Differentially Branched Ester Amine Quadpolymers with Amphiphilic and pH-Sensitive Properties for Efficient Plasmid DNA Delivery

Abstract

Development of highly effective nonviral gene delivery vectors for transfection of diverse cell populations remains a challenge despite utilization of both rational and combinatorial driven approaches to nanoparticle engineering. In this work, multifunctional polyesters are synthesized with well-defined branching structures via A2 + B2/B3 + C1 Michael addition reactions from small molecule acrylate and amine monomers and then end-capped with amine-containing small molecules to assess the influence of polymer branching structure on transfection. These Branched poly(Ester Amine) Quadpolymers (BEAQs) are highly effective for delivery of plasmid DNA to retinal pigment epithelial cells and demonstrate multiple improvements over previously reported leading linear poly(beta-amino ester)s, particularly for volume-limited applications where improved efficiency is required. BEAQs with moderate degrees of branching are demonstrated to be optimal for delivery under high serum conditions and low nanoparticle doses further relevant for therapeutic gene delivery applications. Defined structural properties of each polymer in the series, including tertiary amine content, correlated with cellular transfection efficacy and viability. Trends that can be applied to the rational design of future generations of biodegradable polymers are elucidated.

Keywords: branched polymer, plasmid DNA; nonviral, gene delivery; polymeric nanoparticle; transfection.

Figure 1.

Synthesis of Branched poly(Ester Amine) Quadpolymers (BEAQ). A) Diacrylate monomer B7 and triacrylate monomer B8 were mixed with side-chain monomer S4 to synthesize a series of BEAQs with increasing triacrylate mole fraction and degree of branching. B) Linear polymers possess two end-cap structures per molecule (red), while each triacrylate monomer in branched polymers results in an additional end-cap moiety for every branch point. C) One-pot synthesis of acrylate terminated base polymers was performed at 90°C and 200 mg/mL in DMF for 24 hours. Polymers were then end-capped with monomer E6 at room temperature for one hour to yield the final product.

Figure 2.

Polymer properties influenced by triacrylate mole fraction. A) Predicted properties of partition coefficient (logP) and distribution coefficient (logD) for differentially branched polyesters. B) Titration of BEAQs. C) Effective pKa value of maximum buffering point between pH 4.5–8.5 of differentially branched PBAEs. D) Competition binding assay of polymer and Yo-Pro-1 iodide at low pH. (n=3 wells, mean ± SEM) E) DNA binding in low pH buffer normalized to PBAE tertiary amine content. F) Difference in degree of binding between pH 5 and pH 7.4 calculated as a function of total amines per bp DNA. G) Competition DNA binding assay in isotonic, neutral buffer. (n=3 wells, mean ± SEM) H) DNA binding in isotonic, neutral buffer normalized to secondary amine content. I) TEM image of 20% triacrylate mole fraction polymer nanoparticles.

Figure 3.

In vitro transfection with BEAQs in 10% serum media. HEK239T cells A) percent transfection efficacy, B) normalized geometric mean expression C) viability and D) fluorescence micrograph of cells transfected to express eGFP with the 20 w/w ratio, 50% triacrylate mole-fraction BEAQ. ARPE-19 cells E) percent transfection efficacy, F) normalized geometric mean expression, G) viability and H) fluorescence micrograph of cells transfected to express eGFP with 20 w/w, B8–20% triacrylate mole-fraction BEAQ. (Scale bars 200 μm. n = 4 wells, mean ± SEM).

Figure 4.

The effect of transfection conditions on BEAQs. High serum (50%) transfection of A) HEK293T and B) ARPE-19 cells with 20 w/w nanoparticles (normal DNA dose of 600 ng/well in 96 well plates). Low nanoparticle dose transfection with 40 w/w nanoparticles of C) HEK293T (5 ng) and D) ARPE-19 (10 ng) doses in 384 well plates. (n = 4 wells, mean ± SEM, statistical markings show results of one-way ANOVA with Dunnett corrected multiple comparisons tests to the linear polymer)

Figure 5.

Lysosome colocalization assessment with confocal microscopy. A) Cells transfected with B8–0% and B8–50% at low (20 w/w) and high (40 w/w) nanoparticles and assessed by confocal microscopy show statistically significant differences in the degree of lysosome colocalization. Assessed by one-way ANOVA with Dunnett corrected multiple comparisons to the B8–50%: 40 w/w conditions. B) Representative 2D scattergrams of HEK293T cells at 24 hours post-treatment using 20 w/w nanoparticles. Region 3 represents colocalized pixel intensities. C) All conditions in both cell lines showed statistically significant (Holm-Sidak corrected multiple t-tests) increases in the degree of lysosome colocalization between 4 hours and 24 hours following transfection. (Bars show mean ± SEM of n>100 cells.) D) Representative maximum intensity projection images of cells transfected with 20 w/w nanoparticles 24 hours following transfection, showing lysosome colocalization in white.

Figure 6.

Nuclear localization of plasmid DNA and expression of eGFP assessed by confocal microscopy. HEK293T cells were transfected 24 hours prior with B8–50%: 20 w/w nanoparticles containing 80% non-coding, Cy5-labeled plasmid DNA and 20% coding eGFP-N1 plasmid DNA. A) Maximum intensity projection demonstrating high level of labeled plasmid DNA remaining in the cells with minimal lysosome colocalization. B) Strong eGFP expression from the 20% of unlabeled plasmid DNA. C) A single z-slice shows Cy5-labeled plasmid DNA localized to the nucleus in select cells (white arrows).

Figure 7.

Correlation between BEAQ properties and viability normalized geometric mean expression. Geometric mean expression plots were normalized to the maximum expression for each polymer and scaled by viability at that w/w ratio for A-E) HEK293T cells and F-J) ARPE-19 cells. Dashed-grey curves show a single quadratic fit of all data points for that cell line with calculated R². Plots showing dotted-grey curves in addition to dashed-grey curves were statistically determined to require two fitted quadratic curves to adequately describe the data.