Limits...
Differential intracellular distribution of DNA complexed with polyethylenimine (PEI) and PEI-polyarginine PTD influences exogenous gene expression within live COS-7 cells.

Doyle SR, Chan CK - Genet Vaccines Ther (2007)

Bottom Line: Finally, while not exclusively dependent, microtubule trafficking and, to a greater extent, mitotic events significantly contributed to PEI facilitated gene expression.PEI facilitated expression is significantly influenced by a mitotic event, which is increased by microtubule organization center (MTOC)-associated localization of PEI polyplexes.PEI-Arg, although enhancing DNA internalization per cell, did not improve gene expression, highlighting the importance of microtubule trafficking for PEI vectors and the impact of the Arg peptide to intracellular trafficking.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Genetics and Human Variation, La Trobe University, Melbourne, Victoria 3086, Australia. s.doyle@latrobe.edu.au

ABSTRACT

Background: Polyethylenimine (PEI) is one of the most efficient and versatile non-viral vectors available for gene delivery. Despite many advantages over viral vectors, PEI is still limited by lower transfection efficiency compared to its viral counterparts. Considerable investigation is devoted to the modification of PEI to incorporate virus-like properties to improve its efficacy, including the incorporation of the protein transduction domain (PTD) polyarginine (Arg); itself demonstrated to facilitate membrane translocation of molecular cargo. There is, however, limited understanding of the underlying mechanisms of gene delivery facilitated by both PEI and PEI-bioconjugates such as PEI-polyarginine (PEI-Arg) within live cells, which once elucidated will provide valuable insights into the development of more efficient non-viral gene delivery vectors.

Methods: PEI and PEI-Arg were investigated for their ability to facilitate DNA internalization and gene expression within live COS-7 cells, in terms of the percentage of cells transfected and the relative amount of gene expression per cell. Intracellular trafficking of vectors was investigated using fluorescent microscopy during the first 5 h post transfection. Finally, nocodazole and aphidicolin were used to investigate the role of microtubules and mitosis, respectively, and their impact on PEI and PEI-Arg mediated gene delivery and expression.

Results: PEI-Arg maintained a high cellular DNA uptake efficiency, and facilitated as much as 2-fold more DNA internalization compared to PEI alone. PEI, but not PEI-Arg, displayed microtubule-facilitated trafficking, and was found to accumulate within close proximity to the nucleus. Only PEI facilitated significant gene expression, whereas PEI-Arg conferred negligible expression. Finally, while not exclusively dependent, microtubule trafficking and, to a greater extent, mitotic events significantly contributed to PEI facilitated gene expression.

Conclusion: PEI polyplexes are trafficked by an indirect association with microtubules, following endosomal entrapment. PEI facilitated expression is significantly influenced by a mitotic event, which is increased by microtubule organization center (MTOC)-associated localization of PEI polyplexes. PEI-Arg, although enhancing DNA internalization per cell, did not improve gene expression, highlighting the importance of microtubule trafficking for PEI vectors and the impact of the Arg peptide to intracellular trafficking. This study emphasizes the importance of a holistic approach to investigate the mechanisms of novel gene delivery vectors.

No MeSH data available.


Intracellular trafficking of PEI- and PEI-Arg polyplexes. Time points were taken on the hour, for the first 5 hours post transfection, and viewed by fluorescent microscopy. Merged images were constructed from individual labelled-PEI-, or labelled-pDNA-, and MitoTracker fluorescent images, and are representative of a typical image obtained from five images taken per time point, from two independent experiments. Scale bar = 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2211466&req=5

Figure 2: Intracellular trafficking of PEI- and PEI-Arg polyplexes. Time points were taken on the hour, for the first 5 hours post transfection, and viewed by fluorescent microscopy. Merged images were constructed from individual labelled-PEI-, or labelled-pDNA-, and MitoTracker fluorescent images, and are representative of a typical image obtained from five images taken per time point, from two independent experiments. Scale bar = 10 μm.

Mentions: Within one hour, labelled pDNA/PEI complexes were seen to be interacting with the cell surface and within the cytoplasmic periphery (Figure 2 – PEI/pDNA-YOYO), with evidence of polyplex aggregation in specific areas of the cell surface, seen in Figure 2 (1 h). Complexes were seen to be internalized at 2 hours, and, within 4 hours, significant localization within the nuclear proximity was observed, highlighted by the accumulated fluorescence observed in the DNA and merged photographs (Figure 2 – PEI/pDNA-YOYO 4 h). The distribution of fluorescence was not uniform around the perinuclear region, but co-localized within a region corresponding to the microtubule organization center (MTOC), providing evidence of microtubule involvement in PEI trafficking. The MTOC is located adjacent to the nucleus, and is indirectly identified in the MitoTracker images (Figure 2) as a ring of densely stained mitochondria surrounding a region of markedly fewer mitochondria.


Differential intracellular distribution of DNA complexed with polyethylenimine (PEI) and PEI-polyarginine PTD influences exogenous gene expression within live COS-7 cells.

Doyle SR, Chan CK - Genet Vaccines Ther (2007)

Intracellular trafficking of PEI- and PEI-Arg polyplexes. Time points were taken on the hour, for the first 5 hours post transfection, and viewed by fluorescent microscopy. Merged images were constructed from individual labelled-PEI-, or labelled-pDNA-, and MitoTracker fluorescent images, and are representative of a typical image obtained from five images taken per time point, from two independent experiments. Scale bar = 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2211466&req=5

Figure 2: Intracellular trafficking of PEI- and PEI-Arg polyplexes. Time points were taken on the hour, for the first 5 hours post transfection, and viewed by fluorescent microscopy. Merged images were constructed from individual labelled-PEI-, or labelled-pDNA-, and MitoTracker fluorescent images, and are representative of a typical image obtained from five images taken per time point, from two independent experiments. Scale bar = 10 μm.
Mentions: Within one hour, labelled pDNA/PEI complexes were seen to be interacting with the cell surface and within the cytoplasmic periphery (Figure 2 – PEI/pDNA-YOYO), with evidence of polyplex aggregation in specific areas of the cell surface, seen in Figure 2 (1 h). Complexes were seen to be internalized at 2 hours, and, within 4 hours, significant localization within the nuclear proximity was observed, highlighted by the accumulated fluorescence observed in the DNA and merged photographs (Figure 2 – PEI/pDNA-YOYO 4 h). The distribution of fluorescence was not uniform around the perinuclear region, but co-localized within a region corresponding to the microtubule organization center (MTOC), providing evidence of microtubule involvement in PEI trafficking. The MTOC is located adjacent to the nucleus, and is indirectly identified in the MitoTracker images (Figure 2) as a ring of densely stained mitochondria surrounding a region of markedly fewer mitochondria.

Bottom Line: Finally, while not exclusively dependent, microtubule trafficking and, to a greater extent, mitotic events significantly contributed to PEI facilitated gene expression.PEI facilitated expression is significantly influenced by a mitotic event, which is increased by microtubule organization center (MTOC)-associated localization of PEI polyplexes.PEI-Arg, although enhancing DNA internalization per cell, did not improve gene expression, highlighting the importance of microtubule trafficking for PEI vectors and the impact of the Arg peptide to intracellular trafficking.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Genetics and Human Variation, La Trobe University, Melbourne, Victoria 3086, Australia. s.doyle@latrobe.edu.au

ABSTRACT

Background: Polyethylenimine (PEI) is one of the most efficient and versatile non-viral vectors available for gene delivery. Despite many advantages over viral vectors, PEI is still limited by lower transfection efficiency compared to its viral counterparts. Considerable investigation is devoted to the modification of PEI to incorporate virus-like properties to improve its efficacy, including the incorporation of the protein transduction domain (PTD) polyarginine (Arg); itself demonstrated to facilitate membrane translocation of molecular cargo. There is, however, limited understanding of the underlying mechanisms of gene delivery facilitated by both PEI and PEI-bioconjugates such as PEI-polyarginine (PEI-Arg) within live cells, which once elucidated will provide valuable insights into the development of more efficient non-viral gene delivery vectors.

Methods: PEI and PEI-Arg were investigated for their ability to facilitate DNA internalization and gene expression within live COS-7 cells, in terms of the percentage of cells transfected and the relative amount of gene expression per cell. Intracellular trafficking of vectors was investigated using fluorescent microscopy during the first 5 h post transfection. Finally, nocodazole and aphidicolin were used to investigate the role of microtubules and mitosis, respectively, and their impact on PEI and PEI-Arg mediated gene delivery and expression.

Results: PEI-Arg maintained a high cellular DNA uptake efficiency, and facilitated as much as 2-fold more DNA internalization compared to PEI alone. PEI, but not PEI-Arg, displayed microtubule-facilitated trafficking, and was found to accumulate within close proximity to the nucleus. Only PEI facilitated significant gene expression, whereas PEI-Arg conferred negligible expression. Finally, while not exclusively dependent, microtubule trafficking and, to a greater extent, mitotic events significantly contributed to PEI facilitated gene expression.

Conclusion: PEI polyplexes are trafficked by an indirect association with microtubules, following endosomal entrapment. PEI facilitated expression is significantly influenced by a mitotic event, which is increased by microtubule organization center (MTOC)-associated localization of PEI polyplexes. PEI-Arg, although enhancing DNA internalization per cell, did not improve gene expression, highlighting the importance of microtubule trafficking for PEI vectors and the impact of the Arg peptide to intracellular trafficking. This study emphasizes the importance of a holistic approach to investigate the mechanisms of novel gene delivery vectors.

No MeSH data available.