Journal: Advanced Science

Article Title: Cyclic Arginine–Glycine–Aspartate‐Decorated Lipid Nanoparticle Targeting toward Inflammatory Lesions Involves Hitchhiking with Phagocytes

doi: 10.1002/advs.202100370

Figure Lengend Snippet: Figure 1. In vivo targeting kinetics studied with dynamic IVM. a–c) Snapshots and enlargements of Movie S1, Supporting Information, showing ring-like cRGD-NE agglomerates (a), as well as cRGD-NE agglomerates associated with circulating “black holes” (b,c). The rings and the “black holes” (indicated with arrowheads) were circular in shape with a diameter of 6–8 µm, which corresponds to the size of circulating neutrophils. d) Snapshots of a dynamic imaging series show binding events in angiogenic vasculature of 1–8 µm-sized cRGD-NE (red) agglomerates, appearing as “steps” in fluorescence versus time plots (each line represents the ROI signal intensity of a single binding event). e) cRAD-NE (red) extravasated gradually in the inflamed tissue. f) The averaged signal as a function of time for cRGD-NE (n = 4, 45 binding events) and cRAD-NE (n = 3). g) Targeting kinetics in angiogenic tumor tissue, observed with dynamic MRI in an earlier study (Δt = 21 s; n = 4 for each curve, Adapted with permission[21]). Scale bars: a–c = 10 µm; d,e = 50 µm. Error bars: f,g = SEM.

Article Snippet: [6] Half of the final NP formulation was conjugated with cRGD peptide (Peptides International, PCI-3699-PI) and the other half with control cRAD peptide (Peptides International, PCI-3959-PI) at 13.5 μg Adv.

Techniques: In Vivo, Imaging, Binding Assay

Journal: Advanced Science

Article Title: Cyclic Arginine–Glycine–Aspartate‐Decorated Lipid Nanoparticle Targeting toward Inflammatory Lesions Involves Hitchhiking with Phagocytes

doi: 10.1002/advs.202100370

Figure Lengend Snippet: Figure 2. Nanoemulsion association with immune cells and angiogenic tissue. a) High-speed imaging (Δt = 1.3 s) revealed both bound (white circles) and circulating cRGD-NE (red) positive cells (blood vessels delineated in yellow). b) cRGD-NE (Atto633-PE; red) accumulated extensively in cell-sized agglomerates in angiogenic vasculature adjacent to the wound (w) at 1 h post-injection. Several of these aggregates were also positive for co-injected cRAD-NE (Rhodamine-PE; green). c) cRGD-NE (red-hot look-up table to visualize colocalization with GFP) colocalizing with GFP positive endothelium (green) next to the wound (w). d) Z-stack with orthogonal projections showed non-endothelial cRGD-NE cell-sized agglomerates up to 24 h post- injection in the angiogenic vasculature (endothelial GFP; green). When cRGD-NE signal was enhanced (white box enlarged and enhanced), cRGD- NE colocalization with endothelium became evident. e) cRAD-NE (red) predominantly accumulated through passive diffusion from the vasculature (endothelial GFP; green), 1 h post-injection. Scale bars: b,c = 100 µm; a,e = 50 µm; d = 10 µm.

Article Snippet: [6] Half of the final NP formulation was conjugated with cRGD peptide (Peptides International, PCI-3699-PI) and the other half with control cRAD peptide (Peptides International, PCI-3959-PI) at 13.5 μg Adv.

Techniques: Imaging, Injection

Journal: Advanced Science

Article Title: Cyclic Arginine–Glycine–Aspartate‐Decorated Lipid Nanoparticle Targeting toward Inflammatory Lesions Involves Hitchhiking with Phagocytes

doi: 10.1002/advs.202100370

Figure Lengend Snippet: Figure 3. Ex vivo characterization of interactions between nanoemulsions and circulating immune cells. a–c) Ex vivo CLSM on immune cells isolated 5 min (a,b) and 10 min (c) post-injection of cRAD-NE (green) and cRGD-NE (red) showed that these cells associated with cRGD-NE to a much higher extent than with cRAD-NE. Cells in gray scale were imaged using transmission mode. (b) and (c) show orthogonal projections of z-stacks demonstrating the NE to be present on the cell membrane and intracellularly. d) Flow cytometry on circulating immune cells isolated 2 h after NE administration revealed myeloid-derived phagocytes to be the dominant population engaging the NE and confirmed that these cells associated with cRGD-NE to significantly higher extent than with cRAD-NE (n = 3; mean ± SEM). Flow cytometry histograms (Figure S5, Supporting Information) show data from representative animals revealing insignificant engagement of the NPs with lymphoid cells. Scale bars: a = 25 µm; b,c = 10 µm. p-values: * < 0.05.

Article Snippet: [6] Half of the final NP formulation was conjugated with cRGD peptide (Peptides International, PCI-3699-PI) and the other half with control cRAD peptide (Peptides International, PCI-3959-PI) at 13.5 μg Adv.

Techniques: Ex Vivo, Isolation, Injection, Transmission Assay, Membrane, Flow Cytometry, Derivative Assay

Journal: Advanced Science

Article Title: Cyclic Arginine–Glycine–Aspartate‐Decorated Lipid Nanoparticle Targeting toward Inflammatory Lesions Involves Hitchhiking with Phagocytes

doi: 10.1002/advs.202100370

Figure Lengend Snippet: Figure 4. Inflammation endothelium targeting by cRGD-LP. a) Snapshots of a dynamic imaging series show binding events in angiogenic vasculature of cRGD-LP (red) positive cells. b) These binding events appear as “steps” in fluorescence versus time plots for cRGD-LP (each line represents ROI signal intensity of a single binding event), while cRAD-LP gradually extravasate in the inflamed tissue. c) Snapshots from high-speed imaging (Δt = 1.3 s) 20 h post co-administration of cRGD-LP (red) and cRAD-LP (green). d) Orthogonal projections of z-stacks of white blood cells isolated 25 min post cRGD-LP (red) and cRAD-LP (green) co-administration. e) 6 h post-injection, colocalization between cRGD-LP (red) and GFP positive endothelium (green) as well as non-endothelial cell-sized cRGD-LP agglomerates bound in the vasculature were observed. f) Z-stack with orthogonal projections showing cRGD-LP (red) colocalization with GFP-positive endothelium (green) at 6 h post-injection. g) Flow cytometry on blood cells isolated 2 h after cRGD-LP (red) and cRAD-LP (blue) administration, revealed myeloid cells associating with cRGD-LP to significantly higher extent than with cRAD-LP (n = 6; mean ± SEM). Flow cytometry histograms (Figure S5, Supporting Information) show data from representative animals and confirm that LP also engaged insignificantly with lymphoid cells. Scale bars: a,c,e = 25 µm; d,f = 10 µm. p-values: * < 0.05, *** < 0.001, **** < 0.0001.

Article Snippet: [6] Half of the final NP formulation was conjugated with cRGD peptide (Peptides International, PCI-3699-PI) and the other half with control cRAD peptide (Peptides International, PCI-3959-PI) at 13.5 μg Adv.

Techniques: Imaging, Binding Assay, Isolation, Injection, Flow Cytometry