Microfluidic manufacturing and development of a liposomal vaccine for Clostridium difficile

Oral vaccines offer significant social and economic advantages over vaccines delivered by parenteral routes including higher patient compliance and ease of administration, allowing vaccines to be given by health workers without medical training. Furthermore, oral immunisation can generate both systemic and localised immune responses, making it ideal for generating effective protection against many enteric pathogens such as the gram-positive spore forming bacterium Clostridium difficile (C. difficile). However, design of an effective vaccine for oral administration remains a significant challenge. In order to reach the relevant sites for the induction of immunological responses, the vaccine must first traverse the hostile environment of the gastrointestinal tract (GIT).;For subunit antigens, the acidic pH, degradative enzymes and bile salts present in the GIT can adversely impact their structural stability. This can limit the effectiveness of the immune response. In addition, the mucosal surface within the gut can impair the transport of the vaccine antigen and limit the accessibility to the underlying antigen presenting cells. In order to circumvent these barriers, delivery systems can be employed. Lipid-based delivery systems are commonly used to deliver therapeutics via the oral route of administration. Of the lipid based systems, liposomes have been widely investigated but tend to be used for parenteral delivery.;Therefore, the aim of the work in this thesis was to explore the use of liposomes for the oral delivery of a C.difficile vaccine. Liposomes generally exhibit low toxicity, enhance interactions with biological membranes and can be incredibly versatile in regards to what compounds they can incorporate (whether they are encapsulated within the aqueous core, within the lipid bilayer or surface associated). However, the manufacture of liposomes has been limited to batch scale production which has inherent risk associated. By employing microfluidics as a manufacturing platform, in conjunction with tangential flow filtration for purification, scalable production can be achieved.;Therefore, this method was employed for the formulation and manufacture of the liposomal vaccine systems. Key production parameters were identified and formulations were selected based upon their physicochemical characteristics and protein loading efficiency. Selected formulations were screened for immunological effectiveness on THP-1 macrophage-like cells regarding antigen processing ability and activation marker expression. Finally, a range of formulations were then administered orally to mice to determine in vivo efficacy.;Enhanced antibody responses (IgG) could be observed when antigen was administered encapsulated within liposomal formulation DSPC:Chol (alongside the potent adjuvant cholera toxin) when compared to free antigen alone; however, poor localised IgA responses were observed for all liposomal formulations tested. The work within this thesis presents a platform for the rapid and scalable manufacture, purification and at-line analysis of liposomal formulations incorporating protein, de-risking the translation of liposomal based protein products from bench to large scale production.Oral vaccines offer significant social and economic advantages over vaccines delivered by parenteral routes including higher patient compliance and ease of administration, allowing vaccines to be given by health workers without medical training. Furthermore, oral immunisation can generate both systemic and localised immune responses, making it ideal for generating effective protection against many enteric pathogens such as the gram-positive spore forming bacterium Clostridium difficile (C. difficile). However, design of an effective vaccine for oral administration remains a significant challenge. In order to reach the relevant sites for the induction of immunological responses, the vaccine must first traverse the hostile environment of the gastrointestinal tract (GIT).;For subunit antigens, the acidic pH, degradative enzymes and bile salts present in the GIT can adversely impact their structural stability. This can limit the effectiveness of the immune response. In addition, the mucosal surface within the gut can impair the transport of the vaccine antigen and limit the accessibility to the underlying antigen presenting cells. In order to circumvent these barriers, delivery systems can be employed. Lipid-based delivery systems are commonly used to deliver therapeutics via the oral route of administration. Of the lipid based systems, liposomes have been widely investigated but tend to be used for parenteral delivery.;Therefore, the aim of the work in this thesis was to explore the use of liposomes for the oral delivery of a C.difficile vaccine. Liposomes generally exhibit low toxicity, enhance interactions with biological membranes and can be incredibly versatile in regards to what compounds they can incorporate (whether they are encapsulated within the aqueous core, within the lipid bilayer or surface associated). However, the manufacture of liposomes has been limited to batch scale production which has inherent risk associated. By employing microfluidics as a manufacturing platform, in conjunction with tangential flow filtration for purification, scalable production can be achieved.;Therefore, this method was employed for the formulation and manufacture of the liposomal vaccine systems. Key production parameters were identified and formulations were selected based upon their physicochemical characteristics and protein loading efficiency. Selected formulations were screened for immunological effectiveness on THP-1 macrophage-like cells regarding antigen processing ability and activation marker expression. Finally, a range of formulations were then administered orally to mice to determine in vivo efficacy.;Enhanced antibody responses (IgG) could be observed when antigen was administered encapsulated within liposomal formulation DSPC:Chol (alongside the potent adjuvant cholera toxin) when compared to free antigen alone; however, poor localised IgA responses were observed for all liposomal formulations tested. The work within this thesis presents a platform for the rapid and scalable manufacture, purification and at-line analysis of liposomal formulations incorporating protein, de-risking the translation of liposomal based protein products from bench to large scale production

Crystallographic properties and elemental migration in two-stage prepared CuIn1−xAlxSe2 thin films for photovoltaic applications

Two-stage fabrication of CuIn1−xAlxSe2 thin films for photovoltaic absorbers using sputtered Cu–In–Al metallic precursors has been investigated. Precursors containing different relative amounts of Al were selenised and their structural and chemical properties characterised. X-ray diffraction (XRD) analyses revealed that the Al was only incorporated into the chalcopyrite structure for precursor composition ratios x = [Al]/([Al] + [In]) ⩾ 0.38, while chemical analysis of the cross-section indicated partial segregation of Al near the back of the film. Precursor films in the range of compositions that yielded no Al incorporation were then selenised at four different temperatures. Glow discharge optical emission spectroscopy, plasma profiling time-of-flight mass spectrometry and XRD analyses provided an insight into the diffusion processes and reactions taking place during the selenisation stage. The effect of post-selenisation annealing without additional Se was also investigated, and led to partial incorporation of the Al into the CuInSe2 lattice but no rediffusion

Microfluidic platforms to study host-microbiome interactions

About three-quarters of human body surface is exposed to dense microbial population along the gastro-intestinal tract (GIT) that accommodates around 80% of immune cells. The GIT is one of the most critical sites for metabolic and immunologic homeostasis in the body. While large-scale genomic analysis and germ-free mice have been widely used, they are limited to capture the dynamic functional interaction between host-microbiome in a humanized setting. in-vitro recapitulation of GIT physiology can be a powerful alternative for hypothesis testing in diseases like IBD and cancer. As a first step, we developed a simple transwell assembly to closely emulate GIT anatomy and the barrier function in a quantitative and analytical manner. The system enables us to sequentially house bacterial cells, a mucus layer, human intestinal epithelial cells and human peripheral blood mononuclear cells (hPBMCs) in a single co-culture platform. Porcine intestine-derived mucin formed a biophysically relevant barrier and also provided in-vivo like biochemical milieu to bacterial cells. Addition of human epithelial cell monolayer on a collagen coated semi-permeable membrane added another level of cellular and biophysical complexity. Finally, by introducing human peripheral blood mononuclear cells (hPBMCs), we simulated host-microbiome interactions and successfully captured responses relevant to gut inflammation. Initial data indicate that bacterial products successfully stimulate hPBMCs. Whereas, mucus and epithelial barriers demonstrated strong immunomodulatory functions. Further investigations are being carried out in order to create models that will incorporate differentiated epithelial cells (villi), physiologically relevant flow and stress conditions along with co-culture of aerobic (mucosal) and anaerobic (luminal) components of the human GIT. Hopefully, these models will enable us to study mechanistic interactions of the microbiome with the host and reveal novel therapeutic targets for diseases associated with the dysregulation of host-microbiome homeostasis in human GIT

Sodium-Glucose Cotransporter-2 Inhibitor and Glucagon-Like Peptide-1 Receptor Agonist Combination Therapy in Type 2 Diabetes: Protocol for a Kidney End Points Real-world Study (COMBi-KID Study)

BACKGROUND: Sodium-glucose cotransporter-2 inhibitors (SGLT2is) and glucagon-like peptide-1 receptor agonists (GLP-1RAs) are both considered to be part of standard care in the management of glycemia in type 2 diabetes. Recent trial evidence has indicated benefits on primary kidney end points for individual drugs within each medication class. Despite the potential benefits of combining SGLT2is and GLP-1RAs for glycemia management, according to national and international guideline recommendations, there is currently limited data on kidney end points for this drug combination. OBJECTIVE: The aims of the study are to assess the real-world effects of combination SGLT2i and GLP-1RA therapies for diabetes management on kidney end points, glycemic control, and weight in people with type 2 diabetes who are being treated with renin-angiotensin system blockade medication. METHODS: This retrospective cohort study will use the electronic health records of people with type 2 diabetes that are registered with general practices covering over 15 million people in England and Wales and are included in the Oxford-Royal College of General Practitioners Research and Surveillance Centre network. A propensity score-matched cohort of prevalent new users of SGLT2is and GLP-1RAs and those who have been prescribed SGLT2is and GLP-1RAs in combination will be identified. They will be matched based on drug histories, comorbidities, and demographics. A repeated-measures, multilevel, linear regression analysis will be performed to compare the mean change (from baseline) in estimated glomerular filtration rate at 12 and 24 months between those who switched to combined therapy and those continuing monotherapy with an SGLT2i or GLP-1RA. The secondary end points will be albuminuria, serum creatinine level, glycated hemoglobin level, and BMI. These will also be assessed for change at the 12- and 24-month follow-ups. RESULTS: The study is due to commence in March 2022 and is expected to be complete by September 2022. CONCLUSIONS: Our study will be the first to assess the impact of combination SGLT2i and GLP-1RA therapy in type 2 diabetes on primary kidney end points from a real-world perspective. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): PRR1-10.2196/34206

Optical spectroscopy studies of Cu2ZnSnSe4 thin films

Cu2ZnSnSe4 thin films were synthesised by selenisation of magnetron sputtered metal precursors. The band gap determined from the absorption spectra increases from 1.01 eV at 300 K to 1.05 eV at 4.2 K. In lower quality films photoluminescence spectra show a broad, low intensity asymmetric band associated with a recombination of free electrons and holes localised on acceptors in the presence of spatial potential fluctuations. In high quality material the luminescence band becomes intense and narrow resolving two phonon replicas. Its shifts at changing excitation power suggest donor–acceptor pair recombination mechanisms. The proposed model involving two pairs of donors and acceptors is supported by the evolution of the band intensity and spectral position with temperature. Energy levels of the donors and acceptors are estimated using Arrhenius quenching analysis

Rapid and scale-independent microfluidic manufacture of liposomes entrapping protein incorporating in-line purification and at-line size monitoring

Within this paper we present work that has the ability to de-risk the translation of liposomes from bench to the clinic. We have used microfluidics for the rapid and scale-independent manufacture of liposomes and have incorporated in-line purification and at-line monitoring of particle size. Using this process, we have manufactured a range of neutral and anionic liposomes incorporating protein. Factors investigated include the microfluidics operating parameters (flow rate ratio (FRR) and total flow rate (TFR)) and the liposome formulation. From these studies, we demonstrate that FRR is a key factor influencing liposome size, protein loading and release profiles. The liposome formulations produced by microfluidics offer high protein loading (20–35%) compared to production by sonication or extrusion (<5%). This high loading achieved by microfluidics results from the manufacturing process and is independent of lipid selection and concentration across the range tested. Using in-line purification and at-line size monitoring, we outline the normal operating range for effective production of size controlled (60–100 nm), homogenous (PDI <0.2) high load liposomes. This easy microfluidic process provides a translational manufacturing pathway for liposomes in a wide-range of applications