Modulation of PEI-Mediated Gene Transfection through Controlling

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Modulation of PEI-mediated gene transfection through controlling cytoskeleton organization and nuclear morphology via nanogrooved topographies Peng-Yuan Wang, Yen-Shiang Lian, Ray Chang, Wei-Hao Liao, Wen-Shiang Chen, and Wei-Bor Tsai ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00617 • Publication Date (Web): 10 Oct 2017 Downloaded from http://pubs.acs.org on October 16, 2017

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ACS Biomaterials Science & Engineering

Modulation of PEI-mediated gene transfection through controlling cytoskeleton organization and nuclear morphology via nanogrooved topographies

Peng-Yuan Wang,1,2 Yen-Shiang Lian,3 Ray Chang,3 Wei-Hao Liao,4 Wen-Shiang Chen,4 Wei-Bor Tsai3* 1. Department of Chemistry and Biotechnology, Swinburne University of Technology, Victoria, Australia 2. Graduate Institute of Nanomedicine and Medical Engineering, College of Biomedical Engineering, Taipei Medical University, Taipei, Taiwan 3. Department of Chemical Engineering, National Taiwan University, Taipei, Taiwan 4. Department of Physical Medicine and Rehabilitation, National Taiwan University Hospital and National Taiwan University, College of Medicine, Taipei, Taiwan * Corresponding Author Wei-Bor Tsai, Tel: +886-2-3366-3996; Fax: +886- 2-2362-3040; email: [email protected]; PYW: 250 Wuxing St., Taipei 11031, Taiwan (R.O.C.) YSL, RC, WHL, WSC, WBT: No. 1, Sec. 4, Roosevelt Rd., Taipei 10617, Taiwan (R.O.C.)

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Abstract The effect of nanotopographies on cell adhesion, migration, proliferation, differentiation and/or apoptosis have been studied over the last two decades. However, the effect of nanotopography on gene transfection of adhered cells is far from understood. One key phenomenon of using nanotopography is mimicry of native cell morphology in vitro such as in alignment of skeletal myoblasts on nanogrooves. The formation of focal adhesions, the cytoskeleton, and the morphology of cell nuclei are altered by underlying nanogrooves, but the role of these changes in gene transfection are not well understood. In this study, C2C12 skeletal myoblasts were transfected using polyethyleneimine (PEI)/DNA complexes on nanogrooved patterns of two groove widths (400 nm and 800 nm) at three depths (50 nm and 400 or 500 nm). The results showed that the deep nanogrooved surfaces (i.e. 400/400 and 800/500) induced formation of aligned, parallel F-actin and elongated nucleus morphology. Gene transfection was also reduced on the deep nanogrooved surfaces. Disruption of F-actin organization using Cytochalasin D (Cyto-D) restored the nuclear morphology accompanied with higher transfection efficiency, demonstrating that the reduction in gene expression on deep nanogrooves was due to cytoskeletal stretching and nucleus elongation. Spatiotemporal images of fluorescent-labelled PEI/DNA complexes showed that endocytosis of PEI/DNA complexes was retarded and DNA trafficking

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into the cell nucleus was reduced. This study demonstrates for the first time the important role of cytoskeletal organization and nuclear morphology in PEI-mediated gene transfection to skeletal myoblasts using nanogrooved patterns. These findings are informative for in vitro studies and could potentially be useful in in vivo intramuscular (IM) administration. Keywords: nanotopographies, grooves, gene transfection, cytoskeleton, nuclear morphology

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1.

Introduction The extracellular matrix (ECM) is composed of various biomacromolecules

which form two-dimensional (2D) or three-dimensional (3D) scaffolds with diverse nano- and micro-structures. The interaction between mammalian cells and these nanostructures has an important impact on cell migration, proliferation, differentiation and/or apoptosis 1. The effects of nanotopographies on mammalian cells have been repeatedly demonstrated in vitro to be dependent on the cell types, materials, and the geometry and size of the nanotopographies involved 2-10. For example, we have demonstrated that skeletal myoblasts align with nanogrooves (450 nm or 900 nm in width), and alignment is controlled by nanogroove depth, i.e. cells align more on deeper nanogrooves 11. After 8 days of differentiation culture, parallel myotubes were formed on the nanogrooves, especially on deeper grooves (900 nm in width/500 nm in depth), while randomly orientated and branched myotubes were formed on flat controls. More importantly, cell nuclei were also elongated and aligned with the nanogrooves, resulting in a series of downstream gene transcription changes 12. Parallel myoblasts and myotubes mimic the native structure of skeletal muscle, which can be further used in tissue engineering, while branched myotubes have less applicability. Gene therapy aims to treat diseases by delivering therapeutic genes into patient

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cells 13. It may involve replacing a faulty gene or introducing a new gene to modify cells to achieve specific clinical purpose. A number of studies have been devoted to constructing non-toxic carriers and enhancing transfection efficiency using non-viral approaches; however, developing carriers with low toxicity and high efficiency remain a challenge 13. Recently, efforts have been made to increase gene transfection efficiency using surface nanotopographies 14-16. However, the effects of cell morphology, cytoskeletal networks, and nucleus shape on in vitro gene transfection have been less discussed despite possibly having tremendous effects on this event 17. The influence of surface nanotopography on endocytosis and cellular transfectability of cultured cells has been studied using both forward (gene delivery from culture media) and reverse (gene delivery from substrate surfaces) transfection approaches 17. However, it is difficult to obtain a general conclusion on the effect of surface nanotopography on gene transfection because diverse transfection protocols, plasmid sizes and carriers (e.g. polyethyleneimine (PEI), liposome, and various nanoparticles) are used. For example, human mesenchymal stem cells (hMSCs) and monkey kidney fibroblasts (COS7) cultured on structured surfaces with micro(2-µm-diameter) and nanopillars (200-nm-diameter) were found to increase internalization of fluorescently labeled dextran 18. When hMSCs were introduced to green-fluorescent protein (GFP)-encoding plasmid with Lipofectamine, the highest

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transfection efficiency was observed in cells on nanopillars, but not nanogrooves. On the other hand, using the same transfection system, higher transfection efficiency of human lung fibroblasts was observed on a specific feature height (i.e. 150 nm) of both nanopillars (500-nm-diameter) and nanogrooves (500-nm-width), but not 560-nm-height features, compared to flat controls 19. Because a variety of cell types, pattern structures, transfection approaches, and protocols have been used, the mechanism of substrate nanotopography-mediated endocytosis and nuclear uptake is still far from understood. In this study, we focused on the in vitro generation of parallel aligned skeletal muscle cells with structural similarity to the native muscle structure, and the effects of nanogroove topography on gene transfection using PEI as a carrier in these cells. It has been shown that the gene transfection efficiency of intramuscular (IM) injection is less effective than in vitro transfection 20. The physical barriers and microenvironment in in vivo situations must be considered. Skeletal myoblasts are aligned on nanogrooves with an elongated morphology and parallel actin filaments which mimic the native muscle structure in vivo 11, 21-22. We hypothesized that cytoskeletal tension and nucleus shape are two key parameters that affect gene transfection. Cells were transfected using PEI/DNA complexes and the mechanism of DNA trafficking was studied. A commercially available PEI system with high and stable gene transfection

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efficiency (>1 x 1011 RLU/mg protein) is used. We focused on the effect of pattern-induced cytoskeleton rearrangement on gene transfection using this system. This study provides useful information on in vitro gene transfection which could be applicable for IM injection.

2.

Materials and Methods

2.1 Materials Polystyrene (PS) was purchased from Nihon Shiyaku Industries, Japan. Polydimethylsiloxane (PDMS; Sylgard 184) was purchased from Dow Corning, USA. jetPEI® (cat# 101-10N) was purchased from Polyplus Transfection, USA. Plasmid (pEGFP-C1 4700 bp and pCI-neo-luc+; 7187 bp) was purchased from BioMed Resource Core of the 1st Core Facility Lab (National Taiwan University, Taiwan). ULYSIS Nucleic Acid Labeling Kits (cat# MP21650) was purchased from Life Technologies, USA. 4’, 6-Diamidino-2-phenylindole (DAPI) was purchased from Invitrogen, USA. Bradford reagent (cat# E530) was purchased from AMRESCO (OH, USA). YOYO-1 (cat# Y-3601) was purchased from Molecular Probe. Cytochalasin D (Cyto-D, cat# C8273), Phalloidin-Tetramethylrhodamine B isothiocyanate (cat# P1951) and other chemicals were purchased from Sigma, USA, unless specified otherwise.

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2.2 Fabrication and characterization of nanogrooved substrates Four nanogrooved patterns were fabricated according to a previous study 23. In brief, nanotextured silicon substrates (groove width/depth (nm): 400/50, 400/400, 800/50, and 800/500) were fabricated by electron beam lithography and dry etching. The silicone rubber molds, PDMS, were made by pouring elastomer and initiator (10:1 mixture) over the silicon masters. The 10% PS solution was dropped onto the PET substrates, and then the PDMS molds were faced down on the PS solution. The PS replicates were dried in the hood overnight. PS patterns were treated with oxygen plasma at 50 sccm, O2 flow rate of 70 mTorr, and power of 30 W for 180 seconds. Nanogrooves were observed using electron scanning microscopy (SEM, JSM-5310, JEOL, Japan) by sputtering gold to 5 nm thickness. The wettability of surfaces treated with O2 plasma was characterized by measuring water contact angle (FTA-125, First Ten Angstroms, Portsmouth, VA, USA) at 0° and 90° from the direction of the nanogrooves. Five measurements were conducted for each sample (n = 5).

2.3 Culture of skeletal myoblasts The mouse skeletal myoblast cell line C2C12 (ATCC #CRL-1772) was purchased from the Food Industry Research and Development Institute (Hsinchu,

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Taiwan) and cultured as described in a previous study 11. Samples were treated with 70% ethanol prior to cell seeding. Cells were passaged continuously in T75 flasks in Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum and high glucose at 37°C in a CO2 incubator containing 5% CO2 and saturated humidity. Harvested cells were seeded on nanopatterned PS placed in 24-well plate at a concentration of 2x104 cells/cm2 in 1 mL culture medium. After 1 day or 3 days of incubation, cells were either characterized or transfected.

2.4 Fluorescent staining of cells After incubation, the cells on the surfaces were rinsed with PBS twice and then fixed by 4% paraformaldehyde solution for 20 minutes. After fixation, samples were rinsed three times with PBS and then permeated with 0.1% PBST (Triton-X 100 in PBS) for 20 minutes. Then, the samples were blocked by 2% bovine serum albumin solution for 20 minutes after rinsing with 0.1% PBST for 10 minutes for 3 times. The F-actin was stained by 500 nM Phalloidin-TRITC solution for 3 hours at 37°C and rinsed with PBS for 3 times. Cell nucleus was stained with 4 µg/mL DAPI solution at 37°C for 20 minutes and rinsed with PBS for 3 times. Images were captured by confocal microscope (TCS SP2, Leica, Germany).

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2.5

Analysis of F-actin and cell nuclei F-actin and cell nuclei were analyzed using ImageJ software as described in a

previous study 11. For cytoskeleton analysis, F-actin images were analysed using ImageJ software with an oval profile plug-in. Fast Fourier transform (FFT) was used to convert the original image data from ‘‘real’’ space into a mathematically defined ‘‘frequency’’ space. The normalized intensity was plotted at an angle from 0° to 360°, where 0° was defined as the direction of the x-axis in a Cartesian coordinate system (2D coordinates). F-actin alignment was described as pixel intensity plotted against alignment angle (0-180°). For F-actin orientation, the sum of pixel intensity between 70°-110° (i.e. 90°±20°) was normalized to total pixel intensity between 0°-180° and presented as a percentage (%). To determine nucleus circularity, the outlines of cell nuclei were manually traced, and were fit to ellipses by ImageJ software. The circularity (Cir) was calculated by the dimensions of the fitted ellipse and defined as (4×π×area)/(perimeter2). A value of 1 indicated a perfect circle, while 0 indicated a line.

2.6 Determination of cell numbers Cells were lysed and the amount of protein (µg) was determined using Bradford reagent (AMRESCO, OH, USA) and ELISA reader (EL800, BIO-TEK, USA)

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according to supplier’s instructions. Cell number was determined using lactate dehydrogenase (LDH) assay according to a previous study 23. The LDH assay was employed by estimating the amount of LDH in cytosol. Samples were rinsed with PBS and lysed with 0.5 mL PBST for 30 min. 60 µL/well LDH working reagent solution (1:1:1 of Lactate solution (36 mg/mL): NAD+ solution (3 mg/mL NAD+, 2.7 mg/mL diaphorase, 0.3 mg/mL BSA, and 12 mg/mL sucrose): INT solution (0.2 mg INT/mL)) was added to the samples and cells, and then incubated for 30 min at 37°C. Finally, the OD values were read at 490 nm. Standard curves were also generated from samples with known cell numbers.

2.7 Gene transfection The pEGFP-C1 (4.7 kbp) plasmid was used for all gene transfection experiments, while the CI-neo-luc+ plasmid was used to quantify the amount of DNA within the nucleus (Supporting Information, Fig S1). Commercial linear jetPEI® (polyplus, USA) was used as the gene transfer carrier. PEI/DNA nano-complexes with N/P ratio = 5 (nitrogen-to-phosphorus) were prepared in 0.15 M NaCl at 37°C for 20 min. C2C12 myoblasts were cultured on nanogrooved surfaces for 3 days, transfected with PEI/DNA complexes containing 1 µg DNA for 24 h, and then lysed. The level of gene transfection efficiency was evaluated by quantifying the intensity of GFP

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translated from plasmid. The total GFP was then determined using a fluorometer (Modulus Single Tube, Turner BioSystem, USA). The total protein content of transfected cells was determined using Bradford reagent. The F-actin inhibitor, Cytochalasin D (Cyto-D), was used to disrupt polymerization of F-actin. Transfection experiments were then processed following the same protocol as above, but with the addition of 1 µM Cyto-D in the culture medium for 4 h prior to transfection experiments.

2.8 Mechanism of DNA uptake DNA was labeled by ULYSIS® Nucleic Acid Labeling Kits (cat# MP21650, Life Technologies, USA). 1 µg DNA was first precipitated by adding 1/10 volume of 3 M sodium acetate to two volumes of absolute ethanol. The solution was frozen at –70°C for 30 minutes and then centrifuged at 12,000 rpm for 15 minutes. The supernatant was decanted and the pellet was washed by 70% ethanol and air dried. The pellet was re-suspended in 20 µL labeling buffer, 1 µL labeling reagent stock solution was added to the DNA solution, and the mixture volume was brought to 25 µL with labeling buffer. The labeling reaction was incubated at 80°C for 15 minutes. The reaction was then stopped by plunging the sample into an ice bath. The sample was centrifuged at 12,000 rpm and the supernatant was decanted.

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After cells were incubated for 3 days, the ULYSIS-labeled DNA(GFP) condensed with jetPEI® was transfected to the cells for 24 h and then lysed. To quantify the quantity of DNA within the nucleus, the images of ULYSIS-labeled DNA was overlapped with DAPI-stained nucleus, and analyzed using ImageJ software. The confocal images were converted into 8-bit grayscale and image threshold was to quantify the green fluorescent intensity. The relative intensity (%) was presented for comparing DNA complexes inside cell nucleus between each sample. To further confirm the mechanism of DNA trafficking, YOYO-labeled PEI/DNA(luc+) was transfected into cells cultured for 24 h and 48 h on the flat and 800/500 surfaces. Labelling was performed according to supplier’s instructions. Spatiotemporal images of PEI/DNA complexes were captured using confocal microscopy (TCS SP5, Leica, Germany).

2.9 Statistical analysis Statistical analysis was performed using GraphPad Instat 3.0 (La Jolla, CA, USA). All experiments were at least repeated three times.

Statistically significant

differences between each group were determined by one-way ANOVA and Student– Newman–Keuls multiple comparison tests. p < 0.05 was considered a significant difference.

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Results 3.1 Surface characterization The production of four nanogrooved surfaces with different widths and depths (400/50, 400/400, 800/50 and 800/500) was confirmed from the SEM images (Figure 1A). The flat and nanogrooved surfaces exhibited different surface wettability (Figure 1B). Prior to oxygen plasma treatment, the water contact angles of the flat surfaces were 90.4°. After oxygen plasma treatment, the water contact angles on the flat substrates decreased to 42.0°, indicating that the surfaces became more hydrophilic. All nanogrooved surfaces exhibited more hydrophilic compared to flat surfaces with water contact angles less than approximately 30.0°. Because the shape of water droplets on the grooved surface were elliptical, the water contact angles vertical and orthogonal to the nanogrooves was measured. Overall, the water contact angles in both directions, i.e. parallel or perpendicular to the grooves, were similar.

3.2 Cell morphology On Day 1, C2C12 cells had attached to the surfaces and showed an aligned morphology on nanogrooved surfaces, while the direction of cell morphology on flat surfaces was random (Figure 2A). On Day 3, the cells were confluent on all surface

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types. An overall cell alignment was still found on the nanogrooved surfaces, while the cells on the flat control grew without specific direction (Supporting Information, Figure S2). F-actin staining on the Day 3 samples indicated that the cells on the nanogrooved surfaces developed parallel and aligned F-actin along the nanogrooves’ direction, while the F-actin within the cells on the flat control was interlaced (Figure 2B). According to analysis using ImageJ software, the majority of the F-actin on the nanogrooved surfaces was parallel to the direction of nanogrooves (i.e. ~90°), while the directional tendency of the F-actin on the flat surface was not obvious (Figure 2C). The percentage of the “aligned” actin filaments (within 90°±20°) was >50% on the two deep nanogrooved surfaces (400/400 and 800/500), ~35% on the two shallow nanogrooved surfaces (400/50 and 800/50), and only 20% on the flat controls (Figure 2D).

3.3 Nuclear circularity The shape of cell nuclei was characterized on Day 1 and Day 3 (Figure 3A and 3B). On Day 1, the cell nuclei were rounded on most of the surfaces (> 95%) except the 800/500 surface (~85%; Figure 3C). After 3 days of incubation, the circularity of the nuclei was significantly decreased as the cell nuclei were elongated on all nanogrooved surfaces except 400/50 but not on the flat control. About 40% of cell

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nuclei were elongated on the two deep nanogrooved surfaces (400/400 and 800/500; Cir > 0.96; Figure 3D) and only 25% of cell nuclei were elongated on 800/50 (Figure 3D). It was found that more than 95% cell nuclei were rounded on 400/50 and flat surfaces (Cir > 0.96).

3.4 Gene transfection efficiency C2C12 myoblasts were cultured on the nanogrooved surfaces for 3 days and then treated with jetPEI®/DNA complexes for 24 h. Plasmid-encoded GFP was expressed after entry of complexes into the nucleus. The gene transfection efficiency was determined after 24 h of transfection by quantifying the GFP intensity of transfected cells (i.e. intensity/protein). The results showed that GFP expression was significantly decreased on nanogrooves in a manner dependent on groove dimensions (Figure 4A). GFP expression relative to flat surfaces (100%) decreased by approximately 73% on 400/400 (~27.7%) and was most decreased on 800/500 (~8.6%, Figure 4A). These results suggested that the efficiency of gene transfection decreases with increasing depth and width of nanogrooves. Cell numbers were further quantified using the LDH method. These results showed that cell numbers were at a similar level (~1.5 × 105/cm2) on all surfaces on Day 3 (Figure 4B). Thus, the cell number would not be

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the main reason for a decrease in expression. As shown previously, cytoskeletal and nuclear morphology changed from day 1 to day 3. We investigated whether the morphological changes in the nucleus were associated with the efficiency of gene transfection. Thus, PEI/DNA complexes were administrated after 1 day or 3 days of culture, and the expression of GFP was analyzed at 24 h after transfection (Figure 5). The results showed that transfection efficiency was similar when cells were transfected after 1 day of culture (Figure 5), when the cells were not confluent on the substrates and cell nuclei were not yet significantly elongated. Again, the transfection efficiency was greatly reduced on the nanogrooved surfaces (~34% on 400/400 vs. 100% on Flat; p < 0.01 ) in comparison to the flat surface at day 3 (Figure 5), when cells were transfected after 3 days of culture, when the cells were confluent and the cell nuclei became elongated on the nanogrooved surfaces.

3.5

Mechanism of PEI/DNA trafficking We investigated whether the delivery of gene carriers was related to the cells

cultured on the nanogrooved surfaces. Thus, we determined the total amount of DNA inside the cells and the relative amount of DNA delivered into nuclei using UYLSIS-labeled DNA after 24h transfection (Figure 6). Our results showed that

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although the amount of intracellular DNA was similar on each type of the surfaces (Figure 6A), the amount of DNA delivered into the nucleus was greatly reduced on nanogrooved surfaces in comparison to flat surfaces, especially in the 800/50 and 800/500 surfaces (p < 0.05, Figure 6B and 6C). To further confirm the mechanism of PEI/DNA trafficking, spatiotemporal images of YOYO-labelled PEI/DNA complexes were captured after 24 h and 48 h (Figure 7). The results showed that the majority of PEI/DNA complexes were located in the cytoplasm. There were more PEI/DNA complexes on the flat samples than on the 800/500 surfaces after 48 h, indicating that endocytosis of PEI/DNA was higher and/or intact DNA was located in the cytoplasm when cells were attached to flat surfaces. We also noted that the distribution of DNA was different between flat and 800/400 surfaces, as there were more dot-like DNA foci on the 800/400 samples.

3.6

Gene transfection during disruption of F-actin We suspected that the low trafficking of DNA into nuclei might be related to

constraints on cytoskeletal arrangement and the deformation of nuclei. Thus, Cytochalasin D (Cyto-D), an F-actin inhibitor, was added into cell culture media after 3 days of culture, prior to the addition of the PEI/DNA complexes. Fluorescent

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imaging of F-actin showed that the cytoskeleton was disrupted by the addition of Cyto-D on both the flat and 800/500 surfaces (Figure 8A). It is interesting to note that many of the nuclei reverted from the aligned and elongated morphology to a random and rounded shape when Cyto-D was added (Figure 8A). The gene expression on 800/500 surfaces was also significantly increased approximately six-fold by the addition of Cyto-D after 24 h transfection (~20% vs. ~118%, p < 0.05, Figure 8B), while the transfection efficiency on the flat control was not significantly affected after Cyto-D was added (~100% vs. ~88%, Figure 8B). This indicated that cytoskeletal and nuclear elongation was the main reason for the decrease in gene delivery.

Discussion Nanoscale structures are found frequently in vivo environments. Patterning of various nanotopographies on biomaterials have also been well-recognized to haves prominent effects on cell adhesion, migration, proliferation, differentiation, and apoptosis in vitro 24-27. We previously showed that nanogrooved patterns can induce cellular alignment and increase the cell-cell fusion efficiency of skeletal myoblasts 11. In this study, we examined the effect of nanogrooves on gene transfection efficacy of skeletal myoblasts, and we explored a possible biological mechanism involved in

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PEI-based gene delivery systems. C2C12 myoblasts were found to align with nanogrooves in a depth-dependent manner, i.e. the deeper the nanogrooves, the more aligned the cells became. Focal adhesions are believed to be key in forcing cells to align along nanogrooves, in a distinct mechanism from cell alignment on microgrooves. A number of studies have shown that focal adhesion-induced mechanotransduction can modulate intracellular signaling pathways and subsequently change cellular functions 28-30. Cytoskeletal organization and cell nucleus morphology were also affected by the focal adhesions. In general, parallel F-actin and elongated cell nucleus were found when cells were guided by the underlying nanogrooves. However, the shape of cell nucleus (i.e. circularity) did not change until day 3, indicating that reshaping of the cell nucleus takes time. Gene transfection via cationic polymers such as polyethyleneimine (PEI) has many advantages, such as a strong DNA condensation capacity, intrinsic endosomal activity, and unique buffering capacity compared to virus-based approaches. However, the efficiency and biocompatibility (i.e. cytotoxicity) of this method both need to be improved 13. To date, PEI and its derivatives are still the most widely studied vectors. Therefore, linear PEI was used in this proof-of-concept study. Due to the unique properties of PEI, release of DNA into cytoplasm from DNA/PEI complexes occurs

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spontaneously via binding to the cell surface, traversing the cell membrane, and endosomal escape 13. In this study, we found that the total amount of DNA within the cytosol was similar among cells cultured on all the examined surface types after 24 h, which is consistent with reports that PEI has a superior ability to penetrate cell membranes. However, when PEI/DNA complexes were used in transfection for 48 h, more DNA were found within the cytoplasm and/or nucleus (Figure 7). It is interesting that at the earlier time point of 24 h, the amount of PEI/DNA complexes within cells was similar between surfaces. Previous reports have also suggested that PEI has excellent endosomal escape properties 31. Thus, our results may suggest that either the endocytosis of PEI/DNA complexes is retarded or the DNA more easily degraded within the cytoplasm on the nanogrooved surfaces. The dot-like DNA foci appearing on the nanogrooved samples could also indicate the DNA cannot escape from endosomes after entry. On the examination of cell nuclei, we found that the nuclear morphology of cells on the nanogrooved surfaces was altered from round to elongated between day 1 and day 3 of culture. Similarly, the transfection efficiency (i.e., the expression of GFP) was greatly decreased in cells on the nanogrooved surface after 3 days of culture, but not 1 day. The amount of DNA within the nuclei was decreased on the nanogrooved surfaces compared to the flat control. Therefore, we hypotheses that the shape or

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circularity of cell nuclei is highly related to DNA trafficking into the nucleus. DNA trafficking into elongated nucleus and stretched cytoskeleton appears to be less efficient. DNA trafficking into the nucleus using non-viral carriers have been proposed via several models, including via nuclear pores or during mitosis 32. Mitosis is a widely accepted model for nuclear trafficking because the size of the nuclear envelope becomes dynamic during this process 33. In this study, however, we found that it is not likely that DNA is small enough for trafficking through nuclear pores (~10 nm). Additionally, cell division was not likely to be the mechanism on DNA entry observed in this study because gene transfection was done after confluence, i.e. at day 3. Therefore, it is highly possible that nuclear deformation and cytoskeletal network can reduce DNA trafficking into the nucleus. This could explain why the efficiency of gene delivery is time-dependent, 14 as nuclear deformation takes time to occur. It has been reported that nanotopographies alter gene expression via nuclear deformation 34. The shape of cells and cell nuclei are controlled by cytoskeletal system including actin filaments (~ 7 nm in diameter), intermediate filaments (~ 8-12 nm in diameter), and microtubules (~25 nm in diameter). The cytoskeleton interweaves within the cytoplasm, determines the cell shape, and generates mechanical stress on the nucleus 7, 35. In this study, cells were forced to align on

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nanogrooves (i.e. 800/500) and formed parallel F-actin, which also affected the organization of cytoskeletal networks. Consequently, this cytoskeleton restructuring results in nuclear deformation, and thus may alter molecular diffusion within cytoplasm. To further understand nanotopographical effects on gene transfection, F-actin was disrupted before gene transfection. Intermediate filaments are stable cytoskeletal components which surround the nucleus and are also responsible for cell structure and organelle localization. Disassembling intermediate filaments alters the resilience of cells 36. Condensed F-actin and intracellular tension may also obstruct the DNA trafficking 37. Our results showed that, with F-actin inhibition, the cytoskeleton of myoblasts on nanogrooved surfaces does not become parallel and aligned with nanogrooves. Cell nuclei also become randomly orientated. Without a well-organized cytoskeleton and elongated cell nucleus, gene transfection efficiency was reversed to similar levels across all surfaces (flat and nanogrooves). This indicates that DNA trafficking into the nucleus is inhibited by cytoskeletal organization and cell nucleus elongation, and this effect can be reversed by disruption of these intracellular structures. Recently, gene transfection to human lung fibroblasts using nanogratings through nuclear deformation has been reported 19. Nuclear volume, proliferation, transfection,

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and collagen production were affected by nanogratings. Furthermore, nuclear deformation was dependent on feature size. When nuclear volume was increased, the proliferation rate, transfection efficiency, and collagen production of the fibroblasts were also increased. Our results echoed the conclusion of this particular study that altering cytoskeleton organization and deformation of cell nuclei modulates gene transfection. Nevertheless, different cell types, DNA carriers, and surface patterns (even chemical patterns) are often relevant in different applications. It remains difficult to obtain a general conclusion on the effects of nanotopography on gene transfection. In this proof-of-concept study, we demonstrated that the PEI delivery system in myoblasts is retarded on nanogrooved patterns where myoblast morphology mimics the native skeletal muscle in vivo. The exact mechanism for the correlation between nucleus deformation and gene transfection needs further investigation. These findings could be important in finding new methods to increase IM injection efficiency, such as reducing muscle stretching.

Conclusion In this study, the effect of nanogrooved topography on PEI-mediated gene transfection to skeletal myoblasts was investigated. As the muscle cells aligned and elongated in the direction of nanogroove patterning, cell morphology mimicked the

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native structure of skeletal muscle. These results are informative for intramuscular (IM) injection. Interestingly, the expression of transfected genes was decreased on nanogrooved surfaces, especially on deeper nanogrooved surfaces (i.e. 400/400 and 800/500). We found that the decrease in the gene expression was due to slower endocytosis of PEI/DNA complexes or shorter survival of DNA within cytoplasm, as well as diminished DNA trafficking into the nuclei on nanogrooved surfaces. Disruption of F-actin structure by Cyto-D on the nanogrooved surfaces restored the shape of nuclei and the expression of GFP, indicating that cell stretching may have a negative effect on PEI based gene delivery. Therefore, modulating the shape of cell nucleus and organization of cytoskeleton is one of the keys to controlling gene transfection in skeletal myoblasts and may explain low gene transfection efficiency in vivo. This study provides evidence for a new mechanism involved in in vitro gene transfection.

Supporting Information: The Supporting Information is available free of charge. The Supporting Information contains plasmid data sheets and phase contract images at day 3.

Acknowledgement

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This research was supported from Ministry of Science and Technology (MOST) of Taiwan (103-2221-E-002-207-MY3). The authors thank National Nano Device laboratories, Hsinchu, Taiwan, for the fabrication of the nano-grooved silicon substrates. The Australian Research Council (ARC) is acknowledged for providing the Discovery Early Career Researcher Award (DECRA: DE150101755) to P.-Y. Wang. Taipei Medical University and MOST of Taiwan are also acknowledged for providing funding support for P.-Y. Wang (TMU105-AE1-B13 and 105-2314-B-038-088-MY2). Authors thank Sheryl Ding for grammatical checking. The authors declare that there is no conflict of interest regarding the publication of this article

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For Table of Contents Use Only Modulation of PEI-mediated gene transfection through controlling cytoskeleton organization and nuclear morphology via nanogrooved topographies

Peng-Yuan Wang,1,2 Yen-Shiang Lian,3 Ray Chang,3 Wei-Hao Liao,3 Wen-Shiang Chen,4 Wei-Bor Tsai3* 1. Graduate Institute of Nanomedicine and Medical Engineering, College of Biomedical Engineering, Taipei Medical University, Taipei, Taiwan 2. Department of Chemistry and Biotechnology, Swinburne University of Technology, Victoria, Australia 3. Department of Chemical Engineering, National Taiwan University, Taipei, Taiwan 4. Department of Physical Medicine and Rehabilitation, National Taiwan University Hospital and National Taiwan University, College of Medicine, Taipei, Taiwan

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Modulation of PEI-Mediated Gene Transfection through Controlling

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