Development of advanced nuclear magnetic resonance methods for biochemical analysis of biofluids and hyperpolarized magnetic resonance spectroscopic imaging for organ-on-chip

Abstract

[eng] The main challenge in modern medicine is to prevent diseases and, when disease strikes, to detect it and cure it efficiently, promptly and cost-effectively (understanding cost as economic burden, patient and relatives emotional distress, and patient’s lifelong side effects, amongst others). To address this ambitious challenge, better understanding of disease onset, progression and regression is needed. Currently, we lack precise and timely tools to measure the metabolic signature of a specific patient and organ non-invasively and in real-time. In fact, there is a lack of sensitive and non-destructive tools to quantify cell metabolomics and kinetics accurately and reliably in the laboratory, to advance our understanding of biochemical changes that occur at cellular and subcellular level.

Nowadays, analytical techniques such as NMR may relate a measurable chemical response to a biological event. An NMR spectrum may quantify hundreds of metabolite concentrations from a target organ in situ (in vivo or in vitro) or in biofluids (e.g. blood ) moreover this fingerprint often changes through physiological and pathological factors. 13C-NMR has been extensively used to follow the fate of several metabolic tracers and understand the metabolism. Recently, a novel non-invasive, chemically-resolved NMR spectroscopic imaging technique has enabled real-time mapping the in vivo spatial distribution and metabolic fate of 13C-enriched endogenous tracers. These tracers have been introduced into an animal, a human or even a chip. The 13C substrate is prepared via a so-called “hyperpolarisation” technique or dynamic nuclear polarisation (DNP), which transfers the near-100% polarisation of electrons in the presence of a magnetic field and at ~1 K temperatures to the nuclei of interest. DNP-MR achieves a 13C spin polarisation ~50,000 greater than at thermal equilibrium, boosting sensitivity sufficiently to enable real-time,

quantitative assessment. A single DNP shot (~seconds) can provide a signal-to-noise ratio (SNR) that is not accessible by any conventional NMR acquisition. Dynamic Nuclear Polarisation has been already applied for clinical studies and the results are promising for early localized cancer diagnosis. However, to conduct a clinical hyperpolarisation trial requires administration of hyperpolarized agents, often via intravenous injection, which some patients may find it uncomfortable or stressful. Clinical trials also involve navigating regulatory approvals, ethical considerations and patient consent, which add complexity to the research process. Lastly, the price of most of the regents is prohibitive, and the cost of each study reaches into several thousand dollars. This thesis explores a new approach to combine microfluidic platforms to mantain biologic in vitro models alive in combination with 13C-hyperpolarized magnetic resonance imaging. Organ-on-a-chip (OoC) devices offer new approaches to metabolic disease modelling and drug discovery by providing biologically relevant models of tissues and organs in vitro integrated with sensing technology. As such, OoC devices have the potential to revolutionise the pharmaceutical industry by enabling reliable and high predictive in vitro testing of drug candidates. The capability to miniaturise microfluidic systems and advanced tissue fabrication procedures have enabled researchers to create multiple tissues on a chip with a high degree of control over experimental variables for high-content screening applications. OoC are widely studied for the ability to engineer precise and controlled conditions to grow and manipulate small biological in vitro systems to study their response to either external stimuli or the metabolism they have by themselves. This approach Is currently focused to replace some of the in vivo studies to improve personalized and precision medicine. The aim of this thesis is to study the reproducibility of hyperpolarization analysis while addressing external factors that impact polarization stability. Also to adapt microfluidic systems for an accurate hyperpolarized contrast agent injection and the corresponding metabolic analysis of the in vitro models in an MRI scanner using 13C magnetic resonance imaging.

[cat] El principal repte de la medicina moderna és prevenir les malalties i, quan apareixen, detectar-les i curar-les de manera eficient, ràpida i econòmica, considerant tant els costos econòmics com l'angoixa emocional del pacient i els seus familiars. Per afrontar aquest repte, és necessària una millor comprensió de l'inici, i la progressió de les malalties. Actualment, manquem d'eines de diagnòstic per medir els sistemas metabòlics d’un organ malalt de manera no invasiva i a temps real.

Tècniques com la Ressonància Magnètica Nuclear (RMN) poden relacionar una resposta química amb un esdeveniment biològic. Un espectre de RMN pot quantificar desenes de concentracions de metabòlits d'un òrgan in situ (in vivo o in vitro) o en biofluids. Més concretament, la ressonància de carboni-13 (13C) s'ha utilitzat per seguir la conversió química de compostos i entendre el comportament metaòlic. Recentment, una tècnica d'imatge espectroscòpica de RMN ha permès fer imatge en temps real de la distribució espacial in vivo i el destí metabòlic d’agents de contrast enriquits amb 13C, com ara el piruvat. Aquests rastrejadors es preparen mitjançant “hiperpolarització” o polarització nuclear dinàmica (DNP), que augmenta la sensibilitat < 10,000 vegades permetent una avaluació quantitativa en temps real.

Els dispositius Organ-on-a-chip (OoC) ofereixen nous enfocaments per a la modelització de malalties metabòliques i el descobriment de fàrmacs mitjançant la provisió de models biològicament rellevants de teixits i òrgans in vitro, integrats amb tecnologia de sensors. Els OoC són àmpliament estudiats per la seva capacitat d'enginyeria de condicions precises i controlades per cultivar i manipular petits sistemes biològics in vitro per estudiar la seva resposta a estímuls externs o el metabolisme propi.

Aquesta tesi explora la combinació de plataformes microfluídiques per mantenir models biològics in vitro vius amb la ressonància magnètica hiperpolaritzada de 13C. L'objectiu és estudiar la reproductibilitat de l'anàlisi d'hiperpolarització, adaptar sistemes microfluídics per a la injecció precisa d'agents de contrast hiperpolaritzats i l'anàlisi metabòlica dels models in vitro en un escàner MRI amb 13C.