Date of Award

Winter 2018

Document type

Thesis

Degree Name

MSc by research (Master of Science by research)

First Supervisor

Dr Ben Ryan

Second Supervisor

Professor Steve Kerrigan

Funder/Sponsor

Ministry of Higher Education in Kuwait

Keywords

Staphylococcus Aureus, Staphylococcal Infections, Nanotechnology

Abstract

Staphylococcus aureus (S. aureus) is an opportunistic pathogen causing a wide variety of infections which range in seriousness. Treatment of S. aureus infections is challenging due its ability to a) form protective biofilms, b) survive intracellularly, and c) the emergence of resistant strains. Upon recovery, patients are at high risk of recurrent infection. We hypothesise that recurrent infection is due to the ability of S. aureus to evade the immune system and administered antibacterials by residing intracellularly. Therefore, the aim of this thesis is to develop a drug delivery strategy which can successfully deliver antibacterials intracellularly and in so doing eliminate the resident bacteria.

In agreement with the literature we found that S. aureus internalises into endothelial cells, within 1 hour (HAoECs). Once internalised we also confirmed that S. aureus has the ability to re-emerge, and hence, in vivo may cause re-infection. Our next objective was to develop a drug delivery system and evaluate its ability to deliver its therapeutic payload intracellularly. Poly(lactic-co-glycolic acid) (PLGA) given its biocompatibility, biodegradability, predictable release kinetics and acceptable regulatory status was used in this study to formulate drug loaded nanoparticles.

Rhodamine B was chosen as a model drug given its autofluorescence and its approximation to the physicochemical characteristics of vancomycin, our ultimate drug of interest. The double emulsion (w1/o/w2) solvent evaporation method was the best suited method to encapsulate rhodamine b into polymeric nanoparticles, given its hydrophilic nature and hence formed the basis of our formulation strategy for both it and vancomycin. Rhodamine B loaded PLGA nanoparticles confirmed the ability of the formulation process to produce nanoparticles of suitable physicochemical haracteristics (Size 507.7 nm ± 35.24; Zeta potential -25.3 mV ± 1.2 & PDI 0.34 ± 0.04, n=3, mean ± SEM) to internalise into HAoECs after 18 hours incubation.

To improve the observed encapsulation efficiencies of vancomycin the effect of different formulation variables was assessed. Our results showed that using a theoretical drug loading of 10% w/w vancomycin to formulate nanoparticles by the double emulsion (w1/o/w2) solvent evaporation method with 0.3 mL inner aqueous phase and 2.5% w/v polyvinyl alcohol in the external aqueous phase produced nanoparticles that displayed the highest encapsulation efficiency (82% w/w ± 12) of vancomycin while retaining the physicochemical characteristics suitable for intracellular drug delivery (Size 339nm ± 5.9; Zeta potential -31.7mV ± 2.4, n=3, mean ± SEM). The nanoparticles were spherical in shape, displayed biphasic drug release kinetics and were proven, in vitro to be non-toxic. Efficacy studies undertaken to assess the effect of the formulated nanoparticles on intracellular S. aureus showed vancomycin loaded PLGA nanoparticles to be the most effective, albeit the difference between groups was not statistically significant.

In conclusion, S. aureus remains a commensal pathogen of concern, with humans at risk of treatment failure due to antibiotic resistance or evasion. As it is hard to develop new antimicrobial classes for clinical use, there is undoubtedly a need for a novel therapy, such as the one described in this thesis which seeks to utilise novel drug delivery strategies to deliver already licenced and indicated therapeutics for the treatment of S. aureus infections.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 4.0 License.

File Size

12.3 MB

Comments

A thesis submitted for the degree of Master of Science from the Royal College of Surgeons in Ireland in 2018.

Available for download on Friday, November 15, 2019

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