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Lipid nanoparticle (LNP) remains the most advanced platform for messenger RNA (mRNA) delivery. To date, mRNA LNPs synthesis is mostly performed by mixing lipids and mRNA with microfluidics. In this study, a cost‐effective microfluidic setup for synthesizing mRNA LNPs is developed. It allows to fine‐tune the LNPs characteristics without compromising LNP properties. It is compared with a commercial device (NanoAssemblr) and ethanol injection and the influence of manufacturing conditions on the performance of mRNA LNPs is investigated. LNPs prepared by ethanol injection exhibit broader size distributions and more inhomogeneous internal structure (e.g., bleb‐like substructures), while other LNPs show uniform structure with dense cores. Small angel X‐ray scattering (SAXS) data indicate a tighter interaction between mRNA and lipids within LNPs synthesized by custom device, compared to LNPs produced by NanoAssemblr. Interestingly, the better transfection efficiency of polysarcosine (pSar)‐modified LNPs correlates with a higher surface roughness than that of PEGylated ones. The manufacturing approach, however, shows modest influence on mRNA expression in vivo. In summary, the home‐developed cost‐effective microfluidic device can synthesize LNPs and represents a potent alternative to NanoAssemblr. The preparation methods show notable effect on LNPs’ structure but a minor influence on mRNA delivery in vitro and in vivo. Lipid nanoparticles remain the clinically established platform for mRNA delivery and mostly synthesized using microfluidic device. In this study, the correlation is presented between production method (commerical/house‐devleloped microfluidic device and ethanol injection), lipid composition (PEGylated lipid and polysarcosine‐conjugated lipid), internal structure, physicochemical properties, and mRNA delivery performance in vitro and in vivo.

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 vivointravenous 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 vivoglioblastoma models.

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.

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 stab2and 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 stab2inhibition or targeting as a general approach for modifying nanoparticle–liver interactions of a wide range of nanomedicines.

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.

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. In this study, a liposome formulation, composed of just two lipids and characterized by phase separation, is described. In vivo, after systemic administration in zebrafish and mice, this formulation is capable of hijacking a triglyceride lipase‐mediated lipid transport pathway resulting in liposome recognition and uptake within specific endothelial cell subsets.

Mesoporous silica nanoparticles (MSNs) have been explored extensively as solid supports for proteins in biological and medical applications. Small (<200 nm) MSNs with ordered large pores (>5 nm), capable of encapsulating therapeutic small molecules suitable for delivery applications in vivo, are rare however. Here we present small, elongated, cuboidal, MSNs with average dimensions of 90 × 43 nm that possess disk-shaped cavities, stacked on top of each other, which run parallel to the short axis of the particle. Amine functionalization was achieved by modifying the MSN surface with 3-aminopropyltriethoxysilane or 3-[2-(2-aminoethylamino)ethylamino]propyltrimethoxysilane (AP-MSNs and AEP-MSNs) and were shown to have similar dimensions to the nonfunctionalized MSNs. The dimensions of these particles, and their large surface areas as measured by nitrogen adsorption–desorption isotherms, make them ideal scaffolds for protein encapsulation and delivery. We therefore investigated the encapsulation and release behavior for seven model proteins (α-lactalbumin, ovalbumin, bovine serum albumin, catalase, hemoglobin, lysozyme, and cytochrome c). It was discovered that all types of MSNs used in this study allow rapid encapsulation, with a high loading capacity, for all proteins studied. Furthermore, the release profiles of the proteins were tunable. The variation in both rate and amount of protein uptake and release was found to be determined by the surface chemistry of the MSNs, together with the isoelectric point (pI), and molecular weight of the proteins, as well as by the ionic strength of the buffer. These MSNs with their large surface area and optimal dimensions provide a scaffold with a high encapsulation efficiency and controllable release profiles for a variety of proteins, enabling potential applications in fields such as drug delivery and protein therapy.

Efficient delivery of drugs to living cells is still a major challenge. Currently, most methods rely on the endocytotic pathway resulting in low delivery efficiency due to limited endosomal escape and/or degradation in lysosomes. Here, we report a new method for direct drug delivery into the cytosol of live cells in vitroand invivoutilizing targeted membrane fusion between liposomes and live cells. A pair of complementary coiled-coil lipopeptides was embedded in the lipid bilayer of liposomes and cell membranes respectively, resulting in targeted membrane fusion with concomitant release of liposome encapsulated cargo including fluorescent dyes and the cytotoxic drug doxorubicin. Using a wide spectrum of endocytosis inhibitors and endosome trackers, we demonstrate that the major site of cargo release is at the plasma membrane. This method thus allows for the quick and efficient delivery of drugs and is expected to have many invitro, ex vivo, and invivoapplications.

Triple-negative breast cancer (TNBC) is a highly aggressive and recurrent type of breast carcinoma that is associated with poor patient prognosis. Because of the limited efficacy of current treatments, new therapeutic strategies need to be developed. The CXCR4-CXCL12 chemokine signaling axis guides cell migration in physiological and pathological processes, including breast cancer metastasis. Although targeted therapies to inhibit the CXCR4-CXCL12 axis are under clinical experimentation, still no effective therapeutic approaches have been established to block CXCR4 in TNBC. To unravel the role of the CXCR4-CXCL12 axis in the formation of TNBC early metastases, we used the zebrafish xenograft model. Importantly, we demonstrate that cross-communication between the zebrafish and human ligands and receptors takes place and human tumor cells expressing CXCR4 initiate early metastatic events by sensing zebrafish cognate ligands at the metastatic site. Taking advantage of the conserved intercommunication between human tumor cells and the zebrafish host, we blocked TNBC early metastatic events by chemical and genetic inhibition of CXCR4 signaling. We used IT1t, a potent CXCR4 antagonist, and show for the first time its promising anti-tumor effects. In conclusion, we confirm the validity of the zebrafish as a xenotransplantation model and propose a pharmacological approach to target CXCR4 in TNBC.

Biallelic inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene predisposes human patients to the development of highly vascularized neoplasms in multiple organ systems. We show that zebrafish vhl mutants display a marked increase in blood vessel formation throughout the embryo, starting at 2 days post-fertilization. The most severe neovascularization is observed in distinct areas that overlap with high vegfa mRNA expression, including the vhl mutant brain and eye. Real-time quantitative PCR revealed increased expression of the duplicated VEGFA orthologs vegfaa and vegfab, and of vegfb and its receptors flt1, kdr and kdr-like, indicating increased vascular endothelial growth factor (Vegf) signaling in vhl mutants. Similar to VHL-associated retinal neoplasms, diabetic retinopathy and age-related macular degeneration, we show, by tetramethyl rhodamine-dextran angiography, that vascular abnormalities in the vhl−/− retina lead to vascular leakage, severe macular edema and retinal detachment. Significantly, vessels in the brain and eye express cxcr4a, a marker gene expressed by tumor and vascular cells in VHL-associated hemangioblastomas and renal cell carcinomas. VEGF receptor (VEGFR) tyrosine kinase inhibition (through exposure to sunitinib and 676475) blocked vhl−/−-induced angiogenesis in all affected tissues, demonstrating that Vegfaa, Vegfab and Vegfb are key effectors of the vhl−/− angiogenic phenotype through Flt1, Kdr and Kdr-like signaling. Since we show that the vhl−/− angiogenic phenotype shares distinct characteristics with VHL-associated vascular neoplasms, zebrafish vhl mutants provide a valuable in vivo vertebrate model to elucidate underlying mechanisms contributing to the development of these lesions. Furthermore, vhl mutant zebrafish embryos carrying blood vessel-specific transgenes represent a unique and clinically relevant model for tissue-specific, hypoxia-induced pathological angiogenesis and vascular retinopathies. Importantly, they will allow for a cost-effective, non-invasive and efficient way to screen for novel pharmacological agents and combinatorial treatments.