A family of bioreducible poly(disulfide amine)s for gene delivery

Abstract

A family of bioreducible poly(disulfide amine)s, which differ in the length of polymethylene spacer [-(CH(2))(n)-] in the main chain and the side chain, has been synthesized. These bioreducible poly(disulfide amine)s exhibit local environment specific degradability and are associated with lower cytotoxicity than branched poly(ethylenimine) (bPEI, 25 kDa). These cationic polymers also show higher buffering capacity and protonation degree than bPEI, facilitating the endosomal escape of carried genetic materials. The transfection efficiency of these agents is oligomethylene length dependent. Poly(cystaminebisacrylamide-spermine) [poly(CBA-SP)], poly(cystaminebisacrylamide-bis(3-aminopropyl)-1,3-propanediamine) [poly(CBA-APPD)], and poly(cyxtaminebisacrylamide-bis(3-aminopropyl)-ethylenediamine) [ploy(CBA-APED)] with longer propylene [-(CH(2))(3)-] side spacer, demonstrate higher transfection efficacy than the counterpart poly(cystaminebisacrylamide-bis(2-aminoethyl)-1,3-propanediamine) [poly(CBA-AEPD)] and poly(cystaminebisacrylamide-triethylenetetramine) [poly(CBA-TETA)], which have shorter ethylene [-(CH(2))(2)-] side spacer. The poly(CBA-SP), poly(CBA-APPD), poly(CBA-APED) with the main chain spacer of -(CH(2))(4)-, -(CH(2))(3)-, -(CH(2))(2)- demonstrate similar transfection efficiency, indicating the length of polymer main chain spacer has less influence on transfection efficiency. However, with the same short ethylene [-(CH(2))(2)-] side spacer, poly(CBA-AEPD), with the longer main chain oligomethylene units [-(CH(2))(3)-], showed relatively higher transfection efficiency than poly(CBA-TETA), having shorter main chain oligomethylene units [-(CH(2))(2)-]. Of these polymeric carriers, poly(CBA-SP) demonstrated the highest transfection in the C2C12 cell line, while poly(CBA-APED) showed the highest transfection in the HeLa cell line. All of these agents showed greater transfection activity than commercialized bPEI 25 kDa. The poly(disulfide amine)s are promising safe and efficient non-viral vectors for gene delivery.

Figure 1

Buffering capacity and protonation behavior of poly(disulfide amine)s. (A) Titration curves of poly(disulfide amine)s. Polymers with 5 mmol amino nitrogen atoms were dissolved in 10 mL of 1.0 M NaCl. Solutions were initially set to pH 11.0 by 0.1 M NaOH and then titrated by 0.01 M HCl to pH 3.0. As a reference, the titration curves of bPEI (25 kDa) and 0.1 M NaCl were also presented. (B) Protonation degree (α) – pH curves of poly(disulfide amine)s in the pH range of 11 to 3. (C) Apparent pKa – (1-α) curves of poly(disulfide amine)s.

Figure 1

Buffering capacity and protonation behavior of poly(disulfide amine)s. (A) Titration curves of poly(disulfide amine)s. Polymers with 5 mmol amino nitrogen atoms were dissolved in 10 mL of 1.0 M NaCl. Solutions were initially set to pH 11.0 by 0.1 M NaOH and then titrated by 0.01 M HCl to pH 3.0. As a reference, the titration curves of bPEI (25 kDa) and 0.1 M NaCl were also presented. (B) Protonation degree (α) – pH curves of poly(disulfide amine)s in the pH range of 11 to 3. (C) Apparent pKa – (1-α) curves of poly(disulfide amine)s.

Figure 2

Average particle sizes of poly(disulfide amine)s/DNA polyplexes at w/w ratios of 1:1, 5:1, 10:1, 20:1 and 30:1. bPEI (25 kDa)/DNA polyplexes were measured at the same w/w ratios for comparison. The experiments were repeated three times and the data were represented as mean value ± standard deviations.

Figure 3

Gel retardation assay of poly(disulfide amine)s/DNA polyplexes at varying w/w ratios at the conditions of (A) without and (B) with DTT incubation (10.0 mM, 37°C, 1 h). Lane assignments correspond to polymer/DNA w/w ratios and represented as: lane 1 (0:1, plasmid DNA); lane 2 (bPEI (25kDa)/DNA 1:1); lane 3 (0.1:1); lane 4 (0.2:1); lane 5 (0.5:1); lane 6 (1:1); lane 7 (2:1); lane 8 (5:1); lane 9 (10:1); lane 10 (20:1); lane 11 (30:1).

Figure 4

Transfection efficiency of poly(disulfide amine)s/pCMV-Luc polyplexes at varying w/w ratios in two different cell lines. (A) HeLa cells: human cervical cancer cell line; (B) C2C12 cells: mouse myoblast cell line. Control: non-treated cells. The transfection efficiency of bPEI (25kDa)/DNA at optimal w/w ratio of 0.6:1 was measured for comparison. The w/w ratios of poly(disulfide amine)s/DNA were 1:1, 5:1, 10:1, 20:1, and 30:1. Transfection experiments were repeated three times and the results were expressed as the relative luminescent unit (RLU) of luciferase reporter gene expression normalized by the total cell protein content in each well as mean values ± standard deviations.

Figure 5

Relative cell viabilities of poly(disulfide amine)s/DNA polyplexes were measured as a function of w/w ratios in C2C12 cells. The polymer/DNA w/w ratios were set as 1:1, 5:1, 10:1, 20:1 and 30:1. The MTT assay of bPEI (25kDa)/DNA at the same w/w ratio was measured for comparison. Cytotoxicity was repeated three times and the data were represented as mean values ± standard deviations.

Figure 6

The cellular uptake of poly(disulfide amine)s/DNA polyplexes in C2C12 cells. Fluorescence histogram intensity correspond to polymer/DNA w/w ratios and represented as: closed grey peak (control, untreated cells); red line (1:1); green line (5:1); blue line (10:1); purple line (20:1); light blue line (30:1); M1 region (M1 gated fluorescent intensity).

Scheme 1

Synthesis of poly(disulfide amine)s using oligoamines and bisacryamide monomers ((poly(CBA-SP), poly(CBA-APPD), poly(CBA-APED), poly(CBA-AEPD) and poly(CBA-TETA)). The synthetic route of poly(CBA-SP) is shown as a representative procedure.