Research paperA freeze-dried formulation of bacteriophage encapsulated in biodegradable microspheres
Introduction
The clinical benefit of bacteriophage therapy in man has been verified by more recent reports of clearance of antibiotic-resistant pathogens following oral dosing [1], and has begun to attract the attention of the wider research community [2]. Bacteriophages have also been reported to treat antibiotic-resistant ear infections in pet dogs, [3] and importantly, a Phase I/IIa clinical trial targeting Pseudomonas aeruginosa infections of the human ear was completed in November 2007 (Biocontrol Ltd., UK). The interest in bacteriophage therapy arises from the increasing problem of antibiotic resistance, particularly of the vancomycin-resistant Pseudomonal and Staphylococcal strains [4], [5]. A good clinical example of this problem, and where bacteriophage may be of clinical potential, is the treatment of chronic lung infections seen in cystic fibrosis (CF) patients. Antibiotic therapy for CF patients is continually under review in response to emerging antibiotic resistance as a consequence of the high selection pressures on bacteria [6]. Chronic P. aeruginosa colonization also leads to the appearance of mucoid strains which grow as biofilms producing an alginate exopolysaccharide [7].
Clinical use of bacteriophage in animals and man commonly appears to involve simple liquid formulations for oral or topical administration [1], [8]. Oral dosing has been shown to be safe in humans; resulting in the maintenance of the natural balance of the gut flora and the absence of phage-specific antibodies in serum samples [9]. However, a theoretical pharmacokinetic study has suggested that the timing of bacteriophage dosing for the treatment of a bacterial infection could be critical: with earlier inoculations being conversely less effective [10]. Given this, there is a strong argument for the development of robust controlled release delivery vehicles for bacteriophages. Furthermore, passive targeting with controlled release of bacteriophages would be beneficial in the treatment of localized bacterial infections; for example, bacteriophages have been encapsulated in biodegradable poly(ester amide) along with an antibiotic as a wound–healing preparation (PhagoBioDerm™, Intralytix Inc.), which is used clinically to treat topical infections of patients with antibiotic-resistant bacteria [11], [12]. Other than this example, however, there would appear a good opportunity to develop further bacteriophage formulations in anticipation of their emerging clinical use.
In this study, we have focussed on biodegradable poly(dl-lactic-co-glycolic acid) (PLGA) microspheres which are approved delivery vehicles for human use and, through various synthetic routes, can be adapted for specific controlled release profiles alongside active or passive drug targeting [13]. Although the majority of commercialized PLGA microsphere formulations are for peptide hormones, there is a well-established research track record of their potential for the controlled release of proteins [14], [15] and DNA vaccines [16], [17]. While the initial ‘burst’ release of encapsulated protein from microspheres undermines the full potential for controlled release [18], this is conversely of benefit to pulmonary delivery where rapid drug deposition must occur due to muco-ciliary clearance and phagocytosis of PLGA particles [19]. The problem of conformational changes to DNA and proteins during emulsification [20], [21] clearly demands that encapsulation of bacteriophages via the double emulsion–solvent evaporation technique must proceed with caution. We have previously shown that the choice of emulsifier influences protein encapsulation and microsphere morphology, [22] and have developed novel dimpled microcapsules suitable for pulmonary delivery of macromolecules [23]. One advantage of freeze-dried powders for lung inhalation is their simple application for dry-powder inhalers. Other potential routes for the pulmonary delivery of bacteriophages include nebulization [24]. In the present study, we have modified the double emulsion–solvent extraction protocol for the fabrication of dimpled microcapsules, in order to investigate the lytic activity, stability and release of bacteriophages selective for Staphylococcus aureus or P. aeruginosa following encapsulation and lyophilization.
Section snippets
Materials
PLGA (50:50 Poly(dl-lactide:glycolide), inherent viscosity 1.05 dl/g, RG5010) was purchased from Purac Biochem, Netherlands. Poly(vinylalcohol) (PVA) (MW 25,000 88% hydrolyzed), gelatin and fluorescein isothiocyanate (FITC) were purchased from Sigma–Aldrich Chemical Company (Dorset, UK). Water was purified to >17 MΩ–cm. Dichloromethane (DCM), analytical grade, was obtained from Fisher Scientific, UK. Pluronic-L92® PPO-PEO-PPO triblock copolymer (MW PPO:3000–3600) was received as a kind gift from
Measurement of the activity of the formulated bacteriophages
It is important to note that the formulation of bacteriophage via emulsification, lyophilization or encapsulation into microspheres will result in either biologically active or inactive bacteriophage. The plaque assay that is described allows enumeration of the lytic activity of a sample of bacteriophages, with each plaque arising from a single infectious bacteriophage when co-incubated with appropriately viable bacterial cells. However, the plaque assay will only yield quantitative data for
Conclusions
We demonstrate that bacteriophages are quite resilient to encapsulation into biodegradable matrices via emulsification and freeze-drying. Optimization of the protocol, for example, substituting supercritical CO2 for organic solvent [41], or the use of typical lyo- and cryo-protectants for proteins/DNA, may increase the shelf-life of the bacteriophage formulation. This work opens the way towards the passive targeting and controlled release of bacteriophages that may facilitate wider clinical
Acknowledgements
The authors thank Fiona McColm for isolation of bacteriophage selective for P. aeruginosa strain 217 M. The authors also thank Prof. Mike Mattey for helpful discussions and David Blatchford for technical assistance with CLSM. U.P. is the recipient of a Royal Thai government scholarship.
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