Transgene expression and local tissue distribution of naked and polymer-condensed plasmid DNA after intradermal administration in mice - PubMed (original) (raw)

Transgene expression and local tissue distribution of naked and polymer-condensed plasmid DNA after intradermal administration in mice

R Noelle Palumbo et al. J Control Release. 2012.

Abstract

DNA vaccination using cationic polymers as carriers has the potential to be a very powerful method of immunotherapy, but typical immune responses generated have been less than robust. To better understand the details of DNA vaccine delivery in vivo, we prepared polymer/DNA complexes using three structurally distinct cationic polymers and fluorescently labeled plasmid DNA and injected them intradermally into mice. We analyzed transgene expression (luciferase) and the local tissue distribution of the labeled plasmid at the injection site at various time points (from hours to days). Comparable numbers of luciferase expressing cells were observed in the skin of mice receiving naked plasmid or polyplexes one day after transfection. At day 4, however, the polyplexes appeared to result in more transfected skin cells than naked plasmid. Live animal imaging revealed that naked plasmid dispersed quickly in the skin of mice after injection and had a wider distribution than any of the three types of polyplexes. However, naked plasmid level dropped to below detection limit after 24h, whereas polyplexes persisted for up to 2 weeks. The PEGylated polyplexes had a significantly wider distribution in the tissue than the nonPEGylated polyplexes. PEGylated polyplexes also distributed more broadly among dermal fibroblasts and allowed greater interaction with antigen-presenting cells (APCs) (dendritic cells and macrophages) starting at around 24h post-injection. By day 4, co-localization of polyplexes with APCs was observed at the injection site regardless of polymer structure, whereas small amounts of polyplexes were found in the draining lymph nodes. These in vivo findings demonstrate the superior stability of PEGylated polyplexes in physiological milieu and provide important insight on how cationic polymers could be optimized for DNA vaccine delivery.

Copyright © 2012 Elsevier B.V. All rights reserved.

PubMed Disclaimer

Figures

Fig. 1

Fig. 1

Chemical structures of branched PEI (A), PAEM (B), and PEG-_b_-PAEM (C).

Fig. 2

Fig. 2

Visual assessment of polyplex stability in injection buffer (5% glucose) (A) and cell medium containing 10% serum (B) – conditions that mimic the in vivo fluid environment. Whereas PEI and PAEM polyplexes experienced much aggregation over time, PEGylated polyplexes remained stable without visible aggregation. Agarose gel electrophoresis of naked DNA and polyplexes before and after incubation in serum-containing medium (C) confirmed the absence of any free, unbound DNA. Scale bar: 100 μm.

Fig. 2

Fig. 2

Visual assessment of polyplex stability in injection buffer (5% glucose) (A) and cell medium containing 10% serum (B) – conditions that mimic the in vivo fluid environment. Whereas PEI and PAEM polyplexes experienced much aggregation over time, PEGylated polyplexes remained stable without visible aggregation. Agarose gel electrophoresis of naked DNA and polyplexes before and after incubation in serum-containing medium (C) confirmed the absence of any free, unbound DNA. Scale bar: 100 μm.

Fig. 3

Fig. 3

Transgene expression in the skin of mice after intradermal injection in the right hind quadriceps region. Each animal received 40 μg of luciferase plasmid either as naked or complexes with various cationic polymers. Shown are representative fluorescence microscopy images of mouse skin cross-sections. Luciferass-expressing cells (green) were detected by a polyclonal antibody against luciferase. Cell nuclei were stained blue (Hoechst). Images of both low (scale bar: 200 μm) and high (scale bar: 50 μm) magnification are presented. White arrows point to luciferase-expressing cells.

Fig. 4

Fig. 4

Tissue distribution of naked plasmid in live animals after intradermal injection. Three mice were each injected with 10 μg of Cy3-labeled DNA in the right hind quadriceps region and were imaged together (A). Plasmid distribution in three injected mice was shown at indicated time points as marked by a white outline (B) and the area of the signal was quantified (C).

Figure 5

Figure 5

Tissue distribution of polyplexes in live animals after intradermal injection. Three mice were each injected with polyplexes containing 10 μg of Cy3-labeled DNA in the right hind quadriceps region and were imaged together. Plasmid distribution in three injected mice was shown at indicated time points as marked by a white outline (A) and the area of signal was quantified (B). *PEGylated polyplexes showed statistically larger area of spreading than PEI and PAEM-based polyplexes (t test, p<0.001).

Fig. 6

Fig. 6

Dermal distribution of naked plasmid at the injection site. Naked plasmid was labeled with Cy3 (red). Reticular dermal fibroblasts, DCs, and macrophages were stained for ER-TR7, CD11c, and F4/80, respectively (blue or green). Area of co-localization between the plasmid and the cell markers was painted in white. Images shown are representative fields of view. Scale bar: 50 μm (for fibroblasts), 100 μm (for DCs and macrophages).

Fig. 7

Fig. 7

Dermal distribution of polyplexes and co-localization with dermal fibroblasts at the injection site at various time points. (A) Representative fluorescence microscopy images showing plasmid labeled with Cy3 (red), and fibroblasts stained for ER-TR7 (blue). Scale bar: 200 μm. (B) The fraction of co-localization defined as the ratio of pixel areas between co-localized plasmid signal and the total plasmid signal. *PEGylated polyplexes showed statistically more co-localization with fibroblasts than PEI and PAEM-based polyplexes (t test, p<0.05).

Fig. 8

Fig. 8

Dermal distribution of polyplexes and co-localization with DCs at the injection site. (A) Representative fluorescence microscopy images showing plasmid labeled with Cy3 (red), and DCs stained for CD11c (blue). Scale bar: 100 μm. (B) The fraction of co-localization defined as the ratio of pixel areas between co-localized plasmid signal and the total plasmid signal. *PEGylated polyplexes showed statistically more co-localization with DCs than PAEM-based polyplexes at 24 h (t test, p<0.05).

Fig. 9

Fig. 9

Dermal distribution of polyplexes relative to different cell types in skin tissue sections examined using confocal fluorescence microscopy at high magnification. The polyplexes shown were PEGylated and injected 24 h before imaging. Scale bar: 20 μm.

References

    1. Foged C, Sundblad A, Hovgaard L. Targeting vaccines to dendritic cells. Pharm Res. 2002;3:229–238. -PubMed
    1. Banchereau J, Briere F, Caux C, Davoust J, Lebecque S, Liu Y, et al. Immunobiology of dendritic cells. Annu Rev Immunol. 2000;18:767–811. -PubMed
    1. Ribas A. Genetically modified dendritic cells for cancer immunotherapy. Curr Gene Ther. 2005;5:619–628. -PubMed
    1. Nguyen DN, Green JJ, Chan JM, Langer R, Anderson DG. Polymeric materials for gene delivery and DNA vaccination. Adv Mater. 2009;21:847–867. -PMC -PubMed
    1. van den Berg JH, Nuijen B, Schumacher TN, Haanen JBAG, Storm G, Beijnen JH, et al. Synthetic vehicles for DNA vaccination. J Drug Targ. 2010;18:1–14. -PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources