Platelet Extracellular Vesicles Loaded Gelatine Hydrogels for Wound Care

Abstract

Platelet extracellular vesicles (pEVs) isolated from clinical-grade human platelet concentrates are attracting attention as a promising agent for wound healing therapies. Although pEVs have shown potential for skin regeneration, their incorporation into wound bandages has remained limitedly explored. Herein, gelatine-based hydrogel (PAH-G) foams for pEVs loading and release are formulated by crosslinking gelatine with poly(allylamine) hydrochloride (PAH) in the presence of glutaraldehyde and sodium bicarbonate. The optimized PAH-G hydrogel foam, PAH_0.24G₃₇, displayed an elastic modulus G' = 8.5 kPa at 37 °C and retained a rubbery state at elevated temperatures. The excellent swelling properties of PAH_0.24G₃₇ allowed to easily absorb pEVs at high concentration (1 × 10¹¹ particles mL^-1). The therapeutic effect of pEVs was evaluated in vivo on a chronic wound rat model. These studies demonstrated full wound closure after 14 days upon treatment with PAH_0.24G₃₇@pEVs. The maintenance of a reduced-inflammatory environment from the onset of treatment promoted a quicker transition to skin remodeling. Promotion of follicle activation and angiogenesis as well as M1-M2 macrophage modulation are evidenced. Altogether, the multifunctional properties of PAH_0.24G₃₇@pEVs addressed the complex challenges associated with chronic diabetic wounds, representing a significant advance toward personalized treatment regimens for these conditions.

Keywords: chronic wounds; hydrogel foam; platelet extracellular vesicles; skin regeneration; wound model.

Figure 1

Human platelet‐derived extracellular vesicles (pEVs) loaded into gelatine‐based hydrogel foams for improved wound healing. Gelatine‐based hydrogel foams are formed by crosslinking gelatine (G) with poly(allylamine hydrochloride) (PAH) using glutaraldehyde in the presence of sodium bicarbonate(NaHCO₃). The PAH_0.24G₃₇ hydrogel foams – with a composition of 37% w/v gelatine, 0.24% w/v PAH, 26.47 mm glutaraldehyde, 89 mm NaHCO₃ – are loaded with pEVs (1 × 10¹¹ particle mL⁻¹) and applied to wounds in an in vivo diabetic rat model.

Figure 2

Physico‐chemical characterization of different PAH‐G hydrogel foams. (A) Storage (G’, circles) and loss moduli (G”, open circles) of PAH_xG₃₇ (x = 0.12, 0.24, 0.36) hydrogel foams composed of 37% w/v gelatine and different PAH contents: 0.12% w/v (black), 0.24% w/v (red) and 0.36% w/v (grey) in the presence of glutaraldehyde (26.47 mm) and NaHCO₃ (89 mMM). Experimental details: 1 Hz, 1% strain, 1.5 mm gap, 1 °C min⁻¹ after 3.5 h immersion in water. The dotted line at 37 °C. (B) Tan delta (G”/G’) plot as a function of temperature extracted from Figure 2A. (C) Images of PAH_xG₃₇ (x = 0.12, 0.24, 0.36) hydrogel foams before and after temperature sweep to 70 °C, (2.9 cm in diameter, 2–3 mm thick).

Figure 3

Gelatine‐based hydrogel foams. (A) Pore size distribution (gaussian fit) of PAH_0.12G₃₇ (black), PAH_0.24G₃₇ (red), and PAH_0.36G₃₇ (grey); insets: corresponding morphologies; The minimum number of pores considered is 50 with n = 3 replicates. (B) Swelling behavior of PAH_0.24G₃₇ in foam and non‐foam forms. Data are represented as mean ± SD, with n = 3 replicates. (C) Stability of PAH_0.24G₃₇ in foam (red square) and non‐foam (circles) states upon water immersion. Data are represented as mean ± SD, with n = 3 replicates.

Figure 4

In vitro biocompatibility of gelatine and PAH_xG₃₇ hydrogel foams. (A) Metabolic activity after 24 h incubation of the pure extracts from hydrogel foams (163 ± 17 mg) with HDFCs‐adGFP (1 × 10⁴ cells/well) using the resazurin fluorescence‐based assay. Data are represented as mean ± SD, with n = 3 replicates, *p ≤ 0.05. (B) Dead staining images of fibroblasts after incubation for 24 h with PAH_xG₃₇ extracts and positive control using Triton^TM X‐100 at 0.25% v/v. The scale bar is 1000 µm. Dead or damaged cells stained using propidium iodide (orange) and GFP‐expressing HDFCs (green).

Figure 5

Loading/release of pEVs (donor 1) into/from PAH_0.24G₃₇ hydrogel foams. (A) concentration of pEVs as determined by NTA in the first ten fractions collected after column purification. Data are represented as mean ± SD, with n = 3 replicates. (B) Total protein concentration in the different fractions using bicinchoninic acid protein assay. Data are represented as mean ± SD, with n = 3 replicates. (C) pEVs size distribution of fraction 5 (donor 1, 3 different purifications). (D) pEVs release from PAH_0.24G₃₇ hydrogel foams loaded with (6.4 ± 0.27) × 10¹⁰ pEVs mL⁻¹ at 37 °C. Data are represented as mean ± SD, with n = 2 replicates.

Figure 6

Influence of pEVs concentration on wound confluence during 30 h treatment of HDFCs‐adGFP. (A) Images of HDCFs‐adGFP cells at 3‐time intervals of treatment (0, 15, and 30 h), treated with medium, full medium as well as two different pEVs concentrations; images were taken with 2.5× (2.75× eff.) objective and GFP filter cube. (B) Wound confluence during 30 h of treatment of HDFCs‐adGFP with medium (blue), full medium (red), and five different pEV concentrations. Data are represented as mean, with n = 4 replicates. (C) Influence of pEV concentration on maximum wound healing rate. Values represent the mean ± SD (n = 4). Ordinary one‐way ANOVA with Dunnett's multiple comparisons test was used to analyze the results. Statistically significant differences were considered for *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.

Figure 7

Comparative analysis of wound healing efficiency in diabetic rats. (A) Dynamic wound healing areas as calculated using ImageJ software from wounds (8 mm, sterile biopsy punch, on the back) treated with PBS (negative control), pEVs (1 × 10¹¹ particles mL⁻¹), PAH_0.24G₃₇ and PAH_0.24G₃₇@pEVs (loaded with 1 × 10¹¹ particles mL⁻¹). (B) Effectiveness of different treatments on wound healing in diabetic rats over a 14‐day period validated by the wound area (%) over time. For PBS (negative control, blue), pEVs (1 × 10¹¹ particles mL⁻¹, red), PAH_0.24G₃₇ (green) and PAH_0.24G₃₇@pEVs (loaded with 1 × 10¹¹ particles mL⁻¹, violet). For the treatment groups, the samples were topically applied to the diabetic wounds on days 0 and 7. Photographic data were collected after cleaning the wounds and before administering the formulation. All in vivo experimental procedures were independently replicated six times (n = 6), and the results are presented as the mean ± standard deviation (SD). Statistical analyses were conducted using GraphPad Prism software. Two‐way ANOVA (Tukey's multiple comparison test) was employed to assess statistical significance, with significance levels denoted as follows: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.

Figure 8

Histological analysis. (A) Haematoxylin and Eosin stained sections of skin tissues and (B) major organs from diabetic rats treated with different formulations. PBS (negative control), pEVs (1 × 10¹¹ particles mL⁻¹), PAH_0.24G₃₇ and PAH_0.24G₃₇@pEVs (1 × 10¹¹ particles mL⁻¹). The images show no visible signs of tissue damage or inflammation.

Figure 9

Microscopic evaluation of inflammatory response. Images of (A) Amplex red assay and (B) IL‐6 immunofluorescence staining assay showing reactive oxygen species (ROS) levels in diabetic wounds treated with PBS, pEVs, PAH_0.24G₃₇, and PAH_0.24G₃₇@pEVs. All in vivo experimental procedures were independently replicated six times (n = 6). Results are presented as the mean ± standard deviation (SD). One‐way ANOVA (Tukey's multiple comparison test) was employed to assess statistical significance, with significance levels denoted as follows: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. The number of ROS species (Amplex red) and the inflammatory response (IL‐6) is minimized by the application of the pEVs‐doped hydrogel.

Figure 10

PAH_0.24G₃₇@pEVs impact on macrophage polarization. Immunofluorescence staining of PBS, pEVs, PAH_0.24G₃₇, and PAH_0.24G₃₇@pEVs treated wounds for (A) CD86 to assess M1 macrophage polarization and (B) CD206 for M2 macrophage assessment. All in vivo experimental procedures were independently replicated six times (n = 6), and the results are presented as the mean ± standard deviation (SD). The one‐way ANOVA (Tukey's multiple comparison test) was employed to assess statistical significance, with significance levels denoted as follows: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. The images reveal a greater presence of anti‐inflammatory macrophages (M2) than inflammatory macrophages (M1) when using the pEVs‐doped hydrogel.

Figure 11

PAH_0.24G₃₇@pEVs enhanced vascular formation and follicle activation. Immunofluorescence staining (A) CD31 and (B) CD34 on wounds treated with PBS, pEVs, PAH_0.24G₃₇, and PAH_0.24G₃₇@pEVs. All in vivo experimental procedures were independently replicated six times (n = 6), and the results are presented as the mean ± standard deviation (SD). One‐way ANOVA (Tukey's multiple comparison test) was employed to assess statistical significance, with significance levels denoted as follows: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. The images reveal the more marked presence of endothelial cells (CD31) and hematopoietic stem cells and vascular progenitor cells (CD34) when using the pEVs‐doped hydrogel.