Bacteriophage delivering MACF hydrogels for targeted antibacterial action in diabetic wound healing

Introduction: Diabetic foot ulcers (DFUs) are a frequently occurring and devastating clinical complication of diabetic patients and are often colonized with multiple strains of pathogenic bacteria, including antibiotic resistant strains. Standard treatment of DFU infection involves systemic or topical application of strong antibiotics, although topical antimicrobials are favored to limit system toxicity. Biofilm formation is common in chronic wounds, up to 78.2% of all chronic non-healing wounds colonized by a biofilm, which can contribute to local hypoxia, impede wound healing processes, and increase antibiotic resistance within. Treatment of biofilms with standard antimicrobials is limited by decreased diffusion through its structural matrix, leading to increased immunity to antibiotics of biofilms. Due to the surge in multidrug antibiotic resistance microbes, new treatment avenues must be explored to effectively combat pathogenic infection and biofilms that otherwise can result in gangrene, sepsis, and subsequent mortality if left untreated. One potential avenue for treatment can be found in bacteria’s natural predator, bacteriophages. Phages are pathogenic-specific viruses that can invade, replicate within, and lyse bacteria while leaving mammalian and non-targeted natural flora intact. Treatment via phages also allows for penetration through the biofilms that commonly form in chronic wounds through production of natural biofilm eradication mechanisms. The overall goal of this work is to explore the ability of our oxygenating fluorinated methacrylamide chitosan (MACF) hydrogels to serve as a synergistic scaffold to maintain and deliver PP7 phage specific to pathogenic targets (e.g., Pseudomonas aeruginosa) in both planktonic and biofilm models and allowing for improved wound healing responses.

Materials and

Methods: Materials synthesis. MACF was made as previously described [1-3], and liquid MACF was mixed with 1X PBS before being loaded with 106 PFU of PP7 phage, and photo-crosslinking to create PP7 impregnated hydrogel dressings.
Release kinetics, bactericidal ability, biofilm development. PP7 saturated MACF hydrogels (MACF-PP7) were incubated at 37 C in 1X PBS. At 1, 2, 4, 6, 8, 24, 48, and 72 h time points, the full volume of PBS was removed and replaced. PBS collections were purified for RNA before quantifying via the RiboGreen (Invitrogen) RNA quantification assay. To test direct bactericidal ability against planktonic bacteria, MACF-PP7 hydrogels were incubated with actively growing P. aeruginosa liquid cultures for 24 hours before quantifying via BacLight bacterial viability (Invitrogen) assays compared to non-embedded phage, standard MACF, and bacterial controls. Not only are phages effective against planktonic bacteria, but they possess the natural abilities to invade biofilms. A P. aeruginosa biofilm model was developed using the colony biofilm method on sterilized polycarbonate filters.
In vitro skin organoid model. Human Dermal Fibroblasts (HDFs) were seeded (2.5 x 105 cells/mL) in a 3% Methacrylated Gelatin (GelMA) matrix and incubated at 37 C. After 3 days, Primary Epidermal Keratinocytes (PEK) were seeded (2 x 105 cells) onto the fibroblast-containing collagen scaffold and incubated for a further 3 days. After 3 days, the constructs were incubated in an air-liquid interface before P. aeruginosa biofilms were applied at the surface. MACF-PP7 hydrogels were applied to biofilms and incubated for 24 hours before biochemical and molecular analyses.

Results, Conclusions, and Discussions: The increased stability of phages when released from hydrogel scaffolds is beneficial to a phage therapy system and maximum release kinetics were quantified to determine appropriate dressing replacement timing. Using RNA quantification, we determined that 100% of PP7 loaded into MACF was released between the first 24 and 48 hours (Figure 1A). This suggests dressings should be replaced every 48 hours for maximum benefit of phage. However, phage released from the hydrogel also need to maintain bactericidal ability. After 24 hours of coculture, bacterial viability assays were completed. MAC hydrogels were also tested as a control for effects of localized oxygenation, and methacrylated hyaluronic acid (MeHA) hydrogels as a control for of our chitosan base polymer, which is known to have a slight antimicrobial effect. MACF-PP7 hydrogels had the most effective reduction in P. aeruginosa load after 24 hours, and MAC-PP7 and MeHA-PP7 based hydrogels were not significantly different from the liquid PP7 control (p < 0.0001). All treatments without phage showed no effect on bacterial viability compared to bacterial controls (Figure 1B). All statistical analysis was completed by one-way ANOVA with Tukey’s post-hoc analysis, n = 3, different letters denote statistical significance. Due to increased effectiveness of MACF-PP7 hydrogels in bactericidal ability against planktonic bacteria, we decided to utilize this treatment moving forward.
We also are developing a human biofilm model to test our phage releasing hydrogels against standard antibacterial effectiveness. P. aeruginosa was again selected, due to its high prevalence in DFUs and its biofilm forming capabilities. Biofilms were visibly formed on polycarbonate filters after 48 hours of culture at 37 C (Figure 1C, scale bar is 1mm). These biofilms will serve as the basis for bactericidal ability testing of MACF-PP7 releasing hydrogels (Figure 1D) and will also be utilized in an in vitro skin organoid model to study the effects of MACF-PP7 hydrogels on bacterial and cell viability (Figure 1E). Phage therapies demonstrate promise towards targeted antibacterial action in vitro and in vivo, and MACF hydrogels can effectively serve as a scaffold for delivery, while increasing diffusion of localized oxygen for improved host cell metabolism and resulting wound healing.

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