Bar graph displaying confluent ARPE-19 cell-sheet transfection efficacy (%GFP+ cells by FACS) of polymer formulations (n = 4) in our library screen.

<p>(A) Transfection efficacy of polymer library formulated at 30 w/w ratio. (B) Transfection efficacy of polymer library formulated at 60 w/w ratio. Optimal formulation B5-S5-E7 at 60 w/w resulted in 44% transfection efficacy as compared to 26% for Lipofectamine 2000, 22% for ExtremeGENE HP DNA, and 8% for branched 25 kDa PEI.</p

Schematic showing polymerization scheme and monomers used.

<p>(A) Diacrylates (“B”) were added to primary-amine containing amino-alcohol side chains (“S”) to form the base polymers. (B) Base polymers were end-capped with amine monomers (“E”) to form the final, end-modified polymers. (C) The base diacrylate (“B”), amino-alcohol side chain (“S”), and end-modifying amines (“E”) used in the polymer library are listed here. (D) The full structure of B5-S5-E7 (1-(3-aminopropyl)-4-methylpiperazine-end-modified poly(1,5 pentanediol diacrylate-co-5-amino-1-pentanol) is shown here.</p

Comparison of base polymer structure with reduction in metabolic activity.

<p>Each bar represents the average toxicity associated with the end-modified polymers that contained the base polymer shown (n = 11; error bar = SEM). Base diacrylate and side chain amino-alcohols are shown from least hydrophobic to most hydrophobic from left to right. (A) Reduction in metabolic activity of 30 w/w formulations averaged over 10 end-modified amines containing the base polymer shown. (B) Reduction in metabolic activity of 60 w/w formulations averaged over 10 end-modified amines containing the base polymer shown. For statistical analysis, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037543#pone-0037543-t001" target="_blank">Table 1</a>.</p

Number-averaged molecular weight versus time of B5-S5-E7 in PBS at 37°C with agitation.

<p>The half-life of the polymer in solution was 4.6 hr (R² = 0.984), and the polymer was almost completely degraded within 1 day.</p

Comparison of base polymer structure with transfection efficacy.

<p>Each bar represents the average transfection efficacy associated with the end-modified polymers that contained the base polymer shown (n = 11; error bar = SEM). Base diacrylate and side chain amino-alcohols are shown from least hydrophobic to most hydrophobic from left to right. (A) Transfection efficacy of 30 w/w formulations averaged over 11 end-modified amines containing the base polymer shown. (B) Transfection efficacy of 60 w/w formulations averaged over 11 end-modified amines containing the base polymer shown. For statistical analysis, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037543#pone-0037543-t001" target="_blank">Table 1</a>.</p

Reduction in metabolic activity following PBAE nanoparticle administration.

<p>Formulations plotted at 0% reduction of metabolic activity here had equivalent or slightly higher metabolic activity than untreated controls. (A) Reduction in metabolic activity post transfection with polymer library formulated at 30 w/w ratio (n = 4). (B) Reduction in metabolic activity post transfection with polymer library formulated at 60 w/w ratio (n = 4).</p

Results of 2-way ANOVA examining the effect of increased hydrophobicity of the side chain with respect to the base diacrylate it is paired with.

<p>NS is non-significant; P<0.05 is *; P<0.01 is **; P<0.001 is ***.</p

Confocal image of RPE/choroid flat mount post subretinal injections; green corresponds to fluorescence due to GFP expression.

<p>Both images were taken with the same camera settings. (A) pDNA alone. (B) pDNA/nanoparticle injection. (C) Relative transcript level to GAPDH (set at 10,000) of GFP mRNA expression after subretinal injection of PBAE eGFP nanoparticles and subretinal injection of naked DNA. Each injection is diplayed as a separate point, and the mean relative transcript level is displayed as a bar. Subretinal injections using lyophilized GFP-PBAE nanoparticles resulted in 1.1±1×10³-fold and 1.5±0.7×10³-fold increased GFP expression in the RPE/choroid and neural retina, respectively, compared to injection of DNA alone (p = 0.003 for RPE/choroid, p<0.001 for neural retina). (D) Relative fluorescence intensity of retinal flat mounts after subretinal injection with GFP nanoparticles and GFP plasmid alone (n = 3).</p

Biophysical characterization of nanoparticles before and after lyophilization.

<p>(A) Hydrodynamic diameter of freshly prepared DNA/B5-S5-E7 particles versus lyophilized particles (n = 3; bars are standard error). (B) Zeta potential of freshly prepared DNA/B5-S5-E7 particles versus lyophilized particles (n = 3; bars are standard error). Differences in particle size (p = 1) and zeta potential (p = 0.05) are not significant.</p

Comparison of transfection efficacy to reduction in metabolic activity of the polymers in the polymer library.

<p>There is an overall trend of increasing cytotoxicity with increasing transfection efficiency (the best-fit line represents a 0.77% decrease in cell metabolic activity with every 1% increase in transfection efficiency of the formulation) but the trend only explains a portion of the results (R² = 0.37). A number of polymer formulations exhibited high transfection efficiencies and low concomitant cytotoxicities.</p