A Novel Graphene Quantum Dot-Based mRNA Delivery Platform

During the last decades, there has been growing interest in using therapeutic messager RNA (mRNA) together with drug delivery systems. Naked, unformulated mRNA is, however, unable to cross the cell membrane and is susceptible to degradation. Here we use graphene quantum dots (GQDs) functionalized with polyethyleneimine (PEI) as a novel mRNA delivery system. Our results show that these modified GQDs can be used to deliver intact and functional mRNA to Huh-7 hepatocarcinoma cells at low doses and, that the GQDs are not toxic, although cellular toxicity is a problem for these first-generation modified particles. Functionalized GQDs represent a potentially interesting delivery system that is easy to manufacture, stable and effective

Multiomics analysis of naturally efficacious lipid nanoparticle coronas reveals high-density lipoprotein is necessary for their function

In terms of lipid nanoparticle (LNP) engineering, the relationship between particle composition, delivery efficacy, and the composition of the biocoronas that form around LNPs, is poorly understood. To explore this we analyze naturally efficacious biocorona compositions using an unbiased screening workflow. First, LNPs are complexed with plasma samples, from individual lean or obese male rats, and then functionally evaluated in vitro. Then, a fast, automated, and miniaturized method retrieves the LNPs with intact biocoronas, and multiomics analysis of the LNP-corona complexes reveals the particle corona content arising from each individual plasma sample. We find that the most efficacious LNP-corona complexes were enriched with high-density lipoprotein (HDL) and, compared to the commonly used corona-biomarker Apolipoprotein E, corona HDL content was a superior predictor of in-vivo activity. Using technically challenging and clinically relevant lipid nanoparticles, these methods reveal a previously unreported role for HDL as a source of ApoE and, form a framework for improving LNP therapeutic efficacy by controlling corona composition.</p

A high-throughput Galectin-9 imaging assay for quantifying nanoparticle uptake, endosomal escape and functional RNA delivery

RNA-based therapies have great potential to treat many undruggable human diseases. However, their efficacy, in particular for mRNA, remains hampered by poor cellular delivery and limited endosomal escape. Development and optimisation of delivery vectors, such as lipid nanoparticles (LNPs), are impeded by limited screening methods to probe the intracellular processing of LNPs in sufficient detail. We have developed a high-throughput imaging-based endosomal escape assay utilising a Galectin-9 reporter and fluorescently labelled mRNA to probe correlations between nanoparticle-mediated uptake, endosomal escape frequency, and mRNA translation. Furthermore, this assay has been integrated within a screening platform for optimisation of lipid nanoparticle formulations. We show that Galectin-9 recruitment is a robust, quantitative reporter of endosomal escape events induced by different mRNA delivery nanoparticles and small molecules. We identify nanoparticles with superior escape properties and demonstrate cell line variances in endosomal escape response, highlighting the need for fine-tuning of delivery formulations for specific applications

Interaction Kinetics of Individual mRNA-Containing Lipid Nanoparticles with an Endosomal Membrane Mimic: Dependence on pH, Protein Corona Formation, and Lipoprotein Depletion

Lipid nanoparticles (LNPs) have emerged as potent carriers for mRNA delivery, but several challenges remain before this approach can offer broad clinical translation of mRNA therapeutics. To improve their efficacy, a better understanding is required regarding how LNPs are trapped and processed at the anionic endosomal membrane prior to mRNA release. We used surface-sensitive fluorescence microscopy with single LNP resolution to investigate the pH dependency of the binding kinetics of ionizable lipid-containing LNPs to a supported endosomal model membrane. A sharp increase of LNP binding was observed when the pH was lowered from 6 to 5, accompanied by stepwise large-scale LNP disintegration. For LNPs preincubated in serum, protein corona formation shifted the onset of LNP binding and subsequent disintegration to lower pH, an effect that was less pronounced for lipoprotein-depleted serum. The LNP binding to the endosomal membrane mimic was observed to eventually become severely limited by suppression of the driving force for the formation of multivalent bonds during LNP attachment or, more specifically, by charge neutralization of anionic lipids in the model membrane due to their association with cationic lipids from earlier attached LNPs upon their disintegration. Cell uptake experiments demonstrated marginal differences in LNP uptake in untreated and lipoprotein-depleted serum, whereas lipoprotein-depleted serum increased mRNA-controlled protein (eGFP) production substantially. This complies with model membrane data and suggests that protein corona formation on the surface of the LNPs influences the nature of the interaction between LNPs and endosomal membranes

Understanding Intracellular Biology to Improve mRNA Delivery by Lipid Nanoparticles

Poor understanding of intracellular delivery and targeting hinders development of nucleic acid‐based therapeutics transported by nanoparticles. Utilizing a siRNA‐targeting and small molecule profiling approach with advanced imaging and machine learning biological insights is generated into the mechanism of lipid nanoparticle (MC3‐LNP) delivery of mRNA. This workflow is termed Advanced Cellular and Endocytic profiling for Intracellular Delivery (ACE‐ID). A cell‐based imaging assay and perturbation of 178 targets relevant to intracellular trafficking is used to identify corresponding effects on functional mRNA delivery. Targets improving delivery are analyzed by extracting data‐rich phenotypic fingerprints from images using advanced image analysis algorithms. Machine learning is used to determine key features correlating with enhanced delivery, identifying fluid‐phase endocytosis as a productive cellular entry route. With this new knowledge, MC3‐LNP is re‐engineered to target macropinocytosis, and this significantly improves mRNA delivery in vitro and in vivo. The ACE‐ID approach can be broadly applicable for optimizing nanomedicine‐based intracellular delivery systems and has the potential to accelerate the development of delivery systems for nucleic acid‐based therapeutics

Molecular Pharmacology and Structure Function Modelling of the Leukotriene B4 Receptor BLT1

Leukotrienes are membrane derived bioactive lipids that play an important role in immune responses by initiating and maintaining the inflammatory responses. Leukotriene B4 (LTB4) released at the site of an inflammatory response attracts, activates and prolongs the life of leukocytes and lymphocytes. LTB4 can bind to two recently cloned cell surface G-protein coupled receptors named BLT1 and BLT2. This thesis focuses on BLT1 and presents the original discovery of the receptor, the development of monoclonal antibodies against the receptor, an investigation of the signal transduction pathways activated by the receptor, and finally, a three-dimensional computer model of the receptor in an active conformation. Using degenerate primers and a homologous screening strategy, a B-cell lymphoblast cDNA library was examined, and a novel receptor was discovered. This thesis presents subsequent experiments involving radioligand binding, assays for intracellular calcium release, and antibodies that identified this protein as a receptor for LTB4. Given the importance of this receptor for inflammation we sought to produce monoclonal antibodies that could be used to easily identify tissues and cells expressing this receptor. Two antibodies were selected based on their superior utility in a number of common immunohistochemical techniques and pharmacological characterisation of these antibodies is presented. Both antibodies could inhibit a variety of functional responses in both cells that endogenously expressed BLT1 and cells that had been transfected with BLT1. Only one of the antibodies (7B1) antagonised the binding of LTB4 however. The other antibody (14F11) was a non-competitive antagonist. Both antibodies were found to require the complex epitopes represented by the tertiary structure of the receptor. The signal transduction mechanisms of BLT1 were examined with a focus on intra-cellular calcium measured in transfected HeLa cells. HeLa cells were used because they are compatible with BLT1 and, unlike most leukocytes, do not express BLT2. BLT1 induces an increase in intracellular calcium concentrations ([Ca2+]i) through both PTX sensitive and insensitive G-proteins. Calcium is initially released from internal stores followed by an influx of extracellular calcium through store-operated channels. This function of the receptor was not sensitive to the redox state of the extracellular environment, but it was sensitive to treatments that modulated membrane cholesterol levels, or membrane physiology. A number of different kinases were also found to have an effect on BLT1 induced changes in [Ca2+]i including protein kinase C (PKC), protein kinase A (PKA) and protein-tyrosine kinases (PTKs). Inhibiting MAP kinases, Rho-associated kinases, or phosphoinositol-3-kinases (PI3K) had no effect on BLT1 induced calcium signalling. In order to explore how LTB4 binds to BLT1 a three-dimensional computer model was created based on a theoretical model of the active conformation of BLT1. LTB4 was then docked into the receptor’s ligand binding pocket and amino acids that could interact with LTB4 were identified. Several receptors with point mutations of key amino acids were then produced and examined using functional and ligand binding experiments to confirm the ligand-receptor interactions suggested by the theoretical studies

Molecular mapping of epitopes involved in ligand activation of the human receptor for the neuropeptide, VIP, based on hybrids with the human secretin receptor

Receptors for the neurotransmitter and neuroendocrine peptides, vasoactive intesinal peptide (VIP) and secretin, both belong to the Type B subfamily of G-protein-coupled receptors. This group is evolutionally as well as structurally distinct from the much larger Type A, or rhodopsin-type, subfamily. We have mapped the ligand-activating epitopes of the human VIP1 receptor by the use of hybrid receptor constructs with the human secretin receptor. Twelve chimeras were synthesized the successively replacing portions of the former receptor with corresponding portions of the latter receptor, or by interchanging the first extracellular loops. Each of the different chimeric receptor DNAs were then expressed in murine reporter cells, and their ability to activate cAMP production was investigated on stimulation with the respective natural peptide ligands. We stimulated the reporter cells with secretion or VIP following transient expression of the receptor chimeras. The experiments indicated that there are two molecular domains of importance for the recognition and activation of these peptides, namely, the inner portion of the extracellular tail and the first extracellular loop of the two receptors

Physiology, pathology and the biomolecular corona:the confounding factors in nanomedicine design

The biomolecular corona that forms on nanomedicines in different physiological and pathological environments confers a new biological identity. How the recipient biological system's state can potentially affect nanomedicine corona formation, and how this can be modulated, remains obscure. With this perspective, this review summarizes the current knowledge about the content of biological fluids in various compartments and how they can be affected by pathological states, thus impacting biomolecular corona formation. The content of representative biological fluids is explored, and the urgency of integrating corona formation, as an essential component of nanomedicine designs for effective cargo delivery, is highlighted