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The advent of lipid nanoparticles (LNPs) containing ionizable cationic lipids has enabled the encapsulation, stabilization, and intracellular delivery of nucleic acid payloads, leading to FDA-approved siRNA-based therapy and mRNA-based vaccines. Other nucleic acid-based therapeutic modalities, including protein replacement and CRISPR-mediated gene knockout and editing, are being tested in clinical trials, in many cases, for the treatment of liver-related diseases. However, to fully exploit these therapies beyond the liver, improvements in their delivery to extrahepatic targets are needed. Towards this end, both active targeting strategies based on targeting ligands grafted onto LNPs and passive targeting relying on physicochemical LNP parameters such as surface composition, charge, and size are being evaluated. Often, the latter strategy depends on the interaction of LNPs with blood components, forming what is known as the biomolecular corona. Here, I discuss potential challenges related to current LNP-based targeting strategies and the studies of the biomolecular corona on LNPs. I propose potential solutions to overcome some of these obstacles and present approaches currently being tested in preclinical and clinical studies, which face fewer biological barriers than traditional organ-targeting approaches. [Display omitted] • Presents an overview of LNP-based strategies for extrahepatic RNA delivery. • Highlights potential challenges with the LNP strategies and approaches to overcome some of them. • Stresses inherent challenges associated with biomolecular corona studies of LNPs. • Highlights that LNPs are a minor subpopulation of nanoparticles in human plasma. • Advocates innovative approaches to drive progress in the LNP field. [ABSTRACT FROM AUTHOR]

Fluorescence-based techniques are prevalent in studies of nanomedicine-targeting to cells and tissues. However, fluorescence-based studies are rarely quantitative, thus prohibiting direct comparisons of nanomedicine-performance across studies. With this Commentary , we aim to provoke critical thinking about experimental design by treating some often-overlooked pitfalls in 'quantitative' fluorescence-based experimentation. Focusing on fluorescence-labeled nanoparticles, we cover mechanisms like solvent-interactions and fluorophore-dissociation, which disqualify the assumption that ' a higher fluorescence readout ' translates directly to ' a better targeting efficacy '. With departure in recent literature, we propose guidelines for circumventing these pitfalls in studies of tissue-accumulation and cell-uptake, thus covering fluorescence-based techniques like bulk solution fluorescence measurements, fluorescence microscopy, flow cytometry, and infrared fluorescence imaging. With this, we hope to lay a foundation for more 'quantitative thinking' during experimental design, enabling (for example) the estimation and reporting of actual numbers of fluorescent nanoparticles accumulated in cells and organs. [Display omitted] • Stresses the importance of more quantitative approaches in fluorescence-based nanomedicine studies • Provides a brief introduction to some fundamental principles of fluorescence in simple language • Treats fluorescence-based measurements in bulk solution, microscopy, flow cytometry and tissue/body imaging • Highlights often-overlooked challenges related to interactions between fluorophores and the biological environment • Presents strategies toward counting actual numbers of nanoparticles in cells and organs [ABSTRACT FROM AUTHOR]

Flow cytometry (FCM) is a popular method used in characterisation of extracellular vesicles (EVs). Circulating EVs are often identified by FCM by exploiting the lipid nature of EVs by staining with Annexin V (Anx5) or lactadherin against the membrane phospholipid phosphatidylserine (PS) and evaluating the specificity of the labels by detergent lysis of EVs. Here, we investigate whether PS labelling and detergent lysis approaches are confounded by lipoproteins, another family of lipid‐based nanoparticles found in blood, in both frozen and fresh blood plasma. We demonstrated that Anx5 and lactadherin in addition to EVs stained ApoB‐containing lipoproteins, identified by the use of fluorophore‐labelled polyclonal ApoB‐antibody, and that Anx5 had a significantly larger tendency for labelling lipoprotein‐bound PS than lactadherin. Furthermore, detergent lysis resulted in a decrease in both EV and lipoprotein events and especially lipoproteins positive for either Anx5 or lactadherin. Taken together, our findings pose concerns to the use of lipid‐based strategies in identifying EVs by FCM and support the use of transmembrane proteins such as tetraspannins to distinguish EVs from lipoproteins. [ABSTRACT FROM AUTHOR]

[Display omitted] Cationic liposomes specifically target monocytes in blood, rendering them promising drug-delivery tools for cancer immunotherapy, vaccines, and therapies for monocytic leukaemia. The mechanism behind this monocyte targeting ability is, however, not understood, but may involve plasma proteins adsorbed on the liposomal surfaces. To shed light on this, we investigated the biomolecular corona of three different types of PEGylated cationic liposomes, finding all of them to adsorb hyaluronan-associated proteins and proteoglycans upon incubation in human blood plasma. This prompted us to study the role of the TLR4 co-receptors CD44 and CD14, both involved in signalling and uptake pathways of proteoglycans and glycosaminoglycans. We found that separate inhibition of each of these receptors hampered the monocyte uptake of the liposomes in whole human blood. Based on clues from the biomolecular corona, we have thus identified two receptors involved in the targeting and uptake of cationic liposomes in monocytes, in turn suggesting that certain proteoglycans and glycosaminoglycans may serve as monocyte-targeting opsonins. This mechanistic knowledge may pave the way for rational design of future monocyte-targeting drug-delivery platforms. [ABSTRACT FROM AUTHOR]

Flow cytometry (FCM) is a high‐throughput fluorescence‐based technique for multiparameter analysis of individual particles, including cells and nanoparticles. Currently, however, FCM does in many cases not permit proper counting of fluorophore‐tagged markers on individual particles, due to a lack of tools for translating FCM output intensities into accurate numbers of fluorophores. This lack hinders derivation of detailed biologic information and comparison of data between experiments with FCM. To address this technological void, the authors here use DNA nanotechnology to design and construct barrel‐shaped DNA‐origami nanobeads for fluorescence/antigen quantification in FCM. Each bead contains a specific number of calibrator fluorophores and a fluorescent trigger domain with an alternative fluorophore for proper detection in FCM. Using electron microscopy, single‐particle fluorescence microscopy, and FCM, the design of each particle is verified. To validate that the DNA bead‐based FCM calibration enabled the authors to determine the number of antigens on a biological particle, the uniform and well‐characterized murine leukemia virus (MLV) is studied. 48 ± 11 envelope surface protein (Env) trimers per MLV is obtained, which is consistent with reported numbers that relied on low‐throughput imaging. Thus, the authors' DNA‐beads should accelerate quantitative studies of the biology of individual particles with FCM. [ABSTRACT FROM AUTHOR]

Bin/amphiphysin/Rvs (BAR) domains are positively charged crescent-shaped modules that mediate curvature of negatively charged lipid membranes during remodeling processes. The BAR domain proteins PICK1, ICA69, and the arfaptins have recently been demonstrated to coordinate the budding and formation of immature secretory granules (ISGs) at the trans-Golgi network. Here, we identify 4 coding variants in the PICK1 gene from a whole-exome screening of Danish patients with diabetes that each involve a change in positively charged residues in the PICK1 BAR domain. All 4 coding variants failed to rescue insulin content in INS-1E cells upon knock down of endogenous PICK1. Moreover, 2 variants showed dominant-negative properties. In vitro assays addressing BAR domain function suggested that the coding variants compromised BAR domain function but increased the capacity to cause fission of liposomes. Live confocal microscopy and super-resolution microscopy further revealed that PICK1 resides transiently on ISGs before egress via vesicular budding events. Interestingly, this egress of PICK1 was accelerated in the coding variants. We propose that PICK1 assists in or complements the removal of excess membrane and generic membrane trafficking proteins, and possibly also insulin, from ISGs during the maturation process; and that the coding variants may cause premature budding, possibly explaining their dominant-negative function. [ABSTRACT FROM AUTHOR]

[Display omitted] Nucleic acid-based therapeutics encapsulated into lipid nanoparticles (LNPs) can potentially target the root cause of genetic skin diseases. Although nanoparticles are considered impermeable to skin, research and clinical studies have shown that nanoparticles can penetrate into skin with reduced skin barrier function when administered topically. Studies have shown that epidermal keratinocytes express the low-density lipoprotein receptor (LDLR) that mediates endocytosis of apolipoprotein E (apoE)-associated nanoparticles and that dermal fibroblasts express mannose receptors. Here we prepared LNPs designed to exploit these different endocytic pathways for intracellular mRNA delivery to the two most abundant skin cell types, containing: (i) labile PEG-lipids (DMG-PEG2000) prone to dissociate and facilitate apoE-binding to LNPs, enabling apoE-LDLR mediated uptake in keratinocytes, (ii) non-labile PEG-lipids (DSPE-PEG2000) to impose stealth-like properties to LNPs to enable targeting of distant cells, and (iii) mannose-conjugated PEG-lipids (DSPE-PEG2000-Mannose) to target fibroblasts or potentially immune cells containing mannose receptors. All types of LNPs were prepared by vortex mixing and formed monodisperse (PDI ∼ 0.1) LNP samples with sizes of 130 nm (±25%) and high mRNA encapsulation efficiencies (≥90%). The LNP-mediated transfection potency in keratinocytes and fibroblasts was highest for LNPs containing labile PEG-lipids, with the addition of apoE greatly enhancing transfection via LDLR. Coating LNPs with mannose did not improve transfection, and stealth-like LNPs show limited to no transfection. Taken together, our studies suggest using labile PEG-lipids and co-administration of apoE when exploring LNPs for skin delivery. [ABSTRACT FROM AUTHOR]

[Display omitted] Lipid nanoparticles (LNPs) play an important role in mRNA vaccines against COVID-19. In addition, many preclinical and clinical studies, including the siRNA-LNP product, Onpattro®, highlight that LNPs unlock the potential of nucleic acid-based therapies and vaccines. To understand what is key to the success of LNPs, we need to understand the role of the building blocks that constitute them. In this Review , we discuss what each lipid component adds to the LNP delivery platform in terms of size, structure, stability, apparent pK a , nucleic acid encapsulation efficiency, cellular uptake, and endosomal escape. To explore this, we present findings from the liposome field as well as from landmark and recent articles in the LNP literature. We also discuss challenges and strategies related to in vitro/in vivo studies of LNPs based on fluorescence readouts, immunogenicity/reactogenicity, and LNP delivery beyond the liver. How these fundamental challenges are pursued, including what lipid components are added and combined, will likely determine the scope of LNP-based gene therapies and vaccines for treating various diseases. [ABSTRACT FROM AUTHOR]

Membrane-active peptides (MAPs) have several potential therapeutic uses, including as antimicrobial drugs. Many traditional methods used to evaluate the membrane interactions of MAPs have limited applicability. Low-throughput methods, such as microscopy, provide detailed information but often rely on fluorophore-labeled MAPs, and high-throughput assays, such as the calcein release assay, cannot assess the mechanism behind the disruption of vesicular-based lipid membranes. Here we present a flow cytometric assay that provides detailed information about the peptide-lipid membrane interactions on single artificial lipid vesicles while being high-throughput (1000–2000 vesicles/s) and based on label-free MAPs. We synthesized and investigated six MAPs with different modes of action to evaluate the versatility of the assay. The assay is based on the flow cytometric readouts from artificial lipid vesicles, including the fluorescence from membrane-anchored and core-encapsulated fluorophores, and the vesicle concentration. From these parameters, we were able to distinguish between MAPs that induce vesicle solubilization, permeation (pores/membrane distortion), and aggregation or fusion. Our flow cytometry findings have been verified by traditional methods, including the calcein release assay, dynamic light scattering, and fluorescence microscopy on giant unilamellar vesicles. We envision that the presented flow cytometric assay can be used for various types of peptide-lipid membrane studies, e.g. to identify new antibiotics. Moreover, the assay can easily be expanded to derive additional valuable information. [Display omitted] • Developed a novel assay to study the modes of action of membrane-active peptides. • Our flow cytometric high-throughput assay relies on label-free peptides. • Fluorescence microscopy, DLS and calcein release studies confirmed the validity of our assay. • Six very different membrane-active peptides were evaluated in our assay. [ABSTRACT FROM AUTHOR]