Lignin-Based Nonviral Gene Carriers Functionalized by Poly[2-(Dimethylamino)ethyl Methacrylate]: Effect of Grafting Degree and Cationic Chain Length on Transfection Efficiency

Abstract

Lignin is a natural renewable biomass resource with great potential for applications, while its development into high value-added molecules or materials is rare. The development of biomass lignin as potential nonviral gene delivery carriers was initiated by our group through the "grafting-from" approach. Firstly, the lignin was modified into macroinitiator using 2-bromoisobutyryl bromide. Then cationic polymer chains of poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA) were grown from the lignin backbone using atom transfer radical polymerization (ATRP) to yield lignin-PDMAEMA graft copolymers (LPs) with branched structure. To gain a deep understanding of the relationship between the nonviral gene transfection efficiency of such copolymers and their structural and compositional factors, herein eight lignin-based macroinitiators with different modification degrees (MDs, from 3.0 to 100%) were synthesized. Initiated by them, a series of 20 LPs were synthesized with varied structural factors such as grafting degree (GD, which is equal to MD, determining the cationic chain number per lignin macromolecule), cationic chain length (represented by number of repeating DMAEMA units per grafted arm or degree of polymerization, DP) as well as the content of N element (N%) which is due to the grafted PDMAEMA chains and proportional to molecular weight of the LPs. The in vitro gene transfection capability of these graft copolymers was evaluated by luciferase assay in HeLa, COS7 and MDA-MB-231cell lines. Generally, the copolymers LP-12 (N% = 7.28, MD = 36.7%, DP = 13.6) and LP-14 (N% = 6.05, MD = 44.4%, DP = 5.5) showed good gene transfection capabilities in the cell lines tested. Overall, the performance of LP-12 was the best among all the LPs in the three cell lines at the N/P ratios from 10 to 30, which was usually several times higher than PEI standard. However, in MDA-MB-231 at N/P ratio of 30, LP-14 showed the best gene transfection performance among all the LPs. Its gene transfection efficiency was ca. 11 times higher than PEI standard at this N/P ratio. This work demonstrated that, although the content of N element (N%) which is due to the grafted PDMAEMA chains primarily determines the gene transfection efficiency of the LPs, it is not the only factor in explaining the performance of such copolymers with the branched structure. Structural factors of these copolymers such as grafting degree and cationic chain length could have a profound effect on the copolymer performance on gene transfection efficiency. Through carefully adjusting these factors, the gene transfection efficiency of the LPs could be modulated and optimized for different cell lines, which could make this new type of biomass-based biomaterial an attractive choice for various gene delivery applications.

Keywords: PDMAEMA; atom transfer radical polymerization; graft copolymer; lignin; nonviral gene carriers; plasmid DNA.

Figure 1

Synthetic procedures for (a) conversion of biomass lignin into lignin-based macroinitiator (LnMI) and (b) synthesis of lignin-based graft copolymer (LP) via Cu(I)-mediated ATRP of DMAEMA.

Figure 2

¹H NMR characterization results for lignin-based macroinitiators (LnMIs) (DMSO-d₆, 400 MHz, 25 °C). Data for LnMI-f and LnMI-h were reported previously [18]. The solvent residual peak (δ 2.5 ppm) and the water peak (δ 3.3 ppm) were labelled in these spectra.

Figure 3

(A) ¹H NMR characterization result for lignin-PDMAEMA graft copolymer LP-02 (DMSO-d₆, 400 MHz, 25 °C). (B) ¹H NMR characterization results for lignin-PDMAEMA graft copolymers LP-04, LP-06, LP-10, and LP-11 in D₂O containing DCl (0.05 mol/L), respectively (400 MHz, 25 °C). For (A), the solvent residual peak (δ 2.5 ppm) and the water peak (δ 3.3 ppm) were labelled. For (B), the solvent residual peak was labelled at δ 4.7 ppm.

Figure 4

Electrophoretic mobility of pDNA in the polyplexes formed with lignin graft copolymers (A) LP-03, (B) LP-05, (C) LP-06, (D) LP-11, (E) LP-12, (F) LP-14, (G) LP-16, (H) LP-17, (I) LP-20 and (J) PEI (25 kDa) at various N/P ratios; Data for (F) LP-14 and (G) LP-16 were reported previously [18].

Figure 5

Relation between N/P ratio at which complete DNA retardation was observed and the nitrogen content (N%) of lignin-PDMAEMA graft polymer. Data for LP-14 and LP-16 were reported previously [18].

Figure 6

Particle size (A) and zeta potential (B) of the polyplexes formed between cationic lignin-PDMAEMA carriers (LP-12, LP-14 and LP-20) and pDNA in comparison with PEI/pDNA polyplexes at various N/P ratios. Data represent mean ± standard deviation (n = 2 for A, n = 3 for B). Data for LP-14 was reported previously [18].

Figure 7

Cell viability assay of lignin-PDMAEMA copolymers in MDA-MB-231 (A) and COS7 (B) cells in comparison with branched PEI (25 kDa). Data represent mean ± standard deviation (n = 6). Data for LP-14 was reported previously [18].

Figure 8

(A) Variation of the relative cell viability with the N content of the lignin-PDMAEMA copolymers in MDA-MB-231 and COS7 cell lines at the copolymer concentration of 25 μg/mL. (B) Variations of the relative cell viability with the degree of polymerization (DP) and molecular weight (Mn) for the selected lignin-PDMAEMA copolymers LP-03, 05 and 06 in MDA-MB-231 and COS7 cell lines at the copolymer concentration of 25 μg/mL. Data for LP-14 was reported previously [18].

Figure 9

In vitro gene transfection efficiency of the complexes of PEI/pDNA in HeLa cells in the presence of serum at different N/P ratios (A). Relative gene transfection efficiency of the complexes of LP/pDNA in HeLa cell line in the presence of serum at N/P of 10 (B), 20 (C) and 30 (D). Data represent mean ± standard deviation (* p < 0.05, *** p < 0.001, n = 2).

Figure 10

In vitro gene transfection efficiency of the complexes of PEI/pDNA in COS7 cells in the presence of serum at different N/P ratios (A). Relative gene transfection efficiency of the complexes of LP/pDNA in COS7 cell line in the presence of serum at N/P of 10 (B), 20 (C) and 30 (D). Data represent mean ± standard deviation (* p < 0.05, ** p < 0.01, n = 2).

Figure 11

In vitro gene transfection efficiency of the complexes of PEI/pDNA in MDA-MB-231 cells in the presence of serum at different N/P ratios (A). Relative gene transfection efficiency of the complexes of LP/pDNA in MDA-MB-231 cell line in the presence of serum at N/P of 10 (B), 20 (C) and 30 (D). Data represent mean ± standard deviation (* p < 0.05, n = 2).