Design of a silver-zinc nanozeolite-based antibiofilm wound dressing activity in an in vitro biofilm model

doi:10.21203/rs.3.rs-4985510/v1

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Research Article

Design of a silver-zinc nanozeolite-based antibiofilm wound dressing activity in an in vitro biofilm model

https://doi.org/10.21203/rs.3.rs-4985510/v1

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Background

Infected wounds are a major health problem as infection can delay wound healing. Wound dressings play an important part in wound care by maintaining a suitable environment that promotes healing. Silver sulfadiazine dressings have been used for preventing infection in burn wounds. Presently, there are many commercial silver dressings that have obtained FDA clearance.

Results

In this study, we report on a novel silver dressing using microporous aluminosilicate zeolites, termed ABF-XenoMEM. Silver and zinc ion are encapsulated in the zeolite supercages. We show that the silver-zinc zeolite (AM30) alone is effective at inhibiting biofilm formation. The encapsulation protects the silver from rapidly precipitating in biological fluids. We exploit the negatively charged zeolite surface to associate positively charged quaternary ammonium ions (quat) with the zeolite. The combination of the AM30 with the quat enhances the antimicrobial activity. The colloidal nature of the zeolite materials makes it possible to make uniform deposits on a commercial extracellular matrix membrane to develop the final dressing (ABF-XenoMEM). The optimum loading of silver, zinc and quat on the dressing was found to be 30, 6 and 220 µg/cm². Using a colony biofilm model, the activity of ABF-XenoMEM is compared with four well-studied silver-based commercial dressings towards mature biofilms of Pseudomonas aeruginosa (PAO1) and methicillin-resistant Staphylococcus aureus (MRSA). Cytotoxicity of the dressings was examined in HepG2 cells using the MTT assay.

Conclusion

This study shows that the ABF-XenoMEM is competitive with extensively used commercial dressings and demonstrates using a colony biofilm model that nanozeolite-entrapped antimicrobials have potential for alleviating biofilm-infected wounds.

Chronic wounds

Quaternary ammonium

Quat

Silver

Zinc

Antimicrobial

Pseudomonas aeruginosa

Methicillin resistant Staphylococcus aureus

MRSA

Biofilm

Antibiofilm

Extracellular polymeric substances (EPS)

Chronic wounds are often associated with vascular, diabetic, and pressure ulcers and can last 2– 12 weeks.(1) They pose a 1–2% lifetime risk (affect 30.9% of the population in developing countries) and lead to reduced quality of life, deaths, and high economic costs.(2–6) In 2014, 15% of Medicare patients in the US had a wound infection, and 4% had surgical site infections.(5, 6) $96.8B is spent on wound care, with about $7.2B for chronic wound care.(6)

Biofilm formation is associated with 78% of chronic and 6% of acute wounds.(7, 8) Biofilms are 3D structures enclosed by a matrix of self-secreted extracellular polymeric substances (EPS). (9–11) Bacteria in biofilms have several defense mechanisms, including the protection offered by the EPS, hypoxia, overexpression of stress genes, and antimicrobial resistant dormant cells.(12, 13) The bacterial microcolonies within these biofilms have altered phenotype compared to their planktonic counterparts and display tolerance to host immune defense and administered antimicrobial agents. Systemic antibiotic therapy, the mainstay treatment of bacterial infections, therefore, is of limited efficacy due to the difficulty of the compounds reaching bacteria enmeshed within the biofilm and the greater tolerance of the cells to the antimicrobial compounds.(9, 14) Chronic wound infections are generally polymicrobial in nature (15–17).

Biofilms in chronic and burn wounds can delay the wound healing process.(18) Thus, approaches to prevent the formation of biofilms, as well as to disrupt biofilms and kill bacteria are necessary. Since biofilms are structurally robust, strategies to remove biofilms involve mechanical and chemical disruption. Mechanical debridement is painful to the patient and can cause damage to healthy tissue and promote the spread of infection.(19) Use of wound dressings to promote wound healing is a common clinical practice.

Silver is effective against both gram-positive and gram-negative bacteria.(2) The silver mechanism of action is mediated through silver ions, which bind to tissues and intracellular proteins (N, O, or S functionalities), bacterial DNA and RNA, respiratory chains, cellular toxicity mediated through ROS, structural changes in cell walls and intracellular and nuclear membranes.(20) Silver has anti-inflammatory effects,(15) is anti-angiogenic (21) and influences the immune response.(22) As an effective antimicrobial, silver is helpful for reduction of infections when used at sub-toxic levels; at higher levels, it can cause discoloration of the skin, known as argyria.(23)

The first silver-based dressing to prevent infection in burn wounds was introduced in 1968 in the form of silver sulfadiazine (SSD).(24, 25) Silver sulfadiazine combines silver and the antimicrobial sulfadiazine and has been shown to reduce microbial burden.(26) SSD needs to be changed twice daily, and SSD use is associated with reports of more patient pain.(27) This has led to introduction of silver dressings with more controlled silver release than SSD dressings and these dressings do not need to be changed as often.(28) Based on the FDA 510K Premarket Information, there are about 123 different brands of silver wound dressings. Early intervention with silver dressing may decrease biofilm formation, though it is still debated what silver dressing can do if biofilms are already established.(29) Dressings that alleviate infection can minimize the effects of other unfavorable features, such as hypoxia that delay wound healing.

Zeolites are crystalline microporous aluminosilicates with well-defined internal porosities at the sub-nanometer and nanometer scale.(30) These internal cages and channels are accessible to ions and molecules. Zeolites can be synthesized with different morphologies. In this paper, we report on the development of a wound dressing referred to as ABF-XenoMEM with several novel features. In designing this dressing, we have used silver ions trapped in a nanosized microporous aluminosilicate zeolite particle. The hypothesis is that the silver ion trapped in the zeolite is not immediately accessible to constituents of wound fluids, and thus the silver ions are not inactivated by precipitation but can be slowly released over time. Because of the zeolite’s role as a protective encapsulant, lower amounts of silver may still be effective, leading to lower cytotoxicity. In addition, the zeolite makes it possible to colocalize zinc ions alongside the silver ions to improve the antibacterial activity. Further development includes use of a positively charged quaternary ammonium compound that can coulombically associate with the negative surface of the zeolite. The zeolite and quat are deposited on an extracellular matrix commercial dressing (XenoMEM™) to form the wound dressing. The antimicrobial properties of the zeolite particles, biofilm inhibition, and examining the fate of the bacteria in mature biofilms is presented, focusing on Pseudomonas aeruginosa (PAO1) and methicillin-resistant Staphylococcus aureus (MRSA). The study concludes with comparison of the ABF-XenoMEM dressing with four commercial dressings (Procellera™, Acticoat™ 7, Promogran Prisma™ and Aquacel® Ag⁺ Extra™) for killing bacteria in established biofilms. The cytotoxicity of all the dressings were compared using the MTT assay. The commercial dressings (Table 1) were chosen because they have a considerable market share amongst silver-based wound dressings.

Table 1

Brief comparisons of the commercial dressings examined in this study.
Product	Manufacturer	Silver Content		Description (Ref)
Promogran Prisma™	3M	Ag: 20 µg/cm²		Sterile, freeze-dried composite of 44% oxidized regenerated cellulose (ORC), 55% collagen, and 1% silver-ORC. Silver-ORC contains 25% w/w ionically bound silver. (67)
Procellera™	Vomaris	Ag: 900 µg/cm² Zn: 300 µg/cm²		Microcurrent generating antimicrobial wound dressing consisting of a matrix of alternating silver (Ag) and zinc (Zn) dots held in position on a polyester substrate by a biocompatible binder. (69)
Aquacel® Ag⁺Extra™	ConvaTec	Ag: 170 µg/cm²		Hydrofiber™ Technology and Ag⁺Technology – a unique ionic silver-containing, antibiofilm formulation. Two layers of a needle-punched nonwoven fleece of sodium silver CMC fibers enhanced with disodium EDTA and benzethonium chloride^†, stitched with a high-purity cellulose thread. (66,67)
Acticoat™ 7	Smith & Nephew	Ag: 1700 µg/cm²	Two rayon/polyester non-woven inner cores laminated between three layers of nanocrystalline silver (NCS) -coated high-density polyethylene mesh, designed to be the barrier against bacterial invasion. (65,66)
ABF-XenoMEM	ZeoVation	Ag: 30 µg/cm² Zn: 6 µg/cm² AM30: 660 µg/cm² BZC: 221.2 µg/cm²	Silver zinc nanozeolite and benzalkonium chloride coated on XenoMEM dressing (porcine peritoneal membrane). XenoMEM is a matrix consisting primarily of type I collagen and contains additional ECM proteins such as laminin, elastin, fibronectin, collagen III and IV, and proteoglycans.

Materials

Zeolite: Nanozeolite material was prepared using our published procedures.(31) Sodium ion-exchanged nanozeolite served as a control sample (FAU30). Transition metals (Ag⁺ and Zn²⁺) were ion-exchanged into the nanozeolite, resulting in a colloidal suspension, henceforth labeled as AM30. In order to prepare micron-sized powders, AM30 suspension was spray dried using OLT-SP2000 Double Separated Spray dryer from Xiamen Ollital Technology Co. Ltd. using 3.5 wt% of AM30 suspension. Benzalkonium chloride (BZC) was obtained from Sigma Aldrich. In the biofilm-suspension experiments (Figure 5), benzalkonium nitrate was used, made by ion-exchange of the BZC with NaNO₃, as reported in the literature.(32) We refer to the benzalkonium ion as quat throughout this study. XenoMEM^™ was obtained from Viscus Biologics Inc. XenoMEM^™ is a wound dressing matrix composed of primary type I collagen and additional extracellular matrix (ECM) proteins, and it is derived from the porcine peritoneal membrane. It retains the native extracellular matrix structure and function by exhibiting a connective tissue side and a basal membrane side indicated by the XenoMEM^™ embossing.

Preparation of ABF-XenoMEM

The bare XenoMEM^™ sample (5 cm x 5 cm dimension) was weighed first. XenoMEM^™ was stretched from four corners with the help of clips for uniform coating (via a syringe) with the basal smooth side (embossed with XenoMEM^™) facing down. The first step was to coat XenoMEM^™ connective side with AM30 suspension. 3.77% AM30 colloidal suspension was sonicated for 15 mins before coating on XenoMEM^™. 438 µL of 3.77 wt% AM30 was uniformly spread on XenoMEM^™ with the target loading of 660 µg/cm² AM30. The coated sample was allowed to dry at room temperature overnight. The second step was to coat AM30-XenoMEM with BZC suspension. 300 µL of 1.85 wt% BZC was uniformly spread on AM30-XenoMEM with the target loading of 221 µg/cm². The dried ABF-XenoMEM is the final sample.

Characterization

Dynamic Light Scattering (DLS) and Zeta Potential: Particle size analysis of FAU30 and AM30 was performed by DLS using Malvern Zetasizer Nano ZS. Nanozeolite and AM30 at 0.1 wt% was prepared by dilution and sonicated for 10 mins before measuring DLS. The mixed (zeolite and quat) sample was transferred to a disposable folding capillary cuvette DTS 1070 and zeta potential was measured using the same instrument.

HRTEM: High resolution transmission electron microscopy (HRTEM) images of nanozeolite were obtained using a FEI Tecnai F20 S/TEM.

XRD: Nanozeolite and AM30 suspensions were centrifuged, and the solids were collected and dried in the oven at 100^oC for 12 hours. Powder X-ray diffraction (XRD) patterns were obtained using Bruker D8 Advance Diffractometer with Cu Kα radiation with 0.5 divergences, 0.02 step size, and 0.5 sec dwell time.

SEM: The morphological characterization of XenoMEM^™ and ABF-XenoMEM was carried out with Hitachi SU-70 Schottky field emission gun scanning electron microscopy (SEM) with energy dispersive X-ray spectroscopy (EDS) capability for elemental analysis.

Elemental analysis: Silver and zinc loading on AM30 was determined by Galbraith Laboratory (Knoxville, TN) after acid dissolution using Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES). Before elemental analysis, the entire sample was digested using 10% nitric acid, 4% hydrofluoric acid, 4% hydrogen peroxide, and 25% hydrochloric acid and then analyzed using the ICP-OES method.

Methods

Silver release into simulated wound fluid (SWF) from ABF-XenoMEM: Simulated wound fluid was prepared by using 0.2922 g sodium chloride, 0.1680 g of sodium hydrogen carbonate, 0.0149 g of potassium chloride, 0.0139 g of calcium chloride, 1.65 g of bovine albumin, and 50 mL deionized water, as described in the literature.(33) 2 g of SWF was added to the vial, and 2 sets of 2 cm x 2 cm of ABF-XenoMEM (total silver 229.7 µg) were submerged completely in the vial and incubated for 24 hours at RT. After 24 hours of incubation, the SWF was retrieved, weighed, and stored in a new vial for silver elemental analysis and DLS measurement. The two sets of ABF-XenoMEM were transferred to a new vial containing a fresh batch of 2 g of SWF and the incubation process started. This process was repeated for 7 days, with samples collected daily. SWFs were sent to Galbraith Laboratory for elemental analysis. Before elemental analysis, the entire SWF sample was digested using 10% nitric acid, 4% hydrofluoric acid, 4% hydrogen peroxide, and 25% hydrochloric acid and then analyzed using the ICP-OES method.

Zeolite release into SWF: SWF preparation and ABF-XenoMEM incubation method were the same as the silver release experiment. Retrieved SWF (undiluted) were analyzed using the DLS instrument. The zeolite signal (~90-100nm) was normalized to the SWF DLS peak at ~10 nm, and the data compared from Day 1 to Day 7.

Zeolite and Quat Association: Various AM30 and quat mixtures were prepared with a fixed AM30 concentration of 1800 µg/ml and the quat concentration varied from 0, 20, 50, 100, 126, 150, 250, 500, and 1000 µg/ ml. For each of these samples, zeta potential measurements were taken to analyze the zeolite charge variations as a function of the quat concentration.

Biological Methods

Bacterial Strains and Media

For the minimum inhibitory concentration (MIC) experiments, E. coli BL-21 were cultured in LB (Luria broth) broth and MRSA (methicillin-resistant Staphylococcus aureus; strain USA300) were cultured in MH2 (Mueller Hinton 2) broth at 37°C under aerobic conditions for 24 hours. For the remaining biological experiments, Pseudomonas aeruginosa (PAO1) (ATCC 4708) and Staphylococcus aureus subsp. aureus MRSA (ATCC 33592) were obtained from the American Type Culture Collection (ATCC). PAO1 were grown in LB media, while MRSA was cultured in tryptic soy broth (TSA), both at 37°C under aerobic conditions.

Minimum Inhibitory Concentration (MIC)

Standard MIC procedure was followed.(34,35) Briefly, 50 µl of LB broth was added to each well in 96-well plate (Agilent Technologies). Nanoparticles were sonicated and brought to concentration of 800 µg/ml in LB broth. 50 µl of nanoparticle (starting concentration 800 µg/ml or 800 ppm) was added to the first well (effectively becoming 400 ppm), mixed 5 times with a pipette, 50 µl was removed and transferred to the adjacent well. This was repeated for 10-step dilution array, with 50 µl being discarded from the last wells. 50 µl of the cell suspension (5x10⁵ cells/ml) was added to each well, halving the concentration of the nanozeolite (except the sterility control). The inoculum was determined to be 5x10⁵ cells/ml by OD_600nm measurements. The 96-well round-bottom plate was placed in a 37^oC incubator for overnight incubation. MBC (minimal bactericidal concentration) was determined for the AM30 sample.(36) Specifically, 10 µl from each well not showing overnight growth in the presence of the nanoparticle/antibiotic was placed in 100 µl of LB and plated on nutrient agar plates using glass beads to spread on plates. Plates were incubated at 37^oC overnight and assessed for bacterial growth. Colonies, if present, were counted. MH2 media was used with MRSA.

Planktonic Bacterial killing Assay

Cultures of PAO1 were inoculated by scraping a loop of frozen bacterial stock and placing it into LB media. These cultures were grown overnight in a shaking 37°C incubator. 0.5 µl of overnight culture was added to 4.995 ml fresh LB (5 ml total) to make the logarithmically growing bacterial inoculum, which results in 1x10⁶ CFU/ml, and allowed to grow for two hours. AM30 particles immediately after being sonicated in the water bath were added to 1-dram vials containing 400 ml of LB media. 100 ml of bacterial inoculum (~1 x 10⁶cells/ml) was then added. Cultures with particles were incubated 120 minutes at 37°C. After the defined period, 50 ml of bacteria with AM30 was placed in 450 ml of broth containing 0.5 wt% thioglycolate, sonicated gently in a water bath for a few minutes. Serial dilutions were made and 100 ml of each was plated on LB agar plates. Plates were incubated overnight at 37°C and counted the next morning.

Biofilm Inhibition and Crystal Violet Assay

Adapted from our laboratory protocol (37), this assay demonstrates the inhibition of biofilm formation following treatment. 500 µl of water bath-sonicated nanozeolite particles was added to 1.5 ml microcentrifuge tube containing 400 µl of LB media. Particles were added, 100 µl of PAO1 bacterial inoculum (~1 x 10⁶cells/ml) was added. Culture was added, the microcentrifuge tube was vortexed and plated onto a 96-well plate at 200 µl. The final concentration of the nanozeolite FAU30 and AM30 was adjusted to 1.25-100 ppm and the quat at 15. 6 ppm. The plate was placed in a 37^oC incubator for 24 hours. The bacterial growth was quantified by measuring the optical density at 600 nm (OD_600nm). The growth media was removed, the wells were gently washed three times with cold tap water. The biofilms in the wells were then heat-fixed at 70°C for 1 hour. Subsequently, 200 µl of 0.1% crystal violet solution in deionized water was incubated in each well for 15 minutes. Excess stain was rinsed off with cold water, and the plates were allowed to air-dry. To dissolve the crystal violet stain, 200 µl of 33% glacial acetic acid was added to each well, and the biofilm content was indicated by the optical density of 590 nm (OD_590nm).

Colony Biofilm Assay

The materials used for this experiment include TSA and LB broths and agar media, PBS, sterile forceps (either autoclaved or flame-sterilized in 70% v/v ethanol) and Cellulose Acetate Membrane Filters (Type 11106, pore size 0.45 µm; © 2024 Sartorius AG). In addition, 100-mm-diameter sterile Petri dishes, a UV light source (e.g., UVP 8-W multiple-ray laboratory lamp), sterile 15-ml tubes with tightly fitting lids, a vortex mixer, and VWR ultrasonic water-bath were used.

To initiate the colony biofilm assay, the bacterium of interest was inoculated in 5 ml cultures and grown overnight to the stationary phase (37,38). Using sterilized forceps, cellulose acetate membranes were placed in a sterile Petri plate, positioned approximately 30 cm from a UV light source, and treated with UV light in a sterile environment in a biosafety cabinet for 10 minutes on each side. Stationary-phase cultures were then diluted in the appropriate medium to approximately 1 × 10⁷ CFU/ml. A membrane, shiny side up, was placed on an agar medium plate without antibiotics, inoculated with 5 μl of the diluted culture, and allowed to dry before incubating the plates upright at 37^oC for 24 h. The membranes were then transferred to fresh agar plates to ensure a continuous nutrient supply for another 24 h. The LB or TSA agar plates contained appropriate nutrients to support bacterial growth. Incubation was carried out for a total of 48 h. 100 μl of SWF (simulated wound fluid) was added on top of the biofilm. Commercial wound dressings were then placed on top of the biofilm. Each wound dressing was pre-wetted with 500 μl of sterile water before use, and a sterile glass 60 mm Petri dish was placed on top to provide pressure. Biofilms were treated for 24 hours. Post-treatment, the membranes and bandages were transferred aseptically to 15-ml tubes containing 10 ml of sterile media neutralizer with 0.5 wt% thioglycolate solution. Samples were vortexed for two pulses of 60 seconds each and sonicated for 15 minutes to ensure the release of all attached bacteria into the wash fluid. The vortexed and sonicated samples were diluted and plated on LB or TSA agar plates, which were then incubated overnight at 37^oC. The average number of colony-forming units (CFU) present in the extract fluid was counted (being reported as CFU/ml considering the entire 10 ml of media used for bacterial extraction) for the dressing and membrane separately (except for ABF-XenoMEM and Promogran Prisma™).

Cytotoxicity Assay

Cytotoxicity assays of the wound dressings were conducted using the MTT assay. Human HepG2 liver epithelial (HB-8065) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in DMEM (Dulbecco’s Modified Eagle’s Medium, ATCC 30-2002) supplemented with 10% (v/v) fetal bovine serum (FBS) as per the manufacturer’s instructions. The cells were seeded in triplicate in 96-well plates and incubated at 37°C in a 5% CO2 atmosphere. The Invitrogen CyQUANT MTT Cell Proliferation Assay Kit was used for the MTT assay. Cells were seeded at a density of 10,000 cells per well in a 96-well plate and incubated for 24 hours to allow adherence (35,39). A 12-mM MTT stock solution was prepared by adding 1 mL of sterile PBS to a 5-mg vial of MTT and dissolving it by vortexing or sonication. DMEM culture medium on the confluent cells was replaced with 100 µL of Dulbecco’s modified Eagle’s medium (DMEM) colorless with no phenol red, and 10 µL of the 12-mM MTT stock solution was added to each well, including a negative control where 10 µL of the MTT stock solution was added to 100 µL of medium. Pieces of wound dressings measuring 0.5 cm x 0.5 cm were placed in the well. The cells were incubated with shaking every 30 minutes for 3 hours and incubated overnight at 37°C. After treatment the wound dressing was removed. All but 25 µL of the medium was removed from the wells, and 50 µL of DMSO was added to each well. The mixture was pipetted up and down thoroughly to mix, incubated at 37°C for 10 minutes in the dark, mixed again, and the absorbance was read at 540 nm.

Statistical Analysis

To assess the statistical significance of the effect of treatment on bacteria, a Student’s t-test was performed for assays comparing treated bacteria to untreated bacteria. For the biofilm experiments, we compared the ABF-XenoMEM dressing with the commercial dressings. The t-test was conducted using GraphPad Prism version 10.2.3 for Windows (GraphPad Software, Boston, Massachusetts, USA, www.graphpad.com). The following p value thresholds and corresponding symbols were used to denote significance: ns: p > 0.05 (not significant), *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, and ****: p ≤ 0.0001. On the figures, p values greater than 0.05 are indicated on the figures as ‘ns’, p values less that 0.05 are indicated with one asterisk (*), p values less that 0.01 with two asterisks (**), p values less than 0.001 are indicated with three asterisks (***), and p values less than 0.0001 are indicated with four asterisks (****). Statistical rigor ensures confidence in the reproducibility and reliability of the findings. To account for multiple comparisons, Bonferroni corrections were applied. The corrected significance level was calculated by dividing the original significance level (0.05) by the number of comparisons (m). The BioRender Program was used for generating the TOC graphic.

Nanozeolite synthesis, characterization and silver/zinc ion-exchange

For nanozeolite synthesis, we followed our previously published procedure. (31) Figure 1S (Supplementary Section figures are noted with a S) shows the powder X-ray diffraction and the HRTEM of the sodium ion-exchanged nanozeolite (referred to henceforth as FAU30, which is the control) along with the dynamic light scattering of a diluted suspension. The X-ray diffraction is indicative of the faujasite framework (Figure 1S top left), and the high-resolution transmission electron microscopy (Figure 1S top right) indicates reasonably uniform particles of 30 nm. The dynamic light scattering indicates average particle size of ~ 90 nm. It is typical for light scattering to provide larger size numbers than HRTEM.(40) These nanozeolites were ion-exchanged with silver and zinc ions from aqueous solutions to produce the silver ion-zinc ion- aluminosilicate material, referred to as AM30. Figure 1a shows the powder X-ray diffraction of AM30, and dynamic light scattering of a diluted solution. The mild ion-exchange conditions have no effect on the basic faujasite structure and the particle size. The AM30 suspensions are indefinitely stable, and Figure 2S shows the picture of a 1000 ppm AM30 aqueous suspension after storage for two years. No particles settle out over this period, indicating that the suspension is a stable colloidal solution, which will be relevant in manufacture of the dressing, as explained below. The amount of silver and zinc ion incorporated into AM30 was determined by ICP-OES and corresponds to 4.35 wt% silver and 0.56% zinc.

Reason for incorporation of zinc:

It is well recognized that silver ion acts as an antimicrobial, and most silver-based wound dressings contain silver ion in the form of salts or metallic silver, often as AgNP.(41) There are reports in the literature that inclusion of zinc ion can increase the antimicrobial activity of the composite, though the exact mechanism is unclear.(42) The zeolite is a unique support,(43) as it can incorporate multiple ions within its porous framework,(30) thus spatially colocalizing mixtures of ions, as is done with preparation of AM30 (Figure 1b). Figure 2a and 2b compare the minimum inhibitory concentration (MIC) values of zinc-exchanged FAU30, silver-exchanged FAU30 and AM30 against E. coli and MRSA bacteria, respectively.(34,36) Black indicates growth of bacteria and white indicates no growth, with partial growth shown as black dots in white. The concentration at the transition between growth and no growth indicates the MIC values. For Ag-FAU 30 and AM30, MIC for E. coli is 1.56 and 0.78 ug/ml (same value for MBC) and MRSA MIC for Ag-FAU30 and AM30 is 12.5 and 6.25 ppm (same value for MBC), respectively. For both bacteria, the MIC of the AM30 is lower by a factor of two as compared to Ag-FAU30, indicating that the inclusion of zinc ions improves the antimicrobial property. Note that for Zn-FAU30, no antimicrobial activity was observed up to concentrations of 200 mg/ml.

Role of the matrix nanozeolite:

Our motivation to use the nanozeolite as a host for the antimicrobial metal ions is because of reports in the literature that silver ions can precipitate in the wound medium and thus be deactivated.(12,15,44) Enclosing the ions in the zeolite supercages makes them less available to the environment. To prove this hypothesis, we carried out an experiment with PAO1 planktonic cells in LB broth, where they were exposed to a 250-ppm suspension of AM30 (10.9 ppm Ag⁺. 0.14 ppm Zn²⁺), and identical amounts of free zinc and silver ions, all in broth (5.75 ppm Zn (NO₃)₂ (Zn- 0.14 ppm) and 15.8 ppm AgNO₃ (Ag- 10.9 ppm). After a 2h exposure, the solution was neutralized with thioglycolate, and the bacteria counted. Figure 3 shows the cell counts. As compared to the control, there was 5-log₁₀ decrease in bacteria counts (CFU/ml) for the AM30, whereas with the same concentration of silver and zinc ions, there was a 2-log₁₀ decrease in CFU/ml. LB broth contains proteins, which can precipitate the silver ions in solution, whereas with the zeolite, the antimicrobial ions are held within the porous framework and not immediately available to the surrounding matrix.

Biofilm Inhibition:

The ability of AM30 to inhibit PAO1 biofilm formation was examined using the crystal violet assay as we have previously published, (37,38) FAU30 was used as the control treatment. PAO1 bacteria in LB broth were placed in 96-well plates, and the AM30 and FAU30 at varying concentrations were introduced into the bacteria. After 24h, the zeolites were removed, the plates were treated with crystal violet, the dye removed and the stained biofilm on the walls of the plates were dissolved with acetic acid, and the absorbance measured at 590 nm. Figure 4a shows that with FAU30 treatment, robust biofilms were still formed at all concentrations ranging from 1.25 to 200 ppm (the biofilms appear more robust, as manifested by the higher absorbance in the presence of the zeolite. It is unclear if the zeolite is promoting the biofilm formation). For AM30, the data in Figure 4b shows that at concentrations greater than 30 ppm, PAO1 biofilm formation was not observed. Thus, we conclude that AM30 is able to significantly inhibit the formation of PAO1 biofilm.

Enhancing the antimicrobial activity of AM30 via association with quat:

Quaternary ammonium compounds, commonly referred to as quats are extensively used as disinfectants.(45) As shown in Figure 5a, quat (benzalkonium ion) has a positive charge. The surface of the zeolite framework is negatively charged and association between zeolite framework surface and cations are well known. The quat is too large to enter the 7.4 Å zeolite supercages and will be coulombically associated with the surface of the zeolite, as schematically represented in Figure 5a. In order to prove this association, zeta potential measurements were made as a function of zeolite: quat ratio. AM30 was maintained at a fixed concentration of 1800 µg/ml, and quat concentration was varied from 0, 20, 50, 100, 126, 150, 250, 500 and 1000 µg / ml; the zeta potentials were -33.6, -27. -23, -10.4, -4.49, +0.8, +10.5, +20.9, + 28.9 mV, respectively. As expected, the zeolite alone had a negative potential (-33.6 mV) since the surface is negatively charged, and as the quat is titrated in, the potentials become more positive, reflecting the positive charge on the quat, and its increasing association with the surface of the zeolite.

We examined if zeolite-quat associations can enhance the biofilm inhibiting activity of AM30. The biofilm-inhibiting activity of AM30 (1.25-5 ppm) with 15.6 ppm of quat was examined with PAO1 in PBS (note that in PBS lower concentrations of AM30 is required to have the antimicrobial effect). Figure 5b shows that with AM30 concentrations greater than 2.5 ppm, biofilm formation was inhibited. Inhibition of biofilm formation did not occur with 15.6 ppm quat. It is not surprising that we do not see any inhibition of biofilm formation with the quat at concentrations of 15 ppm, since the MIC of quat for PA is reported to be 140000 ppm (46), and thus likely none of the bacteria were killed at this concentration. However, upon mixing the 1.25 ppm AM30 and 15.6 ppm quat, there was significant inhibition of biofilm formation.

Wound dressings with AM30 and quat:

To design a wound dressing, we needed a suitable matrix on which to deposit the antimicrobials. We chose to use an extracellular membrane matrix, marketed by Viscus Biologics as XenoMEM^™. Extracellular matrix (ECM) based wound dressings are used in clinical practice.(47,48) XenoMEM^™ is commercially available through Medline Industries (the product is marketed as Puracol^® Ultra ECM). XenoMEM^™ is a decellularized, lyophilized, and sterilized porcine peritoneal membrane containing collagen (Types I, III, IV), fibronectin, elastin, laminin, vascular endothelial growth factor, and fibroblast growth factor 2.(49) Figure 6 shows the SEM of the two sides of the XenoMEM^™ dressing, a basal side and a connective side.

To optimize the concentration of the additives, we prepared three samples, XenoMEM^™ with AM30 (330 µg/cm²) + quat (110 µg/cm²), AM30 (495 µg/cm²) + quat (165 µg/cm²) and AM30 (660 µg/cm²) + quat (221 µg/cm²). AM30 was drop coated on the basal side, dried and followed by coating with the quat. To test the efficacy of these samples, we chose to examine mature PAO1 biofilms grown on a cellulose membrane (48 h growth of biofilm, Figure 7a), following a literature methodology (37,38). The XenoMEM™ with the additives was placed on top of these biofilms resting on a nutrient agar plate (Figure 7c), exposed for 24 hours, and then the cellulose membrane was removed, and added to 10 ml of media with neutralizer. The membrane + media were vortexed for two pulses of 60 seconds each and sonicated for 15 minutes to extract the bacteria. Spot dilutions were performed to calculate CFU/ml. As seen in Figure 8, the AM30 (660 µg/cm²) + quat (221 µg/cm²) essentially destroyed the biofilm and with no bacterial counts in the extract, and so we chose the AM30 (660 µg/cm²) + quat (221 µg/cm²) as our optimized dressing, and henceforth labeled as ABF-XenoMEM.

The antibiofilm activity of the AM30+quat as a combination is evident in Figure 9. Mature biofilms of PAO1 (formed for 48 h) were exposed to XenoMEM^™, AM30 (660 µg/cm²) on XenoMEM, quat (221 µg/cm²) on XenoMEM and ABF-XenoMEM for 24 hours, and both the membrane (M) and the dressing (B) were analyzed for resident bacteria. Only in the case of ABF-XenoMEM, the bacteria are absent in the extracts from both the dressing and the membrane. The combination of AM30+quat is the most effective against mature biofilms.

Characteristics of ABF-XenoMEM:

SEM of ABF-XenoMEM is shown in Figure 10, and at low resolution appears to be uniformly covering the XenoMEM^™. At higher resolution, the zeolite particles can be seen distributed over the XenoMEM^™. With the nanoparticle coating, the coating appears to be robust, and particles do not fall off from the dressing upon handling. Figure 3S shows the elemental map (by EDS) for silver and appears to be uniformly distributed.

There are numerous studies on micron-sized silver zeolites, including availability from commercial sources. We want to point out that such zeolites would not function well as additives for wound dressings. There are two aspects of the AM30 that makes it appealing for this application. First is the indefinite stability of AM30 in aqueous suspensions (Figure 2S), which makes it easy for generating uniform coatings on surfaces by spray/drop coat methods (micron sized particles will settle out). Second, use of micron sized zeolites (formed by spray drying of AM30, Figure 4S) as coatings on the XenoMEM^™ are unstable and fall off (Figure 4S).

For the dressing to be effective, the zeolite particles will need to be released into the wound fluid. To simulate this release, ABF-XenoMEM was placed in SWF, and DLS of the suspension was carried out to get a measure of how much zeolite is being released as a function of time. Figure 5S shows this data, and it appears that most of the release of the particles in the SWF is occurring between days 1-4. In order to get a quantitative estimate of the release, elemental analysis of the extracts was carried out by ICP-OES. It was found that silver release was complete within 4 days, with 39.4% at end of day 1 (61 ppm in the volume of SWF analyzed), 30.7% end of day 2 (44 ppm in the volume of SWF analyzed), 19.8% at end of day 3 (29 ppm in the volume of SWF analyzed), and 10.1% at end of day 4 (15 ppm in the volume of SWF analyzed). Amounts of silver in samples recovered on days 5-7 were below the detection limits of the instrument.

Comparison of ABF-XenoMEM with commercial silver dressings:

Four commercial dressings were chosen for comparison with ABF-XenoMEM. In Table 1, we outline the specifics of these dressings.

Biofilm Studies: To compare the different dressings, we exposed mature biofilms (48 h growth) to the different dressings. Figure 7 shows photographs of the PAO1 and MRSA biofilm grown for 48h on the cellulose matrix. A change from previous experiments (Figures 8,9) is that we put a layer of SWF between the dressing and the biofilm to better represent a wound environment, as reported in previous studies.(12,50) After 24 h exposure to different wound dressings, the dressing and the membrane were separated and the bacteria on each surface was extracted with media and the bacterial colonies in the extract were counted. With the ECM matrix dressings, ABF-XenoMEM and Promogran Prisma™, the dressing could not be completely separated from the cellulose layer, and both were analyzed together for resident bacteria (for ABF-XenoMEM this was not the case without SWF (Figure 9)). Figure 11 shows the results at the conclusion of 24 h exposure to the different wound dressings for PAO1 biofilms (the bacteria on the dressing and membrane are added, the bacteria on the individual dressing and membrane are shown in Figure 6S). All the dressings have a significant effect on reducing the bacterial load for the gram-negative bacterial biofilm. Aquacel^® Ag⁺ Extra^™seems to perform the best in terms of bacterial reduction, then ABF-XenoMEM, which was effective, followed by the Promogran Prisma™, Acticoat^TM 7 and Procellera^™. Figure 12 shows the results at 24 h exposure of the wound dressings for mature MRSA biofilms (48h) (membrane and dressing bacteria are added together, Figure 7S for bacterial counts analysis of the extracts from the dressing and membrane separately). Against MRSA, only the Procellera^™ did not exhibit a significant decrease in the bacterial load. The ABF-XenoMEM performed significantly better than the other dressings against the gram-positive MRSA biofilm, followed in order by Acticoat^™7, Aquacel^® Ag⁺ Extra^™and Promogran Prisma™.

Cytotoxicity

The dressings were compared for their cytotoxicity towards HepG2 cells using the MTT assay (Figure 13).(51) Consistent with literature on silver dressings, all the dressings were cytotoxic, with ~10% or lower cell viability. Within statistical significance, ABF-XenoMEM is less cytotoxic than Acticoat^™7, but more so than Procellera^™ and Aquacel^® Ag⁺ Extra^™ and no significant difference in cytotoxicity as compared to Promogran Prisma^TM.

In this section, we focus on the novel structural aspects of the new dressing ABF-XenoMEM, and its comparison with commercial dressings towards mature PAO1 and MRSA biofilms.

Structural Aspects:

1) Use of nanozeolites as the host for the antimicrobial agents:

The use of silver zeolites for antimicrobial purposes is well known, and there are many commercial sources.(43) However, these commercial zeolites are in the size range of microns or greater. The disadvantage of these larger sized zeolites in this specific application of wound dressing is that these particles would not form a continuous stable layer on the ECM matrix and fall off the (Figure 4S). The zeolites used in this study have a very small size of 30 nm (Figure 1S). These nanozeolites appear to stick to the ECM layer particularly well. The deposition of the stable suspensions of nanozeolites in a uniform manner using solution-based deposition techniques such as spraying and bioprinting become feasible with stable colloidal dispersions. Micron-sized zeolites rapidly sediment in solutions, and it would be difficult to spray deposit these larger particles. The exact nature of the bonding forces between the nanozeolite and ECM layer that leads to stable layers has not been explored in this study, but it is sufficient to have an extended release of the nanozeolite over at least a period of four days (Figure 5S).

2) Silver/zinc encapsulation in nanozeolite: The combination of silver and zinc in the nanozeolite improves the antimicrobial property of the silver for both E. coli and MRSA (Figure 2). Another role of the zeolite is to act as an encapsulant. In complex biological media, the amount of silver necessary to have antimicrobial effect increases significantly.(25,52,53) Proteins and membranes in biological media form complexes with silver ion,(54) and silver deposits are found in clinical wound exudates from patients with venous ulcer.(55) Nutrient media decreases bioavailability of silver by 2 orders of magnitude as compared to water/PBS and level of silver is 5-fold lower in biological fluids as compared to nutrient.(12,52,56)

Figure 3 shows that for comparable levels of silver/zinc ions, the zeolite has a significantly stronger antimicrobial effect than the free ions in a typical culture medium. Use of the zeolite offers protection from precipitation of the encapsulated silver ion versus silver ion in the free form. An advantage to this lack of deactivation is that lower amounts of silver will be needed to exhibit the same antimicrobial effect. It has been noted that even with sustained silver release for chronic wounds, residual infections are still present, indicating that silver may not be reaching the interior of the wounds or that biofilms prevent the antimicrobial effect.(54). Though not proven in this study, it is possible for the colloidal nanozeolite to penetrate deeper into the wound fluid since it is surrounded by a protein corona, as is well known for nanoparticles of this size in biological fluids.(57,58)

3) Biofilm Inhibition: AM30 was very effective in both broth and PBS for inhibiting biofilm formation as analyzed by the crystal violet assay, as shown in Figures 4 and 5. The combination of quat with AM30 has an added effect on the biofilm inhibition.

4) Mature biofilm studies: As shown in Figure 9, the solutions that were used to extract the bacteria from the PAO1 biofilm on the cellulose membrane as well as on the ABF-XenoMEM dressing after 24h of contact had no bacterial colonies, implying that the actives in the ABF-XenoMEM could access the bacteria in the biofilm and kill the bacteria (a reduction of 8.6 log₁₀ CFU/ml as compared to XenoMEM control). AM30 or quat alone on the XenoMEM^™ was not as effective (Figure 9). Zeta potential measurements (reflecting surface charge) of the AM30 shows that the positively charged quat associates with the negatively charged zeolite surface. Coulombic association of zeolite with a quat to create composites that are efficient for solute/drug extraction has been reported.(59) Other studies of zeolite quat composites shows similar trends as reported here, with the negative charge on the zeolite being compensated with increasing amounts of quat, and finally reaching positive potentials, indicating charge reversal and multilayer formation as the quat covers the zeolite surface.(59,60)

However, the quat-AM30 being coulombically associated is still a hypothesis in real wound fluids, since we have not studied how the association changes in a complex biological fluid with other charged species present. However, there is strong evidence that combination of the zeolite and the quat helps in penetrating the biofilm, and the fact that there were no bacteria in the media used to extract cells on the cellulose membrane or the dressing implies killing of the biofilm bacteria (Figure 9). There is a commercial dressing with quat (benzalkonium chloride) dispersed in polyethylene glycol base (1.44 mg quat in 440 mg of PEG, sodium citrate and citric acid) (61) that can be compared to the quat-XenoMEM (221 ug/cm² quat) in Figure 9. We observe a reduction of 2-log₁₀ CFU/ml in PAO1 (48h) biofilm in both dressing and membrane after treatment with quat-XenoMEM as compared to XenoMEM. Several modifications of the quat-PEG dressing (all similar levels of quat) were tested, (61) two of these performed similarly with the quat-XenoMEM shown in Figure 9, but with one of the quat-PEG formulation, the formation of PA biofilm (24h) was completely disrupted, the paper claims that there is some other proprietary material in this dressing.(61) Another study found the quat-PEG commercial dressing exhibited a reduction of 4-log₁₀ decrease in CFU, with the control having 8-log₁₀ CFU for PAO1 biofilms grown in a porcine explant model.(62)

5) Our rationale for the amount of zeolite and quat used in the ABF-XenoMEM dressing was to use the minimum amounts that could penetrate the biofilm and kill the bacteria of mature PAO1 biofilms within a 24-hour period (Figure 8 and 9). The reason for not increasing the concentration of the antimicrobials any further in the dressing was that both silver and quat are cytotoxic, and cytotoxicity increases with increasing concentrations.

6) Choice of the matrix of XenoMEM^™ was made with the rationale that once the biofilm is penetrated by the quat+zeolite and bacteria killed, then the ECM matrix can assist in the wound healing. XenoMEM^™ has been shown to assist wound healing.(49)

Comparison with Commercial Dressings:

Bacteria in biofilms exhibit considerable resistance to antimicrobials as compared to these same bacteria in the form of planktonic cells. (14,63) The antimicrobial effect of silver ion against bacteria in mature biofilms is poor.(64) Considerable effort has gone into designing dressings that can disrupt the biofilm such that the bacteria become accessible, more planktonic-like, so that the antimicrobial silver can destroy the bacteria. We have chosen four such commercial dressings and discussed them in detail below.

Structural Aspects of Commercial Dressings: Table 1 is a brief comparison of the dressings in this study. Acticoat^™7 has silver in the form of Ag nanoparticle (instead of silver ions) deposited on a high-density polyethylene mesh. It has the highest amount of silver (1700 µg/cm²) amongst the dressings examined. A rayon/polyester non-woven inner core is laminated between the three layers of the Ag NP-polyethylene mesh.(65,66) AgNP were reported to be better prophylaxis of infection as compared to silver ion dressings,(15) the nanoparticles have the potential to reach deeper into biofilms.(9) Aquacel^® Ag⁺ Extra^™contains ethylene diamine tetraacetic acid (EDTA), benzenethionium (BT) chloride and silver ion (170 µg/cm²) in a hydrofiber matrix.(66,67) The EDTA is added to sequester ions that help in the structural integrity of the biofilm and the surfactant BT can impact the biofilm architecture.(18,68) Procellera^™ was chosen because it contained both silver (900 µg/cm²) and zinc (300 µg/cm²).(69) Procellera^™ is made up of alternating metallic silver and zinc dots held by a biocompatible binder on a polyester substrate. In the presence of a conducting medium, there is a voltage generated between the two metal of 0.5-0.9 V, with silver acting as the positive terminal. The proposed mechanism of antimicrobial action is that the negatively charged planktonic bacteria is attracted to the positive charge on the silver electrode, and the ensuing contact promotes the killing of the bacteria. Promogran Prisma™ was chosen since it uses an ECM matrix (as does ABF-XenoMEM), and has the lowest amount of silver ion (20 µg/cm²) amongst the dressings examined.(67)ABF-XenoMEM is made by depositing nanoparticles containing silver ion (30 ug/cm²), zinc ion (6 ug/cm²) and quat (221 ug/cm²) onto an ECM matrix. ABF-XenoMEM has silver at slightly higher concentration of silver that Promogran Prisma™ (30 µg/cm² versus 20 µg/cm²).

As noted in the SEM micrographs of Figures 10 and the elemental maps of Figure 3S, the silver is distributed uniformly on the ECM matrix in ABF-XenoMEM. This is in contrast the to the silver distribution of Aquacel^® Ag⁺ Extra^™and Acticoat^™7. With Aquacel^® Ag⁺ Extra^™, SEM shows that the silver ions resides in the background behind the raised stitching, and lack of antimicrobial effect was noted in specific areas of the dressing due to the dead space.(22,44) In Acticoat^™7, isolated silver nanoparticles are observed in the SEM, distributed along the polyethylene net.(44)

Mature biofilm studies: We compared ABF-XenoMEM with four leading commercial silver-based wound dressings for their ability to reduce the bacteria in mature biofilms of PAO1 and MRSA (Figure 11 and 12, Figure 6S and 7S). Our procedure was consistent across all five dressings. Biofilms were grown on cellulose membranes for 48h, covered with SWF (to mimic the presence of wound fluids), and then exposed to the dressings for 24 hours. The arrangement was such that biofilm bacteria can interact with the dressing directly as well as with antimicrobials released into the SWF.(12,52,56) The dressings and the membrane were analyzed for resident bacteria (except for Promogran Prisma™ and ABF-XenoMEM, where the membrane and dressing were stuck in places and could not be cleanly separated). There have been many in vitro biofilm studies using similar methodology on the commercial membranes reported in the literature and they provide a benchmark against the data that we observe and form the basis of this discussion. This type of colony biofilm model is very typical as the first experiment with new dressings. It gives an idea of the comparative performance, but conclusions drawn from such experiments as to performance of the dressings in dealing with real infected wounds cannot be drawn.

ABF-XenoMEM: Twenty-four-hour exposure of a 48h PAO1 biofilm on a cellulose membrane to ABF-XenoMEM resulted in a reduction of 4.6-log₁₀ CFU/ml (compared to the control sample at 9.6-log₁₀ CFU/ml) in the extracts from the membrane and the dressing (note that the result is different from Figure 9 and is possibly due to the inclusion of the SWF in these experiments). With MRSA, there were no detectable bacteria remaining on the membrane plus the dressing, thus a reduction of 9.8-log₁₀ CFU/ml relative to control. With both these experiments, the dressing could not be completely separated from the membrane, and both were analyzed together.

Acticoat^™7: In the present study, this dressing performed better with MRSA biofilms, there was a reduction of 7.3-log₁₀ CFU/ml (bacterial counts in the extract) relative to control of 9.8-log₁₀ CFU/ml, whereas with PAO1, there was a reduction of 3-log₁₀ in CFU/ml (in the extract) relative to control of 9.6-log₁₀ CFU/ml (bacterial counts in CFU/ml in the extracts from the membrane and dressing are added). Figures 6S and 7S show the bacterial counts in CFU/ml in the two extracts for the cellulose membrane and dressing, respectively. Literature studies with Acticoat^™7 provide comparison with data shown in Figures 11 and 12. In a mature mixed-species PA+MSSA biofilm established in a Drip Flow Biofilm Reactor for 3 days on hydroxyapatite coated slides, reductions of 3.42-log₁₀ CFU/cm² (statistically significant) for MSSA and 1.3- log₁₀ CFU/cm² for PA were noted for Acticoat^™7 (controls were between 9-10 log₁₀CFU/cm²) for a 24h treatment.(70) In another study (12), for PA biofilm there was a 4.2-log₁₀ reduction in CFU/peg (control 7.6-log₁₀CFU/peg), and with MRSA biofilm there was a 4.6-log₁₀ reduction in CFU/peg (control 6.2-log₁₀ CFU/peg) at 24-h mark with Acticoat^™7. With PA, the viable cells on dressing were 1.2-log₁₀ CFU/cm² and with MRSA cells 1.8-log₁₀ CFU/cm² at 24-h mark.(12) The higher activity against S. aureus in both studies is consistent with our observation. With biofilms grown on gauze, Acticoat^™7 was found to be ineffective for both MRSA and PA biofilms (grown for 48-h), and not consistent with our observation.(68)

Aquacel^® Ag⁺ Extra^™: In the present study, this dressing performed better against PAO1 biofilms, showing a reduction of 6.4-log₁₀ CFU/ml decrease relative to control of 9.6-log₁₀ CFU/ml and with MRSA, a reduction of 5.3-log₁₀ CFU/ml relative to control of 9.8-log₁₀ CFU/ml (bacterial counts in CFU/ml in the extracts from the membrane and dressing are added). Literature reports with Aquacel^® Ag⁺ Extra^™ against MRSA biofilm (48 h) found a reduction of 2-log₁₀ CFU (control 10 log₁₀CFU) after 24 h exposure, and for PA biofilms (48 h) a reduction of 6-log₁₀ CFU (control 10 log₁₀ CFU) was observed after 24 h.(68)Results showing significantly better activity towards PA biofilms is consistent with the present study. Another study using colony drip-flow reactor with younger PA biofilms (24h), 24 h exposure to Aquacel^® Ag⁺ Extra^™exhibited reduction of 2.6-log₁₀ CFU/ml relative to control gauze (9.24-log₁₀CFU/ml); however, for PA biofilms that were matured for 72 h, the dressing brought about a decrease of only 0.4-log₁₀ CFU/ml (control gauze 9.2-log₁₀CFU/ml).(67) In another CDC reactor biofilm model study, 72 h Staphylococcus aureus (SA) and PA biofilms were exposed to Aquacel^® Ag⁺ Extra^™for 24 h, no bacteria was recovered for both biofilms (a reduction of 4.3-log₁₀ CFU/ml compared to control for SA and a reduction of 6.4-log₁₀ CFU/ml for PA).(71) In a different study, SA and PA biofilms were grown on the Whatman cyclopore circular membrane on nutrient agar plates for 24 h to form colony biofilms. Treatment with Aquacel^® Ag⁺ Extra^™led to a reduction of 1.2-log₁₀ CFU for SA (control 9.2-log₁₀CFU) in 24-h, whereas for PA, no change was observed at 24 h (study was continued for 5 days, and activity towards SA biofilms were superior that PA biofilms), inconsistent with our study.(22)

Procellera^™: In the present study, this dressing did not perform well relative to the others, a reduction of 1-log₁₀ CFU/ml following treatment of PAO1 biofilms relative to control of 9.6-log₁₀ CFU/ml and no change relative to control of 9.8-log₁₀ CFU/ml for MRSA (bacterial counts in CFU/ml in the extracts from the membrane and dressing are added). In the literature, the dressing was found to be effective over a 24 h period against killing many planktonic bacteria isolated from clinical wounds.(72) Using a colony biofilm reactor, clinical wound isolates were introduced onto cellulose filter membranes, bacteria were circulated for 72 hours and the resident bacteria on the membrane and dressing was analyzed. A reduction of 2-log₁₀ CFU/ml was noted for PA (with blank polyester control of 8-log₁₀CFU/ml) and reduction of <1-log₁₀ CFU/ml decrease for SA with 6-log₁₀ CFU/ml for control gauze. This procedure is slightly different from what is being practiced in this paper; however, the extent of bacterial decrease for PA and MRSA biofilms following treatment (1-2 log₁₀CFU/ml) in this study were of similar levels as the biofilm reactor study.(73)

Promogran Prisma™: In the present study, a reduction of 3.6-log₁₀ CFU/ml relative to control of 9.6-log₁₀ CFU/ml was observed for treated PAO1 biofilms, and reduction of 4.8-log₁₀ CFU/ml for MRSA biofilm relative to control of 9.8-log₁₀ CFU/ml (extracts were prepared from the dressing and membrane combined). From a study using a colony drip-flow reactor, PA biofilms were grown for 24 h, and upon 24 h exposure to dressing, a reduction of 2.77-log₁₀ in CFU/ml relative to control gauze with 9.24-log₁₀ CFU/ml was observed. These results for PA are consistent with our observations.(67)

Cytotoxicity:

Silver toxicity towards prokaryotes and eukaryotes are well studied.(41,74) Cytotoxicity towards HepG2 cells as evaluated by the MTT assay was quite pronounced for all the dressings (~5-10% cell viability). Within statistical significance, Procellera^™, Aquacel^® Ag⁺ Extra^™were less cytotoxic than ABF-XenoMEM. (Figure 13). Acticoat^™7 with the highest silver content shows the most pronounced cytotoxicity and was significantly more toxic than ABF-XenoMEM. A previous study reported that extracts from Acticoat^™7 and Aquacel^® Ag⁺ Extra^™exhibited comparable toxicity towards keratinocytes by MTT assay.(44) In designing ABF-XenoMEM, we attempted to keep the silver concentration low so as not to disrupt the healing process. However, inclusion of quat increases the cytotoxicity as noted with quat-PEG dressings.(63)

The new wound dressing ABF-XenoMEM proposed in this paper has the following characteristics: storage of the silver in nanozeolite so that it does not precipitate in the wound fluid, exploiting the microporous nature of the zeolite to spatially co-locate zinc ion with the silver ion to enhance the antimicrobial activity of silver, inclusion of positively charged quats with the negatively charged zeolite to promote the anti-biofilm activity of the dressing, and extended release of the antimicrobial silver over 4 days. Overall, we can conclude that the ABF-XenoMEM is competitive with the commercial dressings in decreasing PAO1, and MRSA bacteria encapsulated in biofilms in an in vitro colony biofilm model and comparable in cytotoxicity. In vitro results are a promising start for this new type of wound dressing and are a common practice in this field. Future studies will focus on polymicrobial biofilms, ex-vivo assays and for animal models and human clinical studies to demonstrate the real potential of this novel nanozeolite dressing for wound healing of the biofilm-colonized wounds.

MRSA: methicillin-resistant Staphylococcus aureus; PAO1: Pseudomonas aeruginosa O1; SA: Staphylococcus aureus; FAU30: nanozeolite; AM30: silver ion+ zinc ion nanozeolite; quat: quaternary ammonium ion; ABF-XenoMEM: silver-zinc nanozeolite+quat on XenoMEM^™ dressing; SWF: simulated wound fluid; B: wound dressing; M: cellulose membrane.

Ethics approval and consent to participate:

This study did not involve human participants or animals, so ethics approval and consent to participate were not required.

Consent for publication:

All authors give consent for publication.

Availability of data and materials:

The datasets and materials used in this study are available from the corresponding author on reasonable request.

Competing interests:

ZeoVation is a startup company that grew out of the research at The Ohio State University (OSU), Prabir K. Dutta, Bo Wang and Sweta Shrestha are supported by ZeoVation, Inc. and have financial interests in ZeoVation.

Funding:

This work was supported by the United States-National Science Foundation through the Small Business Innovation Research program [grant number – 2025819], and Defense Health Agency (DHA) Restoral funding.

Authors contributions

Professors M.V.H. and P.K.D. conceived the idea for these experiments. S.A.A., M.T., S.S., and B.W. performed all the experiments. All authors contributed to the writing of the manuscript.

Acknowledgements

We thank Mr. Pete Gingras of Viscus Biologics for discussions related to XenoMEM^TM.

Supplementary Information

Figures related to the nanozeolite, elemental mapping of the active metal ions on the dressing, comparison of nano and micron-sized zeolite for coating, dynamic light scattering, and bacterial counts analysis on the wound dressing and cellulose membrane are included in the Supplementary Section.

Disclaimers

1) Any opinions, findings and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation. 2) The views expressed in this abstract are those of the author and do not necessarily reflect the official policy of the position of the Defense Health Agency, Department of Defense, or the United States Government. The experiments reported herein comply with the Animal Welfare Act and per the principles outlined in the “Guide for Care and Use of Laboratory Animals Resources,” National Research Council, National Academy Press, 1996. ABF-XenoMEM is an experimental product being developed by ZeoVation Inc. and is not a commercial product. Neither the Defense Health Agency nor any other component of the Department of Defense or the US government has approved, endorsed, or authorized this product for promotion, service, or activity.

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Competing interest reported. ZeoVation is a startup company that grew out of the research at The Ohio State University (OSU), Prabir K. Dutta, Bo Wang and Sweta Shrestha are supported by ZeoVation, Inc. and have financial interests in ZeoVation.

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Design of a silver-zinc nanozeolite-based antibiofilm wound dressing activity in an in vitro biofilm model

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