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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.


Effect of microtubule disrupting agent, nocodazole, on microtubule morphology and PEI polyplex trafficking. Untreated (A) and nocodazole treated cells (D) displaying normal and disrupted microtubules, highlighted by anti-β-tubulin visualized using FITC secondary antibody. Nucleus stained with DAPI (B, E) and merged immunofluorescence displayed as (C, F). Distribution of PEI/pDNA-YOYO in untreated (G) and nocodazole treated (J) cells with MitoTracker staining (H, K). Merge MitoTracker and PEI/pDNA-YOYO in untreated (I) and nocodazole treated (L) cells. Scale bar = 10 μm. Images represent typical result of two individual slides viewed for each sample, analyzed 5 h post transfection.
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Figure 3: Effect of microtubule disrupting agent, nocodazole, on microtubule morphology and PEI polyplex trafficking. Untreated (A) and nocodazole treated cells (D) displaying normal and disrupted microtubules, highlighted by anti-β-tubulin visualized using FITC secondary antibody. Nucleus stained with DAPI (B, E) and merged immunofluorescence displayed as (C, F). Distribution of PEI/pDNA-YOYO in untreated (G) and nocodazole treated (J) cells with MitoTracker staining (H, K). Merge MitoTracker and PEI/pDNA-YOYO in untreated (I) and nocodazole treated (L) cells. Scale bar = 10 μm. Images represent typical result of two individual slides viewed for each sample, analyzed 5 h post transfection.

Mentions: The microtubule depolymerizing agent nocodazole was utilized to investigate potential association between microtubules and active PEI intracellular trafficking. Microtubules within nocodazole-treated cells were visualized by immunofluorescence using fluorescent microscopy to examine the impact of disrupted microtubules on PEI trafficking (Figure 3). Untreated mitochondrial, PEI/pCH110-YOYO, and merged images depict the accumulation of PEI at the MTOC-associated region. The microtubule controls, showing immunofluorescence of microtubules, nucleus, and merged images highlight a filamentous-microtubule structure throughout the cell and an intense staining of microtubule accumulation within a defined MTOC-associated perinuclear region. Nocodazole-treated cells containing PEI/pDNA-YOYO polyplexes display a random distribution of labelled complexes throughout the whole cell, as compared to controls that display MTOC-associated fluorescence accumulation. Furthermore, the filamentous morphology of mitochondria stained with MitoTracker was seen to be disrupted, becoming evenly distributed around the nucleus. The nocodazole-treated cells displayed disrupted microtubules, highlighted by the loss of the filamentous structure and replaced by a uniform fluorescence throughout the cell. Randomly dispersed fluorescence of PEI polyplex distribution was observed in nocodazole-treated cells (Figure 3E), as compared to the predictable PEI accumulation at the MTOC-associated region in non-treated cells (Figure 3B). The significant differences in morphology of microtubules and PEI accumulations observed between the treated and untreated cells, strongly indicated an albeit indirect correlation between the disruption of microtubules and loss of PEI trafficking.


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)

Effect of microtubule disrupting agent, nocodazole, on microtubule morphology and PEI polyplex trafficking. Untreated (A) and nocodazole treated cells (D) displaying normal and disrupted microtubules, highlighted by anti-β-tubulin visualized using FITC secondary antibody. Nucleus stained with DAPI (B, E) and merged immunofluorescence displayed as (C, F). Distribution of PEI/pDNA-YOYO in untreated (G) and nocodazole treated (J) cells with MitoTracker staining (H, K). Merge MitoTracker and PEI/pDNA-YOYO in untreated (I) and nocodazole treated (L) cells. Scale bar = 10 μm. Images represent typical result of two individual slides viewed for each sample, analyzed 5 h post transfection.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Effect of microtubule disrupting agent, nocodazole, on microtubule morphology and PEI polyplex trafficking. Untreated (A) and nocodazole treated cells (D) displaying normal and disrupted microtubules, highlighted by anti-β-tubulin visualized using FITC secondary antibody. Nucleus stained with DAPI (B, E) and merged immunofluorescence displayed as (C, F). Distribution of PEI/pDNA-YOYO in untreated (G) and nocodazole treated (J) cells with MitoTracker staining (H, K). Merge MitoTracker and PEI/pDNA-YOYO in untreated (I) and nocodazole treated (L) cells. Scale bar = 10 μm. Images represent typical result of two individual slides viewed for each sample, analyzed 5 h post transfection.
Mentions: The microtubule depolymerizing agent nocodazole was utilized to investigate potential association between microtubules and active PEI intracellular trafficking. Microtubules within nocodazole-treated cells were visualized by immunofluorescence using fluorescent microscopy to examine the impact of disrupted microtubules on PEI trafficking (Figure 3). Untreated mitochondrial, PEI/pCH110-YOYO, and merged images depict the accumulation of PEI at the MTOC-associated region. The microtubule controls, showing immunofluorescence of microtubules, nucleus, and merged images highlight a filamentous-microtubule structure throughout the cell and an intense staining of microtubule accumulation within a defined MTOC-associated perinuclear region. Nocodazole-treated cells containing PEI/pDNA-YOYO polyplexes display a random distribution of labelled complexes throughout the whole cell, as compared to controls that display MTOC-associated fluorescence accumulation. Furthermore, the filamentous morphology of mitochondria stained with MitoTracker was seen to be disrupted, becoming evenly distributed around the nucleus. The nocodazole-treated cells displayed disrupted microtubules, highlighted by the loss of the filamentous structure and replaced by a uniform fluorescence throughout the cell. Randomly dispersed fluorescence of PEI polyplex distribution was observed in nocodazole-treated cells (Figure 3E), as compared to the predictable PEI accumulation at the MTOC-associated region in non-treated cells (Figure 3B). The significant differences in morphology of microtubules and PEI accumulations observed between the treated and untreated cells, strongly indicated an albeit indirect correlation between the disruption of microtubules and loss of PEI trafficking.

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.