Cytotoxicity and toxicoproteomics analysis of thiazolidinedione exposure in human-derived cardiomyocytes

Thiazolidinediones (TZDs) (e.g., pioglitazone and rosiglitazone), known insulin sensitiser agents for type II diabetes mellitus, exhibit controversial effects on cardiac tissue. Despite consensus on their association with increased heart failure risk, limiting TZD use in diabetes management, the underlying mechanisms remain uncharacterised. Herein, we report a comprehensive in vitro investigation utilising a novel toxicoproteomics pipeline coupled with cytotoxicity assays in human adult cardiomyocytes to elucidate mechanistic insights into TZD cardiotoxicity. The cytotoxicity assay findings showed a significant loss of mitochondrial adenosine triphosphate production upon exposure to either TZD agents, which may underpin TZD cardiotoxicity. Our toxicoproteomics analysis revealed that mitochondrial dysfunction primarily stems from oxidative phosphorylation impairment, with distinct signalling mechanisms observed for both agents. The type of cell death differed strikingly between the two agents, with rosiglitazone exhibiting features of caspase-dependent apoptosis and pioglitazone implicating mitochondrial-mediated necroptosis, as evidenced by the protein upregulation in the phosphoglycerate mutase family 5–dynamin-related protein 1 axis. Furthermore, our analysis revealed additional mechanistic aspects of cardiotoxicity, showcasing drug specificity. The downregulation of various proteins involved in protein machinery and protein processing in the endoplasmic reticulum was observed in rosiglitazone-treated cells, implicating proteostasis in the rosiglitazone cardiotoxicity. Regarding pioglitazone, the findings suggested the potential activation of the interplay between the complement and coagulation systems and the disruption of the cytoskeletal architecture, which was primarily mediated through the integrin-signalling pathways responsible for pioglitazone-induced myocardial contractile failure. Collectively, this study unlocks substantial mechanistic insight into TZD cardiotoxicity, providing the rationale for future optimisation of antidiabetic therapies

Novel application of synchrotron x-ray computed tomography for ex-vivo imaging of subcutaneously-injected polymeric microsphere suspension formulations

Purpose: Subcutaneously or intramuscularly administered biodegradable microsphere formulations have been successfully exploited in the management of chronic conditions for over two decades, yet mechanistic understanding of the impact of formulation attributes on in vivo absorption rate from such systems is still in its infancy. Methods: Suspension formulation physicochemical attributes may impact particulate deposition in subcutaneous (s.c.) tissue. Hence, the utility of synchrotron X-ray microcomputed tomography (μCT) for assessment of spatial distribution of suspension formulation components (PLG microspheres and vehicle) was evaluated in a porcine s.c. tissue model. Optical imaging of dyed vehicle and subsequent microscopic assessment of microsphere deposition was performed in parallel to compare the two approaches. Results: Our findings demonstrate that synchrotron μCT can be applied to the assessment of microsphere and vehicle distribution in s.c. tissue, and that microspheres can also be visualised in the absence of contrast agent using this approach. The technique was deemed superior to optical imaging of macrotomy for the characterisation of microsphere deposition owing to its non-invasive nature and relatively rapid data acquisition time. Conclusions: The μCT method outlined in this study provides a novel insight into the relative distribution of vehicle and suspended PLG microspheres following s.c. injection. A potential application for our findings is understanding the impact of injection, device and formulation variables on initial and temporal depot geometry in pre-clinical or ex-vivo models that can inform product design

Using narrative medicine via storytelling to improve outcomes in breast cancer patients

Breast cancer (BC) is the principle cause of cancer related deaths in females worldwide. It creates a significant burden on global healthcare, and therefore, remains a priority for the advancement of care toward these patients Narrative medicine (NM) is a technique involving the understanding and utilisation of the patient experience to improve healing. Its therapeutic potential is evident, but has yet to be fully utilised and transferred into a pharmacy settin

Understanding protein corona formation under shear flow conditions

Nanoparticles are small colloidal particles in the 1-100 nm size range. Polymeric nanoparticles are routinely explored for the development of novel drug delivery systems due to their unique and customizable physical and chemical characteristics. Upon introduction to protein-containing medium, nanoparticles will spontaneously adsorb proteins onto their surface and form what is known as the ‘protein corona’. The protein corona leads to changes in the physical and chemical parameters of nanoparticles, which subsequently alters their biological fate (cellular uptake, biodistribution). With most nanoparticles intended for intravenous administration, it is crucial to understand the impact of biological shear flow conditions on protein corona formation and how the protein corona influences their colloidal stability

The application of field-flow fractionation to the analysis of nanomedicines

Thecombination of field-flow fractionation with powerful leading-edge detectors canbe applied to the measurement of nanomaterial physicochemical properties, and thecreation of harmonized robust measurement protocols. The Multiscale MetrologySuite (MMS) at the University of Strathclyde is a unique internationallyleading facility combining multiple leading-edge field flow fractionation modalities(electric, asymmetric and centrifugal) with in-line Raman, inductively-coupledplasma (ICP) mass spectrometry and multimodal detector capability. Using exemplarcase studies, we demonstrate the application of various FFF hyphenations for the analysis of a diverse materials portfolio. One of the goals for the MMS isto raise the standards of traditional academic analytical support underpinningpioneering academic engineering, physical and life sciences research exploringnovel materials as diagnostics and therapeutics

Utilising storytelling to reveal the untold stories of breast cancer

Breast cancer is the most commonly diagnosed female cancer in the UK(1). A 3 part workshop in August 2022 invited ≥ 20 patient participants to recount their narratives of breast cancer using storytelling. The goal of this event was to create a safe environment in which patients’ perceptions of their cancer journey could be shared and documented

The development of a novel pipeline for polymeric nanoparticle characterization following incubation under shear flow conditions

Nanoparticles are small colloidal particles with a dimension between 1-100 nm in size. Polymeric nanoparticles are routinely explored for the development of novel medicines for unmet clinical need due to their unique properties. However, there is currently a high attrition rate for bench-clinic translation, and this may be due to a lack of understanding of the behaviour of nanoparticles under physiologically-relevant conditions. Upon administration to protein-containing medium, nanoparticles will spontaneously adsorb proteins onto their surface and form what is known as the ‘protein corona’ (figure 1) . Protein corona formation leads to changes in the physical and chemical parameters of nanoparticles, which subsequently alters their biological fate (cellular uptake, biodistribution). With most nanoparticles intended for intravenous administration, it is therefore crucial to characterize the impact of biological shear flow conditions on nanoparticle-protein interactions and how this impact their colloidal stability. Aims & Objectives: 1)To develop a robust pipeline for the reproducible characterization of nanoparticles following protein corona formation under physiologically relevant shear flow conditions. 2) To use a range of nanoparticle isolation and analytical techniques to measure the impact of sample handling conditions on particle parameters using model nanoparticles (polystyrene latex)

The role of biological sex in pre-clinical (mouse) mRNA vaccine studies

In this study, we consider the influence of biological sex-specific immune responses on the assessment of mRNA vaccines in pre-clinical murine studies. Recognising the established disparities in immune function attributed to genetic and hormonal differences between individuals of different biological sexes, we compared the mRNA expression and immune responses in mice of both biological sexes after intramuscular injection with mRNA incorporated within lipid nanoparticles. Regarding mRNA expression, no significant difference in protein (luciferase) expression at the injection site was observed between female and male mice following intramuscular administration; however, we found that female BALB/c mice exhibit significantly greater total IgG responses across the concentration range of mRNA lipid nanoparticles (LNPs) in comparison to their male counterparts. This study not only contributes to the scientific understanding of mRNA vaccine evaluation but also emphasizes the importance of considering biological sex in vaccine study designs during pre-clinical evaluation in murine studies

Orthogonal pipelines for lipid nanoparticle evaluation

Ribonucleic acid (RNA) drugs pose promising candidates for gene therapy in treatment resistant conditions and rare diseases. The FDA approval of siRNA Onpattro® in 2018 mRNA-LNP Spikevax® and Comirnaty® COVID-19 vaccinations in 2021[1] ignited research interests as these were the first siRNA and mRNA candidates to utilize lipid nanoparticles (LNPs) as a drug delivery platform. As the RNA-LNP research field is rapidly growing, robust, high-resolution separation techniques coupled to in-line detectors are required to analyze particle key quality attributes and accelerate the successful clinical translation of RNA-LNP therapies. Asymmetric-Flow Field Flow Fraction (AF4) and Size Exclusion Chromatography (SEC) are robust approaches for the characterization of oligo-LNPs [2, 3]. AF4 utilizes perpendicular field induction and particle diffusion-based separation, whereas SEC uses LNP-stationary phase interactions for separation. The goal of this study was to develop separation pipelines for the high-resolution analysis of LNPs. Briefly, we prepared (6Z,9Z,28Z,31Z)-heptatriaconta6,9,28,31-tetraen-19-yl-4-(dimethylamino)- butanoate:cholesterol: 1,2-distearoyl-sn-glycero-3-phosphocholine: 1,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (MC3:CHOL:DSPC:DMG-PEG2000) LNPs and 8-[(2- hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester:cholesterol: 1,2-distearoyl-sn-glycero-3-phosphocholine: 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (SM-102:CHOL:DSPC:DMG-PEG2000) using microfluidics at 50:38.5:10:1.5 mol% ratio. Here, we performed AF4, combined with in-line dynamic light scattering, multi-angle light scattering, and UV detection. Using these detectors, we measured key particle quality attributes including particle size, polydispersity index (PDI), and shape factor. The properties were evaluated alongside oligo-LNP samples that had not been subjected to separation. Manufacture of LNPs using microfluidics-based analysis led to PolyA MC3-LNPs in the 56.5 nm ± 1.2 nm size range with a corresponding PDI of 0.12 ± 0.02, and PolyA SM-102-LNPs of 48.5 nm ± 1.1 nm with a PDI of 0.10 ± 0.01. Our findings show the presence of sub-populations within LNP samples, which cannot be detected using routine particle metrology techniques such as nanoparticle tracking analysis and dynamic light scattering. Our results highlight the need for developing more high-resolution approaches for the analysis of LNPs and linking these to input materials and process parameter design

Nanoparticle isolation from biological media for protein corona analysis : the impact of incubation and recovery protocols on nanoparticle properties

Nanoparticles are increasingly implemented in biomedical applications, including the diagnosis and treatment of disease. When exposed to complex biological media, nanoparticles spontaneously interact with their surrounding environment, leading to the surface-adsorption of small and bio- macromolecules- termed the "corona". Corona composition is governed by nanoparticle properties and incubation parameters. While the focus of most studies is on the protein signature of the nanoparticle corona, the impact of experimental protocols on nanoparticle size in the presence of complex biological media, and the impact of nanoparticle recovery from biological media has not yet been reported. Here using a non-degradable robust model, we show how centrifugation-resuspension protocols used for the isolation of nanoparticles from incubation media, incubation duration and shear flow conditions alter nanoparticle parameters including particle size, zeta potential and total protein content. Our results show significant changes in nanoparticle size following exposure to media containing protein under different flow conditions, which also altered the composition of surface-adsorbed proteins profiled by SDS-PAGE. Our in situ analysis of nanoparticle size in media containing protein using particle tracking analysis highlights that centrifugation-resuspension is disruptive to agglomerates that are spontaneously formed in protein containing media, highlighting the need for in situ analytical methods that do not alter the intermediates formed following nanoparticle exposure to biological media. Nanomedicines are mostly intended for parenteral administration, and our findings show that parameters such as shear flow can significantly alter nanoparticle physicochemical parameters. Overall, we show that the centrifugation-resuspension isolation of nanoparticles from media significantly alters particle parameters in addition to the overall protein composition of surface-adsorbed proteins. We recommend that nanoparticle characterization pipelines studying bio-nano interactions during early nanomedicine development consider biologically-relevant shear flow conditions and media composition that can significantly alter particle physical parameters and subsequent conclusions from these studies