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Results: 5

1.
Fig. 1

Fig. 1. From: Development of viral nanoparticles for efficient intracellular delivery.

(A) Structure of CPMV; surface rendered model highlighting the asymmetric unit consisting of the S protein (green) and L protein (blue). (B) CPMV asymmetric unit; reactive Lys side chains are highlighted in red. (C) Bioconjugation of CPMV with 4FB followed by reaction with R5 peptide.

Zhuojun Wu, et al. Nanoscale. ;4(11):3567-3576.
2.
Fig. 4

Fig. 4. From: Development of viral nanoparticles for efficient intracellular delivery.

Temperature/energy-dependent uptake of CPMV formulations. Confocal microscopy of HeLa cells and CPMV formulations (red). The cell membrane is labeled with wheat germ agglutinin (green), and the nucleus is labeled with DAPI (blue). Scale bar = 10 µm. Cells were incubated for 3 h with 106 VNPs per cell at 4 °C (left) or 37 °C (right). (A) CPMV, (B) CPMV–R5L, (C) CPMV–R5H. Imaging was performed using a Biorad 2100 confocal microscope with a 60× oil objective. Images were analyzed using ImageJ.

Zhuojun Wu, et al. Nanoscale. ;4(11):3567-3576.
3.
Fig. 3

Fig. 3. From: Development of viral nanoparticles for efficient intracellular delivery.

Uptake of VNPs into human cervical cancer cells (HeLa). (A) Evaluation of particle uptake by flow cytometry. Cells were either untreated or treated with 0.1% (w/v) pronase for 3 h. Experiments were conducted using 105 CPMV particles per cell and repeated at least twice; triplicate samples were analyzed, such that 10 000 events were recorded. (B) Fluorescence confocal microscopy of HeLa cells and CPMV formulations (red). Cell membrane is labeled with wheat germ agglutinin (green), and nucleus is labeled with DAPI (blue). Single plane images were analyzed using ImageJ.

Zhuojun Wu, et al. Nanoscale. ;4(11):3567-3576.
4.
Fig. 2

Fig. 2. From: Development of viral nanoparticles for efficient intracellular delivery.

Characterization of CPMV labeling with the biotinylated R5 peptide. (A) Size exclusion chromatography of wild-type CPMV, CPMV–R5L and CPMV–R5Hat 280 nm. (B) ECL dot blot of purified CPMV particles. The number of biotin labels per particle was determined using standardized biotin concentrations and Chemidoc XRS software. (C) Native gel electrophoresis of intact CPMV particles (10 µg) using a 0.8% (w/v) agarose gel. Particles were visualized under UV light. Lane 1 = CPMV, 2 = CPMV–4FB, 3 = CPMV–R5H, 4 = CPMV–PFB, 5 = CPMV–R5L. (D) SDS–PAGE of CPMV particles (10 µg) using a 4–12% Bis-Tris gel and western blotting using streptavidin–alkaline phosphatase to detect the N-terminal biotin tag of the R5 peptide. (E) Zeta potential of CPMV wild type, CPMV–R5L and CPMV–R5H formulations.

Zhuojun Wu, et al. Nanoscale. ;4(11):3567-3576.
5.
Fig. 5

Fig. 5. From: Development of viral nanoparticles for efficient intracellular delivery.

Subcellular fate of CPMV formulations in HeLa cells studied by confocal microscopy. CPMV formulations (red), endolysosomes are labeled with Lamp-1 (green), and nuclei are labeled with DAPI (blue). The overlay of endolysosomes and VNP signals is shown in yellow. (A) Time course and translocation of CPMV formulation over 10 hours. Scale bar = 10 µm. Images were analyzed using ImageJ. (B) Three-dimensional reconstruction of z-sectional data at time point 120 min. Images were recorded at a step size of 0.3 µm. Data were reconstructed using Imaris software. Scale bar = 1 µm. (C) Colocalization analysis of eight representative cells for each CPMV formulation showing the percentage of total internalized CPMV particles colocalizing with the endolyosomes, error bars represent the averages values of standard deviations of all analyzed cells. Co-localization analysis was performed using LSM Examiner software.

Zhuojun Wu, et al. Nanoscale. ;4(11):3567-3576.

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