OBJECTIVE: Develop technology that stabilize bacteriophage (phage) cocktails for long-term storage and use in austere environments. The proposed material solution would aid in improving effectiveness of phage therapy, and ease of use by ensuring long-term stability of phage at a range of temperatures (-20 to 45oC). Phage spray drying, packaging in nanoparticle, hydrogel polymer matrix, or a combination thereof, or other relevant technologies will be considered. DESCRIPTION: Projected warfare dynamics during multi-domain operations (MDO) will require prolonged care (PC) and stabilization of injuries at the point of injury due to extended evacuation times. Under these circumstances, the potential for life threating infections increases, and the need for solutions that prevent and treat wound infections and stabilize the wound is crucial. The global spread of multidrug-resistant organisms (MDRO) creates challenges for all levels of care within the health care system. Military personnel wounded in combat are susceptible to infection by MDROs at a higher rate than the civilian population due to combat wounds typically being larger and accompanied by bone and soft tissue disruption, localized ischemia, as well as severe hemorrhage. The rising rates of MDROs coupled with multiple failures to develop novel classes of antibiotics has renewed interest in alternative treatments to combat MDRO wound infections including the use of phage therapies. Phage preparations are stable for years with refrigerated storage, but gradually lose activity when stored at ambient temperature. Therefore, methodologies and materials that help maintain phage stability at a range of temperatures and promote ease of use are needed to facilitate application of phage treatments during PC and at the point of injury where refrigeration is not available. In addition, materials that would aid in phage delivery to the site of injury (superficial wounds, deep wounds with bone and soft tissue disruption, severe hemorrhage, burns, etc.) and ensure sustained-release of phage would be desirable. The following features will be critical to consider when proposing phage stabilization and delivery technology or materials: 1. A universal methodology or material aiding in phage stabilization at ambient and extreme temperatures for a period of six months to one year. The technologies to be considered, but not limited to, are spray drying, encapsulation in micro or nanoparticles, and incorporation into the hydrogels. 2. Stabilized phage delivered as a powder or as a solution that is easily applied directly to the injury. 3. Multi component hydrogel or nanoparticle based bandage, injectable, etc. 4. Biocompatible natural or synthetic material, non-toxic i.e. not causing any irritation or damage to the tissue. 5. Hemostatic property is desirable for the phage stabilization material. 6. Sustained release of phages from material over time ensuring prolonged phage treatment of the injury. 7. Combination delivery of both phage and antibiotic simultaneously would be advantageous. 8. Ease of use at point of injury. PHASE I: should focus on development and validation of a universal technology for the stabilization of a wide variety of phage types to permit storage at a wide range of temperatures (-20oC to 45oC) for a period of one months. A. This could be achieved by phage drying, encapsulation in micro- or nanoparticles, emulsification in oil particles, crosslinking to stabilizing molecules like Polyethylene glycol (PEG) or any combination of those methods. If drying method was selected, excipients like trehalose, gelatin, mannitol, sucrose, or amino acids need to be tested to select ones that work best with the wide range of different types of phages at varying temperatures. If identification of universal excipients for high and low temperatures becomes challenging, two preparations for -20oC to 0oC, and 1oC to 45 C could be considered. Excipients that will be used for testing should be nontoxic, not cause irritation to the tissue, and should be on the list of chemicals approved by FDA for human use. B. Alternatively, if phages stabilized by encapsulation in particles of choice or directly into the hydrogels, the phage stability and release from these materials need to be tested at different temperatures. Use of materials that are nontoxic, biocompatible and biodegradable is preferred. Materials with tenability of phage release will have an advantage. Flexibility to use hydrogels as injectable dressing or bandage will be a plus. Once the method or material for phage stabilization is selected, the period of stability needs to be verified, and should be at minimum of one months for phase I. At the end of this phase, methods and materials for phage stabilization and materials or working prototype for phage delivery should be demonstrated. Technology needs to be tested and validated with different types of phages. PHASE II: Stability experiments should continue for the duration of phase II. After selecting the best stabilization method for a wide range of phage, the means of phage delivery needs to be identified. The final phage stabilization proof-of-concept prototype applicator for stabilized phage delivery needs to be selected and tested. This could be a small lightweight spray on device or nebulizer. Dried phage could also be stored in sealed pouches or vials to protect from harmful effects of humidity, and mixed with the carrier solution or hydrogel just before application to the wound. This could give some flexibility to add different component or filler depending on the type of wound i.e. apply to the bandage or use as injectable dressing for deep wound treatment. The system could be designed in such way that dry phage is rehydrated just before use. Sustained phage release for 1-3 days is desirable for phage delivery. PHASE III DUAL USE APPLICATIONS: Efforts during this phase should address the US Army Medical Materiel Development Activity and the office of Warfighter Protection and Acute Care small business. This phase should demonstrate evidence of commercial viability of the product. Accompanying application instructions, simplified procedures, and training materials should be drafted in a multimedia format for the use and integration of the product into the market. The end-state for this product is a commercially viable technology that will enable application of phage therapy for prevention or acceleration of healing of wound infections and will aid in warfighter health protection efforts within the Department of Defense (DoD). The final product should provide phage stability and activity of a year or greater. This product could also be broadly applicable to the civilian health care system for wound care, especially against multi-drug resistant infections. The Contractor may collaborate with the Walter Reed Army Institute of Research (WRAIR) team in optimizing and validating system. Potential funding pathways include MTEC and JWMRP. REFERENCES: 1. TRADOC Pamphlet 525-3-1: The U.S. Army in Multi-Domain Operations 2028. (United States Army Training and Doctrine Command, 2018). Or https://wrairintranet.army.mil/WRAIRCares/toolchest/HHC/2019_army_modernization_strategy_final.pdf#search=MDO 2. Jonczyk E., Klak M, Miedzybrodski R, Gorski A. The influence of external factors on bacteriophages review. Folia Microbiol. 2011; 56: 191-200. 3. Malik, D.J., et al., Formulation, stabilization and encapsulation of bacteriophage for phage therapy. Adv. Colloid Interface Sci, 2017. 249: p. 100-133. 4. Kaur, P.; Gondil, V.S.; Chhibber, S. A novel wound dressing consisting of PVA-SA hybrid hydrogel membrane for topical delivery of bacteriophages and antibiotics. Int. J. Pharm. 2019, 572, 118779. 5. Barros, J.A.R.; Melo, L.D.R.d.; Silva, R.A.R.d.; Ferraz, M.P.; Azeredo, J.C.V.d.R.; Pinheiro, V.M.d.C.; Cola o, B.J.A.; Fernandes, M.H.R.; Gomes, P.d.S.; Monteiro, F.J. Encapsulated bacteriophages in alginate-nanohydroxyapatite hydrogel as a novel delivery system to prevent orthopedic implant-associated infections. Nanomed. Nanotechnol. Biol. Med. 2020, 24, 102145 6. Wroe, J.A., C.T. Johnson, and A.J. Garcia, Bacteriophage delivering hydrogels reduce biofilm formation in vitro and infection in vivo. J Biomed Mater Res A, 2020. 108(1): p. 39-49. KEYWORDS: Bacterial infections, multidrug resistance, phages as antibacterial therapy, therapeutic phage, phage delivery material, phage stabilization, delivery application, spray drying, hydrogels, nanoparticle, alginate, chitosan, controlled release, biodegradable
