Next-generation Antimicrobial Peptides (AMPs) incorporated nanofibre wound dressings

The skin is the most exposed organ and, therefore, vulnerable to injury and wounds (Nguyen & Soulika, 2019). Wound healing is a complex tissue repair process, and failing to manage it could result in the formation of scars (Landén et al., 2016; Takeo et al., 2015). Tissue repair involves the partial tissue regeneration involving restitution of tissue components during the wound healing process (Atkin et al., 2019; Gonzalez et al.., 2016). Wound healing is a dynamic process consisting of four phases: inflammation, proliferation, DOI: 10.1002/mds3.10144


| INTRODUC TI ON
The skin is the most exposed organ and, therefore, vulnerable to injury and wounds (Nguyen & Soulika, 2019). Wound healing is a complex tissue repair process, and failing to manage it could result in the formation of scars (Landén et al., 2016;Takeo et al., 2015).
Tissue repair involves the partial tissue regeneration involving restitution of tissue components during the wound healing process (Atkin et al., 2019;Gonzalez et al.., 2016). Wound healing is a dynamic process consisting of four phases: inflammation, proliferation, DOI: 10.1002/mds3.10144 and remodelling (Eming et al., 2014;Ghomi et al., 2019). All four phases should occur in proper sequence in an ideal healing process, as interruptions can result in delayed wound healing and failure to return to the native aesthetic and functional form (Dreifke et al., 2015). The ideal wound dressing should be non-toxic (Jalili Tabaii & Emtiazi, 2018), non-allergic (Negut et al., 2018), promote absorption of wound exudates (Dumville et al., 2016), provide a gaseous exchange, protect against bacterial infection (Dabiri et al., 2016), be easy to apply and remove (Uzun, 2018), and allow for adequate hydration (Hasatsri et al., 2018). Current wound dressing methods such as hydrogels (Kamoun et al., 2017) can maintain a moist wound environment but are not ideal for very wet wounds (Caló & Khutoryanskiy, 2015) where it can lead to infection due to excess moisture (Pilehvar-Soltanahmadi et al., 2017). Hydrocolloidal wound dressings have been successful against bacteria but the dressing may adhere and displace the wound and cause trauma upon removal (Miguel et al., 2019). Thus, a new generation of wound dressing materials (Sood et al., 2014) is expected to control higher moisture levels, and provide sustained release of biologically active ingredients (Georgescu et al., 2017), which will enhance the healing of wounds (Gizaw et al., 2018).
Fibres in the nanometre range are an excellent class of materials for different applications , including biomaterials (Shahriar et al., 2019) for tissue engineering (Adam et al., 2013;Ye et al., 2019), drug delivery (Al-Enizi et al., 2018), air and water filtration systems , and wound healing . Nanofibres can be functionalized into wound dressings due to their distinctive properties; such as a high surface area to volume ratio  and high porosity that allows for both the protection of the wound site and exudate movement (Han & Ceilley, 2017), which is crucial for optimal recovery (Mir et al., 2018). For increased antibacterial resistance, for settings such as hospitals wound treatment is required that can address issues related to the currently available dressings (Illangakoon et al., 2017).
However, among all the approaches, pressurized gyration was selected in this work because of its ability to mass-produce-bandage-like meshes with simple preparation and enhanced processing controls (Alenezi et al., 2019).
Nanofibres loaded with antimicrobial agents such as antibiotics (Thakkar & Misra, 2017), graphene (Lasocka et al., 2019;Matharu et al., 2018), nanoparticles (Hassiba et al., 2017;Troncoso & Torres, 2020) and natural antimicrobial substances (Simoes et al., 2018) are a valuable way to target bacterial species by reducing wound infections (Stone et al., 2018) potentially enhancing the healing process. Nanofibres can release antimicrobial agents with greater efficacy due to their large surface area (Kenry, 2017;Nguyen et al., 2013). The goal is to improve the healing process which would speed up regeneration (Saghazadeh et al., 2018) and reduce scarring (Rahimnejad et al., 2017) by incorporating novel antimicrobial agents into nanofibrous materials. Nanofibres are also able to imitate the extracellular matrix (ECM) (Jun et al., 2018) to provide a suitable cellular niche to accelerate wound repair (Rezk et al., 2018).
Antimicrobial peptides are short generally cationic peptides (Chou et al., 2019) with an amphipathic structure (Agarwal et al., 2016) that are active against certain bacteria, fungi and viruses (Ebenhan et al., 2014;Mahlapuu et al., 2016). The mechanism of antibacterial action is related to their ability to adjust membrane permeability, which destructs the membrane structure of the pathogen. One particular property that makes AMPs effective towards multidrug-resistant bacteria strains  is in its wide-scale multitargeted action (Joo et al., 2016;Zharkova et al., 2019). Usually, AMPs exhibit a net positive charge with a high ratio of hydrophobic amino acids that allows peptides to selectively bind to negatively charged bacterial membranes (Lei et al., 2019;Zhen et al., 2019) to either disrupt the membrane (Kumar et al., 2018), or to enter the bacterium and inhibit intracellular functions (Yazici et al., 2016). Peptides have low toxicity (Yin et al., 2017), high specificity (Lombardi et al., 2019), and are readily synthesized or modified (Tesauro et al., 2019). GH12 is one of the well-established antimicrobial peptides (Y. Wang et al., 2019). Previously, we have engineered a derivative of GH12, GH12-M2 (GLLWHLLHHLLH_GSGGG_K) and showed its antimicrobial activity (Xie et al., 2019). We have also shown that computationally designed AMP2 (KWKRWWWWR) prevents S. epidermidis growth at a very low concentration (Fjell et al., 2011;Yucesoy et al., 2015). AMPs are compatible with the mechanical, functional and structural properties of most polymers and are extremely biocompatible and biodegradable (Sharma et al., 2015). Nanofibres provide a viable means of delivery for AMPs while providing excellent structural features.
In this study, we investigate the viability of PEO-AMP nanofibres to represent an ideal release system to deliver AMPs for wound healing applications. We designed and produced M2 (type 1 AMP), and AMP2 (type 2 AMP) and integrated PEO nanofibres to make wound dressings. A wound dressing material can be created by loading peptides into water-soluble polymers that are efficient in their release. Such materials at the wound site can provide protection while also releasing active antimicrobial support. Thus, the growth of microorganisms is regulated by the antibacterial agents embedded in the structure of the fibre (Morais et al., 2016). We obtained promising results for both of PEO-M2 (type 1 AMP) and PEO-AMP2 (type 2 AMP) nanofibres on S. epidermidis, which will be an effective release mechanism for wound healing applications.

| Materials
Polyethylene oxide (PEO, M w 200,000 gmol −1 ) was purchased from Sigma-Aldrich (Poole, UK). Two AMPs were used including GH12-COOH-M2 (M w 1932 Da) (type 1 AMP) and AMP2 (M w 1517.8 Da) (type 2 AMP) and were synthesized at the Tamerler Laboratory at University of Kansas, USA (section 2.2). Distilled water was used as the solvent for all of the polymer solutions in this study.

| Antimicrobial peptide synthesis and purification
GH12-M2 (M2) (type 1 AMP) and AMP2 (type 2 AMP) AMPs were synthesized by solid-phase peptide synthesis (SPPS) using an AAPPTec Focus XC benchtop peptide synthesizer. Fmoc-rink amide resin with a 0.56 mmol/g substitution factor was used for both peptides. applied to the deprotected resin and mechanically mixed under nitrogen gas for 45 min to couple the amino acids to the resin. The resin was washed with DMF after the addition of amino acid, and the protocol was repeated for each subsequent amino acid. The resins with synthesized peptides were then dried with ethanol following synthesis to extract residual DMF. The peptides were cleaved from the resin, and the sidechains were deprotected using Reagent K (TFA/ thioanisole/ H2O/ phenol/ ethanedithiol (87.5:5:5:2.5)) and precipitated by cold ether. RP-HPLC was used to purify crude peptides, which were then lyophilized and stored at −20°C.

| Preparation of polymer and peptide solutions
PEO was prepared at different concentrations, and from preliminary testing, it was found that 15 w/v% produced maximal fibre yield.
The following peptide concentrations: 35, 70, 105, 140 and 175 μg/ ml, were added to the PEO polymer solutions. The solutions were F I G U R E 1 (a) Schematic diagram illustrating the pressurized gyration set-up and three key phases leading to fibre production; (b) schematic illustration of the antibacterial assessment protocol for AMP integrated PEO nanofibres magnetically stirred to obtain a homogenous solution and stored at 4°C for 48 h. 15 w/v% PEO solution which was used as a negative control was also prepared and magnetically stirred for 24 h.

| Pressurized gyration
The PG set-up shown in Figure 1a was used to spin the fibres in this study. The set-up consists of an aluminium cylindrical vessel (60 mm diameter, 35 mm height) with a total of 24 orifices on the wall of the vessel, each opening is 0.5 mm in diameter. The bottom is connected to a DC motor that generates a rotational speed of up to 36,000 rpm, and the top is connected to a constant nitrogen gas stream (N 2 ) that generates a flow pressure between 0.1 -0.3 MPa.
Before spinning, the polymer solution is poured on the inside of the rotating device. Solvent selection can greatly influence the morphology of the fibres.
High speed and flow pressure cause the polymer solution to erupt as a jet which elongates and thins. As the fibres thin, the solvent evaporates which results in solid fibre formation. In this study, a water-soluble polymer was used as water has a low volatility which allows the solution to elongate for longer, creating thinner strands than what would otherwise have been produced by using more volatile solvents. Furthermore, water has complete biocompatibility in vivo and for wound healing applications. In this work, the maximum rotation speed routinely available at the present time was used as it increases the centrifugal force, and as such, forcing the liquid out the perforations with a greater kinetic energy, which results in thinner fibres. When increasing the pressure, fibres with a smaller diameter are generated due to the gas pressure providing better elongation conditions. Finer diameter fibres are more desirable in this research as they correspond to a more intense antimicrobial release ability largely due to an increased porosity and higher available surface area.

| Fibre characterization
The fibre morphology was analysed using a Hitachi HN004 (Hitachi, Japan) SEM, that operated at an accelerating voltage of 5 kV. The samples were gold sputter coated using Quorum Q1500R ES (Quorum Technologies Ltd., UK) for 90 s before being loading on to the microscope stage. From the digital micrographs, the average fibre diameters were calculated using ImageJ software. In this case, 100 fibre strands were measured at random, and the average was estimated and plotted on histograms using Origin Pro computer software.

| Fluorescence and polarization imaging
Polarization contrast images were captured on a Zeiss Axioplan2 (Zeiss, Germany) microscope, with a 20×NA0.5 objective. Images were captured as brightfield, or polarization contrast with a pair of crossed polarizers, and two-quarter waveplate circular polarizers at 45 degrees. Images were captured with a Zeiss Axiocam HRc (Zeiss, Germany). Birefringence occurs when polarized light passes through an anisotropic crystalline lattice.

| Fourier transform infrared spectroscopy (FTIR)
The infrared spectra of nanofibre samples were recorded on a Fourier transform infrared spectroscopy (FTIR) spectrometer (PerkinElmer Spectrum-400, UK) between 4000 and 450 cm −1 with a resolution of 4 cm −1 .

| Peptide de novo structure modelling
Secondary structure models were developed using the PEP-FOLD 3.5 online server (Lamiable et al., 2016;Shen et al., 2014). PEP-FOLD 3.5 generates 3D structural peptide conformations between 5-50 amino acids long and creates PDB models for the best five structures. The AMP sequences were input, and 200 simulations were performed, assuming aqueous conditions and at a neutral pH. The models were grouped and categorized using sOPEP (optimized potential for efficient structure prediction). The most likely model was imported to UCSF chimera for visualization and recolouring.

| Antimicrobial activity
Staphylococcus epidermidis (ATCC 2988) was cultured according to the ATCC protocol in Nutrient broth (NB). Rehydrated frozen stock F I G U R E 2 Graph showing the effect of applied gas pressure on the fibre diameters using M2 peptides (type 1 AMP) and PEO at low concentrations (35 µg/ml)

| Spinning fibres
The PEO-AMP and PEO-control solutions were spun at 36,000 rpm using the PG apparatus. During initial testing, nanofibres were gen-

| Fibre characterization
The SEM images depict the formed fibres as fine fibre strands with a smooth surface topography (Figure 3). It is emphasized that there is a decrease in fibre size distribution as a result of an increase in the AMP concentration, where uniformity is a highly desired characteristic in wound healing applications.
The average fibre diameter for (M2) peptides at 75 µg/ml is 256 nm ± 100 nm (Figure 3g,h); however by increasing the AMP concentration to 105 µg/ml, the average fibre diameter is reduced to 230 nm ± 67 nm (Figure 3i,j). Further increasing the AMP concentration to 140 µg/ml, the average fibre diameter decreased to 212 nm ± 59 nm (Figure 3k,l). Moreover, using a different peptide, (AMP2), similar effects on nanofibre size distribution were observed. At 105 µg/ml to 140 µg/ml (AMP2), the average fibre diameter decreases from 232 nm ± 69 nm (Figure 3m,n) to 209 nm ±57 nm (Figure 3o,p), respectively. In the case of PEO nanofibres with no AMP, the average fibre diameter at 0.3 MPa was 335 nm ±182 nm (Figure 3q,r). Therefore, it is established that by increasing the AMP concentration in PEO nanofibres, the average fibre diameter decreases and results in a higher surface area to volume ratio. When adjusting the image to a 1 µm scale, it is obvious that the peptides appear on the surface by the dotted texture. In Figure 4d, the peptides are fused on the surface of the AMP2 (type 2 AMP) fibres with an enlarged view of one sector at a scale bar corresponding to 10 µm. The surface topography of the fibres appears smooth, and the peptides on the enlarged view appear dotted. It is assumed that the AMPs are embedded within the polymer matrix and also on the surface of the PEO nanofibres.  The hydrophobic ratio was determined using the APD to calculate and predict tool.

| Fourier transform infrared spectroscopy (FTIR) analysis
at 1650 cm −1 is assumed to correspond to the addition of the peptide, while retaining the bulk chemical properties of the control PEO fibres.

| Antimicrobial peptide structures
AMP integrated fibres were produced using M2 (type 1 AMP) and AMP2 (type 2 AMP) peptides. PEP-FOLD 3.5.8 was used to obtain the secondary structure peptide models from the amino acid sequences (Lamiable et al., 2016) (Figure 6). Physical and chemical properties for each of the AMPs were determined using the ExPASy ProtPram server (Gasteiger et al., 2005) (Table 1). The hydrophobic ratio was calculated using the Antimicrobial Peptide Database (APD) Calculate and Predict tool (Wang et al., 2015). Characteristic properties of AMPs include cationic charge, amphipathicity, hydrophobicity and α-helix secondary structure (Koehbach & Craik, 2019).
Previously, AMP2 has been linked to a titanium binding domain through an engineered spacer as a chimeric peptide in order to bring antimicrobial function to the titanium medical implants (Yucesoy et al., 2015). Chimeric peptides with incorporated AMP2 demonstrated successful antibacterial function against S. epidermidis.
However, this AMP has not been previously investigated as an agent incorporated in polymeric nanofibres designed to address infectionrelated challenges associated with wound healing. It was determined through de novo structure modelling that AMP2 has the α-helix secondary structure frequently observed in other AMPs with strong activity against pathogens such as S. epidermidis (Wisdom et al., 2016).
Moreover, the cationic charge and moderate hydrophobic ratio support the use of this peptide as an antimicrobial agent suitable for incorporation into polymeric nanofibres. The M2 peptide model shows mostly α-helix secondary structure with a positive charge; however, the hydrophobic ratio is lower than the hydrophobic ratio of AMP2. This could support AMP2 functioning more effectively as an AMP compared to M2 (Wisdom et al., 2020).  indicates that the most significant bacterial reduction was obtained at an AMP concentration of 105 µg/ml PEO-AMP2.

| CON CLUS I ON S AND FUTURE PROS PEC TS
Pressurized gyration method was used to produce PEO-nanofibre mesh with AMPs. We incorporated two different AMPs into the PEO F I G U R E 7 (a) Antimicrobial effect of PEO fibres loaded with different concentrations of M2 peptides (type 1 AMP) at 105-175 μg/ml against Staphylococcus epidermidis evaluated with AlamarBlue cell viability assay, and (b) Comparison of antimicrobial effect of PEO fibres loaded with M2 and AMP2 peptides (type 1 and type 2 AMPs) at 105 μg/ml against S. epidermidis nanofibres at varying concentrations (35 µg/ml-175 µg/ml) to investigate the highest bacterial reduction for S. epidermidis, which is a common bacterium associated with wound related complications.
The fabricated nanofibres demonstrated a high efficacy against S. epidermidis. The assessment of bacterial viability indicated a significant decrease in bacterial population with an increase in the AMP concentration. Both M2 and AMP2 peptides confirmed promising bacterial reduction; however, the greatest bacterial reduction was achieved with 105 µg/ml PEO-AMP2 nanofibres. Loading antibacterial peptides with water-soluble polymers such as PEO is an effective delivery method for the rapid release of antimicrobial ailment into a wound site. Furthermore, using water-based solvent systems also allows more environmentally friendly approaches while also ensuring maximal biocompatibility in open wound scenarios. The work presented here shows that AMPs incorporated into nanofibres are a promising design choice as biological dressings for the next generation of wound healing products. PG has now evolved further to make core-sheath fibres . Here the sheath contains a functional material which can be attached to a polymer while the core is a strong polymer. Thus, the AMPs can be in the PEO sheath and this will further enhance our strategy to manufacture wound healing meshes.