RESEARCH GROUP: Pharmaceutical Science & Practice
Project Title:
Development and characterisation of antibody-conjugated, antibiotic-loaded nanoparticles
targeting the flagella of C. difficile.
Supervisor(s): Dr Nigel Ternan, Prof Paul McCarron and Dr M Tambuwala
Contact Details: [email protected] , Room W0073, ext. 23063
Level: PhD
Background to the project:
C. difficile is a flagellate bacterium (Figure 1) that propels itself towards the intestinal cell wall by rotating a whip-like
organelle, known as a flagellum; upon reaching the gut epithelium which it produces cytotoxins that damage the gut
epithelium and lead to diarrhoea. The resulting disease, known as C. difficile infection (CDI) is potentially fatal.
Figure 1. Illustration (upper) of a
Clostridium difficile bacterium and its
rotating flagellum, which brings about
locomotion. Lower image shows attached
nanoparticles on the flagellum, which
restricts rotation and forward motion.
C. difficile colonises the human gastrointestinal tract after the normal gut microbiota has been disrupted by e.g. broad
spectrum antibiotics. C. difficile is the most common cause of infectious diarrhoea worldwide (Rupnik et al., 2009),
leading to four times as many deaths as MRSA in Europe alone; In Ireland, the number of cases of CDI is higher than
that in Europe (7.3, versus 4.1 cases per 10k patient days), with a mortality rate of 10% (ECDC, 2015) Largely
indiscriminate use of antibiotics over the past 20 years has led to the emergence of hypervirulent strains of C. difficile
(He et al., 2010; 2013) that cause a more severe disease. Worryingly, the current stock of antibiotics is becoming less
effective and innovative strategies are needed to tackle microbial infections, including C. difficile infection (Kelly and
Lamont, 2008, O’Neil, 2016).
A yet to be tested approach to tackle C. difficile infection consists of depriving the bacterium of its primary source of
locomotion: immobilising its flagellum. This should reduce the spread of infection. This can be achieved by attaching
a mass of nanoparticles to its flagellum. The nanoparticles can also carry a payload of antibiotic for localised delivery,
thus resulting in greater effectiveness in the therapeutic approach.
Work recently funded at Ulster University, under a Proof of Principle scheme (POP U535, 2015-16), has established
that we can load metronidazole into fluorescently labelled nanoparticles. We have shown that these nanoparticles
can release their payload over a 6-hour period and that their surface can be modified in a way that permits conjugation
of FliC antibodies.
Our overarching hypothesis is that nanoparticles with antibiotic payloads that attach themselves to the C. difficile
bacterium are an effective therapeutic means against infection. The overall aim of the project is to make
nanoparticles with an outer coating of antibodies that attach to flagellin, which is the structural component of the C.
difficile flagellum, and load these NP with antibiotic to specifically target C. difficile. We have divided this aim into 6
objectives:
1. Preparation of Nanoparticles (type, class),
2. Loading of Nanoparticles,
3. Preparation of nanoparticles loaded, and not loaded and conjugated with FliC antibodies,
4. Preparation of nanoparticles (loaded and not loaded), conjugated with FliC antibodies and fluorescent tags,
5. Evaluating motility of C. difficile with attached nanoparticles,
6. Evaluating loaded attached nanoparticles on C. difficile
Objectives of the research project :
Year 1. Delivery system design
Objective 1 – Preparation of Nanoparticles. the first objective of this work will be to use emulsion-based preparation
procedures to prepare nanoparticles with optimal size and surface charge. One key premise of this work, which relies
on interference of motility, will depend of effect attachment to a bacterial structure of defined diameter. Therefore,
we will investigate the effect of particle size as particles that lie outside an optimal size distribution are unlikely to bind
effectively, being either too bulky or of insufficient mass. Our preliminary data show that modification of formulation
parameters enable a range of sizes to be produced. Other factors, such as surface charge and size distribution will be
investigated in a similar manner.
Objective 2 – Loading of Nanoparticles. the second objective will be to load these nanoparticles with model drugs, to
include metronidazole. This work will include determination of loading, release and stability. We have release data
for metronidazole, but we intend to broaden the work to include other key therapeutic agents. This will improve our
understanding of how localised release of antimicrobial agents affect bacterial kill and will enable us to assess the
effectiveness of our system in more complicated media, such as broth media and ex vivo model culture systems for C.
difficile.
Year 2. Visualisation and binding studies
Objective 3 – Preparation of nanoparticles loaded, and not loaded and conjugated with FliC antibodies. this
objective will development the appropriate chemistry on the nanoparticle to allow antibody binding. Our preliminary
data is based on previous published work that shows the polymer we use for nanoparticle manufacture can be
activated to allow antibody attachment. However, attachment is not fully characterised and this objective will assess
the preservation of function follow conjugation. The activity of the Ab will be assessed using immunological assaybased procedures, which will determine antibody viability following attachment.
Objective 4 - Preparation of nanoparticles with fluorescent tags. This objective will begin by loading appropriate
fluorescent labels into the nanoparticle and using this to visualise attachment to the flagella. Previous results shows
that labelling is effective with Nile Red, but we intend to use other lipophilic dyes and investigate what effect this has
on drug loading and antibody attachment. We intend to produce visualisation of high standard, demonstrating
nanoparticle attachment and showing where on the bacterial body the nanoparticles attach.
Year 3 Virulence and motility studies
Objective 5 – Evaluating motility of C. difficile. this objective will use standard microbiological techniques to verify
reductions in motility of C difficile following specific targeting of antibiotic loaded NP to this organism’s flagella.
Critically, we have constructed a mutant strain of C. difficile which is non motile and does not possess flagella (Jain,
2010) and this will be essential to our demonstration of the targetability of the antibody-NP conjugates to this cellular
organelle. We also have access to other non-motile strains of C. difficile though existing research collaborations with
the University of Nottingham and the Wellcome Trust Sanger Institute.
Objective 6 - Evaluating loaded attached nanoparticles. Confirmation of our hypotheses, vis a vis targeting of drugloaded NP to specific bacterial strains opens the door to the development of other, specifically targeted drug delivery
systems for microbial pathogens. Thus, the work to be carried out in this PhD project specifically aligns with the recent
UKRC themes in the area of antimicrobial resistance and antibiotic stewardship, specifically “reducing unnecessary
antimicrobial use” and “accelerating therapeutics”.
Methods to be used :
1. This project will make use of Emulsion-Based Manufacturing methods to manufacture nanoparticles. These
methods are commonly cited in the literature and are multi-variant. This provides a considerable degree of
flexibility to design methods that provide the required particulate specifications needed to enable the objectives
of the work to be realised.
2. This work will use Freeze drying, Centrifugation, Dialysis and Filtration methods to process nanoparticles.
Adaptation of these methods are unique to nanoparticle formulations and will provide the student with an
excellent understanding of the difficulties generally encountered during their manufacture.
3. The work will use Formulation Adaption methods, such as solvent-based sobulibisation, to place active drug
substances and fluorescent dyes into the nanoparticle matrix. Effective loading of payloads into nanoparticles is
challenging and these methods will teach the student how standard methods can be adapted accordingly.
4. Diffusion Analysis methods will be used to determine release kinetics and verify the stability of dyes loaded into
the nanoparticles. This method will also provide the student with information on entrapment efficiency of the
drugs used in this study and determination of drug release kinetics. This will verify pharmaceutical effectiveness
of the delivery system and ensure exposure times are defined.
5. Analytical methods will be used, to include HPLC, fluorescence and UV spectroscopy, together with Dynamic Light
scattering and photon correlation spectroscopy.
6. Conjugation Chemistry will be used to activate nanoparticulate surfaces and prepare nanoparticles for more
sophisticated manipulation.
7. Immunoassay will be used to verify the preservation of antibody activity
8. Fluorescence Microscopy will be used to produce images which verify nanoparticle attachment.
9. Standard Microbiological methods will be used, such as motility assays in agar tubes, serial dilution, plate
counting and inoculation.
Skills required of applicant :
A 2:1 (Hons.) degree in Pharmacy, Pharmaceutical Science, Biomedical Sciences, Microbiology or closely related
discipline is preferable.
The student will be fully trained, including courses in experimental design, statistical analysis, and critical thinking, as
well as in basic microbiology, molecular biology, microscopy/ immunostaining.
Experience in basic microbiology, biochemistry and histology would be an advantage, as would knowledge of/ability
in molecular biology, microscopy and immunostaining procedures. The ability to use bioinformatics tools for sequence
analysis is desirable, as is experience of working in a research laboratory. An interest in chemistry is desirable but not
essential.
References :
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Rupnik M, Wilcox MH, Gerding DN (2010). Clostridium difficile infection: new developments in epidemiology
and pathogenesis. Nat Rev Microbiol. 7(7):526-36. doi: 10.1038/nrmicro2164.
European Centre for Disease Prevention and Control. European Surveillance of Clostridium difficile
infections. Surveillance protocol version 2.2. Stockholm: ECDC; 2015.
Kelly, CP & LaMont JT (2008) Clostridium difficile — More Difficult Than Ever. N Engl J Med 359:1932-1940
doi: 10.1056/NEJMra0707500.
He M, et al., (2013) Emergence and global spread of epidemic healthcare-associated Clostridium difficile. Nat
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Genet. 2013 Jan;45(1):109-13. doi: 10.1038/ng.2478.
He M, et al., (2010) Evolutionary dynamics of Clostridium difficile over short and long time scales. Proc Natl
Acad Sci U S A. 107(16):7527-32. doi: 10.1073/pnas.0914322107.
O’Neill (2016) https://amr-review.org/sites/default/files/160525_Final%20paper_with%20cover.pdf
Jain (2010) A functional genomics investigation of clinically relevant heat-stress in Clostridium difficile strain
630. Ph.D. Thesis, University of Ulster.
Jain S, Graham C, Graham RL, McMullan G, Ternan NG. (2011) Quantitative proteomic analysis of the heat
stress response in Clostridium difficile strain 630. J Proteome Res. 10(9):3880-90. doi: 10.1021/pr200327t.
Ternan NG, Jain S, Srivastava M, McMullan G. (2012) Comparative transcriptional analysis of clinically
relevant heat stress response in Clostridium difficile strain 630 PLoS One. 7(7):e42410. doi:
10.1371/journal.pone.0042410.
Ternan NG, Jain S, Graham RL, McMullan G. (2014) Semiquantitative analysis of clinical heat stress in
Clostridium difficile strain 630 using a GeLC/MS workflow with emPAI quantitation. PLoS One. 9(2):e88960.
doi: 10.1371/journal.pone.0088960.