Journal: ACS nano

Article Title: A Programmable DNA Origami Nanospring That Reports Dynamics of Single Integrin Motion, Force Magnitude and Force Orientation in Living Cells.

doi: 10.1021/acsnano.2c12545

Figure Lengend Snippet: Figure 1. Design of the 4HB DNA NS structure. (a) A cylinder model of the 4HB square lattice (see cross-section). Each cylindrical rod represents a single DNA double helix. Two insertion and deletion sites that produce a superhelical strain are shown in blue and red, respectively. (b) CanDo, a 3D DNA origami structure prediction server,24 predicted a coil structure. Single-stranded DNA (ssDNA) handles projecting out from both ends are shown as red and orange lines, respectively. The complementary ssDNA antihandles are modified with adhesive peptide (cRGDfK, blue hexagon) to the extracellular domain of integrins and biotin (black circle) to attach the NS to the bottom of the dish. (c) An AFM image of the 4HB-NS. The shape was flexible enough to measure single integrin traction force but also showed repetitive bending or expected loops. Scale bar, 200 nm.

Article Snippet: For positive control experiments (cell adhesion with biotinylated cRGDfK), after neutravidin was washed out, 200 μL of biotinylated cRGDfK (1−150 nM in DNA origami buffer, Peptides International, PCI-3697-PI) was added on the glass-bottomed dish, and the mixture was incubated for 10 min. Unbound biotinylated cRGDfK was washed out using a DNA origami buffer.

Techniques: Modification, Adhesive

Journal: ACS nano

Article Title: A Programmable DNA Origami Nanospring That Reports Dynamics of Single Integrin Motion, Force Magnitude and Force Orientation in Living Cells.

doi: 10.1021/acsnano.2c12545

Figure Lengend Snippet: Figure 2. Visualizing NS extensions by single integrins. (a) A typical fluorescence image of Cy3 NS (red) and GFP-paxillin (white) expressed in HFFs and observed by TIRFM. The boxed area is expanded in (b). Scale bar, 10 μm for (a) and 2 μm for (b). The right cartoon in (b) shows NSs attached with integrins via cRGDfK and the glass surface via the biotin−avidin system. (c) A histogram of flattening NS fluorescence spots located outside the cells. The flattening was calculated as 1 −σshort/σlong, where σshort and σlong, respectively, denote the standard deviation for the short and long axes when the fluorescence spot was fit to a 2D Gaussian function. (d, e) Histograms of flattening NS fluorescence spots within (d) and outside FAs underneath cells (e). (n = 370, 428, and 292 molecules from 6, 6, and 5 cells for (c, d, e), respectively.) (f, g) Time trajectories of the angle between the long axis of NS and the horizontal direction of the picture for the spots undergoing Brownian motion (f) and stretching within FAs (g). Each color represents an individual NS.

Article Snippet: For positive control experiments (cell adhesion with biotinylated cRGDfK), after neutravidin was washed out, 200 μL of biotinylated cRGDfK (1−150 nM in DNA origami buffer, Peptides International, PCI-3697-PI) was added on the glass-bottomed dish, and the mixture was incubated for 10 min. Unbound biotinylated cRGDfK was washed out using a DNA origami buffer.

Techniques: Fluorescence, Avidin-Biotin Assay, Standard Deviation

Journal: ACS nano

Article Title: A Programmable DNA Origami Nanospring That Reports Dynamics of Single Integrin Motion, Force Magnitude and Force Orientation in Living Cells.

doi: 10.1021/acsnano.2c12545

Figure Lengend Snippet: Figure 5. NS force sensor indicated dynamics of loaded force and single integrin motions in living cells. (a−c) Representative traces showing the length of the NS (top), loaded force on the NS (middle), and angle of the NS (bottom) for an integrin-bound NS. The force trace was calculated from the NS length. The thin lines indicate raw data; the bold lines indicate moving averages. (d, e) The colors of the traces correspond to (a−c), and the gradations of the colors correspond to the time course. Representative traces showing a single integrin motion tethered with an NS (d) and the force vector (e) in polar coordinates. Colors correspond to (a−c). (f) Histograms of the loaded force distributions for cRGDfK-labeled NS (red, n = 59 molecules; N = 23 cells) and cRGDfK-less NS (blue, n = 138 molecules; N = 24 cells) underneath the cells.

Article Snippet: For positive control experiments (cell adhesion with biotinylated cRGDfK), after neutravidin was washed out, 200 μL of biotinylated cRGDfK (1−150 nM in DNA origami buffer, Peptides International, PCI-3697-PI) was added on the glass-bottomed dish, and the mixture was incubated for 10 min. Unbound biotinylated cRGDfK was washed out using a DNA origami buffer.

Techniques: Plasmid Preparation, Labeling

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