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. 2016 Apr 29;21(5):572.
doi: 10.3390/molecules21050572.

An Electrostatically Self-Assembled Ternary Nanocomplex as a Non-Viral Vector for the Delivery of Plasmid DNA into Human Adipose-Derived Stem Cells

Sun-Hee Cho  1 , Young-Woock Noh  2 , Mi Young Cho  3 , Yong Taik Lim  4
Affiliations

An Electrostatically Self-Assembled Ternary Nanocomplex as a Non-Viral Vector for the Delivery of Plasmid DNA into Human Adipose-Derived Stem Cells

Sun-Hee Cho et al. Molecules. .

Abstract

In this study, we developed electrostatically self-assembled ternary nanocomplexes as a safe and effective non-viral vector for the delivery of plasmid DNA (pDNA) into human adipose-derived stem cells (hASCs). Although polyethylenimine (PEI) polymers initially showed excellent performance as gene delivery carriers, their broad use has been limited by cytotoxicity resulting from their strong positive charge. To reduce the cytotoxicity, we utilized anionic hyaluronic acid (HA) as a corona layer material for pDNA/PEI binary nanocomplexes. HA was also introduced to increase the targeting efficiency of pDNA/PEI nanocomplexes because HA has can bind CD44 that is highly expressed on the surface of hASCs. We confirmed that the addition of HA changed the surface charge of pDNA/PEI nanocomplexes from positive to negative. The pDNA/PEI/HA ternary nanocomplexes showed high transfection efficiency and low cytotoxicity compared with commercially available products. When hASCs were pretreated with HA to passivate CD44, the transfection efficiency of pDNA/PEI/HA nanocomplexes was significantly reduced. These results suggest that HA that can act as a targeting ligand to CD44 contributed to the improved transfection of pDNA into hASCs. Our novel pDNA/PEI/HA nanocomplexes may be used as an effective non-viral pDNA delivery system for hASCs.

Keywords: adipose derived stem cells; hyaluronic acid; non-viral vector; self-assembly; ternary nanocomplex.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Schematic illustrations for the synthesis of electrostatically self-assembled pDNA/PEI/HA ternary nanocomplexes (pDNA (blue line): plasmid DNA, PEI (green line): polyethylenimine, HA (red line): hyaluronic acid).
Figure 2
Figure 2
SEM images of (a) pDNA/PEI; and (b) pDNA/PEI/HA (1:8:20).
Figure 3
Figure 3
The size stability of each nanocomplex after incubation for 24 h. * p < 0.05, n.s = not significant.
Figure 4
Figure 4
(a) Gel retardation assay of nanocomplexes. 1. Naked pDNA; 2. pDNA/PEI (1:8); 3. pDNA/PEI/HA (1:8:5); 4. pDNA/PEI/HA (1:8:10); and 5. pDNA/PEI/HA (1:8:20); (b) Viability of hASCs treated with each nanocomplex formulation as measured by MTS assay (n = 3). hASCs were incubated with various nanocomplexes for 24 h and cell viability was measured. ** p < 0.01 vs. control. n.s = not significant vs. control.
Figure 5
Figure 5
Flow cytometry profiles of non-transfected hASCs (black) and hASCs transfected with pCXLE-EGFP using each nanocomplex (red) and X-tremeGENE (green) (a) at 24 h and (b) 48 h after treatment. Black line = control, red line = complex, green line = X-tremeGENE; (c) Percentage of EGFP-expressing hASCs after transfection by each nanocomplex. Data are presented as means ± S.E. (n = 4), * p < 0.05, ** p < 0.01 vs. X-tremeGENE.
Figure 6
Figure 6
Fluorescence images of hASCs after transfection with each nanocomplex at 24 h. (Ex 490/20 and 360/40; Em 526/36 and 455/50).
Figure 7
Figure 7
Expression of CD44 in hASCs evaluated by (a) flow cytometry and (b) fluorescence image. Scale bar = 40 µm. (c) Competition assay to investigate the effect of HA pretreatment on the transfection efficiency of pDNA/PEI/HA (left) and pDNA/PEI/γ-PGA (right) ternary nanocomplexes at 24 or 48 h. ■ = without HA pretreatment, □ = with HA pretreatment. Data are presented as means ± S.E. (n = 4), * p < 0.05, n.s = not significant vs. untreated control.
Figure 8
Figure 8
Schematic illustration of CD44-mediated endocytosis and delivery of pDNA into a stem cell using our self-assembled complexes. (Blue line: plasmid DNA, green line: PEI, red line: HA).
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