Hyperbranched polymers for in vivo multimodal molecular imaging

For the development of the next generation of polymeric nanomedicines, it is crucial to gain a fundamental understanding of their behaviour and interactions with and within biological systems. Moving beyond in vitro models, into in vivo models, earlier in the development process will greatly aid in the advancement of the next generation of nanomedicines. By moving to whole animal models, our understanding of these systems progresses beyond cell targeting and uptake, to developing mechanisms for how these materials will distribute through tissues and their pharmacokinetic profile. This information is important for truly assessing the performance of a nanomedicine. One possible set of tools for obtaining this information is molecular imaging. Molecular imaging is a field of research dedicated to the real time monitoring of biological processes in vivo, without the use of invasive techniques such as biopsies and dissections. Molecular imaging has been used extensively to follow the in vivo behaviour of a labelled material. This is advantageous because the performance of a single material in one subject can be monitored and mapped against the progression of disease. It can help to provide the pharmacokinetic information necessary for preclinical development of nanomedicines. Nanomedicines can be designed to combine molecular imaging with targeting molecules and therapeutic agents to create a theranostic, which can be used for simultaneous imaging and treatment of disease. This thesis aims to synthesise novel multimodal molecular imaging agents based on a hyperbranched polymer architecture, and to gain a deeper understanding of how these materials behave in vivo. To achieve this, biocompatible hyperbranched polymers with defined architectures were synthesised using RAFT polymerisation techniques. These materials were extensively characterised using a wide range of spectroscopic techniques to thoroughly understand their physical and chemical properties. A variety of synthetic strategies were investigated for functionalising both the α- and ω-chain ends of these polymers with multiple imaging ligands to form multimodal imaging agents. Far-red and near-infrared fluorophores provided for fluorescence imaging and radiometal chelators allowed for positron emission tomography (PET) imaging. These hyperbranched polymer systems were first evaluated as molecular imaging agents in C57 BL/6J mice using whole animal fluorescence and PET-CT imaging. It was shown that the rate of excretion was dependent on the size and level of branching of the hyperbranched polymer cores. The larger more highly branched material showed extended circulation times, making it suitable for use as a passive targeting agent for cancer. It was demonstrated in a murine model for melanoma, that the material showed significant uptake within the tumour after 24 hours and that the material was not cleared from the tissue within 72 hours. To gain a deeper understanding of the behaviour of these materials in vivo, PET imaging was combined with gadolinium contrast enhanced MRI, in order to gain both molecular and physiological information. Using this technique, we were able to show that while a folic acid targeted hyperbranched polymer did accumulate in the tumour tissue, its distribution was concentrated in highly vascularised areas of the tumour. This is the first time that this phenomenon has been demonstrated at a macroscopic level, in a living animal. This has important implications for using these materials as theranostics, because heterogeneous distribution of the nanomaterial, and therefore delivery of a therapeutic, can lead to ineffective treatment of the cancer and thus lead to tumour recurrence. In further development of these imaging agents into theranostics, targeting of the hyperbranched polymers by conjugating single chain fragment antibodies (scFv) was explored. Two potential routes to improve efficiency of conjugation were investigated. Both approaches used novel bifunctional oligoethylene glycol (OEG) linkers to introduce the required chemical functionality to either the hyperbranched polymer or scFv. The first approach utilised a heterobifunctional OEG which was synthesised with a pentafluorophenol ester at one end for coupling with amines and an ω-azide group at the other end to allow for the copper catalysed Huigsen 1,3-dipolar cycloaddition reactions. This linker was first attached to the scFv via activated ester chemistry, to provide the necessary azide functionality for coupling of the scFv to the alkyne end groups of the hyperbranched polymer. The second route used an enzymatic cross coupling approach using the sortase enzyme. In order to achieve this, a triglycine functionalised OEG ligand was synthesised and attached to the hyperbranched polymer. The triglycine could then be used as a substrate for enzymatic cross coupling to scFv bioengineered to possess the required recognition sequence (LPETG). Despite both OEG linkers being demonstrated to be able to undergo conjugation to both the hyperbranched polymers and scFv independently, further optimisation is required to achieve conjugation of the two macromolecules

Cerego-an adaptive tool for teaching first year students chemistry. AKA: Students don’t like textbooks

Problem First year chemistry students need to learn the “language” of organic chemistry in order to be able to understand and interact with the material taught in organic chemistry courses. This involves learning to recognise different representations of a wide range of different functional groups, systematic procedures for naming molecules, how to describe the structure of different chemical species and how to describe how a reaction proceeds. This can be an overwhelming task, in particular for students with limited previous chemistry experience. This material may not be examined explicitly, but without these skills the students cannot attempt to learn and complete assessment on more complicated and difficult concepts and theories. Traditionally these concepts have been taught using lectures and workshops, augmented with material provided in the textbook and online using Blackboard. Plan In our first year organic chemistry unit an online adaptive learning tool, Cerego, was used to augment the learning of nomenclature and chemical group recognition. Cerego allowed students to access the material on any internet enabled device (computer, tablet, smartphone etc.) at any time that was convenient for them. The tool provided feedback on their progress, reminders to continue refreshing their memories, as well as providing analytics for the teaching team. Action The content was only briefly introduced in a one hour lecture and students were directed to use Cerego and their text book as the main tools for independent study of nomenclature and chemical functional groups. Brief questions about nomenclature were included at the start of some lectures and workshops to gauge students’ progress, as the semester progressed. Reflection Results from a short survey was used to measure the effectiveness of Cerego. The feedback was generally positive overall, with all students finding the software at least ‘somewhat useful’, with almost half describing it as their ‘preferred study method for this material’. Students provided comments that they enjoyed the repetition and ease of access. Some students became very engaged with the tool, finding it ‘rather addictive’. One student even commented with ‘love how it can replace Candy Crush’. The major criticisms of Cerego was that there wasn’t enough material in the module, and that it only teaches at a relatively basic memorization level. Students really enjoy this digital and interactive style of learning, so as a community we need to be developing much more effective tools to engage students on a much higher level conceptually

Structural elucidation of hydroxy fatty acids by photodissociation mass spectrometry with photolabile derivatives

© 2020 John Wiley & Sons, Ltd. Rationale: Eicosanoids are short-lived bio-responsive lipids produced locally from oxidation of polyunsaturated fatty acids (FAs) via a cascade of enzymatic or free radical reactions. Alterations in the composition and concentration of eicosanoids are indicative of inflammation responses and there is strong interest in developing analytical methods for the sensitive and selective detection of these lipids in biological mixtures. Most eicosanoids are hydroxy FAs (HFAs), which present a particular analytical challenge due to the presence of regioisomers arising from differing locations of hydroxylation and unsaturation within their structures. Methods: In this study, the recently developed derivatization reagent 1-(3-(aminomethyl)-4-iodophenyl)pyridin-1-ium (4-I-AMPP+) was applied to a representative set of HFAs including bioactive eicosanoids. Photodissociation (PD) mass spectra obtained at 266 nm of 4-I-AMPP+-modified HFAs exhibit abundant product ions arising from photolysis of the aryl–iodide bond within the derivative with subsequent migration of the radical to the hydroxyl group promoting fragmentation of the FA chain and facilitating structural assignment. Results: Representative polyunsaturated HFAs (from the hydroxyeicosatetraenoic acid and hydroxyeicosapentaenoic acid families) were derivatized with 4-I-AMPP+ and subjected to a reversed-phase liquid chromatography workflow that afforded chromatographic resolution of isomers in conjunction with structurally diagnostic PD mass spectra. Conclusions: PD of these complex HFAs was found to be sensitive to the locations of hydroxyl groups and carbon–carbon double bonds, which are structural properties strongly associated with the biosynthetic origins of these lipid mediators

Synthesis of 19F nucleic acid–polymer conjugates as real-time MRI probes of biorecognition

Polymer–DNA conjugates in which one nucleic acid strand contains fluorine-substituted nucleobases have been prepared and characterised. The efficacy of these novel 19F nucleic acid–polymer conjugates as sensitive and selective in vitro reporters of DNA binding events is demonstrated through a number of rapid-acquisition MR sequences. The conjugates respond readily and in a sequence specific manner to external target oligonucleotide sequences by changes in hybridisation. In turn, these structural changes in polymer–nucleotide conjugates translate into responses which are detectable in fluorine relaxation and diffusion switches, and which can be monitored by in vitro Spin Echo and DOSY NMR spectroscopy. Although complementary to conventional FRET methods, the excellent diagnostic properties of fluorine nuclei make this approach a versatile and sensitive probe of molecular structure and conformation in polymeric assemblies

Mass spectrometry-directed structure elucidation and total synthesis of ultra-long chain (O-acyl)-ω-hydroxy fatty acids

The (O-acyl)-ω-hydroxy FAs (OAHFAs) comprise an unusual lipid subclass present in the skin, vernix caseosa, and meibomian gland secretions. Although they are structurally related to the general class of FA esters of hydroxy FAs (FAHFAs), the ultra-long chain (30-34 carbons) and the putative -substitution of the backbone hydroxy FA suggest that OAHFAs have unique biochemistry. Complete structural elucidation of OAHFAs has been challenging because of their low abundance within complex lipid matrices. Furthermore, because these compounds occur as a mixture of closely related isomers, insufficient spectroscopic data have been obtained to guide structure confirmation by total synthesis. Here, we describe the full molecular structure of ultra-long chain OAHFAs extracted from human meibum by exploiting the gas-phase purification of lipids through multistage MS and novel multidimensional ion activation methods. The analysis elucidated sites of unsaturation, the stereochemical configuration of carbon-carbon double bonds, and ester linkage regiochemistry. Such isomer-resolved MS guided the first total synthesis of an ultra-long chain OAHFA, which, in turn, confirmed the structure of the most abundant OAHFA found in human meibum, OAHFA 50:2. The availability of a synthetic OAHFA opens new territory for future investigations into the unique biophysical and biochemical properties of these lipids

Shining a light on bioorthogonal photochemistry for polymer science

Bioorthogonal chemistry is revolutionizing the fields of biological chemistry and nanomedicine, providing tools to actively probe and perturb native biochemical processes. Photochemistry provides the opportunity to actively and non‐invasively control bioorthogonal reactions, providing sophisticated optochemical tools. Despite the opportunities in bioorthogonal photochemistry, there remain many significant challenges to the clinical translation of current research. This review aims to provide an overview of these challenges and highlight recent examples from the literature that are providing revolutionary solutions to overcoming these barriers. It will highlight new photochemical systems that can be triggered by near infrared light in aqueous solutions and have been demonstrated to function in complex biological systems, including in living animals. It will cover diverse classes of photochemical reactions including photopolymerization, uncaging, conjugation, and photoswitching. The discussion will detail how new approaches are being integrated into polymers or highlight unexploited opportunities. This review intends to showcase how the unique synergy of bioorthogonal photochemistry and polymer science provides vast opportunities in the fields of biomaterials, nanomedicine, and theranostics. This will hopefully provide inspiration to material scientists to integrate bioorthogonal photochemistry into new adaptable materials and ensure translation to solve clinical challenges

Molecular imaging with polymers

Polymers open up new possibilities in the field of molecular imaging, allowing sensitive and robust agents that can be imaged over long periods of time. This review highlights some recent advances in polymeric molecular imaging agents in both (pre)clinical and emerging applications

Activating discipline specific thinking with adaptive learning : A digital tool to enhance learning in chemistry

In tertiary science education, students are encouraged to engage in discipline specific thinking, to learn their chosen subject. The challenge for educators is engaging all students equitably, despite their educational backgrounds and depth of discipline specific knowledge. Personalising learning in the context of large-scale tertiary courses can only be achieved by using digital technologies. In the context of chemistry education, this project has investigated how an adaptive learning technology can effectively and consistently engage students in discipline specific thinking, by personalising their learning pathway. Adaptive learning has been integrated into a foundational chemistry subject and through quantitative analysis there is empirical evidence to support the benefit adaptive learning has on outcomes, in both the short and long term. This study shows adaptive learning can equitably meet the needs for all students and can lead to improvements in educational behaviour beyond grades. The evidence supports adaptive learning as one critical tool for chemistry educators, and educators in other disciplines of science, to include in their suite of pedagogical strategies to meet the needs of all their students.</p