Cytocompatibility and Cellular Internalization of PEGylated âClickable

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Cytocompatibility and Cellular Internalization of PEGylated “Clickable” Nucleic Acid Oligomers Alex J Anderson, Erica B. Peters, Alex Neumann, Justine Wagner, Benjamin Fairbanks, Stephanie J. Bryant, and Christopher N. Bowman Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00162 • Publication Date (Web): 26 Apr 2018 Downloaded from http://pubs.acs.org on April 26, 2018

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Cytocompatibility and Cellular Internalization of PEGylated “Clickable” Nucleic Acid Oligomers Alex J. Anderson1, Erica B. Peters, Alex Neumann, Justine Wagner, Benjamin Fairbanks, Stephanie J. Bryant1,2,3*, Christopher N. Bowman1,2,3 1

Department of Chemical and Biological Engineering, University of Colorado, Boulder, CO 80303 2

Material Science and Engineering Program, University of Colorado, Boulder, CO 80303 3

BioFrontiers Institute, University of Colorado, Boulder, CO 80303

Abstract. The recently developed synthetic oligonucleotides referred to as “Click” Nucleic Acids (CNAs) are promising due to their relatively simple synthesis based on thiol-X reactions with numerous potential applications in biotechnology, biodetection, gene silencing, and drug delivery. Here, the cytocompatibility and cellular uptake of rhodamine tagged, PEGylated CNA copolymers (PEG-CNA-RHO) were evaluated. NIH 3T3 fibroblast cells treated for 1 hour with 1, 10 or 100 µg/mL PEG-CNA-RHO maintained an average cell viability of 86%, which was not significantly different from the untreated control. Cellular uptake of PEG-CNA-RHO was detected within 30 seconds and the amount internalized increased over the course of 1 hour. Moreover, these copolymers were internalized within cells to a higher degree than controls consisting of either rhodamine tagged PEG or the rhodamine alone. Uptake was not affected by

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temperature (i.e., 4°C or 37°C), suggesting a passive uptake mechanism. Subcellular colocalization analysis failed to indicate significant correlations between the internalized PEGCNA-RHO and the organelles examined (mitochondria, endoplasmic reticulum, endosomes and lysosomes). These results indicate that CNA copolymers are cytocompatible and are readily internalized by cells, supporting the idea that CNAs are a promising alternative to DNA in antisense therapy applications.

Introduction Nucleic acids represent one of the most powerful, functional biological materials due to their ability to control and direct biological function at all levels and to transmit genetic information from one generation to the next. These functions are possible solely because of the specific nucleotide sequence of each polymer and the nucleobase’s ability to recognize and hybridize with its complementary base. These characteristics are what has made nucleic acids a popular topic in both material science and biotechnology. Specifically, the ability of DNA to recognize and bind to highly specific genetic sequences has led to its use as antisense agents in gene therapy. Significant reductions in protein expression, and thereby a modulation of disease symptoms, have been achieved by delivering oligomeric DNA that is complementary to targeted sequences of either DNA or mRNA1–3. While gene therapy has been a major area of research4,5, the field has largely been hindered by the inefficient cellular delivery of DNA, instability of foreign DNA in biological environments, and the limited scale of DNA synthesis. Several techniques have been developed to circumvent these shortcomings. One common strategy is to condense antisense oligonucleotides into complexed nanoparticle delivery


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systems6. This strategy has been shown to increase transfection efficiency as well as protect them from extracellular degradation. However, these nanoparticles have a complicated mechanism of action, often require the use of cytotoxic polymers7, and once the oligonucleotides are released into the cytosol, they are vulnerable to intracellular degradation8,9. To prevent oligonucleotide degradation, the nucleotide structure can be chemically modified such as in phosphorothioate nucleic acids (PS-DNA) or peptide nucleic acids (PNAs). By altering or eliminating the phosphodiester bond, PS-DNA, PNAs, and other types of modified nucleic acids have proven to be nuclease resistant10–12. In some cases, they have been shown to have higher transfection efficiencies13. However, like DNA synthesis, the assembly of most modified nucleic acids oligomers relies on a complicated solid-phase strategy14,15, which is low yield and difficult to purify, time consuming, and can require the use of hazardous chemicals and solvents16. In contrast, recently reported “Click” Nucleic Acids (CNAs) are nucleic acid oligomers synthesized by taking advantage of thiol-mediated click reactions17. CNA monomers bare a resemblance to DNA nucleotides in that they contain a pendent nucleobase; however, they differ in that the backbone contains a thioether structure that arises from the thiol-X reactions that are used to form the CNA. By employing conditions that favor the correct thiol-X reaction, carefully designed CNA monomers are polymerized into CNA polymer chains quickly and under ambient settings, making them an interesting and attractive alternative form of oligomeric nucleobases. CNA structure eliminates the phosphodiester bond, rendering it invisible to both endo- and exonucleases18,19. Furthermore, while complete specific and arbitrary sequence control is still out of reach, sets of orthogonal thiol-X reactions can be used to build nucleic acid sequences that are polymerized in a single step to yield repetitive sequences of nucleotides. Thus, CNAs have the potential to be used for antisense gene therapy strategies, either as the antisense agent itself for


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treatment of diseases caused by repeat expansions20 or as a complexation agent for DNA delivery21. In addition, they may offer many advantages over current antisense agents, such as nuclease resistance and a more facile synthesis. However, before CNAs can be realized for such applications, it is necessary to understand whether they will be compatible with biological systems. This study investigated cytocompatibility, cell uptake properties, and interactions with subcellular organelles of CNAs in vitro. Copolymers of CNA and poly(ethylene glycol) (PEG) were synthesized to create water soluble CNA conjugates. PEGylated CNAs functionalized with a fluorophore were incubated with cells and cytocompatibility was determined by measuring cell metabolism. Cellular internalization was assessed by visualizing up-take with fluorescence microscopy. Finally, immunofluorescent techniques were employed to study subcellular colocalization of CNA copolymers upon internalization. Experimental PEG-CNA-RHO and PEG-RHO conjugate Synthesis. The thymine CNA monomer was synthesized as previously described16. Photo-initiated copolymerization was carried out by dissolving thiolated poly(ethylene glycol) (PEG-SH) (MW 2000 g/mol, Sigma Aldrich) at 100 mM and thymine CNA monomer at 1 M in DMF containing 0.01 wt. % 2,2-Dimethoxy-2-phenylacetophenone (DMPA). After irradiation with 365 nm light for 15 minutes, the resulting material was precipitated into cold diethyl ether. The supernatant was decanted leaving the solid copolymer (PEG-CNA), which was dried and resuspended in DI H2O. After agitating the polymer suspension overnight, the insoluble polymer fraction was spun down and the supernatant was collected and lyophilized, yielding water soluble PEG-CNA whose presence was confirmed by 1H NMR (Bruker AV-III 400 MHz) and GPC (Tosoh HLC®-


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8320GPC). In all cases, GPC samples were run in DMSO using two detectors, Refractive Index (RI) and Ultraviolet (UV). Average molecular weights were calculated using PMMA standards. PEG-CNA was then dissolved at 500 µM in PBS in a scintillation vial and dithiols were reduced with tris(2-carboxyethyl)phosphine (TCEP) (Chem-Impex International) for 20 minutes while the solution was purged with inert gas. Then, under an inert atmosphere, 2.5 eq. of Rhodamine Red (RHO) C2 maleimide dissolved in DMSO (7.4 mM) was added to the stirring PEG-CNA solution and allowed to react for 2-3 hours in the dark. The reaction solution was then dialyzed in the dark with a 1 kDa MWCO against DI H2O for 2 days (until the diasylate appeared colorless) yielding a pink fluorescent solution. The dialyzed PEG-CNA-RHO was then lyophilized and analyzed by 1H NMR and GPC. The extent of labelling by the dye was determined by UV-VIS. The lyophilized PEG-CNA-RHO powder was then reconstituted at a concentration of 1 mg/mL in PBS. This solution was sterile filtered with a 0.22 µm, PVDF, sterile syringe filter and frozen in aliquots. PEG-RHO was synthesized in a similar manner by reacting PEG-SH (MW 2000 g/mol) with Rhodamine Red C2 maleimide. Once the fluorescent molecule was introduced, care was taken to protect the compound from exposure to light. Cell Culture. NIH 3T3 fibroblast cells obtained from ATCC were cultured in DMEM growth media supplemented with 10% FBS and 1% antibiotics at 37°C and 5% CO2. Cells were passaged every 3-5 days or when 90% confluency was reached with 0.5% trypsin-EDTA and replating at a 1:10 ratio. Cell viability. Viability was assessed by an MTT assay. Briefly, cells were seeded in a 96 well plate at 800 cells/well and allowed to attach and grow for 48 hours. Cells were then treated with varying


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concentrations of PEG-CNA-RHO and PEG-RHO conjugate (0-100 µg/mL) in triplicate for 1 hour. A positive control was performed by adding a small punch of a powdered latex glove to the wells. After treatment, cells were washed with PBS and cultured in fresh media for another 48 hours. The assay was performed by replacing the media in each well with 100 µL of fresh media and adding 10 µL of 12 mM MTT to each well after which the plate was incubated at 37°C and 5% CO2 for 4-6 hours. The resulting crystal precipitates were dissolved by adding 100 µL of 0.1 g/mL sodium dodecyl sulfate in 0.1 M HCl after which the solution’s absorbance was measured at 570 nm. Cellular uptake of fluorescent conjugates. Cells were plated in a high quality, glass bottom 96 well plate (ibidi® µ-Plate) at a concentration of 4,000-7,000 cells/well and allowed to adhere and grow overnight at 37°C and 5% CO2. The culture medium was replaced with 135 µL of fresh media to which 15 µL of the 1 mg/mL PEG-CNA-RHO solution was added. The plate was swirled gently to allow mixing. After incubation for 1 hour, the cells were washed with PBS, and fresh media was added to the wells. Time dependent cellular uptake and localization. Cells were plated and treated with PEG-CNA-RHO as described previously, but with varying incubation times from 30 seconds to 6 hours. After incubation, the cells were washed with PBS and immediately fixed with 4% paraformaldehyde for 10 minutes at room temperature. After washing with PBS, cells were incubated with fresh PBS overnight at 4°C, protected from light. Immunofluorescence. Cells were plated and treated with PEG-CNA-RHO the same way as before. After treatment, fresh media was added to the wells and the cells were immediately imaged to verify uptake.


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Cells were then fixed with 4% paraformaldehyde for 20 minutes at room temperature, after which they were washed twice with PBS. Cells were permeabilized with 0.5% Triton X-100 for 5 minutes at room temperature, washed twice with PBS, and then blocked with 3% BSA for 60 minutes at room temperature. After rinsing with PBS, cells were stained with primary antibodies (Endoplasmic Reticulum: calreticuluin antibody, rabbit anti-mouse, Novus Biologicals®, used at 1:90 dilution in 3% BSA; Mitochondria: ATPB antibody, mouse monoclonal, Abcam®, used at 1:500 dilution in 3% BSA; Endosome: EEA1 antibody, mouse monoclonal, R&D Systems ®, used at 1:50 dilution in 3% BSA) for 1 hour at room temperature. After rinsing wells three times with PBS, all traces of the primary antibody were removed by incubating with 0.01% Tween-20 for 1 hour at room temperature. The cells were washed with PBS twice and then stained with the fluorescent secondary antibody (AlexaFluor 488, donkey anti-mouse, Invitrogen™, used at 1:200 dilution in 3% BSA; AlexaFluor 647 donkey anti-rabbit, Invitrogen™, used at 1:200 dilution in 3% BSA) for 1 hour at room temperature in the dark. After washing with PBS three times, Factin was stained with and phallotoxins (Phalloidin 488 and Phalloidin 647, Invitrogen™, used at 1:60 dilution in 3% BSA) for 20 minutes at room temperature. Cells were washed with PBS twice, and then stained with Hoechst 33342 (Invitrogen™, used at 1 µg/mL in 3% BSA) for 5 minutes at room temperature. Finally, cells were washed with PBS twice and stored in PBS at 4°C until imaged. Fluorescence microscopy. Cells with fluorescent components were imaged on a Nikon Spinning Disc Confocal microscope. To evaluate the extent of cellular uptake, a 40x air objective was used. For single cell images, a 100x oil immersion objective was used. For all cases, multiple images across


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multiple replicate wells were taken. Reported images are representative of the corresponding experimental conditions. All image analysis was completed with the ImageJ software. Statistical Analysis. Data presented are means of replicates (n, indicated in figure captions) with error bars representing standard deviations. Statistical analysis for the MTT assay and cell uptake assay was performed using a One-Way ANOVA with respect to cell viability and average fluorescence intensity per cell, respectively. Statistical significance was defined at the 95% confidence limit (p < 0.05). In cases where significant differences were present, comparisons were made using Tukey’s Post-Hoc analysis to identify differences between groups. Results and Discussion To begin evaluating CNAs’ cytocompatibility, linear, PEGylated CNAs were synthesized to increase the hydrophilicity of the CNA chain. Due to the lack of charge and hydrophilic groups on the CNA backbone, CNA homopolymers exhibit poor water solubility. It has also been shown that the addition of PEG to biomaterials can help increase the material’s bioavailability22, protect the material from degradation23, and stabilize nucleic acid hybridization interactions24. Photoinitiated copolymerization of a thiolated PEG (molecular weight of 2000 g/mol) and thymine CNA monomers yielded a linear block-copolymer, named PEG-CNA. This type of copolymerization was chosen as the preferred method of synthesis because it is a simple, onestep reaction and because it results in a thiol end group on the CNA block which can subsequently be functionalized with a fluorophore. Gel permeation chromatography of this copolymer showed an increase in molecular weight from the PEG-SH, suggesting successful copolymerization, along with a low-molecular weight peak that is attributed to the cyclized monomer (Supporting Figure S1). The water-soluble fraction of the block-copolymer was


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isolated, and GPC and 1H NMR analysis confirmed that this copolymer contained an average of 5 thymine repeat units per chain (as obtained by peak integration with respect to the PEG methoxy peak) (See Supporting Figure S5). It should be noted that the degree of polymerization calculated here is less than our original molar excess of thymine monomer (10:1, thymine monomer:PEG-SH). This behavior is because the cyclized monomer that is observed in the GPC trace, unreacted CNA homopolymers, and copolymers with higher degrees of polymerization are less likely to be solubilized by the water due to their high CNA content. Finally, a fluorophore (Rhodamine Red C2 maleimide) was conjugated to the sulfhydryl terminated end of the CNA chain, to yield the PEG-CNA-RHO conjugate. Effective conjugation was confirmed by 1H NMR analysis and UV-VIS (Supporting Figure S6 and S7). The GPC trace of the resulting compound showed that the major peak is maintained and that many low molecular weight peaks from the initial copolymerization disappear (Supporting Figure S8). Experiments were conducted using the strategy summarized in Scheme 1. Internalization experiments were performed by incubating cells with known concentrations of PEG-CNA-RHO, fluorescent PEG (PEG-RHO), or rhodamine dye alone (RHO), followed by subsequent washing. In this study, NIH 3T3 fibroblast cells were employed, which have been used previously to investigate cytocompatibility and cell uptake25–27. Scheme 1. In vitro strategy for evaluating the cytocompatibility of PEGylated CNA copolymers.


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The cytocompatibility of PEG-CNA-RHO was determined by the MTT assay, which measures cellular metabolism. Since metabolic activity is directly proportional to cell number, the MTT assay is an indirect measurement of cell viability, or how many cells survive after being treated with a molecule of interest. Concentrations of PEG-CNA-RHO ranging from 0 to 100 µg/mL were incubated with NIH 3T3 cells for a period of one hour. Over the concentration range tested, cell metabolic activity was not statistically different from the un-treated control (Figure 1) indicating cytocompatibility. As a positive control, cells were incubated with a powdered latex glove for the same amount of time, which reduced cell metabolism to ~20%28. Cells were also treated with PEG-RHO in the same concentration range as above. Cell viability after treatment with PEG-CNA-RHO was not statistically different from the viability of cells treated with PEGRHO, which indicates that the CNA block does not add any significant toxicity.


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Figure 1. An MTT assay confirms the low toxicity of the synthetic conjugates delivered in this study. Tukey’s post hoc comparison failed to identify significant differ-ences in viability at a 95% confidence limit. (n = 3, error bars represent standard deviations). To examine the effect of the PEG-CNA-RHO copolymer structure on cell uptake, cells were incubated with PEG-CNA-RHO, fluorescent PEG (PEG-RHO), and the fluorescent dye alone (RHO). Figures 2 shows the extent of cellular uptake of representative samples after treatment with the three types of molecules. The bright red fluorescent spots seen in the first panel of Figure 2 are cells that have internalized PEG-CNA-RHO. In contrast, the lack of fluorescence in the other panels (PEG-RHO and RHO alone, respectively) indicates that comparatively less uptake was observed. Semi-quantitative analysis confirms that cells incubated with PEG-CNARHO exhibited a statistically higher average fluorescence intensity than cells treated with PEGRHO or RHO alone. These results suggest that the presence of CNA in the copolymer facilitates uptake by cells.


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Figure 2. Cellular internalization of PEG-CNA-RHO, PEG-RHO, and RHO. Cells were treated with 100 µg/mL of conjugate or an equimolar amount of rhodamine for 1 hr, washed, fixed and imaged by fluorescence microscopy. A significantly greater average fluorescence intensity per cell after washing was determined for cells treated with PEG-CNA-RHO (*=p < 0.001) than for cells treated with PEG-RHO or RHO controls, indicating CNA dependent cellular uptake. (n>1000, error bars represent standard deviations) Uptake kinetics were investigated by incubating cells with 100 µg/mL of PEG-CNA-RHO for various times ranging from 30 seconds to 1 hour. This concentration was chosen because it was shown to be cytocompatible as well as visible with microscopy. Representative confocal microscopy images and semi-quantitative image analysis show an increase in cell-associated fluorescence over time, which are shown in Figure 3. All further experiments were conducted with a 1 hour incubation step because it appears that PEG-CNA-RHO uptake levels are easily visualized on this time scale.


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Figure 3. Incubating cells for increasingly longer times leads to an increased average cellassociated fluorescence. Representative images show cell fluorescence at 30 seconds, 10 minutes, 30 minutes, and 1 hour. Fluorescent intensity appears to begin to level off on the order of hours. (n>30, error bars represent standard deviations) Subcellular localization of the CNA copolymers was studied through immunofluorescence microscopy. In short, cells were treated with a known concentration of PEG-CNA-RHO (red fluorescence) for 1 hour, fixed, and then stained with organelle-specific fluorophore-antibody conjugates (green fluorescence). By measuring the presence and intensity of red fluorescence against green fluorescence for each pixel of an image, a semi-quantitative degree of colocalization is determined in the form of the Pearson coefficient or Mander’s coefficient29,30. This phenomenon is also visually identified in an overlay image by the presence of yellow pixels. In all, four organelles were stained: endosomes, endoplasmic reticulum, mitochondria, and lysosomes. Additionally, nuclei and actin were stained to aid in cellular identification. Resulting overlay images of individual cells are shown in Figure 4. The localization of PEG-CNA-RHO (red fluorescence) in each image shows a similar motif: the presence of small punctate areas of high fluorescence amid a mostly diffuse pattern within the cell. The small punctate areas within


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these images could suggest the sequestration of PEG-CNA-RHO by an organelle, or could simply be aggregation of the polymer in the highly complex intracellular environment. One phenomenon to mention is that in some images it appears that the PEG-CNA-RHO and organelles look to be negatively correlated. This behavior is seen in the images staining for mitochondria, lysosomes, and the endoplasmic reticulum and are indicated by the white arrows. It should also be noted that there is evidence of PEG-CNA-RHO in the nucleus, which is seen by the magenta color in the images in Figure 4. Previous studies have shown that nuclear uptake of PS-DNA (and to some extent, natural phosphodiester linked DNA) occurs to an extent31,32. However, some reports have warned that this result could be an artifact of the fixation and permeabilization process33.

Figure 4. Three representative images of immunofluorescence microscopy images highlight the cellular localization of PEG-CNA-rho conjugates. Cells were treated with 100 µg/mL of PEGCNA-rho conjugates for 1 hr, then fixed, permeabilized, and stained. Blue represents the nucleus


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(Hoechst 33342), red represents the PEG-CNA-RHO conjugate, and green represents either endosomes, the endoplasmic reticulum, mitochondria, or lysosomes. Areas of yellow indicate colocalization between the PEG-CNA-rho conjugate and the indicated organelle. Areas of magenta indicate colocalization between the PEG-CNA-rho conjugate and the nucleus. To calculate the Pearson and Mander’s coefficients, the JACoP ImageJ plugin was used to determine the overlap between the green and red channels of the overlay images. The Pearson coefficient generally measures how well the intensities of each channel are correlated for every pixel of an image. The Mander’s coefficient takes this one step further and can differentiate between overlap of the green channel with the red, or the red channel with the green. For both coefficients, only values close to 1 indicate significant colocalization, while all other values do not allow conclusions to be drawn34. In all images, colocalization coefficients were no higher than 0.4, suggesting that no conclusions can be made regarding colocalization between PEGCNA-RHO and any of the organelles examined. However, this does not mean that there is no colocalization between PEG-CNA-RHO and subcellular organelles. It is possible that significant interactions are occurring, but the uncertainty associated with immunofluorescence techniques does not allow for its detection. For instance, given that the CNA block contains repeats of thymine, we might expect the copolymer to bind to structures containing adenine residues, like the poly-A tail of mRNA. However, the immunofluorescence techniques here do not allow for the detection of such specific interactions. To identify the mechanism by which uptake of CNA occurs, experiments were performed at 4°C or 37°C to probe passive and active transport mechanisms, respectively. At 4°C, active endocytotic uptake mechanisms are severely limited due to the lack of energy (i.e. ATP production)35, but passive transport pathways are not. Thus, the relative extent of uptake that is


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observed at 4°C provides information about whether uptake is passive or active. Cells were incubated at either 4°C or 37°C and then treated with PEG-CNA-RHO. As seen in Figure 5, the difference in average cellular fluorescence was determined to be insignificant between 4°C or 37°C, suggesting that uptake is primarily through a passive mechanism (i.e. free or facilitated diffusion).

Figure 5. Temperature dependent uptake of PEG-CNA-RHO. Cells were incubated with 100 µg/mL of PEG-CNA-RHO for 1 hour at either 4°C or 37°C. Cellular uptake of PEG-CNA-RHO was not significantly affected by incubation temperature, suggesting a passive uptake mechanism. It is likely that some of these results are explained by unique structural elements of the CNAs. First, the PEG-CNA-RHO exhibits block amphiphilicity due to the hydrophilic PEG block and hydrophobic CNA block. It is possible that the hydrophobic CNA block is facilitating interaction with the phospholipids within the cell membrane, leading to increased translocation. Previous reports of similarly structured amphiphilic molecules have indeed shown that increasing hydrophobicity leads to greater cell uptake36. Further, studies on amphiphilic cell-penetrating peptides (CPPs) have concluded that insertion of the CPPs hydrophobic region into the cell membrane is crucial for the initial steps of membrane translocation37,38. It is also worth noting that like many other amphiphilic molecules, these polymers self-assemble into particles or micelles in solution (Supporting Figure S10). There have been other reports of self-assembled


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structures that are internalized by energy-independent pathways; however, the exact mechanisms are not fully understood.39,40. In addition, CNAs maintain a net neutral charge under biological conditions, which could also affect their interaction with the cell membrane. It has been shown that electrostatics affect the hydrophobic partitioning of amphiphiles into lipid-bilayer membranes38,41. This behavior suggests that neutral CNAs have an increased association with the cell membrane and ultimately increased uptake compared to negatively charged DNA, which experiences electrostatic repulsion between the negatively charged backbone of DNA and the negatively charged cell membrane surface42,43. Conclusions CNAs have been developed as an alternative approach to modifying nucleic acids. Their ability to participate in thiol-X reactions allows for facile synthesis of nucleic acid oligomers. Similar oligomers have been shown to have effective antisense characteristics in gene therapy applications. In the present study, the evaluation of CNA-based copolymers as potential antisense oligonucleotides was initiated by evaluating their cytocompatibility and cellular internalization characteristics. CNA copolymers were determined to be non-toxic to cells at concentrations up to 100 µg/mL. Additionally, PEG-CNA-RHO was taken up by cells within 1 hour and to a greater extent than PEG-RHO and RHO alone and that the extent of uptake was not affected by temperature, suggesting a passive, CNA dependent uptake mechanism. It is possible that this uptake is because the lipophilic CNA block allows for better interaction with the lipid cell membrane. The distribution of PEG-CNA-RHO within cells exhibited both diffuse and punctate patterns with no apparent specific organelle colocalization. These results show that CNAs are cytocompatible, readily taken up by cells, and thus warrant future studies to assess their potential as a synthetically simple alternative to current antisense approaches.


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ASSOCIATED CONTENT Supporting Information.1H NMR of intermediates for PEG-CNA-RHO and PEG-RHO synthesis (PEG-SH, thymine thiol-ene monomer, PEG-CNA, PEG-CNA-RHO, PEG-RHO); GPC traces of intermediates; UV-VIS of PEG-CNA-RHO and method of calculating extent of labelling by fluorescent dye; DLS data showing size distribution of particles formed (file type: PDF). AUTHOR INFORMATION Corresponding Author. * Email: [email protected] Author Contributions. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT This work was completed with support from an NSF MRSEC grant (DMR 1420736) and from a US Department of Education GAANN Fellowship to Alex Anderson. E.B.P. was supported by the NIH T32 National Institutional Research Service Award T32 HL07670. The imaging work was performed at the BioFrontiers Institute Advanced Light Microscopy Core. Spinning disc confocal microscopy was performed on Nikon Ti-E microscope supported by the BioFrontiers Institute and the Howard Hughes Medical Institute. ABBREVIATIONS CNA, “Clickable” Nucleic Acid; PEG, poly(ethylene glycol); PEG-CNA-RHO, fluorescent clickable nucleic acid copolymer; PEG-RHO, fluorescent poly(ethylene glycol) polymer; RHO, rhodamine


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