Multifunctional triblock copolymers for intracellular messenger RNA delivery

Abstract

Messenger RNA (mRNA) is a promising alternative to plasmid DNA (pDNA) for gene vaccination applications, but safe and effective delivery systems are rare. Reversible addition-fragmentation chain transfer (RAFT) polymerization was employed to synthesize a series of triblock copolymers designed to enhance the intracellular delivery of mRNA. These materials are composed of a cationic dimethylaminoethyl methacrylate (DMAEMA) segment to mediate mRNA condensation, a hydrophilic poly(ethylene glycol) methyl ether methacrylate (PEGMA) segment to enhance stability and biocompatibility, and a pH-responsive endosomolytic copolymer of diethylaminoethyl methacrylate (DEAEMA) and butyl methacrylate (BMA) designed to facilitate cytosolic entry. The blocking order and PEGMA segment length were systematically varied to investigate the effect of different polymer architectures on mRNA delivery efficacy. These polymers were monodisperse, exhibited pH-dependent hemolytic activity, and condensed mRNA into 86-216 nm particles. mRNA polyplexes formed from polymers with the PEGMA segment in the center of the polymer chain displayed the greatest stability to heparin displacement and were associated with the highest transfection efficiencies in two immune cell lines, RAW 264.7 macrophages (77%) and DC2.4 dendritic cells (50%). Transfected DC2.4 cells were shown to be capable of subsequently activating antigen-specific T cells, demonstrating the potential of these multifunctional triblock copolymers for mRNA-based vaccination strategies.

Figure 1

Representative molecular weight distributions obtained by gel permeation chromatography for intermediate macroCTAs and final triblock copolymer (DPE1) synthesized via RAFT polymerization.

Figure 2

Representative ¹H-NMR (CDCl₃) spectrum of a triblock copolymer (DPE1). Monomer compositions were determined by integration of peaks i, vi, xxi, and (iii, x, xvi, xxii).

Figure 3

Hemolytic activity of polymers at 20 μg/mL mass concentration of the DEAEMA-co-BMA block. Polymers were incubated with red blood cells at the indicated pH values for 1 h at 37 °C. Hemolytic activity was quantified based on absorbance measurements for hemoglobin release. Activities are normalized relative to a Triton X-100 positive control and DPBS negative control and are from a single representative experiment conducted in triplicate ± standard deviation.

Figure 4

mRNA polyplex stability against heparin displacement. Polyplexes were incubated with equal volumes of heparin solutions at the indicated concentrations for 15 min at room temperature prior to agarose gel electrophoresis.

Figure 5

In vitro transfection efficiencies in (A) RAW 264.7 macrophages and (B) DC2.4 dendritic cells. Lipofectamine 2000 (LF) was used as a positive control. Cells were treated with mRNA polyplexes for 4 h, then analyzed by flow cytometry for GFP expression. Data are from a single representative experiment conducted in triplicate ± standard deviation. In (A), DPE2 and DPE3 are significantly different than all other treatments and each other (p < 0.05). In (B), DPE1, DPE2, and DPE3 are significantly different than all other treatments, and DPE3 is significantly different than DPE1 (p < 0.05). (C) Fluorescence micrograph of GFP expression in RAW 264.7 macrophages (blue, Hoechst nuclear stain) 4 h post-transfection. Scale bar is 50 μm.

Figure 6

In vitro cytotoxicity of polyplexes in RAW 264.7 macrophages. Cells were treated with mRNA polyplexes for 24 h prior to cell viability analysis via alamarBlue (Invitrogen) assay. Viabilities are normalized relative to untreated cells. Data are from a single representative experiment conducted in triplicate ± standard deviation.

Figure 7

B3Z T cell activation by transfected DC2.4 dendritic cells. DC2.4 cells were treated with mRNA polyplexes encoding ovalbumin for 4 h, then cocultured with B3Z T cells for 24 h. T cell activation was then quantified based on β-galactosidase activity. Responses are normalized relative to cells treated with 0.1 μM SIINFEKL peptide as a positive control and are from a single representative experiment conducted in triplicate ± standard deviation. DPE1, DPE2, and DPE3 are significantly different than LF, and DPE1 is significantly different than all other treatments (p < 0.05).

Scheme 1

RAFT-mediated synthesis of a D-P-E triblock copolymer composed of discrete cationic DMAEMA, hydrophilic PEGMA, and endosomolytic DEAEMA-co-BMA segments.