Alginate-based hydrogel, methods and uses thereof

Abstract

The present disclosure relates to a hydrogel for use in tissue engineering / regeneration comprising a polymeric alginate solution forming a hydrogel matrix and a pharmaceutical amount of an antimicrobial peptide, wherein the antimicrobial peptide is entrapped / embedded in the hydrogel matrix. The method to obtain said hydrogel, as well as the uses thereof are also described.

Description

D E S C R I P T I O NALGI NATE-BASED HYDROGEL, METHODS AND USES THEREOFTECHNICAL FIELD

[0001] The present disclosure relates to the field of medicine, in particular tissue engineering and regenerative medicine. Namely the use of a biocompatible alginate- based hydrogel loaded with antimicrobial peptides (AMPs) to foster an environment conducive to tissue repair. The disclosed hydrogel promotes a microenvironment that exhibits combined anti-inflammatory, reactive oxygen species (ROS)-scavenging, pro- angiogenic, and collagen-inducing benefits, thereby facilitating effective wound healing in complex wounds such as diabetic foot ulcers.BACKGROUND

[0002] Diabetic foot ulcers (DFUs) are a severe and prevalent complication of diabetes, mainly driven by a combination of hyperglycemia, peripheral neuropathy and peripheral vascular disease [1], These conditions disrupt the complex and dynamic process of wound healing, fostering an environment prone to microbial growth, further increasing recurrence rates, and limiting the success of treatments. DFUs affect approximately 30% of patients with diabetes during their lifetime, often leading to prolonged hospitalizations and even amputations in severe cases. The risk of recurrence is also common, with 40% of patients experiencing recurrence within one year, 60% within three years, and 65% within five years. Moreover, patients with DFUs face a 2.5-fold higher risk of death within five years and a twofold increased risk of death within ten years compared to those without DFUs. The high incidence, recurrence, and mortality associated with DFUs underscore the urgent need for advanced therapeutic approaches that can effectively promote wound healing and tissue regeneration, while preventing infection and inflammation.

[0003] Despite significant advances in research, managing DFUs remains a significant clinical challenge due to their complex and multifaceted nature. Effective therapeuticapproaches should address glycemic control, revascularization, local wound management, and infection control, while also incorporating lifestyle modifications.

[0004] Overcoming infection remains a key challenge in managing DFUs. Antimicrobial peptides (AMPs) offer a promising alternative to traditional antibiotics by targeting microbial membranes or intracellular components like DNA, reducing the risk of antimicrobial resistance [2], Due to their inherent positive charge, AMPs permeabilize negatively charged microbial membranes, leading to cell lysis while their hydrophobicity facilitates membrane destabilization and cell depolarization. Furthermore, AMPs modulate the host immune system by stimulating chemokine and cytokine production, and thereby activating immune cells [3], Ultimately, certain AMPs also promote wound healing by enhancing cell proliferation, angiogenesis, and collagen - all hallmarks of effective tissue regeneration [4], The AMP hBD-2 is one of the most predominant AMP in human skin, known for its broad-spectrum antimicrobial activity, including against the most predominant pathogens in DFUs, e.g. Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Candida spp. [4], Besides, hBD-2 has also demonstrated ability to promote cell proliferation and migration [4], Another example is the AMP PP4-3.1, which is a hybrid construct, where the cosmeceutical pentapeptide-4 (PP4), recognized for its collagen-inducing properties, is covalently linked to the peptide 3.1, which has demonstrated antimicrobial activity against both Gram-positive and Gram-negative bacteria [18,19],

[0005] AMPs may yet present some drawbacks such as low stability and potential toxicity, as well as altered expression level and functionality under diabetic conditions as happens with the endogenous AMP hBD-2 [5], To address several of these limitations, biomaterials have emerged as potential delivery vehicles for AMPs, offering a more controlled therapeutic approach [6, 7], Alginate is an example of a widely studied biomaterial that shows several advantages to be used as a wound dressing for the delivery of AMPs. Indeed, the anionic nature of the alginate polymer is associated with excellent biocompatibility, hydrophilicity, and outstanding swelling / absorbing capacity, all suitable traits to be applied as wound dressings [7], Moreover, alginate can be formulated to form a protective barrier that preservesmoisture and facilitates ion exchange with wound exudate and blood, creating an optimal and conducive environment for healing [8],

[0006] These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure.GENERAL DESCRIPTION

[0007] The present disclosure relates to a biocompatible alginate-based hydrogel that, when loaded with antimicrobial peptides (AMPs), fosters an environment conducive to tissue repair by offering combined anti-inflammatory, ROS-scavenging, pro-angiogenic, and collagen-inducing benefits for complex wounds.

[0008] The present disclosure relates to a hydrogel for tissue regeneration comprising alginate forming a hydrogel matrix and a pharmaceutical amount of an antimicrobial peptide, wherein the antimicrobial peptide is entrapped / embedded in the hydrogel matrix. Surprisingly, the disclosed hydrogel is a pH stable hydrogel and exhibits a sustained peptide release profile for over 72 hours.

[0009] In an embodiment, the disclosed hydrogel allowed for a controlled release of antimicrobial peptides, in particular a sustained release for over three days.

[0010] In an embodiment, the hydrogel of the present disclosure, comprises 2-4 % (w / v) of alginate, preferably 2.5 to 3 % (w / v), more preferably 3 % (w / v); and 0.1 to 10 pg / mL of the antimicrobial peptide, preferably 0.5 to 2 pg / mL of the antimicrobial peptide, more preferably 1 pg / mL.

[0011] In an embodiment, the antimicrobial peptide is selected from a list consisting of: human |3-defensin 1, human |3-defensin 2, human |3-defensin 3, PP4 3.1, human cathelicidin antimicrobial peptide (LL-37), and mixtures thereof. In a preferred embodiment, the antimicrobial peptide is selected from a list consisting of: human |3- defensin 1, human |3-defensin 2, human |3-defensin 3, human cathelicidin antimicrobial peptide (LL-37), and mixtures thereof; more preferably the antimicrobial peptide is selected from a list consisting of: human |3-defensin 1, human |3-defensin 2, human |3- defensin 3, and mixtures thereof; even more preferably, the antimicrobial peptide is human |3-defensin 2.

[0012] In an embodiment, the hydrogel polymer matrix comprises a porous structure with pore diameter ranging from 0.5 nm to 20 nm measured by scanning electron microscopy; preferably ranging from 2 nm to 6 nm.

[0013] In an embodiment, the alginate hydrogel polymer matrix has an initial storage modulus ranging from 3.58 ± 0.35 kPa to 14.79 ± 1.69 kPa and / or an initial loss modulus ranging from 0.12 ± 0.02 kPa to 1.54 ± 0.24 kPa, measured by rheology.

[0014] In an embodiment, the alginate hydrogel polymer matrix swelling ratio ranges from 29.37 ± 1.44 to 34.36 ± 3.55, obtained by subtracting the weight of the dry hydrogel disk to the weight of the swollen hydrogel disk, divided by the weight of the dry hydrogel disk.

[0015] In an embodiment, the alginate hydrogel polymer matrix mesh size ranges from 45 nm to 115 nm, preferably from 50 nm to 108 nm, even more preferably from 50.98 ± 2.86 nm to 107.05 ± 5.35 nm, measured by rheology.

[0016] In an embodiment, the hydrogel further comprises a crosslinking agent, in particular an ionic crosslinking agent.

[0017] In an embodiment, the crosslinking agent is selected from a list consisting of calcium sulphate, barium chloride, strontium chloride, magnesium chloride, zinc chloride, aluminum chloride, and ferric chloride. In a preferred embodiment the crosslinking agent is calcium sulphate slurry.

[0018] In an embodiment, the crosslinking ratio, i.e. the ratio of crosslinked sites to total active sites, ranges from 1:3 to 1:1, more preferably is 4:5.

[0019] In an embodiment, the hydrogel matrix comprises a mixture of alginates with different molecular weights; preferably at least a low molecular weight alginate and a high molecular weight alginate.

[0020] In an embodiment, the alginate molecular weight ranges from 100-300 kDa; preferably wherein the low molecular weight alginate ranges from 120-150 kDa and the high molecular weight alginate ranges 240-260 kDa.

[0021] In an embodiment, the weight ratio of low molecular weight alginate and high molecular weight alginate solutions ranges from 1:4-4:1, more preferably 3:4-l:4.

[0022] In an embodiment, the hydrogel of the present disclosure further comprises an active compound; preferably wherein the active compound is selected from a list consisting of: anti-inflammatory, antipyretic, antibiotic, antimicrobial peptide, or mixtures thereof.

[0023] In an embodiment, the hydrogel of the present disclosure is for use in medicine or veterinary.

[0024] In an embodiment, the hydrogel of the present disclosure is for use in wound treatment, wound tissue regeneration and / or wound healing.

[0025] In an embodiment, the hydrogel of the present disclosure is for use in the treatment of chronic wounds, more preferably diabetic foot ulcers.

[0026] Another aspect of the present disclosure relates to an article comprising the hydrogel of the present disclosure.

[0027] In an embodiment, the article is a mesh, a membrane, scaffold, a fiber, a disc, or a patch.

[0028] Another aspect of the present disclosure is the use of the disclosed product for the manufacture of a medicament for wound treatment, wound tissue regeneration and / or wound healing.

[0029] In an embodiment, the use of the disclosed product is for the manufacture of a medicament for the treatment of chronic wounds, more preferably diabetic foot ulcers.

[0030] Another aspect of the present disclosure relates to a method for treating or preventing chronic wounds, more preferably diabetic foot ulcers in a subject, the method comprising administering the hydrogel of the present disclosure.

[0031] An aspect of the present disclosure relates to a method for obtaining the hydrogel of the present disclosure: obtaining a low molecular weight alginate solution and high molecular weight alginate solution; preferably in ultrapure water; mixing the low molecular weight and high molecular weight alginate solutions within interconnected syringes;crosslinking with a crosslink agent / gelling agent; adding an antimicrobial peptide, either separately or combined, with a low or high molecular weight alginate solution.

[0032] In an embodiment, distinct AMPs can be incorporated in alginate-based hydrogel matrix, namely human p-defensins 1-3, human cathelicidin antimicrobial peptide, PP4-3.1, and mixtures thereof. By incorporating AMPs, alginate-based dressings can further enhance antimicrobial and wound-healing properties by providing sustained peptide release and localized activity at the wound site, hence being suitable as a promising tool for the effective management and treatment of DFUs and other chronic or infected wounds. In particular, alginate-based hydrogel comprising low and high molecular weight alginate polymers surprisingly showed sustained release of AMPs, shield them from degradation, and support tissue regeneration.

[0033] The management of diabetic foot ulcers (DFUs) is a major clinical challenge due to their complex, multifactorial ethology involving chronic infection, persistent inflammation, impaired angiogenesis, and oxidative stress. Current therapies, including topical antibiotics, growth factors, and passive dressings, fail to effectively address these multitargeted pathological processes. Moreover, conventional antimicrobials often lead to antibiotic resistance and cytotoxicity, while AMPs, though potent, are unstable and rapidly degraded under physiological conditions, limiting their therapeutic potential.

[0034] There is a clear unmet need for a biocompatible, multifunctional wound dressing capable of providing sustained antimicrobial activity, inflammation modulation, and tissue regeneration support within the hostile microenvironment of complex wounds such as DFUs. The disclosed hydrogel overcomes these limitations by integrating AMPs within a structurally stable alginate hydrogel, ensuring prolonged bioactivity, moisture retention, and controlled peptide release. This synergistic composition effectively restores the healing balance in DFUs, thereby reducing infection, promoting tissue repair, and minimizing the risk of amputation.BRI EF DESCRI PTION OF THE DRAWI NGS

[0035] The following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of invention.

[0036] Figure 1: Development and characterization of alginate-based hydrogels for AMP delivery. (A) Schematic representation of hydrogel preparation. (B) Representative picture of the produced alginate-based hydrogel disks. Rheological analysis of the hydrogels showing (C) storage moduli (G'), (D) loss moduli (G"), (E) tan(delta), (F) swelling ratios (Q), and (G) mesh sizes, over different time points and pH levels (6, 7, and 8) mimicking wound healing conditions. (H) Scanning electron microscopy of the alginate-based hydrogels. Scale bars - 200nm (picture inlet) and lpm. (I) Pore size distribution histogram of the alginate-based hydrogels, with indication of the average pore diameter. (J) Degradation profiles, over different time points and pH levels (6, 7, and 8) mimicking wound healing conditions. (K) Cumulative AMP release (%) determined through the Pierce Micro BCA™ Protein Assay Kit at the different time points. The mean is represented by the central line, and the shaded areas denote the error envelope representing the standard deviation (SD).

[0037] Figure 2: In vitro biocompatibility and cell migration of AMP-loaded hydrogels. Biocompatibility was assessed using MTT assay on (A) HaCaT and (B) HDFa cells after 24 hours of incubation. Cell viability below 70% is considered an indication of a cytotoxic effect (indicated by red dotted line). Representative images of scratch assays on (C, D) HaCaT and (E) HDFa cells at time points 0, 24, and 48 hours, and at a 10x magnification. Scale bars - 200 pm. Quantitative analysis of gap closure using scratch assay on (F, G) HaCaT and (H) HDFa cells over 48 hours of incubation. Bars in plots represent mean with individual measurements denoted by scatter points and the error bars indicate standard deviation (SD) (A and B). Gap closure (F-H) is presented as mean ± standard deviation (SD). Statistical analysis was conducted using one-way ANOVA followed by Holm Sidak's multiple comparisons test. Asterisk(s), and section, hash and double dagger sign(s) specify statistically significant differences between the following conditions: ** control versus hBD-2 (p < 0.01), § control versus PP4-3.1 (p < 0.05), # hBD-2 versus PP4-3.1 (p < 0.05), and tt hBD-2 versus hBD-2:PP4-3.1 (p < 0.01).

[0038] Figure 3: In vivo efficacy of hBD-2 hydrogels in wound healing. (A) Representative images of wound healing progression over time for each group (days 0, 3, 7, and 10). Scale bars - 5mm. (B) Wound closure (% of initial wound area) was measured using acetate assessments. (C) Wound microbial load was determined from topical gauze collection. (D) Representative histological images for each group stained with hematoxylin and eosin (top) and Masson's trichrome (bottom). Scale bars - 200pm. Black triangles indicate increased collagen deposition in wounded area. Ep: epidermis; GT: granulation tissue. (E) The epidermal thickness index was calculated by dividing the average epidermal thickness in wounded skin by the average epidermal thickness in uninjured skin, then multiplying by 100. (F) Epidermal thickness in the wounded area. (G) Granulation tissue thickness in the wounded area. (H) The granulation tissue thickness index was calculated by dividing the average granulation tissue thickness in wounded skin by the average epidermal thickness in uninjured skin, then multiplying by 100. (I) Collagen fiber quantification (% of control) in the wounded area. (J) Overall histology score quantification. Parametric data (B) are presented as mean ± standard error of the mean (SEM), while histological data (E-H) are shown as bars representing mean values, with individual measurements indicated by scatter points, and error bars reflecting standard deviation (SD). Microbial load data (C) is represented as a median with an interquartile range, with individual measurements represented by scatter points. Statistical analysis was conducted using two-way ANOVA followed by Dunnett's multiple comparisons test for wound closure and microbial load data, and one-way ANOVA followed by Holm Sidak's multiple comparisons test for histological data. Asterisks and hash signs denote statistically significant differences between the following conditions: # control versus blank hydrogels (## p < 0.01, and #### p < 0.0001), and * control versus hBD-2 hydrogels (*p < 0.05, **p < 0.01, and ****p < 0.0001).

[0039] Figure 4: In vivo anti-inflammatory effects of hBD-2 hydrogels at day 10 postinjury. Representative images of (A) Ml-like macrophages and (B) M2-like macrophages, both indicated by white arrows. Scale bars - 20pm (picture inlets) and 100pm. Quantification of (C) Ml-like macrophages, (D) M2-like macrophages, and (E) M1 / M2 ratio. Representative images of (F) CD3+ cells (indicated by white arrows), (G)interleukin-6 (IL-6), and (H) monocyte chemoattractant protein-1 (MCP-1). Scale bars - 20pm (picture inlets) and 100pm. (I) Quantification of CD3+ cells per field at the wound site. (J) Percentage of IL-6 expression levels relative to the control group. (K) Percentage of MCP-1 expression levels relative to the control group. Bars represent mean ± standard error of the mean (SEM) for parametric data and median with interquartile range for non-parametric data, both with individual measurements denoted by scatter points. Statistical analysis was conducted using one-way ANOVA followed by Holm Sidak's multiple comparisons test or Kruskal-Wallis test followed by Dunn's multiple comparisons test, depending on the data distribution. Asterisk(s) specify statistically significant differences between conditions. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

[0040] Figure 5: In vivo effects of hBD-2 hydrogels on wound healing progression at day 10 post-injury. Representative images of (A) dihydroethidium assay (DHE), (B) Ki67+cells (indicated by white arrows), (C) CD31+cells (indicated by white arrows), (G) matrix metalloproteinase-9 (MMP-9), and (H) collagen type 1 alpha 1 (COL1A1). Scale bars - 20pm (picture inlets) and 100pm. (D) Percentage of DHE expression levels relative to the control group. (E) Number of Ki67+cells per field at the wound site. (F) Number of CD31+ cells per field at the wound site. (I) Percentage of MMP-9 expression levels relative to the control group. (J) Percentage of COL1A1 expression levels relative to the control group. Bars represent the mean with individual measurements denoted by scatter points and the error bars indicate the standard error of the mean (SEM). Statistical analysis was conducted using one-way ANOVA followed by Holm Sidak's multiple comparisons test. Asterisk specifies statistically significant differences between conditions. *p < 0.05.DETAILED DESCRIPTION

[0041] The present disclosure relates to a hydrogel for tissue engineering / regeneration comprising a polymeric alginate solution forming a hydrogel matrix and a pharmaceutical amount of an antimicrobial peptide, wherein the antimicrobial peptide is entrapped / embedded in the hydrogel matrix. The method to obtain said hydrogel, as well as the uses thereof are also described.

[0042] The alginate-based hydrogels of the present disclosure showed suitable elasticity and stiffness, supporting a nanometric porosity for AMP loading, and pH stability.

[0043] In one embodiment, the alginate-based hydrogels of the present disclosure were developed as a biocompatible delivery system for exogenous AMPs, designed to protect these peptides from proteolytic degradation and ensure sustained release, thus increasing their functional capacity. As example, two distinct AMPs with dual antimicrobial and wound-healing properties (hBD-2 and PP4-3.1) were used to load onto the hydrogels, individually or in combination. A representative picture of the produced alginate-based hydrogel disks is shown in Figure IB. Rheological characterization of the hydrogels was performed to measure certain viscoelastic parameters, including G' and G" moduli, tan(delta), swelling ratio (Q), mesh size ( ;), and degradation profiles. The hydrogels showed significant elasticity and stiffness, fostering a nanometric porous network suitable for AMP loading (Figure 1C-G). Specifically, high initial G' (14.79 ± 1.69 kPa) and low initial Q (29.37 ± 1.44) values were observed, indicative of notable stiffness (Figure 1C and F). Slight decreases in G' were observed over time across pH 6 to 8, consistent with the pH range during wound healing [9], while increases in Q values were also noted (Figure 1C and F). Correspondingly, the mesh size of the hydrogels increased over time under alkaline conditions, with values ranging between 50.98 ± 2.86 nm and 107.05 ± 5.35 nm (Figure 1G). Scanning electron microscopy confirmed the nanometric porous structure, showing similar pore diameters between samples (average pore diameter of 5.16 ± 6.17 nm), despite some heterogeneity of the pore size distribution (Figure 1H and I). Furthermore, the degradation rates across varying pH levels remained relatively stable, with values between 92.81 ± 9.35% and 108.99 ± 10.41% (Figure 1J). Finally, evaluation of AMP release using a peptide surrogate showed sustained release for over three days, with around half of the loaded content (51.91 ± 5.14%) released by 12 hours (Figure IK).

[0044] In an embodiment, AMP-loaded hydrogels of the present disclosure exhibited biocompatibility and promoted gap closure in vitro.

[0045] In an embodiment, the AMP-loaded hydrogels biocompatibility and cell migration were assessed in vitro using HaCaT and HDFa cells. Both AMP-loaded and blank hydrogels displayed outstanding biocompatibility, with over 95% cell viability in both cell types (Figure 2A and B). In addition, hBD-2 hydrogels promoted gap closure, showing a tendency for an increase in HaCaT and HDFa migration by more than 1.13- fold and 1.08-fold, respectively, after 24 hours compared to free AMPs, blank hydrogels and the synthetic peptide PP4-3.1 hydrogels (Figure 2C-H). Notably, cell migration was sustained for up to 48 hours in the AMP-loaded hydrogels, while free AMPs exhibited no further cell migration after 24 hours (Figure 2F-H). Hydrogels loaded with hBD-2 were further used for in vivo application, due to the superior cell migration promoted by those hydrogels after 24 hours compared to free AMPs and blank or PP4-3.1 hydrogels (Figure 2F-H).

[0046] In an embodiment, hBD-2 hydrogels of the present disclosure accelerated wound closure and reduced microbial load in a streptozotocin (STZ)-induced diabetic mouse model.

[0047] To further evaluate the efficacy of the hydrogels, in another embodiment a STZ-induced diabetic mouse model of wound healing was used. The hBD-2 hydrogels significantly accelerated wound closure at days 3, 7, 8, and 9 (p<0.05) compared to the gauze control (Figure 3A and B), and further showed enhanced re-epithelialization and tissue remodelling (Figure 3D). The hBD-2 hydrogels further showed a tendency for an increase by 1.25-fold in wound healing rates on days 5, 6, 8, 9, and 10, compared to blank hydrogels (Figure 3A and B). In addition, hBD-2 hydrogels decreased the microbial load by 30-fold on day 7 compared to blank hydrogels (p<0.05) (Figure 3C). On day 10, a tendency for a decrease by 9.2-fold compared to the control and by 80- fold compared to blank hydrogels in the microbial load was further observed (Figure 3C). Moreover, animals treated with both hBD-2 and blank hydrogels displayed a reduction in the ETI by more than 1.50-fold and 1.75-fold, respectively, compared to the control (Figure 3E). The hBD-2 hydrogels also led to the smallest epidermal thickness (more than 1.15-fold) compared to control and blank hydrogels (Figure 3F). Besides, animals treated with hBD-2 hydrogels led to the smallest granulation tissue thickness (more than 1.07-fold) compared to control and blank hydrogels, showing theclosest granulation tissue thickness index to 100% (97.86 ± %) (Figure G and H). Furthermore, hBD-2 hydrogels led to the highest collagen content (more than 1.09- fold) compared to control and blank hydrogels (Figure 31). Finally, using the histology scoring system

[0010] , it was observed that hBD-2 hydrogels displayed the highest histology score, 1.2-fold higher than the gauze control and blank hydrogels (Figure 3J).

[0048] In an embodiment, hBD-2 hydrogels of the present disclosure showed antiinflammatory properties in skin wounds of diabetic mice.

[0049] Another aspect of the present disclosure relates to anti-inflammatory effects of hBD-2 hydrogels. These anti-inflammatory effects were evaluated at day 10 post-injury via immunohistochemical analysis of wound tissue. In particular, Ml and M2-like macrophages were quantified using CD68 and TNF-a or CD68 and CD206 markers, respectively. A statistically significant decrease of Ml-like macrophages was observed in the hBD-2 hydrogel group (p<0.01) compared to the control group (Figure 4A and C), while no significant increase of M2-like macrophages was observed (Figure 4B and D). Blank hydrogels also denoted a decrease of Ml-like macrophages compared to the control group, despite not being statistically significant (Figure 4A and C). No changes in M2-like macrophages were observed in this group when compared to the control group (Figure 4B and D). Both hBD-2 and blank hydrogel groups led to a decrease in the M1 / M2 ratio of macrophage polarization (p<0.001), with the hBD-2 hydrogel group showing the most significant decrease (Figure 4E). In addition, the number of CD3- positive lymphocytes was decreased in both hBD-2 and blank hydrogel groups (p<0.05) compared to the control group (Figure 4F and I). Lastly, the pro-inflammatory IL-6 and MCP-1 markers were also investigated, but no significant changes in expression levels were observed among the experimental groups (Figure 4G, H, J, and K). However, the hBD-2 hydrogel group revealed a slight decrease by more than 1.15-fold in IL-6 expression levels compared to the gauze control and blank hydrogels (Figure 4G and J).

[0050] In an embodiment, hBD-2 hydrogels of the present disclosure reduced oxidative stress and promoted angiogenesis and type I collagen expression in skin wounds of diabetic mice.

[0051] In another embodiment, the levels of oxidative stress, angiogenic response and type I collagen expression were evaluated at day 10 post-injury to further explore thehydrogels' effects on wound healing. In particular, ROS levels were measured using the DHE assay, and both hBD-2 and blank hydrogel groups significantly reduced DHE expression levels (p<0.0001 and p<0.001, respectively) compared to the control group, with the hBD-2 hydrogel group showing the most tangible decrease (Figure 5A and D). Proliferation was further assessed by quantification of Ki67-positive cells. The hBD-2 hydrogels increased the number of Ki67-positive cells by more than 1.5-fold compared to the gauze control and blank hydrogels (Figure 5B and E). In turn, neovascularization, assessed by CD31-positive cells, was significantly increased in both hBD-2 and blank hydrogel groups (p<0.01 and p<0.05, respectively), with the hBD-2 hydrogels displaying the most significant increase (Figure 5C and F).

[0052] In another embodiment, wound remodelling and maturation were evaluated via MMP-9 or COL1A1 expression levels relative to the control group. No statistically significant changes were observed in the content of MMP-9 among the experimental groups. However, the hBD-2 hydrogel group slightly increased by more than 1.2-fold MMP-9 expression levels compared to the gauze control and blank hydrogels (Figure 5G and I). Moreover, COL1A1 expression levels were significantly increased in the hBD- 2 hydrogel group (p<0.05) compared to the gauze control, whereas no changes were observed for the blank hydrogel group (Figure 5H and J).

[0053] The disclosed alginate-based hydrogels loaded with antimicrobial peptides are suitable for use in compositions or dressings intended to enhance or support the healing of chronic diabetic wounds. These hydrogels were designed to provide sustained release of AMPs, shield them from degradation, and support tissue regeneration. In one embodiment, the AMP hBD-2 was used, due to its antimicrobial activity in disrupting the microbial membranes

[0016] , Surprisingly the hydrogels of the present disclosure promoted superior cell migration and demonstrated great biocompatibility in vitro. Moreover, the hBD-2 hydrogels showed prolonged AMP release, reduced inflammation and promoted angiogenesis and collagen deposition in an STZ-induced diabetic mouse model. Altogether, these findings show that alginate- based hydrogels loaded with AMPs improve chronic diabetic wound management.

[0054] In another embodiment, the alginate hydrogels of the present disclosure exhibited favourable viscoelastic properties and a porous nanostructure that supportssustained AMP release, making them well-suitable for wound healing applications. In the state of art, ionic crosslinked alginate hydrogels presented similar stiffness and subsequent nanometric mesh size. The nanometric porous network of the alginate hydrogels is likely to play a critical role in mediating the release profile of the AMPs. Surprisingly the hydrogels of the present disclosure exhibit a controlled release of AMPs over three days, in contrast to the more rapid release profile described by other studies, where more than 60% of the AMP initially loaded was released within 8 hours with no significant release after that. This extended delivery over time is appropriate to reduce the inflammatory phase that often lasts more than three days in diabetic wounds [9], and to foster the progress of subsequent phases of the wound healing process. In another embodiment, comparing the release profiles of the hydrogel of the present disclosure to those reported in the state of art, it is important to consider how different gelation schemes influence hydrogel properties. Ionic gelation in alginate hydrogels can be achieved through distinct internal and external mechanisms, influencing crosslinking kinetics, mechanical properties, and hydrogel homogeneity. Decisively, these differences in gelation methodology directly affect the release profile of encapsulated cargo and should be wisely considered when developing delivery systems.

[0055] In another embodiment, in vitro biocompatibility of the alginate hydrogels of the present disclosure was tested with HaCaT and HDFa cells further ensuring the nontoxic nature of the hydrogels, both blank and AMP-loaded. The biocompatibility of alginate hydrogels is well established and previous studies showed similar results as described in the present disclosure. Besides, hBD-2 hydrogels showed a tendency for an enhancement of HaCaT and HDFa migration after 24 hours compared to free AMPs, blank hydrogels and PP4-3.1 hydrogels. Striking, cell migration was sustained up to 48 hours with AMP-loaded hydrogels, contrary to free AMPs that exhibited stationary migration after 24 hours. Surprisingly, it was observed a synergistic effect between the alginate polymeric matrix and the AMPs, capable of facilitating cell migration. A similar observation has been reported with endogenous AMPs LL-37 and hBD-2, which stimulated in vitro wound closure in HaCaT cells, though only LL-37 showed continued effects beyond 24 hours [5],

[0056] In one embodiment, the in vivo effects of the hydrogels of the present disclosure were assessed by STZ-induced diabetic mouse model of wound healing. This model is widely accepted due to the shared pathophysiological features of DFUs in both type 1 and type 2 diabetes. Notably, the STZ-induced diabetic mice replicate key characteristics observed in human diabetic skin wounds, such as reduced peripheral nerve function, blood vessel numbers, granulation tissue formation, and collagen composition. In this model, hBD-2 hydrogels significantly accelerated wound closure compared to the control throughout the experimental period, while the blank hydrogels showed a significant effect only at days 3 and 7. This aligns with previously reported findings that AMPs, such as p-defensins, enhance wound healing by targeting the antimicrobial effects on pathogens and stimulating local immune responses [5], In another embodiment, hBD-2 hydrogels reduced overall microbial load on day 7 compared to blank hydrogels, with a similar decrease observed on day 10. In the state of art, it is known that germ-free mice, lacking commensal skin microbiota, experienced faster and scarless healing; while other studies suggested that local skin commensal microorganisms may develop pathogenic behaviours in microenvironments of chronic wounds, further impairing wound healing. Nevertheless, the observed increase in the blank hydrogel group suggests that, in the absence of antimicrobial agents, the hydrogel stiffness and water content could promote microbial growth. Surprisingly, the hBD-2 hydrogels of the present disclosure further exhibited the best overall histology score with appropriate re-epithelialization and granulation tissue formation, as well as higher collagen content compared to the other experimental groups, indicating a mature wound healing progress. Although blank hydrogels also improved these parameters, the effects were less pronounced, suggesting a synergistic effect between the alginate-based hydrogel and the AMP hBD- 2. Similar findings were reported, where an alginate hydrogel containing the AMP Chol-37(F34-R), yet using a much higher AMP concentration (200 pg / mL) than us, showed the best wound appearance in a murine model of Pseudomonas aeruginosa- infected wounds, but with no diabetes, exhibiting clear hierarchical structure between the epidermis and dermis, compared to other controls. Altogether, these results emphasize the in vivo applicability and efficacy of the hBD-2 alginate hydrogels of the present disclosure for wounds in diabetes.

[0057] A persistent state of inflammation often drives wound healing impairment in patients with diabetes. Several pro-inflammatory cytokines, namely TNF-a, IL-6, and MCP-1, act as key players in the recruitment of cells such as neutrophils and macrophages to the wound site to stimulate an immune response either for clearance of cellular debris from damaged skin cells or for infection control. For normal wound healing to proceed, a shift from a pro- to an anti-inflammatory environment is essential. In another embodiment, the hBD-2 hydrogels of the present disclosure significantly increased M2-like (anti-inflammatory) macrophages relative to Ml-like (pro-inflammatory) macrophages, leading to a decrease of the M1 / M2 ratio of macrophage polarization. Both hBD-2 and blank hydrogels induced a decrease in the number of lymphocytes. The Ml-to-M2-like macrophage shift, accompanied by the decrease of lymphocytes, is fundamental for attenuating the inflammatory environment and subsequently enable wound healing progression. In addition, no major changes in IL-6 and MCP-1 expression levels were observed between the experimental groups, despite the hBD-2 hydrogels revealing a slight decrease in the IL- 6 expression levels. These findings suggest that the hBD-2 hydrogels of the present disclosure support and enhance a transition towards a more pro-reparative stage of wound healing.

[0058] Chronic wounds are further exacerbated by the overproduction of ROS, leading to cell damage, disrupted angiogenesis and altered extracellular matrix remodelling. In one embodiment, the hBD-2 hydrogels of the present disclosure led to a decrease in DHE expression levels, indicating a ROS-scavenging effect. Similar results were observed with the blank hydrogels, yet to a lower extent, supporting the synergistic effect between the alginate-based hydrogel and the AMP hBD-2. This outcome is likely to be correlated with the decreased inflammation in treated wounds, enabling favourable conditions for tissue repair. In the state of art, it is reported the critical role of maintaining a balanced level of ROS to enhance effective wound healing and infection control. For instance, an immunoregulatory hydrogel with controlled hyperthermia-augmented oxygenation and ROS-scavenging properties was developed to tackle the detrimental cycle caused by hypoxia and ROS overproduction that results in inflammatory dysregulation and subsequent impaired healing. In contrast, anotherapproach utilizes an ATP-activated spatiotemporally controlled hydrogel prodrug system, which exploits bacteria-secreted ATP to trigger ROS generation. This is achieved through the loaded indole-3-acetic acid prodrug and a nanozyme, resulting in potent antibacterial effects.

[0059] In another embodiment, the hBD-2 hydrogels of the present disclosure increased neovascularization, cell proliferation, COL1A1 deposition, as previously observed by the Masson's trichrome staining, and MMP-9 production, thus confirming the evidence of a progression towards the final phases of wound healing. Local cellular proliferation, accompanied by the formation of new blood vessels, is of utmost importance for adequate re-epithelialization and provision of nutrients, oxygen and immune cells to the healing tissue. Moreover, despite high levels of MMP-9 are often associated with chronic wounds, the presence of MMP-9 is yet essential for the breakdown of disorganized old collagen, predominantly of type III, and subsequent replacement by the stronger type I collagen, allowing appropriate remodelling. In the state of art, it has been reported the development of multifunctional hydrogels, such as the cationic hydrogels prepared by chemically cross-linking trans-1,4- cyclohexanediamine with l,3-dibromo-2-propanol using a condensation reaction. These hydrogels demonstrated pro-angiogenic, pro-collagen, anti-inflammatory, and antibacterial effects in 5. aureus-infected wounds in diabetic rats.

[0060] In one embodiment, the hydrogels of the present disclosure exhibit the following key therapeutic properties: non-toxicity, exudate absorption, moistening activity, anti-inflammatory activity, antimicrobial activity, ROS reduction, proangiogenesis, collagen deposition, and wound healing.

[0061] The present disclosure relates to safe and effective hBD-2 alginate hydrogels for promoting wound healing both in vitro and in vivo.

[0062] A particular aspect of the present disclosure is the synergistic combination of the AMP hBD-2's bioactivity and the intrinsic properties of the alginate hydrogels. This synergistic combination fosters an environment conducive to tissue repair by offering anti-inflammatory, ROS-scavenging, pro-angiogenic, and collagen-inducing benefits. These hydrogels of the present disclosure show promise as a novel multifunctional therapeutic option for complex wounds such as diabetic foot ulcers.Materials and Methods

[0063] Alginate polymers were purchased from Nova Matrix, Norway. Furthermore, the AMP hBD-2 was purchased from ProSpec-Tany TechnoGene Ltd, Israel, whereas the peptide PP4-3.1 was produced at the Peptide and Peptide-Nucleic Acids Synthesis Facility (POP-UP) of the Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, Portugal.

[0064] The primary antibodies anti-rabbit interleukin-6 (IL-6) and anti-rat Kiel 67 (Ki67) were purchased from Thermo Fisher Scientific, MA, U.S., whereas the primary antibodies anti-rat CD31 and anti-rabbit matrix metalloproteinase-9 (MMP-9) were obtained from Millipore, MA, U.S. Moreover, the primary antibodies anti-rabbit monocyte chemoattractant protein-1 (MCP-1), anti-rat CD206, and anti-goat collagen type I alpha 1 (COL1A1) were obtained from Santa Cruz, CA, U.S. Furthermore, the primary antibody anti-rat TNF-a was purchased from Bio-Rad AbD Serotec Ltd, CA, U.S., whereas the primary antibodies anti-rabbit CD3 and CD68 were purchased from Abeam Pic, Cambridge, UK. Additionally, the normal goat serum was obtained from Life Technologies, CA, U.S. The secondary antibodies goat anti-rat Alexa Fluor 594, antirabbit Alexa Fluor 488, and anti-rabbit Alexa Fluor 594, as well as the secondary antibody rabbit anti-goat Alexa Fluor 488, were obtained from Abeam Pic, Cambridge, UK. The 4',6-diamidino-2-phenylindole (DAPI) and dihydroethidium (DHE) probes were acquired from Sigma-Aldrich, MO, U.S.

[0065] All the remaining reagents were purchased from Sigma-Aldrich, MO, U.S. or from VWR, Portugal.

[0066] In one embodiment for the formulation and loading of the hydrogels of the present disclosure, MVG alginate, containing a higher G-block content (> 60% as specified by the manufacturer) was used, including a high molecular weight (HMW) LF20 / 40 and a low molecular weight (LMW) LF10 / 60 polymer. The molecular weights of the HMW and the LMW polymers are of ~250 kDa and ~120-150 kDa, respectively. In particular, alginate polymers used for in vitro and in vivo assays were prepared under sterile and aseptic conditions.

[0067] In one embodiment for the formulation of the hydrogels, several input variables were explored for their effects on modulating alginate hydrogel properties, namely storage and loss moduli, swelling capacity and correspondingly mesh size. This included the ratio of LMW and HMW polymer, the ratio of gelling agent, the total polymer volume fraction, and the percentage of oxidation modification of polymer. The input variables were then optimized via the response surface predictions generated by the Design of Experiment (DoE) software to achieve the fundamental goal of AMP loading and subsequent sustained release over time.

[0068] In another embodiment, LMW and HMW alginates were reconstituted in ultrapure water to a final concentration of 3% (w / v) polymer alginate solution. The LMW and HMW alginate solutions were then mixed within interconnected syringes in a proportion of 75 / 25 (LMW / HMW), followed by a mixture with a gelling agent, a calcium sulfate slurry at a ratio of 4:5, to ionically crosslink the polymer chains. The mixture was rapidly dispensed onto a glass plate set with 1-mm spacers, sandwiched with another glass plate, and incubated for at least 25 min at room temperature (RT). Afterwards, disks were punched out using a 6-mm diameter biopsy punch (Utilmedica - Produtos Medicos Hospitalares, Portugal), and left for swelling / stabilizing in phosphate buffered saline containing calcium and magnesium ions (PBS++) for at least 3 hours at 37°C, 5% CO2.

[0069] In another embodiment, for the loading of AMPs onto the hydrogels of the present disclosure, AMPs with distinct antimicrobial and wound healing properties (hBD-2 and PP4-3.1) were previously added, either separately or combined, to the syringe with the LMW alginate solution to a final concentration of 1 pg / mL. A schematic representation of the hydrogel preparation protocol is illustrated in Figure 1A. A new set of hydrogels was prepared for each experiment.

[0070] In one embodiment, the rheological characterization of the alginate hydrogel disks was carried out using a HR-3 Discovery Hybrid Rheometer (TA Instruments, U.S.). In particular, to measure the viscoelastic properties of the alginate hydrogels, the disks were first swollen, as described above, and excess fluid was removed with a damp kimwipe. Afterwards, the hydrogel disks were placed between parallel plates (axial force holding on ~0.1) in the rheometer for determination of their initial storage (G')and loss (G") moduli, as well as their respective tan(delta). The disks were then strained over a range within 0.1-5% at a frequency of 1 Hz and values for G' and G" were obtained within the linear viscoelastic region. At least 9 points were used to obtain an average G' or G" value for each disk and then an average G' or G" value was reported for each hydrogel condition (n=3). In order to measure changes in G' and G" moduli over time and across different pH conditions, the hydrogel disks were immersed in PBS at pH=6, 7, and 8. During this process, the disks were incubated at 37 °C, and 5% CO2, and measurements were recorded at the following time points: 0, 1, 6, 12, 24, and 72 hours.

[0071] In one embodiment, the equilibrium swelling ratios (Qs) of the hydrogels of the present disclosure were determined as previously described. Briefly, after swelling in PBS++and the removal of excess fluid, the mass of the hydrogel disk was determined. The hydrogel disks were then frozen and lyophilized, afterwards the dry weight was obtained. The swelling ratio was defined as:where Ws is the weight of the swollen hydrogel disk and Wd is the weight of the dry hydrogel disk.

[0072] In one embodiment, the initial mesh sizes ( ;) of the hydrogel disks were calculated using the measured values of G' and Q immediately after swelling, as previously described. In detail, the molecular weight between crosslinks (Me) was determined by:where cpis the total concentration of the polymer solution (g / m3), R is the gas constant (8.314 m3Pa mol1K-1), and T is the temperature at which the measurement was performed (296.15 K). The polymer volume fraction (V2) was then calculated as:where ppis the density of the alginate polymer (1.601 g cm-3) and q is the density of water (1.0 g cm'3).

[0073] Finally, the mesh size was obtained by:where Mris the molecular weight of the monomer units (194 g / mol for mannuronic acid or guluronic acid), L is the carbon-carbon bond length of the monomer unit (5.15 A), and Cnis the characteristic ratio (Cn= 0.021Mn+ 17.95 calculated as for alginate).

[0074] In one embodiment the degradation profiles of the hydrogels of the present disclosure were further determined. Briefly, after swelling in PBS++and the removal of excess fluid, the hydrogel disks were frozen and lyophilized. Afterwards, the dry hydrogel disk weight was obtained at the following time points: 0 (initial dry weight), 1, 6, 12, 24, and 72 hours. The degradation profiles were then defined as the dry hydrogel disk weight (%) at each time point relative to the initial dry hydrogel disk weight.

[0075] In one embodiment, scanning electron microscopy was performed. In particular, alginate hydrogel disks were lyophilized for 24 hours, mounted on carbon tape and coated with a thin layer of gold, as previously described. Afterwards, the disks were placed in a vacuum chamber and imaged with the Zeiss Merlin high- resolution field emission gun - scanning electron microscope (FEG-SEM) with a Gemini II column (Carl Zeiss AG, Germany). Automated pore size distribution was determined using the image processing Fiji software (Fiji is just ImageJZ version 2.9.0 / 1.53t) from SEM image regions of hydrogel disks made from two different batches (duplicates), as well as the average pore diameter (in nm).

[0076] In one embodiment, in vitro release kinetics was performed. The quantification of the AMP release from the hydrogels was determined using the Pierce Micro BCA™ Protein Assay Kit (Pierce, Rockford, IL, U.S.). For that, a surrogate AMP (cathelicidin antimicrobial peptide LL-37) with similar characteristics, i.e. molecular weight, net charge, and hydrophobicity, to the hBD-2, but with wider availability, was used to assess the in vitro release kinetics of the produced hydrogels. This AMP was purchasedfrom Kaneka Eurogentec S.A., Belgium. Briefly, after swelling in PBS++, the AMP-loaded hydrogels were immersed in sterile water and incubated at 37°C. Supernatants were then collected for subsequent quantification of cumulative AMP release at following time points: 1, 3, 6, 12, 24, and 72 hours, using the Micro BCA Protein Assay Kit.

[0077] Immortalized human keratinocyte (HaCaT) cells were purchased from CLS Cell Lines Service GmbH (Eppelheim, Germany), whereas primary human dermal fibroblasts, adult (HDFa) (AG08469 cells) were purchased from the Coriell Cell Repositories (Camden, NJ, U.S.). In one embodiment, the AG08469 cells (designated normal 8469) were isolated from a 38-year-old healthy male donor. HaCaT and HDFa cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% of fetal bovine serum (FBS) and 1% of Penicillin-Streptomycin and DMEM supplemented with 15% of FBS, 1% of Penicillin-Streptomycin, and 25mM glucose, respectively. The cell cultures were maintained at 37°C in a humidified atmosphere with 5% of CO2.

[0078] In one embodiment, the cell viability was measured by MTT assay. In particular, HaCaT and HDFa cells were seeded at a density of lxio5cells / mL per well in a 24-well plate and allowed to proliferate. Upon reaching 95% confluency, the cells were incubated with free AMPs (1 pg / mL), blank hydrogels or AMP-loaded hydrogels (1 pg / mL) for 24 hours. A sterile gauze was used as the negative control, while the swelling solution (PBS++) was used as the vehicle control. The colorimetric 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was then performed to assess cytotoxicity in these cells. Accordingly, the soluble tetrazolium salt MTT solution (0.5 mg / mL, final concentration) was added into each well following incubation for 1 hour in HaCaT cells or for 3 hours in HDFa cells. Formed formazan crystals were then dissolved by adding 300 pL of acidic isopropanol (0.04 NHCI in isopropanol). Afterwards, 200 pL of solution from each well was transferred to a 96- well plate, and the absorbance was measured at 570 nm, with a 620 nm reference filter, in a microplate reader (Spectramax plus 384, Molecular Devices, CA, U.S.). Each sample was analyzed in duplicate.

[0079] In one embodiment, a scratch assay assessed cellular migration in response to various stimuli. In particular, HaCaT and HDFa cells were seeded at a density of 2xl05cells / mL per well in a 24-well plate and allowed to proliferate. Once the cells reached 95% confluency, a "scratch" was made with a 200 pL pipette tip following a straight line. The remaining debris and media were removed, then the wells were washed with 1 mL of PBS, and 1 mL of DMEM supplemented with 2% of FBS and 1% of Penicillinstreptomycin was added to reduce cell proliferation. The 24-well plate was then incubated for at least 30 minutes before acquiring the first images. The images were obtained, in the same area, with the phase-contrast inverted microscope Carl Zeiss Axio Observer Zl, 10x objective, at 0, 24, and 48 hours after the initial scratch. Immediately after capturing the initial images, the cells were incubated with free AMPs (1 pg / mL), blank hydrogels or AMP-loaded hydrogels (1 pg / mL) up to 48 hours. Cells without any incubation were used as the negative control, whereas the positive control was obtained by using DMEM supplemented with 10% of FBS and 1% of Penicillin-Streptomycin for HaCaT cells and DMEM supplemented with 15% of FBS, 1% of Penicillin-Streptomycin, and 25mM glucose for HDFa cells, instead of DMEM supplemented only with 2% of FBS. The cell migration was calculated as a percentage of the gap closure compared to the initial scratch area, using the image acquisition Zen Blue Pro software (version 2021) and the image processing Fiji software (Fiji is just lmageJ2 version 2.9.0 / 1.53t).

[0080] In one embodiment, thirteen 7 / 8-week-old male C57BL / 6J mice (Charles River Laboratories, Saint Germain Nuelles, France) weighing 25-30 g were housed in certified local facilities. All animals were submitted to a two-week acclimation period to prevent stress-induced responses. The animals were housed at normal RT under a 12-hour light / dark cycle, with water and pellet food ad libitum. All the experimental protocols involving animals were approved by the animal research ethics committee of the Center for Neuroscience and Cell Biology (ORBEA_213_2019 / 28082019) and the Faculty of Medicine of the University of Coimbra and conducted following the European Directive 2010 / 63 / EU and the Portuguese Decree-Law (113 / 2013) for the use of animals in scientific research.

[0081] In one embodiment, type 1 diabetes mellitus was induced by five consecutive low-dose intraperitoneal injections of streptozotocin (STZ) (50 mg / kg body weight) in a saline solution, at daily intervals to reduce p-cell capacity, as previously described.Mice were considered diabetic when blood glucose levels, measured by the Accu- Check Aviva glucometer one week after the last STZ injection, were higher than 250 mg / dL. The animals were maintained diabetic for a period of 6 to 8 weeks before the wound induction procedure to mimic the chronicity of the condition. During this period, blood glucose levels were regularly checked, and insulin (1-2 lU / mL) was administered via IP injection if needed to avoid weight loss. Before the wound induction (around 30 min before), all animals were administered with a subcutaneous injection of buprenorphine (0.1 mg / kg) for mitigation of pain due to posterior surgical procedures. After this time, animals were anesthetized with isoflurane, and the dorsal hair of the mice was shaved and then removed with a depilatory cream. This region was then carefully cleaned with water and 70% ethanol, and a povidone-iodine antiseptic solution was locally applied before the surgical procedure. Afterwards, two 6 mm diameter full-thickness wounds were created with a biopsy punch in each animal. The animals were then divided into three experimental groups according to the different wound treatments: gauze moistened with a saline solution (n=4), blank hydrogels (n=4), and hBD-2 hydrogels (n=5). Treatments were covered with Tegaderm (3M Health Care) and applied on days 0 and 3. Animals were sacrificed at day 10, then the wounded skins were harvested and included in optimal cutting temperature (OCT) gel and frozen in dry ice or incubated in buffered 4% paraformaldehyde (PFA).

[0082] In one embodiment, the progress of wound healing was evaluated by acetate tracing daily and by photography acquisition at days 0, 3, 7 and 10. Automated wound size quantification was performed from the acetate measurements using the image processing Fiji software (Fiji is just lmageJ2 version 2.9.0 / 1.53t).

[0083] In one embodiment, microbial load was evaluated. In particular, sterile gauzes moistened with 40 pL of a saline solution were placed over the animals' unwounded skin (day 0) and each wound (days 3, 7, and 10) for 1 min to collect the associated microbiota. The gauzes were transferred to 2 mL eppendorf tubes with 1 mL of tryptic soy broth (TSB) medium. Before plating, the gauzes were well squeezed to release the microorganisms. Afterwards, 50 pL of the obtained suspensions were plated in petri dishes containing TSB solid medium to allow microbial growth, in an incubator at 37°C,for 10 days. On day 10, the number of colonies were counted, and the microbial load was expressed as the number of colony-forming units (CFUs) / mL of suspension.

[0084] Histopathological analysis was performed. In one embodiment, sections with 5 pm thickness of paraffin-embedded skin samples were cut using a microtome and stained using hematoxylin and eosin (H&E) (Thermo Fisher Scientific, MA, U.S.) or Masson-Goldner's trichrome (MT) staining kits (Carl Roth, Germany), according to manufacturers' protocols. Samples were then observed in the Carl Zeiss Axio Imager Z2 upright widefield microscope, with a 10x objective. The image acquisition process was conducted using the Zen Blue software (version 2012), while the image analysis process was performed using the image processing Fiji software (Fiji is just lmageJ2 version 2.9.0 / 1.53t). Epidermal thickness index (ETI) was calculated as the average thickness of epidermis in wounded skin divided by the average thickness of epidermis in uninjured skin and multiplied by 100, while the epidermal thickness was measured in three different regions of each wound bed and is presented as the average thickness (in pm). Moreover, granulation tissue thickness was measured in three different regions of each wound bed and is presented as the average thickness (in pm), while the granulation tissue thickness index (ETI) was calculated as the average thickness of granulation tissue in wounded skin divided by the average thickness of granulation tissue in uninjured skin and multiplied by 100. Besides, collagen fiber quantification was determined as the percentage of collagen in the wound bed relative to control wound samples. Lastly, a histology scoring system previously established and described by Vyver et al.

[0010] was used. In brief, this scoring system for murine cutaneous wounds evaluates key parameters in each phase of healing to establish an overall histology score, ranging from 0 (open / unhealed wound) to 12 (completely healed wound with no scarring). Those parameters include re-epithelization (none - 0; partially - 1; total - 2), epithelial thickness index (hypoplasia - 0; hypertrophy - 1; normal - 2), keratinization (no - 0; yes - 2), granulation tissue (thin with less than 100 pm - 0; thick with more than 100 pm - 1; presence of intact dermis - 2), remodelling (none - 0; partial with the presence of either collagen deposition or dermal white adipose tissue - 1; complete with the presence of all dermal white adipose tissue, skinappendages, and panniculus carnosus regeneration - 2), and scar elevation index (hypoplasia - 0; hypertrophy - 1; normal - 2).

[0085] In an embodiment, immunohistochemistry was used to analyse the pattern of several inflammatory cytokines (IL-6 and MCP-1) and cells (Ml and M2-like macrophages and T lymphocytes), types of collagen (COL1A1) and other important factors in proliferation (Ki67+ cells), tissue remodelling (MMP-9), and neovascularization (CD31+ cells). Cryosections with 10 pm thickness of skin samples included in OCT gel were cut using a cryostat. The skin cryosections were then fixed in ice-cold acetone and stained for detection of the above described parameters. Samples were examined in the Carl Zeiss LSM 710 confocal microscope, with a 40x objective. The image acquisition process was conducted using the Zen Black software (version 2012), while the image analysis process was performed using the image processing Fiji software (Fiji is just lmageJ2 version 2.9.0 / 1.53t).

[0086] In an embodiment, dihydroethidium (DHE) assay was performed to assess the presence of ROS. In particular, skin samples included in the OCT gel were cut into cryosections with 10 pm thickness using a cryostat. The skin cryosections were stained with the DHE probe solution (IX PBS with 10 pM DHE) to detect reactive oxygen species (ROS) levels. Samples were observed in the Carl Zeiss LSM 710 confocal microscope, with a 40x objective. The image acquisition process was conducted using the Zen Black software (version 2012), while the image analysis process was performed using the image processing Fiji software (Fiji is just lmageJ2 version 2.9.0 / 1.53t). The presence of ROS was determined as the percentage of DHE expression levels relative to the control group.

[0087] In an embodiment, statistical analysis was performed and data were tested for normality using the Shapiro-Wilk test. For the in vitro studies assessing cell viability and migration, one-way ANOVA followed by Holm Sidak's multiple comparisons test was performed as data met parametric assumptions. Specifically relevant to the in vivo studies, the rationale behind the sample size per experimental group was determined based on previous preliminary work and ethical concerns. For in vivo wound closure and microbial load measurements, two-way ANOVA followed by Dunnett's multiple comparisons test was used to determine the effects of treatment and time. Additionalwound-healing parameters were analysed with either one-way ANOVA (with Holm- Sidak's post hoc test) or Kruskal-Wallis (with Dunn's post hoc test) based on data distribution. Data are presented as mean ± SD or mean ± SEM for parametric data and median (interquartile range, QI - Q3) for non-parametric data. Statistical significance was set at p < 0.05 and is reported in the figure legends or main text where feasible and appropriate. Moreover, significance markers are used in the figures to indicate group differences and detailed descriptions of the makers Including p-values are provided in figure legends. All statistical analyses were performed using IBM SPSS version 28 (SPSS Inc., Chicago, IL, U.S.), while plots were generated using GraphPad Prism version 8 (GraphPad Inc., La Jolla, CA, U.S.).

[0088] This work was financed by the European Regional Development Fund (ERDF) through the Centro 2020 Regional Operational Programme under project CENTRO-Ol- 0145-FEDER-000012 (HealthyAging2020) and through the COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation, and Portuguese national funds via FCT - Funda^ao para a Ciencia e a Tecnologia, under projects POCI- 01-0145-FEDER-007440, DL57 / 2016 / CP1448 / CT0024 (E.C.L.), Ph.D. Scholarships 2020.04990.BD (J.D.S.) and 2023.01320.BD (D.C.), contractDOklO.54499 / 2022.08044. CEECIND / CP1724 / CT0004 (A.G.), UIDB / 04539 / 2020,UIDP / 04539 / 2020, and LA / P / 0058 / 2020. In addition, J.D.S. was also funded by the Fulbright Scholarship for Research with the support of FCT - Funda^ao para a Ciencia e a Tecnologia, LP. 2021 / 2022 (21-073) and by the award Bolsa Luis Marques 2021 from SPD - Sociedade Portuguesa de Diabetologia. Moreover, this work was also supported by the American Heart Association, under grant # 19IPLOI34760654 (E.A.S.).

[0089] This work received support and help from the FCT - MCTES, under projects LA / P / 0008 / 2020 DOI 10.54499 / LA / P / 0008 / 2020, UIDP / 50006 / 2020 DOI10.54499 / UIDP / 50006 / 2020 and UIDB / 50006 / 2020 DOI 10.54499 / UIDB / 50006 / 2020 (A.G. and P.G.).

[0090] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and / or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in differentembodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also to be understood that unless otherwise indicated or otherwise evident from the context and / or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

[0091] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof.

[0092] The above-described embodiments are combinable.

[0093] The following claims further set out particular embodiments of the disclosure.

[0094] References[1] Schaper NC, van Netten JJ, Apelqvist J, Bus SA, Hinchliffe RJ, Lipsky BA. IWGDF Practical guidelines on the prevention and management of diabetic foot disease (2019 update). Diabetes Metab Res Rev 2020;36. https: / / doi.org / 10.1002 / dmrr.3266.[2] da Costa JP, Cova M, Ferreira R, Vitorino R. Antimicrobial peptides: an alternative for innovative medicines? Appl Microbiol Biotechnol 2015;99:2023-40. https: / / doi.org / 10.1007 / s00253-015-6375-x.[3] Petkovic M, Vangmouritzen M, Mojsoska B, Jenssen H. Immunomodulatory properties of host defence peptides in skin wound healing. Biomolecules 2021;ll. https: / / doi.org / 10.3390 / biomll070952.[4] Da Silva J, Leal EC, Carvalho E. Bioactive antimicrobial peptides as therapeutic agents for infected diabetic foot ulcers. Biomolecules 2021;11:1-21. https: / / doi.org / 10.3390 / biomlll21894.[5] Gonzalez-Curiel I, Trujillo V, Montoya-Rosales A, Rincon K, Rivas-Calderon B, De Haro-Acosta J, et al. 1,25-dihydroxyvitamin D3 induces LL-37 and HBD-2 production in keratinocytes from diabetic foot ulcers promoting wound healing: An in vitro model. PLoS One 2014;9:1-10. https: / / doi.org / 10.1371 / journal.pone.0111355.[6] Da Silva J, Leal EC, Carvalho E, Silva EA. Innovative Functional Biomaterials as Therapeutic Wound Dressings for Chronic Diabetic Foot Ulcers. Int J Mol Sci 2023;24. https: / / doi.org / 10.3390 / ijms24129900.[7] Barbu A, Neamtu B, Zahan M, lancu GM, Bacila C, Miresan V. Current Trends in Advanced Alginate-Based Wound Dressings for Chronic Wounds. J Pers Med 2021;ll:890. https: / / doi.org / 10.3390 / jpmll090890.[8] Stoica AE, Chircov C, Grumezescu AM. Nanomaterials for Wound Dressings: AnUp-to-Date Overview. Molecules 2020;25:2699. https: / / doi.org / 10.3390 / molecules25112699.[9] Moura LIF, Dias AMA, Leal EC, Carvalho L, de Sousa HC, Carvalho E. Chitosan- based dressings loaded with neurotensin— an efficient strategy to improve early diabetic wound healing. Acta Biomater 2014;10:843-57. https: / / doi.Org / https: / / doi.org / 10.1016 / j.actbio.2013.09.040.

[0010] Vyver M Van De, Boodhoo K, Frazier T, Hamel K, Kopcewicz M, Levi B, et al. Histology Scoring System for Murine Cutaneous Wounds 2021;30:1141-52. https: / / doi.org / 10.1089 / scd.2021.0124.

Claims

C L A I M S1. A hydrogel for tissue regeneration comprising alginate forming a hydrogel matrix and a pharmaceutical amount of antimicrobial peptide, wherein the antimicrobial peptide is entrapped / embedded in the hydrogel matrix.

2. The hydrogel according to the previous claim 1, comprising:2-4 % (w / v) of alginate, preferably 2.5 to 3 % (w / v); and0.1 to 10 pg / mL of the antimicrobial peptide, preferably 0.5 to 2 pg / mL of the antimicrobial peptide.

3. The hydrogel according to any of the previous claims, wherein the antimicrobial peptide is selected from a list consist of: human p-defensin 1, human p-defensin 2, human p-defensin 3, human cathelicidin antimicrobial peptide, and mixtures thereof.

4. The hydrogel according to any of the previous claims, wherein the hydrogel polymer matrix comprises a porous structure with pore diameter ranging from 0.5 nm to 20 nm measured by scanning electron microscopy; preferably ranging from 2 nm to 6 nm.

5. The hydrogel according to any of the previous claims, wherein the alginate hydrogel polymer matrix has an initial storage modulus ranging from 3.58 ± 0.35 kPa to 14.79 ± 1.69 kPa and / or an initial loss modulus ranging from 0.12 ± 0.02 kPa to 1.54 ± 0.24 kPa, measured by rheology.

6. The hydrogel according to any of the previous claims, wherein the alginate hydrogel polymer matrix swelling ratio ranges from 29.37 ± 1.44 to 34.36 ± 3.55.

7. The hydrogel according to any of the previous claims, wherein the alginate hydrogel polymer matrix mesh size ranges from 45 nm to 115 nm, preferably from 50 nm to 108 nm, measured by rheology.

8. The hydrogel according to any of the previous claims, wherein the hydrogel further comprises a crosslinking agent, in particular an ionic crosslinking agent.

9. The hydrogel according to any of the previous claims, wherein the crosslinking agent is selected from a list consisting of calcium sulphate, barium chloride, strontium chloride, magnesium chloride, zinc chloride, aluminium chloride, and ferric chloride.

10. The hydrogel according to any of the previous claims, wherein the crosslinking ratio ranges from 1:3 to 1:1, more preferably is 4:5.

11. The hydrogel according to any of the previous claims, wherein the hydrogel matrix comprises a mixture of alginates with different molecular weights; preferably at least a low molecular weight alginate and a high molecular weight alginate.

12. The hydrogel according to any of the previous claims, wherein the alginate molecular weight ranges from 100-300 kDa; preferably wherein the low molecular weight alginate ranges from 120-150 kDa and the high molecular weight alginate ranges 240-260 kDa.

13. The hydrogel according to any of the previous claims, wherein the weight ratio of low molecular weight alginate and high molecular weight alginate solutions ranges from 1:4-4:1, more preferably 3:4-l:4.

14. The hydrogel according to any of the previous claims, further comprising an active compound; preferably wherein the active compound is selected from a list consisting of: anti-inflammatory, antipyretic, antibiotic, antimicrobial peptide, or mixtures thereof.

15. The hydrogel according to any of the previous claims for use in medicine or veterinary.

16. The hydrogel according to any of the previous claims, for use in wound treatment, wound tissue regeneration and / or wound healing.

17. The hydrogel according to any of the previous claims, for use in the treatment of chronic wounds, more preferably diabetic foot ulcers.

18. An article comprising the hydrogel according to any of the previous claims.

19. The article according to the previous claim, wherein the article is a mesh, a membrane, scaffold, a fiber, a disc, or a patch.

20. The use of product according to any of the previous claims for the manufacture of a medicament for wound treatment, wound tissue regeneration and / or wound healing.

21. The use of product according to the previous claim for the manufacture of a medicament for the treatment of chronic wounds, more preferably diabetic foot ulcers.

22. A method for treating or preventing chronic wounds, more preferably diabetic foot ulcers in a subject, the method comprising administering the hydrogel according to any of the previous claims.

23. A method for obtaining the hydrogel as described in any of the previous claims comprising the following steps: obtaining a low molecular weight alginate solution and high molecular weight alginate solution; preferably in ultrapure water; mixing the low molecular weight and high molecular weight alginate solutions within interconnected syringes; crosslinking with a crosslink agent / gelling agent; adding an antimicrobial peptide, either separately or combined, with a low or high molecular weight alginate solution.

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