Development of antibody-functionalised biomaterials for cancer immunotherapy

Protein-loaded biomaterials are generating considerable interest due to the growing importance of immunotherapy and tissue engineering in modern medicine. The complex and fragile structures of many therapeutic proteins require advanced delivery methods and careful optimisation of formulation and manufacturing conditions. Electrohydrodynamic processes (EHD) are material fabrication techniques in which a polymer solution is drawn by the influence of an electrical field to produce solid micro-and nano scale fibres and particles. Multiple approaches have been proposed for the incorporation of therapeutic proteins in electrospun scaffolds, including surface functionalization and coaxial electrospinning. These concepts are introduced in Chapter 1. This PhD thesis explores the EHD fabrication, functionalisation with therapeutically relevant proteins, and characterization of polycaprolactone (PCL) materials, with the aim of developing a platform protein delivery technology. Several protein loading methodologies were investigated on electrospun nanofibres and electrosprayed microparticles for applications relevant to regenerative medicine. The experimental procedures used are detailed in Chapter 2. Chapter 3 explored the feasibility of surface functionalisation of electrospun PCL nanoscaffolds with proteins using perfluorophenyl azide chemistry. Examples of the conjugated biomolecules explored include bovine serum albumin, catalase and antibodies (infliximab and OKT3). The covalently conjugated catalase released from the fibres at a much slower rate than physically adsorbed catalase, revealing this approach to be suitable for safe and effective attachment of proteins to hydrocarbon-based biomaterials. The next application explored is the fabrication of surface-functionalised anti-CD3 antibody-modified electrosprayed PCL microparticles, with the ultimate goal of achieving targeted T cell activation when the particles are injected intratumorally. Formulations were prepared by electrospraying PCL particles and subsequent surface functionalization using perfluorophenylazide chemistry (Chapter 4) and by using “click” chemistry to conjugate protein to azide-functionalised PCL (Chapter 5). The developed formulations were extensively characterised with in vitro T cell activation models. It was found that T cell activation can be achieved following stimulation with biomimetic electrosprayed microparticles prepared using either of the explored bioconjugation methods. The final results chapter, Chapter 6, discusses the fabrication and characterisation of electrospun PCL patches loaded with a checkpoint inhibitor monoclonal antibody, ipilimumab. The coaxial electrospinning technology offers a simple solution for the fabrication of antibody-loaded biocompatible scaffolds that can be easily implanted at the desired site of action and release the therapeutic cargo in a sustained fashion. It was also found that electrospinning monoclonal antibodies near to their isoelectric point leads to improved process stability and enhanced protein encapsulation

Protein encapsulation by electrospinning and electrospraying

Given the increasing interest in the use of peptide- and protein-based agents in therapeutic strategies, it is fundamental to develop delivery systems capable of preserving the biological activity of these molecules upon administration, and which can provide tuneable release profiles. Electrohydrodynamic (EHD) techniques, encompassing electrospinning and electrospraying, allow the generation of fibres and particles with high surface area-to-volume ratios, versatile architectures, and highly controllable release profiles. This review is focused on exploring the potential of different EHD methods (including blend, emulsion, and co−/multi-axial electrospinning and electrospraying) for the development of peptide and protein delivery systems. An overview of the principles of each technique is first presented, followed by a survey of the literature on the encapsulation of enzymes, growth factors, antibodies, hormones, and vaccine antigens using EHD approaches. The possibility for localised delivery using stimuli-responsive systems is also explored. Finally, the advantages and challenges with each EHD method are summarised, and the necessary steps for clinical translation and scaled-up production of electrospun and electrosprayed protein delivery systems are discussed

The blending effect of natural polysaccharides with nano-zirconia towards the removal of fluoride and arsenate from water

Nano-zirconia (ZO) was synthesized using a microwave-assisted one-pot precipitation route. Two biopolymers, chitosan (CTS) and carboxymethyl cellulose were blended with ZO at different w/w ratios. The formulation with 30% w/w chitosan (ZO-CTS) was found to give enhanced uptake of F− and As(V). ZO and the most effective ZO-CTS system were characterized using Fourier transform infrared spectroscopy, scanning electron microscopy, X-ray diffraction and X-ray photoelectron spectroscopy. These confirmed the formation of a composite system containing nanoparticles of 50 nm in size, in which ZO was present in the amorphous form. It was observed that the combination of ZO with CTS improved the F− and As(V) adsorption capacity most notably at pH 5.5. Fluoride adsorption by ZO-CTS followed the Freundlich isotherm model, with an adsorption capacity of 120 mg g−1. Adsorption of As(V) by ZO-CTS could be fitted with both the Langmuir and Freundlich isotherm models and was found to have a capacity of 14.8 mg g−1. Gravity filtration studies conducted for groundwater levels indicated the effectiveness of ZO-CTS in adsorbing As(V) and F− at a pH of 5.5. The ability of the ZO-CTS in removing Cd(II) and Pb(II) was also investigated, and no such enhancement was observed, and found the neat ZO was the most potent sorbent here

Development of ibuprofen-loaded electrospun materials suitable for surgical implantation in peripheral nerve injury

The development of nerve wraps for use in the repair of peripheral nerves has shown promise over recent years. A pharmacological effect to improve regeneration may be achieved by loading such materials with therapeutic agents, for example ibuprofen, a non-steroidal anti-inflammatory drug with neuroregenerative properties. In this study, four commercially available polymers (polylactic acid (PLA), polycaprolactone (PCL) and two co-polymers containing different ratios of PLA to PCL) were used to fabricate ibuprofen-loaded nerve wraps using blend electrospinning. In vitro surgical handling experiments identified a formulation containing a PLA/PCL 70/30 molar ratio co-polymer as the most suitable for in vivo implantation. In a rat model, ibuprofen released from electrospun materials significantly improved the rate of axonal growth and sensory recovery over a 21-day recovery period following a sciatic nerve crush. Furthermore, RT-qPCR analysis of nerve segments revealed that the anti-inflammatory and neurotrophic effects of ibuprofen may still be observed 21 days after implantation. This suggests that the formulation developed in this work could have potential to improve nerve regeneration in vivo

Advanced Formulation Approaches for Proteins

Proteins and peptides are highly desirable as therapeutic agents, being highly potent and specific. However, there are myriad challenges with processing them into patient-friendly formulations: they are often unstable and have a tendency to aggregate or degrade upon storage. As a result, the vast majority of protein actives are delivered parenterally as solutions, which has a number of disadvantages in terms of cost, accessibility, and patient experience. Much work has been undertaken to develop new delivery systems for biologics, but to date this has led to relatively few products on the market. In this chapter, we review the challenges faced when developing biologic formulations, discuss the technologies that have been explored to try to overcome these, and consider the different delivery routes that can be applied. We further present an overview of the currently marketed products and assess the likely direction of travel in the next decade

Encapsulation of pharmaceutical and nutraceutical active ingredients using electrospinning processes

10.3390/nano11081968Nanomaterials118196

Co-Processed Excipients for Dispersible Tablets–Part 1: Manufacturability

Co-processed excipients may enhance functionality and reduce drawbacks of traditional excipients for the manufacture of tablets on a commercial scale. The following study aimed to characterise a range of co-processed excipients that may prove suitable for dispersible tablet formulations prepared by direct compression. Co-processed excipients were lubricated and compressed into 10.5-mm convex tablets using a Phoenix compaction simulator. Compression profiles were generated by varying the compression force applied to the formulation and the prepared tablets were characterised for hardness, friability, disintegration and fineness of dispersion. Our data indicates that CombiLac, F-Melt type C and SmartEx QD100 were the top 3 most suitable out of 16 co-processed excipients under the conditions evaluated. They exhibited good flow properties (Carr’s index ˂ 20), excellent tabletability (tensile strength > 3.0 MPa at 0.85 solid fraction), very low friability (< 1% after 15 min), rapid disintegration times (27–49 s) and produced dispersions of ideal fineness (< 250 μm). Other co-processed excipients (including F-Melt type M, Ludiflash, MicroceLac, Pharmaburst 500 and Avicel HFE-102) may be appropriate for dispersible tablets produced by direct compression providing the identified disintegration and dispersion risks were mitigated prior to commercialisation. This indicates that robust dispersible tablets which disintegrate rapidly could be manufactured from a range of co-processed excipients