Journal: Molecular pharmaceutics

Article Title: Spatio-Temporal Cellular Imaging of Polymer-pDNA Nanocomplexes Affords In Situ Morphology and Trafficking Trends

doi: 10.1021/mp400115y

Figure Lengend Snippet: Three-dimensional (3D) volume rendering of polyplexes comprised of Tr455 polymer complexed with FITC-pDNA fluorescence (pseudo colored in blue) and a HeLa cell nucleus labeled with DAPI (pseudo colored in yellow) at 24 hours post transfection. (A) Zoomed out view of a single cell showing the nucleus (pseudo colored yellow) and polyplexes (pseudo colored blue). (B) A close-up view of the indicated part of the cell from ‘A’. Various globular morphologies of the polyplexes can be observed near the nucleus. (C) A ‘top view’ of the polyplexes shown in ‘C’. (D) Illustration of the polyplex confocal imaging parameters studied herein with the 4D imaging techniques. (E) The intracellular mechanisms studied herein with the polyplexes: (i) intracellular polyplex volume; (ii) intracellular inter-polyplex distance and (iii) intracellular polyplex distance from the nucleus. Also, analysis was performed to study 3D colocalization of polyplexes with intracellular organelles such as (i) endosomes (Rab 5), (ii) lysosomes (Rab 7) and the nucleus. In addition, endocytosis inhibition studies were performed to understand and compare polyplex internalization and trafficking.

Article Snippet: The primary antibodies for Rab 5 (specificity: detects Rab 5A, Rab 5B and Rab 5C) and Rab 7 (specificity: detects Rab 7a) were purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA.

Techniques: Fluorescence, Labeling, Transfection, Imaging, Inhibition

Journal: Molecular pharmaceutics

Article Title: Spatio-Temporal Cellular Imaging of Polymer-pDNA Nanocomplexes Affords In Situ Morphology and Trafficking Trends

doi: 10.1021/mp400115y

Figure Lengend Snippet: Three-dimensional reconstructed images of cells transfected with polyplexes formulated with Cy5-pDNA and (A) jetPEI™, (B) Glycofect™, (C) Tr455 and (D) Tr477. With each polymer type, the cells were fixed after 4, 8, 12, and 24 hours. The nucleus is shown in gold and the polyplexes (detected by imaging Cy5-labeled plasmid DNA) are shown in blue. Note: In the time series A-D, the nucleus was set as transparent to view colocalizing polyplexes. The polyplexes colocalizing with the nucleus appear ‘light blue/white’ in color. The controls were cells only, FITC only, DAPI only, Rab 5 primary antibody only, Rab 7 primary antibody only and secondary antibody only. The parameters measured were: (1) volume (μm3) of polyplexes and was determined by the volume of a voxel multiplied by the number of voxels in each physical volume; (2) distance of the polyplex/pDNA complexes from the surface of the nucleus (μm).

Article Snippet: The primary antibodies for Rab 5 (specificity: detects Rab 5A, Rab 5B and Rab 5C) and Rab 7 (specificity: detects Rab 7a) were purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA.

Techniques: Transfection, Imaging, Labeling, Plasmid Preparation

Journal: Molecular pharmaceutics

Article Title: Spatio-Temporal Cellular Imaging of Polymer-pDNA Nanocomplexes Affords In Situ Morphology and Trafficking Trends

doi: 10.1021/mp400115y

Figure Lengend Snippet: Colocalization of polyplexes with Rab 5 proteins, a marker of early endosomes, at time points of 4, 8, 12, and 24 hours. Images A-D show confocal images of HeLa cells showing polyplex colocalization (monitored by imaging Cy5-pDNA) with Rab 5 protein for early endosomes at 4, 8, 12 and 24 hour timepoints for polyplexes formed with Cy5-pDNA and (A) jetPEI™, (B) Glycofect™, (C) Tr455 and (D) Tr477. The nucleus is pseudo colored yellow, the polyplexes (monitored by imaging Cy5-pDNA) are pseudo colored blue, and Rab 5 is pseudo colored in red. The z-axis is directed into the plane of image, which is shown in the x-y plane. The parameter measured was 3D colocalization of the polyplex/pDNA with Rab 5 (early endosome marker in arbitrary units), which was determined by the total of overlapping volumes, rendered from the fluorescence, between the two channels.

Article Snippet: The primary antibodies for Rab 5 (specificity: detects Rab 5A, Rab 5B and Rab 5C) and Rab 7 (specificity: detects Rab 7a) were purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA.

Techniques: Marker, Imaging, Fluorescence

Journal: Molecular pharmaceutics

Article Title: Spatio-Temporal Cellular Imaging of Polymer-pDNA Nanocomplexes Affords In Situ Morphology and Trafficking Trends

doi: 10.1021/mp400115y

Figure Lengend Snippet: 3D volume colocalization of polyplexes with intracellular trafficking proteins: (A) Rab 5, a marker for early endosomes and (B) Rab 7, a marker for lysosomes. The colocalization is sum of intensity of overlapping voxels (in arbitrary units). The reported values within each plot were not significant at an alpha value of 0.05.

Article Snippet: The primary antibodies for Rab 5 (specificity: detects Rab 5A, Rab 5B and Rab 5C) and Rab 7 (specificity: detects Rab 7a) were purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA.

Techniques: Marker