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PLGA-based gene delivering nanoparticle enhance suppression effect of miRNA in HePG2 cells.

Liang GF, Zhu YL, Sun B, Hu FH, Tian T, Li SC, Xiao ZD - Nanoscale Res Lett (2011)

Bottom Line: The N/P ratio (ratio of the polymer nitrogen to the DNA phosphate) 6 of the PLGA/PEI/DNA nanocomplex displays the best property among various N/P proportions, yielding similar transfection efficiency when compared to Lipofectamine/DNA lipoplexes.Moreover, nanocomplex shows better serum compatibility than commercial liposome.Therefore, these results demonstrate that PLGA/PEI nanoparticles are promising non-viral vectors for gene delivery.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, 210096, China. zdxiao@seu.edu.

ABSTRACT
The biggest challenge in the field of gene therapy is how to effectively deliver target genes to special cells. This study aimed to develop a new type of poly(D,L-lactide-co-glycolide) (PLGA)-based nanoparticles for gene delivery, which are capable of overcoming the disadvantages of polyethylenimine (PEI)- or cationic liposome-based gene carrier, such as the cytotoxicity induced by excess positive charge, as well as the aggregation on the cell surface. The PLGA-based nanoparticles presented in this study were synthesized by emulsion evaporation method and characterized by transmission electron microscopy, dynamic light scattering, and energy dispersive spectroscopy. The size of PLGA/PEI nanoparticles in phosphate-buffered saline (PBS) was about 60 nm at the optimal charge ratio. Without observable aggregation, the nanoparticles showed a better monodispersity. The PLGA-based nanoparticles were used as vector carrier for miRNA transfection in HepG2 cells. It exhibited a higher transfection efficiency and lower cytotoxicity in HepG2 cells compared to the PEI/DNA complex. The N/P ratio (ratio of the polymer nitrogen to the DNA phosphate) 6 of the PLGA/PEI/DNA nanocomplex displays the best property among various N/P proportions, yielding similar transfection efficiency when compared to Lipofectamine/DNA lipoplexes. Moreover, nanocomplex shows better serum compatibility than commercial liposome. PLGA nanocomplexes obviously accumulate in tumor cells after transfection, which indicate that the complexes contribute to cellular uptake of pDNA and pronouncedly enhance the treatment effect of miR-26a by inducing cell cycle arrest. Therefore, these results demonstrate that PLGA/PEI nanoparticles are promising non-viral vectors for gene delivery.

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GFP expression in HepG2 cells transfected with different transfection reagents. (A-D) Fluorescent and bright-field images of green fluorescent protein expression in HepG2 cells for PLGA-based polyplexes (N/P ratio 6) with control 25 kDa PEI (N/P ratio 5) and lipo2000. (A) naked DNA, (B) PEI, (C) PLGA/PEI, (D) lipo2000. The images were obtained at magnification of 100×. (E) Transfection efficiency of the nanocomplex determined by flow cytometry analysis at different N/P ratio (n = 3). The transfection reagents/DNA complexes were prepared at their optimal condition. (F) Transfection efficiency of the nanocomplex determined by flow cytometry analysis at 2 μg pDNA mixed with different volume of nanocomplex (4 μg/μl). The nanoparticles/DNA complexes were prepared at optimal N/P ratio (n = 3).
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Figure 3: GFP expression in HepG2 cells transfected with different transfection reagents. (A-D) Fluorescent and bright-field images of green fluorescent protein expression in HepG2 cells for PLGA-based polyplexes (N/P ratio 6) with control 25 kDa PEI (N/P ratio 5) and lipo2000. (A) naked DNA, (B) PEI, (C) PLGA/PEI, (D) lipo2000. The images were obtained at magnification of 100×. (E) Transfection efficiency of the nanocomplex determined by flow cytometry analysis at different N/P ratio (n = 3). The transfection reagents/DNA complexes were prepared at their optimal condition. (F) Transfection efficiency of the nanocomplex determined by flow cytometry analysis at 2 μg pDNA mixed with different volume of nanocomplex (4 μg/μl). The nanoparticles/DNA complexes were prepared at optimal N/P ratio (n = 3).

Mentions: It is clear that gene delivery is dependent on DNA/vector uptake efficiency. Fluorescent proteins, such as GFP, are usually used to label non-viral vectors for measuring the uptake efficiency. In this study, nanoparticle/pGFP-miR-26a complexes were employed for the assessment of the transfection efficiency in HepG2 cells. Firstly, HepG2 cells were transfected by 2 μg of pDNA complexed with 100 μl polymer at different nanoparticles/pDNA N/P ratios. The nanocomplexes were further evaluated for their transfection efficiency in cells by screening the GFP signals with flow cytometry. The results showed that transfection efficiency reached the optimum at nanoparticles/DNA N/P ratio 6 and further decreased till the N/P ratio reached 9 (Figure 3E). It is suggested that insufficient surface potential of complexes at low N/P ratios resulted in lower GFP expression, whereas at high N/P ratios, induction of complexes undoubtedly result in cytotoxicity because of the excessive PEI in the nanocomplex.


PLGA-based gene delivering nanoparticle enhance suppression effect of miRNA in HePG2 cells.

Liang GF, Zhu YL, Sun B, Hu FH, Tian T, Li SC, Xiao ZD - Nanoscale Res Lett (2011)

GFP expression in HepG2 cells transfected with different transfection reagents. (A-D) Fluorescent and bright-field images of green fluorescent protein expression in HepG2 cells for PLGA-based polyplexes (N/P ratio 6) with control 25 kDa PEI (N/P ratio 5) and lipo2000. (A) naked DNA, (B) PEI, (C) PLGA/PEI, (D) lipo2000. The images were obtained at magnification of 100×. (E) Transfection efficiency of the nanocomplex determined by flow cytometry analysis at different N/P ratio (n = 3). The transfection reagents/DNA complexes were prepared at their optimal condition. (F) Transfection efficiency of the nanocomplex determined by flow cytometry analysis at 2 μg pDNA mixed with different volume of nanocomplex (4 μg/μl). The nanoparticles/DNA complexes were prepared at optimal N/P ratio (n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 3: GFP expression in HepG2 cells transfected with different transfection reagents. (A-D) Fluorescent and bright-field images of green fluorescent protein expression in HepG2 cells for PLGA-based polyplexes (N/P ratio 6) with control 25 kDa PEI (N/P ratio 5) and lipo2000. (A) naked DNA, (B) PEI, (C) PLGA/PEI, (D) lipo2000. The images were obtained at magnification of 100×. (E) Transfection efficiency of the nanocomplex determined by flow cytometry analysis at different N/P ratio (n = 3). The transfection reagents/DNA complexes were prepared at their optimal condition. (F) Transfection efficiency of the nanocomplex determined by flow cytometry analysis at 2 μg pDNA mixed with different volume of nanocomplex (4 μg/μl). The nanoparticles/DNA complexes were prepared at optimal N/P ratio (n = 3).
Mentions: It is clear that gene delivery is dependent on DNA/vector uptake efficiency. Fluorescent proteins, such as GFP, are usually used to label non-viral vectors for measuring the uptake efficiency. In this study, nanoparticle/pGFP-miR-26a complexes were employed for the assessment of the transfection efficiency in HepG2 cells. Firstly, HepG2 cells were transfected by 2 μg of pDNA complexed with 100 μl polymer at different nanoparticles/pDNA N/P ratios. The nanocomplexes were further evaluated for their transfection efficiency in cells by screening the GFP signals with flow cytometry. The results showed that transfection efficiency reached the optimum at nanoparticles/DNA N/P ratio 6 and further decreased till the N/P ratio reached 9 (Figure 3E). It is suggested that insufficient surface potential of complexes at low N/P ratios resulted in lower GFP expression, whereas at high N/P ratios, induction of complexes undoubtedly result in cytotoxicity because of the excessive PEI in the nanocomplex.

Bottom Line: The N/P ratio (ratio of the polymer nitrogen to the DNA phosphate) 6 of the PLGA/PEI/DNA nanocomplex displays the best property among various N/P proportions, yielding similar transfection efficiency when compared to Lipofectamine/DNA lipoplexes.Moreover, nanocomplex shows better serum compatibility than commercial liposome.Therefore, these results demonstrate that PLGA/PEI nanoparticles are promising non-viral vectors for gene delivery.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, 210096, China. zdxiao@seu.edu.

ABSTRACT
The biggest challenge in the field of gene therapy is how to effectively deliver target genes to special cells. This study aimed to develop a new type of poly(D,L-lactide-co-glycolide) (PLGA)-based nanoparticles for gene delivery, which are capable of overcoming the disadvantages of polyethylenimine (PEI)- or cationic liposome-based gene carrier, such as the cytotoxicity induced by excess positive charge, as well as the aggregation on the cell surface. The PLGA-based nanoparticles presented in this study were synthesized by emulsion evaporation method and characterized by transmission electron microscopy, dynamic light scattering, and energy dispersive spectroscopy. The size of PLGA/PEI nanoparticles in phosphate-buffered saline (PBS) was about 60 nm at the optimal charge ratio. Without observable aggregation, the nanoparticles showed a better monodispersity. The PLGA-based nanoparticles were used as vector carrier for miRNA transfection in HepG2 cells. It exhibited a higher transfection efficiency and lower cytotoxicity in HepG2 cells compared to the PEI/DNA complex. The N/P ratio (ratio of the polymer nitrogen to the DNA phosphate) 6 of the PLGA/PEI/DNA nanocomplex displays the best property among various N/P proportions, yielding similar transfection efficiency when compared to Lipofectamine/DNA lipoplexes. Moreover, nanocomplex shows better serum compatibility than commercial liposome. PLGA nanocomplexes obviously accumulate in tumor cells after transfection, which indicate that the complexes contribute to cellular uptake of pDNA and pronouncedly enhance the treatment effect of miR-26a by inducing cell cycle arrest. Therefore, these results demonstrate that PLGA/PEI nanoparticles are promising non-viral vectors for gene delivery.

No MeSH data available.


Related in: MedlinePlus