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Tertiary-amine functionalized polyplexes enhanced cellular uptake and prolonged gene expression.

Lo CW, Chang Y, Lee JL, Tsai WB, Chen WS - PLoS ONE (2014)

Bottom Line: Its in-vitro and in-vivo effects on the transfection efficiency and the expression duration were evaluated.However, effective transfection only occurred in the US groups in vivo.Lower weight ratios, e.g., 0.25, exhibited better in-vivo expression for at least 45 days.

View Article: PubMed Central - PubMed

Affiliation: Department of Physical Medicine and Rehabilitation, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan, ROC.

ABSTRACT
Ultrasound (US) has been found to facilitate the transport of DNA across cell membranes. However, the transfection efficiency is generally low, and the expression duration of the transfected gene is brief. In this study, a tertiary polycation, Poly(2-(dimethylamino) ethyl methacrylate) (PDMAEMA), was used as a carrier for US-mediated gene transfection. Its in-vitro and in-vivo effects on the transfection efficiency and the expression duration were evaluated. A mixture of pCI-neo-luc and PDMAEMA was transfected to cultured cells or mouse muscle by exposure to 1-MHz pulse US. A strong expression of luciferase was found 10 days after the transfection in vitro regardless of US exposure. However, effective transfection only occurred in the US groups in vivo. The transfection ability depended on the weight ratio of PDMAEMA to DNA, and was different for the in-vitro and in-vivo conditions. Lower weight ratios, e.g., 0.25, exhibited better in-vivo expression for at least 45 days.

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In vitro transgenic expression of PEI/DNA nanoparticles at an N/P ratio of 5 and PDMAEMA/DNA nanoparticles at a weight ratio of 10 with or without US exposure.The PEI mediated gene expression on day 1 was significantly higher than that of PDMAEMA regardless of US exposure. However, there was no difference after 7 days. *P<0.05 vs. PDMAEAM.
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pone-0097627-g004: In vitro transgenic expression of PEI/DNA nanoparticles at an N/P ratio of 5 and PDMAEMA/DNA nanoparticles at a weight ratio of 10 with or without US exposure.The PEI mediated gene expression on day 1 was significantly higher than that of PDMAEMA regardless of US exposure. However, there was no difference after 7 days. *P<0.05 vs. PDMAEAM.

Mentions: The level of gene expression was evaluated by transfecting the mixture of 1((g plasmid DNA with PEI or PDMAEMA. As shown in Figure 4, the levels of in-vitro gene expression obtained by PEI with or without US are an order of magnitude higher than those obtained by PDMAEMA regardless of US exposure on Day 1 and Day 3. After seven days, the levels of luciferase intensity for PEI groups decreased and showed no significant difference to the PDMAEMA groups. This result suggests that, for in vitro condition, PEI and PDMAEMA had comparable efficiency for gene transfection regardless of US exposure. However, without the help of PEI or PDMAEMA, the expression levels of luciferase for naked DNA were less than 102 RLU. In this study, PDMAEMA-incorporated DNA complexes with polycation molecular weights ranging from 1.9 kDa (12 repeat units) to 6.1 kDa (39 repeat units) were investigated for gene delivery. Preliminary studies showed that, with its relatively low molecular weight, PDMAEMA was less effective as a gene delivery vector (data not shown). Due to the low positive charge of its polymer chain, low molecular weight PDMAEMA is incapable of effectively condensing DNA. The transfection ability of polyplexes at different weight ratios of nanoparticles to DNA could also be explained by the abovementioned charge theory. In the in vitro studies shown in Figure 2, PDMAEMA with higher weight ratios (5 and 10) bind more DNA. This finding supports our previous in vitro studies on PEI [25]. Higher DNA binding ability secured higher in vitro transgene expression (Figure 3A). In addition, the best binding ability of DNA by low molecular weight PDMAEMA(1.9 kDa, 12 repeat units) is less than 80% (data not shown). On the other hand, the binding ability of DNA by PEI at an N/P ratio of 5 and PDMAEMA at a weight ratio of 10 is almost 100% (Figure 2). PEI/DNA at an N/P ratio of 5 and PDMAEMA/DNA at a weight ratio of 10 also provide the best transfection efficiency and expression duration (at least 10 days) for in vitro gene transfection (Figure 4).


Tertiary-amine functionalized polyplexes enhanced cellular uptake and prolonged gene expression.

Lo CW, Chang Y, Lee JL, Tsai WB, Chen WS - PLoS ONE (2014)

In vitro transgenic expression of PEI/DNA nanoparticles at an N/P ratio of 5 and PDMAEMA/DNA nanoparticles at a weight ratio of 10 with or without US exposure.The PEI mediated gene expression on day 1 was significantly higher than that of PDMAEMA regardless of US exposure. However, there was no difference after 7 days. *P<0.05 vs. PDMAEAM.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4020921&req=5

pone-0097627-g004: In vitro transgenic expression of PEI/DNA nanoparticles at an N/P ratio of 5 and PDMAEMA/DNA nanoparticles at a weight ratio of 10 with or without US exposure.The PEI mediated gene expression on day 1 was significantly higher than that of PDMAEMA regardless of US exposure. However, there was no difference after 7 days. *P<0.05 vs. PDMAEAM.
Mentions: The level of gene expression was evaluated by transfecting the mixture of 1((g plasmid DNA with PEI or PDMAEMA. As shown in Figure 4, the levels of in-vitro gene expression obtained by PEI with or without US are an order of magnitude higher than those obtained by PDMAEMA regardless of US exposure on Day 1 and Day 3. After seven days, the levels of luciferase intensity for PEI groups decreased and showed no significant difference to the PDMAEMA groups. This result suggests that, for in vitro condition, PEI and PDMAEMA had comparable efficiency for gene transfection regardless of US exposure. However, without the help of PEI or PDMAEMA, the expression levels of luciferase for naked DNA were less than 102 RLU. In this study, PDMAEMA-incorporated DNA complexes with polycation molecular weights ranging from 1.9 kDa (12 repeat units) to 6.1 kDa (39 repeat units) were investigated for gene delivery. Preliminary studies showed that, with its relatively low molecular weight, PDMAEMA was less effective as a gene delivery vector (data not shown). Due to the low positive charge of its polymer chain, low molecular weight PDMAEMA is incapable of effectively condensing DNA. The transfection ability of polyplexes at different weight ratios of nanoparticles to DNA could also be explained by the abovementioned charge theory. In the in vitro studies shown in Figure 2, PDMAEMA with higher weight ratios (5 and 10) bind more DNA. This finding supports our previous in vitro studies on PEI [25]. Higher DNA binding ability secured higher in vitro transgene expression (Figure 3A). In addition, the best binding ability of DNA by low molecular weight PDMAEMA(1.9 kDa, 12 repeat units) is less than 80% (data not shown). On the other hand, the binding ability of DNA by PEI at an N/P ratio of 5 and PDMAEMA at a weight ratio of 10 is almost 100% (Figure 2). PEI/DNA at an N/P ratio of 5 and PDMAEMA/DNA at a weight ratio of 10 also provide the best transfection efficiency and expression duration (at least 10 days) for in vitro gene transfection (Figure 4).

Bottom Line: Its in-vitro and in-vivo effects on the transfection efficiency and the expression duration were evaluated.However, effective transfection only occurred in the US groups in vivo.Lower weight ratios, e.g., 0.25, exhibited better in-vivo expression for at least 45 days.

View Article: PubMed Central - PubMed

Affiliation: Department of Physical Medicine and Rehabilitation, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan, ROC.

ABSTRACT
Ultrasound (US) has been found to facilitate the transport of DNA across cell membranes. However, the transfection efficiency is generally low, and the expression duration of the transfected gene is brief. In this study, a tertiary polycation, Poly(2-(dimethylamino) ethyl methacrylate) (PDMAEMA), was used as a carrier for US-mediated gene transfection. Its in-vitro and in-vivo effects on the transfection efficiency and the expression duration were evaluated. A mixture of pCI-neo-luc and PDMAEMA was transfected to cultured cells or mouse muscle by exposure to 1-MHz pulse US. A strong expression of luciferase was found 10 days after the transfection in vitro regardless of US exposure. However, effective transfection only occurred in the US groups in vivo. The transfection ability depended on the weight ratio of PDMAEMA to DNA, and was different for the in-vitro and in-vivo conditions. Lower weight ratios, e.g., 0.25, exhibited better in-vivo expression for at least 45 days.

Show MeSH
Related in: MedlinePlus