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Specific delivery of oligonucleotides to the cell nucleus via gentle compression and attachment of polythymidine Zhong Chen, Huize Li, Lei Zhang, Carrie Ka Yee Lee, Lok Wai Cola Ho, Cecilia Ka Wing Chan, Hongrong Yang, and Chung Hang Jonathan Choi ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b11391 • Publication Date (Web): 15 Jul 2019 Downloaded from pubs.acs.org on July 19, 2019

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ACS Applied Materials & Interfaces

Specific delivery of oligonucleotides to the cell nucleus via gentle compression and attachment of polythymidine

Zhong Chen†,‡,#, Huize Li†,#, Lei Zhang†,‡, Carrie K. Lee†, Lok Wai Cola Ho†, Cecilia Ka Wing Chan§, Hongrong Yang†, and Chung Hang Jonathan Choi†,‡,*

Department of Biomedical Engineering, ‡Shun Hing Institute of Advanced Engineering, and

†

§

Department of Surgery, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong.

#

These authors (Z.C. and H.L.) contributed equally.

* To whom correspondence should be addressed. E-mail: [email protected].

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ABSTRACT Non-viral delivery of nucleic acids to the cell nucleus typically requires chemical methods that do not guarantee specific delivery (e.g., transfection agent) or physical methods that may require extensive fabrication (e.g., microfluidics) or an elevated pressure (e.g., 105 Pa for microneedles). We report a method of delivering oligonucleotides to the nucleus with high specificity (relative to the cytosol) by synergistically combining chemical and physical approaches. Particularly, we demonstrate that DNA oligonucleotides appended with a polythymidine [poly(T)] segment (chemical) profusely accumulate inside the nucleus when the cells are under gentle compression imposed by the weight of a single glass coverslip (physical; ~2.2 Pa). Our “compression-cumpoly(T)” delivery method is simple, can be generalizable to three “hard-to-transfect” cell types, and does not induce significant levels of cytotoxicity or oxidative stress to the treated cells provided the use of suitable compression times and oligonucleotide concentrations. In bEnd.3 endothelial cells, compression-aided intranuclear delivery of poly(T) is primarily mediated by importin β and nucleoporin 62. Our method significantly enhances the intranuclear delivery of antisense oligonucleotides to bEnd.3 cells and the inhibition of two target genes, including a reporter gene encoding the enhanced green fluorescent protein (EGFP) and an intranuclear lncRNA oncogene [metastasis associated lung adenocarcinoma transcript 1 (MALAT1)], when compared to delivery without gentle compression or poly(T) attachment. Our data underscore the critical roles of pressure and nucleotide sequence on the intranuclear delivery of nucleic acids.

KEYWORDS specific delivery, compression, cell nucleus, polythymidine, oligonucleotides, importin β


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INTRODUCTION Delivery of nucleic acids to the nucleus not only supports investigations into intracellular biological mechanisms at the genetic level,1–3 but also paves the way for treating diseases by regulating the expression of target genes.4,5 Intranuclear delivery of nucleic acids is challenging due to the need to penetrate both the cell membrane and nuclear membrane. In particular, the nuclear membrane is only permeable to ions and molecules of no larger than 10 nm in diameter.6,7 Passage of larger cargoes up to 39 nm in diameter through the nuclear pore complex (NPC) to the nucleus is typically energy-dependent and requires their interaction with nuclear transport proteins such as importin β (Imp β).8 Conventional non-viral methods of intranuclear delivery fall under two major categories, physical and chemical.9 Physical methods utilize electrical10 or mechanical forces to create transient pores on the cell and nuclear membranes for delivering nucleic acids. However, they entail laborious handling (e.g., microinjection11), extensive fabrication (e.g., microneedles12 and microfluidics13), or operation at pressures on the order of 104–105 Pa (e.g., gene gun14 and microneedles15). For instance, Mann et al. delivered oligonucleotides to the cell nuclei of the myocardium ex vivo by infusing a saphenous vein at a fluid pressure of 100 mmHg (13,300 Pa) for 10 min,16 yet this method involves cannulation. Han et al. delivered single guide RNAs and Cas9 proteins to the cytosol by flowing MCF7 cancer cells through a microfluidic device with constrictions at a fluid pressure of 70 psi (4.8 × 105 Pa) to disrupt the cell membrane.17 While this method empowered genome editing, it necessitates repeated passage of cells through the device and 7 d of recovery. Ding et al. developed a microfluidic device for the intranuclear delivery of plasmid DNAs into HeLa cancer cells by collective mechanical squeezing (for disrupting the cell membrane) and application of an electrical field (for disrupting the nuclear membrane).18 This


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device demands a high fluid pressure (1.5 × 105 Pa) as well as elaborate design components such as electrodes and constrictions. Note that applying pressures on the order of 102–103 Pa to cells is already sufficient to affect cellular functions. For instance, applying a compressive stress of 100 Pa to C2C12 myoblasts for 10 h can damage 35% of the cell population.19 Exerting a compressive stress of 5.8 mmHg (770 Pa) on multiple cancer cell types for 16 h can increase their invasiveness in the tumor microenvironment.20 For chemical methods, nucleic acids are mixed with chemical compounds (e.g., transfection agents21,22 and calcium phosphate23) or conjugated to biomolecules (e.g., cell-penetrating peptides,24,25 nuclear localization signals,8 aptamers targeted to cell surface proteins,26 and transcription factors27) for delivering nucleic acids. For example, Branden et al. showed that attachment of SV40 nuclear localization signals to oligonucleotides can lead to their specific delivery to the nucleus.28 Recent chemical methods also entailed the use of non-cationic bionanomaterials as carriers of nucleic acids, due to their ability to abundantly enter cells without the aid of transfection agents.29,30 Huo et al. used ultrasmall gold nanoparticles of 2 nm in diameter to deliver triplex-forming oligonucleotides that bind to the c-myc promoter. They observed accumulation of these nanoparticles in the nucleus and downregulation of c-myc7. Sprangers et al. prepared liposomal spherical nucleic acid (SNA) nanoconstructs that contain phosphorothioate (PS)-modified antisense oligonucleotides for transfecting cancer cells,31 taking advantage of the observations that SNAs enter the cell via Class A scavenger receptors32,33 and that PS-modified oligonucleotides enter the nucleus by diffusion34 or a pathway mediated by the Ras-related nuclear protein.35 They detected intranuclear accumulation of the antisense oligonucleotides and downregulation of an intranuclear gene. Yet, the specificity of intranuclear delivery (relative to the cytosol) realized by both nanoparticle-based methods is low or unclear.


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We report a new and simple method of delivering DNA oligonucleotides with high specificity to the nucleus by synergistically integrating non-viral physical and chemical approaches. Specifically, we covalently append a polythymidine [poly(T)] segment to the oligonucleotide cargo to be delivered, followed by incubating cells with the poly(T)-appended oligonucleotides under gentle compression exerted by the weight of a glass coverslip (Figure 1A). On the chemical front, we choose poly(T) in light of a previous report that documented the intranuclear residency of poly(T) after being passively internalized by L6 rat muscle cells.36 This observation inspired us to design a poly(T) segment to be inserted at the end of the oligonucleotide cargo and to conveniently synthesize this poly(T)-appended oligonucleotide cargo by automated solid-state synthesis. On the physical front, we recently reported that C2C12 myoblasts, when subject to a compressive stress lower than 300 Pa, can internalize sub-25 nm nanoparticles by up to 5 times more than uncompressed cells without significant loss of cell viability.37 In this work, we demonstrate that gentle compression, at a pressure 103–104 times lower than many existing physical methods, enhances the specificity of intranuclear delivery relative to the cytosol. Our “compression-cumpoly(T)” delivery method is compatible with conventional cell culture vessels, takes several hours, does not require the aid of specialized instrumentation or transfection agents, and is generalizable to three different mammalian cell types that are difficult to be transfected by non-viral methods (bEnd.3 endothelial cells, Kera-308 keratinocytes, and RAW264.7 macrophages).38–40 By tuning three experimental parameters (applied pressure, compression time, and oligonucleotide concentration), we achieve specific intranuclear delivery of poly(T) oligonucleotides of 30 bases long (denoted T30) without inducing severe cytotoxicity or oxidative stress to the three cell types after 48 h of recovery. By employing a genetic knockdown approach, we prove that Imp β and nucleoporin 62 mediate the transport of T30 to the nucleus of bEnd.3 cells. We finally apply our


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“compression-cum-poly(T)” method to enhance the intranuclear delivery of antisense DNA oligonucleotides to bEnd.3 cells and the inhibition of two target genes, namely (1) the gene encoding the enhanced green fluorescent protein (EGFP) and (2) an intranuclear lncRNA oncogene called metastasis associated lung adenocarcinoma transcript 1 (MALAT1).

RESULTS AND DISCUSSIONS Spatial distribution of poly(T) in the culture dish In our preliminary studies, we incubated bEnd.3 endothelial cells, pre-seeded in flat-bottom 24well plates, with OptiMEM that contains 2.5 µM T30 oligonucleotides bearing a Cyanine 3 (Cy3) molecule at the 3’ end (T30-Cy3). OptiMEM, a type of cell culture medium commonly used for formulating nucleic acids for transfection studies,41 contains nutrients to maintain the growth of cells. The advantage of formulating oligonucleotides in OptiMEM lies on its reduced amounts of serum proteins (e.g., nuclease) that may degrade the DNA to be transfected. Immediately after adding T30-Cy3 to the cells, we subjected the cells to gentle compression afforded by the weight of a single glass coverslip (~2.2 Pa, see Equation S1 for calculation). The commercially available round coverslip bears a diameter of 12 mm, small enough to be conveniently deposited into and removed from the round well of most commercially available 24-well plates (with a well diameter of ~15.6 mm) yet large enough to rest atop most cells seeded in the well. After 5 h of compression, we removed the coverslip and oligonucleotide-containing medium, washed the cells with phosphate-buffered saline (PBS) to remove the serum proteins, and imaged the cells in different regions of the well by fluorescence microscopy (Figure 1B and S1). For the uncompressed cells seeded outside of the compressed region, we noticed weak Cy3 fluorescence. The weak Cy3 fluorescence in bEnd.3 cells is consistent with a previously reported method of gene transfection


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called “gymnotic delivery”,42 characterized by the entry of μM concentrations of oligonucleotides to cells without the aid of compression or a transfection agent. (Note that gymnotic delivery does not guarantee entry to the cell nucleus.) For the compressed cells, we observed contrasting levels of Cy3 fluorescence in different regions of the compressed area. Specifically, we detected intense Cy3 fluorescence inside cells that are seeded in the periphery of the compressed region (denoted “coverslip periphery”), which accounts for ~44% of the total compressed area. On the contrary, the cells seeded at the center of the compressed region (denoted “central region”) are not appreciably fluorescent. After compression, we removed the coverslip and stained the cells by calcein-AM. The intense intracellular calcein fluorescence in the central region confirms that the cells remained viable after compression (Figure S2), excluding the possibility that the weak Cy3 fluorescence in the central region stems from limited cellular uptake due to poor cell viability. By contrast, staining the cells pre-treated with organic solvents for 20 min (which are presumably dead) did not show detectable calcein fluorescence (Figure S3). As the coverslip is generally smooth (Figure S4) and impermeable to liquid, when the coverslip compresses an incompressible fluid (i.e., DNA-containing culture medium) along the vertical (z) direction, the fluid will be forced to flow radially outward (along the r direction) in cylindrical coordinates. Owing to this fluid mechanical phenomenon known as “squeezed flow”, the periphery of the compressed area will contain more DNA-containing fluid than the central region of the compressed area.43 Therefore, we posited that the coverslip limits the accessibility of the DNA-rich medium to the cells seeded in the central region. To test this hypothesis, in a separate experiment, we incubated bEnd.3 cells with medium containing Hoechst 33342, a fluorescent chemical stain for the nuclei of living cells (Figure S5). After 10 min of coverslip-aided compression, we removed the coverslip and examined the spatial distribution of Hoechst 33342. In line with our T30-Cy3 data, we detected Hoechst


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33342 fluorescence exclusively in the coverslip periphery but not the central region, evidence of fluid inaccessibility at the central region. As a positive control, cells in the uncompressed region also emit Hoechst 33342 fluorescence. Thus, fluid accessibility explains the more efficient intracellular delivery of T30 to the coverslip periphery than the central region. Future studies will focus on improving the fluid accessibility to the central region by developing porous, biocompatible materials for compression-aided delivery. Intracellular distribution of poly(T) in compressed cells To identify the intracellular location of the strong Cy3 fluorescence, we incubated bEnd.3 cells with T30-Cy3 under coverslip-aided compression for 5 h, followed by rinsing away the DNA and staining the compressed cells with 4',6-diamidino-2-phenylindole (DAPI) (Figure 1C). By confocal microscopy, we detected pronounced overlapping of the DAPI and Cy3 fluorescence with a Mander’s colocalization coefficient of over 0.8, evidence of intranuclear accumulation of T30Cy3 in cells in the coverslip periphery. [The Mander’s coefficient (M) measures the colocalization of objects in dual-colored fluorescence images, whereby M=1 indicates perfect colocalization and M=0 indicates no colocalization.44 Previously, we utilized the Mander’s coefficient to characterize the intracellular location of nanoparticles as revealed in confocal images.45] By manual counting, we identified Cy3 fluorescence in the nuclei of 56% ± 3% (n=150) of cells in the coverslip periphery. By multiplying the areal fraction of the compressed region with strong Cy3 fluorescence (i.e., coverslip periphery) by the fraction of cells with intranuclear accumulation of T30-Cy3 in the periphery, we estimate that the total intranuclear delivery efficiency of all cells under compression in the well is roughly 24.5%. By contrast, by incubating bEnd.3 cells with T30-Cy3 for 5 h without compression, we only detected limited colocalization of the DAPI and Cy3 fluorescence (with a Mander’s coefficient of lower than 0.1). Besides bEnd.3 endothelial cells, we further confirmed


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by confocal imaging that coverslip-aided compression supports the delivery of T30-Cy3 into the nuclei of Kera-308 keratinocytes and RAW264.7 macrophages (Figure 1C). Upon incubation with T30-Cy3 under compression for 5 h, we detected Cy3 fluorescence in the nuclei of 43% ± 6% (n=150) of the Kera-308 keratinocytes and 62% ± 1% (n=150) of the RAW264.7 macrophages in the coverslip periphery, evidence of the generalizability of our delivery method. Next, we chose bEnd.3 cells for two additional collections of characterization studies because they attach the bottom of the culture vessel without growing into clusters and show an elongated cell morphology that is amenable to microscopy. [As bEnd.3 endothelioma cells express low levels of thrombospondin-1 (a natural inhibitor of angiogenesis46) and form vascular tumors in mice,47 we will showcase the utility of our “compression-cum-poly(T)” method by delivering antisense oligonucleotides against an endogenous cancer gene to the nuclei of bEnd.3 cells (Figure 8)]. In the first collection of studies, we incubated bEnd.3 cells with T30-Cy3 under compression for 5 h, followed by staining the compressed cells with LysoTracker® Green DND-26, a fluorescent stain for acidic compartments like late endosome and lysosome. Confocal images show moderate colocalization between the signals of T30-Cy3 and LysoTracker (with a Mander’s coefficient of less than 0.4), suggesting that T30-Cy3 do not strongly accumulate in acidic compartments after compression (Figure S6). By incubating cells with T30-Cy3 for 5 h without compression, however, we detected strong colocalization between T30-Cy3 and LysoTracker (with a Mander’s coefficient of higher than 0.8), indicating substantial localization in acidic compartments. These results prove that coverslip-aided compression facilitates the redistribution of T30-Cy3 away from the acidic compartments inside bEnd.3 cells. Concurrently, we showed by flow cytometry that the Cy3 mean fluorescence intensity (MFI) of the compressed bEnd.3 cells is 20.2% ± 1.9% higher than that of the uncompressed cells upon incubation with T30-Cy3 for 5 h (Figure 1D); that is, coverslip-aided


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compression enhances the cellular uptake of T30-Cy3. As a negative control, cells untreated with T30-Cy3, with or without compression, have negligible values of intracellular Cy3 MFI. In our second collection of studies, we confirmed the compression-aided delivery of T30-Cy3 to the nucleus of bEnd.3 cells by performing three extra types of imaging experiments. For our first imaging method, after incubating bEnd.3 cells with T30-Cy3 under compression for 5 h, we stained the cells with DAPI and employed confocal imaging to capture z-stack consecutive optical slices of a representative cell in the coverslip periphery (Figure 2A). By three-dimensional (3D) confocal imaging, we observed that the intracellular MFI of both Cy3 and Hoechst 33342 signals are overlapping and are the highest in the central region of the z-stack (Figure 2A). MFI measurements of the z-stack show that the center of the cell emits the strongest Cy3 and Hoechst 33342 fluorescence. Z-stack confocal images that portray the center of the cell clearly show accumulation of T30-Cy3 inside the nuclei (Figure S7). We collected another set of z-stack images of a bEnd.3 cell incubated with T30-Cy3 under compression and stitched the optical slices back together to reconstruct the three-dimensional (3D) distribution of intracellular fluorescence (Figure 2A). Notably, we detected ~6-fold stronger Cy3 fluorescence in the nucleus than in the cytosol, evidence of specific intranuclear delivery of T30-Cy3. For our second imaging method, we employed super-resolution structured illumination microscopy (SIM) to image bEnd.3 cells incubated with T30-Cy3 under coverslip-aided compression for 5 h (Figure 2B). After staining the compressed cells with DAPI, we observed punctate patches of intense Cy3 fluorescence at a XY spatial resolution of 120 nm48 inside the nuclear membrane. The fluorescence of Cy3 does not significantly overlap with the fluorescence of DAPI (which stains intranuclear chromatin49), proof of accumulation of T30-Cy3 inside the nucleoplasm. For our third imaging method, we utilized super-resolution stimulated emission depletion (STED) microscopy to prove definitively the


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accumulation of T30-Cy3 inside the nucleus after incubating bEnd.3 cells with T30-Cy3 under compression for 5 h. As the XY spatial resolution of the Cy3 fluorescence due to STED imaging is ~50 nm,50 the limited colocalization between the Cy3 fluorescence and the DAPI-stained chromatin further confirms the accumulation of T30-Cy3 in the nucleoplasm (Figure 2C). Poly(T)-dependent intranuclear delivery We investigated whether coverslip-aided compression may facilitate the intranuclear delivery of other types of oligonucleotides besides T30-Cy3. To start with, we considered the effect of fluorophore type (Figure 2D). We incubated bEnd.3 cells with T30 oligonucleotides attached to a fluorescein isothiocyanate (FITC) molecule at their 3’ end (T30-FITC) for 5 h. Without coverslipaided compression, we detected FITC fluorescence primarily in the cytosol but not the nucleus, in agreement with past reports on the “gymnotic delivery” of µM concentrations of oligonucleotides into cells without the aid of transfection agents.42,51 With compression, however, confocal imaging reveals strong FITC fluorescence in the nuclei of 52% ± 5% (n=150) of the cells in the coverslip periphery without evident accumulation in the cytosol (or 22.3 % of the compressed cells in the well), consistent with our T30-Cy3 imaging data. (We also did not detect considerable FITC signals in the central region.) In another control experiment, we treated bEnd.3 cells with free FITC molecules under compression and detected weak intracellular fluorescence and limited intranuclear accumulation. This result verifies that the intranuclear FITC fluorescence observed in the compressed cells results from T30 but not the fluorophore. To examine the generalizability of our bEnd.3 data to other cell types, we repeated our delivery studies with Kera-308 cells (Figure S8) and RAW264.7 cells (Figure S9) by incubating them with T30-FITC for 5 h. For the compressed cells, we detected specific intranuclear accumulation of FITC fluorescence for the compressed cells in the coverslip periphery but not the central region. Without compression, the


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FITC fluorescence is localized in the cytosol without considerable accumulation in the nucleus. As a negative control, we did not detect appreciable levels of fluorescence when both cell types were incubated with free FITC molecules for 5 h. We then addressed the effect of oligonucleotide sequence on compression-aided intranuclear delivery (Figure 2E–F). In our first experiment, we incubated bEnd.3 cells with either T30-Cy3 or DNA oligonucleotides containing 30 repeating adenosines and a Cyanine 3 (Cy3) dye at the 3’ end (A30-Cy3) under compression for 5 h. Confocal images show that T30-Cy3 more efficiently accumulates in the cell nuclei than A30-Cy3. The intracellular location of A30-Cy3 is mostly cytosolic, indicating the sequence-specific nature of intranuclear accumulation. Flow cytometry data reveals 2-fold higher cellular uptake of T30-Cy3 than A30-Cy3. In another experiment, we prepared a random DNA sequence of 18 bases long (Random18; sequence information in Table S1) and another DNA sequence that includes Random18 and 12 extra thymidines attached to its 3’ end (Random18-T12). Attaching a T12 segment to Random18 pronouncedly facilitates intranuclear accumulation and increases the cellular uptake of Random18 by ~70%. Note that, at a constant oligonucleotide length of 30 bases, the Cy3 MFI of T30-treated cell samples is still the highest, followed by cells treated with Random18-T12 and A30. This trend agrees with the T content of their respective DNA sequences. Our data indicate that poly(T) plays a critical role in the specific intranuclear delivery of DNA oligonucleotides by coverslip-aided compression. Effect of pressure on intranuclear delivery and cell viability Previous reports pointed to cellular injury when cells are under compression. For instance, application of a compressive stress of 400 Pa to C2C12 myoblasts for 3 h significantly reduced their viability by over 50%.19 In this work, we optimized the pressure to balance intranuclear delivery and cell viability. Upon compression-aided delivery of T30-Cy3 into bEnd.3 cells for 5 h,


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we stained the cells with calcein-AM to correlate the intracellular distribution of Cy3 fluorescence and calcein fluorescence as a function of pressure. In parallel, we used flow cytometry to quantify the change in cellular Cy3 MFI (an indicator of cellular uptake of T30-Cy3) upon compression. To explore the effect of pressure on intranuclear delivery and cell viability, we incubated bEnd.3 cells with T30-Cy3 for 5 h under compression by loading a polydimethylsiloxane (PDMS) disk of 12 mm in diameter and different regularly increasing thicknesses on top of the coverslip (Figure S10), setting the range of pressure from 2.2 Pa (no PDMS disk added) to ~50 Pa (coverslip loaded with a PDMS disk of 6 mm thick; Equation S2). The number of cells with Cy3-positive nuclei in the coverslip periphery significantly decreases when our applied pressure increases from 2.2 Pa to 50 Pa (Figure 3A and S11). Flow cytometry measurements also show the highest Cy3 MFI after 5 h compression cells at 2.2 Pa (Figure 3B). A higher pressure also inflicts more severe damage to the cells. In this set-up, most cells remain viable after 5 h of compression at applied pressures below 10 Pa, as evidenced by the intracellular calcein fluorescence overlapping with intranuclear Cy3 fluorescence. When pressure exceeds 20 Pa, only a small fraction of cells still simultaneously exhibits Cy3 and calcein fluorescence; most compressed cells display strong intranuclear Cy3 fluorescence without detectable intracellular calcein fluorescence. Our data suggest that the optimal pressure for achieving intranuclear delivery while maintaining cell viability is 1–10 Pa for bEnd.3 cells. Still, we acknowledge the limitation of our set-up that imposing a higher pressure more significantly reduces the accessibility of T30-Cy3 to cells (and hence the resultant value of Cy3 MFI). For example, elevating the applied pressure from 2.2 Pa to 20 Pa reduces the value of L (width of the coverslip periphery; Figure 1B) from 1.5 mm to 1.0 mm and the areal fraction of the coverslip periphery region from 43.8% to 30.6% (Figure S5 and S12). Effect of compression time on intranuclear delivery and cell viability


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Time-lapse live confocal imaging of bEnd.3 cells incubated with T30-Cy3 under coverslip-aided compression reveals significant intranuclear accumulation of T30-Cy3 within the first 2 h of compression (Figure S13). By using fluorescence imaging to track bEnd.3 cells incubated with T30-Cy3 under compression for various durations of time, we detected Cy3 fluorescence in the nucleus after 5 min of compression and the gradual intensification in intranuclear Cy3 fluorescence to after 24 h of compression. (For these studies on uptake kinetics, note that we did not use regular intervals of compression time because we wish to investigate how early the oligonucleotides start entering the cell nucleus by our “compression-cum-poly(T)” method. By literature precedent, DNA-coated nanoparticles rapidly enter endothelial cells within the first hour of incubation, yet they enter cells at a more moderate pace at later time points.32,52 Therefore, we included 0.1 h, 0.25 h, and 0.5 h as early time points to observe the efficiency of delivering T30-Cy3 into the cell and cell nucleus by flow cytometry and fluorescence imaging.) The fraction of cells with Cy3-positive nuclei in the coverslip periphery rises from 2.8% ± 1.3% after 5 min of compression to 79.4% ± 1.9 % after 24 h of compression (Figure 3C and S14). Flow cytometry data corroborate that the Cy3 MFI of the compressed cells sharply increases by 17.7-fold as the duration of compression increases from 5 min to 24 h (Figure 3D). The cells in the coverslip periphery are largely viable after 5 h of compression, as shown by the strong intracellular calcein fluorescence overlapping with the strong intranuclear Cy3 fluorescence. After 12 h of compression, whereas most cells exhibit Cy3 and calcein fluorescence, we noticed the emergence of a small fraction of cells with intranuclear Cy3 fluorescence but not intracellular calcein fluorescence. After 24 h of compression, our images depict prevalent spatial separation of T30-Cy3 and calcein fluorescence, suggesting that most compressed cells with abundant amounts of T30-Cy3 in the nuclei are injured or dead.


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In conclusion, 5 h is the optimal duration of compression that balances intranuclear delivery and cell viability for bEnd.3 cells. (Figure 4A contains additional images that bolster this conclusion.) Effect of oligonucleotide concentration on intranuclear delivery and cell viability In this set of studies, we increased the concentration of oligonucleotides regularly by 2–3 times at each sample point. By using the alamarBlue assay, we confirmed that T30 at μM concentrations does not induce significant cytotoxicity to uncompressed bEnd.3 cells (Figure S15). By fluorescence microscopy, we observed increased intranuclear Cy3 fluorescence when we incubated bEnd.3 cells with higher concentrations of T30-Cy3 under compression for 5 h (Figure 3E and S16). Corroborating flow cytometry data show that the Cy3 MFI of treated cells increases with oligonucleotide concentration and reaches its maximal value when the concentration is 5 µM (Figure 3F). Staining by calcein-AM did not reveal severe loss in cell viability due to the concurrent application of μM concentrations of T30-Cy3 and coverslip-aided compression, as shown by the strong calcein-AM fluorescence inside the cells for all concentrations tested. In summary, incubation with micromolar concentrations (2.5–5 µM) of poly(T) under a mild pressure (1–10 Pa) for several hours (2–5 h) is optimal for the specific intranuclear delivery of poly(T) without drastically reducing the viability of bEnd.3 cells. We further proved that, for Kera-308 and RAW264.7 cells, a compression duration of 5 h and an oligonucleotide concentration of 2.5 µM are suitable parameters to achieve specific intranuclear delivery of T30-Cy3 without severe attenuation in cell viability (Figure 4B–C). Effect of the “compression-cum-poly(T)” treatment on inducing cellular oxidative stress A lingering concern over the feasibility of our “compression-cum-poly(T)” delivery method is hypoxia. That is, the cells may not have access to sufficient amounts of oxygen during coverslipaided compression, causing the generation of reactive oxygen species (ROS) inside the cells.53 In


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our first set of studies, we incubated bEnd.3 cells with 2.5 μM T30 under compression for 5 h, followed by measuring the cellular levels of ROS and staining the treated cells with CellROX Green, a cell-permeant dye that is weakly fluorescent in its reduced state but emits intense green fluorescence when oxidized by ROS.54 By measuring the fluorescence of the cells by flow cytometry, we detected a significant increase in green fluorescence of the compressed cells when compared to the untreated cells (Figure 5A–B). To benchmark our “compression-cum-poly(T)” method against existing methods of transfection, we measured the cellular level of ROS induced by the transfection of bEnd.3 cells with T30 via Lipofectamine, a commercially available transfection agent. Notably, provided the same treatment time of 5 h, the cellular level of ROS generated by our “compression-cum-poly(T)” method is not significantly different than that generated by Lipofectamine-aided delivery. In our second set of studies, we incubated bEnd.3 cells with T30 according to our “compression-cum-poly(T)” method and Lipofectamine-aided delivery for 5 h, removed the oligonucleotides, and allowed the cell samples to recover for another 48 h. Flow cytometry analysis reveals that the cellular levels of ROS generated by both methods of delivery subsided to that of untreated cells. To test the generalizability of our observations, we repeated both sets of studies above on two additional cell types (Kera-308 keratinocytes in Figure 5C–D and RAW 264.7 macrophages in Figure 5E–F) and obtained similar results to those derived from bEnd.3 cells. Our “compression-cum-poly(T)” method gives rise to higher intracellular ROS production to a level comparable to Lipofectamine-aided delivery, but such an increase in ROS level is reversible upon long-term cell recovery for all three cell types tested. Our data suggest limited long-term oxidative stress imposed to the cells due to our delivery method. At this point, we sought to interpret our previous data on cell viability in Figure 3 based on the argument of cellular oxidative stress. By literature precedent, imposing compression on myoblasts


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induces cellular injury (possibly due to the breakdown of the cell membrane and cytoskeleton), and the degree of injury typically increases with the duration of compression.55 To address the effect of compression time on cellular oxidative stress, we assessed the level of ROS associated with bEnd.3 cells that were compressed for 5 h or 24 h while incubated with the same concentration of T30 (2.5 μM), followed by staining the cells with CellROX Green (Figure S17). Our confocal imaging data revealed a significant enhancement in CellROX Green fluorescence for the cell samples treated for 24 h than those treated for 5 h, proof of significantly higher cellular levels of ROS induced by prolonged compression and consistent with our observations in Figure 3C-D. In addition, based on our published data, incubation of cells with high concentrations of DNA-coated nanoparticles (>100 μg/mL) reduces the viability of C166 endothelial cells, MOVAS smooth muscle cells, and RAW 264.7 macrophages.52 To probe the effect of oligonucleotide concentration on cellular oxidative stress, we assessed the level of ROS associated with bEnd.3 cells that were incubated with 2.5 μM T30 or 10 μM T30 while under compression for 5 h, followed by staining the cells with CellROX Green (Figure S17). Our confocal imaging data revealed a significant enhancement in CellROX Green fluorescence for the cell samples treated with 10 μM T30 than those treated with 2.5 μM T30, proof of significantly higher cellular levels of ROS induced by an elevated DNA concentration and in line with our observations in Figure 3E–F. Pathway for the cellular uptake of poly(T) under coverslip-aided compression We next examined the pathway that governs the transport of poly(T) through the cell membrane, the first major barrier between extracellular space and the nucleus, when bEnd.3 cells are under coverslip-aided compression. To do so, we pre-treated the cells with a series of pharmacological inhibitors of common endocytosis pathways without compression for 1 h, followed by incubating the cells with culture medium containing T30-Cy3 and inhibitors under compression for 5 h. We


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confirmed that bEnd.3 cells, compressed or uncompressed, remain largely viable when they are treated with these blockers for 1 h (Figure S18). Fluorescence images reveal that pre-treatment with dynasore (a blocker of dynamin-mediated uptake) and a mixture of sodium azide (NaN3) and 2- deoxyglycose (blockers of energy-dependent uptake) severely attenuated the cellular uptake of T30-Cy3, as evidenced by the weak Cy3 fluorescence in the cytosol or nucleus (Figure S19). Flow cytometry data show 58.0% ± 3.3% and 44.0% ± 4.8% drop in Cy3 MFI after pre-treatment with dynasore and NaN3/2-DG, suggesting involvement of energy and dynamin in the uptake of poly(T) by compressed cells. For bEnd.3 cells that were treated with chlorpromazine (a blocker of clathrinmediated endocytosis) and cytochalasin D (a blocker of actin filaments), we observed a modest reduction in Cy3 MFI by 15.2% ± 1.5% and 16.6% ± 3.3%, respectively, suggesting weaker involvement of clathrin and actin in cellular uptake. Additionally, we performed the same blocking studies with bEnd.3 cells that were uncompressed during cellular uptake (Figure S20). In agreement with the data for the compressed cells, our data reveal 79.3% ± 1.4% and 80.4% ± 0.3% reduction in the uptake of T30-Cy3 by the uncompressed bEnd.3 cells upon pre-treatment with dynasore and NaN3/2-DG, respectively, implying dynamin-mediated uptake and energy-dependent uptake, respectively. (For energy-dependent uptake, internalization of biomolecules requires the consumption of energy, typically in the form of ATP.56 One may inhibit energy-dependent uptake by incubating cells at 4 °C or with medium that contains an ATPase inhibitor, like NaN3.57 For dynamin-dependent uptake, dynamin, a membrane scission protein, is recruited to a budding vesicle that contains the biomolecule to be internalized by the cell. Scission of the vesicle at the cell membrane allows for the release of the vesicle into the cytoplasm.58) We also found 42.3% ± 1.8% and 51.3% ± 3.1% reduction in uptake by the uncompressed cells upon pre-treatment with


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chlorpromazine and cytochalasin D, respectively. These data suggest stronger involvement of clathrin and actin in the uptake of poly(T) by uncompressed bEnd.3 cells. Pathway for the intranuclear trafficking of poly(T) under coverslip-aided compression We investigated the pathway of intranuclear transport of poly(T) across the nuclear membrane, the second major barrier between extracellular space and the nucleus. In our ensuing pharmacological inhibition studies, we verified that bEnd.3 cells remain mostly viable following simultaneous coverslip-aided compression and treatment with the inhibitors of intranuclear trafficking (Figure S21). Initially, we considered cell cycle-dependent transport, because the temporary breakdown of the nuclear membrane during cell division may facilitate the passage of poly(T) to the nucleus.59 As microtubules play an important role in cell division, we pre-treated bEnd.3 cells with nocodazole without compression for 1 h (which disrupts the formation of microtubules60) before incubating them with T30-Cy3 under compression for 5 h. Yet, fluorescence images reveal predominant accumulation of T30-Cy3 in the nucleus of nocodazole-treated cells. When compared to those cells subject to identical conditions of compression without pharmacological inhibition, we detected by manual counting a mere 1.7% drop in the fraction of cells with significant intranuclear Cy3 fluorescence in the coverslip periphery upon treatment with nocodazole. Flow cytometry analysis, which measures the fluorescence of the entire cell, did not show any appreciable drop in intracellular Cy3 MFI, indicating minimal reduction in cellular uptake. Such data led us to exclude the possibility of cell cycle-dependent entry to the nucleus. We then considered the role of Imp β, which can bind to the NPC61 and regulate the transport of biomolecules and nanostructures62 between the cytoplasm and nucleus.63 We pre-treated bEnd.3 cells with ivermectin (an inhibitor of importin α/β-mediated nuclear import64,65) and importazole (an inhibitor of Imp β-mediated nuclear import66) for 1 h, followed by incubating the cells with


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T30-Cy3 under compression for 5 h. Fluorescence images show that pre-treatment by both inhibitors led to predominantly cytosolic accumulation with limited intranuclear entry (Figure 6A). Pre-treatment by ivermectin and importazole reduced the number of cells with significant intranuclear accumulation in the coverslip periphery by 91.7% ± 2.6% and 90.9% ± 2.7%, respectively, when compared to cells under identical conditions of compression but not treated with pharmacological inhibitors (Figure 6B). Flow cytometry analysis, while unable to capture the spatial redistribution of T30-Cy3 from the nucleus to cytosol, still reveals 25.7% ± 3.4% and 32.8% ± 7.5% reduction in intracellular Cy3 MFI after importazole and ivermectin treatment, respectively (Figure S22). This ~30% drop in cellular uptake of T30-Cy3 due to pharmacological inhibition corroborates our previous observation that coverslip-aided compression increases the cellular uptake of T30-Cy3 by 20.2% ± 1.9% (Figure 1D). Moreover, we used Lipofectamine 2000 to transfect bEnd.3 cells with siRNAs that specifically silence the expression of Imp β62 and nucleoporin 62 (Nup62), a constituent protein located near the central channel of the NPC67 that can bind with Imp β.68,69 By western blotting, we validated the attenuated expression of both proteins due to RNA interference (RNAi)-mediated inhibition (Figure S23). Next, we incubated these genetically silenced cells with T30-Cy3 under compression for 5 h and assessed its degree of intranuclear delivery. Fluorescence images reveal predominantly cytoplasmic accumulation of T30-Cy3 with limited intranuclear delivery (Figure 6C). Relative to the cells under identical conditions of compression but treated with non-targeting control siRNAs (siCtrl), genetic knockdown of Imp β and Nup62 drastically attenuated the number of cells in the coverslip periphery with significant intranuclear Cy3 fluorescence by 88.4% ± 2.0% and 87.1% ± 1.1%, respectively (Figure 6D). By flow cytometry, we noticed that genetic knockdown of Imp β and Nup62 attenuated the cellular uptake of T30-Cy3 by 26.7% ± 6.2% and 24.5% ± 7.0%,


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respectively (Figure S24), consistent with our flow cytometry data that show ~30% reduction in uptake for the cells inhibited by importazole and ivermectin. As a negative control, we confirmed that Lipofectamine-aided transfection of bEnd.3 cells with siCtrl did not noticeably influence the intranuclear accumulation of Cy3 fluorescence and overall Cy3 MFI of cells when compared to cells under identical conditions of compression but not treated with siRNAs (Figure S24). These data suggest that Imp β and Nup62 mediate the compression-aided intranuclear delivery of poly(T), although Imp β and Nup62 are not critical for cellular uptake of poly(T). We used super-resolution SIM to visualize the interaction between poly(T) and Imp β upon incubation of bEnd.3 cells with T30-Cy3 under compression for 2 h. By immunofluorescence staining for Imp β, we observed small, discontinuous patches of Cy3 fluorescence moderately colocalized with those of Imp β (with a Mander’s coefficient of 0.75) on the nuclear membrane, evidence of interaction between T30-Cy3 and Imp β (Figure 6E). In an analogous experiment based on uncompressed bEnd.3 cells, SIM images reveal spots of Cy3 fluorescence outside of the nucleus that are barely colocalized with the Imp β fluorescence on the nuclear membrane (Figure S25). The small Mander’s colocalization coefficient of 0.21 indicates weak correlation between poly(T) and Imp β for uncompressed cells, corroborating Figure 1 that intranuclear delivery of poly(T) requires gentle compression. These SIM data show that Imp β mediates compression-aided intranuclear delivery of poly(T) in bEnd.3 cells, consistent with our genetic knockdown data. Intranuclear delivery of antisense DNA oligonucleotides against EGFP We sought to use our “compression-cum-poly(T)” method to promote the intranuclear delivery of antisense oligonucleotides and gene inhibition. To ascertain a suitable length of a poly(T) segment to be appended to an antisense oligonucleotide (typically around 20-30 bases long), we firstly probed the effect of nucleotide length on the intranuclear accumulation of poly(T). In this


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connection, we incubated bEnd.3 cells with Cy3-labeled poly(T) of various lengths between 12 bases (T12-Cy3) and 120 bases (T120-Cy3) under coverslip-aided compression for 5 h. Representative confocal micrographs reveal intranuclear accumulation of poly(T) shorter than 80 bases long (T80-Cy3); on the contrary, poly(T) longer than 80 bases such as T100-Cy3 and T120Cy3 are generally not localized to the nuclei (Figure 7A and S26). Flow cytometry data show that T30-Cy3 enters cells the most among all nucleotide lengths tested (Figure 7B). T12-Cy3 and T20Cy3 exhibit less intracellular accumulation than T30-Cy3, possibly due to insufficient amounts of T in their sequences. As the length of poly(T) increases from 30 to 120 bases, we observed an inverse relationship between nucleotide length and cellular uptake. Next, we chose an 18-base long antisense DNA sequence that targets the gene encoding EGFP (AS-EGFP; sequence information in Table S1) for intranuclear delivery. EGFP is a reporter protein routinely used for evaluating the efficiency of transfecting cells with antisense oligonucleotides.70 Recall from Figure 7B that the most optimal length of poly(T) for compression-aided intranuclear delivery from 25 bases (between T20 and T30) to 45 bases (between T30 and T60). Therefore, we strategically appended short poly(T) segments of 6, 12, or 24 bases long to the 3’ or 5’ end of ASEGFP (sequence information in Table S1), keeping the total length of the poly(T)-appended ASEGFP sequence between 25 and 45 bases long. [The poly(T) segment appended to the AS-EGFP sequence only aids its intranuclear delivery for silencing the expression of EGFP. To our knowledge, the poly(T) segment does not target any known gene or protein.] After incubating bEnd.3 cells with Cy3-labeled, poly(T)-appended AS-EGFP under compression for 5 h, we observed by confocal imaging significantly enhanced intranuclear accumulation (Figure 7C) and cellular uptake (Figure 7D) of poly(T)-appended AS-EGFP when the length of poly(T) segment


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increases from 6 bases to 24 bases. Such an observed effect of poly(T) length holds regardless of the site of attachment at the 3’ or 5’ end of the AS-EGFP strand (Figure S27). After that, we addressed whether improved intranuclear accumulation of AS-EGFP due to combined coverslip-aided compression and poly(T) attachment translates to enhanced inhibition of EGFP. In this set of experiments, we used Lipofectamine to transfect bEnd.3 cells with EGFPexpressing plasmids on Day 1. On Day 3, we incubated the EGFP-expressing bEnd.3 cells with 2.5 μM AS-EGFP appended to various lengths of short poly(T) segments (6, 12, or 24 bases) to its 3’ end under compression for 5 h. Subsequently, we removed the coverslip and DNA-containing medium, and replenished the cells with fresh DNA-free medium for recovery. On Day 5, we assessed the EGFP fluorescence of the cells by flow cytometry (Figure 7E). We verified that, without delivering AS-EGFP or poly(T)-appended AS-EGFP into the EGFP-expressing bEnd.3 cells, expression of EGFP on Day 5 was not significantly lower than that on Day 3 (Figure S28). Relative to untreated EGFP-expressing cells, the mean levels of reduction in EGFP expression for the cells treated with AS-EGFP, AS-EGFP-T6, AS-EGFP-T12, and AS-EGFP-T24 under compression were 18.8% ± 4.0%, 25.8% ± 1.9%, 27.6% ± 5.4%, and 41.3% ± 6.0%, respectively. We believe that 41% is a notable level of reduction in EGFP expression, because Rosi et al. showed that treating EGFP-expressing endothelial cells with SNA nanostructures bearing the same antisense DNA sequence [but without poly(T) modification] at similar DNA amounts to our delivery method (~1 nmol) merely led to 11% of reduction in mean EGFP expression.71 In other words, our data show that the simultaneous attachment of the poly(T) segment to AS-EGFP and coverslip-aided compression significantly improved antisense-mediated inhibition of EGFP. As control experiments, we treated EGFP-expressing bEnd.3 cells with poly(T)-appended AS-EGFP sequences at the same DNA concentration (2.5 μM) but without coverslip-aided compression (i.e.,


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gymnotic delivery). In this case, the mean levels of EGFP knockdown due to gymnotic delivery of AS-EGFP, AS-EGFP-T6, AS-EGFP-T12, and AS-EGFP-T24 were 1.4% ± 7.4%, 12.6% ± 2.5%, 13.8% ± 3.5%, and 15.7% ± 11.9%, respectively, statistically insignificant relative to untreated cells. Of note, appending the T24 segment to the 5’ end of AS-EGFP (T24-AS-EGFP) instead of 3’ end (AS-EGFP-T24) did not significantly affect the efficiency of EGFP knockdown (Figure 7F), in line with our observation that the site of poly(T) attachment did not drastically influence intranuclear accumulation (Figure 7C and S27). To benchmark our “compression-cum-poly(T)” method against existing methods of transfection, we measured the level of gene inhibition afforded by Lipofectamine (Figure S29). We transfected EGFP-expressing bEnd.3 cells with 25 nM AS-EGFP or AS-EGFP-T24 by using Lipofectamine without compression for 5 h, followed by 48 h of recovery. Per the recommendation by the manufacturer, our chosen DNA concentration for Lipofectamine-aided delivery is 100-fold lower than that used in our delivery method. Using elevated concentrations of DNA (in the μM range) necessitates proportionately higher amounts of Lipofectamine, causing severe cytotoxicity (data not shown). Lipofectamine-aided delivery of AS-EGFP, AS-EGFP-T24, and T24-AS-EGFP (sequence information in Table S1) led to 15.5% ± 1.7%, 4.5% ± 3.4%, and 3.6% ± 8.9% reduction in mean EGFP expression, respectively, not statistically significant relative to untreated cells yet consistent with previous observations by Rosi et al.71 Appending the T24 segment did not lead to higher gene inhibition via Lipofectamine-aided delivery. Our data echo the hard-to-transfect nature of bEnd.3 cells.38 To derive some insights into these observations, we captured extra confocal images of bEnd.3 cells that were (1) transfected with Cy3-labeled AS-EGFP-T24 under coverslipaided compression and (2) transfected with Cy3-AS-EGFP via Lipofectamine (Figure S30). These confocal images show that, while our “compression-cum-poly(T)” method resulted in specific


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intranuclear accumulation of Cy3 fluorescence, Lipofectamine-aided transfection typically gave rise to random patches of Cy3 fluorescence with no obvious specificity to the nucleus of bEnd.3 cells. In conclusion, our data illustrate the roles of gentle compression and poly(T) attachment in synergistically promoting not only the intranuclear delivery of antisense oligonucleotides but also the inhibition of an exogenous target gene that encodes a reporter protein. Intranuclear delivery of antisense DNA oligonucleotides against MALAT1 Cellular uptake of antisense oligonucleotides and their diffusion to the cell nucleus is an established mechanism of action of antisense-mediated gene inhibition.72 Recent evidence pointed to the robust activity of RNase H1-dependent antisense oligonucleotides in both the cytosol and nucleus of cells.73 That being said, we acknowledge that inhibition of EGFP does not necessarily happen in the nucleus. To more convincingly correlate the (1) level of intranuclear accumulation of antisense oligonucleotides to the (2) resultant level of gene inhibition, we further extended our “compression-cum-poly(T)” method to promote the delivery of antisense oligonucleotides to the nuclei of bEnd.3 endothelioma cells and the inhibition of MALAT1, a lncRNA cancer gene highly expressed in the cell nucleus.74 A predictor of cancer metastasis,75 MALAT1 is upregulated in various types of cancer (e.g., hepatocellular carcinoma76 and prostate cancer77) and linked to the proliferation of cancer cells78 and growth of blood vessels.79 As mentioned above, the poly(T) segment serves to aid the intranuclear delivery of the antisense oligonucleotides against MALAT1 (AS-MALAT1).80 To our knowledge, poly(T) does not target any known gene or protein. On Day 1, we incubated bEnd.3 cells with 2.5 μM AS-MALAT1, AS-MALAT-1 appended to a T24 segment at its 3’ end (AS-MALAT1-T24) or 5’ end (T24-AS-MALAT1), or a control scrambled sequence (AS-MALAT1-scramble) under coverslip-aided compression for 5 h. (See sequence information in Table S1.) Subsequently, we removed the coverslip and DNA-containing


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medium, and replenished the cells with fresh DNA-free medium for recovery. On Day 3, we extracted the RNA from the treated cells and measured the expression levels of MALAT1 by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). By appending T24 to the 3’ or 5’ end of AS-MALAT1 and applying compression, we detected by confocal imaging more abundant delivery of AS-MALAT1 to the nuclei of bEnd.3 cells (Figure 8A). qRT-PCR analysis reveals that the mean expression levels of MALAT1 for the cells treated with AS-MALAT1-T24, T24-AS-MALAT1 under compression were significantly reduced by 46.6 % ± 19.3% and 44.8% ± 9.7%, respectively, when compared to untreated control cells (Figure 8B). [As Sprangers et al. previously showed that the knockdown efficiency of MALAT1 in A549 lung cancer cells depends heavily on the concentration of antisense oligonucleotides added,31 we believe that it is possible to improve the knockdown efficiency of MALAT1 by incubating bEnd.3 cells with higher amounts of poly(T)-appended AS-MALAT1 under compression. Yet, we showed that adding an excessively high concentration of DNA to the cells induced cytotoxicity (Figure 3E-F) and oxidative stress to the cells (Figure S17).] Conversely, treating the cells with AS-MALAT1 (without T24 appended) under compression only led to a modest reduction in MALAT1 expression level by 11.8% ± 8.9%. We finally benchmarked the levels of MALAT1 inhibition due to our “compression-cum-poly(T)” method against Lipofectamine-mediated delivery. Like our previous studies on EGFP inhibition (Figure 7E–F) and per the recommendation by the manufacturer, we incubated bEnd.3 cells with Lipofectamine and only 25 nM AS-MALAT1, 100 times lower than the concentration used in our “compression-cum-poly(T)” method, lest we compromise cell viability by incubating the cells with μM concentrations of DNA and disproportionately high amounts of Lipofectamine. By qRTPCR analysis, we observed 26.4% ± 28.9% of MALAT-1 inhibition when the cells were transfected with AS-MALAT1 via Lipofectamine when compared to untreated cells, lower the mean level of


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MALAT1 inhibition for those cells treated with AS-MALAT1-T24 under compression (~45%). By confocal imaging, bEnd.3 cells that were incubated with Cy3-labeled AS-MALAT1-T24 under compression exhibited specific accumulation of Cy3 fluorescence inside the nucleus. On the contrary, we detected random patches of Cy3 fluorescence with no specificity to the nucleus for cells that were transfected with Cy3-AS-MALAT2 via Lipofectamine (Figure S31). These data showcase the utility of our “compression-cum-poly(T)” method to promote the intranuclear delivery of antisense oligonucleotides and the inhibition of an intranuclear cancer gene.

CONCLUSION We have proven that poly(T)-appended DNA oligonucleotides abundantly accumulate inside the nuclei of three different “hard-to-transfect” mammalian cell types (i.e., brain endothelial cells, keratinocytes, and macrophages) with high specificity (relative to the cytosol) when the cells are under gentle compression afforded by the weight of a glass coverslip for several hours. Compression and DNA sequence are pivotal parameters for intranuclear delivery. Applying a pressure of 1–10 Pa (several orders of magnitude lower than that used in many existing non-viral physical methods) to cells is sufficient to direct the delivery of DNA to the nucleus that are otherwise inaccessible to uncompressed cells. As our method of intranuclear delivery requires the attachment of poly(T) segments, future studies will focus on elucidating the molecular interactions between poly(T)-rich oligonucleotides and the nucleus. Our “compression-cum-poly(T)” delivery method is simple and compatible to conventional culture vessels. Careful adjustment of applied pressure, compression time, and oligonucleotide concentration is crucial for effective intranuclear delivery without severely compromising cell viability or inducing cellular oxidative stress during compression. At the mechanistic level, both


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Imp β and Nup62 mediate the transport of poly(T) to the nucleus of bEnd.3 endothelial cells under gentle compression. Finally, we have applied our “compression-cum-poly(T)” method to promote the delivery of antisense DNA oligonucleotides to the nuclei of bEnd.3 endothelioma cells and the inhibition of an exogenous reporter gene (EGFP) as well as an intranuclear lncRNA cancer gene (MALAT1). In terms of intranuclear delivery and gene inhibition, our method outperforms the delivery of the same antisense DNA oligonucleotides in cases without gentle compression or poly(T) attachment, for both target genes tested. Future investigations will focus on enhancing intranuclear delivery of other types of nucleic acids (e.g., siRNAs and ribozymes) that target other genes and gene products as well as delivery to other cell types (e.g., non-adherent, different morphologies, and different nucleus-to-cytosol ratios).

EXPERIMENTAL SECTION Compression-mediated intranuclear delivery. Cells were seeded in flat-bottom 24-well plates (SPL Life Sciences) at a density of 2×105 cells per well. When cells reached 70-80% confluency, complete DMEM was removed, and the cells were rinsed with phosphate-buffered saline (PBS; pH = 7.4) twice. 0.25 mL of DNA oligonucleotides (2.5 μM in OptiMEM) was added to each well. A round coverslip of 12 mm in diameter (Marienfeld Superior), pre-sterilized by sonication in 75% ethanol for 1 h and pre-rinsed with PBS, was immediately placed on top of the cells. Upon various durations of compression, the coverslip and DNA containing-medium were removed, and cells were washed twice with PBS to remove the excess DNA. Intracellular distribution of DNA. Cells were fixed with 0.2 mL of 4% paraformaldehyde (PFA) in PBS for 10 min, rinsed with PBS for three times and then stained with 0.2 mL of 5 µg/mL 4’,6-diamidine-2’-phenylindole dihydrochloride (DAPI; Sigma-Aldrich) in PBS for 5 min. Cells


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were washed with PBS for an additional three times and observed under an Olympus FV1000 confocal microscope. The excitation laser and emission filters for DAPI are 405 nm and 470 nm, respectively. The excitation laser and emission filters for Cy3 are 543 nm and 620 nm, respectively. Estimation of intranuclear delivery efficiency. Over 100 cells in multiple images taken in different coverslip peripheral areas of the same sample were analyzed. Intranuclear delivery efficiency was determined by counting the percentage of cells with significant intranuclear fluorescence based on three times of manual counting. Statistical significance was calculated by one-way ANOVA with Tukey post-hoc. 3D Confocal imaging. Seeded in 35 mm confocal dishes (SPL Life Sciences), cells were stained with 0.25 mL of 0.01 mg/mL Hoechst 33342 for 10 min, rinsed with PBS, and incubated with 0.25 mL of 2.5 μM Cy3-labeled DNA (in OptiMEM) for 5 h under coverslip compression. A representative cell with profuse intranuclear accumulation of Cy3-labeled DNA was picked. A Zstack series of images, obtained by focusing the top and bottom of the cell with a separation distance of 0.57 µm between two adjacent slices, were collected for 3D reconstruction by using the NIS-Elements AR software (Nikon). Quantification of fluorescence intensity in the nuclei and cytosol of bEnd.3 cells collected from 3D confocal microscopy was also performed by using the NIS-Elements AR software (Nikon). SIM. Cells were grown in 35 mm confocal dishes. After incubation with 0.25 mL of 2.5 µM Cy3-labeled DNA under coverslip compression for 5 h, cells were fixed in 4% PFA for 10 min at room temperature (RT), washed with PBS, and stained with DAPI for 5 min. Cells were imaged by a Nikon N-SIM microscope using the 3D-SIM mode and a 100× 1.49 NA oil-immersion objective. The excitation and emission wavelengths of DAPI are 395-415 nm and 435-485 nm,


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respectively. The excitation laser and emission filters for Cy3 are 556-566 nm and 570-640 nm, respectively. 3D-SIM images were reconstructed by using the NIS-Elements AR software (Nikon). STED microscopy. Cells were grown in 35 mm confocal dishes. After incubation with 0.25 mL of 2.5 µM Cy3-labeled DNA under compression for 5 h, cells were fixed in 4% PFA for 10 min at RT, washed with PBS, and stained with DAPI for 5 min. Confocal imaging of DAPI fluorescence and STED imaging of Cy3 fluorescence was performed on a Leica TCS SP8 STED microscope equipped with an HC PL APO CS2 100x/1.40 oil objective. Images were acquired by using a 200 Hz bidirectional scanner, with a 405 nm diode laser and a STED laser (775 nm) detected with hybrid detectors in the range of 422–501 nm and 729–794 nm, respectively. Pixel size was 20–50 nm for all confocal and STED images acquired. Laser powers were optimized for each sample. Cell viability. The treated cells (pre-seeded in a 24-well plate) were rinsed with PBS twice, and incubated with 0.2 mL of 0.5 μg/mL calcein-AM (Life Technologies) in PBS for 10 min at 37 °C. After removing the working solution and washing with PBS twice, cells were imaged by a Nikon Ti-E motorized inverted fluorescence microscope. The excitation and emission filters for calceinAM are 465-495 nm and 515-555 nm, respectively. Cellular oxidative stress. The treated cells (pre-seeded in a 24-well plate) were incubated with 0.2 mL of staining solution [5 μM CellROX Green Reagent (Invitrogen; C10444) formulated in OptiMEM] for 30 min at 37 °C. Cells were rinsed three times with PBS, trypsinized, collected by centrifugation at 4,000 rpm for 10 min, resuspended in PBS, and analyzed by a BD FACSVerse flow cytometer. By tuning the forward and side scattering parameters to eliminate dead cells and debris, 10,000 gated events were collected for analysis. Cells were excited by a laser at 488 nm, and the emitted fluorescence detected by using a 527/33 nm bandpass filter. Analytical gates were set such that less than 5% of the untreated cells exceeds the gate and falls in the CellROX Green-


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positive region. Data were analyzed by one-way ANOVA with Tukey post-hoc by the IBM SPSS software (Version 19). Data represent mean ± SD from three independent experiments. Cellular uptake of DNA by flow cytometry. Seeded in a 24-well plate, cells were incubated with fluorescently labeled DNA oligonucleotides at different concentrations for different durations of time under coverslip compression. As a negative control, cells were untreated or incubated with the same DNA oligonucleotides but without coverslip compression. After treatment, cells were washed with PBS twice, trypsinized, collected by centrifugation, and resuspended in PBS for analysis by a BD FACSVerse flow cytometer. By tuning the forward and side scatter parameters to eliminate dead cells and debris, 10,000 gated events were collected for analysis. Fluorescence emission was detected from the fluorescence channel (586/42 nm) for Cy3. Triplicate counts were made for each treatment. The mean fluorescence intensity after coverslip compression was compared to the control groups after deducting the background fluorescence of the untreated cells. Antisense-based knockdown of EGFP. Seeded in 24-well plates at a density of 3×105 cells per well 24 h in advance, bEnd.3 cells were incubated with 0.25 mL of transfection medium. Formulated in OptiMEM, the transfection medium consists of 7 μg/mL Lipofectamine 2000 and 2.5 μg/mL pEGFP-N1 DNA plasmid [which contains the EGFP gene; Clontech]. After 5 h, the transfection medium was switched to complete DMEM for overnight recovery. The cells were then cultured in DMEM supplemented with 0.5% FBS for 24 h. Next, the EGFP-expressing cells were divided into different experimental groups and treated with 0.25 mL of 2.5 μM AS-EGFP in OptiMEM for 5 h, with or without coverslip compression. In some groups, the AS-EGFP sequence contains an extra T6, T12, or T24 segment at its 3' or 5' end. As control experiments, EGFPexpressing cells were transfected with 0.25 mL of OptiMEM containing 25 nM AS-EGFP or ASEGFP-T24 and 1.5 μL of Lipofectamine 2000. After 5 h of treatment, for all experimental groups,


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the coverslip and DNA-containing medium were removed. Cells were left to be recovered in complete medium at 37 °C overnight and cultured in DMEM supplemented with 0.5% FBS for another 24 h. Cells were trypsinized, collected by centrifugation at 4,000 rpm for 10 min, resuspended in PBS, and analyzed by a BD FACSVerse flow cytometer. (Refer to the “Cellular Oxidative Stress” section above for detailed settings of flow cytometry.) Relative EGFP expression in each treatment group was calculated by dividing the percentage of EGFP-positive cells in the treated group by the percentage of EGFP-positive cells in the untreated group and multiplying by 100%. Data were analyzed by one-way ANOVA with Tukey post-hoc by the IBM SPSS software (Version 19). Data represent mean ± SD from three independent experiments. Antisense-based knockdown of MALAT1. Seeded in 24-well plates at a density of 3×105 cells per well 24 h in advance, bEnd.3 cells were divided into different experimental groups and treated with 0.25 mL of 2.5 μM AS-MALAT1 in OptiMEM for 5 h, with or without coverslip compression. In some groups, the AS-MALAT1 sequence contains an extra T24 segment at its 3' or 5' end. As control experiments, bEnd.3 cells were transfected with 0.25 mL of OptiMEM containing 25 nM AS-MALAT1 or AS-MALAT1-T24 and 1.5 μL of Lipofectamine 2000. After 5 h of treatment, for all experimental groups, the coverslip and DNA-containing medium were removed. Cells were left to be recovered in complete medium at 37 °C overnight and cultured in DMEM supplemented with 0.5% FBS for another 24 h. For all samples, RNA was extracted from the treated cells by using the Trizol Reagent (Invitrogen) per the manufacturer's protocol. RNA concentration was measured by using a NanoDrop™ One spectrophotometer. The extracted RNA was reverse transcribed into cDNA by using the RevertAid First Strand cDNA Synthesis Kit per the manufacturer’s instructions (Thermo Fisher Scientific). qPCR was performed by using ready-to-use TaqMan primers and probes (Applied Biosystem) specific for MALAT1 (Mm01227912_s1) and glyceraldehyde-3-


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phosphate dehydrogenase (GAPDH; Mm99999915_g1). The relative level of MALAT1 expression was normalized to that of GAPDH and the relative expression levels were calculated with the 2-Ct method. Data were analyzed by one-way ANOVA with Tukey post-hoc by the IBM SPSS software (Version 19). Data represent mean ± SD from three independent experiments.

ASSOCIATED CONTENT Supporting Information Synthesis of oligonucleotides, cell culture, transport of fluid under coverslip compression, staining with LysoTracker, gentle compression at different pressures, live cell imaging, cell viability by the alamarBlue assay, cellular oxidative stress by the CellROX Green assay, determining the pathway of trafficking by pharmacological blocking, determining the pathway of trafficking by RNA interference (RNAi), immunofluorescence staining western blotting, calculation of pressure exerted by the weight of a glass coverslip on the cells beneath, calculation of areal fraction of coverslip periphery with significant intranuclear accumulation of DNA, and intracellular distribution of poly(T) due to transfection via Lipofectamine. The Supporting Information is available free of charge on the ACS Publications website http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Email: [email protected] ORCID Chung Hang Jonathan Choi: 0000-0003-2935-7217 Author Contributions


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#These authors (Z.C. and H.L.) contributed equally. Notes The authors declare no competing financial interests.

ACKNOWLEDGMENTS This work was in part supported by a General Research Fund (Project No.: 14302916) from the Research Grants Council (RGC) and a National Natural Science Foundation of China (NSFC)/RGC Joint Research Scheme grant (Project No.: N_CUHK434/16). It was supported by the Chow Yuk Ho Technology Centre for Innovative Medicine at The Chinese University of Hong Kong (CUHK). C.H.J.C. acknowledges a Croucher Startup Allowance and a Croucher Innovation Award from the Croucher Foundation. Z.C. and L.Z. are funded by the Shun Hing Institute of Advanced Engineering at CUHK (Project No.: BME-8115047). We thank Ada Kong (School of Life Sciences, CUHK) for guidance in flow cytometry, Gary Lai (Department of Physics, CUHK) for guidance in SIM, and Freddie Kwan (School of Life Sciences, CUHK) for guidance in confocal imaging. We thank Chun Kit Choi, Yifei Yao, and Yee Ting Elaine Chiu (Department of Biomedical Engineering, CUHK) for discussions.

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Figure 1. Intranuclear delivery of polythymidine oligonucleotides [poly(T)] via coverslip-aided compression. (A) Schematic illustration of the experimental setup. Seeded in a 24-well plate, cells were incubated with fluorescently labeled poly(T) under coverslip compression for several hours. (B) After coverslip-aided compression of 2.5 µM Cy3-labeled T30 DNA (T30-Cy3) for 5 h, the bEnd.3 cells in the coverslip periphery (~44% of the area under compression by the coverslip) exhibit strong Cy3 fluorescence, but those in the central region of the coverslip and the uncompressed region do not. (C) After compression, the coverslip was removed and the


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compressed cells were stained with DAPI. By confocal imaging, the Cy3 fluorescence is localized in the nuclei of bEnd.3, Kera-308, and RAW 264.7 cells. (Blue: DAPI; Red: T30-Cy3; Purple: merged). Without compression, the Cy3 fluorescence does not notably colocalize with the nuclei. The Mander’s colocalization coefficients between Cy3 and DAPI are displayed in yellow. (D) By flow cytometry analysis, the Cy3 mean fluorescence intensity (MFI) of the compressed bEnd.3 cells is ~20% higher than that of uncompressed cells. 100 a. u. (arbitrary units) refers to the intracellular MFI resulting from a reference study that entails the incubation of bEnd.3 cells with 2.5 µM of T30-Cy3 for 5 h with coverslip compression. Cells untreated with T30-Cy3, with or without compression, have limited values of Cy3 MFI. Statistical comparisons were analyzed by the one-way ANOVA with Tukey post-hoc. **P < 0.01. ****P < 0.0001. Error bar indicates standard deviation resulting from three independent experiments.


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Figure 2. Additional evidence of coverslip-aided delivery of poly(T) to the cell nuclei. (A) (Panel 1) A representative 3D reconstructed confocal image of a bEnd.3 cell incubated with T30-Cy3 (red) under compression shows pronounced accumulation in the nucleus (blue, Hoechst 33342). (Panel 2) Cy3 MFI of the 26 z-stack confocal images is the highest at the center of a representative bEnd.3 cell that was stained with Hoechst 33342 (blue) incubated with T30-Cy3 (red) for 5 h under coverslip compression. 100 a. u. refers to the intracellular MFI from a reference z-stack image with the highest MFI for Cy3 or Hoechst 33342. (Panel 3) Selected snapshot z-stack cross-sectional confocal images from the same representative cell as described in Panel 2. After coverslip-aided delivery for 5 h, T30-Cy3 (red) accumulates in the nucleoplasm of bEnd.3 cells by super-resolution


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(B) structured illumination microscopy (SIM) and (C) stimulated emission depletion (STED) microscopy. Blue: DAPI. (D) By confocal imaging, untreated bEnd.3 cells do not show appreciable fluorescence. For the cells incubated with T30-FITC for 5 h without compression, the FITC fluorescence (green) is localized in the cytosol but not the nucleus (blue, DAPI). For those incubated with T30-FITC under coverslip compression, the FITC fluorescence is mostly localized in the nucleus. Incubating the cells with free FITC molecules under compression did not yield appreciable fluorescence. (E) By coverslip-aided delivery for 5 h, T30-Cy3 more effectively accumulates in the nucleus (blue, DAPI) than A30-Cy3. Cytosolic localization of A30-Cy3 indicates sequence-specific intranuclear accumulation of DNA. Attaching a T12 segment to the 3’ end of an 18-base long random DNA sequence (Random18-Cy3) to form a 30-base sequence (Random18-T12-Cy3) enhances intranuclear delivery. Red: Cy3-labeled DNA. (F) Flow cytometry data reveal more Cy3 fluorescence in bEnd.3 cells that were incubated with “T-rich” sequences under coverslip compression. Statistical comparisons were analyzed by the t test. Error bars denote standard deviations resulting from triplicate experiments. *P < 0.05, ***P < 0.001.


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Figure 3. Optimizing the compression-aided intranuclear delivery of poly(T) for bEnd.3 cells. (A) Cells were largely viable with significant intranuclear accumulation of T30-Cy3 (red) after 5 h of compression (~2.2 Pa), as evidenced by the strong calcein fluorescence (green). Compression at higher pressures, by adding defined weights on top of the coverslip, severely reduced intranuclear delivery and cell viability. (B) Cy3 MFI of cells as a function of pressure after 5 h of compression. (C) Compression (2.2 Pa) over 12 h led to significant intranuclear delivery of T30-Cy3 at the expense of cell viability, as revealed by the presence of cells with strong intranuclear Cy3


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fluorescence yet weak intracellular calcein fluorescence. (D) Cy3 MFI of cells as a function of compression time. (E) Upon incubation with T30-Cy3 at different concentrations under compression for 5 h, staining by calcein-AM did not reveal much loss in viability. Incubation with T30-Cy3 at 2.5 µM (and higher concentrations) led to significant intranuclear delivery. (F) Cy3 MFI of cells as a function of T30-Cy3 concentration after 5 h of compression. Error bars denote standard deviation resulting from triplicate experiments. For all flow cytometry data (Figure 2B, 2D, and 2F), 100 a. u. (arbitrary units) refers to the intracellular MFI resulting from a reference study that entails the incubation of bEnd.3 cells with 2.5 µM of T30-Cy3 for 5 h with coverslip compression (2.2 Pa). One-way ANOVA with Tukey post-hoc was used for all statistical comparisons. **P < 0.01, ***P < 0.001, ns: not significant.


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Figure 4. Effect of the “compression-cum-poly(T)” treatment on cell viability. (A) bEnd.3, (B) Kera-308, and (C) RAW264.7 cells were incubated with 2.5 µM T30-Cy3 (red) under coverslipaided compression (2.2 Pa) for 5 h. The coverslip was then removed, and the compressed cells were stained with calcein-AM. T30-Cy3 mostly localizes in the cell nuclei (arrow) for all cell types. The cells remain largely viable, as evidenced by the intense intracellular calcein fluorescence (green).


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Figure 5. Effect of the “compression-cum-poly(T)” treatment on inducing cellular oxidative stress. (A)–(B) bEnd.3, (C)–(D) Kera-308, and (E)–(F) RAW264.7 cells were incubated with 2.5 µM T30


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under coverslip-aided compression for 5 h and stained by CellROX Green. Representative flow cytometry data of the treated cells reveal significantly higher levels of reactive oxygen species (ROS) than the untreated cells. As control studies, transfecting these cell types with 25 nM T30Cy3 via Lipofectamine for 5 h also led to statistically higher cellular levels of ROS than untreated cells, but the resultant level of ROS was similar to that due to our “compression-cum-poly(T)” method. For our “compression-cum-poly(T)” method and Lipofectamine-aided transfection, the levels of ROS of the treated cells subsided to the levels of untreated cells after 48 h of recovery. One-way ANOVA with Tukey post-hoc was used for statistical comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant.


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Figure 6. Mechanism for the intranuclear delivery of poly(T) under coverslip-aided compression in bEnd.3 cells. (A) After 5 h of compression, T30-Cy3 (red) mostly accumulated in the cytosol when bEnd.3 cells were pre-treated with importazole and ivermectin; on the contrary, T30-Cy3


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mostly resided in the nuclei (DAPI; blue) upon pre-treatment with nocodazole. (B) With all cell samples under compression, pre-treatment with importazole and ivermectin reduced the uptake of T30-Cy3 by 90.9% ± 2.7% and 91.7 ± 2.6%% in the coverslip periphery, respectively, relative to cells not treated with pharmacological inhibitors. Pre-treatment with nocodazole did not reduce cellular uptake drastically. (C) After 5 h of compression, T30-Cy3 mostly accumulated in the cytosol when bEnd.3 cells were treated with siRNA that specifically silences the expression of importin β (siImpβ) and nucleoporin 62 (siNup62); by contrast, T30-Cy3 mostly resided in the nuclei when the cells are treated with non-targeting siRNA (siCtrl). (D) With all cell samples under compression, RNAi-mediated genetic knockdown of Imp β and Nup62 attenuated the uptake of T30-Cy3 under compression by 88.4% ± 2.0% and 87.1% ± 1.1%in the coverslip periphery, respectively, relative to the cells treated with non-targeting control siRNA (siCtrl). Transfection of bEnd.3 cells with siCtrl did not significantly influence the uptake of T30-Cy3. Error bars denote standard deviations resulting from triplicate experiments. One-way ANOVA with Tukey post-hoc was used for all statistical comparisons. ****P < 0.0001, ns: not significant. (E) Intranuclear localization of T30-Cy3 inside compressed bEnd.3 cells by super-resolution SIM. Mander’s colocalization coefficient between T30-Cy3 (red) and Imp β (green) is displayed in yellow.


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Figure 7. Intranuclear delivery of antisense DNA oligonucleotides against EGFP (AS-EGFP) by our “compression-cum-poly(T)” method. (A)–(B) Confocal images and flow cytometry data


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reveal an optimal oligonucleotide length that maximizes delivery of Cy3-labeled poly(T) (red) to the nucleus (DAPI; blue) of bEnd.3 cells upon 5 h of compression. Purple color indicates accumulation of poly(T) in the nucleus. 100 a. u. (arbitrary units) refers to the intracellular MFI resulting from a reference study that entails the incubation of bEnd.3 cells with 2.5 µM T30-Cy3 for 5 h with compression. (C)–(D) Confocal images and flow cytometry data show the enhanced delivery of Cy3-labeled AS-EGFP (red) to the nucleus of bEnd.3 cells (blue) with longer poly(T) segments attached to AS-EGFP after 5 h of compression. 100 a. u. (arbitrary units) refers to the intracellular MFI resulting from a reference study that entails the incubation of bEnd.3 cells with 2.5 µM of AS-EGFP for 5 h with compression. (E) Knockdown of EGFP due to coverslip-aided intranuclear delivery of AS-EGFP appended with a poly(T) segment of various lengths to the 3’ end. After 5 h of compression, the EGFP-expressing bEnd.3 cells were recovered for 48 h before measuring their fluorescence by flow cytometry. EGFP knockdown due to compression-aided delivery is higher than that achievable without compression, and knockdown increases with the length of poly(T) appended. (F) Knockdown of EGFP due to coverslip-aided intranuclear delivery of AS-EGFP with T24 appended to the 3’ or 5’ end. For (E) and (F), “100%” expression refers to the expression level of untreated EGFP-expressing cells (calculated based on the fraction of EGFPpositive cells). Level of statistical significance labeled on top of each treatment bar (without bracket) was made with reference to untreated EGFP-expressing bEnd.3 cells (the leftmost bar). Level of statistical significance labeled on top of a bracket was made by comparing the data between the two bars indicated by the bracket. One-way ANOVA with Tukey post-hoc was used for all statistical comparisons. *** P < 0.001, ns: not significant. Error bars denote standard deviations resulting from triplicate experiments.


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Figure 8. Intranuclear delivery of antisense DNA oligonucleotides against MALAT1 to bEnd.3 cancer cells by our “compression-cum-poly(T)” method. (A) Confocal images show more abundant delivery of Cy3-labeled AS-MALAT1 (red) to the nucleus of bEnd.3 cells (blue) with the attachment of T24 to the 3’ or 5’ end of the sequence after 5 h of compression. Transfection with Cy3-AS-MALAT1 (red) via Lipofectamine did not lead to specific accumulation in the nuclei. (B) Knockdown of MALAT1. After 5 h of compression, the bEnd.3 cells were recovered for 48 h before measuring the expression of MALAT1 by qRT-PCR. MALAT-1 knockdown due to compression-aided delivery was higher when T24 was attached to AS-MALAT1. MALAT1 expression was normalized to that of GAPDH. “100%” expression refers to the expression level of untreated bEnd.3 cells. Level of statistical significance labeled on top of each treatment bar was made with reference to untreated bEnd.3 cells (the leftmost bar). One-way ANOVA with Tukey post-hoc was used for all statistical comparisons. ** P < 0.01, ns: not significant. Error bars denote standard deviations resulting from triplicate experiments.


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