Development of a wound dressing based on modified polyurethane foam to localize bacteriophage action in the wound

Introduction

The problem of antimicrobial resistance is one of the main challenges of modern medicine, which necessitates the search for alternative methods of combating bacterial infections. Bacteriophages represent a promising approach in the treatment of infectious diseases, particularly in the therapy of wound infections [1]. However, the use of bacteriophages in clinical practice is associated with a number of technological challenges, the key ones being the methods of their delivery to the site of infection and maintaining their therapeutic concentration in the area of clinical interest [2].

The effectiveness of topical phage therapy is determined by adherence to principles developed on the basis of recent experimental and clinical studies. One of the main requirements is the creation of a phage concentration in the area of clinical interest exceeding the threshold value of C = 10⁸-10⁹ PFU/mL, which is 100 times higher than the concentration of target bacteria [2]. To achieve this goal, the use of polymeric materials as bacteriophage carriers holds promise [3].

Polyurethane foam is a potential material for the creation of wound dressings due to its porous structure, elasticity, and biocompatibility [4]. However, native polyurethane foam possesses hydrophobic properties, which limits its application for wound dressings requiring active absorption of exudate. Surface modification of polyurethane foam is a promising approach for imparting hydrophilicity, adhesive, and antibacterial properties to the material [5, 6, 7].

Oxidative polymerization of catecholamine derivatives was used as the modification reaction, resulting in the formation of a biomimetic coating on the surface with unique properties [7]. The presence of free hydroxyl and amino groups provides adhesive properties and the possibility of immobilizing biologically active agents, including antibiotics and bacteriophages [8].

The objective of this study was to develop a prototype wound dressing based on modified polyurethane foam for the immobilization of bacteriophages and their controlled release in the wound.

Materials and Methods

The following materials were used in the study: technical polyurethane foam (Sintez OKA); staphylococcal bacteriophage "Staphylophage" manufactured by the branch of JSC NPO "Microgen" in Nizhny Novgorod "IMBIO," a sterile purified filtrate of phage lysates of bacteria of the genus Staphylococcus with an Appelman activity of not less than 10⁻⁵; tris-(hydroxymethyl)-aminomethane (Pharmpur® Ph Eur, BP, Scharlab, Spain); isotonic NaCl solution (OOO "Grotex," Russia); Staphylococcus aureus culture, clinical strain No. 229 from the working collection of the bacteriological laboratory of the University Clinic "PIMU," isolated from a patient with burn wounds; GRM agar.

Modification of polyurethane foam. Plates were cut from technical polyurethane foam measuring 2×2×0.5 cm. Modification of polyurethane foam plates was carried out by oxidative polymerization of catecholamine derivatives. A 0.01 M solution of tris-(hydroxymethyl)-aminomethane was prepared, to which dopamine hydrochloride was added at a concentration of 2 mg/mL. The pH of the solution was adjusted to 8.6 to initiate polymerization. Polyurethane foam samples were completely immersed in the resulting solution and kept at room temperature in air under natural lighting for 24 hours. After polymerization, the samples were washed with distilled water and then dried in a drying oven at 45°C for 4 hours. The success of modification was assessed visually by the characteristic color change of the material to dark brown and black-brown (according to the RAL classification), with possible uneven coloration ("marbling").

Determination of physicochemical properties

Determination of absorption capacity by mass. Polyurethane foam samples of standard shape (parallelepiped plates) were weighed on an analytical balance (m₀, g) and immersed in distilled water for 10 minutes. After removing excess water, the samples were reweighed (m₁, g). Absorption capacity was calculated using the following formula:

B = (m₁ - m₀) / m₀ × 100% (1), where

B — absorption capacity of the material by mass, %;

m₁ — mass of the material sample after water absorption, g;

m₀ — mass of the initial material, g.

All measurements were performed in six replicates.

Determination of absorption capacity by volume. The initial volume of the sample (V₀) was measured, after which the sample was completely saturated with distilled water. The amount of absorbed liquid (Vₐᵦₛ) was determined by the difference in volumes, and the absorption coefficient was calculated using the following formula:

K = (Vₐᵦₛ / V₀) × 100%, where

K — water absorption coefficient of the material, %;

Vₐᵦₛ — volume of liquid absorbed by the material sample, cm³/mL;

V₀ — initial volume of the sample, cm³/mL.

All measurements were performed in six replicates.

Determination of porosity. Samples were weighed (m₀, g), completely saturated with distilled water, and reweighed (m₁, g). Porosity was calculated using the following formula:

P = (m₁ - m₀) / V_H₂O × 100%, where

P — porosity of the material, %;

m₀ — mass of the initial material sample, g;

m₁ — mass of the material sample after complete saturation with liquid, g;

V_H₂O — volume of water required for complete saturation of the sample.

All measurements were performed in six replicates.

Determination of droplet imbibition time. A drop of distilled water with a volume of 10 µL was applied to the surface of the sample using a microdispenser. The time for complete absorption of the drop was recorded with a stopwatch. For all indicators, the result was the arithmetic mean of at least five measurements.

Dialysis of bacteriophages by the Kruvchinsky method. The kinetics of bacteriophage release were studied by the Kruvchinsky dialysis method through a semipermeable membrane. A polyurethane foam plate saturated with bacteriophage was placed in a dialysis tube closed at one end with a cellulose membrane. The tube was immersed in isotonic NaCl solution and incubated at 37°C. Samples were taken at 1, 3, 5, and 8 hours of dialysis, with the medium volume adjusted to the initial value after each sampling.

Assessment of bacteriophage viability on modified polyurethane foam substrate. Polyurethane foam samples were saturated with bacteriophage in an amount equal to ½ of the maximum absorption volume. The saturated samples were placed in Petri dishes with GRM agar, onto which a suspension of S. aureus microorganisms at a concentration of 1×10⁵ CFU/mL (corresponding to an optical density of 0.5-0.6 units on the McFarland scale) had been previously applied. The dishes were incubated at 37°C for 24 hours. The degree of lysis was assessed on a five-point scale:

• "—" — absence of lytic activity;

• "+" — low activity;

• "++" — lysis zone with a large number of secondary growth colonies;

• "+++" — lysis zone with single secondary growth colonies;

• "++++" — clear lysis zone without secondary growth colonies;

The procedure was performed in three replicates to ensure the representativeness of the data obtained and to minimize random errors.

Analysis of bacteriophage lytic activity kinetics. A suspension of S. aureus at a concentration of 1×10⁵ CFU/mL (corresponding to an optical density of 0.5-0.6 units on the McFarland scale) was prepared and evenly applied to dried Petri dishes with GRM agar. From the samples obtained during dialysis (1, 3, 5, and 8 hours), 20 µL of dialysate was taken and applied to the dishes. Sampling was performed in triplicate for each experimental time point. The dishes were incubated at 37°C for 24 hours, after which the degree of lysis was assessed.

Determination of bacteriophage titer by the Appelman method. Twelve sterile bacteriological test tubes with 4.5 mL of sterile nutrient broth were prepared. Into the 1st test tube, 0.5 mL of the studied phage was introduced with a sterile pipette. The contents of the test tube were mixed, and 0.5 mL from the 1st test tube was transferred to the 2nd, mixed, and from the 2nd test tube transferred to the 3rd, and so on up to the 10th inclusive. From the 10th test tube, 0.5 mL was removed. The 11th and 12th test tubes served as controls (test tube 11 — microorganism growth control, containing 4.5 mL of broth and 0.2 mL of broth culture of S. aureus; test tube 12 — nutrient broth sterility control, containing 4.5 mL of broth without the addition of bacterial culture of S. aureus and bacteriophage). Bacteriophage dilutions from -10 to -10,000,000,000 (10⁻¹ — 10⁻¹⁰) were obtained. Next, 0.2 mL of 18-24-hour broth culture of S. aureus bacteria sensitive to the titrated bacteriophage was added to all 10 test tubes of the prepared dilution series. The rack with test tubes was incubated in a thermostat at 37°C. Results were recorded after 18-20 hours. The titer was considered to be the maximum dilution of bacteriophage at which complete lysis of the phage-sensitive S. aureus culture was observed.

Statistical analysis. Upon confirmation of normal distribution, parametric comparison of data (t-test for two independent samples) was used for analysis. If the data did not conform to normal distribution, the nonparametric method of sample comparison (Mann-Whitney test) was used. Significance of differences was determined at a significance level of p < 0.05.

Results and Discussion

Effect of modification on the physicochemical properties of polyurethane foam. The results of absorption capacity assessment are presented in Table 1. By mass, the absorption capacity of polyurethane foam (PU) was 3459.6 ± 197.8%, while that of modified polyurethane foam (MPU) was 3395.7 ± 205.26%. By volume, the value for PU was 217 ± 22.72%, and for MPU — 213.42 ± 23.3%. The tendency for a slight decrease in moisture capacity of MPU is associated with a reduction in pore size due to adsorption of polymerization products of catecholamine derivatives, which is consistent with porosity data (Table 1).

Table 1. Comparison of the absorption capacity of the original and modified polyurethane foam

Notes: PU — polyurethane foam; MPU — modified polyurethane foam.

The results of porosity determination are presented in Table 2. For PU, the average porosity was 97.04 ± 0.86%, while for MPU it was 95.36 ± 4.29%. The decrease in porosity of MPU can be explained by the adsorption of oxidative polymerization products of catecholamine derivatives on the pore surface, which leads to a reduction in their effective diameter.

Table 2. Porosity of the original and modified polyurethane foam

The imbibition time of one drop of distilled water with a volume of 10 µL is an indicator of material hydrophilicity. For PU, the average absorption time was 53 min 48 s ± 14 min 6 s, which indicates pronounced hydrophobic properties of the material. After modification of PU, the absorption time was statistically significantly reduced to 4.3 ± 2.44 seconds (p < 0.05), indicating that the material acquired pronounced hydrophilic properties.

The obtained results of physicochemical property assessment demonstrate that modification radically changes the surface properties of PU, transforming the hydrophobic material into a modified one with hydrophilic properties (MPU). This is of great importance for application as a wound dressing, as it ensures effective absorption of wound exudate.

Assessment of bacteriophage viability and lytic activity on polyurethane foam plates. MPU demonstrated maximum lytic activity ("++++") at all dialysis time points (1, 3, 5, and 8 hours), indicating complete preservation of bacteriophage viability. For PU, maximum activity was observed only in the first 3 hours, while at 5 and 8 hours of dialysis a decrease in activity was noted ("++" and "++", respectively) with the formation of a lysis zone with a large number of secondary growth colonies (Table 3).

Table 3. Comparison of the degree of bacterial lysis by dialysate of polyurethane foam and modified polyurethane foam

The obtained results indicate that modification not only does not reduce but actually promotes the preservation of bacteriophage viability during their immobilization and subsequent release.

Bacteriophage titration. The results of bacteriophage titer determination by the Appelman method for PU and MPU are presented in Tables 4 and 5.

Table 4. Titration of bacteriophage in polyurethane foam dialysate

Table 5. Titration of bacteriophage in dialysate of modified polyurethane foam

The titration data show that in bacteriophage samples released from MPU, complete lysis of the bacterial culture was observed at all studied dilutions (up to 10⁻⁵) at all time points. This indicates the preservation of high bacteriophage lytic activity at a level of not less than 10⁵ PFU/mL.

In the case of PU, after only 3 hours of dialysis the bacteriophage titer decreased to 10³, after 5 hours — to 10², and by 8 hours growth of S. aureus was observed at all dilutions, indicating almost complete loss of lytic activity.

The obtained results demonstrate that MPU ensures long-term preservation of viability and lytic activity of immobilized bacteriophages, which is critically important for application as a wound dressing. The modifying coating on PU likely creates a protective microenvironment for bacteriophages, protecting them from denaturation and loss of activity.

Study Limitations

A limitation of this study was its in vitro nature. Static, as well as simple dynamic in vitro models, do not account for active transport of substances, metabolic activity of tissues, or the influence of enzyme systems. Unlike a living organism, there is no blood flow or lymphatic drainage here, which constantly maintain a concentration gradient. The model is also limited by the use of standardized buffer solutions. In real conditions, diffusion is affected, in addition to pH, by the viscosity of biological media and the presence of specific proteins that bind active substances and alter their transport properties. The semipermeable membranes used have a fixed pore size and a specific chemical composition. They cannot mimic the complex structure of biological barriers, their selective permeability, the presence of ion channels, receptors, and transport systems. This leads to the fact that the rate of substance release in vitro may differ significantly from actual bioavailability.

Conclusion

The conducted study demonstrated the effectiveness of polyurethane foam modification for creating wound dressings with immobilized bacteriophages. Modification leads to a radical change in the surface properties of the material, transforming hydrophobic polyurethane foam into a hydrophilic one: the imbibition time of a water drop is reduced from 53 min to 4.3 s, which ensures effective absorption of wound exudate.

The key result of the work is the establishment of MPU's ability to maintain the viability and lytic activity of immobilized bacteriophages. Appelman titration showed that the modified material ensures the preservation of bacteriophage activity at a level of not less than 10⁵ PFU/mL for 8 hours of dialysis, whereas for PU almost complete loss of activity was observed by this time.

PU modification not only improves the hydrophilicity of the material but also imparts adhesive, antibacterial, antioxidant, and other properties, which further enhances the therapeutic potential of the developed dressing through effective immobilization of bacteriophages and their controlled release into the wound environment.

Thus, MPU represents a promising platform for creating biomimetic wound dressings capable of ensuring effective delivery of bacteriophages to the site of infection and maintaining their therapeutic concentration. The developed material may find application in the treatment of infected wounds, particularly those caused by antibiotic-resistant staphylococcal strains.

Promising directions for further research include studying the kinetics of phage release under conditions simulating the wound environment, as well as conducting preclinical trials of the effectiveness of the developed dressing on experimental models of infected wounds.