Systematic Study of a Library of PDMAEMA-Based, Superparamagnetic Nano-Stars for the Transfection of CHO-K1 Cells

Abstract

The introduction of the DNA into mammalian cells remains a challenge in gene delivery, particularly in vivo. Viral vectors are unmatched in their efficiency for gene delivery, but may trigger immune responses and cause severe side-reactions. Non-viral vectors are much less efficient. Recently, our group has suggested that a star-shaped structure improves and even transforms the gene delivery capability of synthetic polycations. In this contribution, this effect was systematically studied using a library of highly homogeneous, paramagnetic nano-star polycations with varied arm lengths and grafting densities. Gene delivery was conducted in CHO-K1 cells, using a plasmid encoding a green fluorescent reporter protein. Transfection efficiencies and cytotoxicities varied systematically with the nano-star architecture. The arm density was particularly important, with values of approximately 0.06 arms/nm² yielding the best results. In addition, a certain fraction of the cells became magnetic during transfection. The gene delivery potential of a nano-star and its ability to render the cells magnetic did not have any correlations. End-capping the polycation arms with di(ethylene glycol) methyl ether methacrylate (PDEGMA) significantly improved serum compatibility under transfection conditions; such nano-stars are potential candidates for future in vivo testing.

Keywords: ATRP; CHO cells; EGFP; PDEGMA; PDMAEMA; cellular uptake; gene delivery; magnetic nanoparticles; polycation; transfection.

Figure 1

Average hydrodynamic radii (R_h) and zeta potentials of polyplexes prepared at the indicated N/P ratio. Data represent mean ± S.E.M., n ≥ 3.

Figure 2

DNA melting temperature (T_M) as function of the N/P-ratio (A) polyplexes prepared in HBG, (B) polyplexes prepared in aqueous 150 mM NaCl solution. Lines serve as guides. Data represent mean ± S.E.M., n ≥ 2. Standard deviations are given for all data points but may be too small for viewing.

Figure 3

pH of the polyplex solution as a function of the N/P-ratio. Polyplexes were prepared either in HBG (empty symbols) or aqueous 150 mM NaCl solution (filled symbols). Lines serve as a guide. Data represent mean ± S.E.M., n ≥ 2.

Figure 4

Transfection efficiencies (), magnetic cell fractions (), and viabilities (○) determined 26 h post transfection. Data represent mean ± S.E.M., n ≥ 3. In the case of the viabilities, statistically significant differences (p < 0.05) compared to mock transfected cells (97.6% ± 2.5%, n ≥ 30) are denoted by #.

Figure 5

Influence of contact time on transgene-expressing and magnetic cell fractions. Cells were transfected with P4 at N/P 15. Data represent one experiment carried out in duplicate, with random experimental error shown.

Figure 6

Enhanced green fluorescent protein (EGFP) expression in magnetic and non-magnetic cell fractions compared to the controls (no separation). Cells were transfected with P4 (N/P 20), separated 24 h post transfection (t = 0) by magnetically-assisted cell sorting and placed into separated cultures. Data represent one experiment carried out in duplicate, with random experimental error shown.

Figure 7

EGFP expression in the respective magnetic, non-magnetic and control cell cultures. Initial separation by magnetic cell sorting took place 26 h after transfection (P4, N/P = 20). The individual cultures were again sorted 22 h later. Data represent one experiment carried out in duplicate with the random experimental error.

Figure 8

Correlogram between nano-star/polyplex characteristics and the cellular responses (viability, transfection efficiency, magnetism). Positive correlations are indicated in blue, negative ones in red. No correlation coefficients were calculated for parameters, which showed no statistically relevant differences (p > 0.01).

Figure 9

Transfection efficiencies (), magnetic cell fractions (), and viabilities (○) determined at 26 h post transfection. Data represent mean ± S.E.M., n ≥ 3.

Figure 10

Average hydrodynamic radii (R_h) and zeta potentials of polyplexes prepared with DB. Data represent mean ± S.E.M., n ≥ 3.

Figure 11

Effect of serum on transfection efficiencies of DB (N/P 20), P3 (N/P 20), and P4 (N/P 15). Data represent one experiment carried out in duplicate, with random experimental error shown.

Figure 12

Transfection efficiency of DB in the presence of fetal calf serum as a function of the N/P ratio. Bars: percentage of EGFP-positive cells, circles: viabilities of cells. Data represent one experiment carried out in duplicate, with random experimental error shown.