Studying the fate and action of a designed therapeutic protein-nanomaterial in vivo utilizing a novel correlative cryo-3D-SIM and cryo soft X-ray tomography approach

Abstract

En esta tesis se utilizó una novedosa estrategia correlativa combinando crio microscopía de luz estructurada en 3D (cryo-3D-SIM) y crio tomografía de rayos X blandos (cryo-SXT) para estudiar el destino y la acción de un híbrido proteína-nanomaterial terapéutico. Esta estrategia puede ser especialmente beneficioso para localizar moléculas específicas en el entorno celular nativo en bio-nanomedicina.

Revealing the intracellular location of novel therapeutic agents is paramount for the understanding of their effect at the cell ultrastructure level. During this thesis project we focused on a novel protein-nanomaterial hybrid (Prot-NM) that was designed to exhibit antifibrotic properties. This Prot-NM has shown great promise in mitigating an excess deposition of collagen during myocardial fibrosis. Here, a novel correlative cryo 3D imaging approach was utilized to further investigate the fate and action of this protein. Cardiac fibrosis is a health condition that affects millions of people worldwide and with the main cause being age, incidence for this condition has seen a steady increase over the last few decades. Because of that, it is essential that new therapeutic agents are developed in order to prevent the need for invasive surgery, which is currently the most common intervention. Therapeutic intervention is very challenging because of the complexity of intra- and inter-cellular signalling. Hsp90 is an important protein-folding mediator, called a chaperone, and current inhibitors target its ATP binding site which has a severe effect on the overall cellular homeostasis. As an alternative, our designed Prot-NM targets the C-terminal end of the Hsp90 molecule, which is a protein binding site used for initiating various signalling cascades. By blocking this site collagen production is reduced and its normal chaperone function is maintained. This therapeutic protein was designed based on a common protein domain found in nature, the tetratricopeptide repeat. Apart from its designed Hsp90 binding domain, the Prot-NM has an additional stabilizing domain containing a small gold nanocluster (AuNC). This AuNC increases the overall stability of the TPR-protein, increases cellular uptake rates compared to previous studies.Furthermore, two variants of this Prot-NM were tested, with and without the Hsp90 binding domain, in order to investigate the effect of the AuNC at the cellular level. The cells that were utilized during this study were primary mouse cardiac fibroblasts, and the immortalized variant thereof, NIH-3T3 cells. In order to localize this Prot-NM in vivo, a novel correlative light and X-ray tomography approach was proposed. After an unsuccessful hybrid approach using room temperature confocal microscopy and cryo soft X-ray tomography, the workflow had to be adapted and it was decided to go for an all-cryo approach utilizing the new cryo-3D- structured illumination microscope (SIM) at B24 of the Diamond Synchrotron (UK) which only recently opened for user-access. As this microscope was specifically designed for correlative work with cryo-SXT, sample preparation and imaging proved to be straightforward. However, due to its novelty, no fiducialisation strategy, nor correlation protocols were available at the time. The development of a working protocol for the complete process became therefor a large part of this thesis work. After refining the sample preparation, we were able to collect the data that allowed us to unambiguously localize the Prot-NM within the 3D cellular space of both vitrified cell-types. This newly established correlative workflow placed the fluorescent visible light signal from the Prot-NM within specific multivesicular bodies and showed distinct differences when comparing the two cell-types. This correlative super-resolution and X-ray imaging strategy joins high specificity, by the use of fluorescence, with high spatial resolution at 30 nm (half pitch) provided by cryo-SXT in whole cells, without the need of staining or fixation, and can be of particular benefit to locate specific molecules in the native cellular environment in bio-nanomedicine research. The data presented in this manuscript highlights the development of this new workflow, and its ability to shed light on complex drug-cell interactions. The Prot-NM studied here has shown great promise in preventing myocardial fibrosis events without inducing side effects in primary cardiac mouse fibroblasts.