Staff member

Cells tightly regulate membrane composition to maintain homeostasis and adapt to perturbations. Disruptions in lipid metabolism, such as those occurring in Gaucher disease (GD), can disturb this balance. In GD, excess glucosylceramide (GlcCer) accumulates in lysosomes, altering overall lipid metabolism and potentially impacting membrane composition. This study investigates how GlcCer influences lipid distribution and the local mechanical properties of model lipid bilayers composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), egg sphingomyelin (SME), cholesterol (Chol) and GlcCer in varying ratios, using atomic force microscopy and force spectroscopy. In simple bilayers (DOPC and DOPC:Chol), GlcCer forms rigid, unstable domains with high lateral packing. In contrast, in complex DOPC:Chol:SME membranes, higher GlcCer concentrations are accommodated but significantly alter domain organization and nanomechanical properties. Excess GlcCer preferentially partitions into segregated domains, sequestering cholesterol and SME, thereby reshaping lipid distribution with potential implications for membrane curvature, protein diffusion and localization.

JTD Keywords: Afm, Atomic force microscopy, Atomic-force microscopy, Force spectroscopy, Functional roles, Gaucher disease, Glucosylceramide, Lipid membranes, Membrane structure, Models, Nanomechanical properties, Nanomechanics, Neutral glycosphingolipids, Phase-diagram, Spectroscopy, Sphingomyelin, Supported lipid-bilayers

Targeting therapeutic nanocarriers (NCs) to endothelial receptors favours transport across the blood-brain barrier (BBB), a main obstacle to access the brain. While these strategies compel validation in animals, quantitative sub-cellular resolution is non-viable in vivo. BBB-on-chip (BBB-oC) devices can help. Illustrating this, we used a BBB-oC comprising a lateral channel containing a human brain endothelial lining and a central chamber containing hydrogel-embedded pericytes and astrocytes. We studied NCs targeting intercellular adhesion molecule-1 (ICAM-1), a cell-surface protein overexpressed in pathology and involved in CAM-mediated transport. Brain access was validated in vivo after injection of NCs coated with anti-ICAM-1 vs. control IgG. ICAM-1 expression was verified in vitro using endothelial cells, pericytes, and astrocytes (756-, 511-, 690-fold over non-specific signal under TNF alpha). VE-cadherin presence and lack of dextran leakage demonstrated a restrictive BBB-oC barrier. Data showed endothelial targeting of anti-ICAM-1 NCs (428 NCs/cell at 1 h), uptake (60% of cell-interacting NCs), and transcytosis (90%; 24 h) downregulated by a CAM-pathway inhibitor (88% decay; 1 h). Non-transcyosed NCs trafficked to lysosomes, while transcytosed NCs interacted with pericytes and astrocytes (2643 NCs/cell; 24 h) and entered them (90% of transcytosed NCs). This BBB-oC represents a valuable model to evaluate ICAM-1-mediated transcytosis, complementing animal studies.

JTD Keywords: Blood-brain barrier, Design, Drug-delivery, Endothelial and basolateral interactions, Endothelium, Icam-1 targeting, Icam-1-targeted nanocarriers, Nanoparticles, Organ-on-a-chip, Targeted nanocarriers, Transcytosis mechanism, Transport

Drug targeting can be achieved by coupling drugs or their carriers to affinity molecules, mostly antibodies (Abs), which recognise specific protein targets. However, most proteins are not expressed in an exclusive configuration but as various isoforms. Hence, selected targeting molecules may fail to target with enough efficiency in clinical trials, which is overlooked. We illustrate this by targeting intercellular adhesion molecule 1 (ICAM-1), a cell-surface protein overexpressed in many pathologies. Most ICAM-1 targeting studies used Ab R6.5, which binds ICAM-1 domain 2 (D2). Yet, literature and our data show that D2 is frequently absent among ICAM-1 isoforms. We thus produced a battery of five new Abs (B4, B6, B11, C12 and G2) and tested their ability to recognise both full-length and -D2 ICAM-1. In solution, all Abs recognised both ICAM-1 forms (from 5.3 x 1011 to 4.2 x 1012 sum intensity/well). Coating them on nanocarriers (NCs) rendered G2 specific against -D2 ICAM-1 (4.2 x 106 NCs/well) while other Abs kept their dual recognition (from 6.4 x 106 to 2.2 x 107 NCs/well). All Abs induced NC intracellular uptake in respective cells (from 42% to 85%) and displayed good cross-species reactivity (from 4.4 x 1011 to 2.6 x 1012 sum intensity/well). These Abs represent valuable tools to target ICAM-1 and illustrate a new targeting paradigm that may improve classical strategies.

JTD Keywords: Adhesion, Antibody-targeted nanocarriers, Cross-species reactivit, Design, Domai, Endothelial delivery, Enlimomab, Icam-1, Icam-1 isoforms, Intercellular adhesion molecule 1, Nanocarriers, Nanoparticles, New recombinant antibodies, Pecam-1, Targeting and intracellular trafficking

Intercellular adhesion molecule-1 (ICAM-1) is a membrane protein whose expression is enhanced at pathological sites, supporting drug delivery using nanocarriers (NCs). Any of its five extracellular domains (D1 to D5) can be targeted, yet most NC studies have used antibody (Ab) R6.5, which targets domain D2. While this provided efficient NC targeting and intracellular transport, literature indicates the absence of D2 in about 50 % of ICAM-1 isoforms expressed in mouse models. In this study, we verified the presence of ICAM-1 isoforms lacking D2 in human cells at both mRNA and protein levels, supporting the need to test Abs targeting other ICAM-1 domains. We developed a new cell model specifically lacking ICAM-1 D2 and compared R6.5 to Abs targeting D1 (Ab 15.2), D3D4 (Ab G-5), and D5 (Ab H-4). Abs G-5 and H-4 showed best targeting results, for which they were coated on model polymeric NCs. Compared to non-specific IgG NCs, both anti-ICAM-1 formulations targeted recombinant cells expressing human ICAM-1 lacking D2 and also primary cells naturally expressing the whole ICAM-1 isoform pattern observed. Both formulations were efficiently internalized by cells and trafficked to lysosomes, as previously observed for ICAM-1-targeting systems. Furthermore, NCs coated with either one of these two Abs showed good cross-species reactivity, being amenable for future pre-clinical testing. Therefore, Abs G-5 or H-4 are good options to provide ICAM-1 targeting without missing ICAM-1 isoforms lacking D2, present in human.

JTD Keywords: Adhesion molecule-1 icam-1, Anti-icam-1 antibody, Antibody-targeted nanocarriers, Design, Different receptor epitopes, Domai, Endothelial delivery, Enlimomab, Icam-1 extracellular domains, Icam-1 isoforms, Identification, Intercellular adhesion molecule 1, Monoclonal-antibodies, Nanoparticles, Targeting and endocytosi, Transport

ASM deficiency in Niemann-Pick disease type A results in aberrant cellular accumulation of sphingomyelin, neuroinflammation, neurodegeneration, and early death. There is no available treatment because enzyme replacement therapy cannot surmount the blood-brain barrier (BBB). Nanocarriers (NCs) targeted across the BBB via transcytosis might help; yet, whether ASM deficiency alters transcytosis remains poorly characterized. We investigated this using model NCs targeted to intracellular adhesion molecule-1 (ICAM-1), transferrin receptor (TfR), or plasmalemma vesicle-associated protein-1 (PV1) in ASM-normal vs. ASM-deficient BBB models. Disease differentially changed the expression of all three targets, with ICAM-1 becoming the highest. Apical binding and uptake of anti-TfR NCs and anti-PV1 NCs were unaffected by disease, while anti-ICAM-1 NCs had increased apical binding and decreased uptake rate, resulting in unchanged intracellular NCs. Additionally, anti-ICAM-1 NCs underwent basolateral reuptake after transcytosis, whose rate was decreased by disease, as for apical uptake. Consequently, disease increased the effective transcytosis rate for anti-ICAM-1 NCs. Increased transcytosis was also observed for anti-PV1 NCs, while anti-TfR NCs remained unaffected. A fraction of each formulation trafficked to endothelial lysosomes. This was decreased in disease for anti-ICAM-1 NCs and anti-PV1 NCs, agreeing with opposite transcytosis changes, while it increased for anti-TfR NCs. Overall, these variations in receptor expression and NC transport resulted in anti-ICAM-1 NCs displaying the highest absolute transcytosis in the disease condition. Furthermore, these results revealed that ASM deficiency can differently alter these processes depending on the particular target, for which this type of study is key to guide the design of therapeutic NCs.© 2023. Controlled Release Society.

JTD Keywords: asm deficiency, blood-brain barrier, delivery, determines, drug, endocytosis, enzymes, icam-1, lysosomal storage disease, mechanisms, nanoparticles, natural-history, niemann-pick disease type a, pv-1, receptor-mediated transcytosis, trafficking, transferrin receptor, Asm deficiency, Blood-brain barrier, Blood–brain barrier, Drug carriers, Drug nanocarriers, Humans, Icam-1, Icam-1-targeted nanocarriers, Intercellular adhesion molecule-1, Lysosomal storage disease, Niemann-pick disease type a, Niemann-pick disease, type a, Niemann-pick diseases, Pv-1, Receptor-mediated transcytosis, Transferrin receptor

Lysosomes play a central role in cellular homeostasis and alterations in this compartment associate with many diseases. The most studied example is that of lysosomal storage disorders (LSDs), a group of 60 + maladies due to genetic mutations affecting lysosomal components, mostly enzymes. This leads to aberrant intracellular storage of macromolecules, altering normal cell function and causing multiorgan syndromes, often fatal within the first years of life. Several treatment modalities are available for a dozen LSDs, mostly consisting of enzyme replacement therapy (ERT) strategies. Yet, poor biodistribution to main targets such as the central nervous system, musculoskeletal tissue, and others, as well as generation of blocking antibodies and adverse effects hinder effective LSD treatment. Drug delivery systems are being studied to surmount these obstacles, including polymeric constructs and nanoparticles that consti-tute the focus of this article. We provide an overview of the formulations being tested, the diseases they aim to treat, and the results observed from respective in vitro and in vivo studies. We also discuss the advantages and disadvantages of these strategies, the remaining gaps of knowledge regarding their per-formance, and important items to consider for their clinical translation. Overall, polymeric nanocon-structs hold considerable promise to advance treatment for LSDs.(c) 2023 Elsevier B.V. All rights reserved.

JTD Keywords: cellular and animal models, enzyme replacement therapy, lysosomal storage disorders, nanoemulsions, nanoparticles, Beta-glucuronidase deficiency, Blood-brain-barrier, Cellular and animal models, Central-nervous-system, Drug delivery systems, Enzyme replacement therapy, Feline gm1 gangliosidosis, Human acid sphingomyelinase, Human alpha-galactosidase, Humans, Lysosomal storage diseases, Lysosomal storage disorders, Lysosomes, Mucopolysaccharidosis type-ii, Nanoemulsions, Nanoparticles, Neuronal ceroid-lipofuscinosis, Niemann-pick-disease, Pluripotent stem-cells, Polymer-based drug delivery systems, Polymers, Tissue distribution