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. 2020 Dec 29;117(52):32919-32928.
doi: 10.1073/pnas.2016860117. Epub 2020 Dec 14.

Quinine copolymer reporters promote efficient intracellular DNA delivery and illuminate a protein-induced unpackaging mechanism

Craig Van Bruggen  1 , David Punihaole  1 , Allison R Keith  2 , Andrew J Schmitz  1 , Jakub Tolar  2 , Renee R Frontiera  3 , Theresa M Reineke  3
Affiliations

Quinine copolymer reporters promote efficient intracellular DNA delivery and illuminate a protein-induced unpackaging mechanism

Craig Van Bruggen et al. Proc Natl Acad Sci U S A. .

Abstract

Polymeric vehicles that efficiently package and controllably release nucleic acids enable the development of safer and more efficacious strategies in genetic and polynucleotide therapies. Developing delivery platforms that endogenously monitor the molecular interactions, which facilitate binding and release of nucleic acids in cells, would aid in the rational design of more effective vectors for clinical applications. Here, we report the facile synthesis of a copolymer containing quinine and 2-hydroxyethyl acrylate that effectively compacts plasmid DNA (pDNA) through electrostatic binding and intercalation. This polymer system poly(quinine-co-HEA) packages pDNA and shows exceptional cellular internalization, transgene expression, and low cytotoxicity compared to commercial controls for several human cell lines, including HeLa, HEK 293T, K562, and keratinocytes (N/TERTs). Using quinine as an endogenous reporter for pDNA intercalation, Raman imaging revealed that proteins inside cells facilitate the unpackaging of polymer-DNA complexes (polyplexes) and the release of their cargo. Our work showcases the ability of this quinine copolymer reporter to not only facilitate effective gene delivery but also enable diagnostic monitoring of polymer-pDNA binding interactions on the molecular scale via Raman imaging. The use of Raman chemical imaging in the field of gene delivery yields unprecedented insight into the unpackaging behavior of polyplexes in cells and provides a methodology to assess and design more efficient delivery vehicles for gene-based therapies.

Keywords: biomaterial; drug delivery; gene editing; gene therapy; polymer.

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Conflict of interest statement

Competing interest statement: A US Provisional Application (62/536,427) on this work has been filed.

Figures

Fig. 1.
Fig. 1.
Mechanisms underlying unique plasmid binding and release mechanisms of QCRs. (A) Poly(quinine-co-HEA) self-assembles with pDNA at low pH (3–4) to form polyplexes and compacts the plasmid (B) via electrostatic forces as well as binding via π-stacking interactions. (C) Dilution in cell media causes aggregation of the polyplexes, which promotes sedimentation of plasmid to the cell surface. The fluorescent particles (λex = 350 nm, λem = 450 nm) are uptaken (D), in part, by macropinocytosis. (E) Raman imaging is used to quantify release of pDNA by exposure to intracellular proteins.
Fig. 2.
Fig. 2.
Synthesis and characterization of QCRs and controls. (A) Free-radical copolymerization scheme used in this study to incorporate quinine, consisting of a bicyclic quinuclidine head group and fluorescent quinoline ring (λex = 350 nm, λem = 450 nm), into copolymers with comonomers such as HEA (blue). (B) Structural properties of polymers used in biological studies as determined by 1H NMR and SEC. (C) Quinine was copolymerized with various acrylamides including HEAm, and HEA was copolymerized with canonical cationic comonomers such as DMAEMA.
Fig. 3.
Fig. 3.
Characterization of QCR–DNA binding. (A) Dye-exclusion assay monitors pDNA compaction in polyplexes (N/P = 6) formed in acidic aqueous media (pH 3 to 4). The fluorescence of PicoGreen intercalation in polyplexes is normalized to the fluorescence from free pDNA. (B) The polyplex solution in A is diluted first in cell media (DMEM) followed by addition of FBS (10% vol/vol). The change in relative PicoGreen fluorescence upon sequential addition of solutions is used to calculate the percent decrease in compact pDNA between steps. (C) (i) Raman spectrum of unbound poly(quinine-co-HEA) polymer under acidic solution conditions and (ii) difference spectrum highlighting the spectral shift that occurs in the quinoline ring mode of poly(quinine-co-HEA) due to DNA intercalation. The difference in the quinoline ring mode frequency (3 cm−1) between intercalated and deintercalated quinine can be used to monitor poly(quinine-co-HEA) polyplex unpackaging. (D) Schematic of polyplex aggregation in serum-free DMEM prior to exposure to cells (defined as formulation time). (E) Plot showing the hydrodynamic diameter of poly(quinine-co-HEA) polyplexes (N/P = 8) over time after addition of serum-free DMEM. Data for A, B, and E are represented as the mean ± SD (n = 3).
Fig. 4.
Fig. 4.
Fluorescence of intracellular polyplexes containing quinine and size-dependent activity. (A) After the “formulation time” (Fig. 3D) to achieve the desired mean particle diameter, the poly(quinine-co-HEA) polyplexes were incubated with HeLa cells for a defined time (defined as “cell incubation time”). (B and C) Images of fixed HeLa cells 48 h posttransfection with pZsGreen and poly(quinine-co-HEA) (N/P = 8) at (B) 4× magnification with poly(quinine-co-HEA) (blue) and (C) 40× magnification. (D) Images of HeLa cells fixed 6 h posttransfection with poly(quinine-co-HEA) at various formulation and cell incubation times. The sample names [i, ii] are derived from (i) formulation time (Fig. 3D) and (ii) cell incubation time (Fig. 4A). The left image in each pair is an overlay of transmission and polymer (blue), and the right image is of polymer only. (Scale bars, 10 µm.) (E) Bar graph showing the percent transfection efficiency of live cells (as determined by flow cytometry) and relative cell viability (as determined by CCK-8 cell counting kit) 48 h posttransfection. (F) HeLa cells transfected with the conditions shown for sample [30,15] in E were incubated with endocytosis inhibitors. Incubation with DMA (macropinocytosis inhibitor) gives a statistically significant reduction in transfection indicating macropinocytosis contributes to successful transfection of aggregated polyplexes. Data for E and F are represented as the mean ± SD (n = 3); *P < 0.05.
Fig. 5.
Fig. 5.
QCR variant of poly(quinine-co-HEA) containing 14% quinine efficiently transfects a variety of cell types. (A and B) The transfection efficiency in the delivery of pZsGreen (4.7-kb plasmid) to adherent cell lines (HeLa and HEK 293T), keratinocytes, and suspension cell line K562 was improved significantly by using poly(quinine-co-HEA) compared to Lipofectamine 2000 as determined by flow cytometry 48 h posttransfection. (C) The cell viability of HEK 293T cells was determined 48 h posttransfection via CCK-8 assay. (D) Efficient transfection of keratinocytes using a large plasmid (10-kb, pZsGreen) was maintained using poly(quinine-co-HEA). Data in A–D are represented as the mean ± SD (n = 3).
Fig. 6.
Fig. 6.
Representative Raman images of HeLa cells and QCR polyplex particles after (A) 4 h, (B) 24 h, and (C) 48 h posttransfection. Cells (green) were visualized using the integrated intensity of the protein Amide I band (1,660 cm−1), while polyplex particles (magenta) were visualized by the quinoline ring mode of quinine (1,369 cm−1). The overlay of these images shows the presence of both intracellular and extracellular particles at all timepoints. (Scale bars, 5 µm.)
Fig. 7.
Fig. 7.
Results from PCA of Raman hyperspectral images for HeLa cells 48 h posttransfection. (A) PC 1 score map showing the relative concentration of quinine moieties in poly(quinine-co-HEA) polyplexes that are DNA-intercalated. (B) PC 1 loading vector showing the 1,369-cm−1 quinoline ring stretching mode spectral signature indicative of quinine–DNA intercalation. (C) PC 3 score map showing the relative concentration of quinine moieties in polyplexes that are deintercalated from pDNA. (D) PC 3 loading vector showing the 1,372-cm−1 quinoline ring stretching mode indicative of quinine deintercalation. (E) Raman image showing the relative concentration of protein distributed in the cells. The PC score maps shown in A and C were used to determine (F) the percent deintercalation for every pixel of the polyplex particles. (F, Inset) A magnified region containing a polyplex with a ring-shaped unpackaging behavior. (G) Correlates the percent deintercalation with respect to the relative concentration of protein as a function of the normalized distance (R/r) from the centroids of polyplex particles inside cells (see SI Appendix for details). (Scale bars, 5 µm.)
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