Encapsulation of bacteriophages, bacteriocins and endolysins in pH-responsive formulations for targeted-delivery and controlled release in the gastrointestinal tract

The emergence of multi-drug resistant bacteria has created an urgent need for new and alternative viable antibacterial therapies. With the antibiotic development pipeline for new classes of drugs experiencing a decline in new successful treatments coming to market, a focus towards evaluating alternatives has increased significantly over recent years. Specifically, bacteriophage (phage) therapy has sparked renewed interest with the application of living, bacteria specific viruses for infection control. Other narrow spectrum antibiotics comprise bacteriocins and endolysins, both of which have proved promising in vitro. There are major advantages of phage and protein antibiotic therapy in comparison to using conventional broad-spectrum antibiotics, such as the ability of phages and bacteria/phage derived proteins to evolve alongside the target bacterial strain and the possibility of microbiome engineering. However, limitations include the sensitivity of phages and proteins to processing conditions encountered during their manufacture as well as the detrimental impact on viability upon exposure to environmental stresses experienced e.g. during transit to the site of infection. These include acid and enzyme exposure in the stomach for gastrointestinal applications and the impact of the host’s immune system on phage and protein pharmacodynamics. Hence, there is an imminent need to explore encapsulation approaches of each potential antibiotic into targeted delivery and controlled release formulations.

In this PhD thesis, initial research into bacteriophage processing enabled high titres (1010 PFU/ml) of phages to be produced using a batch mode of production (shake flasks). The process of membrane emulsification was selected for encapsulation of enteric phages building on previous published work using this process. Firstly, the parameters of droplet production were investigated to confirm the suitable parameters required for the controlled production of droplets with a mean diameter of ~100μm. Subsequently, an E. coli specific Podovirus was encapsulated using three different pH-responsive formulations due to incorporation of either Eudragit L100-55, L100 or S100 polymers. Phage encapsulation in each formulation was tested to confirm different pH-dependent release characteristics suitable for targeting different sections of the GI tract (e.g. human duodenum, jejunum, ileum, and colon). The encapsulated phages in the microcapsules withstood acid exposure and showed good stability upon storage over 6 months under refrigerated conditions.

Membrane emulsification in pH-responsive formulations was used to encapsulate an E. coli specific bacteriocin (colicin Ia and E9). Initially, a process for the overexpression and purification of colicin was developed yielding protein at a suitably high concentration (100mg/ml) for encapsulation. Incubation time prior to addition of IPTG, and IPTG concentration were parameters that were varied enabling sufficient amounts of colicins E9 and Ia (~80-100mg) per 5 litre batch to be produced. Subsequently, the colicins were encapsulated using membrane emulsification and tested in vitro to confirm protein yields within the microcapsules. An in vivo murine E. coli colonisation model was utilised to evaluate the delivery of encapsulated colicin to the mouse caecum as a proof-of-principal study. Significant reductions in CFU were observed in the colicin treatment group of mice (in comparison with the untreated group) verifying that the encapsulated colicin in the pH-responsive microcapsules reached the site of infection. A significant proportion of mice within the treatment group displayed no E. coli colonisation following 3 days of treatment.

Looking from a different perspective, antibiotic resistance genes are passed through the food chain and are specifically exacerbated within the meat production industry. Antibiotics are routinely overused worldwide during meat production to prevent the spread of disease through animal populations and, in some countries, continue to be used as a growth promoter. The use of phages added to animal feed could help facilitate a slowing down of the rate of resistance gene emergence, reducing carriage and transmission of AMR bacteria, as well as for the successful treatment of enteric bacterial infections. Incorporating phages in animal feed requires consideration of processing stresses such as hot extrusion encountered during animal feed pellet production. Phages were formulated into core-shell pH-responsive capsules with the aim to design capsules that allow the phages to withstand wet thermal heat treatment and extrusion. The encapsulated phages withstood exposure to 95⁰C for up to 120s, in comparison with just 15s without encapsulation. The encapsulated phages in the core-shell capsules survived acid exposure for 2 hours at pH 1 with no significant change in phage titre.

Finally, a novel phage endolysin with specific lytic activity against clinical C. difficile strains was encapsulated in pH-responsive capsules to protect against gastric acidity and enzymatic damage. The endolysin displayed lytic activity against three strains of C. difficile in vitro using turbidity and CFU reduction assays. Concentrations as low as 3.8μg/ml enabled a decrease in optical density and a modest reduction in viable bacterial counts. A concentration dependent rate of lysis was observed, with higher concentrations facilitating rapid rates of bacterial killing. The in vivo activity of the endolysin was evaluated using a Galleria Mellonella infection model in which the larvae were force-fed C. difficile followed by endolysin treatment after hours of infection. The G. mellonella Health Index Score was utilised to determine the severity of C. difficile infection at discrete timepoints throughout the infection period (Loh et al., 2013). Results indicated higher larvae survival rates after 2 doses of endolysin treatment as well as improved Health Index Scores, suggesting a less severe infection.

The results presented in this thesis demonstrate a number of different encapsulation approaches and pH-responsive formulations suitable for a range of novel biotherapeutics currently under development as alternatives to conventional antibiotics.