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Using inositol as a biocompatible ligand for efficient transgene expression.

Zhang L, Bellis SL, Fan Y, Wu Y - Int J Nanomedicine (2015)

Bottom Line: The three resulting PG6-PEI-INO polymers have an increased number of INO ligands per molecule.Studies further revealed that extracellular adenosine triphosphate (eATP) can inhibit the transgene efficiency of PG6-PEI-INO polymers, as compared with PEI and PG6-PEI that were not conjugated with inositol.Our work unveiled the possibility of using inositol as an effective ligand for transgene expression.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, People's Republic of China.

ABSTRACT
Transgene transfection techniques using cationic polymers such as polyethylenimines (PEIs) and PEI derivatives as gene vectors have shown efficacy, although they also have shortcomings. PEIs have decent DNA-binding capability and good cell internalization performance, but they cannot deliver gene payloads very efficiently to cell nuclei. In this study, three hyperbranched polyglycerol-polyethylenimine (PG6-PEI) polymers conjugated with myo-inositol (INO) molecules were developed. The three resulting PG6-PEI-INO polymers have an increased number of INO ligands per molecule. PG6-PEI-INO 1 had only 14 carboxymethyl INO (CMINO) units per molecule. PG6-PEI-INO 2 had approximately 130 CMINO units per molecule. PG6-PEI-INO 3 had as high as 415 CMINO units approximately. Mixing PG6-PEI-INO polymers with DNA produced compact nanocomposites. We then performed localization studies using fluorescent microscopy. As the number of conjugated inositol ligands increased in PG6-PEI-INO polymers, there was a corresponding increase in accumulation of the polymers within 293T cell nuclei. Transfection performed with spherical 293T cells yielded 82% of EGFP-positive cells when using PG6-PEI-INO 3 as the vehicle. Studies further revealed that extracellular adenosine triphosphate (eATP) can inhibit the transgene efficiency of PG6-PEI-INO polymers, as compared with PEI and PG6-PEI that were not conjugated with inositol. Our work unveiled the possibility of using inositol as an effective ligand for transgene expression.

No MeSH data available.


Inhibition of extracellular ATP on transgene activity of PG6-PEI-INO.Notes: (A) The 293T cells were treated with (a) PEI25k/pEGFP-C1 (w/w =1.3), (b) PG6-PEI/pEGFP-C1 (w/w =7), and (c) PG6-PEI-INO 3/pEGFP-C1 (w/w =7) for 96 hours, respectively. Extracellular ATP was used at 1.7 μg per mL culture medium. Scale bar: 10 μm. (B) EGFP-positive 293T cell ratios mediated by polymers/DNA/ATP. Plasmid DNA (pEGFP-C1) was used at 1.3 μg per mL culture medium. The cells were treated with the polymers/DNA/ATP for 44 hours (a) and 96 hours (b), respectively.Abbreviations: ATP, adenosine triphosphate; INO, myo-inositol; PEI, polyethylenimine; PG6, polyglycerol.
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f8-ijn-10-2871: Inhibition of extracellular ATP on transgene activity of PG6-PEI-INO.Notes: (A) The 293T cells were treated with (a) PEI25k/pEGFP-C1 (w/w =1.3), (b) PG6-PEI/pEGFP-C1 (w/w =7), and (c) PG6-PEI-INO 3/pEGFP-C1 (w/w =7) for 96 hours, respectively. Extracellular ATP was used at 1.7 μg per mL culture medium. Scale bar: 10 μm. (B) EGFP-positive 293T cell ratios mediated by polymers/DNA/ATP. Plasmid DNA (pEGFP-C1) was used at 1.3 μg per mL culture medium. The cells were treated with the polymers/DNA/ATP for 44 hours (a) and 96 hours (b), respectively.Abbreviations: ATP, adenosine triphosphate; INO, myo-inositol; PEI, polyethylenimine; PG6, polyglycerol.

Mentions: The influence of eATP on transfection with PG6-PEI-INO 3/pEGFP-C1, PG6-PEI/pEGFP-C1, and PEI25k/pEGFP-C1 at optimized weight ratios was analyzed. With the supplementation of eATP, cells transfected with the PG6-PEI-INO 3/pEGFP-C1 reagent declined to <1%. Transfection with PEI25k/pEGFP-C1 was not apparently influenced by eATP (Figure 8). Compared with PG6-PEI-INO, the influence of eATP on PG6-PEI was much smaller. Since eATP did not reduce the transfection activity of PEI25k or PG6-PEI apparently, we deduced that the transgene activity of PG6-PEI-INO 3 should be related to certain intracellular pathways, rather than weakening of DNA-binding activity.


Using inositol as a biocompatible ligand for efficient transgene expression.

Zhang L, Bellis SL, Fan Y, Wu Y - Int J Nanomedicine (2015)

Inhibition of extracellular ATP on transgene activity of PG6-PEI-INO.Notes: (A) The 293T cells were treated with (a) PEI25k/pEGFP-C1 (w/w =1.3), (b) PG6-PEI/pEGFP-C1 (w/w =7), and (c) PG6-PEI-INO 3/pEGFP-C1 (w/w =7) for 96 hours, respectively. Extracellular ATP was used at 1.7 μg per mL culture medium. Scale bar: 10 μm. (B) EGFP-positive 293T cell ratios mediated by polymers/DNA/ATP. Plasmid DNA (pEGFP-C1) was used at 1.3 μg per mL culture medium. The cells were treated with the polymers/DNA/ATP for 44 hours (a) and 96 hours (b), respectively.Abbreviations: ATP, adenosine triphosphate; INO, myo-inositol; PEI, polyethylenimine; PG6, polyglycerol.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4403686&req=5

f8-ijn-10-2871: Inhibition of extracellular ATP on transgene activity of PG6-PEI-INO.Notes: (A) The 293T cells were treated with (a) PEI25k/pEGFP-C1 (w/w =1.3), (b) PG6-PEI/pEGFP-C1 (w/w =7), and (c) PG6-PEI-INO 3/pEGFP-C1 (w/w =7) for 96 hours, respectively. Extracellular ATP was used at 1.7 μg per mL culture medium. Scale bar: 10 μm. (B) EGFP-positive 293T cell ratios mediated by polymers/DNA/ATP. Plasmid DNA (pEGFP-C1) was used at 1.3 μg per mL culture medium. The cells were treated with the polymers/DNA/ATP for 44 hours (a) and 96 hours (b), respectively.Abbreviations: ATP, adenosine triphosphate; INO, myo-inositol; PEI, polyethylenimine; PG6, polyglycerol.
Mentions: The influence of eATP on transfection with PG6-PEI-INO 3/pEGFP-C1, PG6-PEI/pEGFP-C1, and PEI25k/pEGFP-C1 at optimized weight ratios was analyzed. With the supplementation of eATP, cells transfected with the PG6-PEI-INO 3/pEGFP-C1 reagent declined to <1%. Transfection with PEI25k/pEGFP-C1 was not apparently influenced by eATP (Figure 8). Compared with PG6-PEI-INO, the influence of eATP on PG6-PEI was much smaller. Since eATP did not reduce the transfection activity of PEI25k or PG6-PEI apparently, we deduced that the transgene activity of PG6-PEI-INO 3 should be related to certain intracellular pathways, rather than weakening of DNA-binding activity.

Bottom Line: The three resulting PG6-PEI-INO polymers have an increased number of INO ligands per molecule.Studies further revealed that extracellular adenosine triphosphate (eATP) can inhibit the transgene efficiency of PG6-PEI-INO polymers, as compared with PEI and PG6-PEI that were not conjugated with inositol.Our work unveiled the possibility of using inositol as an effective ligand for transgene expression.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, People's Republic of China.

ABSTRACT
Transgene transfection techniques using cationic polymers such as polyethylenimines (PEIs) and PEI derivatives as gene vectors have shown efficacy, although they also have shortcomings. PEIs have decent DNA-binding capability and good cell internalization performance, but they cannot deliver gene payloads very efficiently to cell nuclei. In this study, three hyperbranched polyglycerol-polyethylenimine (PG6-PEI) polymers conjugated with myo-inositol (INO) molecules were developed. The three resulting PG6-PEI-INO polymers have an increased number of INO ligands per molecule. PG6-PEI-INO 1 had only 14 carboxymethyl INO (CMINO) units per molecule. PG6-PEI-INO 2 had approximately 130 CMINO units per molecule. PG6-PEI-INO 3 had as high as 415 CMINO units approximately. Mixing PG6-PEI-INO polymers with DNA produced compact nanocomposites. We then performed localization studies using fluorescent microscopy. As the number of conjugated inositol ligands increased in PG6-PEI-INO polymers, there was a corresponding increase in accumulation of the polymers within 293T cell nuclei. Transfection performed with spherical 293T cells yielded 82% of EGFP-positive cells when using PG6-PEI-INO 3 as the vehicle. Studies further revealed that extracellular adenosine triphosphate (eATP) can inhibit the transgene efficiency of PG6-PEI-INO polymers, as compared with PEI and PG6-PEI that were not conjugated with inositol. Our work unveiled the possibility of using inositol as an effective ligand for transgene expression.

No MeSH data available.