Journal: Science Advances

Article Title: In vivo membrane engineering traps Gd-based MRI contrast agents for detecting microhepatocellular carcinoma

doi: 10.1126/sciadv.aec9913

Figure Lengend Snippet: SPD1 comprises two peptides [SPDa, Ac-K(PpIX)-FF-DHLASLWWGTEL; SPDb, Ac-K(PpIX)-FF-AEEA-C(MAL-PEG 4 -DBCO)], which are composed of four discrete functional domains: (i) DHLASLWWGTEL, a GPC3-targeted motif; (ii) FF, a β sheet motif; (iii) PpIX, a fluorescent dye; (iv) DBCO, a click chemistry group. SPD1 self-assembles into nanoparticles, and SPD1 and Gd-DOTA-N 3 are administered via two sequential injections. SPD1 nanoparticles (first intravenous administration) first accumulate and transform into fibrillar networks on the cell membrane of high GPC3-expressing orthotopic liver tumor through intermolecular π-π stacking between FF. The fibrillar networks, functionalized with DBCO, trapped Gd-DOTA-N 3 (following a second intravenous administration) in the extracellular space via a bioorthogonal reaction and immobilized it in an ordered arrangement, thereby enhancing the T 1 -weighted signal for the precise detection of micro-HCC. Schematic elements were created by eBioart.

Article Snippet: SPD1 and SPD2 nanoparticles (50 μM) incubated in the presence or absence of human GPC3 recombinant protein (NovoProtein, C414) at molar concentration ratio of 1000:1 for 24 hours at room temperature, and then CD spectra of peptide nanoparticle solutions were acquired using a CD spectrometer (Chirascan, Applied Photophysics, UK).

Techniques: Functional Assay, Membrane, Expressing

Journal: Science Advances

Article Title: In vivo membrane engineering traps Gd-based MRI contrast agents for detecting microhepatocellular carcinoma

doi: 10.1126/sciadv.aec9913

Figure Lengend Snippet: ( A and B ) UV-vis absorption spectra (A) and fluorescence (FL) emission spectra (B) of PpIX (excitation: 405 nm) following the gradual addition of H 2 O (from 0 to 99.5%) to a DMSO solution of SPD1 nanoparticles. a.u., arbitrary units. ( C ) CAC of SPD1 nanoparticles was determined using pyrene as a fluorescent probe. ( D ) Representative TEM image of self-assembled SPD1 nanoparticles (50 μM) in aqueous solution. ( E ) TEM images showing the initial SPD1 nanoparticles and nanofibers transformed from SPD1 nanoparticles (50 μM) after incubation with human GPC3 protein [molecular weight (MW) ≈ 61.6 kDa] at varying molar ratios. h, hours. ( F ) TEM images showing the initial SPD1 nanoparticles and nanofibers transformed from SPD1 nanoparticles (50 μM) after incubation with human GPC3 protein at different time points. The molar ratio of human GPC3 protein/SPD1 was ~1:1000. ( G ) CD spectra of SPD1 nanoparticles (50 μM) before and after incubation with human GPC3 protein (1:1000 molar ratio) for various durations, showing the secondary structure transition. mdeg, millidegrees. ( H ) FTIR spectra of SPD1 nanoparticles (50 μM) before and after 24 hours of incubation with GPC3 (1:1000 molar ratio), highlighting a shift in the amide I band consistent with β sheet formation. ( I ) Molecular simulation of the transformation of SPD1 into complex (i.e., SPD1 nanoparticles) in a water box based on the hydrophobic core of PpIX molecules, along with a hydrophilic corona formed by GPC3-targeted ligands. ( J ) Molecular docking simulation for SPD1 and GPC3. Rectangle: the possible binding sites between SPD1 and GPC3. ( K ) MD simulation of fibrillar transformation of SPD1 generated at t = 250 ns after interaction with GPC3. Rectangle: the interaction forces of the hydrogen bond and π-π stacking. All experiments were independently repeated three times with consistent and reproducible results.

Article Snippet: SPD1 and SPD2 nanoparticles (50 μM) incubated in the presence or absence of human GPC3 recombinant protein (NovoProtein, C414) at molar concentration ratio of 1000:1 for 24 hours at room temperature, and then CD spectra of peptide nanoparticle solutions were acquired using a CD spectrometer (Chirascan, Applied Photophysics, UK).

Techniques: Fluorescence, Transformation Assay, Incubation, Molecular Weight, Circular Dichroism, Binding Assay, Generated

Journal: Science Advances

Article Title: In vivo membrane engineering traps Gd-based MRI contrast agents for detecting microhepatocellular carcinoma

doi: 10.1126/sciadv.aec9913

Figure Lengend Snippet: ( A ) SEM-EDX elemental mapping of the Gd-DOTA-N 3 +SPD1+GPC3 probe. False-color maps show the spatial distribution of carbon (C, red), oxygen (O, cyan), nitrogen (N, green), and Gd (magenta), confirming uniform Gd incorporation and colocalization with organic matrix elements. Scale bars, 25 μm. All experiments were independently repeated three times with consistent and reproducible results. ( B ) Schematic illustration of the covalent conjugation attachment of Gd-DOTA-N 3 to SPD1 nanofibers via SPAAC click chemistry, designed to enhance r 1 relaxivity. ( C ) TEM images showing GPC3-modified gold (Au) nanoparticles alone or after incubation with SPD1 or SPD2 nanoparticles (50 μM, 24 hours). Scale bars, 50 nm. ( D ) T 1 -weighted images and pseudocolor T 1 maps comparing Gd-DOTA, Gd-DOTA-N 3 , Gd-DOTA-N 3 +SPD2-GPC3 (noncovalent incubation, molar ratio of 1000:1, 24 hours) for 6 hours, and Gd-DOTA-N 3 +SPD1-GPC3 (bioorthogonal conjugation, molar ratio of 1000:1, 24 hours) for 6 hours. Experiments were independently repeated three times, with consistent results observed across replicates. ( E ) r 1 relaxivity of Gd-DOTA (purple), Gd-DOTA-N 3 (pink), Gd-DOTA-N 3 +SPD2+GPC3 (blue), and Gd-DOTA-N 3 +SPD1+GPC3 (light green). Data are presented as means ± SD ( n = 3 independent experiments). ( F ) Room-temperature VSM showing the enhanced paramagnetic signal from Gd-DOTA-N 3 +SPD1+GPC3 (light green), compared to Gd-DOTA-N 3 +SPD2+GPC3 (blue), Gd-DOTA-N 3 (pink), and Gd-DOTA (purple).

Article Snippet: SPD1 and SPD2 nanoparticles (50 μM) incubated in the presence or absence of human GPC3 recombinant protein (NovoProtein, C414) at molar concentration ratio of 1000:1 for 24 hours at room temperature, and then CD spectra of peptide nanoparticle solutions were acquired using a CD spectrometer (Chirascan, Applied Photophysics, UK).

Techniques: Conjugation Assay, Modification, Incubation

Journal: Science Advances

Article Title: In vivo membrane engineering traps Gd-based MRI contrast agents for detecting microhepatocellular carcinoma

doi: 10.1126/sciadv.aec9913

Figure Lengend Snippet: ( A ) Representative Western blot analysis of GPC3 expression in HepG2, WRL-68, and Hepa1-6 cells ( n = 3 independent experiments). ( B ) Quantitation of relative GPC3 protein level from (A). Data are presented as means ± SD ( n = 3). Statistical analysis was performed using one-way ANOVA with a Tukey’s post hoc test. ( C ) Flow cytometry analysis of surface GPC3 expression in HepG2, WRL-68, and Hepa1-6 cells. ( D ) Representative IHC images of GPC3 expression (brown) in tumor tissues from orthotopic Hepa1-6 tumor-bearing mice. Scale bar, 50 μm. ( E ) CLSM images of HepG2 and Hepa1-6 cells treated with SPD1 or SPD2 nanoparticles (50 μM; red fluorescence) for 6 hours. Scale bars, 20 μm. ( F ) Time-dependent CLSM imaging of HepG2 cells treated with SPD1 nanoparticles (50 μM) showing membrane-localized fibrillar transformation. Scale bars, 20 μm. ( G ) CLSM analysis of HepG2 cells sequentially incubated with SPD1 or SPD2 nanoparticles (50 μM, 6 hours; red) and FITC-labeled anti-GPC3 antibody (green; 1:200; Abcam, #ab207080). Colocalization (yellow) indicates specific binding of SPD1 to membrane-bound GPC3. Fluorescence intensity and colocalization were quantified using MATLAB. Data are presented as means ± SD ( n = 3); n.s., not significant (one-way ANOVA with Tukey’s post hoc test). Scale bars, 20 μm. ( H ) SEM images of untreated HepG2 and WRL-68 cells or incubated with SPD1 or SPD2 nanoparticles (50 μM, 6 hours). Magnified insets highlight membrane-associated fibrillar structures. ( I ) TEM images of untreated HepG2 cells (top) and those treated with SPD1 nanoparticles (50 μM, 24 hours; bottom). Red arrows indicate membrane-associated nanofibers. Scale bars, 500 nm. ( J ) SEM images showing the persistence of SPD1-derived fibrillar networks on HepG2 cells at 6, 24, and 72 hours posttreatment (50 μM). Scale bars, 2 μm. All experiments were independently repeated three times with consistent and reproducible results.

Article Snippet: SPD1 and SPD2 nanoparticles (50 μM) incubated in the presence or absence of human GPC3 recombinant protein (NovoProtein, C414) at molar concentration ratio of 1000:1 for 24 hours at room temperature, and then CD spectra of peptide nanoparticle solutions were acquired using a CD spectrometer (Chirascan, Applied Photophysics, UK).

Techniques: Western Blot, Expressing, Quantitation Assay, Flow Cytometry, Fluorescence, Imaging, Membrane, Transformation Assay, Incubation, Labeling, Binding Assay, Derivative Assay

Journal: Science Advances

Article Title: In vivo membrane engineering traps Gd-based MRI contrast agents for detecting microhepatocellular carcinoma

doi: 10.1126/sciadv.aec9913

Figure Lengend Snippet: ( A ) Representative CLSM images of HepG2 cells incubated with SPD1 nanoparticles (red, 50 μM) for 6 hours, followed by treatment with FITC-N 3 (10 to 50 μM, green) for an additional 6 hours. Merged yellow fluorescence indicates successful copper-free click conjugation between DBCO and N 3 on the cell membrane. Cells treated with SPD2+FITC-N 3 (50 μM) served as the nontargeted controls, showing minimal colocalization. Scale bars, 20 μm. ( B ) Time-dependent kinetics of bioorthogonal conjugation quantified by BCA protein assay and ICP-MS. HepG2 cells were pretreated with SPD1 or SPD2 nanoparticles (50 μM, 6 hours), followed by incubation with Gd-DOTA-N 3 (50 μM) for 0.5, 1, 6, or 12 hours. Cells treated with Gd-DOTA-N 3 alone served as baseline controls. ( C ) T 1 -weighted MR images of HepG2 cells treated with Gd-DOTA (50 μM), Gd-DOTA-N 3 (50 μM), or sequentially with SPD1 or SPD2 (50 μM, 6 hours) followed by Gd-DOTA-N 3 (50 μM, 6 hours). ( D ) Quantitative r 1 relaxivity under the corresponding treatment conditions in (C). ( E ) r 1 relaxivity of HepG2 cells preblocked with anti-GPC3 antibody (5 μg/ml, 12 hours; Abcam, #ab207080) before SPD1 treatment (50 μM, 6 hours) followed by Gd-DOTA-N 3 (50 μM, 6 hours). ( F to H ) Cell viability of WRL-68 (F), HepG2 (G), and Hepa1-6 (H) cells after sequential treatment with SPD1 or SPD2 for 6 hours followed by Gd-DOTA-N 3 (50 μM, 6 hours). Cell viability was quantified using the CCK-8 assay. Data are presented as means ± SD { n = 3 for [(A) to (E)]; n = 6 for [(F) to (H)]}. Statistical significance was performed using one-way ANOVA followed by Tukey’s post hoc test. P < 0.05 was considered statistically significant; n.s., not significant. All experiments were independently repeated three times with consistent results.

Article Snippet: SPD1 and SPD2 nanoparticles (50 μM) incubated in the presence or absence of human GPC3 recombinant protein (NovoProtein, C414) at molar concentration ratio of 1000:1 for 24 hours at room temperature, and then CD spectra of peptide nanoparticle solutions were acquired using a CD spectrometer (Chirascan, Applied Photophysics, UK).

Techniques: Incubation, Fluorescence, Conjugation Assay, Membrane, Bicinchoninic Acid Protein Assay, CCK-8 Assay