Antimicrobial fibers

Abstract

- 98 - Abstract The invention relates to new electrospun fiber materials of a biodegradable polymer loaded with antimicrobial peptides. At least about 50% of the electrospun 5 antimicrobial peptides are trapped within the fibers allowing the materials to maintain an antibacterial and an antibiofilm activity during at least 3-days after application. The materials of the invention may be used to treat mucosa and skin conditions such as infected wounds.

Description

[0001] Antimicrobial Fibers

[0002] Field of the invention

[0003] The present invention relates to the fields of Biotechnology, Tissue Engineering, Pharmaceutical Sciences, and Medicine. In particular, the invention relates to an antimicrobial peptide electrospinning composition, antimicrobial fibers material produced using such an electrospinning composition, and a preparation method thereof.

[0004] Background of the invention

[0005] Skin wounds are all, to some extent, microbiologically contaminated and hard to treat wound infection may develop for patients having deficient immune system (e.g. diabetic, elderly, immunocompromised persons) or infected with highly virulent pathogens. It has been estimated that 1.5 - 2 million people in Europe suffer from acute or chronic wounds (Lindholm, Searle and Searle, 2016) and 3.5 billion people worldwide suffer from oral diseases (oral wounds and wound infections). Biofilm formation is one of the main issues associated with chronic wounds (Wolcott, Rhoads and Dowd, 2008; Cowan, 2011; Percival et al., 2012; Larsen and Fiehn, 2017). The healthcare costs related to chronic wound infections are also a burden for society.

[0006] The treatment of chronic skin wounds and oral wounds requires a multidisciplinary approach involving surgical removal of the biofilm and then the application of suitable antimicrobial treatment and wound dressing. At present, traditional topical antimicrobial / antibacterial formulations for wounds are used and often systemic administration of antibiotics is performed (Lee, Kuti and Nicolau, 2005). However, these topical dosage forms require frequent applications and are ineffective in the presence of wound exudate, biofilm, or enzymes (Siddiqui and Bernstein, 2010; Sankar et al., 2011), making topical treatment extremely challenging. As for systemic administration of antibiotics, there is an important risk of toxicity during treatment and, at the same time, insufficiently low drug levels in the wound (Bowler, Duerden and Armstrong, 2001; Wolcott, Rhoads and Dowd, 2008; Boateng and Catanzano, 2015). Furthermore, with the global development of antibiotics resistance, the use of antibiotics is getting less and less effective. It is thus crucial to better protect and heal wounds and to inhibit the formation of biofilm. Antimicrobial peptides (AMPs) are potent, broad-spectrum antimicrobials which have demonstrated a great potential as novel therapeutic agents. Since microbial membranes are their primary targets, less resistance has been reported than with antibiotics (Boparai and Sharma, 2020). AMPs could thus be key components of improved wound dressings. The main disadvantage of AMPs is their low stability (Zhang, 2021) and toxicity when dosage is increased. Novel carrier systems and nanotechnological approaches are thus needed for their successful and controlled delivery.

[0007] Rajasekharan et al (US 2020 / 0237857) have developed a solid antimicrobial hydrogel comprising a crosslinkable amphiphilic component with an AMP covalently linked to one of its domains. This antimicrobial hydrogel is thus suitable for sustained performances as AMPs are immobilized on it. However, only one synthetic AMP has been used so far with this system, suggesting a lack of universality. Moreover, hydrogels structural properties differ a lot from the skin's extracellular matrix.

[0008] Electrospinning is a well-known and easily scalable method of production of nano- or microfibers presenting a number of advantages for wound treatment including their resemblance to the natural extracellular matrix (ECM), their high surface area to volume ratio, their tunable porosity and their ability to load active ingredients. Novel treatment strategies have been developed to fight bacterial wound infections including advanced local preparations produced by electrospinning (Torres-Martinez et al., 2018a). For example, Restrata®, Phoenix® and Nanotrix® are approaching or have already reached the market. These systems comprise synthetic polymers and are developed for the treatment of chronic skin wounds or as temporary skin substitutes. However, they do not contain any drug.

[0009] It has recently been shown that electrospun fibers can be functionalized with AMPs (Felgueiras and Amorim, 2017), with promising effects on wound healing. However, despite great promises, no AMP-loaded electrospun fiber matrix for wound infection prevention and treatment has yet reached the market, underlying the difficulties that need to be overcome in order to develop effective and stable AMP-loaded wound dressings. Indeed, the selection of a suitable composition and electrospinning conditions is very challenging as AMPs functionalities need to be preserved and their antibacterial activity maintained for several days (Lanno et al., PCT / EP2022 / 076874; Ramos et al., 2023). Depending on the polymer and solvent, different electrospun fiber matrices with different properties can be obtained (Preem et al., 2017; Zupancic et al., 2018; Akhmetova et al., 2020; Lanno et al., 2020; Ramos et al., 2020). The drug release behavior from electrospun matrices largely depends on the used polymers (Preem et al., 2017, 2019; Zupancic et al., 2018; Ramos et al., 2020) and solvents (Lanno et al., 2020). Similarly, the mechanical properties of the electrospun matrices differ dramatically depending on the materials used. Attempts so far have led to the degradation of AMPs during electrospinning or poor drug efficiency after 24h. This is the case in recent publications describing electrospun AMPs (Kielholz et al., 2022) and electrospun fiber matrices (US20220096706 and EP3946144).

[0010] Traditional / monoaxial electrospinning, while simpler to use and more cost- effective, is particularly challenging for AMPs as solvents suitable for both AMPs and polymers are difficult to find. To overcome that difficulty, most research teams have relied on coaxial or emulsion electrospinning, allowing to use hydrophilic polymers or mixtures of hydrophilic and hydrophobic polymers (Kielholz et al. 2022; Yu et al., 2021; Amariei et al., 2018). Unfortunately, the results obtained are disappointing (less than 24 h efficiency). Some polymer mixtures (e.g. PAA-PVA blends) even required additional heat-treatment and cross-linking steps to keep the polymers stable enough in an aqueous environment.

[0011] The inventors have previously developed a method (Lanno et al. PCT / EP2022 / 076874) which can be used to incorporate unstable AMPs. However, even if AMPs are incorporated without major drug loss, they are quickly released from the fibers (cf. p. 62 of the present application). Such fiber matrices can thus be used as drug delivery systems, but not for prolonged antimicrobial effect.

[0012] There is thus still a strong need to develop new AMP loaded electrospun fiber mats (also called matrices) that could be used for wound dressing with a sustained antibacterial activity over several days. It is especially desired to develop a method of production of such mats that would be compatible with monoaxial electrospinning. The present invention meets these and other needs.

[0013] Summary of the invention

[0014] The inventors have developed new antimicrobial fiber materials comprising electrospun fibers (e.g. microfibers) of a biodegradable polymer loaded with antimicrobial peptides (AMPs). Surprisingly, a significant part of the AMPs is trapped within the fibers, allowing the material to have a prolonged antibacterial and antimicrobial effect that lasts at least 7 days, with demonstrated wound healing properties. The inventors also discovered that their antimicrobial fiber material is also efficient against biofilm, which is a decisive advantage.

[0015] Importantly, the invention allows the local treatment of wound infections, which enables to use higher drug doses than with systemic treatment while reducing side effects (Manzo et al., 2018). It is also important to stress that the electrospun matrices of the invention have suitable mechanical properties for use as wound dressings and can be sterilized or disinfected without modifying their key properties or efficiency.

[0016] The antimicrobial fiber material of the invention has been obtained with a new AMP composition for electrospinning. This composition comprises a solution of a biodegradable aliphatic polymer, an AMP and a polar organic solvent (all as described herein). It is suitable for all AMPs and can be used with most electrospinning methods, including monoaxial electrospinning. The composition of the invention ensures the preservation of the functionalities of the AMPs after electrospinning.

[0017] For the composition development, it was needed to select suitable polymer(s) and its(their) concentrations together with suitable solvent(s) that enable successful electrospinning. The solvent needs to be able to dissolve (or disperse) AMPs as well as the polymer(s). The concentrations of AMP(s) and polymer(s), as well as the electrospinning process and environmental parameters need to be fine-tuned in order to obtain the most homogeneous, stable and smooth drug-loaded fibers with appropriate AMP content and preserved biological activity.

[0018] Thus, in a first aspect, the invention relates to an antimicrobial fiber material comprising electrospun fibers of a biodegradable polymer loaded with antimicrobial peptides (AMPs), wherein at least about 50% of the AMPs are trapped within the fibers. Preferably, at least about 55%, more preferably at least about 60%, still preferably at least about 65%, even more preferably at least about 70% of the AMPs are trapped within the fibers. Preferably, the trapped AMPs essentially remain within the fibers for at least 1 day, preferably at least 2 days, more preferably at least about 3 days, even more preferably at least about 7 days under normal conditions of use of the material. Preferably, the nontrapped AMPs are essentially released from the fibers in no more than a day, preferably in no more than 10 h, even more preferably in no more than 1 h, under normal conditions of use of the material. Without being bound by theory, this immediate release of nontrapped active agent, followed by long-acting effect from trapped AMP is believed to give an enhanced wound-treatment effect.

[0019] In a preferred embodiment, the fibers have been electrospun in the presence of a polar organic solvent. Preferably, the fibers have a diameter comprised between about 0.01 pm and about 10 pm, more preferably between about 0.08 pm and about 10 pm, even more preferably between about 0.1 pm and about 5 pm.

[0020] In another preferred embodiment, the material comprises between about 0.1% and about 20% in weight of AMPs, preferably between about 1% and about 10% in weight of AMPs.

[0021] In yet another preferred embodiment, the material is not toxic, preferably not toxic to eukaryotic cells.

[0022] In some embodiments, the electrospun fibers are further loaded with at least one antibiotic (AB). The antimicrobial fiber material therefore may comprise at least one AB as referred to with respect to any aspect described herein.

[0023] In a second aspect, the invention also relates to a method of electrospinning AMPs comprising the following steps: a) preparing a solution of a biodegradable polymer, a polar organic solvent, and an AMP; b) electrospinning the solution obtained at step a).

[0024] Preferably, step a) last between about 12 h and about 24 h.

[0025] In a preferred embodiment, step a) comprises the four following sub-steps: ai) adding a biodegradable polymer to a polar organic solvent;

[0026] 32) mixing the solution obtained at step ai); as) adding an AMP to the solution of step as);

[0027] 34) mixing the solution obtained at step as).

[0028] Preferably, step as) lasts between about 12 h and about 36 h (preferably between about 12h and about 24 h). Preferably, step 34) lasts between about 10 min to about 4 h. Preferably, step as) is done under continuous mixing of the solution obtained at step as). Preferably, step b) occurs between about 1 min and about 10 min after step a). In a variant of the second aspect, the invention also relates to a method of electrospinning AMPs comprising the following steps: a) preparing a solution of a biodegradable polymer, a polar organic solvent, and an AMP; b) electrospinning the solution obtained at step a).

[0029] Preferably, step a) last between about 12 h and about 24 h.

[0030] In a preferred embodiment of this variant, step a) comprises the four following substeps: ai) Adding a biodegradable polymer and AMP to a polar organic solvent;

[0031] 32) Mixing the mixture obtained in step ai.;

[0032] Preferably, step 32) lasts between about 12 h and about 48 h. Preferably, step 32) lasts between about 15 to about 30 h, most preferably between about 16 hours and about 24 hours. This variant may be used preferentially where the AMP concentration is above around 4%, (e.g. 4 to 20%).

[0033] In a preferred embodiment, the solution of step a) comprises between about 0.1% and about 20%, preferably between about 0.5% and about 15%, more preferably between about 1% and about 10% in weight of AMPs.

[0034] In a particular embodiment, the method further comprises a step of sterilization of the material obtained at step b. Preferably, the sterilization technique is selected from the group consisting in gamma irradiation, UV treatment, and a combination thereof, more preferably gamma-irradiation.

[0035] In a particular embodiment, the solution prepared in step a) further comprises at least one AB.

[0036] Step a) may comprise the following steps: ai) adding at least one biodegradable polymer to a polar organic solvent;

[0037] 32) mixing the solution obtained at step ai);

[0038] 33) adding an AMP and an AB to the solution of step 32);

[0039] 34) mixing the solution obtained at step 33). In an alternative embodiment, the solution prepared in step a) further comprises a least one antibiotic (AB) and step a) may comprise the following steps: ai) Adding a biodegradable polymer, AB and AMP to a polar organic solvent;

[0040] 32) Mixing the mixture obtained in step ai;

[0041] Preferably, step 32) lasts between about 12 h and about 48 h. Preferably, step 32) lasts between about 15 to about 30 h, most preferably between about 16 hours and about 24 hours.

[0042] In a third aspect, the invention still relates to an AMP composition for electrospinning comprising a solution of a biodegradable polymer, an AMP and a polar organic solvent. Preferably, the solution comprises between about 0.1% and about 20% in weight of AMPs, preferably between about 1% and about 10% in weight of AMPs.

[0043] In a particular embodiment where AB are present in the solution in step a), it is preferred if the concentration of AB is less than that of AMP. Preferably, the solution comprises between about 0.1% to about 20% by weight of ABs, preferably between about 0.5% to 7% by weight of ABs, especially 1.0% to 5.0% by weight of ABs. The preferred ratio of AMP to AB in the solution in step a) is 10:1 to 1:1, especially 5:1 to 1:1, such as about 2:1 AMP to AB.

[0044] In a preferred embodiment, the biodegradable polymer according to the first, second and third aspect of the invention is a cyclic or an aliphatic polymer, preferably an aliphatic polymer. Preferably, said biodegradable polymer is a polyester, preferably an aliphatic polyester, selected from the group consisting in polycarbonates, polylactides, polycaprolactones, polyglycolides, polyhydroxyalkanoates, poly(dioxanone)s, poly(lactide- co-glycolide), poly(butyrolactone)s, poly(valerolactone)s, and a combination thereof, more preferably selected from the group consisting in Poly(P-butyrolactone) (PBL), Poly(£- caprolactone-co-lactide) (PCLA), Poly(3-hydroxybutyrate) (PHB), Poly(3-hydroxyvalerate) (PHV), Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), Poly(methyl methacrylate) (PMMA), Polyglycolide (PGA), Poly( lactic acid) (PLA), Poly( L-lactide) (PLLA), Poly(D, L-lactic acid) (PDLA), E-polycaprolactone (PCL), a copolymer of PDLA and PCL (PLC) such as a PLC with a molar ratio of PDLA on PCL of about 70 / 30 (i.e. 70 / 30 PLC), poly(trimethylene carbonate) (PTMC), a derivative thereof and a combination thereof, even more preferably a polyester selected from the group consisting in PDLA, PCL, PLC, such as 70 / 30 PLC, a derivative thereof and a combination thereof. In another preferred embodiment, the AMP according to the first, second and third aspect of the invention is selected from the group consisting in maximin H5, dermcidin, cecropins, andropin, moricin, ceratotoxin, melittin, magainin, dermaseptin, bombinin, esculentins, buforin II, CAP18, abaecin, drosocin, apidaecin, attacin, prophenin, indolicidin, brevinins, such as brevinin-1, protegrin, tachyplesins, defensins, drosomycin, bacitracin, boceprevir, dalbavancin, daptomycin, enfuvirtide, oritavancin, teicoplanin, telaprevir, telavancin, vancomycin, guavanin 2, bacteriocin, peptaibols, plectasin, hydramacin, aurelin, mastoparan, thioester-containing protein 1, aurein, cathelicidins, reglll peptides, nisin, copsin, diptericin, pleurocidin, D- pleurocidin, L-temporin, LL-37, colistin, gramicidin, a derivative thereof and a combination thereof. Preferably, the AMP is a cationic antimicrobial peptide, preferably selected from the group consisting in pleurocidin, D-pleurocidin, L-temporin, LL-37, colistin, bacitracin, gramicidin, daptomycin, a derivative and a combination thereof. Alternatively, the AMP is selected from the group consisting in pleurocidin, L-temporin, LL-37, colistin, daptomycin, a derivative and a combination thereof, preferably colistin or a derivative thereof.ln yet another preferred embodiment, the polar organic solvent according to the first, second and third aspect of the invention is selected from the group consisting in dimethylformamide, acetonitrile, acetone, hexamethylphosphoric triamide, N,N- diethylacetamine, 4-methylmorpholine-N-oxide monohydrate, N-methylpyrrolidinone, dimethylsulfoxyde (DMSO), tetrahydrofurane (THF), dioxan, methylcarbonate, fluoroalcohols, a derivative, and a combination thereof, preferably the polar organic solvent is a fluoroalcohol. Preferably, the fluoroalcohol is selected from the group consisting in trifluoroethanol (2,2,2-trifluoroethanol; TFE), hexafluoroisopropanol (l,l,l,3,3,3-hexafluoro-2-propanol; HFIP), perfluoro-tert-butanol (1,1, 1,3,3, 3-hexafluoro- 2-(trifluoromethyl)-2-propanol; PFTB), 2-fluoroethanol, and a combination thereof, preferably the fluoroalcohol is HFIP. In a particular embodiment, the AMP composition for electrospinning additionally comprises at least one antibiotic (AB). Suitable ABs are described herein.

[0045] In a fourth aspect, the invention further relates to the use of the composition according to the third aspect for electrospinning AMPs.

[0046] In a preferred embodiment, electrospinning according to the first, second, third and fourth aspect of the invention is not perfusion electrospinning. Preferably, electrospinning is monoaxial electrospinning. In a fifth aspect, the invention yet relates to an antimicrobial fiber material obtained by the method of electrospinning according to the second aspect, or by the use of the composition according to the fourth aspect.

[0047] In a preferred embodiment, the material according to the first aspect or the fifth aspect of the invention has an antimicrobial activity, preferably an antibacterial activity. Preferably, the antimicrobial activity, preferably antibacterial activity, is of at least about a 3 log reduction. Preferably, the material according to the first aspect or the fifth aspect of the invention has an antibiofilm activity. Preferably, the antibiofilm activity is of at least about a 3 log reduction. Preferably, the antimicrobial activity and / or antibiofilm activity last at least about 3 days, preferably at least about 7 days, optionally at least about 14 days under normal conditions of use of the material.

[0048] In a sixth aspect, the invention further relates to an antimicrobial fiber mat (also referred to as a fiber matrix - the terms are used equivalently herein) comprising a material according to the first and fifth aspects of the invention having a flat structure and a thickness comprised between about 0.01 mm and about 5 mm, preferably between about 0.08 mm and about 0.1 mm. Preferably, the mat has a hardness comprised between about 200 g and about 1000 g, more preferably between about 300 g and about 800 g, even more preferably about 500 g. Preferably, the mat has a deformation at hardness comprised between about 2 mm and 20 mm, more preferably between about 4 mm and about 15 mm, even more preferably about 10 mm. Preferably, the mat has a hardness work done comprised between about 10 mJ and 60 mJ, more preferably between about 20 mJ and about 50 mJ, even more preferably about 35 mJ.

[0049] In particular embodiments where the antimicrobial fiber mat comprises both AMP and AB, it may be referred to as a "combination mat".

[0050] In a seventh aspect, the invention further concerns an antimicrobial multilayer system comprising a mat according to the sixth aspect of the invention and further comprising at least one secondary dressing. Preferably, at least one secondary dressing is absorptive and / or non-adhesive.

[0051] In a preferred embodiment, the antimicrobial multilayer system comprises at least two secondary dressings, a first secondary dressing on one side of the mat comprising an absorptive and / or adhesive layer and a second secondary dressing on the other side of the mat comprising a backing layer. Preferably, at least a part of one of the sides of the mat is free of any additional dressing, optionally one of the sides of the mat is free of any additional dressing.

[0052] In another preferred embodiment, the material according to the first or fifth aspect, the mat according to the sixth aspect or the multilayer system according to the seventh aspect further comprise a chelating agent. Preferably, the chelating agent is selected from the group consisting in Dimercaprol (BAL), ethylenediaminetetraacetic acid (EDTA) salts such as calcium EDTA or sodium EDTA, succimer (DMSA), penicillamine, trientine hydrochloride, deferoxamine mesylate, deferiprone, deferasirox, pentetate calcium trisodium (Ca-DTPA), pentetate zinc trisodium (Ca-DTPA), prussian blue (Radiogardase), and a combination thereof, preferably the chelating agent is an EDTA salt, more preferably sodium EDTA. Preferably, the chelating power induced by the chelating agent is equivalent to the chelating power induced by sodium-EDTA at a concentration comprised between about 0.1 mM and about 10 mM in the microenvironment of the material.

[0053] In an eighth aspect, the invention also concerns the use of the material according to the first or fifth aspect, of the mat according to the sixth aspect, or of the multilayer system according to the seventh aspect for the treatment of a subject in needs thereof.

[0054] In an embodiment, the material, mat or multilayer system is sterilized prior to its application to the subject. Preferably, the sterilization technique is selected from the group consisting in gamma irradiation, UV treatment, and a combination thereof, more preferably gamma-irradiation.

[0055] In a preferred embodiment, the treatment is a topical treatment, preferably a topical treatment of the epithelium, more preferably a topical treatment of the skin or of a mucosa, such as the oral mucosa. Preferably, the treatment is a treatment of a skin condition, more preferably selected from the group consisting in a wound, a cancer such as a skin cancer, acne, atopic dermatitis, psoriasis, ichthyosis, scars and dull skin, such as epidermolysis bullosa, skin burns, plastic surgery support or skin implants support (for example skin grafts), or a combination thereof.

[0056] In a particularly preferred embodiment, the treatment is a wound treatment, more preferably an infected wound treatment. Preferably, the treatment is a skin wound treatment, more preferably an infected skin wound treatment. The infection is preferably a bacterial, viral or fungal infection, more preferably a bacterial infection, even more preferably a bacterial infection with biofilm. The wound may be a chronic wound, such as chronic non-healing ulcers. The wound may also be an acute wound, such as traumatic acute wounds.

[0057] In a preferred embodiment, the material, the mat or the multilayer system is removed or replaced after between 1 and about 14 days, preferably after between 1 and about 7 days, more preferably after between 2 and about 4 days, even more preferably after about 3 days. Preferably, a new material, mat or multilayer system is applied to the subject when the previous one is removed as many times as necessary, for example until full recovery.

[0058] In still another preferred embodiment, the subject is a human or a non-human animal, preferably a mammal, more preferably a mammal selected from the group consisting in dogs, cats, horses, cows, pigs, sheep, goats, rabbits, hamsters, guinea pigs, hens, chickens, ducks and non-human primates. Preferably, the subject is a human.

[0059] Finally, in a ninth aspect, the invention concerns an antimicrobial fiber material according to the first aspect formed or formable by a method according to the second aspect.

[0060] Brief Description of the drawings

[0061] Figure 1: Schematic summary of the invention. Key: AMP- antimicrobial peptide.

[0062] Figure 2: Scanning electron microscopy (SEM) images showing the morphology of antimicrobial peptide (AMP) loaded electrospun fibers.

[0063] Figure 3: Scanning electron microscopy (SEM) images showing the morphology of antimicrobial peptide colistin 4% (COL)-loaded electrospun fiber matrices in different formulations (PDLA, PCL, PLC and PDLA+PCL). Square indicates 10p.m.

[0064] Figure 4: Scanning electron microscopy (SEM) images of AMP-loaded electrospun matrices: COL 8% + PCL (A); COL 8% + PLC (B); COL 4% + PDLA + PCL (50:50) (C); COL 4% + PDLA + PCL (70:30) (D); COL 8% + PDLA (AA:FA) (E); PLE 4% + PCL (F); BAC 8% + PCL (G) showing the morphology of the electrospun fibers.

[0065] Figure 5: Scanning electron microscopy (SEM) images of AMP+AB-loaded electrospun matrices COL 2.6 % + CIP 1.3% + PDLA+ PCL (A); COL 2.6 % + CLIN 1.3% + PDLA+ PCL (B); COL 2.6 % + CEF 1.3% + PDLA+ PCL (C) and AB-loaded matrices CIP 4% + PDLA (D); CIP 8% + PDLA (E); and CLIN 8% + PDLA (F) as controls, showing the morphology of the electrospun fibers.

[0066] Figure 6: Antimicrobial peptides pleurocidin (PLE) and colistin (COL) degradation in 1 x PBS buffer at 37 °C based on HPLC experiment data. Key: COL - colistin; PLE - pleurocidin.

[0067] Figure 7: (A) Growth inhibition of E. coli in biorelevant medium (DMEM / Ham-12 + 10% FBS) after 24 h. (B) Matrix sterility after 24 h in vitro experiment with E. coli. The data are represented as a mean of three biological replicates (N=3).

[0068] Figure 8: (C) Growth inhibition of 5. aureus in biorelevant medium (DMEM / Ham-12 + 10% FBS) after 24 h. (D) Matrix sterility after 24 h in vitro experiment with 5. aureus. The data are represented as a mean of three biological replicates (N=3).

[0069] Figure 9: (E) Growth inhibition of P. aeruginosa in biorelevant medium (DMEM / Ham-12 + 10% FBS) after 24 h. (F) Matrix sterility after 24 h in vitro experiment with P. aeruginosa. The data are represented as a mean of three biological replicates (N=3). The data are represented as a mean of three biological replicates (N=3).

[0070] Figure 10: The log reduction values with standard deviation (N =3 ) of different antimicrobial wound matrices against E. coli, P. aeruginosa, and 5. aureus illustrating the killing efficacy for various formulations.

[0071] Figure 11: Mean and standard deviation (N=3) of the log-transformed number of E. coli DSM1103 CFUs detected on PLE 8% + PDLA and PDLA control matrices after modified ASTM E218-18 assay. **p<0.01, unpaired t-test.

[0072] Figure 12: Mean and standard deviation (N =2-3) of the log-transformed number of E. coli DSM1103 CFUs detected on different electrospun matrices in ASTM E218-18 assay after soaking in PBS at 37 °C for 1, 3 or 7 days. The dotted line represents the detection limit.

[0073] Figure 13: Antibiofilm activity of COL-loaded electrospun matrices (COL 4%) by measuring the number of biofilm bacteria after 24 h on top of pig skin (ex vivo wound infection model). A - C Number of biofilm bacteria after 24 h using ES fibrous matrices. E. coli DSM 1103 was used for testing. Results are shown in logarithmic scale as a number of colony forming units (CFU), with standard deviation bars (n=3). Experiments were performed using at least 3 technical replicates. Key: A. formulation PDLA + HFIP + COL 4%; B. PCL + HFIP+ COL 4%; C. PLC + HFIP+ COL 4%. Hatched bars indicate COL containing wound matrices.

[0074] Figure 14: Antimicrobial peptide colistin (COL) contents before sterilization and after sterilization treatment measured by HPLC. Different formulations tested A- PDLA + HFIP + COL 4%; B- PCL + HFIP + COL 4%; C- PLC+ HFIP + COL 4%. Results are shown with standard deviation bars (n=3). Statistical significance is shown *, p<0.05. Experiments were performed using at least 3 technical replicates.

[0075] Figure 15: European Pharmacopoeia sterility testing. Key from left to right: negative control, positive control, and ES fibrous wound matrix in a sterility test.

[0076] Figure 16: Samples of matrices electrospun from mixtures of PDLA (5%) and PCL (9%) solutions in HFIP after 3 weeks of being in biorelevant aqueous conditions; from the left: 50 / 50 weight ratio mixture and 70 / 30 weight ratio mixture.

[0077] Figure 17: Mechanical properties (hardness; deformation at hardness; hardness work done) and thickness of antimicrobial peptide (AMP) colistin (COL) loaded electrospun fiber matrices (not gamma sterilized) (N=3; error bars= SD). Key: Hatched bars indicate COL containing wound matrices (COL 4%). A. formulation PDLA + HFIP + COL 4%; B. PCL + HFIP + COL 4%; C. PLC + HFIP + COL 4%.

[0078] Figure 18: Results from direct cell viability assay on Baby Hamster Kidney fibroblast cells (BHK-21). Results are shown for both gamma sterilized and non-sterilized samples and for all different formulations (A- PDLA + HFIP + COL 4%; B- PCL + HFIP + COL 4%; C- PLC+ HFIP+ COL 4%) at timepoint zero and after 24 h. Experiments were performed using at least 3 technical replicates.

[0079] Figure 19: CCK-8 activity of cells treated with fiber matrix extracts for 24 hours and control cells. The dotted line marks the CCK-8 activity of control cells (also shown in the last column).

[0080] Figure 20: Antibacterial effects of different wound dressings on Staphylococcus aureus in chronic rat wound infection model after 24 or 72 hours of treatment. Values shown as the reduction of bacterial loads in percentages from the average bacterial load of untreated wounds in comparable timepoints. Control treatments (commercial preparation SuprasorbX) is also shown (N=1...6). Colistin (COL) + PCL matrix (7.1%) using AA:FA solvent system, COL+PDLA matrix (8%) using DMF as a solvent and nisin+PCL (8%) matrix using HFIP as a solvent were also prepared for comparison.

[0081] Figure 21: Wound size changes. (A) Initial dermatome wound size in cm2measured immediately after wounding. (B) Remaining wound size from initial wound after 24 or 72 hours of 5. aureus-infected wounds without treatment. (C) Remaining wound size from initial wound after treatment for 24 or 72 hours. Commercial Suprasorb X + PHMB shown as control. Remaining wound sizes shown as percentages from initial wound (100%) based on wound area image analysis.

[0082] Figure 22: Heatmap of wound severity scores based on histopathology reports after 24 and 72 hours of treatment. The shade represents wound severity, with darker shades indicating more severe wounds and lighter shades indicating less severe ones. The maximum possible score is 12. Untreated wounds and commercial Suprasorb X + PHMB are shown as controls in Italics. X - no data.

[0083] Detailed Description of the Invention

[0084] The inventors have developed an antimicrobial fiber material comprising electrospun fibers of a biodegradable aliphatic polymer loaded with antimicrobial peptides (AMPs). At least about 30% of said AMPs are trapped within the fibers.

[0085] Definitions

[0086] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0087] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the present application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, representative methods and materials are herein described.

[0088] As used herein, the terms "a", "an", and "the" refer to "one or more" when used in this application, including the claims. Thus, for example, reference to "a polymer" includes mixtures of one or more such compounds, two or more such compounds, and the like.

[0089] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about". As used herein, the term "about" when referring to a measurable value such as an amount of weight, time, dose, etc. is meant to encompass in one example variations of ± 20%, preferably ± 10%, more preferably ± 5%, alternatively ± 2%, still alternatively ± 1% and yet alternatively ± 0.1% from the specified amount. For example, "about 20" may include all the values ranging from ± 5% of 20, i.e. a range of values from 19 to 21. The term consisting "substantially" of, as used herein may mean having no more than 10% (e.g. by weight) of other materials, such as 5 wt% or less thereof, preferably 1 wt% or less. Corresponding, terms such as "substantially free" as used herein refer to something have no more than 10 % (e.g. by weight) of the indicated component, such as 5 wt% or less thereof, preferably 1 wt% or less thereof, especially 0.1 wt% or less thereof. All % are by weight unless indicated otherwise.

[0090] As used herein, the term "antimicrobial" refers to an agent that kills or stops the growth of microorganisms.

[0091] As used herein, the term "antibacterial" refers to an agent that kills or stops the growth of bacteria.

[0092] Antimicrobial (e.g. antibacterial) activity can be measured as a reduction in living microbial (e.g. bacterial) cells following contact for a period of, for example, 1 hour or 24 hours.

[0093] Antimicrobial, such as antibacterial, effectiveness is according to the invention expressed in log reduction value, i.e. the logarithm (base 10) of the ratio of the initial microbial count to the final microbial count. For example, a 3 log reduction means that the material has reduced the microbial population by 99.9%. A 3 log reduction is often used as a threshold in standards and guidelines for a significant antimicrobial activity.

[0094] As used herein, the term "antibiofilm" refers to the ability of certain agents to slow down, stop or prevent the formation or growth of complex microbial communities called biofilms.

[0095] Antibiofilm effectiveness is according to the invention expressed in log reduction value, i.e. the logarithm (base 10) of the ratio of the count of biofilm bacteria in a given sample to the count of biofilm bacteria in a control sample. The term "given sample" as used herein may referto the material according to the invention and control sample to the same material without AMPs. The count of biofilm bacteria thus refers to the count of biofilm bacteria present on the surface or in contact with the material according to the invention. For example, a 3 log reduction means that the material has a population of biofilm bacteria corresponding to 99.9% of the control population.

[0096] As used herein the terms "antimicrobial peptide (AMP)" and "host defense peptide" are equivalent and refer to a diverse group of peptides that are potent, broadspectrum antimicrobial agents. As used herein, the term "polymer" refers to a chain of chemically bonded monomers, the term "polyester" refers to a category of polymers that contain the ester functional group in every repeat unit of their main chain, the term "polycarbonate" refers to polyesters of carbonic acids, the term "polylactic acid", "poly(lactic acid)" and "polylactide" are equivalent and refer to polyesters having repetition of (C3H4O2) as the backbone of their main chain, the term "polyhydroxyalkanoates" refers to a group of polyesters composed of hydroxyalkanoate monomers and produced by microorganisms, the term "polycaprolactones" refers to polyesters formed by ringopening polymerization of E-caprolactone under the catalysis of metal anion complex catalysts, the term "polyglycolides" refers to polyesters formed by ring-opening polymerization of glycolide monomers, the term "poly(dioxanone)s" refers to polyesters of multiple repeating ether-ester units, the term "poly(lactide-co-glycolide)" refers to polyesters that are copolymers of poly lactic acid(s) and poly glycolic acid(s), the term "poly(butyrolactone)s" refers to polyesters composed of butyrolactone monomers, the term "poly(valerolactone)s" refers to polyesters made from valerolactone monomers through the ring-opening polymerization of 6-valerolactone.

[0097] As used herein, the term "synthetic" refers to polymers made by chemical synthesis.

[0098] As used herein, the term "hydrophobic" refers to polymers that do not mix with water.

[0099] As used herein, the term "aliphatic" refers to organic compounds containing carbon and hydrogen joined together in straight chains, branched chains, or non-aromatic rings.

[0100] As used herein, the term "biodegradable" refers to the ability of a material to decompose after interactions with biological elements such as bacteria, thereby avoiding pollution.

[0101] As used herein, the term "bioresorbable" refers to the ability of a material to get degraded in the place of its use. For a dressing, it allows not to remove it and to let it degrade on site.

[0102] As used herein, the term "biodurable" refers to the ability of a material to resist the body's conditions (corrosion, oxidation, or other forms of biological degradation) and to keep its shape and properties. As used herein, the term "biocompatible" refers to the ability of a material to be compatible with living tissues or a living system by not being toxic, injurious, or physiologically reactive and not causing immunological rejection.

[0103] As used herein, the term "polar organic solvent" refers to carbon-based solvents having a dipole moment.

[0104] As used herein, the term "fluoroalcohols" refers to organofluorine compounds consisting of an alcohol functional group with at least one C-F bond.

[0105] As used herein, the term "electrospinning" refers to a fiber production method that uses electrical forces to draw charged threads of polymer solutions for producing nanofibers with diameters ranging from nanometers to micrometers.

[0106] As used herein, the term "chelating agent" refers to chemical compounds whose structures permit the attachment of their two or more donor atoms (or sites) to the same metal ion simultaneously thereby producing one or more ring-like complexes.

[0107] According to the invention, the chelation capacity of a chelating agent is the amount (measured in moles or grams) of metal ions that can be effectively bound by one mole or one gram of the chelating agent, respectively.

[0108] Antimicrobial fiber material

[0109] In a first aspect, the invention relates to an antimicrobial fiber material comprising electrospun fibers of a biodegradable polymer loaded with antimicrobial peptides, wherein at least about 30% of the antimicrobial peptides are trapped within the fibers.

[0110] The antimicrobial fiber material of the invention comprises electrospun fibers.

[0111] As used herein the term "fiber" refers to nanofibers or microfibers having a diameter comprised between about 0.001 pm and about 50 pm, preferably between 0.01 pm and 10 pm, more preferably between about 0.08 pm and about 10 pm, even more preferably between about 0.1 pm and about 5 pm.

[0112] The fibers of the material according to the invention have preferably a smooth surface and homogeneous fiber diameter sizes (narrow size distribution) within the material. For example, the diameter of fibers in the material may not vary more than about 20%, preferably about 15%, more preferably about 10%, even more preferably about 5% from the average diameter size of the fibers within said material. The fibers according to the invention are electrospun fibers. Such fibers have been obtained by electrospinning of a polymer. Any electrospinning technique, well-known from the man skilled in the art, can be used to produce the electrospun fibers of the invention. However, it is advantageous and preferred to use monoaxial electrospinning. Indeed, monoaxial electrospinning is a simple, cost effective and easily scalable method. As used herein the terms "monoaxial electrospinning", "axial electrospinning", and "uniaxial electrospinning" are equivalent and refer to a technique that produces ultra-fine polymeric fibers from a single solution or melt, involving the use of a capillary nozzle from which the polymer solution or melt is pushed out in a high-voltage electric field, where the material is stretched and thinned into solid fibers and collected on a ground metal surface. Other electrospinning techniques can be used, including needle-based and needleless electrospinning techniques, preferably electrospinning techniques selected from the group consisting in coaxial, multijet, magnetic fluid, roller, centrifugal, porous tube, bubble, triaxial, conical wire coil, ball, disk, wet (3D), cone, spiral coil, ultrasound- enhanced, and microfluidics electrospinning as well as electrospinning related methods of solution blow-spinning (Daristodle JL et al.) and halospinning (Martens et al. US11697892B2)(x). These electrospinning techniques are well known from the man skilled in the art and their descriptions can be found in Keriouz A et al, and Lanno et al. (PCT / EP2022 / 076874). In a preferred embodiment, the electrospinning technique according to the invention is not perfusion electrospinning. Such a technique is well known from the man skilled in the art and a description of it can be found in Phaneuf M.D. et al (US2006020023). Among others, it can be noted that with perfusion electrospinning, there is a uniform coating of the liquid mixture (i.e. the liquid being electrospun) onto the surface of the mandrel (i.e. the collector of the electrospinning set-up). On the opposite, with the electrospinning technique of the invention, preferably monoaxial electrospinning, no liquid mixture is collected on the collector, fibers formation occurs on the fly towards the collector, and solid fibers are collected onto the collector as a uniform layer.

[0113] The polymer according to the invention is a biodegradable polymer. Optionally, the polymer of the invention may be bioresorbable, biodurable, and / or biocompatible. The biodegradable polymer of the invention may be a synthetic polymer. Preferably, the polymeraccording to the invention is hydrophobic. The polymeraccording to the invention may be a cyclic or an aliphatic polymer, it is preferably an aliphatic polymer. In a preferred embodiment, the biodegradable polymer according to the invention is a polyester, preferably an aliphatic polyester. The biodegradable polymer according to the invention may thus be selected from the group consisting in polycarbonates, polylactides, polycaprolactones, polyglycolides, polyhydroxyalkanoates, poly(dioxanone)s, poly(lactide-co-glycolide), poly(butyrolactone)s, poly(valerolactone)s, and a combination thereof. The biodegradable polymer according to the invention is preferably a polylactide. The biodegradable polymer according to the invention is preferably an aliphatic polyester selected from the group consisting in Poly(P-butyrolactone) (PBL), Poly(£-caprolactone-co- lactide) (PCLA), Poly(3-hydroxybutyrate) (PHB), Poly(3-hydroxyvalerate) (PHV), Poly(3- hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), Poly(methyl methacrylate) (PMMA), Polyglycolide (PGA), Poly(lactic acid) (PLA), Poly(L-lactide) (PLLA), Poly( D, L-lactic acid) (PDLA), E-polycaprolactone (PCL), a copolymer of PDLA and PCL (PLC) such as a PLC with a molar ratio of PDLA on PCL of about 70 / 30 (i.e. 70 / 30 PLC), poly(trimethylene carbonate) (PTMC), a derivative thereof and a combination thereof. More preferably, the aliphatic polymer according to the invention is a polyester selected from the group consisting in PDLA, PCL, PLC, such as 70 / 30 PLC, a derivative thereof and a combination thereof. The biodegradable polymer for use in any aspect of the present invention preferably comprises or consists of any of the polymers mentioned herein or any mixtures thereof.

[0114] In some cases, the biodegradable polymer is free or substantially free from polyethers, such as polyethylene oxide (PEO), polyethylene glycol (PEG), polypropylene glycol (PPG), polyoxymethylene (POM) or polypropylene oxide (PPO).

[0115] In preferred embodiments, the biodegradable polymer is free or substantially free from proteins (such as gelatin) and polysaccharides (such as starch and / or cellulose).

[0116] The antimicrobial fiber material according to any aspect or embodiment of the present invention is preferably free or substantially free of any non-biodegradable polymer material. Therefore, the electrospun fibers according to any aspect or embodiment of the present invention are preferably free or substantially free from any non-biodegradable polymer. Non-biodegradable polymers are not limited, and include any polymer or oligomer which cannot be broken down into benign materials. Examples of non- biodegradable polymers include homopolymers or copolymers comprising: polyethylene terephthalate (PET), polyurethane (PU), polyethylene (PE), polypropylene (PP), polyvinyl chloride (PVC) and / or polystyrene (PS). The material according to the invention may comprise between about 2% and about 50%, such as 2% and 35% (e.g. between about 2% and about 20%) in weight of polymer, preferably between about 3% and about 15% in weight of polymer, more preferably between about 4% and about 12% in weight of polymer. In a most preferred embodiment, the biodegradable polymer according to the invention is a polyester selected from the group consisting in PDLA, PCL, PLC, such as 70 / 30 PLC, a derivative thereof and a combination thereof and constitutes between about 3% and about 15%, more preferably between about 4% and about 12% in weight of the material. For example, the material according to the invention may comprise between about 7% and about 12%, preferably about 10%, in weight of PLC as the polymer, the material according to the invention may comprise between about 4% and about 8%, preferably about 5%, in weight of PDLA as the polymer, and the material according to the invention may comprise between about 7% and about 12%, preferably about 10%, in weight of PCL as the polymer.

[0117] The electrospun fibers according to the invention are loaded with antimicrobial peptides (AMPs). Any AMP known from the man skilled in the art can be used in the material of the invention.

[0118] AMP's are well known in the art and have been subject to reviews such as Zhang et al. 2021. By 2021, the AMP database [Data Repository of Antimicrobial Peptides (DRAMP), http: / / dramp.cpu-bioinfor.org / ] had reported 3791 AMP, any of which may be used in the various aspects of the present invention. In one embodiment, the AMP used in the present invention may be one or more AMP listed in the Data Repository of Antimicrobial Peptides. Typical AMPs for use herein have a net charge of +2 to +9 or -1 to -8 and / or contain 10-100 amino acids. Some AMPs contain the amino terminal copper and nickel (ATCUN) binding motif.

[0119] A first subgroup of AMPs suitable for use in the invention have a net charge range of -1 to -8 and contain 5 to 70 amino acid residues. Their structure may include a-helical peptides and / or cyclic cystine knots.

[0120] A second subgroup comprises cationic a-helical AMPs, which may be small peptides with less than 40 amino acids in length and / or may carry a net charge of +2 to +9 and / or may have the C-terminus amidated. Such AMPs may contain over 50% (by number) hydrophobic amino acids. A third subgroup of AMPs suitable for use in the invention is cationic -sheet AMP, which typically contain 2-8 cysteine residues forming 1-4 pairs of intramolecular disulfide bonds.

[0121] A fourth subgroup of AMPs suitable for use in the invention is the extended cationic AMPs, which may contain specific amino acids such as arginine, proline, tryptophan, glycine and histidine. Such AMPs may lack regular secondary structures.

[0122] The AMPs for use in any aspect of the present invention may be any AMP described herein including any of the first to fourth groups or any combination thereof.

[0123] The AMP according to the invention may be a cationic AMP, such as a linear cationic a-helical AMP or a cationic AMP enriched in proline, arginine, phenylalanine, glycine, or tryptophan, an anionic AMP, or a combination thereof. The AMP according to the invention may have been isolated from mammals, amphibians, microorganisms, insects, or a combination thereof. Without being limitative, the AMP according to the invention may be selected from the group consisting in maximin H5, dermcidin, cecropins, andropin, moricin, ceratotoxin, melittin, magainin, dermaseptin, bombinin, esculentins, buforin II, CAP18, abaecin, drosocin, apidaecin, attacin, prophenin, indolicidin, brevinins, such as brevinin-1, protegrin, tachyplesins, defensins, drosomycin, bacitracin, boceprevir, dalbavancin, daptomycin, enfuvirtide, oritavancin, teicoplanin, telaprevir, telavancin, vancomycin, guavanin 2, bacteriocin, peptaibols, plectasin, hydramacin, aurelin, mastoparan, thioester-containing protein 1, aurein, cathelicidins, reglll peptides, nisin, copsin, diptericin, pleurocidin, D-pleurocidin, L-temporin, LL-37, colistin, gramicidin, a derivative thereof and a combination thereof. Preferably, the AMP according to the invention is a cationic AMP, more preferably selected from the group consisting in pleurocidin, D-pleurocidin, L-temporin, LL-37, colistin, for example colistin sulphate, bacitracin, for example bacitracin zinc, gramicidin, daptomycin, a derivative and a combination thereof, more preferablythe AMP according to the invention is selected from the group consisting in pleurocidin, L-temporin, LL-37, colistin, such as colistin sulphate, daptomycin, a derivative and a combination thereof. The AMP according to the invention may also be selected from the group consisting in pleurocidin, L-temporin, colistin, such as colistin sulphate, daptomycin, a derivative and a combination thereof. In a particular embodiment, the AMP according to the invention is colistin, preferably colistin sulphate, or a derivative thereof. The AMP for use in any aspect of the present invention preferably comprises or consists of any of the AMPs mentioned herein or any mixtures thereof.

[0124] It is generally preferred if the AMP according to any embodiment or aspect of the invention is selected from a ribosomal AMP or non-ribosomal AMP. Ribosomal AMPs include, but are not limited to: L-Temporin, LL-37 and pleurocidin (PLE). Non-ribosomal AMPs include, but are not limited to, any of the following: bacitracin (BAC), gramicidin (GRA), colistin (COL) and daptomycin (DAP). Mixtures of ribosomal AMPs and / or non- ribosomal AMPs may be used.

[0125] In some embodiments, it is preferred that the AMP according to any embodiment or aspect of the invention is free or substantially free of daptomycin (DAP). A preferred list of non-ribosomal AMPs therefore may include bacitracin (BAC), gramicidin (GRA), colistin (COL) or a combination thereof.

[0126] In some cases, it is preferred that the AMP according to any embodiment or aspect of the present invention is free or substantially free from bacteriocins. Bacteriocins are not limited, but include peptides produced by bacteria. Such peptides include peptides ribosomally synthesized in bacteria, such as mundticin ST4SA, plantaricin 423, nisin, pediocin, leucocin and / or enterocin.

[0127] It is generally preferred that the AMP is a natural peptide, that is, a peptide occurring in nature. Therefore, in some cases, the AMP is free or substantially free from any artificially created peptides (i.e. synthetic peptides not found in nature). In one embodiment, the AMP may be at least one naturally occurring AMP extracted and / or purified from a natural source. In an alternative embodiment, the AMP may be a naturally occurring peptide made synthetically. Evidently, mixtures of extracted and synthetic AMP may be used.

[0128] The material according to the invention may comprise between about 0.1% and about 40% (e.g. between about 0.1% and about 30%, or between about 0.1% and about 20%) in weight of AMPs, preferably between about 0.5% and about 15% in weight of AMPs, more preferably between about 1% and about 10% in weight of AMPs, even more preferably between about 2% and about 8% in weight of AMPs. In a particularly preferred embodiment, the AMP according to the invention is selected from the group consisting in pleurocidin, L-temporin, LL-37, colistin, such as colistin sulphate, daptomycin, gramicidin, bacitracin a derivative and a combination thereof, and constitutes between about 1% and about 10%, preferably between about 2% and about 8% in weight of the material.

[0129] In a very preferred embodiment, the biodegradable polymer according to the invention is a polyester selected from the group consisting in PDLA, PCL, PLC, such as 70 / 30 PLC, a derivative thereof and a combination thereof, and the AMP according to the invention is selected from the group consisting in pleurocidin L-temporin, LL-37, colistin, such as colistin sulphate, daptomycin, gramicidin, bacitracin a derivative and a combination thereof, preferably said polymer constitutes between about 3% and about 15%, more preferably between about 4% and about 12% in weight of the material, and said AMP constitutes between about 1% and about 10%, preferably between about 2% and about 8% in weight of the material.

[0130] The AMPs loaded in the electrospun fibers according to the invention can either be trapped within the fibers or non-trapped AMPs.

[0131] At least about 30% of the AMPs loaded in the fibers of the material during electrospinning are trapped within the fibers. Preferably, at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% of the AMPs are trapped within the fibers. In a preferred embodiment at least about 50% of the AMPs according to the invention are trapped within the fibers of the material. In a most preferred embodiment at least about 60% of the AMPs according to the invention are trapped within the fibers of the material.

[0132] As used herein, the term "trapped" refers to AMPs that will essentially remain within the fibers under normal conditions of use of the material for at least 1 day, preferably at least 2 days, more preferably at least about 3 days, still preferably at least about 7 days, yet preferably at least about 14 days, even more preferably at least about 21 days.

[0133] In some cases, some or all of the "trapped" AMPs will remain within the fibers indefinitely under normal conditions (i.e. will be released only (or substantially) by degradation of the polymer component ratherthan by diffusion out of the polymer). Such "permanently trapped" AMP will remain in the polymer under suitable conditions (e.g. held at 37°C in excess PBS) for at least 1 month, preferably at least 3 months. In one embodiment, at least 20% (e.g. 20 to 99%) of the AMP originally incorporated will be permanently trapped. This may preferably be at least 30% or at least 50%. The term "normal conditions of use" refers to biorelevant use as a dressing such as application of the material according to the invention to the skin or a mucosa of a subject, with optionally a temperature comprised between about 35°C and about 42°C, preferably about 37°C, and eventually a volume of exudate not exceeding 12 000g / m2 / day, preferably not exceeding 6000g / m2 / day. As used herein, the term "essentially remain within the fibers" means that no more than about 15%, preferably no more than about 10%, more preferably no more than about 5%, even more preferably no more than about 1% of the trapped AMPs will leak out of the fibers of the material during the time period considered.

[0134] In an alternative embodiment, the term "at least about x% of the AMPs are trapped within the fibers", x being comprised between about 30 and about 95, as used herein refers to a percentage of AMPs that will remain within the fibers for at least 1 day, preferably at least 2 days, more preferably at least about 3 days, still preferably at least about 7 days, yet preferably at least about 14 days, even more preferably at least about 21 days under normal conditions of use of the material.

[0135] No more than 70% of the AMPs loaded in the fibers of the material during electrospinning are not trapped within the fibers. Preferably, no more than about 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% of the AMPs are not trapped within the fibers. In a preferred embodiment, the non-trapped AMPs are essentially released from the fibers in no more than about a day, preferably in no more than about 10 h, even more preferably in no more than about 1 h, under normal conditions of use of the material. In a particular embodiment, the non-trapped AMPs are released from the fibers in no more than about 10 min, preferably no more than about 5 min, even more preferably no more than about 1 min, under normal conditions of use of the material. As used herein, the term "essentially released from the fibers" means that no more than about 15%, preferably no more than about 10%, more preferably no more than about 5%, even more preferably no more than about 1% of the non-trapped AMPs will remain in the fibers of the material at the end of the time period considered.

[0136] A test which can readily be used to establish what proportion of the antimicrobial peptides are trapped within the fibers for an appointed time is as follows: a) The total antimicrobial peptide content of a sample of antimicrobial fiber material is measured as a first amount; b) The sample of antimicrobial fiber material is placed in biorelevant buffer media (for example lxPBS (pH 7.4), DMEM + 10% FBS, DMEM+ 0.2 mM EDTA, LB + 0.2 mM EDTA); c) The antimicrobial fiber material is maintained in a closed vessel at 36 to 38°C for the appointed time (as described herein, for example for about 24 h, about 3 days or about 7 days); d) After the appointed time, the amount of antimicrobial peptide which remains in the sample of antimicrobial fiber material is measured as a second amount, for example by dissolving the fiber material in an organic solvent and measuring the peptide content, for example by HPLC; e) The proportion of antimicrobial peptides trapped within the fibers is calculated as to proportion of the second amount to the first amount and typically expressed as a percentage of the second amount to the first amount.

[0137] To test whether antimicrobial peptides (AMPs) are "trapped" as such, the test period can be short, such as about 1 hour, or about 4 hours, but the test period is preferably of about 24 h, about 3 days or about 7 days. Other test periods can be appropriate as disclosed herein (e.g. longer periods such as 1 month to test for "permanently trapped" AMP. Such a test may be used as a substitute for "normal conditions of use" in any appropriate aspect or embodiment of the invention disclosed herein.

[0138] To establish what proportion of the antimicrobial peptides are "non-trapped" within the fibers for an appointed time, the proportion of trapped antimicrobial peptides forthat appointed time can be subtracted to 100 %. Such a way of calculation may be used as a substitute for "normal conditions of use" in any appropriate aspect or embodiment of the invention disclosed herein.

[0139] In a particular embodiment, the AMPs present in the material according to the invention are essentially trapped within the fibers. It can be considered that all AMPs of a given material are essentially trapped within its fibers at a given time if the amount of AMPs released from the fibers at that time is of no more than about 15%, 10%, 5%, or l%.ln some particularly beneficial cases, the trapped AMPs can remain in the material of the invention for at least 1 week yet still exhibit antibacterial activity. In one embodiment, the AMPs may exhibit antibacterial activity while remaining trapped in the polymer fibers. The present invention therefore is not limited to embodiments where it is required for the trapped AMPs to slowly be released from the fiber in order to exhibit an antimicrobial effect.

[0140] It is to be noted that AMPs released in the vicinity of the material can diffuse, undergo degradation or be adsorbed onto the material of the invention.

[0141] In another embodiment according to the invention, the trapped and non-trapped AMPs remain within the fibers of the material after electrospinning and under normal conditions of storage until the material starts to be used. The term "normal conditions of storage" refers to a period of no more than about 2 years, preferably no more than about 1 year, even more preferably no more than about 6 months at a temperature of no more than about 30°C, preferably no more than about 25°C, more preferably no more than about 20°C, still preferably no more than about 10°C, even more preferably no more than about 4°C, eventually the storage temperature can be under 0°C, and with a relative humidity of no more than about 20%, preferably no more than about 10%, more preferably no more than about 5%, even more preferably a relative humidity of about 0%.

[0142] In a preferred embodiment, the electrospun fibers according to the invention have been electrospun with a polar organic solvent. In a preferred embodiment, the polar organic solvent according to the invention is selected from the group consisting of a polar organic solvent selected from an amide solvent, a nitrile solvent, a ketone solvent, an alcohol solvent, a morpholine solvent, a morpholine oxide solvent, a sulphoxide solvent, a furan solvent, a dioxane solvent, a fluoroalcohol solvent and mixtures thereof, preferably a solvent comprising at least one fluoroalcohol solvent. While the polar organic solvent is not present in the material, or only as traces, its use during the electrospinning process has a key impact on the ability of the fibers to trap AMPs. Traces of polar organic solvents according to the invention may be present in the material, preferably the amount of polar organic solvent in the material is of no more than about 0.5%, preferably no more than 0.1%, more preferably no more than 0.01%, even more preferably no more than 0.001%. Polar organic solvents are well known from the man skilled in the art and any polar organic solvent can be used to electrospin the fibers according to the invention. In a preferred embodiment, the polar organic solvent according to the invention is selected from the group consisting in dimethylformamide, acetonitrile, acetone, hexamethylphosphoric triamide, N,N-diethylacetamine, 4-methylmorpholine-N-oxide monohydrate, N- methylpyrrolidinone, dimethylsulfoxyde (DMSO), tetrahydrofurane (THF), dioxan, methylcarbonate, fluoroalcohols, a derivative, and a combination thereof. Preferably, the polar organic solvent according to the invention is a fluoroalcohol. In an even preferred embodiment, the polar organic solvent according to the invention is a fluoroalcohol selected from the group consisting in trifluoroethanol (2,2,2-trifluoroethanol; TFE), hexafluoroisopropanol (l,l,l,3,3,3-hexafluoro-2-propanol; HFIP), perfluoro-tert-butanol (l,l,l,3,3,3-hexafluoro-2-(trifluoromethyl)-2-propanol; PFTB), 2-fluoroethanol, a derivative, and a combination thereof. In a most preferred embodiment, the solvent according to the invention is HFIP. In one embodiment, the polar organic solvent is not a carbonate solvent. In one embodiment, the polar organic solvent is not methylcarbonate. In a preferred embodiment therefore, the polar organic solvent is free or substantially free from carbonate solvents such as methylcarbonate. In some cases, the polar organic solvent is free or substantially free from amide solvents (e.g. dimethylformamide (DMF)) and / or sulfoxide solvents (e.g. dimethylsulfoxide (DMSO)). The polar solvent may be free or substantially free of DMF and / or DMSO, especially free or substantially free of DMF.

[0143] In some cases, the polar organic solvent comprises an acidic solvent. Suitable acidic solvents include, but are not limited to: acetic acid, formic acid, propanoic acid, butanoic acid, phosphoric acid, citric acid, oxalic acid, p-toluenesulfonic acid or combinations thereof. It is particularly preferred if the acid solvent comprises a mixture of acetic acid and formic acid, especially in a ratio of 10:1 to 1:1, especially 5:1 to 2:1, such as 3:1. In some cases the polar organic solvent is selected from either a fluoroalcohol or an acidic solvent (e.g. as described herein in each case). Since the nature of the polar organic solvent can affect the structure of the electrospun fibers, in one embodiment, the electrospun fibers are formed or formable by electrospinning from a polar organic solvent or mixture thereof (such as a polar organic solvent as described herein). In one embodiment, the electrospun fibers are formed or formable by electrospinning from a polar organic solvent selected from an amide solvent, a nitrile solvent, a ketone solvent, an alcohol solvent, a morpholine solvent, a morpholine oxide solvent, a sulfoxide solvent, a furan solvent, a dioxane solvent, a fluoroalcohol solvent and mixtures thereof, preferably a solvent comprising at least one fluoroalcohol. In one embodiment, the electrospun fibers are formed or formable by electrospinning from a polar organic solvent selected from the group consisting of dimethylformamide, acetonitrile, acetone, hexamethylphosphoric triamide, N,N-diethylacetamine, 4-methylmorpholine-N-oxide monohydrate, N- methylpyrrolidinone, dimethylsulfoxide (DMSO), tetrahydrofurane (THF), dioxan, methylcarbonate, trifluoroethanol (2,2,2-trifluoroethanol; TFE), hexafluoroisopropanol (l,l,l,3,3,3-hexafluoro-2-propanol; HFIP), perfluoro-tert-butanol (1,1, 1,3,3, 3-hexafluoro- 2-(trifluoromethyl)-2-propanol; PFTB), 2-fluoroethanol and mixtures thereof. The polar organic solvent for use in any aspect of the present invention preferably comprises or consists of any of the solvents mentioned as suitable herein, or any mixtures thereof.

[0144] In a preferred embodiment, the antimicrobial fiber material according to the invention has an antimicrobial activity, preferably an antibacterial activity, and / or an antibiofilm activity, preferably the antimicrobial fiber material according to the invention has an antibacterial activity and an antibiofilm activity.

[0145] The antimicrobial activity and / or the antibiofilm activity of the antimicrobial fiber material according to the invention may last at least about 2 days, preferably at least about 3 days, more preferably at least about 5 days, even more preferably at least about 7 days under normal conditions of use of the material. Optionally, the antimicrobial activity and / or the antibiofilm activity of the antimicrobial fiber material according to the invention last at least about 14 days, alternatively at least about 21 days under normal conditions of use of the material. Preferably, therefore, the material according to any aspect of the present invention has antimicrobial and / or antibacterial and / or antibiofilm activity whilst the AMP is trapped.

[0146] It is preferable that the antimicrobial activity and any other appropriate feature of the antimicrobial fiber material of the invention be tested under normal conditions of use of the material. As an alternative, where "normal conditions of use" are specified, the antimicrobial fiber material of the invention may be tested by a method corresponding to that set out above for the testing of the proportion of the antimicrobial peptides that are trapped within the fibers for an appointed time.

[0147] For example, is as follows: a) The antimicrobial peptide content of a sample of antimicrobial fiber material is measured as a first amount; b) The sample of antimicrobial fiber material is placed in biorelevant buffer media (for example lxPBS (pH 7.4), DMEM + 10% FBS, DMEM+ 0.2 mM EDTA, LB + 0.2 mM EDTA); c) The antimicrobial fiber material is maintained in a closed vessel at 36 to 38°C for the appointed time (as described herein); d) After the appointed time, the amount of antimicrobial peptide which remains in the sample of antimicrobial fiber material is measured as a second amount for example by dissolving the fiber material in an organic solvent and measuring the peptide content, for example by HPLC; e) The proportion of antimicrobial peptides trapped within the fibers is calculated as to proportion of the second amount to the first amount and typically expressed as a percentage of the second amount to the first amount.

[0148] The antimicrobial activity, preferably antibacterial activity, and / or the antibiofilm activity, is of at least about a 3 log reduction, preferably at least about a 4 log reduction, alternatively at least about a 5 log reduction.

[0149] In another embodiment, the material according to the invention is sterile.

[0150] In yet another embodiment, the material according to the invention is not toxic, preferably not toxic to eukaryotic cells. As used herein, the term "not toxic" refers to materials that are not toxic to humans or other non-human animals, preferably mammals. As used herein, the terms "not toxic to eukaryotic cells" or "biocompatible to eukaryotic cells" are equivalent and refer to materials that do not decrease eukaryotic cells viability and / or do not decrease the metabolic activity of eukaryotic cells. The metabolic activity of eukaryotic cells can be evaluated by an MTS activity test.

[0151] Toxicity and non-toxicity as indicated herein will typically be at the relevant amounts and concentrations indicated herein for the relevant aspects and embodiments of the invention.

[0152] In still another embodiment, the material according to the invention further comprises a chelating agent. Preferably, said chelating agent is selected from the group consisting in Dimercaprol (BAL), ethylenediaminetetraacetic (EDTA) salts such as calcium EDTA or sodium EDTA, succimer (DMSA), penicillamine, trientine hydrochloride, deferoxamine mesylate, deferiprone, deferasirox, pentetate calcium trisodium (Ca-DTPA), pentetate zinc trisodium (Ca-DTPA), prussian blue (Radiogardase), and a combination thereof, more preferably the chelating agent is an EDTA salt, even more preferably the chelating agent is sodium EDTA. The chelating power induced by the chelating agent according to the invention may be equivalent to the chelating power induced by sodium- EDTA at a concentration comprised between about 0.1 mM and about 10 mM, preferably about 0.2 mM, in the microenvironment of the material. Alternatively, the chelating capacity of the chelating agent is in the range of O.lmM to about 10 mM of metal ions per liter in the microenvironment of the material. As used herein, the term "microenvironment" refers to the biological environment that is in direct contact with the material when applied to a human. The chelating agent according to the invention may be present in the fibers of the material. Alternatively, the chelating agent according to the invention may be present in the material but not in the fibers, for example, the chelating agent may be adsorbed on the surface of the fibers.

[0153] In still another embodiment, the material according to any aspect of the invention may further comprise at least one antibiotic (AB).

[0154] The at least one antibiotic is not particularly limited, but may be selected from any of the following: aminoglycosides, ansamycins, carbacephems, carbapenems, cephalosporins, such as the cephalosporins of first, second, third, fourth and fifth generation, lincosamides, macrolides, monobactams, nitrofurans, oxazolidinones, penicillins, quinolones, fluoroquinolones, sulfonamides, tetracyclines, nitrofurans, a derivative thereof and a combination thereof. Preferably, the antibiotic is selected from the group consisting in amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, streptomycin, spectinomycin, geldanamycin, herbimycin, rifaximin, loracarbef, ertapenem, doripenem, imipenem, cilastatin, meropenem, cefadroxil, cefazolin, cephradine, cephapirin, cephalothin, cefalexin, cefaclor, cefoxitin, cefotetan, cefamandole, cefmetazole, cefonicid, loracarbef, cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, moxalactam, ceftriaxone (e.g. ceftriaxone disodium hemi(heptahydrate)) , cefepime, ceftaroline fosamil, ceftobiprole, clindamycin (e.g. clindamycin hydrochloride, clindamycin phosphate, clindamycin base), lincomycin, azithromycin, clarithromycin, erythromycin, roxithromycin, telithromycin, spiramycin, fidaxomicin, aztreonam, furazolidone, nitrofurantoin, linezolid, posizolid, radezolid, tedizolid, amoxicillin, ampicillin, azlocillin, dicloxacillin, flucioxacillin, mezlocillin, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, temocillin, ticarcillin, ciprofloxacin (e.g. ciprofloxacin HCI, ciprofloxacin base), enoxacin, gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nadifloxacin, nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin, temafloxacin, mafenide, sulfacetamide, sulfadiazine, silver sulfadiazine, sulfadimethoxine, sulfamethizole, sulfamethoxazole, sulfanilimide, sulfasalazine, sulfisoxazole, trimethoprimsulfamethoxazole, sulfonamidochrysoidine, demeclocycline, doxycycline, metacycline, minocycline, oxytetracycline, tetracycline, clofazimine, dapsone, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide, rifampicin, rifabutin, rifapentine, streptomycin, arsphenamine, chloramphenicol, fosfomycin, fusidic acid, metronidazole, mupirocin, platensimycin, quinupristin, dalfopristin, thiamphenicol, tigecycline, tinidazole, trimethoprim, teixobactin, malacidins, halicin, difurazone, furazolidone, nifurfoline, nifuroxazide, nifurquinazol, nifurtoinol, nifurzide, nitrofural, nitrofurantoin, ranbezolid, furaltadone, furazidine, furaginum, furylfuramide, nifuratel, nifurtimox, fanft, azitromycin, nitrofurazone, enrofloxacin, ozenoxacin, meclocycline, a derivative and a combination thereof. Alternatively, the antibiotic is selected from the group consisting in clindamycin, azitromycin, tetracycline, ciprofloxacin, erythromycin, mupirocin, nitrofurazone, fusidic acid, sulfacetamide, gatifloxacin, levofloxacin, chloramphenicol, cefazolin, imipenem, cilastatin, ampicillin, gentamycin, moxifloxacin, doxycycline, streptomycin, tigecycline, enrofloxacin, neomycin, ozenoxacin, meclocycline, metronidazol , rifampicin, a derivative and a combination thereof, preferably chloramphenicol or a derivative thereof.

[0155] In an especially preferred embodiment, the antibiotic (AB) is selected from the class of fluoroquinolones, macrolides and / or cephalosporins, more preferably, at least one of ciprofloxacin (including hydrochloride), clindamycin (including hydrochloride and phosphate) and ceftriaxone (including disodium hemi(heptahydrate)). Interestingly, when at least one antibiotic is included in the antimicrobial fiber material according to any aspect of the present invention, a high encapsulation effect can be achieved for both the AMP and the AB, and the material can offer antibacterial activity against various strains of bacteria. Moreover, in cases where the antimicrobial fiber material comprises a combination of AMP and AB, it is possible to achieve an initial burst release of AB whilst at least 50% of the AMP are trapped within the fibers (e.g. trapped as described herein). Without being bound by theory, when the antimicrobial fiber materials comprise both AMP and AB, a strong antibacterial effect can be observed when relatively small amounts of the active agents are encapsulated in the fibers. The combination of AB and AMP in the fibers may demonstrate a synergistic effect, where antibacterial activity is unexpectedly high.

[0156] Method of electrospinning

[0157] In a second aspect, the invention also concerns a method of electrospinning antimicrobial peptides comprising the following steps: a) preparing a solution of a biodegradable polymer, a polar organic solvent, and an antimicrobial peptide; b) electrospinning the solution obtained at step a).

[0158] The solution of step a) comprises a biodegradable polymer. Optionally, the biodegradable polymer of the method of the invention may be bioresorbable, biodurable, and / or biocompatible. Preferably, the polymer according to the method of the invention is hydrophobic. The polymer according to the method of the invention may be synthetic. The polymer according to the method of the invention may be a cyclic or an aliphatic polymer, it is preferably an aliphatic polymer. In a preferred embodiment, the biodegradable polymer according to the method of the invention is a polyester, preferably an aliphatic polyester. The biodegradable polymer may thus be selected from the group consisting in polycarbonates, polylactides, polycaprolactones, polyglycolides, polyhydroxyalkanoates, poly(dioxanone)s, poly(lactide-co-glycolide), poly(butyrolactone)s, poly(valerolactone)s, and a combination thereof. The biodegradable polymer according to the invention is preferably a polylactide (such as a polylactide homopolymer and / or at least one polylactide copolymer (e.g. copolymerized with at least one other polyester subunit, such as any polyester indicated herein including polyglycolide and / or polylactone)). The biodegradable polymer according to the invention is preferably an aliphatic polyester selected from the group consisting in Poly(P-butyrolactone) (PBL), Poly(E-caprolactone-co-lactide) (PCLA), Poly(3-hydroxybutyrate) (PHB), Poly(3- hydroxyvalerate) (PHV), Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), Poly(methyl methacrylate) (PMMA), Polyglycolide (PGA), Poly(lactic acid) (PLA), Poly(L- lactide) (PLLA), Poly(D,L-lactic acid) (PDLA), E-polycaprolactone (PCL), a copolymer of PDLA and PCL (PLC) such as a PLC with a molar ratio of PDLA on PCL of about 70 / 30 (i.e. 70 / 30 PLC), poly(trimethylene carbonate) (PTMC), a derivative thereof and a combination thereof. More preferably, the aliphatic polymer according to the invention is a polyester selected from the group consisting in PDLA, PCL, PLC, such as 70 / 30 PLC, a derivative thereof and a combination thereof (e.g. a mixture and / or copolymer thereof).

[0159] The solution of step a) according to the method of the invention comprises between about 2% and about 20% in weight of polymer, preferably between about 3% and about 15% in weight of polymer, more preferably between about 4% and about 12% in weight of polymer. In a most preferred embodiment, the biodegradable polymeraccording to the method of invention is a polyester selected from the group consisting in PDLA, PCL, PLC, such as 70 / 30 PLC, a derivative thereof and a combination thereof and constitutes between about 3% and about 15%, more preferably between about 4% and about 12% in weight of the solution of step a). For example, the solution of step a) according to the method of the invention may comprise between about 7% and about 12%, preferably about 10%, in weight of PLC as the polymer, the material according to the invention may comprise between about 4% and about 8%, preferably about 5%, in weight of PDLA as the polymer, and the material according to the invention may comprise between about 7% and about 12%, preferably about 10%, in weight of PCL as the polymer.

[0160] The solution of step a) according to the method of the invention further comprises a polar organic solvent. Polar organic solvents are well known from the man skilled in the art and any polar organic solvent can be used in step a) of the method of the invention. In a preferred embodiment, the polar organic solvent according to the method of invention is selected from the group consisting in dimethylformamide, acetonitrile, acetone, hexamethylphosphoric triamide, N,N-diethylacetamine, 4-methylmorpholine-N-oxide monohydrate, N-methylpyrrolidinone, dimethylsulfoxide (DMSO), tetrahydrofurane (THF), dioxan, methylcarbonate, fluoroalcohols, a derivative, and a combination thereof. Preferably, the polar organic solvent according to the method of the invention is a fluoroalcohol. More preferably, the polar organic solvent according to the method of the invention is a fluoroalcohol selected from the group consisting in trifluoroethanol (2,2,2- trifluoroethanol; TFE), hexafluoroisopropanol (l,l,l,3,3,3-hexafluoro-2-propanol; HFIP), perfluoro-tert-butanol (l,l,l,3,3,3-hexafluoro-2-(trifluoromethyl)-2-propanol; PFTB), 2- fluoroethanol, a derivative, and a combination thereof. Even more preferably, the polar organic solvent according to the method of the invention is HFIP. The solution of step a) according to the method of the invention also comprises an

[0161] AMP. Any AMP known from the man skilled in the art can be used with the method of the invention. The AMP according to the method of the invention may be a cationic AMP, such as a linear cationic a-helical AMP or a cationic AMP enriched in proline, arginine, phenylalanine, glycine, or tryptophan, an anionic AMP or a combination thereof. The AMP according to the method of the invention may have been isolated from mammals, amphibians, microorganisms, insects, or a combination thereof. Without being limitative, the AMP according to the method of the invention may be selected from the group consisting in maximin H5, dermcidin, cecropins, andropin, moricin, ceratotoxin, melittin, magainin, dermaseptin, bombinin, esculentins, buforin II, CAP18, abaecin, drosocin, apidaecin, attacin, prophenin, indolicidin, brevinins, such as brevinin-1, protegrin, tachyplesins, defensins, drosomycin, bacitracin, boceprevir, dalbavancin, daptomycin, enfuvirtide, oritavancin, teicoplanin, telaprevir, telavancin, vancomycin, guavanin 2, bacteriocin, peptaibols, plectasin, hydramacin, aurelin, mastoparan, thioester-containing protein 1, aurein, cathelicidins, reglll peptides, nisin, copsin, diptericin, pleurocidin, D- pleurocidin, L-temporin, LL-37, colistin, gramicidin, a derivative thereof and a combination thereof. Preferably, the AMP according to the method of the invention is a cationic AMP, more preferably selected from the group consisting in pleurocidin, D-pleurocidin, L- temporin, LL-37, colistin, for example colistin sulphate, bacitracin, for example Zn- bacitracin, gramicidin, daptomycin, a derivative and a combination thereof, even more preferably the AMP according to the invention is selected from the group consisting in pleurocidin, L-temporin, LL-37, colistin, such as colistin sulphate, daptomycin, a derivative and a combination thereof. The AMP according to the invention may also be selected from the group consisting in pleurocidin, L-temporin, colistin, such as colistin sulphate, daptomycin, a derivative and a combination thereof. In a particular embodiment, the AMP according to the method of the invention is colistin, preferably colistin sulphate, or a derivative thereof.

[0162] The solution of step a) according to the method of the invention comprises between about 0.1% and about 30% (e.g. 0.1% to 20%) in weight of AMPs, preferably between about 0.5% and about 15% in weight of AMPs, more preferably between about 1% and about 10% in weight of AMPs. In a highly preferred embodiment, the AMP according to the invention is selected from the group consisting of pleurocidin, D- pleurocidin, L-temporin, LL-37, colistin (such as colistin sulphate), daptomycin, gramicidin, bacitracin a derivative and a combination thereof, most preferably from pleurocidin, L- temporin, LL-37, colistin, such as colistin sulphate, daptomycin, a derivative and a combination thereof, and constitutes between 0.5% and 20% (e.g. about 0.5% and about 15%), more preferably between about 1% and about 10% in weight of the solution of step a).

[0163] In a very preferred embodiment, the biodegradable polymer according to the method of invention is a polyester selected from the group consisting in PDLA, PCL, PLC, such as 70 / 30 PLC, a derivative thereof and a combination thereof, the polar organic solvent according to the method of invention is a fluoroalcohol selected from the group consisting in trifluoroethanol (2,2,2-trifluoroethanol; TFE), hexafluoroisopropanol (l,l,l,3,3,3-hexafluoro-2-propanol; HFIP), perfluoro-tert-butanol (1,1, 1,3,3, 3-hexafluoro- 2-(trifluoromethyl)-2-propanol; PFTB), 2-fluoroethanol, a derivative, and a combination thereof, preferably HFIP, and the AMP according to the invention is selected from the group consisting in pleurocidin, L-temporin, LL-37, colistin, such as colistin sulphate, daptomycin, a derivative and a combination thereof, preferably said biodegradable polymer constitutes between about 3% and about 15%, more preferably between about 4% and about 12% in weight of the solution of step a), and said AMP constitutes between about 0.5% and about 15%, more preferably between about 1% and about 10% in weight of the solution of step a).

[0164] In a particular embodiment where AB are present in the solution in step a), it is preferred if the concentration of AB is less than that of AMP. Preferably, the solution comprises between about 0.1% to about 20% by weight of ABs, preferably between about 0.5% to 7% by weight of ABs, especially 1.0% to 5.0% by weight of ABs. The preferred ratio of AMP to AB in the solution in step a) is 10:1 to 1:1, especially 5:1 to 1:1, such as about 2:1 AMP to AB.

[0165] The step a) of the method according to the invention may last between about 12 h and about 24 h, preferably between about 15 h and about 21 h, more preferably between about 17 h and about 19 h, even more preferably about 18 h.

[0166] In a preferred embodiment, step a) comprises the four following sub-steps: ai) adding a biodegradable polymer to a polar organic solvent;

[0167] 32) mixing the solution obtained at step ai); as) adding an antimicrobial peptide to the solution of step 32);

[0168] 34) mixing the solution obtained at step as). or ai) adding a biodegradable polymer and an antimicrobial peptide to a polar organic solvent;

[0169] 32) mixing the solution obtained at step ai).

[0170] Mixing of step 32) and 34) may be achieved by any technical mean well known by the man skilled in the art. For example, mixing can be achieved with a magnetic stirrer. Step 32) may last between about 12 h and about 24 h, preferably between about 15 h and about 21 h, more preferably between about 17 h and about 19 h, even more preferably about 18 h. Step 34) may last between about 10 min and about 4 h, preferably between about 15 min and about 3 h, more preferably between about 20 min and about 2 h, even more preferably about 30 min.

[0171] In a particular embodiment, step as) is done under continuous mixing of the solution obtained at step 32).

[0172] In step b) according to the method of the invention, the solution obtained at step a) is electrospun. Electrospinning techniques are well known by the man skilled in the art, who can choose into a wide range of electrospinning techniques to implement the method of the invention, including needle-based and needleless electrospinning techniques. For example, the electrospinning technique to be used with the method according to the invention may be selected from the group consisting in monoaxial, multijet, magneticfluid, roller, centrifugal, porous tube, bubble, conical wire coil, ball, disk, wet (3D), cone, spiral coil, ultrasound-enhanced electrospinning. These methods are described in Keriouz A et al. Preferably, the electrospinning technique according to the method of the invention is monoaxial electrospinning. Monoaxial electrospinning is a simple, cost effective and easily scalable method. Preferably, monoaxial electrospinning is performed with a 23 G needle with a preferred volume comprised between about 4 mL and about 9 mL, a voltage preferably comprised between about 6.9 kV and about 15.2 kV, a preferred flow rate comprised between about 0.8 mL / h and about 2.5 mL / h, a distance from the collector preferably comprised between about 11 cm and about 18 cm, a temperature preferably comprised between about 22 °C and about 26 °C, and a relative humidity preferably comprised between about 19 % and about 36%. Evidently, the skilled worker will be able to scale up these conditions to larger scales as needed.

[0173] In another preferred embodiment, the electrospinning technique according to the method of the invention is not perfusion electrospinning. Perfusion electrospinning is a technique well known by the man skilled in the art and described in Phaneuf M.D. et al (US2006020023). Among others, it can be noted that perfusion electrospinning comprises a step of forming a uniform coating of the liquid mixture (i.e. the electrospun liquid) onto the surface of the mandrel (i.e. the collector of the electrospinning set-up). On the opposite, with the electrospinning technique according to the method of the invention, no liquid mixture is collected on the collector, the fibers are formed on the fly towards the collector and solid fibers are collected onto the collector, preferably as a uniform layer.

[0174] In another preferred embodiment, the electrospinning technique according to the method of the invention is not suspension electrospinning. It is therefore preferred that the biodegradable polymer, polar organic solvent and antimicrobial peptide are selected to allow suitable dissolution.

[0175] In a preferred embodiment, the step b) of the method according to the invention occurs between about 30 s and about 20 min, preferably between about 1 min and about 10 min, even more preferably about 5 min after step a).

[0176] In a particular embodiment, the method of the invention further comprises a step of washing the material obtained at step b) of the method in order to remove the antimicrobial peptides that are not trapped within the fibers. Preferably, said material is washed by immersion in a medium during between about 1 min and about 24 h, preferably between about 10 min and about 10 h, even more preferably about 1 h. The washing medium is preferably selected from the group consisting in a saline solution, a PBS solution or distilled water, more preferably PBS solution.

[0177] In a preferred embodiment, the method according to the invention further comprises a step of sterilization or disinfection of the material obtained at step b) of the method, before or after the optional step of washing, preferably before the optional step of washing. As used herein, the term "sterilization" refers to the process of eliminating all the microorganisms present in the material of the invention. As used herein, the term "disinfection" refers to the process of reducing or eliminating the harmful microorganisms present in the material of the invention. Any sterilization or disinfection techniques, well known from the man skilled in the art, can be used to sterilize or disinfect the material obtained at step b) of the method. Preferably, the sterilization or disinfection technique to be used in the method according to the invention is selected from the group consisting in ethanol bath, gamma irradiation, UV treatment, ethylene oxide gas, vaporized hydrogen peroxide, chlorine dioxide gas, vaporized peracetic acid, nitrogen dioxide, electron beam, plasma treatment, hydrostatic pressure, and a combination thereof, preferably the sterilization or disinfection technique to be used in the method according to the invention is selected from the group consisting in ethanol bath, gamma irradiation, UV treatment, and a combination thereof, more preferably gamma-irradiation. Preferably, the material obtained at step b) of the method according to the invention is sterilized with a radiation of about 30 kGy.

[0178] In a particular embodiment, at least about 15%, preferably at least about 25%, more preferably at least about 50%, even more preferably at least about 60% of the antimicrobial peptides of the solution of step a) remain in the material after the electrospinning step b).

[0179] In another particular embodiment, at least about 30%, preferably at least about 45%, more preferably at least about 55%, even more preferably at least about 65% of the AMPs obtained at step b) remain in the material after the washing step.

[0180] AMP Composition

[0181] In a third aspect, the invention also concerns an antimicrobial peptide composition for electrospinning comprising a solution of a biodegradable polymer, an antimicrobial peptide and a polar organic solvent.

[0182] The biodegradable polymer, the antimicrobial peptide, the polar organic solvent and their proportions are as described in the second aspect of the invention, i.e. the Method of electrospinning section herein above of the present application.

[0183] Use of the composition

[0184] In a fourth aspect, the invention further concerns the use of the composition as described in the third aspect of the invention, i.e. AMP composition of the present application (e.g. as described herein above), for electrospinning antimicrobial peptides. Product obtained by the method or use

[0185] In a fifth aspect, the invention yet concerns an antimicrobial fiber material obtained by the method of electrospinning according to the second aspect of the invention, i.e. Method of electrospinning herein above of the present application, or by the use of the composition according to the fourth aspect of the invention, i.e. Use of the composition of the present application (e.g. as described herein above).

[0186] In a preferred embodiment, said antimicrobial fiber material has an antimicrobial activity, preferably an antibacterial activity, and / or an antibiofilm activity. Preferably, said antimicrobial fiber material has an antibacterial activity and an antibiofilm activity.

[0187] The antimicrobial activity and / or the antibiofilm activity of said antimicrobial fiber material last at least about 2 days, preferably at least about 3 days, more preferably at least about 5 days, even more preferably at least about 7 days under normal conditions of use of the material. Optionally, the antimicrobial activity and / or the antibiofilm activity of said antimicrobial fiber material last at least about 14 days, alternatively at least about 21 days under normal conditions of use of the material.

[0188] The antimicrobial activity, preferably antibacterial activity, and / or the antibiofilm activity of said antimicrobial fiber material is of at least about a 3 log reduction, preferably at least about a 4 log reduction, alternatively at least about a 5 log reduction.

[0189] In another embodiment, the antimicrobial fiber material according to the invention is sterile.

[0190] In yet another embodiment, the antimicrobial fiber material according to the invention is not toxic, preferably not toxic to eukaryotic cells.

[0191] In another embodiment, the antimicrobial fiber material according to the invention further comprises a chelating agent. Preferably, said chelating agent is selected from the group consisting in Dimercaprol (BAL), ethylenediaminetetraacetic (EDTA) salts such as calcium EDTA or sodium EDTA, succimer (DMSA), penicillamine, trientine hydrochloride, deferoxamine mesylate, deferiprone, deferasirox, pentetate calcium trisodium (Ca-DTPA), pentetate zinc trisodium (Ca-DTPA), prussian blue (Radiogardase), and a combination thereof, more preferably the chelating agent is an EDTA salt, even more preferably the chelating agent is sodium EDTA. The chelating power induced by the chelating agent according to the invention may be equivalent to the chelating power induced by sodium- EDTA at a concentration comprised between about 0.1 mM and about 10 mM, preferably about 0.2 mM, in the microenvironment of the material. Alternatively, the chelating capacity of the chelating agent is in the range of 0.1 mM to about 10 mM of metal ions per liter in the microenvironment of the material.

[0192] The chelating agent according to the invention may be present in the fibers of the material. Alternatively, the chelating agent according to the invention is present in the material but not in the fibers, for example, the chelating agent may be adsorbed on the surface of the fibers.

[0193] Antimicrobial fiber mat (matrix)

[0194] In a sixth aspect, the invention also concerns an antimicrobial fiber mat comprising a material according to the first aspect of the invention, i.e. as described in the antimicrobial fiber material section herein above of the present application, or a material according to the fifth aspect of the invention, i.e. the Product obtained by the method or use section herein above of the present application and having a flat structure.

[0195] In a preferred embodiment, the antimicrobial fiber mat according to the invention has a thickness comprised between about 0.01 mm and about 5 mm, preferably between about 0.08 mm and about 0.1 mm.

[0196] In another embodiment, the antimicrobial fiber mat according to the invention has a hardness comprised between about 200 g and about 1000 g, preferably between about 300 g and about 800 g, more preferably about 500 g. As used herein, the term "hardness" refers to the ability of a material to withstand force without deformation, scratching, penetration, and indentation, i.e. its ability to maintain its physical features even in the face of applied force. It is expressed in grams, referring to how many grams of weight the material withstands without deformation. Hardness can be measured by any method well known by the man skilled in the art, for example with a texture analyser, for example to perform a puncture test (the deformation is thus puncturing / penetration). The hardness is calculated as a Force necessary to attain a given deformation or Load and can be detected at the highest peak during compression depending on the method.

[0197] In still another embodiment, the antimicrobial fiber mat according to the invention has a deformation at hardness comprised between about 2 mm and 20 mm, preferably between about 4 mm and about 15 mm, more preferably about 10 mm. As used herein, the term "deformation at hardness" refers to how much the material deforms before breaking when force equal to the hardness is applied. It is a good indicator of the flexibility of a material. It can be measured with a texture analyser and puncture testing experiments.

[0198] In yet another embodiment, the antimicrobial fiber mat according to the invention has a hardness work done comprised between about 10 mJ and 60 mJ, preferably between about 20 mJ and about 50 mJ, more preferably about 35 mJ. As used herein, the term "hardness work done" refers to the energy required to deform a material during a hardness test (work required to break the sample). It can be measured with a texture analyser and puncture testing experiments.

[0199] The mat according to the invention can be used as a monolayer dressing.

[0200] Antimicrobial multilayer system

[0201] In a seventh aspect, the invention also concerns an antimicrobial multilayer system comprising a mat according to the sixth aspect of the invention, i.e. the antimicrobial fiber mat section herein above of the present application, and further comprising at least one secondary dressing. As such, the antimicrobial multilayer system can be used as a multilayer dressing.

[0202] The mat according to the invention can be used in any kind of dressing. Dressings are well-known from the man skilled in the art. In an embodiment, the at least one secondary dressing according to the invention is absorptive. Alternatively, the at least one secondary dressing according to the invention is non-adhesive. Preferably, the at least one secondary dressing according to the invention is absorptive and non-adhesive. In another embodiment, the at least one secondary dressing is adhesive and optionally absorptive.

[0203] In another embodiment, the antimicrobial multilayer system according to the invention comprises at least two secondary dressings. Preferably, a first secondary dressing located on one side of the mat and a second secondary dressing located on the other side of the mat. Preferably, the first secondary dressing comprises an absorptive and / or adhesive layer, and / or the second secondary dressing comprises a backing layer.

[0204] As used herein, the term backing layer refers to the outermost protective layer of a wound dressing. Its main roles are to protect the underlaying dressing, i.e. the mat according to the invention, for example from moisture and / or contamination, to provide a structural support and to enhance its functionality. In a preferred embodiment, the backing layer has the same electrospun polymer composition without AMPs.

[0205] In yet another embodiment, at least a part of one of the sides of the mat of the antimicrobial multilayer system according to the invention is free of any additional dressing, optionally one of the sides of the mat is completely free of any additional dressing.

[0206] In still another embodiment, the antimicrobial multilayer system according to the invention further comprises a chelating agent. Preferably, said chelating agent is selected from the group consisting in Dimercaprol (BAL), ethylenediaminetetraacetic (EDTA) salts such as calcium EDTA or sodium EDTA, succimer (DMSA), penicillamine, trientine hydrochloride, deferoxamine mesylate, deferiprone, deferasirox, pentetate calcium trisodium (Ca-DTPA), pentetate zinc trisodium (Ca-DTPA), prussian blue (Radiogardase), and a combination thereof, more preferably the chelating agent is an EDTA salt, even more preferably the chelating agent is sodium EDTA. The chelating power induced by the chelating agent according to the invention may be equivalent to the chelating power induced by sodium-EDTA at a concentration comprised between about 0.1 mM and about 10 mM, preferably about 0.2 mM, in the microenvironment of the material. Alternatively, the chelating capacity of the chelating agent is in the range of O.lmM to about 10 mM of metal ions per liter in the microenvironment of the material.

[0207] The chelating agent according to the invention may be present in the fibers of the mat of the antimicrobial multilayer system. Alternatively, the chelating agent according to the invention is present in the antimicrobial multilayer system but not in the fibers, for example, the chelating agent may be adsorbed to the surface of the fibers or may be in a secondary dressing.

[0208] Use for treatment & Treatment

[0209] In an eighth aspect, the invention also concerns the use of the antimicrobial fiber material according to the first aspect of the invention, i.e. antimicrobial fiber material described above in the present application, of the antimicrobial fiber material according to the fifth aspect of the invention, i.e. product obtained by the method or use described above in the present application, of the antimicrobial fiber mat according to the sixth aspect of the invention, i.e. antimicrobial fiber mat described above in the present application, or of the antimicrobial multilayer system according to the seventh aspect of the invention, i.e. antimicrobial multilayer system described above in the present application, for the treatment of a subject in needs thereof.

[0210] As used herein, the term "treatment" refers to any act directing at improving the medical status of a subject. The treatment may thus improve or reduce the cause or certain symptoms of a medical condition, or eradicate the cause or certain symptoms of a medical condition. The treatment may also have the effect of preventing or slowing down the progression of a medical condition. As used herein, the term "prevention" refers to any act directed at preventing or delaying the appearance of a medical condition.

[0211] The invention also concerns the use of the antimicrobial fiber material according to the first aspect of the invention, i.e. antimicrobial fiber material described above in the present application, of the antimicrobial fiber material according to the fifth aspect of the invention, i.e. product obtained by the method or use described above in the present application, of the antimicrobial fiber mat according to the sixth aspect of the invention, i.e. antimicrobial fiber mat described above in the present application, or of the antimicrobial multilayer system according to the seventh aspect of the invention, i.e. antimicrobial multilayer system described above in of the present application, to treat a subject in needs thereof.

[0212] The invention still concerns the use of the antimicrobial fiber material according to the first aspect of the invention, i.e. antimicrobial fiber material described above in the present application, of the antimicrobial fiber material according to the fifth aspect of the invention, i.e. product obtained by the method or use described above in the present application, of the antimicrobial fiber mat according to the sixth aspect of the invention, i.e. antimicrobial fiber mat described above in the present application, or of the antimicrobial multilayer system according to the seventh aspect of the invention, i.e. antimicrobial multilayer system described above in of the present application, for the preparation of a dressing intended for the treatment of a subject in need thereof.

[0213] In a preferred embodiment, the treatment according to the invention is a topical treatment, preferably a topical treatment of the epithelium, more preferably a topical treatment of the skin or of a mucosa, such as the oral mucosa.

[0214] The invention finally concerns a method of treatment of a subject in needs thereof comprising the application of an antimicrobial fiber material according to the first aspect of the invention, i.e. antimicrobial fiber material described above in the present application, of an antimicrobial fiber material according to the fifth aspect of the invention, i.e. product obtained by the method or use described above in the present application, of an antimicrobial fiber mat according to the sixth aspect of the invention, i.e. antimicrobial fiber mat described above in the present application, or of a antimicrobial multilayer system according to the seventh aspect of the invention, i.e. antimicrobial multilayer system described above in the present application, to the subject, preferably to the epithelium of a subject, more preferably to the skin or a mucosa, such as the oral mucosa, of the subject.

[0215] In a preferred embodiment, the treatment according to the invention is a treatment of a skin condition preferably selected from the group consisting in a wound, a cancer such as a skin cancer, acne, atopic dermatitis, psoriasis, ichthyosis, scars and dull skin, such as epidermolysis bullosa, skin burns, plastic surgery support or skin implants support, or a combination thereof.

[0216] In an alternative embodiment, the treatment according to the invention is a treatment of a condition of a mucosa, preferably an oral mucosa condition, more preferably selected from the group consisting in a wound, Sjogren's disease, Lihhen planus, leukoplakia, oral ulcers e.g. Aphthous stomatitis, and periodontitis.

[0217] In a preferred embodiment, the treatment is a wound treatment, more preferably an infected wound treatment. The treatment according to the invention may thus be a skin wound treatment, more preferably an infected skin wound treatment. The treatment according to the invention may also be a mucosa, such as an oral mucosa, wound treatment, more preferably an infected mucosa, such as an oral mucosa, wound treatment. As used herein, the term "infection" or "infected" refers to a bacterial, viral or fungal infection, preferably to a bacterial infection, such as an antibiotics resistant bacterial infection, more preferably to a bacterial infection with biofilm.

[0218] In another preferred embodiment, the treatment according to the invention is a chronic wound treatment, such as chronic non-healing ulcers. It may be a skin chronic wound treatment or a mucosa, such as an oral mucosa, chronic wound treatment.

[0219] In still another preferred embodiment, the treatment according to the invention is an acute wound treatment, such as traumatic acute wounds. It may be a skin acute wound treatment or a mucosa, such as an oral mucosa, acute wound treatment. The antibacterial fiber material, the antibacterial fiber mat or the antimicrobial multilayer system according to the invention may be washed prior to its application to the subject. The antibacterial fiber material, the antibacterial fiber mat or the antimicrobial multilayer system according to the invention is preferably washed by immersion in a medium. Said washing may last between about 1 min and about 24 h, preferably between about 10 min and about 10 h, even more preferably about 1 h. The washing medium according to the invention is preferably selected from the group consisting in saline solutions, PBS and distilled water.

[0220] The antibacterial fiber material, the antibacterial fiber mat or the antimicrobial multilayer system according to the invention may be sterilized or disinfected prior to its application to the subject, eventually after being washed. Sterilization and disinfection techniques are well known from the man skilled in the art and any sterilization or disinfection technique can be used with this invention. However, the sterilization or disinfection technique according to the invention is preferably selected from the group consisting in an ethanol bath, gamma irradiation, UV treatment, and a combination thereof, preferably gamma-irradiation.

[0221] In a preferred embodiment, the antibacterial fiber material, the antibacterial fiber mat, or the antimicrobial multilayer system according to the invention is removed or replaced after between 1 and about 21 days, preferably after between 1 and about 14 days, more preferably after between 1 and about 7 days, still preferably after between 2 and about 4 days, even more preferably after about 3 days. Preferably, a new antibacterial fiber material, antibacterial fiber mat, or antimicrobial multilayer system according to the invention is applied to the subject when the previous one is removed, and as many times as necessary, for example until full recovery of the subject. For example, the antibacterial fiber material, antibacterial fiber mat, or antimicrobial multilayer system according to the invention may be replaced 2, 3, 4, 5 ,6, 7, 8, 9, 10 times.

[0222] The subject according to the invention is a human or a non-human animal, preferably a mammal, more preferably a mammal selected from the group consisting in dogs, cats, horses, cows, pigs, sheep, goats, rabbits, hamsters, guinea pigs, hens, chickens, ducks and non-human primates. Preferably, the subject according to the invention is human. EXAMPLES

[0223] Material & Method Materials

[0224] Polymers. Biodegradable high-quality polymers of DL-lactide (PDLA, Purasorb® PDL 20, MW 406 000 g / mol; chemical name: poly[3,6-dimethyl-l,4-dioxane-2, 5-dione]), copolymer of L-lactide and E-Caprolactone in a 70 / 30 molar ratio (PLC, Purasorb PLC7015, Purac Corbion, The Netherlands, inherent viscosity midpoint of 1.5 dL / g, MW 202 000 g / mol; chemical name: (3S-cis)-3,6-dimethyl-l,4-dioxane-2, 5-dione, polymer with 2- oxepanone), polymer E-polycaprolactone (PCL, Purasorb PC 12, Purac Corbion, MW 115 000-135 000 g / mol; chemical name: poly[2-oxepanone]) as hydrophobic polymers were purchased from Corbion. PEO (polyethylene oxide), SENTRY ™ POLYOX™ WSR 1105- LEO NF Grade, DOW Chemicals, MW 900 000 g / mol; chemical name: a-hydro-oj- hydroxypoly(oxyethylene)) was used as a hydrophilic polymer together with PLC fortesting the in-situ microfluidic electrospinning method of preparation of AMP-loaded matrices (Lanno et al., PCT / EP2022 / 076874). Low melt S agarose (AppliChem) was used for making the microcapsules.

[0225] Antimicrobial peptides (AMPs). Bacitracin Zn salt (BAC), gramicidin (GRA), daptomycin (DAP) were all obtained from Thermo Fisher Scientific. Colistin sulphate (COL) (C4461; also named Polymyxin E; according to manufacturer consisting of a mixture of Polymyxin El and E2) was obtained from Sigma. All other antimicrobial peptides (LL-37, pleurocidin (PLE), its analogue D-pleurocidin (D-PLE), and temporin L (TEMP)) were purchased from Cambridge Research Biomedicals (Cleveland, UK) as desalted grade (crude). All peptides were amidated at the C-terminus. Nisin (Lactococcus lactis, > 900IU / mg), was obtained from Merck Millipore.

[0226] Antibiotics (ABs) Ciprofloxacin (CIP, PubChem CID: 2764), clindamycin hydrochloride (CLIN, PubChem CID: 131632848), and ceftriaxone disodium hemi(heptahydrate) (CEF, PubChem CID: 124202479) were purchased from Sigma-Aldrich Inc. (Germany).

[0227] Solvents. 1,1,1,3,3,3-hexafluoroisopropanol (HFIP, 99.5%) purchased from Apollo Scientific was applied as a solvent in electrospinning. Other solvents used for peptide purification and high-performance liquid chromatography (HPLC) analyses were acetonitrile, trifluoroacetic acid (TFA), phosphoric acid, and methanol. These were - M - purchased from Sigma-Aldrich and were of reagent grade and used as received without any further purification. Methylcarbonate (Me2CO3; Sigma) and distilled water were used for in situ microfluidic electrospinning. / V, / V-Dimethylformamide (DMF, anhydrous, 99.8%) purchased from Sigma-Aldrich, and acetic acid (AA, 99.8 - 100.5%) and formic acid (FA, >98%) purchased from Honeywell Fluka were used as solvents in electrospinning of control matrices.

[0228] Buffers, growth media. Sodium sulphate and potassium dihydrogen phosphate from Sigma were used to make solutions for HPLC. Mueller-Hinton broth (MHB) was purchased from Thermo Fisher Scientific, Oxoid (Basingstoke, UK). All bacteria were grown in BDTM DifcoTM Lennox lysogeny broth (LB) (Becton, Dickinson and Company, Le Point de Claix, France) culture medium. For the biofilm studies Dulbecco's Modified Eagle Medium DMEM / F-12 (Sigma-Aldrich, Gillingham, United Kingdom), without L-glutamine and phenol red was used together with 10% (v / v) heat inactivated fetal bovine serum (FBS, Sigma-Aldrich, Sao Paulo, Brazil). For some experiments the media were supplemented with ethylenediaminetetraacetic acid (EDTA) from Sigma. Phosphate buffered saline (1 x PBS) pH adjusted to 7.4 was used in this study. For European Pharmacopoeia (Ph.Eur, 10.0) sterility testing (monograph 2.6.1.), two different dehydrated culture media soya-bean casein digest medium (also known as Tryptic Soy Broth (TSB), Sigma-Aldrich, Bangalore, India) and fluid thioglycolate medium LAB025 (Lab M Limited, Lancashire, United Kingdom) were used. To create anaerobic conditions anaerobic gas generation bags BD GasPakTM, EZ Anaerobe Container System (Benex Limited, Dublin, Ireland) were used. For eukaryotic cell study Glasgow Modified Essential Medium (GMEM) (GibcoTM, Thermo Fisher, USA) supplemented with 10% fetal bovine serum (FBS) (GibcoTM, Thermo Fisher, USA), 2% tryptose phosphate broth (TPB) (GibcoTM, Thermo Fisher, USA), 2% IM HEPES buffer (Corning Inc., Corning, USA), 100 pg / mL penicillin, and 100 pg / mL streptomycin (GibcoTM, Thermo Fisher, USA) was used.

[0229] Bacteria. Strains utilized in bacterial tests (liquid cultures) included E. coli DSM1103, 5. aureus DSM2569 and P. aeruginosa DSM1117, relevant for skin and wound infections purchased from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) and stored at -80°C. E. coli DSM1103 was also used for antibiofilm efficacy testing (Lorenz et al. 2023). Additionally, Acinetobacter baumannii AYE, ATCC 17978 and Pseudomonas aeruginosa PAO1, NCTC 13437 along with Escherichia coli NCTC 12923 were selected as relevant multiresistant bacteria to study the potential antimicrobial activity of the electrospun AMP-loaded matrices. These bacteria were obtained through the National Collection of Type Cultures (NCTC). For sterility testing E. coli laboratory strain MG1566 (ATCC 700926) for aerobic conditions and Fusobacterium nucleatum spp polymorphum for anaerobic conditions were used as positive controls. These bacterial strains were purchased from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) and stored at -80°C.

[0230] Eukaryotic cells. Baby Hamster Kidney fibroblast cell line (BHK-21) was used to test the safety and biocompatibility of the prepared AMP-loaded matrices.

[0231] Methods

[0232] Antimicrobial peptides (AMPs) purification

[0233] The crude AMPs purchased were further purified using reverse phase chromatography with a Waters SymmetryPrep C8, 7 mm, 19 x 300 mm column. A gradient of H2O - TFA 0.1% and acetonitrile - TFA 0.1% was used for this purpose. The collected phase was then centrifuged, immersed in liquid.

[0234] Preparation of electrospinning solutions

[0235] For the preparation of ES solutions, hydrophobic polymer PDLA 5% (wt), PLC 10% (wt) or PCL 10% (wt) solutions in HFIP were prepared. For testing of polymer mixtures, two different polymer solutions were prepared (5% PDLA and 9% PCL solutions) and then mixed together by weight and then left stirring on the magnetic stirrer for approximately for 1.5 hours. We tested three different mixtures with ratios, 50% PCL + 50% PDLA, 34% PCL + 66% PDLA, and 30% PCL + 70% PDLA. 70:30 and 50:50 mixtures were selected for further studies with AMP, whereas then the ES solutions were not prepared by mixing separately prepared PDLA and PCL solutions but both polymers were weighed in a single vial and dissolved together in a solvent. All formulations were produced with AMP inside the fibers and without AMP pristine polymeric fibers as control matrices. All polymer solutions were prepared in 20 mL glass vials, solution amount was 6 mL for monoaxial needle-based ES. All ES solutions were left on the magnetic stirrer to stir for 18 ± 2 h. All formulations were produced with various AMPs (ribosomal or non-ribosomal) inside the fibers. Different peptides were used, namely ribosomal cationic AMPs LL-37, pleurocidin (PLE), D-PLE, L-temporin (TEMP) and non-ribosomal cationic AMPs bacitracin (BAC); gramicidin (GRA); colistin (COL), and daptomycin (DAP). The used peptides with their theoretical amount are provided in Table 1. Peptide was added into the polymer solution 20 min, 2h or up to 20 h prior ES. Alternatively, polymer and AMP (and AB if used) were added together to the solvent and stirred for 20 min to 24 hours. Peptides concentrations were also varied depending on the peptide, and for some peptides different concentrations were tested (0.5, 1, 4 vs 8%).

[0236] For in-situ microfluidic electrospinning, PLC solution (12% w / w) in MeCO3 with PEO (0.3% w / w) was prepared (solution 1). This polymer solution was kept on a magnetic stirrer for one-day prior use. PLC solution with PEO needed heating during dissolving, therefore the solutions were heated on a heating plate at 40°C with constant stirring for 24 h prior electrospinning. Before electrospinning the solutions were allowed to cool down to room temperature. For the preparation of microcapsules, 0.625% agarose solution in distilled water was prepared (solution 2). Solutions were prepared in vials and left on heated (90°C ± 2° C) magnetic stirrer overnight. AMP-loaded electrospinning solutions were prepared as previously reported (Lanno et al., PCT / EP2022 / 076874). PLC+PEO solution and agarose solution were prepared and an antimicrobial peptide (COL) was added to the agarose solution. COL (0.151 g) was added while preparing 0.625% agarose aqueous solution (ad 10 g) and stirred on magnetic stirrer for 1 h prior electrospinning.

[0237] Fibers comprising antibiotics (AB) in combination with COL (COL / CIP, COL / CLIN, COL / CEF) were also prepared. Initially, the electrospinning solution was prepared from two different polymers in HFIP. Both polymers were weighed to obtain a mixture of two polymers in 5050 ratio. Briefly, 10% (w / w) of PCL was weighed (1.7 g) and 5% (w / w) of PDLA (0.85 g) was weighed and approximately 10 mL of HFIP added on top (adjusted if needed) and the weight of added solvent registered. The total amount of antimicrobial agents was 3.9% (w / w), which consisted of 2.6% of AMP (COL) and 1.3% of AB (CIP; CLIN;CEF). The solution was stirred until full dissolution of the polymers and drugs. Electrospinning was conducted as previously described and the exact electrospinning process and environmental parameters are given in Table 2. Table 1. All antimicrobial peptide (AMPs) loaded formulations (with ribosomal and non-ribosomal AMPs) for monoaxial needle-based electrospinning (ES).

[0238] Electrospinning (ES) ES was performed using an ESR2OORD robotized ES system (NanoNC, Seoul,

[0239] Republic of Korea) using monoaxial needle-based set-up. ES process parameters were varied to achieve homogenous and reproducible fiber formation. For different formulations (AMPs, polymers and solvents) slightly different ES process parameters were used, but the used ranges were as follows:

[0240] • Solution volume: 4 - 9 mL

[0241] • Needle: 23 G • Voltage: 6.9 - 15.2 kV

[0242] • Flow rate: 0.8 - 2.5 mL / h

[0243] • Distance from collector: 11 - 18 cm

[0244] • Temperature: 22 - 26 °C

[0245] • Relative humidity: 19 - 36 % Process was repeated at least in duplicate to understand the reproducibility of the electrospinning.

[0246] Table 2. ES process parameters for producing AMP loaded fiber matrices. In all conditions a needle of 23 G was used, the collector was a half or full roller, the rotation speed of the collector was always of 25 rpm.

[0247]

[0248] A plastic syringe (10 mL, inner diameter 15.89 mm), supplemented with a blunt needle (23 G) was loaded with a polymeric solution with or without active ingredient (AMP). Polymer solution volume varied from 4 - 9 mL depending on the collector size (half roller 29 x 10 cm or full roller 29 x 20 cm).

[0249] For fiber collection a roller collector (diameter 9 cm, width 10 or 20 cm, rotating at 25 rpm) covered with aluminum foil was used. Ready samples were put into Ziploc bags. Electrospun AMP-loaded fiber matrices and pristine control matrices were stored protected from the light in a desiccator above silica gel (low relative humidity, RH) and under vacuum at room temperature (RT) (21 ± 2 °C) to avoid humidity induced changes in the matrices. Table 3. ES process parameters for producing pristine fiber matrices. In all conditions a needle of 23 G was used, the collector was a full roller, used at a rotation speed of 25 rpm. For in-situ microfluidic electrospinning, ES was performed using an ESR200RD robotized ES system using monoaxial set-up and attached to microfluidic device, as described in Lanno et al (PCT / EP2022 / 076874). Shortly, for microfluidics, a chip made of polydimethylsiloxane (PDMS) was prepared. The three-channel (two inputs and one output) connection was built of channels of a square-section, with overall channel diameter of 0.40 mm, and a microcapsule forming section diameter of 0.1 mm. Microcapsule forming section ratios of 1 x 1 x 1 cm were used. The microcapsules are formed in the T junction of the microfluidic device and exit from the outlet channel towards the needle attached to the exit tube. The ES electrode is attached to the needle.

[0250] The following ES conditions were used for AMP-loaded matrix preparation: 0.9 mL / h for the PLC+ PEO solution (solution 1) and at the same time 0.2 mL / h for the AMP- consisting agarose solution. The viscosity of PLC / PEO solution was as previously 784 cP; (Torque 54,5%; Temperature: 22°C; Speed 5 RPM).

[0251] Total of 2.7 mL PLC / PEO solution and 0.6 mL agarose + COL solution was electrospun on a metal collector plate covered with aluminum folio (ES lasted for 3 h). Plastic syringes (3 mL) were used and single use blunt needle B. Braun of 18G (inner diameter of 0.34 mm) was used. ES parameters were voltage of 13 kV and distance between the needle and collector of 11 cm. Humidity varied between 15.3 and 30.8% and temperature between 27.3°C to 31.0°C. To keep the RH in the ES chamber in specified ranges, a dehumidifier (COTES A / S, Denmark) was used. Ready samples on foil were put into Ziploc bags. The samples were kept at RT (21 ± 2 °C) in a desiccator at 0% RH above silica gel to avoid humidity induced changes in the matrices.

[0252] Electrospun matrices characterization

[0253] Morphology

[0254] The morphology of electrospun fiber matrices and fiber diameter analysis were investigated using scanning electron microscopy, SEM (Zeiss EVO 15 MA, Hamburg, Germany). Random areas were selected to study the diameter and surface topography of each matrix. For SEM analysis, the matrices were mounted on aluminum stubs and magnetron-sputter coated with a 3-nm platinum layer in an argon atmosphere prior to microscopy. Images of the top layer were collected and analyzed. Mean fiber diameter was calculated (N=100) and presented together with the standard deviation (SD).

[0255] AMP content and stability

[0256] Mass spectroscopy (MS) analyses were performed to understand the stability of AMPs (PLE) within used solvents for electrospinning and within fibers after electrospinning. MS analysis was conducted using mass spectroscopy (MS) Q Exactive Plus (Thermo Fisher Scientific, Waltham, Massachusetts, USA). MS raw files were processed with MaxQuant software package (1.4.0.8).

[0257] The AMP and / or AB loading into the fiber matrices was analyzed using a high- performance liquid chromatography (HPLC) (LC20, Shimadzu Europa GmbH, Duisburg, Japan). Random 4 cm2pieces were cut and weighted to study not only the content in AMP (or AB), but its distribution across the fiber matrix. All 2x2 cm matrices were dissolved in 1 mL of HFIP and then 4 mL of distilled water added, vortexed and filtered through the PTFE filter with the pore size of 0.45 pM. Alternatively, after dissolving the matrices in 1 mL of HFIP, 2 mL of distilled water was added, vortexed and centrifuged at 15,000 rpm for 5 minutes, after which the supernatant was collected for the analysis.

[0258] For COL analyses HPLC LC20 with PDA detector (wavelength at 215 nm) and Phenomenex Luna C18(2), 250 X 4.6 mm, 5 pm and European Pharmacopoeia method (vol 9) were used. The mobile phase used was a 22:78 V / V mixture of acetonitrile and a solution of sodium sulphate, the pH of latter adjusted to 2.4 using phosphoric acid. The flow rate was 1.0 mL / min, and injection volume was 10 pL.

[0259] For PLE, D-PLE, TEMP and LL-37, the matrices were dissolved in 1 mL of HFIP + 2 mL H2O. For analyses HPLC LC20 with PDA detector (wavelength at 220 nm) and Phenomenex Luna C18(2), 250 X 4.6 mm, 5 pm was used. For mobile phase A 0.1 % (V / V) TFA in water and mobile phase B 0.1% (V / V) TFA in acetonitrile was used. The flow rate was of 1.0 mL / min and the injection volume was 20 pL, the gradient scheme used was: 0- 3 min B 10%; 3-20 min B 10% -> B 80%; 20-25 min B 80 %; 25-29 min B 10%.

[0260] For BAG analyses HPLC LC20 with PDA detector (wavelength at 254 nm) and Phenomenex Luna column size 150 X 4.6 mm, 3 pm was used. The mobile phase used was a 43:100:300:557 V / V / V / V mixture of acetonitrile, solution B, water for chromatography and methanol. Solution B was made in a volumetric flask as follows: 54.4 g of potassium dihydrogen phosphate in water for chromatography was dissolved and diluted to 2000 mL with the same solvent. The pH was adjusted to pH 6.0 with a 34.8 g / L solution of dipotassium hydrogen phosphate and filtered through a membrane filter (nominal pore size 0.45 pm). The flow rate was 1.0 mL / min, and injection volume was 20 pL.

[0261] For DAP analyses HPLC LC20 with PDA detector (wavelength at 262 nm) and Phenomenex Luna C18(2), 250 X 4.6 mm, 5 pm was used. The mobile phase used was a 46:54V / V mixture of acetonitrile-buffer (KH2PO4 20 mM pH = 3.2). The flow rate was 1.0 mL / min, and injection volume was 20 pL.

[0262] For GRA analyses HPLC LC20 with PDA detector (wavelength at 282 nm) and Phenomenex Luna C18(2), 250 X 4.6 mm, 5 pm was used at temperature 50°C. The mobile phase used was a 29:71 V / V mixture of water and methanol. The flow rate was 1.0 mL / min, and injection volume was 20 pL.

[0263] For CLIN analyses HPLC LC20 with PDA detector (wavelength at 210 nm) and column with a stationary phase of octylsilyl silica gel for chromatography R, 250 X 4.6 mm, 5 pm was used. The mobile phase used was a 1:4 V / V mixture of acetonitrile R1 and 13.6 g / L (0.1M) solution of potassium dihydrogen phosphate R previously adjusted to pH 2.5 with phosphoric acid R. The flow rate was 1.0 mL / min and injection volume 10 pL, run time

[0264] 20 minutes. For CIP analyses HPLC LC20 with PDA detector (wavelength at 215 nm) and

[0265] Phenomenex Luna C18(2), 250 X 4.6 mm, 5 nm was used at temperature 40 °C. The mobile phase was a 13:87 V / V mixture of acetonitrile R and 2.45 g / L solution of phosphoric acid R, previously adjusted to pH 3.0 with triethylamine R. The flow rate was 1.0 mL / min and injection volume 20 pL, run time 20 minutes.

[0266] For CEF analyses HPLC LC20 with PDA detector (wavelength at 215 nm) and Phenomenex Luna C18(2), 250 X 4.6 mm, 5 pm was used at temperature 40 °C. The mobile phase was a 13:87 V / V mixture of acetonitrile R and 2.45 g / L solution of phosphoric acid R, previously adjusted to pH 3.0 with triethylamine R. The flow rate was 1.0 mL / min and injection volume 20 pL, run time 20 minutes.

[0267] Nisin

[0268] Drug trapping and leaching from the matrix after incubation for 24 h, 3 days and 1 week

[0269] AMP-loaded matrices (size 2 x 2 cm) were placed into different media, namely DMEM + 10% FBS; DMEM + 0.2 mM EDTA, LB + 0.2 mM EDTA and lxPBS for 24 h at 37°C. Additional leaching test was performed with DMEM + 0.2 mM EDTA and lxPBS and incubation periods of 3 days and 1 week at 37°C. After the incubation period, matrices were rinsed with distilled water, placed into the freezer (-20°C) overnight and then lyophilized for 24 h. Matrices were weighted and dissolved for content analysis in HPLC as described previously (AMP content and stability).

[0270] Bacterial studies for pure AMPs and drug-loaded electrospun fiber matrices (in vitro)

[0271] Checkerboard Assay - antibacterial synergy testing

[0272] A checkerboard assay was performed to evaluate the interaction between COL and selected antibacterial agents. The aim was to confirm that COL does not show antagonism with the ABs loaded into combination matrices: CIP, CLIN or CEF. Stock solutions (1 mg / mL) of each antibacterial agent were prepared in deionized water and subsequently diluted with Mueller-Hinton broth (MHB) to reach the needed concentration. 50 pL of both COL and AB solutions were pipetted into 96-well plates, so the concentration of COL decreased stepwise along the ordinate and concentration of AB decreased stepwise along the abscissa. The final concentrations of COL decreased from 250 pg / mL, 200 pg / mL, 150 pg / mL, 100 pg / mL, 64 pg / mL, 32 pg / mL, to 16 pg / mL. In the last row, no COL was added; instead, 50 pL of MHB was used. From that row, MIC of the AB was obtained. The final concentrations of the AB decreased stepwise from 64 pg / mL, 32 pg / mL, 16 pg / mL, 8 pg / mL, 4 pg / mL, 2 pg / mL, 1 pg / mL, 0.5 pg / mL, 0.25 pg / mL, 0.125 pg / mL, to 0.0625 pg / mL. In the last column, no AB was added, and 50 pL of MHB was used instead. From that column, MIC of COL was obtained.

[0273] An overnight culture of 5. aureus DSM2569 was adjusted to an OD600 of 1.0 (~109CFU / mL) and subsequently diluted 100-fold twice to achieve =105CFU / mL. A 100 pL aliquot of this inoculum was added to each well. Plates were incubated statically at 37 °C for 24 h, after which optical density was measured using a CLARIOstar® Plus plate reader (BMG LABTECH, Germany). To quantify the interaction between the tested ABs, the FIC (fractional inhibitory concentration) index (FICI) was calculated using the following formula:

[0274] FICI < to 0.5 were considered strongly synergistic, values of 0.5 to <1 were considered to indicate weak synergism, indifferent effect was defined when 1 < FIC < 2, and antagonism behaviour for FIC values > 2.

[0275] Antibacterial activity assay - antimicrobial peptides (AMPs) in solution

[0276] The antibacterial activity of the AMPs was assessed through a modified two-fold broth microdilution assay with modal minimal inhibitory concentrations (MICs) generated from at least three biological replicate experiments. The method broadly followed EUCAST methodology, with the exception that in this case the non-cationic-adjusted Mueller Hinton was used. Peptides were diluted in a two-fold dilution in media down a 96-well plate. Bacteria (A. baumanii) were then added, at a starting concentration of 105CFU / mL, from a diluted overnight culture. Plates were incubated for 20 h at 37 °C in static conditions. The OD600 was determined using a Clariostar plate reader (BMG Labtech). The MIC was defined as the lowest concentration where growth was < 0.1 above the background absorbance. Antibacte ria 1 activity assay - antimicrobial peptide (AMP) loaded matrices

[0277] Method I. The antibacterial activity of AMP-loaded electrospun matrices was analyzed using MIC-like assay. The 0.6 cm diameter pieces of the electrospun matrices were directly inserted into the wells of a 96 well plate previously filled with 100 pL bacterial growth media (MHB). The theoretical concentration of AMP in each well was calculated based on each independent matrix weight and this information was used for the preparation of positive controls. Non-loaded control electrospun matrices and a filter paper were used as negative controls. 100 pL of bacteria (P. aeruginosa PAO 1) dispersion was added at a concentration of 105CFU / mL, from a diluted overnight culture. Plates were incubated for 20 h at 37 °C in static conditions. Bacterial growth was visualized against black surface if the visible growth occurred or not.

[0278] Method II. Overnight culture (12-18 h) of E. coli DSM1103, 5. aureus DSM2569 or P. aeruginosa DSM1117 in LB (inoculated by transferring 1 colony from LB plate to 10 mL of LB) was centrifuged (10 min, 3000 rpm) and the pellet was resuspended in a new growth medium (DMEM / F12 without phenol red + 10% FBS). The density of the culture was diluted to ODl.

[0279] Following dilutions were made with the same medium from the bacterial culture:

[0280] 1) The obtained OD1 culture - 10A9 CFU / mL

[0281] 2) lOOx dilution - 10A7 CFU / mL

[0282] 3) lOOx dilution - 10A5 CFU / mL

[0283] 4) lOOx dilution - 10A3 CFU / mL

[0284] ES wound matrices (cut in lxl cm2) were placed into the wells with the bacterial dispersions and incubated for 24 h at 37 °C. The growth inhibition (max log CFU limiting growth) was assessed visually (clear or turbid media) and killing of bacteria (max log CFU killed bacteria) was assessed by plating the media out on LB plate. To check the presence of any viable bacteria attached onto the matrices, the matrices were washed twice with lx phosphate buffered saline (lxPBS) to remove residues of the medium and any bacteria in it and transferred into 1 mL of fresh LB liquid medium on a new 24 well-plate. The wellplate was incubated at 37 °C until the following day when sterility was estimated. Turbidity of the medium indicated the presence of viable bacteria attached onto the matrix, whilst medium remained clear if the matrix was sterile. Preliminary tests in alternative growth media were carried out using a method described above with a few modifications. The matrices were challenged with 1 mL of bacterial dispersions (E. coli DSM1103, 5. aureus DSM2569 or P. aeruginosa DSM1117) at the concentration of 103CFU / mL in either 0.2 mM disodium EDTA dihydrate solution in DMEM / F12 culture medium or 0.2 mM disodium EDTA dihydrate solution in LB. After incubation, the samples were visually inspected to detect turbidity that would indicate bacterial growth and lack of antimicrobial efficacy.

[0285] Method III (ASTM E218-18 assay)

[0286] The method followed the ASTM E2180-18 standard with minor modifications. 0.3% agar slurry was prepared by heating and stirring agar in lxPBS until fully dissolved, then sterilized by autoclaving for 15 min at 121 °C and equilibrated to 37 °C.

[0287] Overnight liquid culture of E. coli DSM1103 was diluted to OD 1 (600 nm). 100 pL of the bacterial dispersion was added to 10 mL portion of agar slurry, resulting in an inoculum of approximately 107CFU / mL. The CFU count was also determined by dilution and plating.

[0288] 1.5x1.5 cm pieces of matrices were placed into tissue culture inserts in 12-well plates (CellCrown™12NX, Scaffdex) and immersed in 3 mL of lxPBS. The matrices were soaked for 1 day, 3 days or 7 days at 37 °C. Subsequently, the matrices were rinsed with distilled water and left to dry without being removed from the inserts. 50 pL of inoculated agar slurry was pipetted onto each piece of matrix, resulting in a final inoculum level of approximately 5xl05CFU / matrix. As a control, 50 pL of inoculated agar slurry was pipetted onto blank ES matrix of either pristine PCL or PDLA. To prevent drying, 1 mL of lxPBS was added to outer empty wells, and plates were sealed in plastic Ziplock bags. Once the slurry had gelled, plates were incubated at 37 °C for 24 h. Two replicates were performed for each matrix and controls.

[0289] After incubation, matrices were removed from the inserts and transferred to Eppendorf tubes containing 1 mL of PBS. The tubes were then sonicated for 1 min and vortexed at maximum speed for 1 min.

[0290] Ex vivo antibiofilm activity assay - antimicrobial peptide (AMP) loaded matrices An antibiofilm effect was studied using a method published by Lorenz et al. 2023. Sterile non-treated flat bottom 24-well-plates (VWR® International, LLC, Shanghai, China) were used and inside one well-based biofilm model was set up as follows. Three sterile filter paper discs cut to fit the well properly (diameter of 13 mm) were placed at the bottom of the well. Dulbecco's Modified Eagle Medium (DMEM / F-12) without L-glutamine and phenol red supplemented with heat-inactivated FBS (10% (v / v)) used as a medium to mimic the wound exudate. 250 pL of medium was added into every well, which was an optimal amount to immerse the filter papers and keep the set up moist. Then 1 x 1 cm pig ear skin samples (cut in aseptic conditions under laminar-flow hood) was positioned on top of the filter papers. Next a dilution from an overnight culture (20 h, 37°C, 200 rpm) of pathogenic E. coli DSM 1103 (ATCC 25922, clinical isolate) in LB was diluted using 1 x PBS to optimal dilution (10E6 CFU / mL) was used. 10 pL of bacterial dispersion was added on top of the pig skin for ex vivo model and immediately after ES wound matrices were added on top. To ensure close and comparable contact between ES wound matrix and pig skin well plate inserts (CellCrownTM 24, Scaffdex Oy, Tampere Finland) were placed on top of ES matrix to obtain tight contact with the skin and to allow proper hydration of the wound matrix. For the biofilm assay, drug-loaded samples were always studied in comparison to pristine wound matrices and to uncovered substrates (pig skin). For that in every assay three samples were covered with drug-loaded ES wound matrices, three with pristine wound matrices and three were left uncovered and incubated as described above.

[0291] The set up were incubated for 24 h at 37°C. For incubation, well plates were closed with parafilm and placed in the Zip-lock bags to avoid drying. After incubation, the wound matrices were removed from the substrate and both substrate and matrix were separately placed into 1 mL of 1 x PBS, washed twice with 1 x PBS and biofilm quantification was carried out as described below. Firstly, planktonic bacteria were removed by washing the samples (S = 1 cm2) twice with 1 mL of 1 x PBS solution and placed into a new buffer (1 mL). Biofilm disruption was carried out by vortexing (Vortex-Genie 2, Scientific Industries) samples for 30 s and sonification (Bandelin Sonorex digital 10 P, operating at 20% of maximum power) for 30 s was used 6 times. When not handling the samples, they were placed on ice. After that 100 pL aliquots were used to make 10-times dilutions and plated on LB agar plates, and incubated overnight at 37°C. Every experiment was carried out in triplicate and the mean value was obtained. Also, technical replicates were used for CFU plating. Data are presented as CFU / cm2that equals CFU of bacteria from biofilm grown on lxl cm sample. In vitro antibiofilm activity assay after 24 h of incubation in biorelevant buffer solution.

[0292] It was studied if the peptide loaded matrices had an antibiofilm activity after being in 1 x PBS buffer for 24 h. For that COL loaded matrices were placed into 1 x PBS buffer (pH 7.4) for 24 h at 37 °C. After that, the same matrices were used in in vitro biofilm assay described in previous paragraphs, but instead of using pig ear skin an ES Gelatine - glucose matrix as an artificial skin was used. Method published by Lorenz et al. 2023.

[0293] Preliminary degradation testing after 1 week

[0294] For COL containing ES matrices a preliminary degradation test was made for one week. A set of 4 cm2square-shape samples (N=3) were cut from the fiber matrices and weighed, then immersed into 10 mL of lxPBS solution statically at 37°C for 1 week. Static conditions were selected in order to mimic the wound environment. After that, the samples were removed from PBS and washed with distilled water and placed on a plastic Falcon Cell strainer sieve (mesh size 40 pm, 70 pm diameter) (Fisher scientific, Thermo Fischer, USA) placed on a 50 mL Falcon tube to remove free surface solution and weighed. Wet samples were lyophilized for 24 h. After 24 h the dry weight of the samples was measured. Weight difference was calculated considering the weight of the matrix before the test and the dry weight of the matrix after the test. The fiber matrices morphology before and after degradation testing was assessed by SEM.

[0295] Furthermore, the shrinking and no shrinking behaviour of the matrices (COL-loaded and pristine polymer matrices) was studied visually by taking images of the matrices and measuring the size of the samples after being 24 h in biorelevant conditions.

[0296] Mechanical analyses

[0297] The mechanical behavior of the ES fiber matrices (non-sterilized and y-sterilized) was studied by Brookfield CT3 Texture Analyzer (Middleboro, MA, USA) equipped with a 10 kg load cell. Puncture test was performed using TexturePro CT software (AMTEK Brookfield, Middleboro, MA, USA). 1.5x1.5 cm pieces of the matrices were used for analysis which were secured between the film support fixture (TA-FSF) and punctures were made with a cylinder probe (TA-42, diameter 3 mm). The target distance 40 mm was used with all the samples with trigger load 5 g and test speed of 2.5 mm / s. All measurements were performed at ambient conditions (temperature of 22 ± 1 °C and RH of 20 ± 2%). Each sample group comprised at least 3-5 specimens. The mean thickness of the fiber matrices varied from 0.05 - 0.1 mm measured using Precision-Micrometer 533.501 (Scalamesszeuge, Dettingen, Germany) with a resolution of 0.01 mm.

[0298] Safety and biocompatibility of antimicrobial peptide (AMP)-loaded fiber matrices

[0299] The direct MTS (a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- (4- sulfophenyl)-2H-tetrazolium) assay or indirect assay using Cell Counting Kit-8 (CCK-8, Proteintech, USA), based on WST-8 (chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4- nitrophenyl)-5- (2,4-disulfobenzene)- 2H-tetrazole monosodium salt), were used to test the safety of the ES matrices. Both assays allow to determine the relative viable cell numbers compared to control cells and thus understand cell viability, proliferation, and cytotoxicity.

[0300] The experiments were conducted using hamster kidney (BHK-21) fibroblasts cell line. Fibroblasts were grown in GMEM supplemented with 10% FBS, 2% TBP, 10 mL 1 M HEPES, 100 pg / mL penicillin, and 100 pg / mL streptomycin and were incubated at 37 °C and 5% CO2.

[0301] Direct assay

[0302] The experiment was conducted on 24-well microtiter plates, and 1.5 x 1.5 cm matrices were cut from different locations from the produced matrix and put into CellCrownTM 24-wellplate inserts (Merck, Sigma-Aldrich, Saint Louis, Missouri, USA). The BHK cells were seeded onto ES matrices using a concentration of 50.000 cells per well in 0.5 mL. Additional 750 pL of medium was added into the wells and incubated together with cells at 37°C and 5% CO2for 24 h. As a control, untreated cells grown on the bottom of 24-well plate wells were used. Background wells were created adding 1.25 mL of fresh medium into the empty wells. After incubation, 125 pL of MTS Cell Proliferation reagent (Biovison, Waltham, Boston, MA, USA) was added into each well. Additional 1 h of incubation in the same conditions was carried out, until color change was visible. 200 pL samples from the 24-well plate was transferred to the 96-well plate, making technical duplicates for each. Then, absorbance was measured using a microplate reader at OD=490 nm. Viability was calculated using control cells grown in the bottom of the 24-wellplate.

[0303] Indirect assay

[0304] The cells were seeded onto 24-well plates at a density of 5 x 104cells / well in 0.5 mL of cell culture medium. Cell density was determined using the trypan blue method and the cell counter Countess 3 (Invitrogen, Thermo Fisher, USA). The cells were incubated at 37°C and 5% C02. After 24 h, the culture media were removed from the wells and replaced by the ES fiber extracts. ES fiber extracts were prepared by incubating ES fiber matrices (1 x 1 cm) in 1 mL of cell culture medium at 37°C and 5% CO2 for 24 h. After another 24 h, CCK-8 activity was measured. A CCK-8 solution was added to the cells growing in the growth medium at a ratio of 1:10 and incubated for an additional 1 h at 37°C and 5% CO2. A medium without cells was used as a control for background absorbance. Samples from 24-well plates were transferred to a 96-well plate, making technical duplicates for each, and diluted four times with PBS after which the absorbance was measured using a microplate reader (Tecan Sunrise, Tecan Group Ltd., Mannedorf, Switzerland) at 450 nm.

[0305] Sterilization and sterility testing

[0306] For sterilization of ES matrices, UV radiation (30 min on both sides) and gamma radiation (30 kGy) were tested. Alternatively, the matrices were disinfected using 70% ethanol (immersion for 1 min). After sterilization or disinfection, European Pharmacopoeia sterility test was performed for control wound matrices to evaluate the effectiveness of the selected methods and the drug content in the AMP-containing ES wound matrices was determined after different treatments using HPLC.

[0307] Gamma-sterilization of electrospun matrices was carried out by Scandinavian Clinics Estonia OU. Irradiation was performed using 6OC0 radiation source at the dose level of ~25 kGys. The drug content in electrospun matrices was subsequently determined by HPLC, comparing gamma-sterilized samples with non-sterilized controls as described above.

[0308] Statistical analyses

[0309] Data are presented as an arithmetic mean (n = 3) ± standard deviation (SD). Statistical analyses were performed using either pairwise t-test or independent t-test, variance was calculated using f-test. CFU data were loglO-transformed before statistical analyses. Log reduction was determined by subtracting the log of the bacterial concentrations in treated samples from the ones quantified in untreated samples (biofilms). Scientifically significant difference was found using p-values and p-value of 0.05 was considered significant. In case of multiple comparisons, the p-values were adjusted, using Holm-Bonferroni method. Figures were prepared using MS Excel, MS PowerPoint or GraphPad Prism 9 or using Biorender software.

[0310] In vivo animal studies In-house developed and optimized chronic in vivo wound infection model in diabetic rats was used for the testing of electrospun AMP-loaded and AMP+AB-loaded wound matrices. Animal studies were conducted in accordance with the Animal Study License Committee approved license no 272 and ARRIVE guidelines. Briefly, diabetic grown-up Wistar male rats (diabetes induction performed using streptozocin 65 mg / kg per weight) were used (age 6-12 weeks) after wounding a rat using a dermatome (thickness of the wound ca 0.5 mm, square size of 10 x 10 mm), an artificial biofilm of 5. aeruginosa DSM2569 pre-grown for 24 h (bacterial concentration reaching 107CFU / biofilm matrix) was applied on the surface of the wound. Two wounds were created on the back of each rat and covered with a transparent breathable non-adhesive film window held in place by kinesiological tape. Both wounds were treated using electrospun AMP-loaded and / or AMP+AB loaded wound matrices. Only AB-loaded wound matrices were used as controls to test the synergy of AMP+AB in electrospun fiber matrices. For some rats, commercially available antimicrobial wound dressing Suprasorb®X + PHMB (polyhexanide) (Lohmann&Rauscher, Germany) was applied on the wounds for comparison. In addition, a transparent breathable non-adhesive film window was applied on top of all the treatments. Wound assessment (incl. Histology and tissue modifications) and tissue CFU counting (LB agar and Tegritol agar), were carried out after 24 h and 72 h of infection. ChatGPT (OpenAI, 2025) was used as a tool to find keyword categories suitable for scoring histopathology reports provided by the Estonian University of Life Sciences. Additional specific keyword categories were added manually. Wounds were scored according to the keyword categories, with each category giving a score value of one (scores between 0 and 12).

[0311] Keyword categories are listed below:

[0312] 1. Detachment of the epidermis (wound, ulceration, erosion)

[0313] 2. Cellular debris (necrosis)

[0314] 3. Extravascular erythrocytes (hemorrhage)

[0315] 4. Eosinophilic fibrillar material (fibrin)

[0316] 5. Infiltration of neutrophilic granulocytes

[0317] 6. Infiltrate of lymphocytes and / or plasma cells 7. Fibrous tissue (granulation tissue)

[0318] 8. Presence of macrophages

[0319] 9. Parakeratotic / hyperkeratotic epidermis

[0320] 10. Presence of bacterial colonies

[0321] 11. Deeper tissues involved (dermis, subcutaneous)

[0322] 12. Other more specific pathological finding

[0323] References:

[0324] OpenAL (2025). ChatGPT (GPT-5) [Large language model].

[0325] Results

[0326] Formulations for electrospinning

[0327] Different formulations were tested consisting of various relevant AMPs for wound infection treatment and wound healing, such as pleurocidin (PLE), D-pleurocidin (D-PLE), temporin-L (TEMP), LL-37, bacitracin (BAG), daptomycin (DAP), gramicidin (GRA), and colistin (COL). PLE, D-PLE, TEMP, LL-37 are ribosomal AMPs and BAC, DAP, GRA, and COL are non-ribosomal AMPs. Preliminary testing of different AMPs to be incorporated into electrospun fibers was conducted for their antibacterial activity in minimum inhibitory concentration (MIC) tests against various wound pathogens. It was confirmed that there are differences in the activity, but all can be used for further testing (e.g. COL MIC was 0.5 (A. baumannii 17978)).

[0328] As different antimicrobial substances can either enhance or antagonize each other's activity, it is important to evaluate their interactions in combination therapy. COL was chosen as a model AMP and was combined with three ABs representing different mechanisms of action: CIP (a DNA synthesis inhibitor), CLIN (a protein synthesis inhibitor), and CEF (a cell wall synthesis inhibitor). A checkerboard assay was performed to obtain preliminary information about their interactions. The results indicated that none of the combinations showed antagonistic effects. The calculated FICI value for COL and CIP was (1.1), and for COL and CLIN (1.3), both indicating an indifferent interaction. The FICI value for COL and CEF was (0.7), suggesting weak synergism. Thus, all tested combinations demonstrated either indifferent or weakly synergistic interactions, suggesting potential for use in combination therapy. For that reason, COL was formulated together with these antibiotics in ES matrices. In addition, different polymers (poly-DL-lactide (PDLA), polycaprolactone (PCL), PDLA / PCL mixture in different weight ratios, copolymer of L- lactide and E-Caprolactone (PLC)), as well as different solvents (organic solvents, acids) were tested for formulation development. Furthermore, the polymer concentrations needed for reproducible ES were optimized to find the best formulation compositions. It was confirmed that although acids can be suitable solvents for most of the selected polymers, it cannot be used with all AMPs due to their chemical degradation in acidic environment (confirmed by MS and HPLC). For example, PLE was degraded in the electrospinning solution even prior electrospinning. HFIP was a suitable solvent for all tested water-insoluble hydrophobic polymers PDLA, PLC and PCL and their mixtures (PDLA+PCL). All solutions for electrospinning were prepared as described in M&M section, for the samples with lower concentration of AMP the selected AMP was added prior electrospinning (dissolution of AMP was confirmed visually) in order to avoid possible degradation during overnight stirring in polymer solutions. Higher AMP concentrations needed longer stirring times and AMP and polymer(s) were dissolved together during overnight stirring. The selection of solvent is one of the key parameters that affects most of the properties and behavior of the matrices. Polymer properties are highly dependent on the solvent used, and solvent also affects the interactions between the polymer and the drug.

[0329] Electrospinning of AMP loaded matrices

[0330] Electrospinning technique is known to be dependent on various material, process and environmental parameters. Therefore, during formulation development we optimized all these parameters and were able to achieve stable and reproducible electrospinning as well as electrospun (ES) matrices. Both ES fiber matrices with AMP as well as control matrices without drug (pristine polymer matrices) were prepared.

[0331] All tested polymer solutions (PDLA, PLC, PCL) and also mixtures of PDLA / PCL solutions were nicely electrospinnable using HFIP. The latter means that during electrospinning no dripping of the solution took place, and the fibers were uniformly collected on the grounded collector plate. All AMPs were tested for preliminary electrospinning experiments. Exact formulations used to produce AMP-loaded ES fiber matrices are listed in Table 1 and exact ES parameters are provided in Tables 2 and 3. All tested formulations were optimized separately, however the best working process parameters had all rather similar mean value ranges as listed below:

[0332] Electrospinning of the formulations: o Solution volume: 4 - 11.5 mL o Needle size: 23 G o Voltage: 6.9 - 15.2 kV o Flow rate: 0.8 - 2.5 mL / h o Distance between the needle and the collector: 11 - 18 cm o Temperature: 22 - 26 °C o Relative humidity: 19 - 60 %

[0333] Characterisation of electrospun AMP loaded matrices

[0334] Morphology

[0335] In addition to the visual observation of the electrospinning process, the morphology of AMP loaded matrices was investigated after electrospinning in order to understand if electrospinning was successful or not. Based on the scanning electron microscopy (SEM) analyses it was seen that all ES formulations showed uniform fibrous structures (Fig 2), although the exact average fiber diameter of the formulations and fiber diameter size distributions varied between different formulations (Fig 3, 4 and 5, Tables 4 and 5).

[0336] Table 4. Fiber diameters of electrospun (ES) antimicrobial peptide (AMP)-loaded matrices prepared using different hydrophobic polymers (PDLA, PCL, PLC and PDLA and PCL mixture) and HFIP as a solvent, unless otherwise mentioned.

[0337] Table 5. Fiber diameters of pristine electrospun (ES) matrices prepared using different hydrophobic polymers (PDLA, PCL, PLC, and PDLA and PCL mixture) and HFIP as a solvent.

[0338] When different AMPs were included into the same PDLA formulation, in general, all fibers were in micrometer range (microfibers) and with uniform fiber diameter. No major ES errors (e.g. beads) were observed confirming suitable and optimized ES conditions. No major differences in the fiber diameter nor morphology of the fibers between the different formulations.

[0339] Results showed that for PDLA formulation the fiber average diameter did not change when adding COL into the formulation (Fig 3). For PLC formulation the added AMP decreased the average fiber diameter which is often seen with drug loaded ES fibers. PCL formulation had the smallest fiber diameter and PLC formulation had the largest average fiber diameter.

[0340] AMP content analysis.

[0341] During the AMP content analysis in ES matrix (using HPLC) it was seen that HFIP formulations with different polymers enabled to preserve the stability and functionality of the peptides during and after electrospinning (Table 6). PLE showed even 100% presence of AMP within the ES fibers and for example COL showed that approximately 73% of AMP was present in the fibers after electrospinning in some formulations, but for other formulations the AMP could be fully encapsulated. For other tested peptides the amount of peptide incorporated into the fibers (encapsulation efficiency, EE%) varied. All tested AMPs were successfully incorporated into the polymer fibers with HFIP as a solvent during monoaxial needle-based electrospinning and the measured AMP concentrations within the fibers were all satisfactory. The average AMP concentrations incorporated within the fibers are shown in Table 6. When AA / FA or DMF were used as solvents for electrospinning, then the encapsulation efficiency was approximately 90% and 100%, respectively. No major differences compared to the results obtained with HFIP as a solvent, AA / FA showed slightly smaller EE values.

[0342] For combination matrices (consisting of AMP and AB), the content of both drugs was measured, unless there was an overlap in the chromatograms and the full analyses were not possible to be performed (e.g. COL+CLIN combination matrix)(Table 6). With other combination matrices (COL+CIP), it was seen that both drugs were successfully incorporated into the fibers. When only AB-loaded matrices were electrospun for comparison, then it was seen that also ABs were successfully incorporated into the ES matrices (Table 6).

[0343] Table 6. Antimicrobial peptide (AMP) theoretical and measured content in electrospun (ES) fibers prepared using HFIP as a solvent.

[0344] Key: NA - not available

[0345] AMP content in ES matrix after incubation in an aqueous environment for specified time- periods- trapping of AMP into the matrix

[0346] COL-loaded and PLE-loaded matrices were put into different media used in bacterial studies and initially kept there for 24 h, in order to understand the possible leaching of AMPs from the matrix in an aqueous environment. The results presented in Table 7 show that both peptides were still incorporated within the ES matrix after incubation, approximately 60-80% of PLE and 60% of COL was present. Table 7. Colistin (COL) and pleurocidin (PLE) content trapped in electrospun (ES) 5%

[0347] PDLA fiber matrices (ES using HFIP as a solvent) before experiment and after being in different media at 37 °C for 24 h.

[0348] Key: NA- not available Table 8 shows that using different polymers for the electrospinning of COL-loaded matrices, COL was securely kept inside the matrix (up to 89% of COL) with all different formulations (COL formulations with PDLA, PCL and PLC). For these experiments even longer time-periods 3 days and 1 week and biorelevant buffer lxPBS or DMEM + 0.2 mM EDTA were used for incubation. No major difference between the behavior of the formulations was observed, as all leaching was completed withing the first 24 h. There seems to be the trend that COL is more tightly bound to PLC+COL matrices, compared to PDLA+COL and PCL+COL matrices, and that higher initial COL concentration (8%) leads to higher released COL values (Table 8). The results showed only some evidence that COL was more secured in PLC+COL fiber matrices in lxPBS whereas in DMEM+ EDTA more COL was leached.

[0349] Table 8. Colistin (COL) content trapped in the electrospun (ES) fiber matrices with different hydrophobic polymers (PDLA, PCL, PLC) (ES using HFIP as a solvent) after being in DMEM + 0.2 mM EDTA or 1 x PBS (pH 7.4) at 37 °C for 3 days and 1 week.

[0350] Key: NA- not available Also, when COL content in aqueous medium (lxPBS) was measured using HPLC, only small amount of COL was detected after 24 h, 3 days and 1 week. However, as soon as AMP is leached from the matrix it will be quickly also degraded and the amount of peptide measured in biorelevant buffer medium (lxPBS) is lower compared to the calculated number based on the amount of peptide left within the matrices. AMPs are known to be very unstable molecules; the latter was also confirmed in these experiments. It was seen that the control sample drug concentrations (AMPs solution in lxPBS at 37 °C) were reduced after 24 h, for PLE the degradation of the peptide was 36 % and for COL the degradation was 29 % (Fig 6). It is expected that AMPs will be chemically degraded at 37 °C (it is known that in the body also fast enzymatic degradation takes place), and therefore the longer the AMP is kept within the fibers the more it is protected, and it can have its activity and functionality.

[0351] ES AMP-loaded matrices and AMP and AB-combination matrices were put into lx PBS for specified time-periods (24 h, 3 days and 1 week) in order to further understand the drug trapping and leaching from the matrix. The results showed that even with slight differences between the exact formulations, all AMP-loaded and AMP and AB-loaded combination matrices still incorporated more than 59% of drugs within the matrix after being in an aqueous environment for 24 h and more than 40% after 3 days in an aqueous environment (Table 9). Table 9. Drug content trapped in the electrospun (ES) fiber matrices (ES using HFIP as a solvent) after being in 1 x PBS (pH 7.4) at 37 °C for 1 day, 3 days and 1 week.

[0352] * Percentage of drug remaining in the matrix relative to the theoretical drug content, calculated from the initially loaded drug amount.

[0353] For a combination matrix consisting of COL and CIP, COL was still more than 50% trapped within the matrix even after 7 days of being in an aqueous environment. Interestingly, CIP release was changed, and most of the CIP was released very quickly after becoming in contact with aqueous environment, and only 17% of CIP was still trapped within the matrix after 7 days in a buffer medium (Table 9). When CIP was alone in the fiber matrix, CIP was also trapped in the fiber matrix approximately 97%. Similar behavior was confirmed with COL 2.6% +CLIN 1.3%+ PDLA + PCL combination matrix (Table 9), confirming that AMP was trapped within the matrix despite that AB was released in aqueous environment.

[0354] Different solvents were used for electrospinning (e.g. AA:FA or DMF) together with the same polymers (PDLA, PCL). It was seen that AMP (COL) was quickly released when DMF was used as a solvent, whereas using AA:FA solvent mixture, the release was minimal. . It was confirmed that in the first case, approximately only 0.1% of COL was still trapped within the fiber matrix (C0L8% + PDLA in DMF) after 24 h in aqueous environment and no COL was detected in the fiber matrices after 3 days and 7 days of experiment, whereas in the latter case, more than 90% of COL remained trapped within the fiber matrix (COL 7.1% + PDLA in AA:FA) (Table 9). Antibacterial effect of AMP-loaded matrices on overnight liquid bacterial culture in growth medium

[0355] In order to understand more the antimicrobial activity of AMP-loaded matrices in vitro, the matrices were tested in Mueller Hinton Broth. COL-loaded formulations showed an antibacterial effect on all three bacterial strains tested in growth medium (MHB) with pH 7.4 (Table 10). DAP matrices showed effect only on 5. aureus tested strain. Other peptides were not effective in this in vitro assay against any tested pathogenic bacteria. Different COL concentrations were tested, and both 4% and 8% COL-loaded matrices were active against all tested pathogens.

[0356] Table 10. AMP-loaded 5% PDLA matrices electrospun using HFIP as a solvent activity against E. coli; S. aureus; P. aeruginosa in bacterial growth medium with pH 7.4.

[0357] *detection limit 7 logCFU; **detection limit 9logCFU; NE - not effective

[0358] Antibacterial effect on overnight liquid bacterial culture - in different biorelevant growth media

[0359] Since the antibacterial effect of some AMP-loaded matrices was not seen in MHB at pH 7.4, we tested other biorelevant media and we observed and proved their antibacterial activity. First, medium comprised of DMEM / F12 + 10% FBS was chosen as it mimics wound exudate and is thus relevant for testing potential wound dressings. The matrices were challenged with different inoculum sizes from 103to 109CFU / mL of three different wound pathogens: E. coli DSM1103, 5. aureus DSM2569 and P. aeruginosa DSM1117. Bacteriostatic and bactericidal effects of the AMP-loaded matrices were thus evaluated . In addition, the sterility of the matrices after being challenged with the bacteria was also tested (Table 11, Fig 7 to 9). Table 11. Bacteriostatic and bactericidal effect and sterility of different AMP- loaded and AMP and AB-loaded combination matrices using HFIP as a solvent, unless otherwise stated. Growth inhibition is expressed as max logCFU values where no visible bacterial growth was detected (detection limit: 7logCFU). Killing efficacy is expressed in log reduction values (detection limit: 8.1 log reduction). Sterility is expressed as max logCFU values where the matrices remain sterile (detection limit: 9logCFU). All tests were performed in triplicate.

[0360] [NS = not sterile; and NE = not effective].

[0361] COL-loaded matrices exhibited antibacterial spectrum primarily against gramnegative bacteria (Table 11 and Figure 7 to 10). Different hydrophobic carrier polymers were tested for COL and although some differences were seen between these formulations, all COL 4% and 8% matrices were highly active against both E. coli and P. aeruginosa, inhibiting the growth of at least 107CFU / cm2. Killing efficacy was slightly better against E. coli compared to P. aeruginosa, but more than 5 log reduction was seen with all COL formulations against these bacteria. Most COL 4% and 8% matrices remained sterile even when challenged with 109CFU / cm2of either E. coli or P. aeruginosa. Activity against gram-positive 5. aureus was mostly present but not as marked. It was seen that increasing COL concentration in a matrix had a positive effect on its antibacterial properties. In addition to COL, PLE 4% + PCL, BAC 8% + PCL and a combination matrix with both COL and CIP (PDLA+PCL) were effective against E. coli, whereas the latter was also effective against 5. aureus and P. aeruginosa. BAC 8% matrix was also effective against 5. aureus, but not against P. aeruginosa. DAP-loaded matrices were active against 5. aureus. Overall, it can be concluded that different hydrophobic carrier polymers can be used for preparing effective AMP-loaded ES matrices. CIP-loaded matrices were also effective against all tested pathogens, with a more marked activity against gram-negative bacteria (Table 11 and Figures 7 to 10). Both 4% and 8% drug loadings proved to be effective, but increasing the drug content also improved antibacterial activity, especially against 5. aureus. Antibacterial activity of CLIN was observed only against 5. aureus. However, the matrix remained sterile even when challenged with the highest 5. aureus concentration.

[0362] The checkerboard assay which indicated weak synergism or indifference between AMP and ABs in our chosen combinations was performed using solutions of the antibacterial agents, whereas our in vitro antibacterial activity tests were conducted with drug-loaded ES matrices. In these matrices, only a small amount of COL was released compared to the relatively rapid release of the ABs. Consequently, the checkerboard assay provided a simplified view of the interactions between the pure compounds in solution, while the antibacterial testing of the ES matrices offered a more realistic understanding of how these agents act within the matrix environment. Notably, the combination of COL and CIP demonstrated substantially higher antibacterial activity in the ES matrices than predicted by the checkerboard results (Table 11).

[0363] As the main mechanism of action of AMP-loaded matrices is related to their surface properties and trapped AMPs, the abovementioned liquid culture assay may underestimate the real antibacterial properties of ES matrices in certain cases. To understand whether this is indeed so, PLE 8% + PDLA matrix that showed negligible antibacterial activity in the liquid culture assay (data not shown) was also tested with a modified version of ASTM E218-18 assay which is as standard method for determining the activity of incorporated antimicrobial agent(s) in polymeric or hydrophobic materials. It was confirmed that PLE 8% + PDLA matrix indeed has antibacterial properties when in direct contact with the bacteria (Figure 11). The number of bacteria detected on the PLE 8% + PDLA matrices was approximately 3 orders of magnitude smaller compared to the number detected on PDLA control fibers.

[0364] As stated before, most of the drug is not released in dissolution testing but remains trapped within the polymeric matrix. To understand whether this gives the matrices prolonged antibacterial properties, a modified version of ASTM E218-18 assay was used after incubation of the matrices in PBS for 1, 3, or 7 days. PLE 4% + PCL, COL 8% + PCL, CIP 4% + PDLA, COL 2.6% + CIP 1.3% + PDLA + PCL, COL 2.6% + CLIN 1.3% + PDLA + PCL, COL 2.6% + CEF 1.3% + PDLA + PCL matrices were chosen to verify the prolonged actions of the matrices in this assay. As controls, blank PCL, PDLA and PCL + PDLA matrices were used. In addition, Nisin 8% + PCL matrix was tested to understand the activity of this matrix in direct contact and prolonged setup. The results are shown in Figure 12.The results revealed no antibacterial action of Nisin 8% + PCL matrices. PLE 4% + PCL matrix retained good antibacterial properties, reducing the number of bacteria about 3 orders of magnitude, which corresponds to 99.9% killing. All other tested drug-loaded matrices retained even stronger antibacterial properties after soaking in PBS for I to 7 days. Compared to controls, the number of bacteria on these drug-loaded matrices was reduced more than 4 orders of magnitude, thus killing more than 99.99% of the bacteria.

[0365] Ethylenediaminetetraacetic acid (EDTA) is a chelating agent widely used in various applications due to its ability to bind metal ions through its carboxylate and amine groups. EDTA is available as two salts: edetate disodium and edetate calcium disodium. Both salts are used as chelating agents, whereas disodium form binds calcium more avidly and is thus unsuitable for systemic treatment. As bacteria adhere to host tissue by bacterial adhesins, which require positive ions (Ca2+, Mg2+, Zn2+) for normal functioning, depriving these ions can lead to reduced bacterial adhesion and thus EDTA solutions have been used for irrigation to reduce bioburden and loosen biofilm. EDTA is also known to improve antibacterial activity and to lower MIC values of many antibacterial agents. EDTA irrigation solutions below 10 mM have been shown to be safe. Thus, we have tested alternative growth media in addition to our artificial wound exudate: DMEM / F12 or LB supplemented with disodium edetate dihydrate (DMEM+EDTA or LB+EDTA) (0.2 mM). We saw that adding EDTA can significantly improve the activity of AMPs (Table 12), whether it is by direct synergistic mechanism of action or by modifying the microenvironment. This was more prominently seen in LB as the concentration of cations is higher in DMEM, and thus, DMEM possibly requires higher EDTA concentration for similar effect. We saw that bacteria were able to grow in the presence of EDTA if there were no other antibacterial agents / matrices in the media, thus the EDTA concentration in these media was too low to be solely responsible for the antibacterial effect. Table 12. AMP loaded matrices activity against E. coli DSM1103; 5. aureus

[0366] DSM2569; P. aeruginosa DSM1117 in different growth media. Matrices that were able to inhibit the growth of 103CFU / mL of initial inoculum size are shown.

[0367] Next, it was decided that both the antibacterial activity as well as antibiofilm activity of the electrospun AMP loaded matrices should be also tested.

[0368] Antibacterial and antibiofilm activity assay

[0369] Selected COL-loaded ES fiber matrix formulations were further tested for their antibiofilm and antibacterial activity using ex vivo wound infection models (Lorenz et al 2023). In an ex vivo antibiofilm assay it was seen that bacterial biofilm formed on pig skin samples covered with pristine control matrices up to 108CFU / cm2, the same number of biofilm bacteria was quantified on top of the pig skin (uncovered sample). Showing and confirming that pristine wound matrices do not have any antibiofilm effect. However, the results show that all COL loaded fibrous wound matrices had the desired activity against biofilm formation (biofilm inhibition) compared to the control (pristine) wound matrices (Fig 13).

[0370] The number of biofilm bacteria was reduced compared to their respective untreated controls for all different formulations tested. All ES matrices showed a reduction in the number of biofilm bacteria approximately 4 log in biofilm formed bacterial numbers (Fig 13, Table 13).

[0371] In Table 13, it is shown that PDLA fiber matrix had the least amount of COL in 1 cm2sized matrix, but it had equally strong antibiofilm effect as other formulations where the drug content was almost 4 times higher. Table 13. The effect of colistin 4% (COL) loaded electrospun (ES) matrix on the number of biofilm bacteria compared to the number of biofilm bacteria of their respective pristine polymer matrices. Matrices ES using HFIP as a solvent.

[0372] Table 13 shows that different formulations consisted of different amount of AMP per 1 cm2 of the matrix. This is because the matrix can be of different thickness with different polymers used and different polymers had different concentrations in the ES solutions (even though the volume for ES as well as AMP concentration in solid state (4 %w / w) were kept constant). Here, lxl cm matrix was weighed and then the amount of AMP was calculated from this (Table 13). These results further emphasize that the main mechanism of action is related to the antibacterial activity of the matrix surface. Regardless of the differences in thickness of the matrices (and hence absolute amount of AMP), the antibacterial activity was similar as the contact area with infected skin was the same.

[0373] The antibiofilm effect of these AMP containing ES matrices was investigated after being incubated in the aqueous biorelevant environment for 24 h. This enabled to remove the possible leached amount of AMP from the environment and test the antibiofilm effect of wound matrices. For that, a modification of the test was performed, and matrices were kept in 1 x PBS buffer for 24 h at 37 °C and then rinsed with pure 1 x PBS and placed in the in vitro biofilm assay. For example, the PDLA + COL matrices reduced the number of biofilm bacteria approximately 4 log. These results show that the formulations preserve their antibiofilm effect even after pre-incubation in aqueous medium which confirms the surface activity of AMP-loaded matrices.

[0374] Sterilization of electrospun AMP loaded matrices and their sterility AMP content after sterilization

[0375] Different polymers, in addition to the ES methods used, affected the amount of AMP within the ES fibers. COL-loaded ES matrices (COL 4%) were tested for sterilization. Different sterilization techniques were tested (e.g. ethanol soaking, y- radiation, UV treatment) (Fig 14). Preliminary testing results showed that all ES matrices showed reduced COL content after UV sterilization or ethanol soaking (except PLC). For using y- irradiation the peptide content in PDLA formulation was reduced (AMP degraded) approximately 50%. Whereas when more hydrophobic polymers (e.g PCL or PLC) were used this helped to avoid the peptide degradation which was less than 20%. The most stable was a formulation with PCL polymer, where COL amount in the fibers was decreased only 4 % from the initial incorporated COL value (Table 14).

[0376] The sterility of ES AMP-loaded matrices was proven using the European Pharmacopoeia Sterility testing method. It was shown that all sterilized AMP-loaded matrices were clean from contamination. (Example of the test results for one formulation, Fig 15).

[0377] Table 14. Antimicrobial peptide (AMP) colistin (COL) content within electrospun (ES) fiber matrices (ES using HFIP as a solvent) and the extent of its degradation after gamma- irradiation

[0378] The stability of AMPs and ABs within ES fiber matrices was tested after gammasterilization of AMP and AB-loaded formulations. As expected, gamma-sterilization induced degradation of drugs within the matrices (Table 15).

[0379] Table 15. Drug degradation in electrospun (ES) matrices following gammasterilization (25 kGy), N=3.

[0380] It was seen that ribosomal AMPs were more unstable during gamma-sterilization compared to the non-ribosomal AMPs whilst the degradation percentage varied from 7% up to 55%. AB were more protected and stable within the matrices; their degradation percentage was between 2% and 18%.

[0381] Preliminary stability testing after 1 week in aqueous medium

[0382] The stability of the ES fibrous wound matrices containing 4% COL was performed.

[0383] The latter enabled to show the stability of fiber matrix (by measuring the weight of the fiber matrix) after 1 week in 1 x PBS buffer at 37 °C. The results showed that overall, no significant weight loss hence relevant matrix degradation was observed after 1 week (Table 16). Table 16. Degradation of colistin (COL) loaded electrospun (ES) fiber matrices with different polymers (ES using HFIP as a solvent) after 1 week in 1 x PBS at 37 °C (weight loss, %).

[0384] However, there were differences in their behavior which was observed visually while handling the samples. After being in contact with aqueous environment, some of the samples shrinked, whereas others preserved their shape and size. This feature is important for the usability of the matrices as wound matrices. For example, 2 x 2 cm square-shaped samples did not change for PCL formulations. But PDLA and PLC samples shrinked. For PDLA + COL it was twice smaller l x l cm. PLC samples also shrinked, but less compared to the PDLA samples. We have also tested and confirmed that the mixtures of the polymers show different shrinking properties compared to the pure materials and 70:30 mixture of PDLA +PCL does not have the shrinking property (similarly to pure the PCL matrices). Figure 16 shows the size of the matrices after being in biorelevant aqueous conditions for 3 weeks. No major weight loss of these matrices with different polymer compositions was confirmed, for both formulations the matrix weight was above 95% from the initial 100% (although slightly higher compared to pure PDLA matrix). Hence, these results show also good potential of PDLA + PCL mixtures used for final application (keeping in mind that other properties are similar to pure PDLA and PCL formulations-antimicrobial and antibiofilm activity etc.). The mechanical properties of the ES fiber matrices enable to understand more about this behavior and find the best working formulation for in vivo testing.

[0385] Mechanical properties of AMP-loaded matrices

[0386] In addition to the observation of the behavior of the matrices in aqueous environment visually, we also measured the mechanical properties of fiber matrices relevant for wound matrices. The mechanical properties of ES fiber matrices are important to be investigated for wound healing applications, as the matrix needs to be easily handleable and comfortable for the patient. Therefore, it is desired that the matrix would be flexible and with enough strength. The COL-loaded matrices as well as non-loaded pristine polymeric matrices were analyzed for their relevant mechanical properties (hardness, deformation at hardness work done) (Fig 17).

[0387] The thickest matrices exhibited the highest elasticity and were also the strongest matrices amongst all the tested formulations. The formulations that consisted of the highest percentage of polymer resulted in thickest matrices after electrospinning. There was a common trend observed that COL-loaded matrices were stronger and more flexible compared to the non-loaded pristine matrices (only PLC matrices were more elastic compared to the COL-loaded PLC matrices). We have seen previously that gamma sterilization increases the hardness and deformation at hardness for PCL and chloramphenicol-loaded PCL matrices (Preem et al. 2019) and that the structure of the matrix and presence of pores (results of different solvents and environment used during electrospinning) also affects the mechanical properties (Lanno et al. 2020; Ramos et al. 2020). All tested formulations, except PCL matrices, showed that gamma sterilization had lowered the elasticity and hardness of the matrices. Only gamma sterilized PCL matrices loaded with COL were stronger compared to the matrices not treated with gamma irradiation.

[0388] Safety and biocompatibility of AMP loaded matrices

[0389] The safety and biocompatibility of COL loaded matrices (COL 4%) was tested using direct MTS cell viability assay or indirect CCK-8 assay using BHK-21 fibroblast cells and for some of the formulations also human primary fibroblast cells were used. These tests enable us to understand the biocompatibility of the matrices when in contact with living eukaryotic cells and draw conclusions about the safety of the wound matrices. Cell viability was calculated in comparison to the cells plated just at the bottom of the plastic well (controls). Safety of the ES matrices was proven as the MTS or CCK-8 activity of the cells (relating with the cell viability) did not decrease compared to the controls (Figures 18 and 19). Moreover, the metabolic activity of the cells treated with the extracts of COL matrices (all formulations), PLE 4% + PCL, BAC 8% + PCL and combination matrix of COL and CLIN was significantly higher (p<0.05). Thus, all tested formulations appeared to be safe and nontoxic to eukaryotic cells. . Comparison of different COL-loaded electrospun fiber matrices with different formulations

[0390] According to the cell attachment and proliferation on fibers measurement results it was seen that all matrices are non-toxic and do not reduce cell viability. When all formulations were tested for other relevant properties, it was seen that different formulations have different advantages and disadvantages. For example, the best working formulations as regards antimicrobial / antibiofilm behavior was a formulation consisting of PDLA, respectively. However, the least degradation of the peptide during sterilization was observed with formulation PCL+HFIP.

[0391] Comparative results with the in situ microfluidic electrospinning method

[0392] COL was successfully incorporated into fibers with the in-situ microfluidic electrospinning method, previously developed (Lanno et al., PCT / EP2022 / 076874) for unstable AMPs. Two different COL concentrations (4% and 8%) were tested. Characterization showed that fibrous matrices were obtained as previously and content analysis revealed that the matrices consisted of approximately 2.93% and 5.6 ± 0.8% of COL, in 4% and 8% COL-loaded matrices, respectively. Therefore, the method can be used to encapsulated unstable AMPs without major drug loss while preserving their stability.

[0393] Leaching of COL was studied in lxPBS, revealing that 72% of the AMPs were released from the fibers within the first 24 h and up to 91% after 3 days. Therefore, on the opposite to the results obtained with the new method of the invention, matrices prepared by in-situ microfluidics electrospinning are not able to trap AMPs within their fibers. The in-situ microfluidics electrospinning fiber matrices can thus be used as drug delivery systems, but not for a sustained activity.

[0394] In vivo animal studies - antibacterial activity, wound healing properties and safety of wound matrices

[0395] The antibacterial and antibiofilm effect of ES AMP-loaded combination matrices were tested in in vivo rat chronic wound infection model. As a control antimicrobial wound dressing Suprasorb was used. For the comparison of formulation effects, COL 8% + PDLA matrix using DMF as a solvent, COL 8% + PCL matrix using AA:FA as a solvent system and NISIN 8%+ PCL using HFIP as a solvent were used. For the comparison of synergistic effects, only AB-loaded fibrous matrices were prepared and tested. The AMP-loaded and AMP and AB-loaded combination matrices exhibit antibacterial activity also in in vivo infected rat wounds (Figure 20).

[0396] In general, all AMP and AB-loaded combination matrices worked well in vivo even with lower antimicrobial agent concentration (lower drug concentrations were used in combination matrices) and all these matrices have more than 50% trapping of the AMPs within the matrices. It was seen that all combination matrices of AMP+AB (COL+ CIP, COL+CLIN and COL+CEF in PDLA +PCL matrix) showed very good antibacterial activity in in vivo experiments. This result confirms the results obtained also from in vitro experiments.

[0397] It was clearly seen that the concentration of AMP and AB had an effect (4% vs 8%), hence higher concentrations of AMP or AB, increased the antibacterial activity compared to lower concentrations of AMP and AB. We also confirmed that the desired antibacterial activity was present up to 72 h of testing, as no major changes in antibacterial activity were observed between 24 h and 72 h for AMP-loaded and AB-loaded matrices. Even slightly better activity was seen for AMP-loaded and AMP+AB loaded wound matrices after 72 h of treatment compared to 24 h.

[0398] Formulation effect

[0399] To understand the effect of formulation composition, different polymers (PCL, PDLA and their mixture, and PLC) were used to prepare the matrices with COL (8%)(non- ribosomal AMP). It was seen that no major differences between these hydrophobic polymer matrices as carriers were detected (Figure 20). PCL vs PDLA matrices that consisted of 8% COL both showed similar antibacterial activity in in vivo experiments after 24 h. The same was confirmed also in in vitro experiments. In case PLE was used (ribosomal AMP), two different polymers were tested (PCL and PDLA), both these formulations showed good and comparable antimicrobial activity against 5. aureus., despite the differences seen in in vitro antibacterial performance (Table 11). Interestingly, the effect of solvents became evident, as when control ES fiber matrices were used (COL+PDLA in DMF), then these matrices showed practically no antibacterial activity in in vivo experiments after 24 h and / or 72 h of experiment (Figure 20). Proving that the fast release of COL from the matrix (detected using HPLC) decreased the matrix's antibacterial and antibiofilm activity in in vivo biorelevant wound models. In comparison, when COL+PDLA matrices were prepared from AA:FA as a solvent, then these matrices showed prolonged (up to 72 h) and strong antimicrobial efficacy in in vivo model (Figure 20). In addition, COL+PDLA matrices made using HFIP as a solvent preserved their antibacterial and antibiofilm activity up to 72 h in in vivo experiments. We have also shown in vitro that the matrices preserve their antimicrobial activity also for 3 days and up to 7 days (Figure 12).).

[0400] Synergy testing in electrospun combination matrices

[0401] Synergy was also tested in ES combination fiber matrices in in vivo animal model where AMP and AB were together encapsulated into the fibers. As a comparison both drugs were used for the treatment alone (AMP-loaded matrices and AB-loaded matrices). Although all combination matrices consisted of less antimicrobial drugs in total, then the antibacterial activity observed was similarly to the single component fiber matrices highly effective (Figure 20). COL+CEF combination matrix had the highest effect after 72 h (Figure

[0402] 20). Although COL+CLIN combination matrices showed very strong antibacterial effect, then CLIN alone in the ES wound matrices showed only small or even negligible antimicrobial activity (Figure 20). This highlights that improved synergistic activity of AMP and ABs was obtained in these ES matrices.

[0403] Size of the wound- wound closing rate

[0404] From photos collected during wound healing it was seen that if no treatment was used, then the wound healing was slower compared to the wounds which were treated with antimicrobial fibrous AMP-loaded matrices and / or AMP+AB-loaded matrices (Figure

[0405] 21).

[0406] If no treatment was used, the wound size increased from 24 h to 72 h, showing that the infection was indeed in wounds causing delayed healing (Figure 21). All covered wounds (despite the wound dressings used for the treatment) decreased the wound size faster compared to the untreated wounds.

[0407] Histopathology analyses and wound scoring

[0408] Tissue samples collected from animals after the experiments revealed that there were major differences between treated wounds and untreated wounds. Based on the image analyses it was seen that infected dermatome wounds show the infiltration of different immune cells and local tissue damage which showed higher scores in histopathology analyses compared to treated wounds (Figure 22). Whilst treated wounds after 72 h of treatment showed significant changes in the wound and signs of wound healing. It is evident that if no treatment is performed then the infection develops further from 24 h to 72 h and wound conditions get worse. All wounds treated with AMP-loaded wound matrices (COL) and AMP+AB-loaded combination wound matrices revealed that wounds had less inflammation and lower histopathology scores were observed.

[0409] Acknowledgements

[0410] Funding of the patent- PRG1507 project. The authors thank the Estonian Research Council and Estonian Ministry of Education and Research for funding. MSc M. Kula vi i r and Prof. K. Kirsimae are thanked for the SEM measurements and Dr Andres Meos for HPLC measurements. Dr Ivo Laidmae is acknowledged for fruitful discussions about electrospinning and Janis Romanopulos and Prof James Mason (King's Colledge London, UK) for the synthesis and providing of ribosomal cationic antimicrobial peptides. Student Lea Vrhovnik, Hanna Marta Nellis, MSc Kelli Randmae, MSc Oksana Gerulis, Dr Mariliis Hinnu and MSc Kaisa Pohako-Palu are thanked for the laboratory experiments.

[0411] References:

[0412] Akhmetova, A.; Lanno, G.-M.; Kogermann, K.; Malmsten, M.; Rades, T.; Heinz, A. (2020). Highly Elastic and Water Stable Zein Microfibers as a Potential Drug Delivery System for Wound Healing. Pharmaceutics, 12 (5). DOI: 10.3390 / pharmaceuticsl2050458.

[0413] Ambekar, R. S. and Kandasubramanian, B. (2019) 'Advancements in nanofibers for wound dressing: A review', European Polymer Journal, 117, pp. 304-336. doi: 10.1016 / J.EURPOLYMJ.2019.05.020.

[0414] Boateng, J. and Catanzano, O. (2015) 'Advanced Therapeutic Dressings for Effective Wound Healing— A Review', Journal of Pharmaceutical Sciences, 104(11), pp. 3653-3680. doi: 10.1002 / JPS.24610.

[0415] Bowler, P. G., Duerden, B. I. and Armstrong, D. G. (2001) 'Wound microbiology and associated approaches to wound management.', Clinical microbiology reviews, 14(2), pp. 244-69. doi: 10.1128 / CMR.14.2.244-269.2001.

[0416] Cowan, T. (2011) 'Biofilms and their management: from concept to clinical reality. Journal of wound care, 20(5), pp. 220, 222-6. doi: 10.12968 / jowc.2011.20.5.220.

[0417] Daristotle, J.L., Behrens, A.M., Sandler, A.D. A Review of the Fundamental Principles and Applications of Solution Blow Spinning. ACS Appl. Mater. Interfaces 2016, 8, 51, 34951-34963 https: / / doi.org / 10.1021 / acsami.6bl2994

[0418] Felgueiras, H. P. and Amorim, M. T. P. (2017) 'Functionalization of electrospun polymeric wound dressings with antimicrobial peptides', Colloids and Surfaces B: Biointerfaces, 156, pp. 133-148. doi: 10.1016 / J.COLSURFB.2017.05.001.

[0419] Fjell, C. D. et al. (2012) 'Designing antimicrobial peptides: form follows function', Nature Reviews Drug Discovery, 11(1), pp. 37-51. doi: 10.1038 / nrd3591.

[0420] Gera, S., Kankuri, E. and Kogermann, K. (2021) 'Antimicrobial peptides - Unleashing their therapeutic potential using nanotechnology', Pharmacology & Therapeutics, p. 107990. doi: 10.1016 / J.PHARMTHERA.2021.107990.

[0421] Lanno, G.-M. et al. (2020) 'Antibacterial Porous Electrospun Fibers as Skin Scaffolds for Wound Healing Applications', ACS Omega, 5(46), pp. 30011-30022. doi: 10.1021 / acsomega.0c04402.

[0422] Larsen, T. and Fiehn, N.-E. (2017) 'Dental biofilm infections - an update', APMIS, 125(4), pp. 376-384. doi: 10.1111 / apm.12688.

[0423] Lee, S. Y., Kuti, J. L. and Nicolau, D. P. (2005) 'Antimicrobial management of complicated skin and skin structure infections in the era of emerging resistance.', Surgical infections, 6(3), pp. 283-95. doi: 10.1089 / sur.2005.6.283.

[0424] Lindholm, C., Searle, Richard and Searle, R (2016) 'Wound management for the 21st century: combining effectiveness and efficiency The impact of wounds on the health system-a growing challenge', International Wound Journal, doi: 10.1111 / iwj.12623.

[0425] Lorenz, K.; Preem, L.; Sagor, K.; Putrins, M.; Tenson, T.; Kogermann, K. Development of In Vitro and Ex Vivo Biofilm Models for the Assessment of Antibacterial Fibrous Electrospun Wound Dressings. Mol. Pharm. 2023, 20, doi:10.1021 / ACS.MOLPHARMACEUT.2C00902.

[0426] Manzo, G. et al. (2018) 'Minor sequence modifications in temporin B cause drastic changes in antibacterial potency and selectivity by fundamentally altering membrane activity', bioRxiv, p. 312215. doi: 10.1101 / 312215.

[0427] Nguyen, L. T., Haney, E. F. and Vogel, H. J. (2011) 'The expanding scope of antimicrobial peptide structures and their modes of action', Trends in Biotechnology. Elsevier Current Trends, pp. 464-472. doi: 10.1016 / j.tibtech.2011.05.001.

[0428] Percival, S. L. et al. (2012) 'A review of the scientific evidence for biofilms in wounds.', Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society, 20(5), pp. 647-57. doi: 10.1111 / j.l524-

[0429] 475X.2012.00836.x. Preem, L. et al. (2017) 'Interactions between Chloramphenicol, Carrier Polymers, and Bacteria-lmplications for Designing Electrospun Drug Delivery Systems Countering

[0430] Wound Infection', Molecular Pharmaceutics, 14(12), pp. 4417-4430. doi: 10.1021 / acs.molpharmaceut.7b00524.

[0431] Preem, L. et al. (2019) Effects and efficacy of different sterilization and disinfection methods on electrospun drug delivery systems. International Journal of Pharmaceutics. 567, 118450, doi:10.1016 / j.ijpharm.2019.118450

[0432] Ramos, C. et al. (2020) High humidity electrospinning of porous fibers for tuning the release of drug delivery systems, International Journal of Polymeric Materials and Polymeric Biomaterials, DOI: 10.1080 / 00914037.2020.1765361.

[0433] Sankar, V. et al. (2011) 'Local drug delivery for oral mucosal diseases: Challenges and opportunities', Oral Diseases, 17(SUPPL. 1), pp. 73-84. doi: 10.1111 / j.l601- 0825.2011.01793.x.

[0434] Siddiqui, A. R. and Bernstein, J. M. (2010) 'Chronic wound infection: facts and controversies.', Clinics in dermatology, 28(5), pp. 519-26. doi: 10.1016 / j.clindermatol.2010.03.009.

[0435] Thenmozhi, S. et al. (2017) 'Electrospun nanofibers: New generation materials for advanced applications', Materials Science and Engineering: B, 217, pp. 36-48. doi: 10.1016 / J.MSEB.2017.01.001.

[0436] Torres-Martinez, E. J. et al. (2018) 'A Summary of Electrospun Nanofibers as Drug Delivery System: Drugs Loaded and Biopolymers Used as Matrices.', Current drug delivery, 15(10), pp. 1360-1374. doi: 10.2174 / 1567201815666180723114326.

[0437] Wolcott, R. D., Rhoads, D. D. and Dowd, S. E. (2008) 'Biofilms and chronic wound inflammation.', Journal of wound care, 17(8), pp. 333-41. doi: 10.12968 / jowc.2008.17.8.30796.

[0438] Zhang QY, Yan ZB, Meng YM, Hong XY, Shao G, Ma JJ, Cheng XR, Liu J, Kang J, Fu CY. Antimicrobial peptides: mechanism of action, activity and clinical potential. Mil Med Res. 2021 Sep 9;8(1):48. doi: 10.1186 / s40779-021-00343-2. PMID: 34496967; PMCID: PMC8425997.

[0439] Zupancic, S. et al. (2018) 'Impact of PCL nanofiber mat structural properties on hydrophilic drug release and antibacterial activity on periodontal pathogens', European Journal of Pharmaceutical Sciences, 122, pp. 347-358. doi: 10.1016 / J.EJPS.2018.07.024.

Claims

Claims1. An antimicrobial fiber material comprising electrospun fibers of a biodegradable polymer loaded with antimicrobial peptides, wherein at least about 50% of the antimicrobial peptides are trapped within the fibers.

2. The material according to claim 1, wherein the fibers have been electrospun in the presence of a polar organic solvent.

3. The material according to claim 1 or 2, wherein at least about 55%, preferably at least about 60%, more preferably at least about 65%, even more preferably at least about 70% of the antimicrobial peptides are trapped within the fibers.

4. The material according to any of the preceding claims, wherein the trapped antimicrobial peptides essentially remain within the fibers for at least 1 day, preferably at least 2 days, more preferably at least about 3 days, even more preferably at least about 7 days under normal conditions of use of the material.

5. The material according to any of the preceding claims, wherein the non-trapped antimicrobial peptides are essentially released from the fibers in no more than a day, preferably in no more than 10 h, even more preferably in no more than 1 h, under normal conditions of use of the material.

6. The material according to any of the preceding claims, wherein the fibers have a diameter comprised between about 0.01 pm and about 10 pm, preferably between about 0.08 pm and about 10 pm, more preferably between about 0.1 pm and about 5 pm.

7. The material according to any preceding claim, additionally comprising at least one antibiotic (AB), preferably wherein the AB is selected from the class of fluoroquinolones, macrolides and / or cephalosporins, more preferably is selected from ciprofloxacin, clindamycin, and / or ceftriaxone.

8. A method of electrospinning antimicrobial peptides comprising the following steps: a) preparing a solution of a biodegradable polymer, a polar organic solvent, and an antimicrobial peptide; b) electrospinning the solution obtained at step a).

9. The method according to claim 8, wherein step a) comprises: i) the four following sub-steps:ai) adding a biodegradable polymer to a polar organic solvent;32) mixing the solution obtained at step ai); as) adding an antimicrobial peptide to the solution of step as);34) mixing the solution obtained at step as); or ii) the two following sub-steps: ai) Adding a biodegradable polymer and AMP to a polar organic solvent; as) Mixing the mixture obtained in step ai.

10. An antimicrobial peptide composition for electrospinning comprising a solution of a biodegradable polymer, an antimicrobial peptide and a polar organic solvent.

11. The material according to any of claims 1 to 7, the method of electrospinning according to claim 8, 9 or the antimicrobial peptide composition according to claim 10, wherein the biodegradable polymer is a cyclic or an aliphatic polymer, preferably an aliphatic polymer.

12. The material according to any of claims 1 to 7 or 11, the method of electrospinning according to any of claims 8, 9 or 11, or the antimicrobial peptide composition according to claim 10 or 11, wherein the aliphatic polymer is a polyester, preferably a polyester selected from the group consisting in polycarbonates, polylactides, polycaprolactones, polyglycolides, polyhydroxyalkanoates, poly(dioxanone)s, poly(lactide- co-glycolide), poly(butyrolactone)s, poly(valerolactone)s, and a combination thereof, more preferably selected from the group consisting in Poly(P-butyrolactone) (PBL), Poly(£- caprolactone-co-lactide) (PCLA), Poly(3-hydroxybutyrate) (PHB), Poly(3-hydroxyvalerate) (PHV), Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), Poly(methyl methacrylate) (PMMA), Polyglycolide (PGA), Poly(lactic acid) (PLA), Poly( L-lactide) (PLLA), Poly(D,L-lactic acid) (PDLA), E-polycaprolactone (PCL), a copolymer of PDLA and PCL (PLC) such as a PLC with a molar ratio of PDLA on PCL of about 70 / 30 (i.e. 70 / 30 PLC), poly(trimethylene carbonate) (PTMC), a derivative thereof and a combination thereof, even more preferably a polyester selected from the group consisting in PDLA, PCL, PLC, such as 70 / 30 PLC, a derivative thereof and a combination thereof.

13. The material according to any of claims 1 to 7, 11 or 12, the method of electrospinning according to any of claims 8, 9, 11 or 12, or the antimicrobial peptide composition according to any of claims 10 to 12, wherein the antimicrobial peptide is selected from the group consisting in maximin H5, dermcidin, cecropins, andropin,moricin, ceratotoxin, melittin, magainin, dermaseptin, bombinin, brevinin-1, esculentins, buforin II, CAP18, abaecin, drosocin, apidaecin, attacin, prophenin, indolicidin, brevinins, such as brevinin-1, protegrin, tachyplesins, defensins, drosomycin, bacitracin, boceprevir, dalbavancin, daptomycin, enfuvirtide, oritavancin, teicoplanin, telaprevir, telavancin, vancomycin, guavanin 2, bacteriocin, peptaibols, plectasin, hydramacin, aurelin, mastoparan, thioester-containing protein 1, aurein, cathelicidins, reglll peptides, nisin, copsin, diptericin, pleurocidin, L-temporin, LL-37, colistin, gramicidin, a derivative thereof and a combination thereof.

14. The material according to any of claims 1 to 7, or 11 to 13, the method of electrospinning according to any of claims 8, 9, or 11 to 13, or the antimicrobial peptide composition according to any of claims 10 to 13, wherein the antimicrobial peptide is a cationic antimicrobial peptide, preferably selected from the group consisting in pleurocidin, L-temporin, LL-37, colistin, bacitracin, gramicidin, daptomycin, a derivative and a combination thereof.

15. The material according to any of claims 1 to 7, or 11 to 14, the method of electrospinning according to any of claims 8, 9, or 11 to 13, or the antimicrobial peptide composition according to any of claims 10 to 14, wherein the antimicrobial peptide is selected from the group consisting in pleurocidin, L-temporin, LL-37, colistin, daptomycin, a derivative and a combination thereof, preferably colistin or a derivative thereof.

16. The material according to any of claims 2 to 7, or 11 to 15, the method of electrospinning according to any of claims 8, 9, or 11 to 15, or the antimicrobial peptide composition according to any of claims 10 to 15, wherein the polar organic solvent is selected from the group consisting in dimethylformamide, acetonitrile, acetone, hexamethylphosphoric triamide, N,N-diethylacetamine, 4-methylmorpholine-N-oxide monohydrate, N-methylpyrrolidinone, dimethylsulfoxyide (DMSO), tetrahydrofurane (THF), dioxan, methylcarbonate, fluoroalcohols, a derivative, and a combination thereof, preferably the polar organic solvent is a fluoroalcohol.

17. The material according to any of claims 2 to 7, or 11 to 16, the method of electrospinning according to any of claims 8, 9, or 11 to 16, or the antimicrobial peptide composition according to any of claims 10 to 16, wherein the fluoroalcohol is selected from the group consisting in trifluoroethanol (2,2,2-trifluoroethanol; TFE), hexafluoroisopropanol (l,l,l,3,3,3-hexafluoro-2-propanol; HFIP), perfluoro-tert-butanol(l,l,l,3,3,3-hexafluoro-2-(trifluoromethyl)-2-propanol; PFTB), 2-fluoroethanol, and a combination thereof, preferably the fluoroalcohol is HFIP.

18. Use of the composition according to any of claims 10 to 17 for electrospinning antimicrobial peptides.

19. The material according to any of claims 1 to 7, or 11 to 17, the method of electrospinning according to any of claims 8, 9, or 11 to 17, the antimicrobial peptide composition according to any of claims 10 to 17, or the use of the composition according to claim 18, wherein electrospinning is not perfusion electrospinning.

20. The material according to any of claims 1 to 7, or 11 to 17, the method of electrospinning according to any of claims 8, 9, or 11 to 17, the antimicrobial peptide composition according to any of claims 10 to 17, or the use of the composition according to claim 18, wherein electrospinning is monoaxial electrospinning.

21. An antimicrobial fiber material obtained by the method of electrospinning according to any of claims 8, 9, 11 to 17, 19 or 20, or by the use of the composition according to any of claims 18 to 20.

22. An Antimicrobial fiber mat comprising a material according to any of claims 1 to 7, 11 to 17, or 19 to 21 having a flat structure and a thickness comprised between about 0.01 mm and about 5 mm, preferably between about 0.08 mm and about 0.1 mm.

23. Use of the material according to any of claims 1 to 7, 11 to 17, or 19 to 21, of the mat according to claim 22, for the treatment of a subject in needs thereof.

24. Use of the material according to claim 23, or of the mat according to claim23for treatment, wherein the treatment is a topical treatment, preferably a topical treatment of the epithelium, more preferably a topical treatment of the skin or of a mucosa, such as the oral mucosa.

25. Use of the material according to claim 23 or 24, or of the mat according to claim 23 or 24 for treatment, wherein the treatment is a treatment of a skin condition preferably selected from the group consisting in a wound, a cancer such as a skin cancer, acne, atopic dermatitis, psoriasis, ichthyosis, scars and dull skin, such as epidermolysis bullosa, skin burns, plastic surgery support or skin implants support, or a combination thereof.

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