Plasma lipid transport and metabolism are essential to ensure correct cellular function throughout the body. Dynamically regulated in time and space, the well-characterized mechanisms underpinning... Show morePlasma lipid transport and metabolism are essential to ensure correct cellular function throughout the body. Dynamically regulated in time and space, the well-characterized mechanisms underpinning plasma lipid transport and metabolism offers an enticing, but as yet underexplored, rationale to design synthetic lipid nanoparticles with inherent cell/tissue selectivity. Herein, a systemically administered liposome formulation, composed of just two lipids, that is capable of hijacking a triglyceride lipase-mediated lipid transport pathway resulting in liposome recognition and uptake within specific endothelial cell subsets is described. In the absence of targeting ligands, liposome-lipase interactions are mediated by a unique, phase-separated ("parachute") liposome morphology. Within the embryonic zebrafish, selective liposome accumulation is observed at the developing blood-brain barrier. In mice, extensive liposome accumulation within the liver and spleen - which is reduced, but not eliminated, following small molecule lipase inhibition - supports a role for endothelial lipase but highlights these liposomes are also subject to significant "off-target" by reticuloendothelial system organs. Overall, these compositionally simplistic liposomes offer new insights into the discovery and design of lipid-based nanoparticles that can exploit endogenous lipid transport and metabolism pathways to achieve cell selective targeting in vivo. Show less
Gold nanorods (GNRs) are versatile asymmetric nanoparticles with unique optical properties. These properties make GNRs ideal agents for applications such as photothermal cancer therapy, biosensing,... Show moreGold nanorods (GNRs) are versatile asymmetric nanoparticles with unique optical properties. These properties make GNRs ideal agents for applications such as photothermal cancer therapy, biosensing, and in vivo imaging. However, as-synthesised GNRs need to be modified with a biocompatible stabilising coating in order to be employed in these fields as the ligands used to stabilise GNRs during synthesis are toxic. An issue is that GNR performance in the aforementioned techniques can be affected by these modified coatings. For example if coatings are too thick then GNR entry into cells, or their sensitivity in sensing applications, can be compromised. Here we show that thiolated peptide amphiphiles (PAs) can act as GNR stabilisers and provide a thin and highly-stable coating under physiologically relevant conditions. Additionally, all tested PAs formed highly ordered (51.8-58.8% β-content), and dense (2.62-3.87 peptides per nm2) monolayers on the GNR surface. Moreover, the PA-coated GNRs demonstrated no cytotoxicity in vitro and, via injection in zebrafish embryos, the behavior and cellular interactions of such PA-coated GNRs were visualised in vivo, in real time, with two-photon (2P) microscopy. Show less
Lipid nanoparticles (LNPs) are the leading nonviral technologies for the delivery of exogenous RNA to target cells in vivo. As systemic delivery platforms, these technologies are exemplified by... Show moreLipid nanoparticles (LNPs) are the leading nonviral technologies for the delivery of exogenous RNA to target cells in vivo. As systemic delivery platforms, these technologies are exemplified by Onpattro, an approved LNP-based RNA interference therapy, administered intravenously and targeted to parenchymal liver cells. The discovery of systemically administered LNP technologies capable of preferential RNA delivery beyond hepatocytes has, however, proven more challenging. Here, preceded by comprehensive mechanistic understanding of in vivo nanoparticle biodistribution and bodily clearance, an LNP-based messenger RNA (mRNA) delivery platform is rationally designed to preferentially target the hepatic reticuloendothelial system (RES). Evaluated in embryonic zebrafish, validated in mice, and directly compared to LNP-mRNA systems based on the lipid composition of Onpattro, RES-targeted LNPs significantly enhance mRNA expression both globally within the liver and specifically within hepatic RES cell types. Hepatic RES targeting requires just a single lipid change within the formulation of Onpattro to switch LNP surface charge from neutral to anionic. This technology not only provides new opportunities to treat liver-specific and systemic diseases in which RES cell types play a key role but, more importantly, exemplifies that rational design of advanced RNA therapies must be preceded by a robust understanding of the dominant nano-biointeractions involved. Show less
The work described in this dissertation contributes to a better mechanistic understanding of nanoparticles in vivo. To achieve that goal, we used the zebrafish as a highly predictive pre-screening... Show moreThe work described in this dissertation contributes to a better mechanistic understanding of nanoparticles in vivo. To achieve that goal, we used the zebrafish as a highly predictive pre-screening model of nanoparticles. This approach enables the investigation of the fundamental behavior of nanoparticles, correlation of the physicochemical properties of the formulated nanoparticles with their biodistribution and identification of important nano-bio interactions. Zebrafish established transgenic lines were used to study specific interactions. In addition, genetically modified zebrafish applying CRISPR/Cas9 were generated. These strategies not only show key mechanistic features of nanoparticles in circulation, but also promote the rational design of more efficient nanoparticles systems.After understanding the fundamental behavior of nanoparticles, this thesis describes the identification of a key interaction between stabilins receptors (expressed in liver sinusoidal endothelial cells) and nanoparticles. Next, the scope is changed to design nano-systems that target specific cell types showing liposomes capable of switching the surface charge in situ and in vivo using light as an external trigger and a rationally designed lipid nanoparticle formulation containing mRNA able to preferentially target the hepatic reticuloendothelial system. In addition, a phase-separated liposomes hijacking a lipase mediated transport to selectively target endothelial lipase in vivo was studied. Show less
Clearance of nanoparticles (NPs) after intravenous injection - mainly by the liver - is a critical barrier for the clinical translation of nanomaterials. Physicochemical properties of NPs are known... Show moreClearance of nanoparticles (NPs) after intravenous injection - mainly by the liver - is a critical barrier for the clinical translation of nanomaterials. Physicochemical properties of NPs are known to influence their distribution through cell-specific interactions; however, the molecular mechanisms responsible for liver cellular NP uptake are poorly understood. Liver sinusoidal endothelial cells and Kupffer cells are critical participants in this clearance process. Here we use a zebrafish model for liver-NP interaction to identify the endothelial scavenger receptor Stabilin-1 as a non-redundant receptor for the clearance of small anionic NPs. Furthermore, we show that physiologically, Stabilin-1 is required for the removal of bacterial lipopolysaccharide (LPS/endotoxin) from circulation and that Stabilin-1 cooperates with its homolog Stabilin-2 in the clearance of larger (~100 nm) anionic NPs. Our findings allow optimization of anionic nanomedicine biodistribution and targeting therapies that use Stabilin-1 and -2 for liver endothelium-specific delivery. Show less
Surface charge plays a fundamental role in determining the fate of a nanoparticle, and any encapsulated contents, in vivo. Herein, we describe, and visualise in real time, light-triggered switching... Show moreSurface charge plays a fundamental role in determining the fate of a nanoparticle, and any encapsulated contents, in vivo. Herein, we describe, and visualise in real time, light-triggered switching of liposome surface charge, from neutral to cationic, in situ and in vivo (embryonic zebrafish). Prior to light activation, intravenously administered liposomes, composed of just two lipid reagents, freely circulate and successfully evade innate immune cells present in the fish. Upon in situ irradiation and surface charge switching, however, liposomes rapidly adsorb to, and are taken up by, endothelial cells and/or are phagocytosed by blood resident macrophages. Coupling complete external control of nanoparticle targeting together with the intracellular delivery of encapsulated (and membrane impermeable) cargos, these compositionally simple liposomes are proof that advanced nanoparticle function in vivo does not require increased design complexity but rather a thorough understanding of the fundamental nano-bio interactions involved. Show less
