Biomimetic substrates for immune cell activation

This thesis describes the fabrication of biomimetic substrates, and their use as tools to probe cellular interactions of key immune cells. Nanoparticles of gold and zinc sulfide have been fabricated, and patterned into nanoarrays. Adaptive (T cell) and innate (NK cell) immune cell responses to nanoscale spacing of ligand-receptor pairs were measured, and the effect of presenting stimulatory ligands on substrates with varying mechanical properties has been tested for T cell responses. The advanced materials in this thesis act to create artificial immune synapses, and probe the effect of these stimuli on engagement and activation of human immune cells. Specifically, block co-polymers were used to form polymer micelles which encapsulate metal ions and form metal or metal compound nanoparticles. Micelles encapsulating metal ions or nanoparticles were formed and deposited onto substrates using Block Co-polymer Micellar Lithography (BCML) to form nanoparticle arrays with controlled inter-particle spacing. Well controlled gold nanoparticle arrays with spacing between 25-150nm have been produced. The technique has been further developed to include fabrication of zinc sulfide particles and nanoarrays. Zinc sulfide nanoparticles showed a unique internal structure with 5nm crystalline domains set in an amorphous matrix and an optical band gap of between 3.88-4.28eV. Nanoparticle arrays were then functionalised with biological ligands, notably antibodies that engage with the NK cell surface receptor CD16, or the T cell TCR/CD3 moiety. The cellular response to these materials was measured, and was sensitive to the nanoscale arrangement of stimulatory ligands; both cell types responded to ligands with 25nm, but not 104nm, inter-ligand spacing. In an alternative approach, spherical PEG hydrogel particles of diameter 5-50μm were formed with controlled rigidity between 3-2000kPa. T cell response as a function of substrate rigidity was tested, and cells showed maximal response to anti-CD3 functionalised substrates with rigidities of 3-5kPa.Open Acces

The early career researcher's toolkit: translating tissue engineering, regenerative medicine and cell therapy products

Although the importance of translation for the development of tissue engineering, regenerative medicine and cell-based therapies is widely recognized, the process of translation is less well understood. This is particularly the case among some early career researchers who may not appreciate the intricacies of translational research or make decisions early in development which later hinders effective translation. Based on our own research and experiences as early career researchers involved in tissue engineering and regenerative medicine translation, we discuss common pitfalls associated with translational research, providing practical solutions and important considerations which will aid process and product development. Suggestions range from effective project management, consideration of key manufacturing, clinical and regulatory matters and means of exploiting research for successful commercialization

The early career researcher's toolkit:translating tissue engineering, regenerative medicine and cell therapy products

Although the importance of translation for the development of tissue engineering, regenerative medicine and cell-based therapies is widely recognized, the process of translation is less well understood. This is particularly the case among some early career researchers who may not appreciate the intricacies of translational research or make decisions early in development which later hinders effective translation. Based on our own research and experiences as early career researchers involved in tissue engineering and regenerative medicine translation, we discuss common pitfalls associated with translational research, providing practical solutions and important considerations which will aid process and product development. Suggestions range from effective project management, consideration of key manufacturing, clinical and regulatory matters and means of exploiting research for successful commercialization

T cell immunoengineering with advanced biomaterials

Recent advances in biomaterials design offer the potential to actively control immune cell activation and behaviour. Many human diseases, such as infections, cancer, and autoimmune disorders, are partly mediated by inappropriate or insufficient activation of the immune system. T cells play a central role in the host immune response to these diseases, and so constitute a promising cell type for manipulation. In vivo, T cells are stimulated by antigen presenting cells (APC), therefore to design immunoengineering biomaterials that control T cell behaviour, artificial interfaces that mimic the natural APC-T cell interaction are required. This review draws together research in the design and fabrication of such biomaterial interfaces, and highlights efforts to elucidate key parameters in T cell activation, such as substrate mechanical properties and spatial organization of receptors, illustrating how they can be manipulated by bioengineering approaches to alter T cell function

Drug delivery across length scales

Over the last century, there has been a dramatic change in the nature of therapeutic, biologically active molecules available to treat disease. Therapies have evolved from extracted natural products towards rationally designed biomolecules, including small molecules, engineered proteins and nucleic acids. The use of potent drugs which target specific organs, cells or biochemical pathways, necessitates new tools which can enable controlled delivery and dosing of these therapeutics to their biological targets. Here, we review the miniaturisation of drug delivery systems from the macro to nano-scale, focussing on controlled dosing and controlled targeting as two key parameters in drug delivery device design. We describe how the miniaturisation of these devices enables the move from repeated, systemic dosing, to on-demand, targeted delivery of therapeutic drugs and highlight areas of focus for the future

Improved delivery of PLGA microparticles and microparticle-cell scaffolds in clinical needle gauges using modified viscosity formulations

Polymer microparticles are widely used as acellular drug delivery platforms in regenerative medicine, and have emerging potential as cellular scaffolds for therapeutic cell delivery. In the clinic, PLGA microparticles are typically administered intramuscularly or subcutaneously, with the clinician and clinical application site determining the precise needle gauge used for delivery. Here, we explored the role of needle diameter in microparticle delivery yield, and develop a modified viscosity formulation to improve microparticle delivery across a range of clinically relevant needle diameters. We have identified an optimal biocompatible formulation containing 0.25% pluronic F127 and 0.25% carboxymethyl cellulose, which can increase delivery payload to 520% across needle gauges 21–30G, and note that needle diameter impacts delivery efficacy. We use this formulation to increase the delivery yield of PLGA microparticles, and separately, PLGA-cell scaffolds supporting viable mesenchymal stem cells (MSCs), demonstrating the first in vitro delivery of this cell scaffold system. Together, these results highlight an optimal formulation for the delivery of microparticle and microparticle-cell scaffolds, and illustrate how careful choice of delivery formulation and needle size can dramatically impact delivery payload

Nanoscale ligand spacing influences receptor triggering in T cells and NK cells.

Bioactive nanoscale arrays were constructed to ligate activating cell surface receptors on T cells (the CD3 component of the TCR complex) and natural killer (NK) cells (CD16). These arrays are formed from biofunctionalized gold nanospheres with controlled interparticle spacing in the range 25-104 nm. Responses to these nanoarrays were assessed using the extent of membrane-localized phosphotyrosine in T cells stimulated with CD3-binding nanoarrays and the size of cell contact area for NK cells stimulated with CD16-binding nanoarrays. In both cases, the strength of response decreased with increasing spacing, falling to background levels by 69 nm in the T cell/anti-CD3 system and 104 nm for the NK cell/anti-CD16 system. These results demonstrate that immune receptor triggering can be influenced by the nanoscale spatial organization of receptor/ligand interactions

Mechanisms of polymer-templated nanoparticle synthesis: contrasting ZnS and Au

We combine solution small-angle X-ray scattering (SAXS) and high-resolution analytical transmission electron microscopy (ATEM) to gain a full mechanistic understanding of substructure formation in nanoparticles templated by block copolymer reverse micelles, specifically poly(styrene)-block-poly(2-vinyl pyridine). We report a novel substructure for micelle-templated ZnS nanoparticles, in which small crystallites (~4 nm) exist within a larger (~20 nm) amorphous organic-inorganic hybrid matrix. The formation of this complex structure is explained via SAXS measurements that characterize in situ for the first time the intermediate state of the metal-loaded micelle core: Zn2+ ions are distributed throughout the micelle core, which solidifies as a unit on sulfidation. The nanoparticle size is thus determined by the radius of the metal-loaded core, rather than the quantity of available metal ions. This mechanism leads to particle size counter-intuitively decreasing with increasing metal content, based on the modified interactions of the metal-complexed monomers in direct contrast to gold nanoparticles templated by the same polymer