Targeted Delivery of Narrow-Spectrum Protein Antibiotics to the Lower Gastrointestinal Tract in a Murine Model of Escherichia coli Colonization.

Bacteriocins are narrow-spectrum protein antibiotics that could potentially be used to engineer the human gut microbiota. However, technologies for targeted delivery of proteins to the lower gastrointestinal (GI) tract in preclinical animal models are currently lacking. In this work, we have developed methods for the microencapsulation of Escherichia coli targeting bacteriocins, colicin E9 and Ia, in a pH responsive formulation to allow their targeted delivery and controlled release in an in vivo murine model of E. coli colonization. Membrane emulsification was used to produce a water-in-oil emulsion with the water-soluble polymer subsequently cross-linked to produce hydrogel microcapsules. The microcapsule fabrication process allowed control of the size of the drug delivery system and a near 100% yield of the encapsulated therapeutic cargo. pH-triggered release of the encapsulated colicins was achieved using a widely available pH-responsive anionic copolymer in combination with alginate biopolymers. In vivo experiments using a murine E. coli intestinal colonization model demonstrated that oral delivery of the encapsulated colicins resulted in a significant decrease in intestinal colonization and reduction in E. coli shedding in the feces of the animals. Employing controlled release drug delivery systems such as that described here is essential to enable delivery of new protein therapeutics or other biological interventions for testing within small animal models of infection. Such approaches may have considerable value for the future development of strategies to engineer the human gut microbiota, which is central to health and disease.

Bacteriophage Encapsulation in pH-Responsive Core-Shell Capsules as an Animal Feed Additive.

Increasing antibiotic resistance in bacteria that cause zoonotic infections is a major problem for farmers rearing animals for food as well as for consumers who eat the contaminated meat resulting in food-borne infections. Bacteriophages incorporated in animal feed may help reduce carriage and infections in animals including chickens and pigs. There are, however, unmet challenges in protecting phages from processing stresses e.g., during animal feed pelleting operations and during transit of phages through the acidic gastric environment. Core-shell capsules were produced using a concentric nozzle and commercially available encapsulation equipment to fabricate capsules with phages formulated in an oil-in-water microemulsion in the core. pH-responsive capsules released the encapsulated phage cargo within 10-30 min triggered by changes in local environmental pH typically found in the lower gastrointestinal (GI) tract of animals. Acid stability of phages exposed to pH values as low as pH 1 was demonstrated. Encapsulated phages were able to withstand exposure to 95 °C wet heat thermal stress for up to 120 s, conditions typically encountered during feed pellet extrusion processing. Free phages were inactivated within 15 s under these conditions. The present study demonstrates that encapsulation of bacteriophages in core-shell pH-responsive capsules with water-in-oil emulsified phages in the core significantly improves phage viability upon exposure to processing and environmental stresses that require consideration during production of animal feed and application in animals for biocontrol. The results from this study should help guide future development of phage formulations suitable for use in animal feed for animal biocontrol applications.

Microencapsulation of Bacteriophages Using Membrane Emulsification in Different pH-Triggered Controlled Release Formulations for Oral Administration.

An E.coli-specific phage was encapsulated in three different pH responsive polymer formulations using the process of membrane emulsification. Small 100 µm capsules were fabricated and shown to afford phages suitable acid protection upon exposure to pH 1.5. Selection of polymer formulations allowed controlled release of phages at pH 5.5, pH 6 and pH 7. Other aspects of phage encapsulation including factors affecting encapsulation yield, release kinetics, acid and storage stability were evaluated. The work presented here would be useful for future evaluation of new therapeutic strategies including microbiome editing approaches allowing pH-triggered release of phages and delivery of encapsulated cargo to different intestinal compartments. The size of the capsules were selected to permit ease of delivery using small bore oral gavage tubes typically used in pre-clinical studies for evaluation of drug substances using small animal vertebrate models such as in mice and rats.

Microencapsulation of Enteric Bacteriophages in a pH-Responsive Solid Oral Dosage Formulation Using a Scalable Membrane Emulsification Process.

A scalable low-shear membrane emulsification process was used to produce microencapsulated Escherichia coli-phages in a solid oral dosage form. Uniform pH-responsive composite microparticles (mean size ~100 µm) composed of Eudragit® S100 and alginate were produced. The internal microstructure of the gelled microcapsules was studied using ion-milling and imaging, which showed that the microparticles had a solid internal core. The microencapsulation process significantly protected phages upon prolonged exposure to a simulated gastric acidic environment. Encapsulated phages that had been pre-exposed to simulated gastric acid were added to actively growing bacterial cells using in vitro cell cultures and were found to be effective in killing E. coli. Encapsulated phages were also shown to be effective in killing actively growing E. coli in the presence of human epithelial cells. Confocal microscopy images showed that the morphology of encapsulated phage-treated epithelial cells was considerably better than controls without phage treatment. The encapsulated phages were stable during refrigerated storage over a four-week period. The process of membrane emulsification is highly scalable and is a promising route to produce industrial quantities of pH-responsive oral solid dosage forms suitable for delivering high titres of viable phages to the gastrointestinal tract.

Sarcopenia is related to physical functioning and leg strength in middle-aged women.

BACKGROUND: In the aging process, loss of muscle is relatively continuous, but the initiation, timing, and amount of muscle loss that relate to functional compromise are poorly described. Also poorly understood is whether strength and functioning in aging are related to the amount of lean mass and its change as well as to the amount of fat mass and its change. METHODS: The purpose of the study was to ascertain whether 3-year lean and fat mass change predicted functional status in 712 African American and Caucasian women, aged 34-58 years. Fat and lean mass were assessed with bioelectrical impedance. Lower leg strength (torque) was measured with a portable isometric chair, and two indices of physical functioning, walking velocity and double support (both feet touching the surface while walking), were measured with an instrumented gait mat. RESULTS: Almost 9% of middle-aged women had at least a 6% loss (>2.5 kg) of lean mass over the 3-year observation period. Women who lost at least 2.5 kg of lean mass had slower walking velocity and less leg strength, although women who simultaneously gained more than 2.5 kg of fat mass (at least 7.5%) did not have less leg strength. Age was significantly associated with less velocity, less leg strength, and more time in double support. CONCLUSIONS: Even in middle-aged women, there is loss of lean mass among almost 1 woman in 10, and this loss of lean mass (sarcopenia) is associated with greater compromise in physical functioning.