Antibacterial and Cellular Response Toward a Gasotransmitter-Based Hybrid Wound Dressing

Abstract

Biological processes such as infection, angiogenesis, and fibroblast proliferation and migration need to be regulated for effective healing of a wound. Failing to do so can delay the overall wound healing and add to the suffering and healthcare cost. Endogenous nitric oxide (NO) is a well-known gasotransmitter in the natural healing process in humans and other mammals. To utilize its inherent ability in the current study, an exogenous NO donor (S-nitroso-glutathione, GSNO) was integrated into a hybrid formulation consisting of a natural polymer (alginate) and a synthetic polymer (poly(vinyl alcohol) (PVA)). The alginate–PVA–GSNO dressings showed a sustained NO release for 72 h that resulted in 99.89 ± 0.40% and 98.93 ± 0.69% eradication of Staphylococcus aureus and Pseudomonas aeruginosa, respectively, which are among the most common causal agents of wound infections. The designed dressings resulted in a 3-fold increase in the proliferation of human endothelial cells when compared with control without GSNO showing its angiogenic potential. In addition, mouse fibroblast cells exposed to leachates from alginate–PVA–GSNO dressings showed significantly higher proliferation when compared to control alginate–PVA showing the NO release from exogenous GSNO in fibroblast proliferation. Fibroblast migration was shown to be much faster with GSNO-based dressings when compared to corresponding control dressings resulting in complete closure of an in vitro wound model within 48 h. The porous dressings also possessed important physical properties such as swelling, water vapor transmission, and moisture content that are desirable for effective wound healing. Overall, this study supports the possibility of using therapeutic alginate–PVA–GSNO dressing to provide a supportive environment for accelerated wound healing.

Graphical Abstract

1. INTRODUCTION

Wounding leads to exposure of subcutaneous tissue that provides a nutritious environment conducive to microbial colonization and proliferation. The presence of bacteria at the wound site downregulates the host immune response, ultimately delaying the natural wound healing process. Inappropriate progression of the healing process in chronic wounds can often make the total healing time unpredictable.^1,2 It can also result in life-threatening complications like pressure sores and diabetic foot ulcers, which can lead to amputation of the affected limb.³ Skin infections contribute to 200 million visits to physicians costing over $350 million annually.⁴ Therefore, controlling infection at the wound site is considered one of the critical parameters in wound healing applications. Antibiotics and silver-based antimicrobial agents that are popularly used as topical ointments for wounded surface are limited by several factors. The bacterial biofilms are protected by extracellular polysaccharide matrix leading to bacterial resistance against antibiotics. This also perpetuates the inflammatory phase of wound healing.⁵ On the other hand, silver nanoparticles have shown to be genotoxic and cytotoxic to host cells.^6,7 Moreover, these antimicrobial agents only target wound infection while other important host cell responses such as angiogenesis and fibroblast proliferation and migration are left to their natural fates. Besides infection, a major challenge during the wound healing process is to rejuvenate the ruptured blood vessels in the wounded tissue (angiogenesis) which is important for the transfer of chemokines, nutrients, and oxygen.⁸ In addition, fibroblast cell proliferation and migration are required for ultimate collagen matrix formation and scarring of the tissue.⁹ Given the complexity of the wound healing process, it is obvious that a holistic approach is needed to target different biological responses desirable for faster wound healing while simultaneously preventing antibiotic resistance and cytotoxicity to host cells.

In the last two decades, nitric oxide (NO) has emerged as a critical player in all four phases of natural wound healing namely hemostasis, inflammation, fibroblast proliferation, and tissue remodeling.¹⁰ Nitric oxide can act against a wide variety of microorganisms: Gram-positive and -negative bacteria, fungus, yeast, and viruses.^11-14 The gaseous nature of NO allows penetration through the matrix in the biofilm, which gives it an extra advantage over antibiotics and silver-based antibacterial strategies. Because of its rapid action, short-half-life, and nonspecific action it does not promote antibacterial resistance. Furthermore, NO is more than just an antibacterial agent due to its proven role in angiogenesis, vasodilation, cell proliferation, inflammation, collagen synthesis, and tissue remodeling.^10,15-18 Thus, mimicking the endogenous NO release by utilizing donor molecules such as S-nitroso-glutathione (GSNO) and integrating it in a polymer system to make the wound dressing can offer great potential in faster healing. S-Nitroso-glutathione is a well-known endogenously produced S-nitrosothiol in humans. Decomposition of GSNO via thermal stimuli, moisture, and metal ions can lead to NO release and the corresponding biological effects. Endogenous GSNO functions range from preventing embolization within the vasculature to modulating angiogenesis by promoting vascular endothelial growth factor (VEGF) production within damaged tissue.^19,20 Along with these functions, GSNO has also been shown to be involved in collagen deposition in cutaneous wound repair, further demonstrating its abilities in wound healing.^21,22

In this work, we have integrated GSNO in a hybrid formulation of alginate and poly(vinyl alcohol) (PVA) and studied the wound healing potential of alginate–PVA–GSNO dressings. Sodium alginate (NaAlg) provides an ideal matrix material for wound dressing applications.^23-25 It is an inexpensive biopolymer that has widely been used in biomedical applications due to its high hydrophilicity and biocompatibility.^26,27 The bioadhesive and biodegradable behavior of alginate films in addition to maintaining a moist environment are important characteristics of an effective wound dressing.^28,29 Unfortunately, natural polymers including alginate, despite possessing great wound healing potential, undergo rapid in vivo degradation by proteases. This is due to poor mechanical strength, which makes it difficult to prolong the diffusion of an encapsulated therapeutic agent.^1,2 In addition, owing to its biocompatibility with tissue and plasma proteins, PVA is a potential candidate for wound healing applications.³⁰ These NO releasing dressing resulted in a 9-fold increase in dermal blood flow via irradiation as a result of NO release. In another study, GSNO was incorporated in polymeric blend of PVA with poly(vinylpyrrolidone (PVP) leading to sustained NO release due to thermal stabalization.³¹ Thus, the hybrid combination of PVA-alginate helps overcome the limitation possessed by natural or synthetic polymer individually.^23,32

The alginate–PVA–GSNO dressings were processed via lyophilization resulting in a porous morphology. This study discusses the fabrication and characterization of NO-releasing alginate–PVA–GSNO dressings and validates the antibacterial, angiogenic, and proliferative and migratory attributes.

2. EXPERIMENT SECTION

2.1. Materials.

Sodium salts of alginic acid, calcium chloride, ethylenediaminetetraacetic acid (EDTA), and sodium chloride were obtained from Sigma-Aldrich (St. Louis, MO). PVA (88% hydrolyzed, M.W. approximately 13 000–23 000) was bought from Acros Organics (New Jersey) and glycerol was bought from Fischer Chemicals (Fair Lawn, NJ). LB broth and LB Agar were obtained from Fisher Bioreagents (Fair Lawn, NJ). Dulbecco’s Modification of Eagle’s medium (DMEM) and trypsin-EDTA were purchased from Corning (Manassas, VA). The Cell Counting Kit-8 (CCK-8) was obtained from Sigma-Aldrich (St Louis, MO). The antibiotic penicillin-streptomycin (Pen-Strep) and fetal bovine serum (FBS) were purchased from Gibco-Life Technologies (Grand Island, NY 14072). l-Glutathione (reduced 98+ %) was purchased from Alfa Aesar (Ward Hill, MA). The bacterial strains Pseudomonas aeruginosa (ATCC 27853) and Staphylococcus aureus (ATCC 5538) as well as the mouse fibroblast cell line (ATCC 1658) were originally obtained from American Tissue Culture Collection (ATCC). Autoclaved phosphate buffered saline (PBS) was used for all in vitro experiments. The Culture-insert 2-well in μ-Dish (35 mm, high) used for cell migration experiment was purchased from ibidi (Martinsried, Germany).

2.2. Material Preparation.

2.2.1. Synthesis of GSNO.

Synthesis of GSNO was performed by modifying a standard protocol.³³ Reduced glutathione (900 mg, 2.93 mmol) was first dissolved in 4 mL of DI water and 1.25 mL of 2 M HCl. The solution was allowed to chill in ice for 10 min before being nitrosated with an equimolar amount of sodium nitrite. The solution was then covered and allowed to cool in an ice bath further for 30 more minutes. Chilled acetone (5 mL) was then slowly added to the solution and allowed to stir for an additional 10 min while still in the ice bath. The GSNO precipitate that formed was then collected by vacuum filtration and further washed with cold acetone and water. The resulting washed product was then allowed to dry under vacuum overnight before being collected and stored in the freezer. The purity of each GSNO batch was tested via NOA peak integration.

2.2.2. Engineering NO-Releasing Wound Dressings. Formulation of the Alginate–PVA Hybrid.

The basic polymeric composite for wound dressings were formulated by a solvent casting method. Sodium alginate (1 g) was slowly added to a conical flask containing 20 mL of deionized water at 40 °C and allowed to dissolve for 2 h. A magnetic stirrer was used to formulate a polymeric dispersion of appropriate consistency. As recommended by existing literature, 0.2 g of PVA was dissolved in 19.6 mL of deionized water, which was simultaneously stirred using a magnetic stirrer and heated to a temperature of 90 °C.³⁴ The temperature of the PVA solution was brought down to around 45–50 °C before adding it to the alginate solution. Both polymeric solutions were blended together for 10 min to obtain a uniform polymeric dispersion. To the alginate–PVA mixture, 0.4 mL of glycerol was added. Finally, it produced a solvent mixture of 40 mL (total) with 2.5% (w/v) sodium alginate, 0.5% (w/v) PVA. One % (v/v) glycerol was added to it as a plasticizer. The resulting formulation was cast into Petri dishes and was kept in the freezer for 3 h.

Cross-Linking of the Wound Dressings and Incorporation of NO Donor.

A 2% (w/v) calcium chloride (CaCl₂) was prepared by dissolving 0.4 g of CaCl₂ in 20 mL of deionized water. In parallel, 30 mg/mL GSNO solution was prepared by using water as a solvent. Finally, CaCl₂ and GSNO were mixed in a 1:1 ratio by adding 5 mL of 2% CaCl₂ and 5 mL of GSNO (30 mg/mL) using a vortex mixer to obtain a uniform solution. The final GSNO concentration was 15 mg/mL in the cross-linking solution. The frozen alginate–PVA polymeric dispersions were submerged in the CaCl₂-GSNO solution and allowed to cross-link for 20 h. For the control wound dressings, the cross-linking was carried out using 2% CaCl₂ but without GSNO.

Lyophilization.

After 20 h of cross-linking, the cross-linked films were lyophilized for 7 h at −80 °C and <1.5 mbar pressure in a Labconco freeze-dryer to create a porous matrix. The alginate–PVA–GSNO dressings were stored in the freezer (−15 °C to 30 °C) before being used in the experiment to avoid any heat stimulated NO release. Figure 1 shows the control (alginate–PVA) and NO-releasing (alginate–PVA–GSNO) dressings.

Figure 1.

Images of control alginate–PVA dressing (A) and NO releasing alginate–PVA–GSNO dressing (B). The shape of the dressings is flexible; a round composite is shown only for representation.

2.3. Material Characterization.

2.3.1. Chemiluminescence-Based NO Release Measurement.

A chemiluminescence-based method was used to measure the NO released from alginate–PVA–GSNO using a Sievers chemiluminescence nitric oxide analyzer (NOA) 280i (Boulder, CO).³⁵ The NOA has the capability to selectively map NO via the reaction of NO with ozone, thereby reducing intervention from other molecules.^36,37 As recommended by a previously published report, small circular films (diameter of a 5/16th inch) were punched out and wrapped in Kimwipes (KIMTECH) to mimic a semimoist wound environment prior to using them for NO flux analysis.³⁸ The samples were dipped in PBS (pH 7.4) containing EDTA and placed at the bottom of the sample holder. The released NO was continuously purged from the sample and swept from the headspace using nitrogen into the chemiluminescence detection chamber. The films were tested for NO release at different time points: 0, 28, 48, and 72 h. The NO profile was measured at each time point until a stable plateau was recorded, and then stored at 37 °C until the next time point and soaked again in wet Kimwipes before NO release analysis to keep the study condition uniform.

2.3.2. Water Permeability, Moisture Content, and Swelling Index. Water Permeability.

The water permeability of the wound dressings was determined using a standard protocol.¹¹ The wound dressings with a diameter of 2 cm were wrapped around the opening of a 10 mL glass vial after filling them with 5 g of dehydrated silica. The weight of the vials (n = 3 each) with wound dressings were reported and then placed in a humid environment established with saturated sodium salt (75% relative humidity, 22 ± 2 °C). The weight of the vials was measured every 24 h over 7 days. A linear curve was plotted between gained weight (dw) and time (dØt) in order to calculate the water vapor permeability K (kg m m⁻² day⁻¹ Pa⁻¹) of the wound dressing, given

$$K=\frac{\frac{\mathrm{d}w}{\mathrm{d}\varnothing t}}{\text{Ap}\times P}$$ (1)

dw = weight gain due to moisture retention (kg), dØt = time point (day), dw/dØt = slope between weight gain and time point (day), Ap is the surface area of the dressing (m²), P is the saturation vapor pressure of water at 22 °C.

Moisture Content.

The moisture content (MC%) was determined by using a recommended protocol.³⁹ After measuring, the thickness and the surface area, the samples’ weights were recorded. Thereafter these samples were dried in a vacuum oven for 24 h at 105 °C and dry weight was recorded. The MC% was calculated by comparing the weights of dressings before and after drying using the following formula

$$\text{MC}\%=\frac{\text{Weight before drying}-\text{Weight after drying}}{\text{Weight before drying}}\times 100$$ (2)

Swelling Index (SI).

To measure the swelling ratio (SR) of the wound dressings, a recommended protocol was slightly modified.³² Samples of dimension 2 × 2 cm² size (n = 3) were weighed and dried in a vacuum oven at 105 °C for an hour. The weight of both control alginate–PVA and NO-releasing alginate–PVA–GSNO were taken again after drying. Thereafter, the samples were swelled in 0.1 M phosphate buffer saline (PBS, pH 7.4) at room temperature. After soaking for an hour, the weight of the dressing was measured again. The SI was calculated as follows

$$\text{SI}(\%)=\frac{\text{Weight after soaking}-\text{Weight after drying}}{\text{Weight after soaking}}\times 100$$ (3)

2.3.3. Morphology of the Dressing Surface and Porosity Analysis. Scanning Electron Microscopy.

Scanning electron microscopy (SEM) is a useful tool to understand the surface characteristics of a polymer. In the present study, microstructure and surface morphology of the wound dressing (before and after NO donor incorporation) were examined using SEM (FEI Inspect F FEG-SEM).³⁵ A total of three samples of each of the control (without GSNO) and alginate–PVA–GSNO were sputter coated with gold–palladium (10 nm) using a Sputter Coater (Leica EM ACE200) after mounting them on a metal stub. An accelerating voltage of 5 kV was used to capture SEM images of the sample at 100× magnification.

Porosity Measurement.

Pore size was determined using ImageJ software from images taken with light microscopy (Thermo Fisher scientific EVOS XL Cell Imaging System). At least 30 pores were used to determine average measurements of the pore diameter of each sample.

2.4. Biological Characterization.

2.4.1. Eradication of Adhered Gram-Positive and Gram-Negative Bacteria.

The designed NO-releasing alginate–PVA–GSNO dressing samples were examined for their antibacterial efficacy against common bacteria responsible for skin infections: Gram-positive Staphylococcus aureus (S. aureus) and Gram-negative Pseudomonas aeruginosa (P. aeruginosa). A modified version of the standard bacterial adhesion test was used to quantify the inhibition of colony forming units per surface area of the dressing (CFU/cm²).^35,37,40,41

Preparation of Bacterial Suspension.

A single isolated colony of bacteria was picked from the precultured LB-agar plate and inoculated into 10 mL of Luria Broth (LB) medium in a 50 mL Eppendorf tube and allowed to incubate at 37 °C for 14 h at a radial shaking speed of 120 rpm. After 14 h, the optical density of the bacteria was measured at 600 nm (OD₆₀₀) using UV–vis spectrophotometer (Thermo Scientific Genesys 10S UV–vis). After this step, bacterial cells were separated from the LB medium by centrifuging the bacterial culture at 2500 rpm for 8 min, the supernatant was discarded and fresh sterile phosphate buffer saline (PBS, pH 7.4) was added. The same procedure was repeated to wash off any remaining traces of LB. This step was repeated twice, and the bacterial cells were ultimately suspended in PBS (without any residual traces of LB medium) to be used in the experiment further. The removal of traces of nutrient medium (LB) and suspension of bacterial strains in the PBS assures that the bacteria would not grow back after being killed by the antibacterial agent and thus allowed a fair comparison between the control (without GSNO) and alginate–PVA–GSNO wound dressings.

Bacterial Inhibition and Its Quantification.

Prior to exposing the wound dressing to the bacterial suspension, the OD₆₀₀ was measured again and diluted with PBS to achieve 10⁸–10¹⁰ CFU/mL using a standard curve, which is representative for infected chronic wounds. Triplicate (n = 3) samples (diameter = 2.4 cm) of both the alginate–PVA–GSNO wound dressing and alginate–PVA (control) were exposed for 24 h to 5 mL of bacterial suspension in a 50 mL tube incubated at 37 °C and 120 rpm. After bacterial exposure, the wound dressings were removed from the bacterial suspension and any unbound or loosely bound bacteria were washed off by rinsing the dressings with 2 mL PBS using a pipet. The dressings with the adhered bacteria were then transferred to 2 mL of fresh PBS and homogenized for 30 s using a vortex mixer in order to detach the bound bacteria into the PBS solution. The resulting bacterial suspension was serially diluted (10⁻¹ to 10⁻⁵) using PBS, plated in premade LB agar Petri dishes (LB agar concentration 40 g/L) and incubated for 20 h at 37 °C. After 24 h, CFUs appeared on the LB agar plate. The CFUs were counted while adjusting the dilution factor for the amount of bacterial suspension. The number of CFU per weight (CFU/mg) of the wound dressings were obtained for both alginate–PVA–GSNO wound dressings and control dressings. Percent bacterial inhibition was calculated relative to the control using the following eq 3

$$\%\text{Bacterial inhibition}=\frac{\left(\frac{\text{CFU}}{\text{mg}}\text{in control dressings}-\frac{\text{CFU}}{\text{mg}}\text{in test (NO) dressings}\right)\times 100}{\frac{\text{CFU}}{\text{mg}}\text{in control dressings}}$$ (4)

2.4.2. Zone of Inhibition (ZOI) Study Using the Agar Diffusion Method.

The ability of the alginate–PVA–GSNO dressings to inhibit bacterial growth beyond the direct point of contact was tested via standard agar diffusion method.^11,40 As proof of concept, S. aureus was used. The strain culture was spread uniformly and aseptically on premade LB agar petridishes. Circular wound dressing disks (diameter: 2.7 cm) of control alginate–PVA and alginate–PVA–GSNO were placed and gently pressed on top of the bacterial culture. The petridishes were then placed in an incubator at 37 °C for 20 h to allow the formation of a ZOI.

2.4.3. Cell Culture.

Mouse fibroblast cells and human umbilical vein endothelial cells (HUVEC) were maintained in a tissue culture grade T-flask. More specifically, fibroblast cells were maintained in Dulbecco Modification of Eagle’s Medium (DMEM with 4.5 g/L glutamine, 10% fetal bovine serum, and 1% pen-strep antibiotics (10,000 units/mL)) and HUVEC cells were maintained in F-12 K medium with ATCC growth supplements kit. Both types of cell were incubated in humidified incubator with 5% CO₂ at 37 °C. The medium was replaced every alternate day until cells were 80% confluent. Thereafter, the cells were detached from the T-flask surface by enzymatically degrading their extracellular matrix layer by treating them with 0.18% trypsin and 5 mM EDTA for 5 min.

2.4.4. Human Endothelial Cell Proliferation.

Angiogenesis facilitated by endothelial cell proliferation plays a vital role in the delivery of nutrients, oxygen and important blood factors at the wound site. HUVEC cells are widely regarded as the in vitro model cells for angiogenesis.⁴² We investigated if GSNO-containing wound dressings can enhance endothelial cell proliferation thus suggesting its contribution to angiogenesis. 10,000 HUVEC cells/mL were seeded in each of the wells of a 96 well plate and allowed to be attached to the plate surface by incubating in 5% CO₂, 37 °C for 24 h. Meanwhile, a sample of wound dressing weighing 10 mg was added to 10 mL F-12K medium for 24 h to collect the dressings released out of the dressing. The F-12 K medium in the 96-well plate was replaced by leachate solution exposing the cells for the next 24 h. Thereafter, 10 μL of CCK-8 dye solution was added and allowed to react with NADH released by viable cells for 4 h leading to the formation of orange color formazan absorbed at 450 nm. A relative increase in cell number was measured when comparing the formazan absorbance of cells exposed to the NO-releasing (test) and non-NO releasing samples (control)

$$\%\text{Cell Viability}=\frac{\text{Absorbance of the test NO dressings}}{\text{Absorbance of the control dressings}}\times 100$$ (5)

2.4.5. Fibroblast Cell Proliferation and Migration.

The effect of leach outs from the wound dressing were tested against fibroblast cell proliferation in DMEM medium. The study was carried out for 24 h in 5% CO₂, 37 °C. Percent cell viability was measured relative to the NO-releasing (test) and non-NO releasing samples (control) as previously described.

The cell migration assay allowed an in vitro evaluation of the wound healing potential of the alginate–PVA–GSNO dressings. A cell migration kit that consists of a disk containing culture inserts (2-well in μ-Dish 35 mm high (ibidi GmbH)) was used for the assay. The cell culture inserts were filled with a culture containing 5000 cells/ml and incubated in 5% CO₂, 37 °C for 24 h for cells to attach and grow in the dish. After 24 h, the medium and cell culture inserts were removed, leaving a linear cell-free zone separating the cells growing on either side. Two milliliters of leachates was added to the disk. As the cell migrated to the cell-free zone, optical images were taken at different time intervals between 0 to 36 h to trace the progress of cell migration.

2.5. Statistical Significance.

All quantitative studies herein are reported as mean ± standard error with n ≥ 3 samples using standard two-tailed t-tests and comparison p-values of <0.05 unless otherwise mentioned.

3. RESULTS AND DISCUSSION

3.1. NO Release Kinetics.

S-nitrosothiols (RSNOs) such as GSNOs are capable of passively releasing NO from heat, moisture, and catalytically in the presence of certain metal ions.^41,43 It is generally assumed that RSNOs decompose by homolytic cleavage of the S─N bond. This process generates a thiyl radical (RS·) and nitric oxide (NO·) gas (Figure 2). The product of RSNO homolytic cleavage, that is, NO, is one of the key players in wound repair. Thus, providing exogenous NO release from the alginate–PVA–GSNO wound dressing will be crucial in achieving faster wound healing. In this study, NO release from alginate–PVA–GSNO dressing was tested via a chemiluminescence nitric oxide analyzer (NOA) over the testing periods: 0, 24, 48, and 72 h at 37 °C. A real-time NO release profile shown in Figure 3A is a representation until plateau formation and the NO release profile over a 72 h period is presented in Figure 3B. An initial NO release of 5.01 ± 0.49 × 10⁻¹¹ mol mg⁻¹ min⁻¹ was achieved in the first few minutes that gradually reduced to 0.54 ± 0.08 × 10⁻¹¹ mol mg⁻¹ min⁻¹ over a 72 h period. The decrease in the NO flux over time could be attributed to homolytic degradation of GSNO in the presence of moisture/heat.

Figure 2.

Illustration of homolytic decomposition of RSNOs via cleavage of the S─N bond. This process generates a thiyl radical (RS·) and nitric oxide (NO·) gas.

Figure 3.

Real-time NO-release profile from the alginate–PVA–GSNO dressings as observed via chemilumensecence-based NOA (A). The NO release from alginate–PVA–GSNO wound dressings for a 72 h period (B). Statistical data is expressed as mean ± standard error of the mean of n = 3 samples.

The ability of these wound dressings to sustain NO release over a 3 day period would also eliminate the need for redressing thus not only reducing the discomfort to the patient but also cutting down the associated wound-care cost. Similar to endogenous NO, it was expected that the NO flux achieved in this study will also positively affect the important steps in wound healing process such as infection, fibroblast proliferation, cell migration, and angiogenesis.¹⁰ To validate these assumptions, microbial, and cell culture studies were carried out as discussed below.

3.2. Surface Morphology and Pore Size Analysis.

Surface electron microscopy was used to measure the morphology of the alginate–PVA–GSNO wound dressings relative to the controls. As shown in Figure 4, the surface of the dressing material was not altered in the presence of GSNO, confirming that GSNO has no negative effect on the porous surface morphology. The porous structure in the wound dressing offers a great advantage in terms of water vapor transmission as well as a gaseous exchange from the wound bed.

Figure 4.

Surface electron microscopy images of dressing samples taken at 300× magnitude alginate–PVA dressing (A) and alginate–PVA–GSNO dressing (B).

Both the alginate–PVA–GSNO dressing and the control (alginate–PVA) exhibited macroporous characteristics. The pore sizes for both films ranged from 400 to 1600 pm. The average pore size of the GSNO dressing was approximately 860 μm and the average pore size of the control dressing was approximately 900 μm. The nonhomogenous, larger pore sizes are comparable to commercial wound dressings such as Cellosorb Adhesive (Urgo Medical Co).⁴⁴ When applied on a wounded surface, it can be expected that the porous structure would result in preventing building up of anaerobic condition on the injured wound while simultaneously cleaning the wound via exudate absorption. The subsequent NO release would eradicate bacteria proliferation and biofilm formation.

3.3. Thickness, Water Vapor Permeability, Moisture Content, and Swelling Index.

The physical properties like thickness, moisture content (MC%), water permeability, and swelling index (SI%) are important parameters that govern the healing potential of a wound dressing. The thickness of the film was measured with a Digimatic Micrometer (Mitutoyo, Japan) and was found to be 0.29 ± 0.001 mm for control and 0.30 ± 0.006 mm for the films with GSNO. Thereafter, water permeability, MC%, and SI% were calculated (n = 3) and results are presented in Table 1. The water permeability of the alginate–PVA–GSNO was found to be lower than that of the control films (without GSNO). In the past, we have shown that incorporating NO donors into natural polymers tends to decrease their water permeability due to closer networking between them.¹¹ The MC% from the total weight of the wound dressings was calculated based on the differences in weight before and after drying the wound dressing for 24 h at 105 °C. The MC% for the alginate–PVA–GSNO dressings (33.06 ± 2.1) was found to be higher as compared to the control films (23.9 ± 3.7). This is in line with the water permeability (K value) results, suggesting that a decrease in water permeability due to the presence of GSNO helped them retain more moisture and hence a higher MC%. The swelling index (SI) studies were done for 24 h using PBS (pH 7.0) and the difference in weight of the wound dressings was compared before and after soaking in PBS. The SI of the alginate–PVA–GSNO was higher (64.55 ± 2.26%) than control films (48.94 ± 3.56%), further strengthening the conclusion that the presence of GSNO resulted in better retention of the moisture in the NO-releasing films compared to the control. This solvent retention might also be possible due to the hydrophilic nature of GSNO. A similar trend resulting in an increase in swelling index after adding antibiotics (not GSNO) in the alginate–PVA wound dressing has also been observed in another report.³²

Table 1.

Physical Characterization of the Wound Dressing in Terms of Thickness, Vapor Permeability, Moisture Content, and Swelling Index

characterization	control (alginate– PVA)	alginate–PVA– GSNO
thickness (mm)	0.29 ± 0.001	0.30 ± 0.006
water permeability (kg mm−2 day−1 Pa−1)	3.85 × 10−2	3.4 × 10−2
moisture content (MC%)	23.9 ± 3.7	33.06 ± 2.1
swelling index (%)	48.94 ± 3.56	64.55 ± 2.26

Combining these physical attributes along with surface morphology and pore size of wound dressing yields interesting conclusions. The water vapor permeability was shown to decrease very slightly because of the increase in water retention by the NO-releasing wound dressing. This theoretically should increase the NO release from the alginate–PVA–GSNO wound dressings over time as moisture leads to homolytic cleavage of GSNO. This would also have positive consequences when such wound dressings are applied to a wound site where wound exudates would be absorbed in the alginate–PVA–GSNO dressing and result in the prolonged supply of NO from the wound dressings. Eventually, an increase in NO flux will benefit the wound healing through its bactericidal effect.^10,38,45 From a clinical perspective, this is of great advantage to reduce infection in the wound exudates which can otherwise cause serious clinical challenges including hyperinflammation and delay the healing process.^46-48 In addition, an increase in NO flux will also positively impact all four phases of wound healing right from homeostasis until the final phase of tissue remodeling.^10,48-50 A controlled water vapor transmission rate has been shown to help in the proliferation and regular function of fibroblast cells.⁵¹

3.4. Antibacterial Efficacy of alginate–PVA–GSNO Dressings.

Nitric oxide eradicates bacteria in a nonspecific way which can be explained through different theories as stated below. The mechanism behind the bactericidal effect of NO seems to involve oxygen and/or its free radicals ultimately leading to the formation of nitrogen radicals.⁵² Because of its gaseous nature, nitric oxide can cross the cell membrane that leads to the activation of sulfur–iron complexes inhibiting respirator chain enzymes.^40,53 The cellular damage caused by GSNO may result from oxidative and nitrosative stress mainly facilitated by oxidation of thiol or nitrosation of thiol groups.⁵⁴ Another accepted mechanism is the NO-mediated inhibition of enzyme activity in bacterial cells. Lipid peroxidation, nitrosation of amines and thiols in the extracellular matrix, tyrosine nitration in the cell wall, and DNA damage also explain the antibacterial mechanisms of NO.⁵⁵

Commercially available advanced wound dressings often contain antibiotics (Septocoll by Biomet Merck; Collatamp by Innocoll) or other antibacterial agents such as silver (e.g., Acticoat by Smith & Nephew, Actisorb by J&J, and Aquacel by ConvaTec), chlorohexidine (Biopatch by J&J), or iodine (Iodosorb by Smith & Nephew). However, there are growing concerns about the emergence of antibiotic-resistant bacterial strains. Worldwide, many strains of S. aureus are resistant against most antibiotics available for treatment.⁵⁶ Topical macromolecules and antibiotics cannot penetrate through biofilms and thereby require a 1000-fold higher dose when compared to freely floating planktonic bacteria.^40,57 This only worsens the existing issue of antibiotic resistance in addition to causing cytotoxicity. Antibiotics can also delay healing if applied indiscriminately to damaged tissue areas, which defies the actual purpose of their application.^6,58,59 The inefficiency of currently available therapeutic agents not only adds to the suffering of the patient but also causes huge healthcare expenses. The gaseous nature of NO allows penetration through the matrix in the biofilm, which gives it an extra advantage over antibiotics and silver-based antibacterial strategies. Moreover, unlike antibiotics NO application would not lead to the emergence of resistant bacterial strains owing to its rapid action, short half-life (<5 s), and endogenous nature.^60-62 From an application point of view, local administration of even smaller doses of NO donor at the wounded site would allow efficient delivery in addition to reducing the emergence of antibiotic resistance.

In this study, the NO-releasing alginate–PVA–GSNO wound dressings were tested for the ability to inhibit S. aureus and P. aeruginosa. The looming threat of incurable S. aureus is a serious concern for wound infections. S. aureus contributes to more than 70% of the skin infections at the surgical site while P. aeruginosa is among the other major pathogens.⁶³ S. aureus and P. aeruginosa are also known to cause biofilm formation in which the bacteria are protected against the inhibitory effect of antibiotics via an extracellular matrix. Bacterial entry in human blood through biofilm can result in septicemia and inflammation. Since these bacteria are frequently found in hospital settings, they can lead to nosocomial infections in the hospital environment.⁶⁴ In the current study, the NO-releasing wound dressings showed 99.89 ± 0.04% bacterial inhibition of Gram-positive S. aureus and 98.93 ± 0.69% inhibition of Gram-negative P. aeruginosa as compared to the control alginate–PVA dressing without the NO donor. In the log scale, this amounts to ~3-log reduction (see Figure 5). This is in line with other studies done on the antibacterial effect of NO releasing polymers for biomedical applications against a wide variety of bacteria including S. aureus, P. aeruginosa, A. baumanni, and E. coli.^11,40,41

Figure 5.

Graphical representation of the bacterial inhibition by NO releasing alginate–PVA–GSNO wound dressing as compared to the control alginate–PVA dressing. Presence of GSNO led to ~3 log reduction in S. aureus and ~2 log reduction P. aeruginosa CFU/mg when compared to control. Statistical data is expressed as mean ± standard error of the mean of n = 3 samples. Values of p < 0.05 were considered statistically significant.

The agar diffusion method showed the ability of NO-releasing wound dressings to eradicate S. aureus beyond the direct point of contact when placed in an incubator at 37 °C for 24 h. As expected, the results showed no ZOI around control alginate–PVA wound dressings whereas a 2.6 cm ZOI was formed in the agar surrounding the alginate–PVA–GSNO dressing (Figure 6). This is due to the gaseous nature of NO that allowed its diffusion in the agar medium. It should be noted that there were no bacteria underneath the dressings as well reinforcing the inhibition of bacteria in direct contact with alginate–PVA–GSNO dressing.

Figure 6.

ZOI in S. aureus exposed to alginate–PVA–GSNO dressings. The control alginate-PVA showed no ZOI showing the bactera inhibition via nitric oxide diffusion to be a preventating startegy to avoid biofilm formation.

The results from both bacterial tests are indicative of how NO based strategies can both prevent the growth of bacteria (via a zone of inhibition) as well as cure an infection via eradicating the attached viable bacteria resulting in reduced CFU/mg. This is in line with studies done on NO releasing composites which showed antibacterial efficacy of NO in different experimental conditions.⁴⁰ Such NO-releasing dressing can also avoid the use of systemic administration of antibiotics during wound treatment that could otherwise lead to their accumulation in key organs such as kidneys while only a very small percent is delivered to the wound.

3.5. In Vitro Proliferation of Endothelial Cells.

Angiogenesis, the process through which new blood vessels from the pre-existing blood vessels demands considerable behavioral activity of endothelial cells including cell proliferation.⁶⁵ Neovasculogenesis occurs at the wound site by the process of angiogenesis and is critical for wound repair due to its important role in the delivery of oxygen, nutrients, and other mediators. NO is a well-known vasodilator involved in the increased contraction of the smooth muscle cells that support angiogenesis.⁶⁶ Major angiogenic factors such as VEGF and transforming growth factor (TGF-β) that are involved in stimulation, promotion, and stabilization of angiogenesis are shown to be regulated in wound healing animal models via NO donors.^38,67

Human umbilical vein endothelial cells (HUVEC) are widely regarded as in vitro modeling cells for angiogenesis.⁴² Thus, to demonstrate that the NO-releasing wound dressing can support angiogenesis, HUVEC cells were used as the representative endothelial cell line in this study. The study was done using a cell viability assay that utilizes a highly water-soluble tetrazolium salt. In the live cells, WST-8 [2(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt] is reduced by dehydrogenases to give formazan (an orange color product), which can be detected at 450 nm. Hence more absorbance at 450 nm is an indicator of increased cell proliferation. The relative cell viability was calculated by adjusting the absorbance corresponding to the well plate (without the dressing leachates) to be 100%. Cells exposed to leachates collected from alginate–PVA–GSNO resulted in in a 3-fold increase in HUVEC cell viability within the first 24 h. When the results from alginate–PVA were compared with the well plate, there was no significant difference in viability. Figure 7 shows the graphical representation of comparative HUVEC proliferation between wound dressings with and without GSNO. The enhanced endothelial proliferation by alginate–PVA–GSNO can be attributed to the NO release resulting from cleavage of S─N bond when GSNO encountered the moisture/temperature at 37 °C. The endogenous release of NO has been previously shown to positively affect angiogenesis.²⁰ This study provides supportive evidence of angiogenesis via an exogenous supply of NO from GSNO within the first 24 h of application. This can potentially accelerate wound healing as endothelial cells form the blood vessels responsible for supplying immune cells, cytokines, and nutrients to the site of the wound.

Figure 7.

Alginate–PVA–GSNO dressings resulted in approximately three times higher viability of endothelial cells when compared to the well plate (with no leachates) as well as alginate–PVA dressings. Statistical data is expressed as mean ± standard error of the mean of n = 8 samples. Values of p < 0.05 were considered statistically significant.

3.6. Fibroblast Cell Proliferation and Migration.

The onset of the proliferative phase overlaps with the inflammatory phase and is marked by fibroblasts entering the wound site. As soon as the injury occurs, the healthy dermal fibroblasts in the vicinity of the wound undergo proliferation and migrate into the provisional matrix of the wound clot to lay the foundation of a collagen-rich matrix.⁶⁸ The increased collagen synthesis by fibroblast at the wounded tissue is achieved via endogenous NO production.⁶⁹ On the basis of these recommendations, we considered it interesting to study the effect of leachate from alginate–PVA–GSNO) on fibroblast proliferation and migration in vitro.

The proliferation assay was done on mouse fibroblast cells using 24 h leachates from alginate–PVA–GSNO dressing using WST-8 dye-based cell viability assay. Formazan formation and absorbance at 450 nm was measured as an indicator of increased proliferation. As shown in Figure 8, the alginate–PVA–GSNO dressing increased the proliferation of fibroblast cells by ~30% as compared to well plate without any wound dressing leachate as well as the leachates from alginate–PVA.

Figure 8.

NO releasing alginate–PVA–GSNO dressings resulted in ~30% increase in fibroblast cell proliferation when compared to the cells grown without leachates (positive control) and/or alginate–PVA dressing. Statistical data is expressed as mean ± standard error of the mean of n = 8 samples. Values of p < 0.05 were considered statistically significant.

Since alginate–PVA and well plates showed similar results, it is evident that the increased fibroblast proliferation was a result of NO release from the leached GSNO in the culture medium. This is in line with the recent study done by Han et al. in which NO-releasing nanoparticles accelerated the fibroblast proliferation leading eventually to collagen deposition thus resulting in faster wound healing in mice.⁷⁰ These results also indicated that the alginate–PVA–GSNO wound dressings possess no relative cytotoxicity toward the cells when compared to the control. Other published reports including ours have shown other NO-releasing materials possess biocompatibility and hemocompatibility in vitro.^41,71

In addition to fibroblast proliferation, the cell migration assay carried with the leachates from the dressings with and without GSNO also yielded interesting results. The real-time progress of cell migration was qualitatively traced using an ibidi kit observed with optical microscopy. The ibidi kit provides a useful alternative for the scratch assay that can otherwise result in a nonuniform cell-free zone. The images were taken at 0, 24, and 48 h (Figure 9). The results showed that the fibroblasts exposed to the extract from alginate–PVA–GSNO wound dressing resulted in much faster cell migration as compared to alginate–PVA control. This led to complete closure of the in vitro wound within the first 48 h. However, the control sample only led to incomplete migration within the first 48 h. Because the only difference between the latter is GSNO, it is evident that NO release via homolytic cleavage of GSNO in moist conditions and physiological temperature resulted in the fibroblast migration eventually closing the wound in vitro.

Figure 9.

Temporal and spatial response of an in vitro wound model toward the leachate collected from control alginate–PVA and alginate–PVA–GSNO dressings within a 48 h period. As clear from the images, the fibroblast migration (B,E) was much faster in cells exposed to GSNO resulting in complete closure (F) of in vitro wound whereas the control wound without GSNO was left ajar (C).

From an application point of view, the in vitro results of fibroblast proliferation and migration provide supportive in vitro evidence for enhanced wound healing as they play a significant role in collagen deposition and tissue remodeling. However, to establish the effectiveness of the alginate–PVA–GSNO wound dressing for commercialization, further in vivo testing in animal models is recommended as well as planned for future investigations by our group.

4. CONCLUSION

In the current study, a bioinspired hybrid alginate–PVA–GSNO dressing was developed and characterized to show its potential in wound healing. The hybrid material that consists of a natural polymer (alginate) and synthetic polymer (PVA) helps overcome limitations possessed by natural or synthetic polymers individually. It also provides an alternative to the use of a separate antibacterial agent in wound dressings by combining the individual wound healing benefits of alginate, PVA, and GSNO in a single hybrid dressing. The alginate–PVA–GSNO dressings allowed a controlled release of NO resulting in a significant reduction in prevalent wound pathogens namely S. aureus and P. aeruginosa. The gaseous nature of NO allowed diffusion through the agar thus preventing bacterial growth and leading to a zone of inhibition. The bacterial adhesion assay showed its ability to eradicate up to 99.89 ± 0.40% reduction in viable bacteria when compared to control, thus qualifying it as both prevention and cure against bacterial infection. This form of local NO administration directly at the wound site would also avoid the toxic buildup of antibiotics in kidneys as seen very often with systemic administration. The results from this study also provided important proof of concepts suggesting that the alginate–PVA–GSNO wound dressing can regulate angiogenesis, fibroblast proliferation, and migration all of which are essential for faster wound healing. Physical characteristics such as moisture content, water vapor transmission, pore size, swelling ratio, surface morphology, and NO release kinetics showed that the alginate–PVA–GSNO material possesses the desired characteristics needed for enhancing the natural wound healing process. The porous matrix allows for absorption of wound exudates, water vapor transmission, and gaseous exchange. Overall, this study supports the possibility of using therapeutic alginate–PVA–GSNO dressing to provide a supportive environment for accelerated wound healing.