Microencapsulation of Clostridium difficile specific bacteriophages using microfluidic glass capillary devices for colon delivery using pH triggered release

Abstract

The prevalence of pathogenic bacteria acquiring multidrug antibiotic resistance is a global health threat to mankind. This has motivated a renewed interest in developing alternatives to conventional antibiotics including bacteriophages (viruses) as therapeutic agents. The bacterium Clostridium difficile causes colon infection and is particularly difficult to treat with existing antibiotics; phage therapy may offer a viable alternative. The punitive environment within the gastrointestinal tract can inactivate orally delivered phages. C. difficile specific bacteriophage, myovirus CDKM9 was encapsulated in a pH responsive polymer (Eudragit® S100 with and without alginate) using a flow focussing glass microcapillary device. Highly monodispersed core-shell microparticles containing phages trapped within the particle core were produced by in situ polymer curing using 4-aminobenzoic acid dissolved in the oil phase. The size of the generated microparticles could be precisely controlled in the range 80 μm to 160 μm through design of the microfluidic device geometry and by varying flow rates of the dispersed and continuous phase. In contrast to free ‘naked’ phages, those encapsulated within the microparticles could withstand a 3 h exposure to simulated gastric fluid at pH 2 and then underwent a subsequent pH triggered burst release at pH 7. The significance of our research is in demonstrating that C. difficile specific phage can be formulated and encapsulated in highly uniform pH responsive microparticles using a microfluidic system. The microparticles were shown to afford significant protection to the encapsulated phage upon prolonged exposure to an acid solution mimicking the human stomach environment. Phage encapsulation and subsequent release kinetics revealed that the microparticles prepared using Eudragit® S100 formulations possess pH responsive characteristics with phage release triggered in an intestinal pH range suitable for therapeutic purposes. The results reported here provide proof-of-concept data supporting the suitability of our approach for colon targeted delivery of phages for therapeutic purposes.

Encapsulated bacteriophages in alginate-nanohydroxyapatite hydrogel as a novel delivery system to prevent orthopedic implant-associated infections

“…35 Besides, it was shown that the presence of nanoHA into the hydrogel system did not affect the phage releasing from hydrogels, with almost all phages being released from the composites, suggesting Alg-nanoHA hydrogels could be used as a suitable phages release delivery system. 31,33,34 Similar phages release profiles were reported from hydroxypropylmethylcellulose, alginate/CaCO 3 or Eudragit® hydrogels for targeted delivery of phages. 9,23,31,34 Biocompatibility and safety are important requirements for local drug delivery systems and their tissue engineering applications.…”

“…31,33,34 Similar phages release profiles were reported from hydroxypropylmethylcellulose, alginate/CaCO 3 or Eudragit® hydrogels for targeted delivery of phages. 9,23,31,34 Biocompatibility and safety are important requirements for local drug delivery systems and their tissue engineering applications. 36 In the present work, good biocompatibility and safety profiles were observed for all hydrogels subcutaneously implanted in a rabbit model.…”

“…32 Several authors have reported the importance of pH-dependence and hydrophilic behavior of Alg network, on drug release mechanisms. 31,33,34 For instance, at basic pH, the swelling of Alg network occurs, due to the ionization of the COOgroups and diffusion of Ca 2+ ions from gelling sites, 31,33,34 allowing thereby the phages permeability. Whereas, under acid conditions the Alg network shrinks, due to the protonation of the COOgroups and increase of cross-linking degree, 31,33,34 holding the phages entrapped in Alg matrices.…”

“…31,33,34 For instance, at basic pH, the swelling of Alg network occurs, due to the ionization of the COOgroups and diffusion of Ca 2+ ions from gelling sites, 31,33,34 allowing thereby the phages permeability. Whereas, under acid conditions the Alg network shrinks, due to the protonation of the COOgroups and increase of cross-linking degree, 31,33,34 holding the phages entrapped in Alg matrices. It should be noted that no detrimental effects on phages viability and functionally were observed after phages delivery at broad range of pH 5 -9, which could be a promising feature for phages delivery at the site of bone infection, where the normal pH is 6.8.…”

Evaluation of the efficiency of using Salmonella Kentucky and Escherichia coli O119 bacteriophages in the treatment and prevention of salmonellosis and colibacillosis in broiler chickens

“…The purified phage lysates were concentrated using polyethylene glycol precipitation method according to Vinner et al . (2017). The concentrated pellets were suspended in 50 µl buffer peptone broth.…”