however, this band was not visible in nanofibers without honey. ⁽⁶, and the incorporation of honey and its chemical interaction with the base polymer was again evidenced. Depending on the application for which the loaded honey fibers and other natural components are to be used, it is not highly recommended that a strong bond is formed between these and the polymers, because the release of the therapeutic components into the environment is difficult, decreasing its effectiveness in the affected area. As such, the study by Sarhan & Azzazy (34)shows that when making an FTIR analysis of fibers, did not evidence the formation of bonds or chemical union between propolis and bee venom with honey fibers, PVA and chitosan. This low interaction between fiber components facilitated the constant release of the apitherapeutics, after the degradation of the polymeric fibers.

An important piece of data is also presented in the research by Paimard, G. et al. ^22,28 with evidence of the incorporation of honey as a core in PVA fibers obtained by coaxial electrospinning. In the spectrum of these core-shell fibers, only the characteristic PVA bands are present, while the honey bands are not visible. This is an interesting way of confirming the successful coating of the polymer towards the honey core and the shell-core arrangement of the fibers.

The correct incorporation of different concentrations of honey in PVA fibers was also evidenced by FTIR analysis in the work of Sarkar R. ²⁸ The successive addition of honey to the fibers was identified as the concentration of honey was higher, by increasing successive band intensity in the spectrum. Tang, Y. et al. ³² also obtained a similar result regarding the increase in the area under the curve of the characteristic bands of honey, as its content was higher in alginate and PVA fibers; due to the increase in honey content from 5 to 20%, the intensity of the honey bands was greater. It is therefore possible to monitor the change in the concentration of honey in fibers through FTIR analysis.

Thermogravimetric analysis (TGA) of fibers with honey

The thermal stability and strength of honey electrospun fibers were also analyzed. In the research by Arunpandian, B. et al. ¹⁰, the bio-nanofibrous membrane of polyurethane, honey and Carica papaya extract presented a greater weight loss compared to polyurethane fibers, due to a higher quantity of hydrophilic molecules, with the researchers reporting that the presence of honey and the fruit extract contribute to the thermogravimetric stabilization of the fibers.

A study conducted by Fatma Nur, P. et al. ¹⁹ indicates that the thermal stability profiles of polyamide 6/honey and composite fibers containing boric acid at different concentrations were observed. three stages of Weight Loss for polyamide 6 fibers. A small and a larger mass loss will be observed in the temperature range of 57 to 250 °C and 343 to 518 °C, respectively. The initial stage represents the evaporation of water with a weight loss of 2.9% in the temperature range of 57 to 250 °C. The second stage, with a significant weight loss of almost 94% at 343-518 °C, was caused by the degradation of the polyamide backbone macromolecule. The third step is formed after the change of atmosphere from N2 to O2 at 600 °C. It also indicates that the addition of honey changed the decomposition behavior of pure polyamide 6 fibers. PA6-HB0 fibers showed four decomposition steps related to the presence of more hydrophilic groups [58]. The first and second degradation stages were formed together between 103 and 216 °C and 216-286 °C with a weight loss of 8.7% corresponding to the loss of water molecules from PA6 and the volatile content of honey. . . It is clear that the PA6 sample exhibited higher thermal stability compared to PA6H-B0 fibers. This determining the lower thermal stability of electrospun polyurethane fibers loaded with honey and Carica papaya fruit extract compared to pure polymeric fibers. The TGA clearly shows that nanofibers made from honey exhibit good thermal stability below 195 °C, which is higher than polyvinyl alcohol/chitosan fibers incorporated into honey (120 °C) ⁶⁵.

The study of Naeimi, A. et al. ¹⁾ determines the thermal characteristics of the PVA/Chit nanocomposite and the PVA/Chit@Nep/honey nanofibers were obtained using TGA. In TGA curing of PVA/Chit nanocomposite, there are two degradation stages. The first weight loss began at 250 °C starting temperature in relation to the melting point of PVA. The second degradation occurs at 350 °C, which corresponds to chitosan. On the other hand, these two steps exist and in TGA of PVA/Chit@Nep/Honey and considering the carbon residue it is shown that the honey component acted as a good carbon insulator in the polymer matrix. In TGA of PVA/Chit@Nep/Honey nanofibers, it appears that the formation of carbon particles of honey layered structure in the network of PVA and chitosan that resembles carbon. In fact, this carbonization-like layer acted as an effective barrier for oxygen permeation improving the carbon residue. The presence of crystalline structure and the great compactness between honey and the Nepeta dschuparensis plant and the PVA/Chit matrix lead to high thermal stability of this bio nanocomposite ⁶⁶.

Morphological analysis of electrospun fibers with honey

Fiber morphology with honey

In this space, real results were captured about the effects that the addition of honey had on the morphology of polymeric fibers and that the researchers analysed by SEM, TEM and AFM microscopies. The researchers obtained smooth fibers from the PICT polymer only, but by incorporating honey, the fibrous surface was more rough. On the other hand, in the same study, the researchers reported that up to a maximum of 20% honey, fibers were formed without beads, with the fiber beads increasing at higher concentrations. It was found that PVA nanofibers without honey presented a homogeneous morphology, but when adding honey at 20% (v/v) the morphology was less uniform, with a wide distribution of diameters ³². Similar results were presented⁴ with a honey concentration of 40%, when beading and spindles formed in the fibers. These fibers were also observed with an AFM microscope presenting smooth and uniform surfaces with a cylindrical structure in all weight ratios ¹⁰. However, when the honey content was at 40%, beads or spindles began to form. In the study by Naeimi, A. et al. ¹, fiber magnifications were obtained by TEM and confirmed the presence of spherical honey particles and Nepeta extract in the fibrous PVA and chitosan meshes. The distribution of the honey particles was random and contributed to a higher roughness in the fibers.

The concentration of honey is an important factor that affects fiber morphology. Sarkar R. et al ²⁸ used in their research minimum honey concentrations such as 0.2 and 0.5%, obtaining PVA and honey fibers with a dense, smooth and homogeneous arrangement and without defects such as beading. When increasing the content of honey there were no significant differences, therefore estimating that the minimum changes in the concentration of honey do not alter the morphology; however, at very high concentrations such as 40%, the effect of the concentration of honey is important. The study by Abou, S. et al. ³³ also reported obtaining fibers with a totally cylindrical structure, a smooth surface and no beads. In this work, the increase in the concentration of honey with values ​​of 10, 20 and 25% did not affect the shape of the fibers. It should be noted that these fibers were also electrospun with pomegranate juice, bee venom and lyophilized honey, components that could influence the homogeneity of the fibers.

On the other hand, the study carried out by Sarkar, R. et al ⁽²⁸ indicates an unusual result when it comes to fibers with honey. Flattened fibers were obtained with a concentration of 1% honey. It is reported that this morphological change is due to the high viscosity of the precursor solution, where the rapid evaporation of the solvent is prevented and consequently the fibers reach the collector wet and flatten on impact.

Porosity of fibers electrospun with honey

The measurement of fiber porosity is very important in wound healing dressings because high porosity allows perspiration, gas exchange between the external environment and that of the wound, and maintains oxygenation and humidity while allowing the diffusion of exudate, and so aids the necessary conditions for correct healing ⁹. The abundance of pores in the fibrous mats promotes the aseptic state of the wound, as it prevents the emergence of infections and attack by bacteria ¹⁰. The dressing must be designed to a specific porosity since the pore size will allow the accommodation and proliferation of cells of a certain size.

In some investigations of electrospinning with honey, the researchers have managed to obtain fibers with adequate porosity to aid in wound healing. For example, the fibrous polyurethane mesh, extract of Carica papaya and honey ¹⁰, presented an average pore size of 12.54 µm and a distribution between 4 and 50 µm. This porosity is optimal for the deposition of fibroblasts because the cells themselves need a range of 5 to 15 µm to proliferate ¹⁰. For their part, the work by Maleki, H. et al. ¹⁴ referes to the manufacture of a dressing of PVA with honey with a porosity value of 81.43%, an optimal percentage to facilitate constant wound healing.

Mechanical properties of fibers with honey

Effect of honey content on the elasticity of electrospun fibers

In the study carried out by Khan, M. et al. ⁶⁴ the percentage of deformation in the fibers was greater with increase in honey content. This percentage ranged from 9 to 45%. The fibers with 15% honey presented satisfactory mechanical properties with 45% elasticity and a Young's Modulus of 2.7 MPa. This indicates that the highest percentage of honey is not always the most suitable for the design of a dressing with good mechanical properties; the balance between tensile strength and elongation capacity making the handling of the wound dressing more comfortable. In agreement with the mechanical tests results from PICT fibers and honey obtained by Khan, M. et al. ⁶⁴, where the fibers with a 15% concentration of honey presented the most adequate mechanical properties, the study carried out by Ping & He ⁽²⁵ states that a high concentration of honey in PVA fibers is needed to achieve satisfactory mechanical properties, that is, greater than 7.5% by weight.

Contact angle and wettability of fibers with honey

The water contact angle measurement is used to evaluate the hydrophilicity of materials, while the water absorption capacity is a fundamental property of the wound dressing, useful in improving the wound recovery process ^10,32. It is possible to verify that the material actually retains moisture and therefore has good wettability, causing a better exudate management and reduction of the time for dermal fibroblasts to adhere, migrate ⁽⁶ and spread through the affected area ²⁰.

Ullah A. et al. ⁶, observed that when electrospinning cellulose acetate with honey, the contact angle decreased and so the fibers were more hydrophilic. Arunpandian, B. et al. ¹⁰ confirmed something similar in their research by adding honey and Carica papaya extract to the polyurethane nanofibers, a decreased contact angle indicating that the fibers are wettable. This behavior is justified by the presence in the solution of a greater number of hydroxyl groups (OH-) and hydrophilic biomolecules in general. Therefore, when adding honey, the hydrophobicity decreased as confirmed by Khan, M. et al. ²

In contrast, Kadakia, P. et al. ²⁰, confirmed an increase in the contact angle when adding Manuka honey to silk fibroin fibers. This generally unexpected effect manifests when the glucose and fructose of the honey, partly responsible for the hydrophilic character of the fibers, are wrapped between the polymer chains without exposure to the fibrous surface. As a consequence, the hydrophobic behavior typical of silk fibroin was maintained, without being significantly affected by the presence of honey. Nevertheless, the honey scaffolds had contact angles of less than 90 °, which indicates that the fibers did have the ability to absorb and retain water.

Degree of swelling of the honey fibers

By means of the swelling capacity of the nanofibers, the scaffold's ability to absorb moisture, that is, exudates from the wound, is evaluated. The swelling behavior also controls the release of active principles or drugs towards the affected area,² it is an indicator of the material's breathability or its ability to allow the flow of water vapor ⁴. This measure is obtained by immersing electrospun fibers in phosphate buffered saline (PBS) at different time periods. Thus, when the fibers come into contact with the saline solution, they absorb moisture and their weight can be measured at the end of the process. This test has been applied in different investigations of fibers from honey and the researchers have obtained the following results: Sarhan, W. et al. ³⁴ designed a fibers based on PVA, chitosan, honey and natural extracts, which presented a good capacity to swell, and so easily adhering to the affected area without the need for biological adherents. Also Sarkar, R. et al.⁷ obtained similar results for PVA fibers and honey at different concentrations. The fibers with 0.5 and 1% honey showed swelling degrees of 5.7 and 6.0 respectively, higher than the fibers with 0.2 and 0% honey, with values ​​of 4.9 and 3.7, meaning that the fibers with a higher percentage of honey had a greater capacity to absorb moisture. Taking into account that PVA is a polymer with good swelling capacity, the polymeric fibers incorporated with honey notably improved this property, due to the high osmolarity of honey due to its high concentration of sugars; therefore, the higher the osmolarity, the greater the capacity to absorb water.

On the other hand, silk fibroin scaffolds incorporated with Manuka honey demonstrated greater water absorption at 28 days, confirming the moisture retention effect that honey attributes to nanofibrous meshes ²⁰; however, not in all cases did honey allow maximum water absorption. In the work of Sarhan, W. et al. ³⁴, nanofibers of sodium alginate, PVA and variable concentrations of honey were obtained. By increasing the amount of honey, the fibers reduced their ability to absorb moisture. This effect is justified because honey is highly soluble in water and therefore the dressing was completely dissolved in an aqueous medium, losing the mesh structure and thus all its ability to retain water ³⁴. Similarly, when manufacturing nanofibers based on PVA, pomegranate juice and honey at different percentages, Abou, S. et al. ³³ found that scaffolds containing honey showed moderate water absorption compared to scaffolds without honey, which presented greater swelling capacity, probably because honey is very soluble in water and caused the degradation of the fibers. To solve this tendency, it might be necessary to subject the fibers to the crosslinking process for a longer time, to make the fibrous structure more resistant, compact and durable within an aqueous environment; however, it can be verified that the result of the swelling capacity of the fibers depends on the interaction of each polymer with the honey in the middle of an aqueous environment.

Fiber weight loss with honey

The weight loss test has also been implemented to characterize nanofibers with honey, since this is an indicator of the release of therapeutic components towards the wound and of the biodegradation of the material, due to the breaking of the polymer chains ¹³. Honey fibers have been susceptible to weight loss due to increased honey content, which more easily allows water to penetrate the fibers, followed by leaching, hydrolysis and dissolution of the polymer.

In three investigations the same results were obtained with honey and PVA fibers ^13,58. These meshes generally exhibited good degradation properties, compared to PVA-only fibrous meshes. As such, the potential of honey-based fibrous materials for their application in dressings that fulfill their therapeutic function is confirmed, and in a moderate time they degrade and do not add to the polluting agents of the environment.

Effect of the solution parameters in obtaining fibers with honey

The effect of the physical characteristics of the electrospinning solution, such as polymer concentration, viscosity and conductivity, has also been studied to analyze its impact on the process of obtaining the fibers with honey. The influence of these solution variables on the ease and success of fiber formation with honey was analyzed.

Polymer concentration in solution with honey

The researchers demonstrated the effect of the polymeric concentration of PET and chitosan in fibers with honey ⁵. They supplied total concentrations of 17 and 19% w/v for either one of the polymers or both. At polymer concentrations below a weight of 19%, discontinuous, rolled and beaded fibers were obtained. By contrast, at a concentration above 19%, the electrospinning process was not carried out because as the viscosity of the solution increased, it was difficult for the solution to be expelled from the syringe ⁵. In the study by Paimard, G. et al. ²¹ also analyzed the effect of polymer concentration on the morphological quality of the fibers. These authors manufactured fibers based on honey and PVA by coaxial electrospinning (Table 7). The polymer solution was used as the covering and the honey as the center or core of the fiber, and at a concentration of 10% PVA w/v, they obtained smooth and uniform nanofibers; by increasing the concentration to 20%, the fiber diameter was greater and the uniformity of the fibers decreased. Even with this result, more experiments were carried out with PVA concentrations greater than 20% w/v. The morphology obtained was of low quality and not very promising ²¹.

As a result, it is evident that the concentration of the polymer directly affects the morphology of the fibers. For this reason, the polymer concentration must be well balanced: not so low in order not to interrupt the formation of the Taylor cone that precedes the formation of the fibers and not so high to allow normal flow of the solution. Therefore, caution and knowledge of the effect of polymer concentration vs. morphology, must be exercised for the design of fibers with honey from polymers such as PET, Chitosan and PVA reported so far in the referenced articles.

Viscosity of the polymer solution with honey

In general, the viscosity of the polymer solution decreased as the honey content increased. In the work by Tang, Y. et al. ³² a solution to be electrospun of sodium alginate and PVA with 20% v/v of honey, presented a very low viscosity of 419 mPa·S, with respect to solutions with a lower honey content. This value led to the formation of bulbs in the fibers, so we can conclude it is necessary to control the viscosity values to have a better follow-up to the fiber morphology.

Maleki, H. and their work group ^(10,19 reported that the appearance of fiber defects is due to the fact that there was an increasing percentage of honey, which caused the polymer chains not to resist the stretching force from high voltage, and caused the fibers to take on a varied morphology throughout, i.e. accumulated polymer and very thin fiber parts.

On the other hand, it was shown that the addition of natural extracts to polymeric solutions with honey is useful to balance the viscosity of the solution and therefore facilitates the electrospinning process ¹⁹. However, if a solution with too low a viscosity is supplied, an unwanted dripping occurs at the tip of the needle, which does not allow the stretching of the jet or the subsequent formation of fibers. In contrast, Sarkar, R. et al. ²⁸ achieved the opposite effect, with the viscosity increasing the higher the concentration of honey added. This little-known effect perhaps depends on the variety of honey, solvent or mixture of solvents as well as the nature and concentration of the polymer used in the research and its specific composition: The addition of honey to the polymer solution can provide different viscosity values ​​and influence the morphology of electrospun fibers in a certain way.

Biological methods applied to evaluate the functionality of electrospun fibers with honey

Different biological methods in vitro and in vivo have been used to evaluate the potential of fibers with honey, in order to:

combat pathogenic microorganisms present in skin wounds, evaluate the ability to contribute to cell regeneration, and determine the antioxidant or anti-inflammatory potential, which all together promote wound healing.

Antibacterial activity of fibers with honey

The antibacterial activity of fibers with honey has been measured using the agar diffusion method, the dynamic contact method and the viable cell count technique.

It is necessary to evaluate the antibacterial and antibiofilm capacity of the materials that have the potential to be applied as dressings, since these must have the ability to combat the proliferation of bacteria in their early stage and prevent the formation of colonies that are precursors of infection, because these factors are also responsible for the chronicity of wounds in soft tissues ¹⁶. In the study carried out by Sarkar R. et al. ⁽²⁸, honey nanofibers with improved antibacterial activity against E. coli were obtained. The antibiofilm test also indicated that the nanofibers were able to reduce E. coli colonization by E. coli by 300%, by increasing the content of honey in the fibers.

In another study it was found by means of the dynamic contact test, that PVA / sodium alginate and honey matrices significantly decreased bacterial growth depending on the dose of the apitherapeutic. In addition, the honey fibers that were incubated for 24 h, showed prominent antibacterial activity, compared with those incubated for 12 h only. This is an indication that the honey had enough time to continuously release and gave good antibacterial properties to the medium. In contrast, the fibers without honey content allowed the rapid development of S. aureus and E. coli after 24 h ³².

Although honey is an efficient antibacterial agent, its properties can be enhanced by enriching the fibers with natural extracts. This is how Abou, S. et al. ³³⁾ achieved a strong antibacterial effect against gram-negative (E. coli), thanks to the incorporation of pomegranate juice in honey fibers and PVA. For their part, in the research by Sarhan, W. et al. ⁶¹, electrospun scaffolds with honey and Allium sativum and Cleome droserifolia extracts provided complete protection against S. aureus and the resistant strain, compared to the commercial scaffold AquacelAg®.

The antibacterial activity of these extracts is due to the presence of thiosulfinates in Allium sativum and sesquiterpenes in Cleome. These components alter the cellular activity of bacteria, blocking their normal functioning.

Tang, Y. et al. ³², when evaluating the antibacterial activity of sodium alginate, PVA and honey fibers through the disk diffusion method, found that they were more active against S. aureus strains than against E. coli. Similarly, Sarhan & Azzazy ⁽³³ found that adding an apitherapeutic propolis agent to honey fibers provided greater activity against S. aureus when compared to the commercial AquacelAg® dressing. These results are consistent as it has been reported that honey has a better antibacterial effect against gram-positive than gram-negative bacteria ³².

Sarhan & Azzazy ⁽³³ found a way to broaden the spectrum of antibacterial effectiveness of fibers with honey by incorporating bee venom. By counting viable cells, they observed that these fibers allowed complete inhibition against E. coli, the honey matrices being effective against gram-negative as well as gram-positive strains. Furthermore, the antibacterial potential of these fibers was similar to that of the commercial AquacelAg® dressing for E. coli, but it showed improved activity against gram-positive strains: S. aureus and MRSA.

Contribution of electrospun fibers with honey in cell viability and tissue regeneration

Cytocompatibility studies have revealed that the addition of honey to polymeric fibers promotes the development of cells on scaffolds. Honey improved the biocompatibility of cellulose acetate nanofibers, by contributing to the mat's affinity with stem cells for their proliferation and propagation. Likewise, PCL fibers loaded with layers of honey were cytocompatible with different cell lines, such as skin fibroblasts and endothelial cells ¹⁶. In the work group of Kadakia, P. ²⁰, honey caused the release of growth factors from human dermal fibroblasts, which totally infiltrated the electrospun silk fibroin meshes. The mats with honey showed a higher cell density after 28 days compared to the controls, and when compared to the commercial scaffold AquacelAg®, ⁽¹⁹ high levels of viability and cell proliferation caused by the polymeric fibers loaded with honey and natural extracts.

Similarly, alginate, PVA and honey fibers showed a value higher than 93% of cell viability, while the fibers without honey showed values ​​below 93%. In the same research, they related the concentration of honey with cell viability. It was found that 10% honey led to a 102% viability; however, by increasing the honey content to 20% the viability percentage dropped to 96%. This result indicates that increasing the concentration of honey is not always favorable for cell development. The study carried out by Abou, S. et al. ³³ also verified the cytocompatibility of PVA nanofibers, pomegranate juice and honey: the scaffolds had a 100% cell viability, verifying that the fiber components did not show cytotoxicity. Therefore, the ability of honey to contribute to cell viability in artificial media that helps to heal affected skin in vivo continues to be confirmed ³². Sarkar R. et al ⁽²⁸ measured the effect of low concentrations of honey in the development of cells, with fibrous PVA meshes without honey showing reduced cellular activity when compared to fibers loaded with 0.5 and 1% honey. These concentrations effectively improved cell behavior. The formation of parts of the cytoskeleton and dispersed morphologies was observed with the fibers at 0.5% honey, confirming the normal growth of the cells. However, it is found that a concentration of honey in average values ​​is the one that optimally contributes to the adhesion of epithelial cells and the subsequent re-epithelialization ²⁸.

Histological evaluation of in vivo wound healing induced by honey fibers

The functionality of electrospun fibers with honey to contribute to wound healing has been evaluated in vivo. Researchers have caused artificial wounds in mice, by means of excision and the fibrous scaffold has been delivered to the affected area. Overall, promising results have been obtained in tissue regeneration, by inhibiting tissue inflammation and promoting skin development.

Ability to induce wound closure was evaluated for the scaffolds ⁽¹⁹ obtained with honey and pomegranate extract. Honey scaffolds potentially increased the percentage of wound closure when compared to PVA-only scaffolds. The healing progress was evaluated for time periods of 3, 5 and 10 days: at day 10 the honey scaffolds obtained an almost complete wound closure percentage, while with the commercial Medihoney® scaffold (composed of calcium alginate and honey) it took 13 days to heal. This is a successful result because the electrospun fibers with honey and extract allowed the total closure of the wound in a shorter time, compared to the commercial scaffold; that is to say, the electrospun fibers with honey and vegetable extracts are highly effective, which could displace commercial scaffolds. In addition to wound sealing, the scaffold obtained produced a good histological result, since it mainly induced collagen deposition, epidermis re-epithelialization and healing, in contrast to the Medihoney® scaffold. However, an even more satisfactory result was obtained when other apitherapeutic agents such as propolis and bee venom were added to the fibers. The apitherapeutic fibers allowed the development of skin very similar to the original. These fibers have a promising potential for the regeneration of the affected tissue, without visual scars.

In turn, in another study ⁽¹⁹ polymeric meshes were manufactured with honey and added with extracts of Allium Sativum or Cleome droserifolia, which allowed a good percentage of wound closure similar to that produced by the commercial silver-based AquacelAg® scaffold. The fibers they obtained are a good alternative as dressings, based on natural compounds compared to commercial scaffolds made from synthetic compounds.

Antioxidant and / or anti-inflammatory activity of electrospun fibers with honey

Phenolic compounds in honey, such as flavonoids, give electrospun fibers witha high antioxidant potential (32); the deficiency in antioxidant enzymes provides the conditions for an oxidizing environment in the wound, affecting skin regeneration. It is important to know how scaffolds act against oxidative stress, due to the excessive exposure of reactive oxygen species and nitrogen, which leads to inflammation, poor healing and chronicity in the wounds ¹³.

It is reported that dressings should be designed to meet the needs of both the wound ⁽¹³ internal and external microenvironment, because most studies have focused on factors related to the external well-being of the wound, such as the correct management of exudates, debridement, permeability and antimicrobial activity; however, the internal need of the wound, such as the control of oxidative stress, is essential for total healing. A piece of research was therefore found that evaluated the antioxidant potential and, consequentially, the anti-inflammatory capacity of honey scaffolds, since the presence of antioxidant molecules is a direct indicator of the anti-inflammatory potential. that fibers may have. Sarkar, R. et al. ⁽²⁸ presented results related to the measurement of the expression level of inflammatory markers Interleukin (IL-6) and Cyclooxygenase-2 (COX-2) against PVA and honey scaffolds. The matrices with 0.5% honey showed the lowest expression of markers, with respect to the fibers loaded at 1%, despite the fact that the latter fibers fought microorganisms more effectively. Controls and PVA fibers without honey demonstrated the highest expression of inflammatory markers. Thus, honey clearly contributes to the elimination of pro-inflammatory markers. Therefore, it is clear that a significant antioxidant activity can be obtained with small concentrations of honey that will be better accepted at the cellular level in the microenvironment.