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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.


Related in: MedlinePlus

Effect of nocodazole and aphidicolin on PEI polyplex mediated GFP expression. Expression of GFP 24 h post transfection in the presence of nocodazole (light bar), or aphidicolin (dark bar), as a percentage of optimal PEI/pDNA mediated GFP expression at N/P 8. Data obtained as detected by FACS 24 h post transfection, and represented as mean values ± SEM (N = 2).
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Figure 5: Effect of nocodazole and aphidicolin on PEI polyplex mediated GFP expression. Expression of GFP 24 h post transfection in the presence of nocodazole (light bar), or aphidicolin (dark bar), as a percentage of optimal PEI/pDNA mediated GFP expression at N/P 8. Data obtained as detected by FACS 24 h post transfection, and represented as mean values ± SEM (N = 2).

Mentions: The relative high PEI-/low PEI-Arg-facilitated expression indicates a correlation with the presence, and absence, of MTOC-associated accumulation respectively, and hence a possible correlation between microtubule-dependent intracellular trafficking of polyplexes and GFP expression. GFP expression analysis of nocodazole treated cells displayed a reduction of more than 20% of maximal PEI expression (Figure 5). This suggests that GFP expression is not exclusively facilitated by microtubule-dependent trafficking. Taking this into consideration, we expected that the transfection efficiency of PEI-Arg would have been greater. As PEI-Arg-facilitated GFP expression averaged 6-fold less GFP-positive cells (Figure 4B), there must be further limitations affecting PEI-Arg that were not directly evident in this study. This however may be further evidence for the retention of PEI-Arg polyplexes within non-endocytic vesicles. We hypothesize that expression of DNA complexed with PEI-Arg entrapped in these vesicles is hampered, even if they had been engulfed within the nucleus during mitosis.


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 nocodazole and aphidicolin on PEI polyplex mediated GFP expression. Expression of GFP 24 h post transfection in the presence of nocodazole (light bar), or aphidicolin (dark bar), as a percentage of optimal PEI/pDNA mediated GFP expression at N/P 8. Data obtained as detected by FACS 24 h post transfection, and represented as mean values ± SEM (N = 2).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Effect of nocodazole and aphidicolin on PEI polyplex mediated GFP expression. Expression of GFP 24 h post transfection in the presence of nocodazole (light bar), or aphidicolin (dark bar), as a percentage of optimal PEI/pDNA mediated GFP expression at N/P 8. Data obtained as detected by FACS 24 h post transfection, and represented as mean values ± SEM (N = 2).
Mentions: The relative high PEI-/low PEI-Arg-facilitated expression indicates a correlation with the presence, and absence, of MTOC-associated accumulation respectively, and hence a possible correlation between microtubule-dependent intracellular trafficking of polyplexes and GFP expression. GFP expression analysis of nocodazole treated cells displayed a reduction of more than 20% of maximal PEI expression (Figure 5). This suggests that GFP expression is not exclusively facilitated by microtubule-dependent trafficking. Taking this into consideration, we expected that the transfection efficiency of PEI-Arg would have been greater. As PEI-Arg-facilitated GFP expression averaged 6-fold less GFP-positive cells (Figure 4B), there must be further limitations affecting PEI-Arg that were not directly evident in this study. This however may be further evidence for the retention of PEI-Arg polyplexes within non-endocytic vesicles. We hypothesize that expression of DNA complexed with PEI-Arg entrapped in these vesicles is hampered, even if they had been engulfed within the nucleus during mitosis.

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.


Related in: MedlinePlus