Reducible HPMA-co-oligolysine copolymers for nucleic acid delivery

Abstract

Biodegradability can be incorporated into cationic polymers via use of disulfide linkages that are degraded in the reducing environment of the cell cytosol. In this work, N-(2-hydroxypropyl)methacrylamide (HPMA) and methacrylamido-functionalized oligo-l-lysine peptide monomers with either a non-reducible 6-aminohexanoic acid (AHX) linker or a reducible 3-[(2-aminoethyl)dithiol] propionic acid (AEDP) linker were copolymerized via reversible addition-fragmentation chain transfer (RAFT) polymerization. Both of the copolymers and a 1:1 (w/w) mixture of copolymers with reducible and non-reducible peptides were complexed with DNA to form polyplexes. The polyplexes were tested for salt stability, transfection efficiency, and cytotoxicity. The HPMA-oligolysine copolymer containing the reducible AEDP linkers was less efficient at transfection than the non-reducible polymer and was prone to flocculation in saline and serum-containing conditions, but was also not cytotoxic at charge ratios tested. Optimal transfection efficiency and toxicity were attained with mixed formulation of copolymers. Flow cytometry uptake studies indicated that blocking extracellular thiols did not restore transfection efficiency and that the decreased transfection of the reducible polyplex is therefore not primarily caused by extracellular polymer reduction by free thiols. The decrease in transfection efficiency of the reducible polymers could be partially mitigated by the addition of low concentrations of EDTA to prevent metal-catalyzed oxidation of reduced polymers.

Fig. 1

Degradation of HPMA-AedpK₁₀ with TCEP. HPMA-AedpK₁₀ was degraded over time in the presence of 25 mM TCEP. Reduced copolymers were applied to a GPC column to track changes in molecular weight at degradation of the copolymer occurred. (a) Reduction was done in the presence of EDTA. The insert indicates the mass fraction of degraded copolymer and free oligolysine. Degraded copolymer eluted between 14 to 17 min, while free oligolysine eluted from 18 to 20 min. The insert shows the mass fraction of peptide and copolymer. Reduction done without EDTA (b) produced three eluted peaks in SEC. The first is a high molecular weight fraction (9 to 12 min), the second peak is degraded copolymer (14 to 17 min), and the third is oligolysine (18 to 20 min).

Fig. 2

Particle sizing of polyplexes by dynamic light scattering (DLS). The effective diameter of polyplexes formulated with HPMA-AhxK₁₀, HPMA-AedpK₁₀, or a 1:1 (w/w) mixture was determined by DLS in water, PBS with an ionic strength of 150 mM, and DMEM medium supplemented with 10% FBS. Data are presented as mean ± S.D., n = 3.

Fig. 3

Polyplex stability in serum via gel retardation assay. Polyplexes complexed with YOYO-1-labeled plasmid DNA and either HPMA-AedpK₁₀, HPMA-AhxK₁₀, or HPMA-Ahx(d)K₁₀ were incubated with various concentrations of serum (50%, 25%, 10%). Free DNA (plasmid DNA not complexed with polymer) was used as a control.

Fig. 4

(a) Transfection efficiency of HPMA copolymers in HeLa, NIH/3T3, and CHO-K1. Polyplexes (N/P 5) of copolymer and luciferase-encoding plasmid DNA were incubated with cells for 4 h in serum-free conditions. Luciferase activity was measured 48 h after transfection and normalized to total protein content in each sample. Cells: untreated controls; bPEI: branched polyethylenimine (25 kD); PLL: poly-L-lysine (12–24 kD); Mixed copolymers: 1:1 (v/v) mixture of HPMA-AhxK₁₀ and HPMA-AedpK₁₀. (b) Polyplex cytotoxicity in HeLa, NIH/3T3, and CHO-K1. Toxicity of the polyplexes was determined by measuring total protein content and designating untreated cells as 100% viable. Data are presented as mean ± S.D., n = 4, (*) p < 0.05, as determined by two-tailed Student’s t-test.

Fig. 5

Uptake of polyplexes in HeLa cells. Luciferase plasmid DNA was labeled with TOTO-3 prior to complexation with HPMA copolymers (HPMA-AhxK₁₀, HPMA-AedpK₁₀, or a 1:1 (w/w) mixture). HeLa cells pretreated with or without 5 mM DTNB in serum-free media for 1 h prior to transfection and then incubated with polyplexes for 30 min in serum-free media with or without 5 mM DTNB. Polyplex uptake was assessed by flow cytometry. DTNB stands for 5,5′-dithiobis-(2-nitrobenzoic acid). Data are presented as mean ± S.D., n = 3.

Fig. 6

Transfection efficiency of HPMA copolymers in HeLa, CHO-K1, and NIH/3T3 in the presence of 1 mM EDTA. Polyplexes (N/P 5) of copolymer and luciferase-encoding plasmid DNA were incubated with cells for 4 h with 1 mM EDTA in serum-free conditions. Luciferase activity was measured 48 h after transfection and normalized to total protein content in each sample. Untreated: untreated controls; Mixed copolymers: 1:1 (w/w) mixture of HPMA-AhxK₁₀ and HPMA-AedpK₁₀. Data are presented as mean ± S.D., n = 3, (*) p < 0.05, as determined by two-tailed Student’s t-test.

Scheme 1

Synthesis of reducible HPMA-co-oligolysine copolymers via reversible-addition fragmentation chain transfer (RAFT) polymerization. Statistical polymers of HPMA and oligolysine were synthesized via RAFT polymerization using ECT as the chain transfer agent and VA-044 as the initiator. A CTA to I ratio of 10 and a degree of polymerization (DP) of 190 was used in all polymerizations. A non-reducible (Ahx) or a reducible (Aedp) linker was used in the synthesis of the oligolysine (K₁₀) peptide in order to introduce biodegradability into the polymer. Peptide monomers were functionalized with a methacrylamido group (Ma) for polymerization.