Engineering biodegradable and multifunctional peptide-based polymers for gene delivery

Abstract

The complex nature of in vivo gene transfer establishes the need for multifunctional delivery vectors capable of meeting these challenges. An additional consideration for clinical translation of synthetic delivery formulations is reproducibility and scale-up of materials. In this review, we summarize our work over the last five years in developing a modular approach for synthesizing peptide-based polymers. In these materials, bioactive peptides that address various barriers to gene delivery are copolymerized with a hydrophilic backbone of N-(2-hydroxypropyl)methacrylamide (HPMA) using reversible-addition fragmentation chain-transfer (RAFT) polymerization. We demonstrate that this synthetic approach results in well-defined, narrowly-disperse polymers with controllable composition and molecular weight. To date, we have investigated the effectiveness of various bioactive peptides for DNA condensation, endosomal escape, cell targeting, and degradability on gene transfer, as well as the impact of multivalency and polymer architecture on peptide bioactivity.

Figure 1

Schematic of peptide-functionalized HPMA copolymers. (A) Random-statistical copolymers of HPMA (red) and pendant bioactive peptides (green) with a linker (blue) between the hydrophilic backbone and the main peptide sequence. (B) Random-statistical copolymers of HPMA and multiple pendant peptides (cationic peptide in green; second bioactive peptide in purple). (C) Diblock copolymer of HPMA and multiple pendant peptides, where the cationic peptide is on one block, and a second bioactive peptide on a different block.

Figure 2

Synthesis of statistical HPMA-oligolysine copolymers by RAFT polymerization. HPMA and methacrylamido-functionalized oligolysine peptides are copolymerized under aqueous RAFT conditions using ethyl cyanovaleric trithiocarbonate (ECT) as the chain transfer agent (CTA) [71] and VA-044 as the initiator. The 6-carbon aminohexanoic acid (Ahx) is used as a linker between the pendant oligolysine peptide and the hydrophilic backbone.

Figure 3

Synthesis of diblock HPMA copolymers containing multiple pendant peptides. RAFT polymerization of HPMA and PDSMA (molecule 1), and then chain-extension with HPMA and oligo(l-lysine) to form the second block (molecule 2). Disulfide exchange between the pyridyl disulfide on 2 and cysteine-functionalized oligo(l-histidine) peptides yields a diblock polymer with two bioactive peptides (molecule 3).

Figure 4

Transfection of stearic acid (SA)-modified p[HPMA-co-K₁₀] polyplexes in HeLa cells. HeLa cells were treated with polyplexes (containing 1 μg plasmid DNA) at charge ratios (N/P) of 3, 5, and 10 for 4 h in serum-free conditions, washed, and replenished with complete media. At 48 h post-transfection, cell lysates were assessed for (A) luciferase reporter gene expression and (B) protein content as an indicator for cytotoxicity. Data are presented as the mean ± S.D., n = 3.

Figure 5

Comparative transfection of p[HPMA-co-K₁₀] polymers (“pHK10”) modified with various endosomal escape modalities in HeLa cells. HeLa cells (3 × 10⁴) were treated with polyplexes (containing 1 μg plasmid DNA) at charge ratios (N/P) of 3, 4, and 5 for 4 h in serum-free conditions, washed, and replenished with complete media. At 48 h post-transfection, cell lysates were assessed for (A) luciferase reporter gene expression and (B) protein content as an indicator for cytotoxicity. pHK10 unmodified: p[HPMA-co-K₁₀]; pHK10 + histidine: p[HPMA-co-K₁₀-co-K₅H₅] [55]; pHK10 mix + 5% melittin: a mixture of 95:5% (v/v) p[HPMA-co-K₁₀]: p[HPMA-co-melittin]-b-[HPMA-co-K₁₀] [56]; pHK10 mix + 15% melittin: a mixture of 85:15% (v/v) p[HPMA-co-K₁₀]: p[HPMA-co-melittin]-b-[HPMA-co-K₁₀]; pHK10 + melittin: p[HPMA-co-melittin]-b-[HPMA-co-K₁₀]; pHK10 + st-sHGP: p[HPMA-co-K₁₀-co-sHGP]; pHK10 + b-sHGP: p[HPMA-co-sHGP]-b-[HPMA-co-K₁₀]. Data are presented as the mean ± S.D., n = 3.