Deconvolving Passive and Active Targeting of Liposomes Bearing LDL Receptor Binding Peptides Using the Zebrafish Embryo Model.

Improving target versus off-target ratio in nanomedicine remains a major challenge for increasing drug bioavailability and reducing toxicity. Active targeting using ligands on nanoparticle surfaces is a key approach but has limited clinical success. A potential issue is the integration of targeting ligands also changes the physicochemical properties of nanoparticles (passive targeting). Direct studies to understand the mechanisms of active targeting and off-targeting in vivo are limited by the lack of suitable tools. Here, the biodistribution of a representative active targeting liposome is analyzed, modified with an apolipoprotein E (ApoE) peptide that binds to the low-density lipoprotein receptor (LDLR), using zebrafish embryos. The ApoE liposomes demonstrated the expected liver targeting effect but also accumulated in the kidney glomerulus. The ldlra-/- zebrafish is developed to explore the LDLR-specificity of ApoE liposomes. Interestingly, liver targeting depends on the LDLR-specific interaction, while glomerular accumulation is independent of LDLR and peptide sequence. It is found that cationic charges of peptides and the size of liposomes govern glomerular targeting. Increasing the size of ApoE liposomes can avoid this off-targeting. Taken together, the study shows the potential of the zebrafish embryo model for understanding active and passive targeting mechanisms, that can be used to optimize the design of nanoparticles.

Zebrafish as outgroup model to study evolution of scavenger receptor class B type I functions.

BACKGROUND AND AIMS: Scavenger receptor class B1 (SCARB1) - also known as the high-density lipoprotein (HDL) receptor - is a multi-ligand scavenger receptor that is primarily expressed in liver and steroidogenic organs. This receptor is known for its function in reverse cholesterol transport (RCT) in mammals and hence disruption leads to a massive increase in HDL cholesterol in these species. The extracellular domain of SCARB1 - which is important for cholesterol handling - is highly conserved across multiple vertebrates, except in zebrafish. METHODS: To examine the functional conservation of SCARB1 among vertebrates, two stable scarb1 knockout zebrafish lines, scarb1 715delA (scarb1 -1 nt) and scarb1 715_716insGG (scarb1 +2 nt), were created using CRISPR-Cas9 technology. RESULTS: We demonstrate that, in zebrafish, SCARB1 deficiency leads to disruption of carotenoid-based pigmentation, reduced fertility, and a decreased larvae survival rate, whereas steroidogenesis was unaltered. The observed reduced fertility is driven by defects in female fertility (-50 %, p < 0.001). Importantly, these alterations were independent of changes in free (wild-type 2.4 ± 0.2 µg/µl versus scarb1-/- 2.0 ± 0.1 µg/µl) as well as total (wild-type 4.2 ± 0.4 µg/µl versus scarb1-/- 4.0 ± 0.3 µg/µl) plasma cholesterol levels. Uptake of HDL in the liver of scarb1-/- zebrafish larvae was reduced (-86.7 %, p < 0.001), but this coincided with reduced perfusion of the liver. No effect was observed on lipoprotein uptake in the caudal vein. SCARB1 deficient canaries, which also lack carotenoids in their plumage, similarly as scarb1-/- zebrafish, failed to show an increase in plasma free- and total cholesterol levels. CONCLUSION: Our findings suggest that the specific function of SCARB1 in maintaining plasma cholesterol could be an evolutionary novelty that became prominent in mammals, while other known functions were already present earlier during vertebrate evolution.

Polysarcosine-based lipid formulations for intracranial delivery of mRNA.

Messenger RNA (mRNA) is revolutionizing the future of therapeutics in a variety of diseases, including neurological disorders. Lipid formulations have shown to be an effective platform technology for mRNA delivery and are the basis for the approved mRNA vaccines. In many of these lipid formulations, polyethylene glycol (PEG)-functionalized lipid provides steric stabilization and thus plays a key role in improving the stability both ex vivo and in vivo. However, immune responses towards PEGylated lipids may compromise the use of those lipids in some applications (e.g., induction of antigen specific tolerance), or within sensitive tissues (e.g., central nervous system (CNS)). With respect to this issue, polysarcosine (pSar)-based lipopolymers were investigated as an alternative to PEG-lipid in mRNA lipoplexes for controlled intracerebral protein expression in this study. Four polysarcosine-lipids with defined sarcosine average molecular weight (Mn = 2 k, 5 k) and anchor diacyl chain length (m = 14, 18) were synthesized, and incorporated into cationic liposomes. We found that the content, pSar chain length and carbon tail lengths of pSar-lipids govern the transfection efficiency and biodistribution. Increasing carbon diacyl chain length of pSar-lipid led up to 4- and 6-fold lower protein expression in vitro. When the length of either pSar chain or lipid carbon tail increased, the transfection efficiency decreased while the circulation time was prolonged. mRNA lipoplexes containing 2.5% C14-pSar2k resulted in the highest mRNA translation in the brain of zebrafish embryos through intraventricular injection, while C18-pSar2k-liposomes showed a comparable circulation with DSPE-PEG2k-liposomes after systemic administration. To conclude, pSar-lipid enable efficient mRNA delivery, and can substitute PEG-lipids in lipid formulations for controlled protein expression within the CNS.

Phase-Separated Liposomes Hijack Endogenous Lipoprotein Transport and Metabolism Pathways to Target Subsets of Endothelial Cells In Vivo.

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 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.

Development of a Combined Lipid-Based Nanoparticle Formulation for Enhanced siRNA Delivery to Vascular Endothelial Cells.

Low transfection efficiency in endothelial cells (EC) is still a bottleneck for the majority of siRNA-based vascular delivery approaches. In this work, we developed a lipid-based nanoparticle (LNP) formulation based on a combination of a permanently charged cationic lipid-DOTAP and a conditionally ionized cationic lipid-MC3 (DOTAP/MC3) for the enhanced delivery of siRNA into EC. Compared with a single DOTAP or MC3-based benchmark LNP, we demonstrated that the DOTAP/MC3 LNP formulation shows the best transfection efficiency both in primary EC in vitro and in endothelium in zebrafish. The high transfection activity of the DOTAP/MC3 LNP formulation is achieved by a combination of improved endothelial association mediated by DOTAP and MC3-triggered efficient siRNA intracellular release in EC. Furthermore, AbVCAM-1-coupled DOTAP/MC3 LNP-mediated siRNARelA transfection showed pronounced anti-inflammatory effects in inflammatory-activated primary EC by effectively blocking the NF-κB pathway. In conclusion, the combination of permanent and ionizable cationic lipids in LNP formulation provides an effective endothelial cell delivery of siRNA.

Investigating 3R In Vivo Approaches for Bio-Distribution and Efficacy Evaluation of Nucleic Acid Nanocarriers: Studies on Peptide-Mimicking Ionizable Lipid.

Formulations based on ionizable amino-lipids have been put into focus as nucleic acid delivery systems. Recently, the in vitro efficacy of the lipid formulation OH4:DOPE has been explored. However, in vitro performance of nanomedicines cannot correctly predict in vivo efficacy, thereby considerably limiting pre-clinical translation. This is further exacerbated by limited access to mammalian models. The present work proposes to close this gap by investigating in vivo nucleic acid delivery within simpler models, but which still offers physiologically complex environments and also adheres to the 3R guidelines (replace/reduce/refine) to improve animal experiments. The efficacy of OH4:DOPE as a delivery system for nucleic acids is demonstrated using in vivo approaches. It is shown that the formulation is able to transfect complex tissues using the chicken chorioallantoic membrane model. The efficacy of DNA and mRNA lipoplexes is tested extensively in the zebra fish (Danio rerio) embryo which allows the screening of biodistribution and transfection efficiency. Effective transfection of blood vessel endothelial cells is seen, especially in the endocardium. Both model systems allow an efficacy screening according to the 3R guidelines bypassing the in vitro-in vivo gap. Pilot studies in mice are performed to correlate the efficacy of in vivo transfection.

Stabilin 1 and 2 are important regulators for cellular uptake of apolipoprotein B-containing lipoproteins in zebrafish.

BACKGROUND AND AIMS: Scavenger receptors form a superfamily of membrane-bound receptors that bind and internalize different types of ligands, including pro-atherogenic oxidized low-density lipoproteins (oxLDLs). In vitro studies have indicated a role for the liver sinusoidal endothelial cell receptors stabilin 1 (stab1) and 2 (stab2) in oxLDL clearance. In this study, we evaluated the potential role of stab1 and stab2 in lipoprotein uptake in zebrafish, an upcoming model for studying cholesterol metabolism and atherosclerosis. METHODS: Lipoproteins were injected in the duct of Cuvier of wild-type (ABTL) or stab1 and stab2 mutant (stab1-/-stab2-/-) zebrafish larvae at 3 days post-fertilization. To examine the effect of stabilin deficiency on lipoprotein and cholesterol metabolism, zebrafish larvae were challenged with a high cholesterol diet (HCD; 4% w/w) for 10 days. RESULTS: Lipoprotein injections showed impaired uptake of both LDL and oxLDL into the vessel wall of caudal veins of stab1-/-stab2-/- zebrafish, which was paralleled by redistribution to tissue macrophages. Total body cholesterol levels did not differ between HCD-fed stab1-/-stab2-/- and ABTL zebrafish. However, stab1-/-stab2-/- larvae exhibited 1.4-fold higher mRNA expression levels of ldlra involved in (modified) LDL uptake, whereas the expression levels of scavenger receptors scarb1 and cd36 were significantly decreased. CONCLUSIONS: We have shown that stabilins 1 and 2 have an important scavenging function for apolipoprotein B-containing lipoproteins in zebrafish and that combined deficiency of these two proteins strongly upregulates the clearance of lipoproteins by macrophages within the caudal vein. Our current study highlights the use of zebrafish as model to study lipoprotein metabolism and liver sinusoidal endothelial cell function.

Regenerating vascular mural cells in zebrafish fin blood vessels are not derived from pre-existing mural cells and differentially require Pdgfrb signalling for their development.

Vascular networks comprise endothelial cells and mural cells, which include pericytes and smooth muscle cells. To elucidate the mechanisms controlling mural cell recruitment during development and tissue regeneration, we studied zebrafish caudal fin arteries. Mural cells colonizing arteries proximal to the body wrapped around them, whereas those in more distal regions extended protrusions along the proximo-distal vascular axis. Both cell populations expressed platelet-derived growth factor receptor ß (pdgfrb) and the smooth muscle cell marker myosin heavy chain 11a (myh11a). Most wrapping cells in proximal locations additionally expressed actin alpha2, smooth muscle (acta2). Loss of Pdgfrb signalling specifically decreased mural cell numbers at the vascular front. Using lineage tracing, we demonstrate that precursor cells located in periarterial regions and expressing Pgdfrb can give rise to mural cells. Studying tissue regeneration, we did not find evidence that newly formed mural cells were derived from pre-existing cells. Together, our findings reveal conserved roles for Pdgfrb signalling in development and regeneration, and suggest a limited capacity of mural cells to self-renew or contribute to other cell types during tissue regeneration.

Anionic Lipid Nanoparticles Preferentially Deliver mRNA to the Hepatic Reticuloendothelial System.

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 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.

Meningeal lymphatic endothelial cells fulfill scavenger endothelial cell function and cooperate with microglia in waste removal from the brain.

Brain lymphatic endothelial cells (BLECs) constitute a group of loosely connected endothelial cells that reside within the meningeal layer of the zebrafish brain without forming a vascular tubular system. BLECs have been shown to readily endocytose extracellular cargo molecules from the brain parenchyma, however, their functional relevance in relation to microglia remains enigmatic. We here compare their functional uptake efficiency for several macromolecules and bacterial components with microglia in a qualitative and quantitative manner in 5-day-old zebrafish embryos. We find BLECs to be significantly more effective in the uptake of proteins, polysaccharides and virus particles as compared to microglia, while larger particles like bacteria are only ingested by microglia but not by BLECs, implying a clear distribution of tasks between the two cell types in the brain area. In addition, we compare BLECs to the recently discovered scavenger endothelial cells (SECs) of the cardinal vein and find them to accept an identical set of substrate molecules. Our data identifies BLECs as the first brain-associated SEC population in vertebrates, and demonstrates that BLECs cooperate with microglia to remove particle waste from the brain.

Together is Better: mRNA Co-Encapsulation in Lipoplexes is Required to Obtain Ratiometric Co-Delivery and Protein Expression on the Single Cell Level.

Liposomes can efficiently deliver messenger RNA (mRNA) into cells. When mRNA cocktails encoding different proteins are needed, a considerable challenge is to efficiently deliver all mRNAs into the cytosol of each individual cell. In this work, two methods are explored to co-deliver varying ratiometric doses of mRNA encoding red (R) or green (G) fluorescent proteins and it is found that packaging mRNAs into the same lipoplexes (mingle-lipoplexes) is crucial to efficiently deliver multiple mRNA types into the cytosol of individual cells according to the pre-defined ratio. A mixture of lipoplexes containing only one mRNA type (single-lipoplexes), however, seem to follow the "first come - first serve" principle, resulting in a large variation of R/G uptake and expression levels for individual cells leading to ratiometric dosing only on the population level, but rarely on the single-cell level. These experimental observations are quantitatively explained by a theoretical framework based on the stochasticity of mRNA uptake in cells and endosomal escape of mingle- and single-lipoplexes, respectively. Furthermore, the findings are confirmed in 3D retinal organoids and zebrafish embryos, where mingle-lipoplexes outperformed single-lipoplexes to reliably bring both mRNA types into single cells. This benefits applications that require a strict control of protein expression in individual cells.

Zebrafish Embryos as a Predictive Animal Model to Study Nanoparticle Behavior in vivo.

A failure to fully understand the complex in vivo behavior of systemically administered nanomedicines has stymied clinical translation. To bridge this knowledge gap, new in vivo tools are needed to rapidly and accurately assess the nearly infinite array of possible nanoparticle designs. Zebrafish embryos are small, transparent, and easily manipulated animals that allow for whole organism visualization of fluorescently labeled nanoparticles in real time and at cellular resolution using standard microscope setups. Furthermore, key nano-bio interactions present in higher vertebrates are fully conserved in zebrafish embryos, making these animal models a highly predictive and instructive addition to the nanomedicine design pipeline. Here, we present a step-by-step protocol to intravenously administer, image, and analyze nanoparticle behavior in zebrafish embryos and highlight key nano-bio interactions within the embryonic zebrafish corresponding to those commonly found within the mammalian liver. In addition, we outline practical steps required to achieve light-triggered activation of nanoparticles within the transparent embryo. Graphic abstract: Zebrafish embryos to study nanoparticle behavior in vivo. Formulation, intravenous administration, imaging, and analysis of nanoparticles.

Stabilin-1 is required for the endothelial clearance of small anionic nanoparticles.

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 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.

Development of curcumin-loaded zein nanoparticles for transport across the blood-brain barrier and inhibition of glioblastoma cell growth.

Glioblastoma (GBM) is a devastating primary brain tumor resistant to conventional therapies. A major obstacle to GBM treatment is the blood-brain barrier (BBB), or blood-glioma barrier, which prevents the transport of systemically administered (chemotherapeutic) drugs into the tumor. This study reports the design of dodecamer peptide (G23)-functionalized polydopamine (pD)-coated curcumin-loaded zein nanoparticles (CUR-ZpD-G23 NPs) that efficiently traversed the BBB, and delivered curcumin to glioblastoma cells. The NPs enhanced the cellular uptake of curcumin by C6 glioma cells compared to free curcumin, and showed high penetration into 3D tumor spheroids. Functionalization of the NPs with G23 stimulated BBB crossing and tumor spheroid penetration. Moreover, the NPs markedly inhibited proliferation and migration and induced cell death in liquid and soft agar models of C6 glioma cell growth. Fluorescence microscopy and flow cytometry studies showed that the CUR-ZpD-G23 NPs increased cellular ROS production and induced apoptosis of C6 glioma cells. Following in vivo intravenous injection in zebrafish, ZpD-G23 NPs demonstrated the ability to circulate, which is a first prerequisite for their use in targeted drug delivery. In conclusion, zein-polydopamine-G23 NPs show potential as a drug delivery platform for therapy of GBM, which requires further validation in in vivo glioblastoma models.

Stab2-Mediated Clearance of Supramolecular Polymer Nanoparticles in Zebrafish Embryos.

Supramolecular polymers are attractive scaffolds for use as nanocarriers in drug delivery thanks to their modularity and easy fabrication; however, a molecular view into their in vivo behavior is lacking. Herein, we prepare fluorescent squaramide-based supramolecular polymer nanoparticles that range from fibers to spheres while maintaining their surface chemistry and near-neutral surface charge by a co-assembly approach involving a sulfo-cyanine-labeled monomer to track their in vivo biodistribution behavior and clearance in optically transparent zebrafish embryos. Evasion of macrophages, localization of the fibrillar aggregates in the caudal vein, and association with scavenger endothelial cells are observed. The interaction of the fibrillar supramolecular nanoparticles with the caudal vein is abrogated in gene-edited zebrafish lacking Stabilin-2, a receptor analogously found in the mammalian liver, providing a molecular view into their interaction with scavenger endothelial cells. We further show that this interaction can be tuned based on the choice of monomer and its resultant self-assembly.

Zebrafish as a preclinical in vivo screening model for nanomedicines.

The interactions of nanomedicines with biological environments is heavily influenced by their physicochemical properties. Formulation design and optimization are therefore key steps towards successful nanomedicine development. Unfortunately, detailed assessment of nanomedicine formulations, at a macromolecular level, in rodents is severely limited by the restricted imaging possibilities within these animals. Moreover, rodent in vivo studies are time consuming and expensive, limiting the number of formulations that can be practically assessed in any one study. Consequently, screening and optimisation of nanomedicine formulations is most commonly performed in surrogate biological model systems, such as human-derived cell cultures. However, despite the time and cost advantages of classical in vitro models, these artificial systems fail to reflect and mimic the complex biological situation a nanomedicine will encounter in vivo. This has acutely hampered the selection of potentially successful nanomedicines for subsequent rodent in vivo studies. Recently, zebrafish have emerged as a promising in vivo model, within nanomedicine development pipelines, by offering opportunities to quickly screen nanomedicines under in vivo conditions and in a cost-effective manner so as to bridge the current gap between in vitro and rodent studies. In this review, we outline several advantageous features of the zebrafish model, such as biological conservation, imaging modalities, availability of genetic tools and disease models, as well as their various applications in nanomedicine development. Critical experimental parameters are discussed and the most beneficial applications of the zebrafish model, in the context of nanomedicine development, are highlighted.

Dynamics of dual-fluorescent polymersomes with durable integrity in living cancer cells and zebrafish embryos.

The long-term fate of biomedical nanoparticles after endocytosis is often only sparsely addressed in vitro and in vivo, while this is a crucial parameter to conclude on their utility. In this study, dual-fluorescent polyisobutylene-polyethylene glycol (PiB-PEG) polymersomes were studied for several days in vitro and in vivo. In order to optically track the vesicles' integrity, one fluorescent probe was located in the membrane and the other in the aqueous interior compartment. These non-toxic nanovesicles were quickly endocytosed in living A549 lung carcinoma cells but unusually slowly transported to perinuclear lysosomal compartments, where they remained intact and luminescent for at least 90 h without being exocytosed. Fluorescence-assisted flow cytometry indicated that after endocytosis, the nanovesicles were eventually degraded within 7-11 days. In zebrafish embryos, the polymersomes caused no lethality and were quickly taken up by the endothelial cells, where they remained fully intact for as long as 96 h post-injection. This work represents a novel case-study of the remarkable potential of PiB-PEG polymersomes as an in vivo bio-imaging and slow drug delivery platform.

Lipid bilayer-coated mesoporous silica nanoparticles carrying bovine hemoglobin towards an erythrocyte mimic.

Hemoglobin (Hb)-loaded mesoporous silica nanoparticles (MSNs) coated with a lipid bilayer (LB-MSNs) were investigated as an erythrocyte mimic. MSNs with a large average pore size (10 nm) act as a rigid core and provide a protective environment for Hb encapsulated inside the pores. The colloidal stability of Hb-loaded MSNs was enhanced upon the application of a lipid bilayer, through fusion of PEGylated liposomes onto the exterior surface of Hb-loaded MSNs. The morphology and mesostructure of the MSNs were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and surface area analysis. The Hb loading capacity (mg/g) in MSNs was studied by thermogravimetric analysis (TGA). UV-Vis absorption spectroscopy revealed that Hb inside MSNs had an identical, but slightly broadened peak in the Soret region compared to free Hb. Furthermore the encapsulated Hb exhibits similar peroxidase-like activity in catalyzing the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) with hydrogen peroxide. The introduction of a supported lipid bilayer (LB) demonstrated the potential to prevent premature Hb release (the burst release decreased from 25.50 ±â€¯0.33% to 6.73 ±â€¯0.83%) and increased the colloidal stability of the Hb-loaded MSNs (hydrodynamic diameter remained ∼250 nm for at least one week). The in vivo systemic circulation and biodistribution of LB-MSNs were studied in optically transparent zebrafish embryos, revealing that LB-MSNs have the potential to act as an erythrocyte mimic in transfusion therapy.

Directing Nanoparticle Biodistribution through Evasion and Exploitation of Stab2-Dependent Nanoparticle Uptake.

Up to 99% of systemically administered nanoparticles are cleared through the liver. Within the liver, most nanoparticles are thought to be sequestered by macrophages (Kupffer cells), although significant nanoparticle interactions with other hepatic cells have also been observed. To achieve effective cell-specific targeting of drugs through nanoparticle encapsulation, improved mechanistic understanding of nanoparticle-liver interactions is required. Here, we show the caudal vein of the embryonic zebrafish ( Danio rerio) can be used as a model for assessing nanoparticle interactions with mammalian liver sinusoidal (or scavenger) endothelial cells (SECs) and macrophages. We observe that anionic nanoparticles are primarily taken up by SECs and identify an essential requirement for the scavenger receptor, stabilin-2 ( stab2) in this process. Importantly, nanoparticle-SEC interactions can be blocked by dextran sulfate, a competitive inhibitor of stab2 and other scavenger receptors. Finally, we exploit nanoparticle-SEC interactions to demonstrate targeted intracellular drug delivery resulting in the selective deletion of a single blood vessel in the zebrafish embryo. Together, we propose stab2 inhibition or targeting as a general approach for modifying nanoparticle-liver interactions of a wide range of nanomedicines.

Endoglin controls blood vessel diameter through endothelial cell shape changes in response to haemodynamic cues.

The hierarchical organization of properly sized blood vessels ensures the correct distribution of blood to all organs of the body, and is controlled via haemodynamic cues. In current concepts, an endothelium-dependent shear stress set point causes blood vessel enlargement in response to higher flow rates, while lower flow would lead to blood vessel narrowing, thereby establishing homeostasis. We show that during zebrafish embryonic development increases in flow, after an initial expansion of blood vessel diameters, eventually lead to vessel contraction. This is mediated via endothelial cell shape changes. We identify the transforming growth factor beta co-receptor endoglin as an important player in this process. Endoglin mutant cells and blood vessels continue to enlarge in response to flow increases, thus exacerbating pre-existing embryonic arterial-venous shunts. Together, our data suggest that cell shape changes in response to biophysical cues act as an underlying principle allowing for the ordered patterning of tubular organs.