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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., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Although lipid nanoparticles (LNPs) have been FDA-approved for mRNA delivery, there is still much to learn about these fascinating multi-component delivery systems. Here, I discuss the presence of "bleb" structures on LNPs and the co-existence of mRNA-empty LNPs in LNP-mRNA-based formulations. Specifically, I discuss key articles on these structural and compositional heterogeneities, whether these features present negative or positive LNP attributes, and how to deal with them in research and quality control settings. Additionally, I present current approaches and propose novel strategies on how to study and quantify bleb and empty LNP structures. With the conflicting views on these features in the literature and limited systematic studies on their impact on safety and efficacy, I hope this Perspective will support current and bring forward new thinking about these matters. I anticipate that novel studies and insights could emerge from these lines of thinking, which could potentially enhance the development of safe and efficient LNP-based drug products that will either embrace, leverage, or mitigate the presence of blebs and empty LNPs. [Display omitted] • Presents and discusses key articles related to bleb and empty LNP structures. • Examines methods for studying and quantifying these features, including proposing new approaches. • Discusses how to deal with these features differently in research and quality control settings. • Emphasizes the significance of developing standard LNP materials to evaluate LNP analysis tools. • Presents different perspectives on whether bleb and empty LNP structures are positive or negative LNP attributes. [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]

For the past two decades, biomimetic high-density lipoproteins (b-HDL) have been used for various drug delivery applications. The b-HDL mimic the endogenous HDL, and therefore possess many attractive features for drug delivery, including high biocompatibility, biodegradability, and ability to transport and deliver their cargo (e.g. drugs and/or imaging agents) to specific cells and tissues that are recognized by HDL. The b-HDL designs reported in the literature often differ in size, shape, composition, and type of incorporated cargo. However, there exists only limited insight into how the b-HDL design dictates their biodistribution. To fill this gap, we conducted a comprehensive systematic literature search of biodistribution studies using various designs of apolipoprotein A-I (apoA-I)-based b-HDL (i.e. b-HDL with apoA-I, apoA-I mutants, or apoA-I mimicking peptides). We carefully screened 679 papers (search hits) for b-HDL biodistribution studies in mice, and ended up with 24 relevant biodistribution profiles that we compared according to b-HDL design. We show similarities between b-HDL biodistribution studies irrespectively of the b-HDL design, whereas the biodistribution of the b-HDL components (lipids and scaffold) differ significantly. The b-HDL lipids primarily accumulate in liver, while the b-HDL scaffold primarily accumulates in the kidney. Furthermore, both b-HDL lipids and scaffold accumulate well in the tumor tissue in tumor-bearing mice. Finally, we present essential considerations and strategies for b-HDL labeling, and discuss how the b-HDL biodistribution can be tuned through particle design and administration route. Our meta-analysis and discussions provide a detailed overview of the fate of b-HDL in mice that is highly relevant when applying b-HDL for drug delivery or in vivo imaging applications. Unlabelled Image • Biomimetic HDL (b-HDL) used for drug delivery vary in size, shape and composition • The biodistribution of the b-HDL variants is to a large extent similar • The b-HDL lipids accumulate primarily in the liver (and tumor) • The b-HDL scaffold accumulates primarily in the kidney and liver (and tumor) • The b-HDL biodistribution is tunable via particle design and administration route [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]