Differential Polymer Structure Tunes Mechanism of Cellular Uptake and Transfection Routes of Poly(β-amino ester) Polyplexes in Human Breast Cancer Cells

Successful gene delivery with nonviral particles has several barriers, including cellular uptake, endosomal escape, and nuclear transport. Understanding the mechanisms behind these steps is critical to enhancing the effectiveness of gene delivery. Polyplexes formed with poly­(β-amino ester)­s (PBAEs) have been shown to effectively transfer DNA to various cell types, but the mechanism of their cellular uptake has not been identified. This is the first study to evaluate the uptake mechanism of PBAE polyplexes and the dependence of cellular uptake on the end group and molecular weight of the polymer. We synthesized three different analogues of PBAEs with the same base polymer poly­(1,4-butanediol diacrylate-<i>co</i>-4-amino-1-butanol) (B4S4) but with small changes in the end group or molecular weight. We quantified the uptake and transfection efficiencies of the pDNA polyplexes formulated from these polymers in hard-to-transfect triple negative human breast cancer cells (MDA-MB 231). All polymers formed positively charged (10–17 mV) nanoparticles of ∼200 nm in size. Cellular internalization of all three formulations was inhibited the most (60–90% decrease in cellular uptake) by blocking caveolae-mediated endocytosis. Greater inhibition was shown with polymers that had a 1-(3-aminopropyl)-4-methylpiperazine end group (E7) than the others with a 2-(3-aminopropylamino)-ethanol end group (E6) or higher molecular weight. However, caveolae-mediated endocytosis was generally not as efficient as clathrin-mediated endocytosis in leading to transfection. These findings indicate that PBAE polyplexes can be used to transfect triple negative human breast cancer cells and that small changes to the same base polymer can modulate their cellular uptake and transfection routes

Uptake and Transfection with Polymeric Nanoparticles Are Dependent on Polymer End-Group Structure, but Largely Independent of Nanoparticle Physical and Chemical Properties

Development of nonviral particles for gene delivery requires a greater understanding of the properties that enable gene delivery particles to overcome the numerous barriers to intracellular DNA delivery. Linear poly­(beta-amino) esters (PBAE) have shown substantial promise for gene delivery, but the mechanism behind their effectiveness is not well quantified with respect to these barriers. In this study, we synthesized, characterized, and evaluated for gene delivery an array of linear PBAEs that differed by small changes along the backbone, side chain, and end group of the polymers. We examined particle size and surface charge, polymer molecular weight, polymer degradation rate, buffering capacity, cellular uptake, transfection, and cytotoxicity of nanoparticles formulated with these polymers. Significantly, this is the first study that has quantified how small differential structural changes to polymers of this class modulate buffering capacity and polymer degradation rate and relates these findings to gene delivery efficacy. All polymers formed positively charged (zeta potential 21–29 mV) nanosized particles (∼150 nm). The polymers hydrolytically degraded quickly in physiological conditions, with half-lives ranging from 90 min to 6 h depending on polymer structure. The PBAE buffering capacities in the relevant pH range (pH 5.1–7.4) varied from 34% to 95% protonatable amines, and on a per mass basis, PBAEs buffered 1.4–4.6 mmol of H⁺/g. When compared to 25 kDa branched polyethyleneimine (PEI), PBAEs buffer significantly fewer protons/mass, as PEI buffers 6.2 mmol of H⁺/g over the same range. However, due to the relatively low cytotoxicity of PBAEs, higher polymer mass can be used to form particles than with PEI and total buffering capacity of PBAE-based particles significantly exceeds that of PEI. Uptake into COS-7 cells ranged from 0% to 95% of cells and transfection ranged from 0% to 93% of cells, depending on the base polymer structure and the end modifications examined. Five polymers achieved higher uptake and transfection efficacy with less toxicity than branched-PEI control. Surprisingly, acrylate-terminated base polymers were dramatically less efficacious than their end-capped versions, in terms of both uptake (1–3% for acrylate, 75–94% for end-capped) and transfection efficacy (0–1% vs 20–89%), even though there are minimal differences between acrylate and end-capped polymers in terms of DNA retardation in gel electrophoresis, particle size, zeta potential, and cytotoxicity. These studies further elucidate the role of polymer structure for gene delivery and highlight that small molecule end-group modification of a linear polymer can be critical for cellular uptake in a manner that is largely independent of polymer/DNA binding, particle size, and particle surface charge

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

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

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

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

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

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

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