Melanoma is one of the most aggressive skin cancers and requires innovative therapeutic strategies to overcome the limitations of conventional therapies. In this work, upconversion nanoparticles coated with mesoporous silica and functionalized with folic acid (UCNP@mSiO2-FA) were developed as a targeted nanocarrier system for the delivery of doxorubicin (DOX). The UCNPs were synthesized via thermal decomposition, coated with mesoporous silica shells, and functionalized with folic acid (FA) to enable receptor-mediated targeting. DOX was then loaded into the mesoporous silica coating by adsorption, yielding UCNP@mSiO2-FA-DOX. The different UCNPs were characterized for size, composition, colloidal stability, and loading and release of DOX. This comprehensive physicochemical characterization confirmed a high DOX loading efficiency and a slightly increased drug release under acidic conditions, mimicking the tumour microenvironment. In vitro assays using four melanoma cell lines (A375, B16-F10, MNT-1, and SK-MEL-28) revealed an excellent biocompatibility of UCNP@mSiO2-FA and a significantly higher cytotoxicity of UCNP@mSiO2-FA-DOX compared to unloaded UCNPs, in a dose-dependent manner. Cell cycle analysis demonstrated G2/M phase arrest after treatment with UCNP@mSiO2-FA-DOX, confirming its antiproliferative effect. Overall, UCNP@mSiO2-FA-DOX represents a promising nanoplatform for targeted melanoma therapy, combining active tumour targeting and enhanced anticancer efficacy.

Novel methods for introducing chemical and biological functionality to the surface of gold nanoparticles serve to increase the utility of this class of nanomaterials across a range of applications. To date, methods for functionalising gold surfaces have relied upon uncontrollable non-specific adsorption, bespoke chemical linkers, or non-generalisable protein-protein interactions. Herein we report a versatile method for introducing functionality to gold nanoparticles by exploiting the strong interaction between chemically functionalised bovine serum albumin (f-BSA) and citrate-capped gold nanoparticles (AuNPs). We establish the generalisability of the method by introducing a variety of functionalities to gold nanoparticles using cheap, commercially available chemical linkers. The utility of this approach is further demonstrated through the conjugation of the monoclonal antibody Ontruzant to f-BSA-AuNPs using inverse electron-demand Diels-Alder (iEDDA) click chemistry, a hitherto unexplored chemistry for AuNP-IgG conjugation. Finally, we show that the AuNP-Ontruzant particles generated via f-BSA-AuNPs have a greater affinity for their target in a lateral flow format when compared to conventional physisorption, highlighting the potential of this technology for producing sensitive diagnostic tests.

An inverse electron demand Diels-Alder reaction between tetrazine and trans-cyclooctene (TCO) holds great promise for protein modification and manipulation. Herein, we report the design and synthesis of a tetrazine-based disulfide rebridging reagent, which allows the site-selective installation of a tetrazine group into disulfide-containing peptides and proteins such as the hormone somatostatin (SST) and the antigen binding fragment (Fab) of human immunoglobulin G (IgG). The fast and efficient conjugation of the tetrazine modified proteins with three different TCO-containing substrates to form a set of bioconjugates in a site-selective manner was successfully demonstrated for the first time. Homogeneous, well-defined bioconjugates were obtained underlining the great potential of our method for fast bioconjugation in emerging protein therapeutics. The formed bioconjugates were stable against glutathione and in serum, and they maintained their secondary structure. With this work, we broaden the scope of tetrazine chemistry for site-selective protein modification to prepare well-defined SST and Fab conjugates with preserved structures and good stability under biologically relevant conditions.

In this study we describe a novel platform for generating functionalised TIPS microparticles for “click” conjugation to various active compounds.

Diseases are multifactorial, with redundancies and synergies between various pathways. However, most of the antibody-based therapeutics in clinical trials and on the market interact with only one target thus limiting their efficacy. The targeting of multiple epitopes could improve the therapeutic index of treatment and counteract mechanisms of resistance. To this effect, a new class of therapeutics emerged: bispecific antibodies. Bispecific formation using chemical methods is rare and low yielding and/or requires a large excess of one of the two proteins to avoid homodimerisation. In order for chemically prepared bispecifics to deliver their full potential, high-yielding, modular and reliable cross-linking technologies are required. Herein, we describe a novel approach not only for the rapid and high-yielding chemical generation of bispecific antibodies from native antibody fragments, but also for the site-specific dual functionalisation of the resulting bioconjugates. Based on orthogonal clickable functional groups, this strategy enables the assembly of functionalised bispecifics with controlled loading in a modular and convergent manner.

Antibody-targeted nanoparticles have shown exceptional promise as delivery vehicles for anticancer drugs, although manufacturability challenges have hampered clinical progress. These include the potential for uncontrolled and random antibody conjugation, resulting in masked or inactive paratopes and unwanted Fc domain interactions. To circumvent these issues, we show that the interchain disulfide of cetuximab F(ab) may be selectively re-bridged with a strained alkyne handle, to permit 'click' coupling to azide-capped nanoparticles in a highly uniform and oriented manner. When compared to conventional carbodiimide chemistry, this conjugation approach leads to the generation of nanoparticles with a higher surface loading of cetuximab F(ab) and with markedly improved ability to bind to the target epidermal growth factor receptor. Moreover, we show that entrapment of a camptothecin payload within these nanoparticles can enhance drug targeting to antigen-expressing pancreatic cancer cells, resulting in superior cytotoxicity versus the conventional nanoformulation. Collectively, this work highlights the critical need to develop refined methods for the construction of targeted nanoparticles that will accelerate their clinical translation through improved performance and manufacturability.

Herein we report the construction of a nanoparticle-based drug delivery system which targets a key regulator in tumour angiogenesis. We exploit a Variable New Antigen Receptor (VNAR) domain, conjugated using site-specific chemistry, to direct poly lactic acid-co-glycolic acid-polyethylene glycol (PLGA-PEG) nanoparticles to delta like canonical Notch ligand 4 (DLL4). The importance of site-specific chemistry is demonstrated.

Use of antibody-nanoparticle conjugates (ANCs) has emerged as a multi-disciplinary strategy for combating cancer - they combine the versatility of nanoparticles and the potential to deliver cargo to cancer cells with the high targeting specificity of surface antibodies to recognise specific biomarkers that are expressed in cancer cells. Several strategies have been employed to graft nanoparticles to antibodies, however, most of them rely on fragile non-covalent interactions or on methods that do not exert control on antibody paratope orientation (e.g. random modification of multiple lysine residues on antibodies). These issues greatly limit ANCs antigen binding capability, reproducibility and, thus, overall efficacy. In this thesis, alternatives strategies of generating ANCs are proposed, regarding antibody orientation on the nanoparticles’ surface through the use of pyridazinedione-based linkers that site-selectively modify disulfide(s) on antibodies. The overall aim is to achieve highly-controlled ANC construction so that these next-generation ANCs can be employed in future cancer treatments. In Chapter 1, an introduction to current protein modification techniques is presented and, in a more biological context, the structure and use of full antibodies and antibody fragments is described. Additionally, an overview of the current biomedical applications of numerous different types of inorganic and organic nanoparticles is introduced. In Chapter 2, the creation of antibody fragment Fab targeted PEG-PLGA nanoparticles is reported. In particular, the generation of Trastuzumab Fab fragments via digestion techniques and a new approach for their attachment to PEG-PLGA nanoparticles and the consequent results of improved antigen binding are described. In Chapters 3 and 4, different proteins are employed for the generation of ANCs, namely Cetuximab Fab (in which cell studies are also performed) and considerably smaller proteins such as variable new antigen receptors (VNARs) via a similar methodology to that employed in Chapter 2. Concluding, an overview of achieved results and future work are covered.

Enabling oriented installation of non-engineered antibody fragments on nanoparticle surfaces to create next-generation antibody–nanoparticle conjugates.

Delivering potent, stable, targeted andin vivoefficacious antibody–drug conjugates (ADCs) using pyridazinedione functional disulfide re-bridging reagents.