Zebrafish (Danio rerio) larva as an in vivo vertebrate model to study renal function.

There is an increasing interest in using zebrafish (Danio rerio) larva as a vertebrate screening model to study drug disposition. As the pronephric kidney of zebrafish larvae shares high similarity with the anatomy of nephrons in higher vertebrates including humans, we explored in this study whether 3- to 4-day-old zebrafish larvae have a fully functional pronephron. Intravenous injection of fluorescent polyethylene glycol and dextran derivatives of different molecular weight revealed a cutoff of 4.4-7.6 nm in hydrodynamic diameter for passive glomerular filtration, which is in agreement with corresponding values in rodents and humans. Distal tubular reabsorption of a FITC-folate conjugate, covalently modified with PEG2000, via folate receptor 1 was shown. Transport experiments of fluorescent substrates were assessed in the presence and absence of specific inhibitors in the blood systems. Thereby, functional expression in the proximal tubule of organic anion transporter oat (slc22) multidrug resistance-associated protein mrp1 (abcc1), mrp2 (abcc2), mrp4 (abcc4), and zebrafish larva p-glycoprotein analog abcb4 was shown. In addition, nonrenal clearance of fluorescent substrates and plasma protein binding characteristics were assessed in vivo. The results of transporter experiments were confirmed by extrapolation to ex vivo experiments in killifish (Fundulus heteroclitus) proximal kidney tubules. We conclude that the zebrafish larva has a fully functional pronephron at 96 h postfertilization and is therefore an attractive translational vertebrate screening model to bridge the gap between cell culture-based test systems and pharmacokinetic experiments in higher vertebrates.NEW & NOTEWORTHY The study of renal function remains a challenge. In vitro cell-based assays are approved to study, e.g., ABC/SLC-mediated drug transport but do not cover other renal functions such as glomerular filtration. Here, in vivo studies combined with in vitro assays are needed, which are time consuming and expensive. In view of these limitations, our proof-of-concept study demonstrates that the zebrafish larva is a translational in vivo test model that allows for mechanistic investigations to study renal function.

Virus-Derived Peptides for Hepatic Enzyme Delivery.

Recently, a lipopeptide derived from the hepatitis B virus (HBV) large surface protein has been developed as an HBV entry inhibitor. This lipopeptide, called MyrcludexB (MyrB), selectively binds to the sodium taurocholate cotransporting polypeptide (NTCP) on the basolateral membrane of hepatocytes. Here, the feasibility of coupling therapeutic enzymes to MyrB was investigated for the development of enzyme delivery strategies. Hepatotropic targeting shall enable enzyme prodrug therapies and detoxification procedures. Here, horseradish peroxidase (HRP) was conjugated to MyrB via maleimide chemistry, and coupling was validated by SDS-PAGE and reversed-phase HPLC. The specificity of the target recognition of HRP-MyrB could be shown in an NTCP-overexpressing liver parenchymal cell line, as demonstrated by competitive inhibition with an excess of free MyrB and displayed a strong linear dependency on the applied HRP-MyrB concentration. In vivo studies in zebrafish embryos revealed a dominating interaction of HRP-MyrB with scavenger endothelial cells vs xenografted NTCP expressing mammalian cells. In mice, radiolabeled 125I-HRP-MyrBy, as well as the non-NTCP targeted control HRP-peptide-construct (125I-HRP-alaMyrBy) demonstrated a strong liver accumulation confirming the nonspecific interaction with scavenger cells. Still, MyrB conjugation to HRP resulted in an increased and NTCP-mediated hepatotropism, as revealed by competitive inhibition. In conclusion, the model enzyme HRP was successfully conjugated to MyrB to achieve NTCP-specific targeting in vitro with the potential for ex vivo diagnostic applications. In vivo, target specificity was reduced by non-NTCP-mediated interactions. Nonetheless, tissue distribution experiments in zebrafish embryos provide mechanistic insight into underlying scavenging processes indicating partial involvement of stabilin receptors.

Toxicity of silver nanoparticles in macrophages.

Silver nanoparticles (nanosilver) are broadly used today in textiles, food packaging, household devices and bioapplications, prompting a better understanding of their toxicity and biological interactions. In particular, the cytotoxicity of nanosilver with respect to mammalian cells remains unclear, because such investigations can be biased by the nanosilver coatings and the lack of particle size control. Here, nanosilver of well-defined size (5.7 to 20.4 nm) supported on inert nanostructured silica is produced using flame aerosol technology. The cytotoxicity of the prepared nanosilver with respect to murine macrophages is assessed in vitro because these cells are among the first to confront nanosilver upon its intake by mammals. The silica support facilitates the dispersion and stabilization of the prepared nanosilver in biological suspensions, and no other coating or functionalization is applied that could interfere with the biointeractions of nanosilver. Detailed characterization of the particles by X-ray diffraction and electron microscopy reveals that the size of the nanosilver is well controlled. Smaller nanosilver particles release or leach larger fractions of their mass as Ag⁺ ions upon dispersion in water. This strongly influences the cytotoxicity of the nanosilver when incubated with murine macrophages. The size of the nanosilver dictates its mode of cytotoxicity (Ag⁺ ion-specific and/or particle-specific). The toxicity of small nanosilver (<10 nm) is mostly mediated by the released Ag⁺ ions. The influence of such ions on the toxicity of nanosilver decreases with increasing nanosilver size (>10 nm). Direct silver nanoparticle-macrophage interactions dominate the nanosilver toxicity at sizes larger than 10 nm.

Impact of non-ionizable lipids and phase mixing methods on structural properties of lipid nanoparticle formulations.

Lipid nanoparticles (LNPs) have been widely investigated for nucleic acid therapeutic delivery, and demonstrated their potential in enabling new mRNA vaccines. LNPs are usually formulated with multi-lipid components and the composition variables may impact their structural properties. Here, we investigated the impact of helper lipids on physicochemical properties of LNPs using a Design of Experiments (DoE) definitive screening design. Phospholipid head group, degree of unsaturation, ratio to cholesterol as well as PEG-lipid content were varied and a series of 14 LNPs were prepared by microfluidic- and solvent-injection mixing. Solvent-injection mixing by a robotic liquid handler yielded 50-225 nm nanoparticles with highly ordered, ∼5 nm inter-lamellar spacing as measured by small angle X-ray scattering (SAXS) and confirmed by cryo-transmission electron microscopy (cryo-EM). In contrast, microfluidic mixing resulted in less ordered, notably smaller (50-75 nm) and more homogenous nanoparticles. Significant impacts of the stealth-lipid DSPE-PEG2000 on nanoparticle size, polydispersity and encapsulation efficiency of an oligonucleotide cargo were observed in LNPs produced by both methods, while varying the phospholipid type and content had only marginal effect on these physicochemical properties. These findings suggest that from a physicochemical perspective, the design space for combinations of helper lipids in LNPs may be considerably larger than anticipated based on the conservative formulation composition of the currently FDA-approved LNPs, thereby opening opportunities for screening and optimization of novel LNP formulations.

Peritoneal dialysis beyond kidney failure?

Compared to extracorporeal modalities, peritoneal dialysis (PD) is less invasive and more cost-effective, wherein blood is dialyzed intra-corporeally against a solution instilled in the peritoneal cavity. Although PD is mainly indicated for patients with end-stage renal failure, it has also been used for several non-renal indications. The aim of this review is to provide an overview of the role of PD beyond kidney failure. The alternative indications of PD include hypothermia, congestive heart failure, hyperammonemia and poisoning with xenobiotics. The use of PD as a treatment for acute pancreatitis and psoriasis was initially proposed but could not be established; these indications are therefore classified as historically relevant. Recent developments have led to a potential application of PD during the management of stroke and as an oxygenation therapy with the use of oxygen carriers. Novel colloid-based dialysates with improved functionality with respect to detoxification and oxygenation are currently underway, though their efficacy has so far only been demonstrated in pre-clinical settings. Finally, insight into potential future developments of PD is given. Characterization studies are proposed to better understand the fate of non-recovered carriers following dialysate removal, their efficacy following multiple administrations and potential immune response to optimize their formulation, enabling their clinical translation.

Liposome-supported enzymatic peritoneal dialysis.

Compared to hemodialysis, peritoneal dialysis represents a more straightforward and less invasive alternative, though current solutions are not as effective. Herein, the feasibility of liposome-supported enzymatic peritoneal dialysis (LSEPD) is explored to increase the functionality of peritoneal dialysis for the model indication acute alcohol poisoning. Enzyme-loaded liposomes (E-Liposomes) containing alcohol metabolizing enzymes, alcohol oxidase and catalase, are developed and their in vitro and in vivo performances investigated. The E-Liposomes outperform the free enzymes in stability, overcoming the thermal instability of alcohol oxidase and enhancing the in vitro ethanol elimination, which is further accelerated by hydrogen peroxide, due to the rapid generation of oxygen by catalase. Compared to the free enzymes, the E-Liposomes exhibit reduced systemic exposure and organ distribution. In a rodent ethanol intoxication model, LSEPD enhances ethanol metabolism as evidenced by an increased acetaldehyde production, ethanol's primary metabolite. In conclusion, LSEPD presents an innovative platform to temporarily enhance xenobiotic metabolism, in view of the improved enzyme stability and peritoneal retention.

Enzyme-Mimetic Antioxidant Luminescent Nanoparticles for Highly Sensitive Hydrogen Peroxide Biosensing.

Hydrogen peroxide (H2O2) is an abundant molecule associated with biological functions and reacts with natural enzymes, such as catalase. Even though direct H2O2 measurement can be used to diagnose pathological conditions, such as infection and inflammation, H2O2 quantification further enables the detection of disease biomarkers in enzyme-linked assays (e.g., ELISA) in which enzymatic reactions may generate or consume H2O2. Such a quantification is often measured optically with organic dyes in biological media that suffer, however, from poor stability. Currently, the optical H2O2 biosensing without organic-dyes in biological media and at low, submicromolar, concentrations has yet to be achieved. Herein, we rationally design biomimetic artificial enzymes based on antioxidant CeO2 nanoparticles that become luminescent upon their Eu3+ doping. We vary systematically their diameter from 4 to 16 nm and study their catalase-mimetic antioxidant activity, manifested as catalytic H2O2 decomposition in aqueous solutions, revealing a strong nanoparticle surface area dependency. The interaction with H2O2 influences distinctly the particle luminescence rendering them highly sensitive H2O2 biosensors down to 0.15 µM (5.2 ppb) in solutions for biological assays. Our results link two, so far, unrelated research domains, the CeO2 nanoparticle antioxidant activity and luminescence by rare-earth doping. When these enzyme-mimetic nanoparticles are coupled with alcohol oxidase, biosensing can be extended to ethanol exemplifying how their detection potential can be broadened to additional biologically relevant metabolites. The enzyme-mimetic nanomaterial developed here could serve as a starting point of sophisticated in vitro assays toward the highly sensitive detection of disease biomarkers.

Gastroresistant oral peptide for fluorescence imaging of colonic inflammation.

The use of molecular markers for inflammation in the gastrointestinal tract could empower optical imaging modalities for early diagnosis and eventually personalized timely treatments. A major hurdle to the widespread use of functional fluorescence imaging is the absence of suitable contrast agents, in particular to be administered via the oral route due to the usual proteolytic susceptibility of the biomarkers. By designing a retro-inverso peptide, starting from a previously described sequence specific for N-cadherin, we achieved resistance to gastrointestinal degradation and even slightly improved specificity towards the target, both in ex vivo and in vivo experimental colitis. Simulations at fundamental molecular level suggested that the introduced retro-inverso modifications did not affect the folding of the peptide, leaving its ability to interact with the binding pocket of the monomeric N-cadherin unaltered, even when fluorescently labeled. Possible further derivatization of this sequence could be envisaged to further extend the potential of the designed retro-inverso peptide as diagnostic or theranostic agent for the oral route.