Enhancing pDNA Delivery with Hydroquinine Polymers by Modulating Structure and Composition

Abstract

Quinine is a promising natural product building block for polymer-based nucleic acid delivery vehicles as its structure enables DNA binding through both intercalation and electrostatic interactions. However, studies exploring the potential of quinine-based polymers for nucleic acid delivery applications (transfection) are limited. In this work, we used a hydroquinine-functionalized monomer, HQ, with 2-hydroxyethyl acrylate to create a family of seven polymers (HQ-X, X = mole percentage of HQ), with mole percentages of HQ ranging from 12 to 100%. We developed a flow cytometer-based assay for studying the polymer-pDNA complexes (polyplex particles) directly and demonstrate that polymer composition and monomer structure influence polyplex characteristics such as the pDNA loading and the extent of adsorption of serum proteins on polyplex particles. Biological delivery experiments revealed that maximum transgene expression, outperforming commercial controls, was achieved with HQ-25 and HQ-35 as these two variants sustained gene expression over 96 h. HQ-44, HQ-60, and HQ-100 were not successful in inducing transgene expression, despite being able to deliver pDNA into the cells, highlighting that the release of pDNA is likely the bottleneck in transfection for polymers with higher HQ content. Using confocal imaging, we quantified the extent of colocalization between pDNA and lysosomes, proving the remarkable endosomal escape capabilities of the HQ-X polymers. Overall, this study demonstrates the advantages of HQ-X polymers as well as provides guiding principles for improving the monomer structure and polymer composition, supporting the development of the next generation of polymer-based nucleic acid delivery vehicles harnessing the power of natural products.

Figure 1

(A) Synthesis scheme and chemical structure of the QCR. (B) Synthesis scheme for the HQ monomer and copolymerization of HQ with HEA using the controlled polymerization method of RAFT.

Figure 2

(A) General scheme for the dye exclusion assay. Intercalation of PicoGreen in pDNA results in bright green fluorescence. Binding of polymer with pDNA and the subsequent compaction leads to exclusion of PicoGreen from the pDNA resulting in decrease in fluorescence intensity. (B) Normalized fluorescence intensities from the dye exclusion assay. For all N/P ratios, higher mole percentage of HQ in polymer leads to stronger binding and compaction of pDNA, as indicated by the gradual decrease in the fluorescence intensity.

Figure 3

Flow cytometric analysis of aggregated polyplex particles. (A) Scheme for forming fluorescently labeled aggregated polyplexes using HQ-X polymers and Cy5-labeled pDNA. The influence of the polymer composition on protein adsorption on the aggregated polyplexes was studied by incubating the aggregated polyplexes with either FBS or Alexa Fluor 488-labeled bovine serum albumin (BSA-AF488). (B) Flow cytometry scatter plots of the aggregated polyplexes formed using different polymers. Cy5 intensity is on the Y-axis, and Alexa Fluor 488 intensity is on the X-axis. (C) Geometric mean fluorescence intensity of Cy5 from the aggregated polyplexes before and after incubation with FBS. Aggregated polyplexes of QCR have higher pDNA loading compared to HQ-12, HQ-17, and even HQ-25. While HQ-X polymers have lower loading capacity for pDNA, their polyplexes were found to be more resistant to protein-mediated payload unpackaging compared to QCR. Statistical significance was evaluated using two-way ANOVA followed by Šídák’s multiple comparisons test (***p ≤ 0.001). (D) Geometric mean fluorescence intensity of Alexa Fluor 488 from the aggregated polyplexes. HQ-X polymers show significantly low protein adsorption compared to QCR. Statistical significance was evaluated using one-way ANOVA followed by Dunnett’s multiple comparisons test (***p ≤ 0.001).

Figure 4

(A) Representative widefield fluorescence images of HEK293T cells, 48 h after transfection with pZsGreen1-N1 plasmid, N/P = 16 (scale bar = 200 μm). Upon successful transfection, the cells produce ZsGreen1 protein that has green fluorescence. (B) Transfection efficiency assessed by quantifying the percentage of live HEK293T cells that are ZsGreen1+, via flow cytometry. Data are the mean of three replicates ± standard deviation. Cells transfected using HQ-44, HQ-60, and HQ-100 did not show green fluorescence even at the 96 h time point.

Figure 5

(A) Cellular uptake of the polyplexes, measured with flow cytometry, based on the fluorescence intensity of HQ-X observed from the cells. Data are the mean of three replicates ± standard deviation. (B) Comparison of fluorescence intensities of HQ-X polymers in 0.05 M acetic acid in water. The amount of polymers in the solution was adjusted for each composition to have [HQ] = 1 mM for all polymers. At lower mole percentages of HQ, the HQ fluorescence can be used to directly quantify the cellular uptake without additional fluorescent tags. However, at higher percentages, self-quenching of HQ fluorescence leads to higher number of false negatives for HQ+ live cells.

Figure 6

Cellular uptake of the polyplexes, measured with flow cytometry, based on the fluorescence intensity of Cy5 (tagged to the pDNA) observed from the cells. Data are the mean of three replicates ± standard deviation.

Figure 7

Top: representative 3D images (left) and contour surface rendering of the 3D confocal images (right) of HEK293T cells, 24 h after transfection, obtained using confocal laser scanning microscopy. Cy5-labeled pDNA was used as the payload at N/P = 16 (outside cytoplasm: gray, inside cytoplasm: yellow). Lysosomes (red) were stained using an anti-LAMP primary antibody and an Alexa Fluor 555-labeled secondary antibody. Nucleus was stained with DAPI (blue). Fluorescence of ZsGreen1 was used as the cytosolic stain (green). For untreated control, HQ-44, and HQ-60, cell cytoplasm was visualized by staining the actin filaments using Alexa Fluor 488-labeled phalloidin. Pearson’s correlation coefficients (PCCs) for colocalization between pDNA and lysosome are mentioned at the top of the respective images. The contour surface renderings were constructed to evaluate distance between nucleus and the Cy5-labeled pDNA present inside the cells. Scale bar 10 μm.