Development of a novel endosomolytic diblock copolymer for siRNA delivery

Abstract

The gene knockdown activity of small interfering RNA (siRNA) has led to their use as target validation tools and as potential therapeutics for a variety of diseases. The delivery of these double-stranded RNA macromolecules has proven to be challenging, however, and in many cases, is a barrier to their deployment. Here we report the development of a new diblock copolymer family that was designed to enhance the systemic and intracellular delivery of siRNA. These diblock copolymers were synthesized using the controlled reversible addition fragmentation chain transfer polymerization (RAFT) method and are composed of a positively-charged block of dimethylaminoethyl methacrylate (DMAEMA) to mediate siRNA condensation, and a second endosomal-releasing block composed of DMAEMA and propylacrylic acid (PAA) in roughly equimolar ratios, together with butyl methacylate (BMA). A related series of diblock compositions were characterized, with the cationic block kept constant, and with the ratio of DMAEMA and PAA to BMA varied. These carriers became sharply hemolytic at endosomal pH regimes, with increasing hemolytic activity seen as the percentage of BMA in the second block was systematically increased. The diblock copolymers condensed siRNA into 80-250 nm particles with slightly positive Zeta potentials. SiRNA-mediated knockdown of a model protein, namely glyceraldehyde 3-phosphate dehydrogenase (GAPDH), in HeLa cells generally followed the hemolytic activity trends, with the most hydrophobic second block (highest BMA content) exhibiting the best knockdown. This pH-responsive carrier designed to mediate endosomal release shows significant promise for the intracellular delivery of siRNA.

Figure 1

Hemolysis of (A) polymers as a function of pH at a concentration of 10 μg/ml and (B) polymer/siRNA complexes of polymers 5–7 at theoretical charge ratios of 1:1 and 4:1 (25 nM siRNA dose which corresponds to polymer concentrations of 0.67 and 2.64 μg/ml, respectively). Hemolytic activity is normalized relative to a positive control, 1% v/v Triton X-100, and the data represent a single experiment conducted in triplicate ± standard deviation.

Figure 2

HeLa cell internalization of FAM-labeled siRNA and polymer/siRNA complexes formed with polymers 4–7 at theoretical charge ratios of 4:1 (after 4 h). Data are from three independent experiments conducted in triplicate, with error bars representing standard error of the mean. Statistical significance was evaluated at a level of p < 0.05 with the following symbols indicating significance versus naked siRNA and P4, P5, P6, and P7 carriers, respectively: *, &, $, #, %.

Figure 3

HeLa cytotoxicity (A) and GAPDH knockdown (B) as a function of siRNA polymer carrier. HeLa cells were transfected with siRNA against GAPDH at 25 nM using polymer/siRNA complexes formulated at theoretical charge ratios of 4:1. (A) After 24 h, cell lysate was collected and assayed for lactate dehydrogenase, a measure of cell viability, and data is shown relative to untreated cells. (B) After 48 h, both protein (black) and mRNA levels (white) were examined using a GAPDH enzyme activity assay and RT-PCR, respectively; the data is shown relative to cells receiving no treatment. Data are from three independent experiments conducted in triplicate with error bars representing standard deviation. Statistical significance was evaluated at a level of p < 0.05 with the following symbols indicating significance versus P1, P2, P3, P4, P5, P6, and P7 carriers, respectively: *, &, $, #, %, @, !.

Figure 4

GAPDH knockdown in HeLa cells was measured using real time RT-PCR 48 h after treatment with complexes as a function of charge ratio (1:1–8:1) (A) and siRNA dose (1–50 nM) (B) with polymer 7 as the carrier at 4:1 charge ratios. Negative control siRNA #1 (Ambion) and a commercially available transfection reagent, HiPerFect (Qiagen), were used as negative and positive controls, respectively. For A, statistical significance was evaluated at a level of p < 0.05 with the following symbols indicating significance versus 1:1, 2:1, 4:1, 8:1, 8:1 (Control), and HiPerFect treatments, respectively: *, &, $, #, %. For B, statistical significance was evaluated at a level of p < 0.05 with the following symbols indicating significance versus 1 nM, 5 nM, 10 nM, 25 nM, 50 nM, and 50 nM (HiPerFect) treatments, respectively: *, &, $, #, %, @.

Scheme 1

RAFT-mediated synthesis of diblock copolymers consisting of a cationic poly(dimethylaminoethyl methacrylate) (DMAEMA, x=58) block and an endosomolytic polyampholyte block incorporating DMAEMA and propylacrylic acid (PAA) in equimolar ratios, and butyl methacrylate (BMA) (y~70).