Synthesis, characterisation, and diffusive properties of functionalised nanomaterials Edward Douglas Hugh Mansfield Submitted as partial fulfilment for the degree of Doctor of Philosophy School of Pharmacy October 2016 “If we knew what it was we were doing, it would not be called research, would it?” Albert Einstein Acknowledgements First and foremost I would like to thank my supervisors; Prof Vitaliy Khutoryanskiy, Prof Adrian Williams (UoR), Dr Patrick Hole and Dr Katy Sillence (Malvern Instruments Ltd). Your help, support, and guidance throughout this project has been invaluable, and I would not have been able to do it without you. Thank you for both your patience and useful tips throughout my time at Reading. I would also like to thank Malvern Instruments Ltd and the University of Reading for funding my project and making it possible. In addition, I would like to thank everyone else who has helped and supported me throughout the past 3 years, most notably Dr Peter Harris (CFAM, UoR), Dr Radoslaw Kowalczyk (CAF lab, UoR), Dr Isabelle Grillo (ILL, Grenoble), Dr Mark Spillman (UoR), and Dr Sarah Rogers (ISIS, UK). Without their help, many elements of this project would not have been able to be carried out. For this you have my eternal gratitude. All my colleagues in LG3 and 169 have made my experience at Reading one of the best times of my life; most notably to Mike Cook, Sam Bizley and Ruairi Ó Brannigán with complex talks about super heroes, computer games, football and science over morning coffee; and Enrica Calò for the Italian and cookery lessons. In addition, I would like to thank my housemates Adam Gadd, Chris Harris, and Adam Jones, for a fun filled 3 years and (mostly) successful staving off of the zombie apocalypse. Special reference to Sam Bizley and Adam Gadd and weekends of playing Mario Kart, getting hit right on the finish line followed up by ensuing rage (classic Pink-Gold Peach); not forgetting the epic banqueting. The help and support of my parents Barbara and Terry (not forgetting Wilf, Benji and Norris) has been second to none throughout every aspect of my life, and I could not have gotten this far without them. I cannot thank you enough for everything you have done for me and how much you mean to me. In addition to my parents, I would like to thank Emily McAlary, whose love and support through over the last 3 years has been phenomenal. Last but not least I would like to thank the lads back home; Bradley Tobin, Ed Greenwood, Adam Metssitane, Jamie Messer, Matt Cook, Matt Watts, Christian Whittaker, Joe Jull, Doug Sanham, and Arthur Bilalov. Holidays, trips and visits have been a welcome break over the past few years. Ed Mansfield, June 2016 i Declaration of original authorship I confirm that this is my own work and the use of all material from other sources has been properly and fully acknowledged Edward Mansfield June 2016 ii Abstract The aim of this thesis was to assess the diffusive properties of functionalised and unfunctionalised nanomaterials in a variety of different media. The main goal was to gain an insight into the fundamental mechanisms underpinning nanoparticle diffusion and how the surface properties of nanoparticles alter their net movement through different environments. Initially a library of polymer-functionalised silica nanoparticles were synthesised and characterised. The polymers chosen were; poly(ethylene glycol) (PEG), poly(2-oxazolines) (POZ) and poly(n-isopropyl acrylamide) (PNIPAM). Firstly, the diffusion of different sized gold nanoparticles was assessed in concentrations of Pluronic F-127, in order to determine how the solution properties affected diffusion. It was found that as the solution undergoes a transition in response to environmental stimuli, there is an increase in diffusion coefficient; however the area they move in becomes more confined (assessed using a bespoke python script written for use with NTA). PNIPAM- and PNPOZ-silica nanoparticles were assessed for their aggregation and diffusion using DLS, NTA, and SANS. It was found that the position of a nitrogen atom in the amide group, present in both polymers, plays a key role in governing how the particles aggregate in solution, which in turn affects how they diffuse through solvents of varying polarities. POZ-silica nanoparticles were assessed for mucus penetration against a positive control of PEGylated nanoparticles. It was found that POZ-silica was effective at enhancing nanoparticle mucus penetration, and the hydrophilicity of these polymers plays a key role in determining the degree of permeation (with methylated POZ significantly more diffusive than propylated POZ). These finding provide valuable insight into some of the molecular mechanisms governing nanoparticle diffusion and how surface chemistry governs these effects. iii List of Publications Original research articles E. D. H. Mansfield, K. Sillence, P. Hole, A. C. Williams and V. V Khutoryanskiy, POZylation: a new approach to enhance nanoparticle diffusion through mucosal barriers. Nanoscale, 2015, 7, 13671–9. E. D. H. Mansfield, V. R. de la Rosa, R. M. Kowalczyk, I. Grillo, R. Hoogenboom, K. Sillence, P. Hole, A. C. Williams, V. V. .Khutoryanskiy. Minor polymer modifications radically alter the diffusion of poly(2-alkyl-2-oxazolines) functionalised nanoparticles through a mucosal barrier. Biomaterials Science, 2016, 4, 1318-1327. Book chapters “Temperature-responsive polymers functionalised to solid-core nanoparticles” to be published in Temperature-responsive polymers: chemistry, properties and plications. To be published by Wiley & Sons, December 2016. iv List of oral presentations UKICRS: (University of Cardiff, April 2016). Does the nature of a poly(2-oxazoline) coat affect the rate of nanoparticle diffusion through mucus? ISIS student meeting: (Rutherford Appleton Laboratories, Harwell. October 2015). Phase transition and diffusive properties of temperature responsive silica nanoparticles M4 Colloids: (University of Bristol. June 2015). POZylation; a new approach to enhance nanoparticle diffusion through mucosal barriers School of Pharmacy Research Day: (University of Reading. January 2015). Rapid mucopenetration of poly(2-oxazoline) functionalised nanoparticles through gastric mucus: A diffusion study ISIS student meeting: (Rutherford Appleton Laboratories, Harwell. October 2014) Probing the thermo-sensitive nature of poly(N-isopropyl acrylamide) functionalised nanoparticles, and its effect on particle size and diffusion M4 Colloids: (University of Cardiff. June 2014). Probing the diffusion of polymer functionalised nanoparticles through gastric mucus UKICRS: (University of Cork, April 2014). Diffusion of polymer-coated nanoparticles through gastric mucin v List of poster presentations ISIS student meeting: (Rutherford Appleton Laboratories, Harwell. October 2015). Phase transition and diffusive properties of temperature responsive silica nanoparticles 42nd Annual meeting for the Controlled Release Society: (Edenborough. July 2015). Probing the diffusive characteristics of silica nanoparticles functionalized with pharmaceutically relevant polymers through gastric mucus UKICRS: (University of Nottingham. April 2015). Altering the diffusive properties of mucoadhesive silica nanoparticles by functionalising them with poly(ethylene glycol) and poly(2-ethyl-2-oxaqzoline) ISIS student meeting: (Rutherford Appleton Laboratories, Harwell. October 2014) Probing the thermo-sensitive nature of poly(N-isopropyl acrylamide) functionalised nanoparticles, and its effect on particle size and diffusion ISIS new-users meeting: (Rutherford Appleton Laboratories, Harwell. February 2014). Diffusion of polymer-coated nanoparticles through gastric mucin M4 Colloids: (University of Bath. July 2013). Measuring the pore size of hydrogels using various sizes of gold nanoparticle vi Table of contents Acknowledgements………………………………………………………………. i Declaration of original authorship……………………………....…………….. ii Abstract……………………………………………………………………………... iii List of publications…………………………………………..…………………… iv List of oral presentations…………………………………..……………………. v List of poster presentations………………………………..…………………… vi List of figures………………………………………………………………………. xi List of tables……………………………………………………………………….. xvi List of abbreviations……………………………………………………………… xviii Chapter 1- Introduction: Functionalised nanomaterials and principles of diffusion 1.1 Introduction…………………………………………………………………………. 1 1.2 Stokes-Einstein equation…………………………………………………………... 4 1.2.1 Stokes law……………………………………………………………………..……. 4 1.2.2 Einstein-Smoluchowski relation………………………………………………….. 5 1.2.3 The Stokes-Einstein equation and its relevance to nanoscience…………….. 5 1.2.4 Limitations of the Stokes-Einstein equation…………………………………….. 6 1.2.5 Techniques…………………………………………………………………………. 8 1.2.5.1 Franz diffusion cells…………………………………………………………… 8 1.2.5.2 Particle tracking techniques………………………………………….……..... 9 1.2.5.3 Diffusion-ordered spectroscopy………………………….…………………... 11 1.3 Why use nanoparticles?...................................................................................... 12 1.3.1 Effect of nanoparticle core……………………………………………..………….. 13 1.3.2 Nanoparticles in the pharmaceutical industry………………………..………….. 14 1.3.2.1 Gold nanoparticles………………………………………………….…………. 14 1.3.2.2 Silica nanoparticles………………………………………………….………… 15 1.3.2.3 Quantum dots………………………………………………………..…………. 15 1.3.2.4 Magnetic nanoparticles………………………………………………………... 16 1.3.2.5 Polymersomes, liposomes, and micelles…………………………….……… 18 1.3.2.6 Virus-like particles……………………………………………………….…….