Experimental and Computational Approaches to Probe the Mechanism of Shape Control in PEG – Polycation / DNA Micelle Assembly

SUMMARY This study aims to identify key parameters that govern the control of PEG–polycation/DNA micellar nanoparticle shape. Using a combined experimental and computational approach, we have identified several important factors, including polymer structure, solvent polarity, and pH during assembly, that allow DNA nanoparticles to adopt different shapes. INTRODUCTION A key challenge for realizing the full therapeutic potential of gene therapy is to control the in vivo transport properties and cellular uptake of nanoparticle-based gene carriers, thus improving the delivery efficiency. Several recent studies have reported that polycation-condensed DNA micelles can assume different shapes, and that shape may be an important factor influencing the DNA delivery efficiency [1,2]. We have previously shown that the shape of polyethylene glycol (PEG)-b-polyphosphoramidate (PPA)/DNA nanoparticles can be tuned by altering solvent polarity during micelle assembly [3]. In this study, we examine the effect of various structural parameters of the PEG-polycations (block and graft copolymers, PEG and polycation block lengths, PPA vs. PEI) and assembly conditions (solvent polarity and PEI charge density) on the shape of assembled DNA nanoparticles. EXPERIMENTAL METHODS PEG was grafted to linear polyethylenimine (lPEI) and PPA polycations at varying degrees. Nanoparticles were formed by pipetting equal volumes of polymer and DNA solution at an N/P ratio of 8, after which the mixture was incubated for 20 min at room temperature before characterization. Particle size and surface charge was characterized using a Malvern Zetasizer Nano ZS90. TEM characterization was carried out according to our published protocol [4]. Molecular dynamics simulations were performed of a semiflexible ring anionic polyelectrolyte and block copolymers comprised of a cationic block and a neutral block, accounting for changes in solvent polarity according to our published methods [3]. RESULTS AND DISCUSSION Similar to the trends observed for the block copolymer system [3], lPEI-g-PEG/DNA and PPA-g-PEG/DNA nanoparticles adopted a worm-like shape in water. When prepared in 4:6 (v/v) dimethylformamide (DMF)/water mixtures, particle shape transitioned to short rods. Further increase of the DMF fraction to 7:3 (v/v) led to a predominantly spherical shape with a small fraction of rods. Decreasing the pH of the particle preparation solution also affected the shape of the lPEI-g-PEG/DNA particles. When prepared at pH 4, particles primarily adopted a rod shape. Reduction of pH to 2.5 led to the formation of highly uniform, 70 nm spheres. A strong correlation between PEG grafting degree and nanoparticle shape was also observed in TEM analysis (Fig. 1). Particles prepared with a low PEG grafting degree adopted a compact spherical morphology, transitioning to rod-like and string-like particle at higher PEG grafting degrees. Particle shape was not noticeably affected by polycation molecular weight or choice of polycation (PPA vs. lPEI). Figure 1: TEM images of (A–C) lPEI9k-gPEG10k/DNA nanoparticles and (D–F) PPA12kg-PEG10k/DNA nanoparticles with 0.5% (A,D), 2% (B,E), and 3% (C,F) PEG grafting degrees . All scale bars represent 200 nm. From the modeling results, an interesting trend was observed when comparing the assembly of DNA nanoparticles with PPA-gPEG copolymers to PPA-b-PEG copolymers. Not only do grafted copolymers allow the same type of nanoparticle shape control as block copolymers with the same PEG block length, the simulations also indicate that grafted copolymers yield nanoparticles with more welldefined shapes, as demonstrated by the more distinct peaks shown in Fig. 2. For cell transfection studies, we have stabilized the shape of nanoparticles using reversible disulfide crosslinks to preserve the particle size and shape in aqueous media at neutral pH. To permit the ability of celland tissue-specific targeting, we have modified the terminal end of PEG, allowing the conjugation of thiol-containing small molecules, peptides, proteins, polymers, and imaging agents. Figure 2: Preliminary molecular dynamics simulation results comparing the effect of PPAgraft-PEG (blue curve) and PPA-block-PEG copolymer (red curve) on DNA micelle shape. CONCLUSION This study identifies the key assembly parameters controlling the shape of DNA nanoparticles, and provides insight into the crucial governing factors for shape control in complex core micelle assembly. This strategy can be applicable to other DNA micelle systems, and enables studies of shape-dependent transport of DNA/polycation nanoparticles in vivo, which can ultimately lead to improved nonviral nanocarriers for gene therapy