Unravelling Heterogeneities in Complement and Antibody Opsonization of Individual Liposomes as a Function of Surface Architecture.

Coating nanoparticles with poly(ethylene glycol) (PEG) is widely used to achieve long-circulating properties after infusion. While PEG reduces binding of opsonins to the particle surface, immunogenic anti-PEG side-effects show that PEGylated nanoparticles are not truly "stealth" to surface active proteins. A major obstacle for understanding the complex interplay between opsonins and nanoparticles is the averaging effects of the bulk assays that are typically applied to study protein adsorption to nanoparticles. Here, a microscopy-based method for directly quantifying opsonization at the single nanoparticle level is presented. Various surface coatings are investigated on liposomes, including PEG, and show that opsonization by both antibodies and complement C3b is highly dependent on the surface chemistry. It is further demonstrated that this opsonization is heterogeneous, with opsonized and non-opsonized liposomes co-existing in the same ensemble. Surface coatings modify the percentage of opsonized liposomes and/or opsonin surface density on the liposomes, with strikingly different patterns for antibodies and complement. Thus, this assay provides mechanistic details about opsonization at the single nanoparticle level previously inaccessible to established bulk assays.

Pay Attention to Biological Nanoparticles when Studying the Protein Corona on Nanomedicines.

The protein corona of nanoparticles has in recent years received considerable attention, and even been postulated to be the missing link in the translation of nanomedicines from benchtop to bedside. We highlight the different types of biological nanoparticles present in blood that need to be considered in the protein corona research field. We map their size, density, and plasma concentrations, and use this information to stress potential challenges related to the isolation of nanomedicines-with a particular focus on liposomes-when using the traditional isolation methods that separate according to size and density.

Unique Calibrators Derived from Fluorescence-Activated Nanoparticle Sorting for Flow Cytometric Size Estimation of Artificial Vesicles: Possibilities and Limitations.

The use of high-throughput flow cytometry to characterize nanoparticles has received increased interest in recent years. However, to fully realize the potential of flow cytometry for the characterization of nanometer-sized objects, suitable calibrators for size estimation must be developed and the sensitivity of conventional flow cytometers has to be advanced. Based on the scattered signal, silica and plastic beads have often been used as flow cytometric size calibrators to evaluate the size of extracellular vesicles and artificial vesicles (liposomes). However, several studies have shown that these beads are unable to accurately correlate scatter intensity to vesicle size. In this work, we present a novel method to estimate the size of individual liposomes in flow cytometry based on liposomal size calibrators prepared by fluorescence-activated cell sorting (FACS), here coined fluorescence-activated nanoparticle sorting (FANS). These calibration liposomes exhibit sizes, structures, and refractive indexes identical to the particles being studied and thus can serve as unique calibrators. First, a sample of polydisperse fluorophore-labeled unilamellar liposomes was prepared and analyzed by flow cytometry. Next, different fractions of the polydisperse liposomes were FANS-sorted according to their fluorescence intensity. Thereafter, we employed nanoparticle tracking analysis (NTA) to evaluate the liposome sizes of the FANS-sorted liposome fractions. Finally, we correlated the flow cytometric readouts (side scatter and fluorescence intensity) of the FANS-sorted liposome fractions with their corresponding size obtained by NTA. This procedure enabled us to translate the liposome fluorescence intensity to the liposome size in nanometers for all detected individual liposomes. We validated the size distribution of our polydisperse liposome sample obtained from flow cytometry in combination with our FANS-calibrators against standard methods for sizing nanoparticles, including NTA and cryo-transmission electron microscopy. This work also highlights the limitation of using the flow cytometric side scattering readout to determine the size of small (30-300 nm) artificial vesicles. © 2019 International Society for Advancement of Cytometry.

The Composition of Reconstituted High-Density Lipoproteins (rHDL) Dictates the Degree of rHDL Cargo- and Size-Remodeling via Direct Interactions with Endogenous Lipoproteins.

The application of reconstituted high-density lipoproteins (rHDL) as a drug-carrier has during the past decade been established as a promising approach for effective receptor-mediated drug delivery, and its ability to target tumors has recently been confirmed in a clinical trial. The rHDL mimics the endogenous HDL, which is known to be highly dynamic and undergo extensive enzyme-mediated remodulations. Hence, to reveal the physiological rHDL stability, a thorough characterization of the dynamics of rHDL in biologically relevant environments is needed. We employ a size-exclusion chromatography (SEC) method to evaluate the dynamics of discoidal rHDL in fetal bovine serum (FBS), where we track both the rHDL lipids (by the fluorescence from lipid-conjugated fluorophores) and apoA-I (by human apoA-I ELISA). We show by using lipoprotein depleted FBS and isolated lipoproteins that rHDL lipids can be transferred to endogenous lipoproteins via direct interactions in a nonenzymatic process, resulting in rHDL compositional- and size-remodeling. This type of dynamics could lead to misinterpretations of fluorescence-based rHDL uptake studies due to desorption of labile lipophilic fluorophores or off-target side effects due to desorption of incorporated drugs. Importantly, we show how the degree of rHDL remodeling can be controlled by the compositional design of the rHDL. Understanding the correlation between the molecular properties of the rHDL constituents and their collective dynamics is essential for improving the rHDL-based drug delivery platform. Taken together, our work highlights the need to carefully consider the compositional design of rHDL and test its stability in a biological relevant environment, when developing rHDL for drug delivery purposes.

Wnt3a nanodisks promote ex vivo expansion of hematopoietic stem and progenitor cells.

BACKGROUND: Wnt proteins modulate development, stem cell fate and cancer through interactions with cell surface receptors. Wnts are cysteine-rich, glycosylated, lipid modified, two domain proteins that are prone to aggregation. The culprit responsible for this behavior is a covalently bound palmitoleoyl moiety in the N-terminal domain. RESULTS: By combining murine Wnt3a with phospholipid and apolipoprotein A-I, ternary complexes termed nanodisks (ND) were generated. ND-associated Wnt3a is soluble in the absence of detergent micelles and gel filtration chromatography revealed that Wnt3a co-elutes with ND. In signaling assays, Wnt3a ND induced ß-catenin stabilization in mouse fibroblasts as well as hematopoietic stem and progenitor cells (HSPC). Prolonged exposure of HSPC to Wnt3a ND stimulated proliferation and expansion of Lin(-) Sca-1(+) c-Kit(+) cells. Surprisingly, ND lacking Wnt3a contributed to Lin(-) Sca-1(+) c-Kit(+) cell expansion, an effect that was not mediated through ß-catenin. CONCLUSIONS: The data indicate Wnt3a ND constitute a water-soluble transport vehicle capable of promoting ex vivo expansion of HSPC.

Evaluation of reconstituted high-density lipoprotein (rHDL) as a drug delivery platform - a detailed survey of rHDL particles ranging from biophysical properties to clinical implications.

During the last decade, and with increasing intensity, the potential for using reconstituted high-density lipoprotein (rHDL) particles to deliver hydrophobic drugs to impaired cells and tissues has been explored. Here, we evaluate various parameters that should be considered when utilizing discoidal rHDL particles as a drug delivery platform. Key parameters such as preparation basics, pronounced statistical variation in drug incorporation across rHDL particles, effects of lipid composition on HDL/rHDL in vivo and vitro dynamics/particle stability, and pharmacokinetic/safety data from rHDL infusion studies in human subjects will be addressed including the innate receptors and native functions of HDL. The broad but detailed information presented in this work could also be deployed in other rHDL-related research. However, the major aim of this review is to point out factors that have the potential to advance rHDL research toward realizing the 'magic bullet' for lipophilic and hydrophilic drug delivery in various clinical contexts.

Anti-CD20 single chain variable antibody fragment-apolipoprotein A-I chimera containing nanodisks promote targeted bioactive agent delivery to CD20-positive lymphomas.

A fusion protein comprising an α-CD20 single chain variable fragment (scFv) antibody, a spacer peptide, and human apolipoprotein (apo) A-I was constructed and expressed in Escherichia coli. The lipid interaction properties intrinsic to apoA-I as well as the antigen recognition properties of the scFv were retained by the chimera. scFv•apoA-I was formulated into nanoscale reconstituted high-density lipoprotein particles (termed nanodisks; ND) and incubated with cultured cells. α-CD20 scFv•apoA-I ND bound to CD20-positive non-Hodgkins lymphoma (NHL) cells (Ramos and Granta) but not to CD20-negative T lymphocytes (i.e., Jurkat). Binding to NHL cells was partially inhibited by pre-incubation with rituximab, a monoclonal antibody directed against CD20. Confocal fluorescence microscopy analysis of Granta cells following incubation with α-CD20 scFv•apoA-I ND formulated with the intrinsically fluorescent hydrophobic polyphenol, curcumin, revealed α-CD20 scFv•apoA-I localizes to the cell surface, while curcumin off-loads and gains entry to the cell. Compared to control incubations, viability of cultured NHL cells was decreased upon incubation with α-CD20 scFv•apoA-I ND harboring curcumin. Thus, formulation of curcumin ND with α-CD20 scFv•apoA-I as the scaffold component confers cell targeting and enhanced bioactive agent delivery, providing a strategy to minimize toxicity associated with chemotherapeutic agents.

Aberrant hetero-disulfide bond formation by the hypertriglyceridemia-associated p.Gly185Cys APOA5 variant (rs2075291).

OBJECTIVE: Apolipoprotein A-V (apoA-V) is a low-abundance plasma protein that modulates triacylglycerol homeostasis. Gene transfer studies were undertaken in apoa5 (-/-) mice to define the mechanism underlying the correlation between the single-nucleotide polymorphism c.553G>T in APOA5 and hypertriglyceridemia. APPROACH AND RESULTS: Adeno-associated virus (AAV) 2/8-mediated gene transfer of wild-type apoA-V induced a dramatic lowering of plasma triacylglycerol in apoa5 (-/-) mice, whereas AAV2/8-Gly162Cys apoA-V (corresponding to the c.553G>T single-nucleotide polymorphism: rs2075291; p.Gly185Cys when numbering includes signal sequence) had a modest effect. Characterization studies revealed that plasma levels of wild-type and G162C apoA-V in transduced mice were similar and within the physiological range. Fractionation of plasma from mice transduced with AAV2/8-G162C apoA-V indicated that, unlike wild-type apoA-V, >50% of G162C apoA-V was recovered in the lipoprotein-free fraction. Nonreducing SDS-PAGE immunoblot analysis provided evidence that G162C apoA-V present in the lipoprotein-free fraction, but not that portion associated with lipoproteins, displayed altered electrophoretic mobility consistent with disulfide-linked heterodimer formation. Immunoprecipitation followed by liquid chromatography/mass spectrometry of human plasma from subjects homozygous for wild-type APOA5 and c.553G>T APOA5 revealed that G162C apoA-V forms adducts with extraneous plasma proteins including fibronectin, kininogen-1, and others. CONCLUSIONS: Substitution of Cys for Gly at position 162 of mature apoA-V introduces a free cysteine that forms disulfide bonds with plasma proteins such that its lipoprotein-binding and triacylglycerol-modulation functions are compromised.

Cationic lipid nanodisks as an siRNA delivery vehicle.

The term nanodisk (ND) describes reconstituted high-density lipoprotein particles that contain one or more exogenous bioactive agents. In the present study, ND were assembled from apolipoprotein A-I, the zwitterionic glycerophospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and the synthetic cationic lipid 1,2-dimyristoyl-3-trimethylammonium-propane (DMTAP). ND formulated at a DMPC:DMTAP ratio of 70:30 (by weight) were soluble in aqueous media. The particles generated were polydisperse, with diameters ranging from ∼20 to <50 nm. In nucleic acid binding studies, agarose gel retardation assays revealed that a synthetic 23-mer double-stranded oligonucleotide (dsOligo) bound to DMTAP containing ND but not to ND formulated with DMPC alone. Sucrose density gradient ultracentrifugation studies provided additional evidence for stable dsOligo binding to DMTAP-ND. Incubation of cultured hepatoma cells with DMTAP-ND complexed with a siRNA directed against glyceraldehyde 3-phosphate dehydrogenase showed 60% knockdown efficiency. Thus, incorporation of synthetic cationic lipid (i.e., DMTAP) to ND confers an ability to bind siRNA and the resulting complexes possess target gene knockdown activity in a cultured cell model.

Chemically synthesized 58-mer LysM domain binds lipochitin oligosaccharide.

Recognition of carbohydrates by proteins is a ubiquitous biochemical process. In legume-rhizobium symbiosis, lipochitin oligosaccharides, also referred to as nodulation (nod) factors, function as primary rhizobial signal molecules to trigger root nodule development. Perception of these signal molecules is receptor mediated, and nod factor receptor 5 (NFR5) from the model legume Lotus japonicus is predicted to contain three LysM domain binding sites. Here we studied the interactions between nod factor and each of the three NFR5 LysM domains, which were chemically synthesized. LysM domain variants (up to 58 amino acids) designed to optimize solubility were chemically assembled by solid-phase peptide synthesis (SPPS) with microwave heating. Their interaction with nod factors and chitin oligosaccharides was studied by isothermal titration calorimetry and circular dichroism (CD) spectroscopy. LysM2 showed a change in folding upon nod factor binding, thus providing direct evidence that the LysM domain of NFR5 recognizes lipochitin oligosaccharides. These results clearly show that the L. japonicus LysM2 domain binds to the nod factor from Mesorhizobium loti, thereby causing a conformational change in the LysM2 domain. The preferential affinity for nod factors over chitin oligosaccharides was demonstrated by a newly developed glycan microarray. Besides the biological implications, our approach shows that carbohydrate binding to a small protein domain can be detected by CD spectroscopy.

Counterions control whether self-assembly leads to formation of stable and well-defined unilamellar nanotubes or nanoribbons and nanorods.

Self-assembly of the amphiphilic π-conjugated carbenium ion ATOTA-1(+) in aqueous solution selectively leads to discrete and highly stable nanotubes or nanoribbons and nanorods, depending on the nature of the counterion (Cl(-) vs. PF6(-), respectively). The nanotubes formed by the Cl(-) salt illustrate an exceptional example of a structural well-defined (29±2 nm in outer diameter) unilamellar tubular morphology featuring π-conjugated functionality and high stability and flexibility, in aqueous solution.

Gene transfer of apolipoprotein A-V improves the hypertriglyceridemic phenotype of apoa5 (-/-) mice.

OBJECTIVE: Apolipoprotein (apo) A-V is a low abundance protein with a profound influence on plasma triacylglycerol levels. In human populations, single nucleotide polymorphisms and mutations in APOA5 positively correlate with hypertriglyceridemia. As an approach to preventing the deleterious effects of chronic hypertriglyceridemia, apoA-V gene therapy has been pursued. METHODS AND RESULTS: Recombinant adeno-associated virus (AAV) 2/8 harboring the coding sequence for human apoA-V or a control AAV2/8 was transduced into hypertriglyceridemic apoa5 (-/-) mice. After injection of 1×10(12) viral genome AAV2/8-apoA-V, maximal plasma levels of apoA-V protein were achieved at 3 to 4 weeks, after which the concentration slowly declined. Complementing the appearance of apoA-V was a decrease (50±6%) in plasma triacylglycerol content compared with apoa5 (-/-) mice treated with AAV2/8-ß-galactosidase. After 8 weeks the mice were euthanized and plasma lipoproteins separated. AAV2/8-apoA-V-transduced mice displayed a dramatic reduction in very low-density lipoprotein triacylglycerol content. Vector generated apoA-V in plasma associated with both very low-density lipoprotein and high-density lipoprotein fractions. CONCLUSIONS: Taken together, the data show that gene transfer of apoA-V improves the severe hypertriglyceridemia phenotype of apoa5 (-/-) mice. Given the prevalence of hypertriglyceridemia, apoA-V gene therapy offers a potential strategy for maintenance of plasma triacylglycerol homeostasis.

A perspective on bleb and empty LNP structures.

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 blebs and empty LNPs. 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.

Lipid nanoparticle-based strategies for extrahepatic delivery of nucleic acid therapies - challenges and opportunities.

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.

Lipid nanoparticles containing labile PEG-lipids transfect primary human skin cells more efficiently in the presence of apoE.

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 âˆ¼ 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.

Deciphering the monocyte-targeting mechanisms of PEGylated cationic liposomes by investigating the biomolecular corona.

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.

Tuning the double lipidation of salmon calcitonin to introduce a pore-like membrane translocation mechanism.

A widespread strategy to increase the transport of therapeutic peptides across cellular membranes has been to attach lipid moieties to the peptide backbone (lipidation) to enhance their intrinsic membrane interaction. Efforts in vitro and in vivo investigating the correlation between lipidation characteristics and peptide membrane translocation efficiency have traditionally relied on end-point read-out assays and trial-and-error-based optimization strategies. Consequently, the molecular details of how therapeutic peptide lipidation affects it's membrane permeation and translocation mechanisms remain unresolved. Here we employed salmon calcitonin as a model therapeutic peptide and synthesized nine double lipidated analogs with varying lipid chain lengths. We used single giant unilamellar vesicle (GUV) calcein influx time-lapse fluorescence microscopy to determine how tuning the lipidation length can lead to an All-or-None GUV filling mechanism, indicative of a peptide mediated pore formation. Finally, we used a GUVs-containing-inner-GUVs assay to demonstrate that only peptide analogs capable of inducing pore formation show efficient membrane translocation. Our data provided the first mechanistic details on how therapeutic peptide lipidation affects their membrane perturbation mechanism and demonstrated that fine-tuning lipidation parameters could induce an intrinsic pore-forming capability. These insights and the microscopy based workflow introduced for investigating structure-function relations could be pivotal for optimizing future peptide design strategies.

Self-assembly and near perfect macroscopic alignment of fluorescent triangulenium salt in spin-cast thin films on PTFE.

Highly fluorescent, discotic trioxatriangulenium dyes were aligned by simple spin-casting on substrates with friction transferred PTFE layers. The fluorescent crystalline thin films show near perfect macroscopic alignment on centimeter large areas directly from spin-casting. Gracing Incidence X-ray Diffraction (GIXD) unambiguously allowed the determination of a long-range order unit cell as well as its orientation with respect to the PTFE fibers. Further analysis of the X-ray data, in conjunction with polarized absorption spectroscopy, suggest a lamellar packing model with alternating layers of alkyl chains and ionic dyes oriented parallel to the substrate. This structure results in a highly anisotropic electrostatic potential around the cationic chromophore, causing significant shifts in energy and orientation of the optical transitions. Thus, the optical properties of the material are, to a large extent, controlled by the position of the otherwise inert PF6(-) counterions. The bright fluorescence from the films is also polarized parallel to the PTFE alignment layer. Doping of the thin films with fluorescent energy acceptor traps shows that efficient exciton migration takes place in the thin films. The excellent exciton transfer capabilities, in conjunction with the perfect alignment, might be of interest in future applications in solar energy harvesting or as thin film sensors.

Fluorescent and highly stable unimodal DMPC based unilamellar vesicles formed by spontaneous curvature.

The formation of uniform and highly stable unilamellar vesicles (ULVs) and the theory behind it are ongoing tasks within the vesicle community. Herein, we report the formation of highly stable, fluorescent, and unimodal 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) based ULVs with an average size of ~100 nm, as determined by cryogenic transmission electron microscopy (cryo-TEM) and dynamic light scattering (DLS). The ULVs are formed by mixing a two-component powder mixture or mixed lipid film of DMPC and 5 mol % of a novel amphiphilic carbenium salt, sodium 2-didecylamino-6,10-bis(N-methyltaruino)-4,8,12-trioxatriangulenium (Na-DSA) in aqueous solution when subjected to shaking. We propose that the high stability and the unimodal size distribution of the 5% DSA ULVs confirmed by DLS studies are a product of spontaneous curvature. UV-vis absorption/emission studies reveal that the structure of DSA promotes a strong interaction between the DMPC and the DSA to take place due to the complementary charge distribution of the DSA and DMPC head groups. The strong interaction may introduce an asymmetric amphiphile composition in the inner and outer leaflet of the bilayer which drives the spontaneous curvature.