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Development of in Vitro Screening Approaches to Optimise Formulation Performance DEVELOPMENT OF IN VITRO SCREENING APPROACHES TO OPTIMISE FORMULATION PERFORMANCE. ANDRZEJ GALLAS, mgr farm. Thesis submitted to the University of Nottingham for the degree of Doctor of Philosophy. September 2013 Abstract. The pharmaceutical industry has been criticised for a lack of innovation associated with the drug discovery and development process, for example when compared with the computer or music industries. In fact, bringing a new medicine to the market requires, on average, the screening of up to 10 000 molecules, an expense in the range of $500 million-$2 billion and roughly 10-15 years of research. Such a situation not only has a direct impact on the health and life expectancy of every single human being on the planet, but also indicates that alternative strategies for drug development should be investigated. In this thesis, studies of direct formulation-membrane interactions, both in a high throughput (HT) manner and at a nanometre scale, were initially identified as an important approach that could offer advantages for in vitro-in vivo correlations of in-man drug behaviours. Subsequently, supported lipid bilayers (SLBs) of physiologically-relevant lipid compositions were indicated as experimental models of preference for pre-clinical drug development. For that reason, the characterisation and assessment of physicochemical and behavioural properties of the model SLBs at a nanometre scale, as well as development of an SLB microarray for HT applications were the focus of this research. Here, the optimisation and characterisation of model lipid films was performed using atomic force microscopy (AFM), time-of-flight secondary ion mass spectrometry (ToF-SIMS) and X-ray photoelectron spectroscopy (XPS). Additionally, the AFM-investigated assessment of the interactions between model SLBs and formulation components (e.g. Pluronics®, siRNA, DNA polyplexes) enabled both the correlation of in vitro observations with literature-reported in vivo performances of the components of interest and the development of hypotheses with regards a number of phenomena in biology. Furthermore, the development of a SLB microarray prototype suitable for HT applications is reported. Directly, this research improves: the understanding of SLB behaviours and experimental investigation at a nanometre scale of the mechanisms of interactions between membranes and: Pluronics®, nucleic acids and their complexes, as well as the technology of SLB microarray development. Indirectly, this research contributes towards the progress in a number of research areas within pharmaceutical sciences, potentially resulting in new scientific disciplines, such as immunolipidomics or nanopharmacology. 2 Acknowledgements. I would like to thank EPSRC, AstraZeneca and University of Nottingham for funding. I would like to thank my supervisors: Dr Stephanie Allen, Prof. Cameron Alexander, Prof. Morgan Alexander, Prof. Martyn Davies and Dr Sanyogitta Puri for help and guidance throughout the project. I would like to thank Prof. Xinyong Chen, Dr David Scurr, Mrs Emily Smith, Dr Andrew Hook, Dr Adam Celiz, Dr Francisco Fernandez-Trillo and other members of the Laboratory of Biophysics and Surface Analysis and the Drug Delivery groups for advice and hands-on help in laboratory. Mamo, Tato, Haniu, Leszku, babciu Zosiu i Mirciu, dziadku Stasiu i Jurku – serdecznie dziękuję za wsparcie, pomoc i wszystko co umożliwiło mi zrobienie tego doktoratu. I would also like to thank Mrs Christine Grainger-Boultby, Mr Paul Cooling, Mrs Julia Crouch, Dr Christina Grindon and Dr Claudia Matz for technical support during my PhD. I would particularly like to thank Sarah, Gökçen, Vanessa, Marek, Alim, Selina and Beppe for friendship and support throughout my time in Nottingham. Finally, I would like thank my assessors: Prof. Clive Roberts and Dr Patrick Gunning for feedback and time they spent on improving this thesis. Andrzej Gallas. 3 List of Contents. Title Page………………………………………………………………………………………………………………………………………………………………1 Abstract…………………………………….…………………………………………………………………………………………………………………….……2 Acknowledgements…………………….………………………………………..………………………………………………………………………………3 List of Contents…………………………………………………………..…………………………………………………………………………………………4 List of Figures…………………………………………………………………………………………..……………………………………………………………7 List of Tables……..………………………………………………………………………………………………………………………………………………..10 Preface………………………………………………………………………………………………………………………………………………………………..11 1 Chapter 1: Introduction and Aims. Supported lipid bilayers as models for pre-clinical development of new drugs…………………..…12 1.1. Abstract………………………………………………………………………………………………………………………..…………………………12 1.2. Introduction……………………………………………………………………………………………………….……………………………………13 1.3. The importance of membrane interactions in pharmaceutical sciences…………………………………….……………14 1.4. Models for membrane interactions: development and types.…………………………………………………………………17 1.5. SLBs as a model for membrane interactions……………………………………………………………………………………………21 1.6. Current limitations associated with SLBs…………………………………………………………………………………………………26 1.7. Conclusions and future prospects.……………………………………………………..……………………………………………………27 1.8. References………………………………………………………………………………………………………………………………………………30 2 Chapter 2: Materials and Methods. 2.1. Materials………………………………………………………………………………………………………………………………………………33 2.2. Methods………………………………………………………………………………………………………………………………………………36 2.2.1. Preparation of supported lipid bilayers……………………………………………………………………………………………..…36 2.2.1.1. Calculation of lipid amounts for liposome preparation……………………………………………………………………………….…36 2.2.1.2. Bilayer self-spreading technique (BSST).…………………………………………………………………………………………………..…36 2.2.1.3. Vesicle deposition technique (VDT).……………………………………………………………………………………..……………………36 2.2.2. Atomic Force Microscopy (AFM) ……………………………………………………………………………………………………….…37 2.2.2.1. Background……………………………………………………………………………………………………………………………………………37 2.2.2.2. Experimental……………………………………………………………………………………………………………………………………….…38 2.2.2.2.1. Basic SLB imaging (chapter 3 and 4).………………………………………………………………………………………………………..…38 2.2.2.2.2. SLB interaction studies (chapter 5)………………………………………………………………………………………………………………38 2.2.2.2.3. SLB air stability studies (chapter 1 and 6) ……………………………………………………………………………………………………39 2.2.2.2.4. SLB microarray imaging (chapter 6) …………………………………………………………………………………………………..………39 2.2.2.2.5. Imaging of Polyphosphonium-DNA polyplexes (chapter 5) ……………………………………………………………………………39 2.2.2.2.6. Preparation and imaging of FluidArray®-like surfaces (chapter 6) ………………………………………………………………..…40 2.2.2.2.7. AFM data analysis……………………………………………………………………………………………………………………………………40 2.2.2.2.7.1. Evaluation of SLB thickness…………………………………………………………………………………………………….…40 2.2.2.2.7.2. Quantification of SLB coverage………………………………………………………………………………………………..…41 2.2.2.2.7.3. Evaluation of particle size…………………………………………………………………………………………………….……41 2.2.3. Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS)………………………………………………………………41 2.2.3.1. Background.……………………………………………………………………………………………………………………………………………41 2.2.3.2. Experimental…………………………………………………………………………………………………………………………………….……42 2.2.3.3. ToF-SIMS: data analysis……………………………………………………………………………………………………………………………42 2.2.4. X-ray Photoelectron Spectroscopy (XPS)………………………………………………………………………………………………43 2.2.4.1. Background……………………………………………………………………………………………………………………………………………43 2.2.4.2. Experimental…………………………………………………………………………………………………………………………………….……44 2.2.4.2.1. XPS: sample preparation……………………………………………………………………………………………………………………..……44 2.2.4.2.2. XPS: data analysis……………………………………………………………………………………………………………………………………45 + + 2.2.4.2.2.1. Calculation of the theoretical lipid content and ratio between: -N (CH3), =N (H2) and –NH- nitrogen groups for lipid film samples analysed with XPS in the N1s region……………………………………………………46 2.2.4.2.2.2. Calculation of theoretical intensity of the P2p peak for lipid film samples analysed with XPS in the P2p region..…………………………………………………………………………………………………………………..47 2.2.5. Dynamic Light Scattering (DLS)……….……………………………………………………………………………………………………47 2.2.5.1. Background……………………………………………………………………………………………………………………………………………47 4 List of Contents. 2.2.5.2. Experimental……………………………………………………………………………………………………………………………………….…48 2.2.5.2.1. DLS: sample preparation………………………………………………………………………………………………………………………..…48 2.2.5.2.2. DLS: data analysis……………………………………………………………………………………………………………………………………48 2.2.6. Piezoelectric inkjet print head technology……………………………………………………………………………………………48 2.2.6.1. Background……………………………………………………………………………………………………………………………………………48 2.2.6.2. Experimental……………………………………………………………………………………………………………………………………….…49 2.2.7. Preparation of Pluronic® solutions (chapter 4 and 6)……………………………………………………………………………50 2.2.8. Preparation of DNA complexes (chapter 5)….………………………………………………………………………………………50 2.2.9. Preparation of text and figures………………………………………………………………………………………………………….…51 2.3. References……………………………………………………………………………………………………………………………………………51 3 Chapter 3: Fabrication and Characterisation of Model SLBs. Advanced surface analysis techniques for the development of a supported lipid bilayer model………….……………………………………………………………………………………………………………………………………………………53 3.1 Abstract……………………………………………………………………………………………………………………………………………….………53 3.2 Introduction…………………………………………………………………………………………………………………………………………………54 3.3 Materials and Methods……………………………………………………………………………………………………………………………..…54 3.4 Results and Discussion…………………………………………………………………………………………………………………………………55 3.4.1 Studying SLB morphology and composition…………………………………………………………………………………………………55
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