(586c) Bulk and Electrospun Phage Poly(Oligoethylene Glycol Methacrylate)-Based Hydrogels Show Antimicrobial Action Toward Pseudomonas Aeruginosa

Background: Hospital-acquired infections (HAIs) represent one of the largest burdens on healthcare systems around the world [2]. A combination of rising anti-microbial resistance (AMR) and improvements to burn wound care strategies has led to the primary cause of morbidity shifting from anoxic injury to infection and sepsis [7]. Due to the rising incidence of AMR, it is imperative to explore alternatives to conventional antibiotics. One such alternative is the use of phage therapy. To deliver phage to topical wounds, hydrogels have been investigated given their capacity to load phage and maintain them in a hydrated state as well as simultaneously absorb and thus manage wound exudate. While some existing combinations of phage with a hydrogel have been reported [1, 3, 5, 8], currently reported hydrogels tend to have very quick release profiles, meaning significant amounts of the phage are wasted and daily changes of the dressing would be required to retain antibiotic effects. The goal of this work was to evaluate the use of an in situ-gelling hydrogel to manage burn wound exudate coupled with loaded phage to prevent bacterial infection, using Pseudomonas aeruginosa as the model organism. It is hypothesized that entrapping phage in a dynamically-crosslinked hydrogel network will allow for longer release profiles but maintained phage activity, thereby facilitating longer efficacy than currently used antibiotic materials.

Methods: 10^{11 –} 10¹³ PFU/mL of vB_Pae-Kakheti25 (phage for P. aeruginosa) was mixed with hydrazide and aldehyde-functionalized poly(oligoethylene glycol methacrylate) (POEGMA), a copolymer system that gels when the two functionalized polymer solutions are mixed to form a reversible hydrazone-crosslinked hydrogel network. The stability of the phage in each of the copolymers was measured by titering phage at various time intervals. To form bulk gels, the copolymer solutions (with phage dosed in the hydrazide-functionalized copolymer) were injected into a disc-shaped silicone mould and allowed to gel overnight at 4 °C. Electrospun gels were formed by mixing each copolymer with 5 wt% poly(ethylene oxide) (M_W = 600 kg/mol) in equal volumes, with phage added to the hydrazide-functionalized POEGMA/PEO solution. Solutions were loaded into a double-barrel syringe fitted with a static mixer and 18G needle, and a stationary flat-disc collector was placed 10 cm away. A 10 kV voltage was applied to the needle and collector, with copolymer solutions extruded at a flow rate of 15 μL/min. Electrospun scaffolds were then punched with a 0.5 in diameter punch .

Both bulk gels and electrospun scaffolds were tested for swelling, phage release, and bacterial load reduction. Electrospun scaffold fibre diameter and orientation were also characterized using scanning and transmission electron microscopy to assess scaffold morphology and phage integration with the scaffold. To measure swelling, gel discs were submerged in SMG, a salt buffer for phage preservation, at ambient conditions. Excess moisture was removed by gentle wicking with a Kimwipe before daily weighing. To measure release, each gel disc was submerged in SMG buffer in a 6-well plate and incubated at 37°C in a static incubator. Both well and well media were changed for each gel every 24 hours for five days, with the recovered samples titered to assess phage concentration using the double agar overlay plaque assay. To test bacterial load reduction, gels were submerged in 3 mL of TSB broth in a 12-well plate. Exponentially growing bacteria were immediately added to each well to a final concentration of ~10⁵ CFU/mL and incubated at 37°C and 90 rpm, with samples collected at various times over 24 hours. Optical density (OD₆₀₀) measurements were used to determine the optimal dilution for immediate use of the standard plate count method.

Results: showed loss of infectivity within 20 minutes at ambient conditions when stored in the aldehyde copolymer solution but high retained infectivity when phage was stored in the hydrazide copolymer solution; as such, phage was loaded only in the hydrazide copolymer prior to gel formation. Bulk hydrogels were formed at two polymer concentrations (10 and 20 wt%) to assess the effect of polymer concentration on phage stability/release and exudate sorption. Electrospun scaffolds were fabricated at a final polymer concentration of 6.67 wt%, a concentration optimized to ensure good fiber formation during the reactive electrospinning process. Bulk gel swelling tests indicated that both hydrogels tested were stable over any relevant time period for practical clinical use (>1 week). While 20 wt% precursor polymer gels were able absorb a higher volume of model exudate (1.6x by weight versus 1.4x by weight after three days), both have significant absorptive capacity for effective wound exudate management. Scanning electron microscopy of the electrospun scaffolds showed an average fiber diameter of 279 ± 47 nm (Figure 1D); however, encapsulation of phage within the gel nanofibers cannot be ruled out as a possible loading mechanism.

Both bulk and electrospun hydrogels facilitated release of bioactive phage. Bulk gels released phage at a rate independent of the polymer concentration used to prepare the hydrogel but with near zero-order release kinetics over three days, a typical period of use for a wound bandage. Phage was incorporated into the bulk gel at a concentration of approximately 8x10⁹ PFU/mL, while between 1x10⁵ to 1x10⁶ PFU/mL of phage was released each day over the three-day observation period. For the electrospun scaffolds, a higher concentration of 3x10¹¹ PFU/mL was loaded into the scaffold and 10⁷ PFU/mL phage was released after 7 hours of incubation. Correspondingly, when incubated with exponentially growing bacteria under regular lab growth conditions, both bulk (Figure 1B) and electrospun (Figure 1C) phage gel discs were able to fully prevent bacterial growth for at least 8 hours, representing a growth inhibition of at least 3-logs.

Discussion: Since bulk POEGMA gels can be formed via in situ gelation, they allow for custom shapes and sizes of gels to be formed directly on the wound. This provides an advantage over currently used pre-cast hydrogel bandages that need to be cut to shape. However, to match the benefits of such a pre-cast hydrogel, our electrospun version of the phage hydrogel can meet these needs.

For phage release tests, SMG buffer was selected to measure phage release as previous testing found phage to be unstable at ambient conditions in PBS buffer. This also provides direction for end use as the shelf life of phage can limit utility of phage-based hydrogels. Based on the titer of phage from the release experiments in SMG buffer, we expected the phage concentration to be approximately two orders of magnitude higher than the bacteria, giving a high likelihood of bacterial load reduction. Correspondingly, we saw a multi-log reduction of the bacterial load in these tests, suggesting that there is room for the gel to tackle higher bacterial loads. The currently tested bacterial load represents a low bioburden wound, and literature indicates that it is unlikely that solely phage-based hydrogels would be able to tackle high bioburden wounds [6]. It should also be noted that P. aeruginosa is known to develop resistance and persistence to single phages rapidly, and so inability to fully lower bacterial load at 24 hours is an expected result. However, in such cases, co-encapsulating an antibiotic within the porous hydrogel structure may address this issue [4], something that has already been demonstrated to be possible with the POEGMA-based hydrogel matrix [9]. The sustained release of phage from these gels (near-zero order release for at least 3 days) represents a more clinically-relevant profile than achievable with current gels [1, 3, 5, 8]. The gel design is also modular in that the encapsulation of multiple types of phage should be achievable via a mix-and-match (or combination) approach for personalized care.

Significance: Our results indicate that phage-containing POEGMA hydrogels have potential for treating infected burn wounds, offering a new potential therapeutic solution (either acting alone or synergistically with other antibiotics) to deal with emerging wound infection challenges.

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