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High Density of Aligned Nanowire Treated with Polydopamine for Efficient Gene Silencing by siRNA According to Cell Membrane Perturbation Baiju G. Nair,† Kyoji Hagiwara,‡,§ Motoki Ueda,†,‡ Hsiao-hua Yu,†,∥ Hsian-Rong Tseng,⊥ and Yoshihiro Ito*,†,‡ †

Nano Medical Engineering Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 3510198, Japan Emergent Bioengineering Material Research Team, RIKEN Centre for Emergent Matter Science, 2-1 Hirosawa, Wako, Saitama 3510198, Japan § Laboratory of Human Science and Engineering, 1-3-1 Minaminagasaki, Toshima-ku, Tokyo 1710052, Japan ∥ Institute of Chemistry, Academia Sinica, 128 Academia Road Sec. 2, Nankang, Taipei 115, Taiwan ⊥ Department of Molecular and Medical Pharmacology, University of California, Los Angeles CNSI, 570 Westwood Plaza, Los Angeles, California 90095, United States ‡

S Supporting Information *

ABSTRACT: High aspect ratio nanomaterials, such as vertically aligned silicon nanowire (SiNW) substrates, are three-dimensional topological features for cell manipulations. A high density of SiNWs significantly affects not only cell adhesion and proliferation but also the delivery of biomolecules to cells. Here, we used polydopamine (PD) that simply formed a thin coating on various material surfaces by the action of dopamine as a bioinspired approach. The PD coating not only enhanced cell adhesion, spreading, and growth but also anchored more siRNA by adsorption and provided more surface concentration for substrate-mediated delivery. By comparing plain and SiNW surfaces with the same amount of loaded siRNA, we quantitatively found that PD coating efficiently anchored siRNA on the surface, which knocked down the expression of a specific gene by RNA interference. It was also found that the interaction of SiNWs with the cell membrane perturbed the lateral diffusion of lipids in the membrane by fluorescence recovery after photobleaching. The perturbation was considered to induce the effective delivery of siRNA into cells and allow the cells to carry out their biological functions. These results suggest promising applications of PD-coated, high-density SiNWs as simple, fast, and versatile platforms for transmembrane delivery of biomolecules. KEYWORDS: high-density nanowires, bioinspired materials, nanotopography, polydopamine, transmembrane delivery, RNA interference

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INTRODUCTION

efficient for probing the intracellular environment, controlling cellular dynamics, and intracellular manipulations.9 Among such nanotools, various types of the ordered array silicon nanowire (SiNW) have been investigated for enhancement of growth and spreading of various cell lines10,11 and delivery of biomolecules into cells.12−14 It is known that the density of nanowires is an important process of the adhesion and size.15 Although the low density nanowires do not support cell proliferation or migration, they can incorporate various biomolecules into the cell by partial membrane penetration.16 However, high-density nanowires provide light adhesion of cells and reversible attachment by conjugation with a thermosensi-

Cellular membranes are one of the obstacles in the transfer of biomolecules to regulate intracellular events in cells.1 Intruding the plasma membrane for delivery of various molecules has been addressed by various conventional methods.2 Nano- or microfabricated substrates have been developed as an extracellular matrix to instantaneously incorporate the molecule of interest from the surface into the cytoplasm. High aspect ratio nanomaterials, such as, nanotubes,3 nanostraws,4 nanowires,5 nanopillars,6 and nanoneedles,7 are versatile substrates with excellent mechanical, optical, and electrical properties for various biological applications. Furthermore, the varieties of high aspect ratio nanomaterials provide an excellent microenvironment for the exchange of a wide range of molecules between cells and substrates.8 These proven nanotools are © XXXX American Chemical Society

Received: April 26, 2016 Accepted: July 6, 2016

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DOI: 10.1021/acsami.6b04913 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces tive polymer.17 We have already successfully performed in vitro and in vivo delivery of exogenous genes encapsulated in supramolecular nanoparticles with a high spatial density of SiNWs.18 High density of SiNWs are considered to be suitable for harmless manipulation of cells, considering the damage of penetration caused by low density of nanowires. However, the previous methods require various functionalization steps to prepare and conjugate the DNA entrapped nanoparticles on SiNWs. In certain studies, nanowires have slowed down cell growth.19 Therefore, herein we employed a simple siRNA-anchoring followed by cell adhesion and growth enhancing method using a mussel inspired surface coating20 of polydopamine (PD). Considering that 3,4-dihydroxy-L-phenylalanine and lysinerich proteins are responsible for the adhesion of mussels to a surface,21 treatment with dopamine carrying both catechol and amino groups was developed as a facile surface treatment for various materials by the group of Messersmith.21 This coating is very stable owing to covalent and noncovalent interactions and has opened up new opportunities for biomedical applications, including drug delivery and biosensing.22 In addition, it is known that the polymerized dopamine coating provides a porous and uniform layer on the surface of high aspect ratio nanostructures.23,24 Therefore, the coating is expected to increase the surface area carrying functional groups for loading siRNA. Our previous study highlighted the use of PD-coated titanium surfaces for covalent coupling of growth factors and proteins for the therapeutic integration of implants,25,26 and demonstrated the adsorption of siRNA on a PD-coated stainless steel surface for prolonged RNA interference.27 Interestingly, all these facts underscore the biocompatibility of PD without any cytotoxic effect and enhancement of the proliferation of various cells.28 The PD-treated materials were reported for investigation of medical devices.29,30 Here, by combining the advantages of high-density SiNWs and PD, we developed an efficient delivery platform siRNA that can knock-down genes effectively (Scheme 1). The interaction of PD-coated SiNWs with cells was considered to result in perturbation or disturbance of the cell membrane, so that the siRNA from the nanowire substrate could efficiently enter the cytoplasm.

Scheme 1. Polydopamine (PD)-Coated Silicon Nanowires (SiNWs) for Incorporation of siRNA into Cellsa

a

When treated with dopamine, siRNA easily bound electrostatically to the surface of SiNWs without any chemical modifications. Upon culturing the cells on the surface of a vertically aligned silicon nanowire substrate, siRNA was easily incorporated into cells by perturbing the cell membrane to silence gene expression with high efficiency.

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RESULTS AND DISCUSSION Dopamine is a versatile natural molecule, and placing a substrate into an alkaline solution of dopamine results in coating of various materials, enabling novel methods to modify various substrates. Initially, we attempted to coat substrates using 2 mg/mL of dopamine for 12 h, which is the conventional condition. As a result, the deposition of large particles was observed as a thick coating on SiNWs. After optimization, we chose 1 mg/mL of dopamine at room temperature for 6 h as the condition to obtain a thin coating on SiNWs (Figure 1a). The transmission electron microscopy (TEM) micrograph in Figure 1c clearly shows a thin coating of PD (3−4 nm) on SiNWs, which was obtained by coating with 1 mg/mL dopamine. The length and diameter of SiNWs were 10−15 μm and ca.100−200 nm, respectively. X-ray photoelectron spectroscopy (XPS) analysis on PD-SiNWs revealed the presence of dopamine by showing a fresh peak of nitrogen (1s) and an elevated peak of carbon (1s) and oxygen (1s) in the wide XPS spectrum (Figure 1d), although the peak of nitrogen was completely absent on the SiNWs without PD treatment. The

Figure 1. Characterization of untreated and PD-coated SiNWs. SEM images of (a) PD-SiNWs and (b) SiNWs. (c) TEM image of PDSiNWs showing the thickness of PD. Insets show magnified images of the PD coating. (d) XPS spectrum of SiNWs (blue) and PD-coated SiNWs (red). O, N, C, and Si represent oxygen, nitrogen, carbon, and silicon, respectively.

peak for silicon (2p) in PD-SiNWs was also slightly reduced compared with SiNWs without PD. This result could be attributed to the dopamine coating that may have decreased the silicon intensity. These results based on a chemical analysis of the PD-coated surface by XPS are in accordance with those reported by Zhang et al.31,32 After loading of siRNA on the surfaces, the amount was quantified using an image analysis method based on measuring the fluorescent intensity.33 A calibration plot was prepared using fluorescence intensity in images of FAM-conjugated B

DOI: 10.1021/acsami.6b04913 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces siRNA that was adsorbed and retained on SiNWs (Figure S1). Using the plot, the amount of siRNA, which specifically adsorbed on each of substrates, was determined (Figure 2). The

Figure 2. Quantification of siRNA on substrates. The amount of siRNA deposited on each substrate was evaluated and compared with the initial concentration of siRNA in solution.(n = 3). *p < 0.05, **p < 0.01, and NS = no significance.

PD coating enhanced the siRNA adsorption compared with the normal surface as reported previously.27 More importantly, we found that a significant amount of siRNA adsorbed onto PDSiNWs in comparison with other substrates, although there were some nonspecific adsorptions of siRNA on untreated SiNWs. At low siRNA concentrations, there were no large differences between the siRNA adsorbed on PD-SiNWs and a normal silicon substrate coated with PD (PD-plain). However, at high concentrations, there was a significant increase (almost 2-fold) in the amount of amount of siRNA that adsorbed onto both surfaces. Considering the surface area of nanowires, the enhancement was not very high, but a significant amount of siRNA was present on the surface. Scanning electron microscopy (SEM) revealed the cell growth accompanying cell division on PD-SiNWs as shown in Figure 3a. Moreover, the SEM images acquired from the side confirmed that cells were well adhered, spread out, and closely interacted with PD-SiNWs (Figure 3b).

Figure 4. Adhesion and spreading of A549-luc cells after 1 h of seeding and the cell growth after 36 h of culture on the substrates. *p < 0.05, **p < 0.01, and ***P < 0.001 n = 3.

substrates.36 In the present study, cell adhesion and growth was significantly affected by high density SiNWs. We observed that the PD coating has a considerably enhancing effect on cell adhesion, spreading, and growth on both plain and SiNWs surfaces (Figure 4). It has been reported that PD-coated surfaces often promoted cell adhesion, spreading, cell differentiation,37 and significantly influence the multipotency of stem cells.38 It was considered that the presence of the functional groups of PD and the serum protein adsorption are the key factors responsible for PD-mediated cell adhesion and spreading.39 In this study, the functional groups generated during PD coating may also augment adsorption of serum proteins on the SiNWs, which further facilitated an increase in cell adhesion and growth. Fluorescent microscopy confirmed the internalization of FAM-labeled siRNA from the PD-SiNWs platform into A549luc cells (Figure 5). The CellMask and Hoechst 33258 specifically stained plasma membrane and nucleus are shown in Figure 5a,c, respectively. FAM-siRNA delivered by PDSiNWs was visible as a green fluorescence that was observed inside the nucleus/cytoplasm (Figure 5b). As shown in Figure 5d, some FAM-siRNAs were colocalized with chromosomal

Figure 3. SEM images of an A549-luc cell grown on PD-SiNW substrate. (a) Top and (b) side views.

Quantitative evaluations of cell behavior between plain and SiNWs were performed as shown in Figure 4. The presence of nanowire significantly increased cell adhesion compared with flat surfaces. Several studies conducted on various surface topographies at the nanoscale level have demonstrated the influence of modified nanostructures on adhesion and spreading of anchorage-dependent cells.34,35 However, a high-density nanowire substrate is often perceived as a flat surface by cells because they are able to move and proliferate on nanowire C

DOI: 10.1021/acsami.6b04913 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

contact with cell surface, was less than that on the plain surface. The silencing effect was considered to be extremely efficient. The process of membrane interaction with nanowires is dependent on many factors. Previous studies highlight the importance of material, diameter, length, and density of nanowires in the cell adhesion process and access the cytoplasm by membrane penetration.42,43 Understanding how the nanowires efficiently delivered the siRNA was investigated from the viewpoint of the plasma membrane disturbance. The plasma membranes exhibit a lateral diffusion of lipids and membrane proteins during various metabolic activities in cells. These dynamic phenomena in a membrane can be observed meticulously by fluorescence recovery after photo bleaching (FRAP) using a fluorescence microscope.44,45 For a clear examination, we carried out FRAP in the membrane of cells on PD-plain and PD-SiNW surfaces using a fluorophore associated with a 12-carbon fatty acyl tail, BODIPY FL C12, as shown in Figure 7. The lipid diffusion coefficient of

Figure 5. Fluorescent microscopy images of A549 luc cells grown on a PD-SiNW substrate. A549 luc cells were treated with (a) CellMask to stain plasma membrane, (b) FAM-siRNA for internalization into cells, (c) Hoechst 33258 to stain nucleus, and (d) Merged image. Scale bar is 20 μm.

DNA. Alternatively, on bare SiNWs, no significant cellular uptake of FAM-labeled siRNA was observed (Figure S2). The gene knockdown assay in A549-luc cells was quantitatively carried out by measuring the production of luciferase. Figure 6 shows that the substrate-mediated delivery

Figure 7. Fluorescence recovery after photobleaching (FRAP) on the membrane of A549 cells on (a) PD-Plain and (b) PD-SiNW surfaces to evaluate the lateral diffusion of lipids. Arrow and dotted circles indicate the membrane disturbed by SiNWs and the area of photobleaching, respectively. (c) FRAP data were recorded as confocal microscopy images at various time intervals: prebleach, bleach, and recovery of fluorescence with respect to time.

Figure 6. Luciferase gene knockdown activity in response to various amounts of siRNA adsorbed onto the surfaces of bare SiNWs, PDPlain, and PD-SiNWs and complexed with PEI (n = 3).

cells on PD-SiNWs is quantitatively obtained by curve fitting on fluorescent recovery using the Axelrod’s equation.46 The membranes of cells on PD-SiNWs under the same experimental conditions as those of the PD-Plain surface showed a tendency to decrease the lipid diffusion coefficient (0.30 ± 0.02 μm2/sec) compared with cells on the PD-plain surface (0.85 ± 0.15 μm2/ sec) (Table 1). Mechanical barriers hinder the lateral diffusion of lipids and proteins through membranes under normal conditions.47 High aspect ratio materials and polymers can also act as mechanical barriers in membranes, resulting in a

is more effective than conventional transfection using soluble polyethylenimine (PEI). In addition, Figure 6 also demonstrated that the highest luciferase gene knockdown was observed on PD-SiNWs at the same concentrations of adsorbed siRNA with comparing on PD-Plain. PD-SiNWs facilitated significantly more gene knockdown activity than plain surfaces. Substrate mediated gene delivery was from a three-dimensional scaffold has many advantages over conventional platforms because they provide enough surface area for cell adhesion, prevent escape of molecules to medium, and increase the opportunity to internalize more molecules.40 Similarly, vertically aligned nanowires have been considered as a novel three-dimensional nanobiointerface,41 and the ability of PDSiNWs to suppress the gene here can be considered as a powerful means to silence a gene at the three-dimensional level. Comparing the direct contact area between the cell and substrate, the amount of bound siRNA on SiNWs, which can

Table 1. Diffusion Coefficient and Diffusion Time Are Extracted from the FRAP Experiments (n = 3)

D

samples

diffusion coefficient (D) μm2/sec

diffusion time (τD) sec

PD-plain PD-SiNWs

0.85 ± 0.15 0.30 ± 0.02

6.5 ± 1 17.05 ± 1.5

DOI: 10.1021/acsami.6b04913 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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water (three times), the substrate was dried under N2. Subsequently, the substrate was hydrophilized by ozone treatment. Finally, the substrate was dipped into various concentrations of 3,4-dihydroxyphenethylamine hydrochloride (dopamine; Sigma-Aldrich) dissolved in Tris-HCl buffer (10 mM, pH 8.5) with gentle shaking for 6 h. The PD-coated substrate was washed with Milli-Q water three times and dried with N2. Freshly prepared substrates were used for each experiment. Surface Characterization. The surfaces were analyzed using Theta Probe angle resolved X-ray photoelectron spectrometer system with monochromatic Al as X-ray source (ARXPS; Thermo Fisher Scientific, U.K.). The data were analyzed by the Thermo Scientific Advantage data system. SEM. The surface morphology was examined on a scanning electron microscope (SEM; JSM6330F, JEOL, Japan) operating at 5 kV. The sample surface was sputter coated with palladium−platinum using a SC-701 Quick coater (Sanyu, Japan) before SEM observation. A detailed investigation of the cell−substrate interface was performed by SEM. A549 cells (human lung adenocarcinoma epithelial cell line) grown on the substrate were first fixed with glutaraldehyde and postfixed with osmium tetroxide. Next, the cells were dehydrated in ethyl alcohol mixtures and finally dried at a critical point using CO2 in a HCP-2 critical point dryer (Hitachi, Japan). Before SEM observations, cell samples were coated with osmium using an OPC80N osmium plasma coater (Filgen, Japan). TEM. To measure the average thickness of PD on SiNWs, we used transmission electron microscope (TEM; JEM-1230, JEOL, Japan). The SiNWs were collected from the substrate using a spatula and dispersed in distilled water. Ten μL of the suspension were applied to a hydrophilized carbon-coated copper grid and dried. SiNWs were observed at 80 kV and images were analyzed. siRNA Conjugation and Quantification. siRNA against the luciferase gene (sense strand, 5′-CUUACGCUGAGUACUUCGATT3′; antisense strand, 5′-UCGAAGUACUCAGCGUAAGTT-3′) was diluted to 1 μg/μL in diethylpyrocarbonate (DEPC)-treated RNADNase free water (Invitrogen Life Technologies, CA, U.S.A.). A 1 cm2 PD-SiNW sample was prepared as described above. Ten microliters of siRNA solution was dropped on to the surface, followed by incubation at 4 °C for 4 h. To estimate the amount of siRNA conjugated on the SiNWs, we acquired images of SiNWs conjugated with 6-carboxyfluorescein-labeled siRNA (FAM-siRNA; Eurofins Operon Huntsville, AL, U.S.A.) using a molecular imager system (FX, Bio-Rad Laboratories, Hercules, U.S.A.). All images were acquired as 16-bit gray images. We acquired fluorescent images of 1−7 μg FAM-siRNA deposited on SiNW substrates. In each image, the intensity of fluorescence corresponded to the concentration of siRNA, which was calculated by ImageJ software. Finally, a standard plot was prepared to analyze the test sample. The fluorescence obtained from the test sample was used to measure the quantity of siRNA conjugated on SiNWs based on the standard plot. Cell Culture. A549 cells stably expressing luciferase gene (A549luc) were grown in Ham’s F12 K Nutrient mixture medium (Gibco, Life Technologies Carlsbad, U.S.A.) supplemented with 10% fetal bovine serum (FBS; MP Biomedicals, CA, U.S.A.). Cells were passaged regularly to maintain exponential growth. After conjugating siRNA, SiNWs were placed in 12-well plates and loaded with cells at 4 × 104 cells/mL. Cell Adhesion and Growth. Cell adhesion was measured by seeding A549-luc cells on each substrate for 1 h. At 1 h after seeding, the substrates were washed gently with PBS to preserve only adhered cells on each substrate. Next, the cells were fixed and processed for SEM observations. Images of all adhered cells were obtained in five randomly chosen fields on each substrate. Finally, the number of cells was enumerated in each sample to evaluate the cell adhesion. Cell spreading was evaluated by counting the ratio of spherical cells and morphologically changed or filopodia-formed cells (Figure S3). Cell growth was calculated by seeding the cells on each substrate, followed by trypsinization after 36 h of incubation. The cell numbers were counted by Tali Image-based Cytometer (Invitrogen, U.S.A.) and enumerated the growth.

significant decrease in lateral diffusion compared with standard materials.48 Moreover, nanowires exert vertical force on the cell membrane, which is opposed by the cytoskeletal network, resulting in disturbances in the cell membrane.49 We considered that SiNWs act as a mechanical barrier and disturb the cell membrane for the free lateral diffusion of lipids in membrane. According to our findings, we revealed a possible mechanism of nanowire−cell interaction as shown in Figure 8.

Figure 8. Possible mechanism of plasma membrane disturbance for efficient transfection through high-density nanowires.

This type membrane perturbation might result in the decrease in the diffusion coefficient and simultaneously increase the diffusion time compared with the cells on the PD-plain surface. Easy access to the cell interior by the nanowire is hampered by the cell membrane and rigid cytoskeleton scaffold, and simultaneous chemical proration is required to overcome such barriers.50 In the case of low density nanowires, they penetrate the cell membrane and transfer the molecules into the cells.14,43 Here, the highly dense PD-coated nanowires perturb the cell membrane, which may offer sufficient passage for siRNA to move across membranes. The tension in the membrane results in the dislocation of lipids in the membrane and may generate enough passage for siRNA to enter cells.

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CONCLUSIONS We developed a nanoplatform consisting of PD and a substrate of high-density SiNWs to efficiently incorporate siRNA into cells by perturbing the cell membrane without any damage. This study demonstrated that a simple and effective substratemediated nanoplatform may be able to increase the transfer of biomolecules and chemicals into cells for genetic modifications, sensing, and reprogramming. This platform may have numerous applications in the field of cell and tissue engineering.

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EXPERIMENTAL SECTION

Fabrication of SiNW Substrates. Vertically aligned SiNW substrates were prepared by a chemical etching process as described previously.17 Briefly, the surface of the silicon substrate was ultrasonicated in acetone and ethanol at room temperature for 10 min and then 5 min to make it more hydrophilic by removing greasy organic materials. Next, the cleaned silicon substrate was boiled in piranha solution (4:1 (v/v) H2SO4/H2O2) and RCA solution 2 (1:1:5 (v/v/v) NH3/H2O2/H2O) for 1 h. Afterward, deionized water was used to clean the silicon substrate for use in the etching process. The entire etching process was carried out at room temperature in a Teflon vessel containing the etching mixture: deionized water, 4.6 M HF, and 0.2 M silver nitrate. Subsequently, the silver film-deposited silicon substrate was removed by immersing in boiling aqua regia (3:1 (v/v) HCl/HNO3) for 15 min. Finally, the substrate was rinsed thoroughly with deionized water, blown dried with N2 and prepared for surface modification. Dopamine Coating. The substrate was treated with piranha solution (1:3 H2SO4:30% H2O2) to clean and remove any organic materials left on the surface. After repeated washing with distilled E

DOI: 10.1021/acsami.6b04913 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces Fluorescent Microscopy. To measure the incorporation of siRNA into cells, we first conjugated FAM-siRNA on PD-SiNWs. Cells grown on the SiNW substrates with FAM-siRNA were stained with CellMask orange plasma membrane stain (Life Technologies, Carlsbad, U.S.A.) and Hoechst 33258 (Invitrogen, U.S.A.). Images showing the internalization of siRNA were obtained under an inverted fluorescence microscope (AxioVert, Zeiss, U.S.A.). Gene Knockdown Assay. For luciferase gene silencing, luciferase production was measured in A549-luc cells cultured on an siRNAcoated substrate for 48 h. After incubation, SiNW substrates were transferred to a fresh 12-well plate, and 200 μL Picagene LT 2.0 luminescence reagent (Tokyo INK, Japan) was added to each well. Luciferase activity was analyzed after 90 min by measuring the luminescence using an Enspire Multimode microplate reader (PerkinElmer, U.S.A.). Relative percentage of luciferase activity was calculated in each substrate (PD-SiNWs, bare SiNWs, and PD-Plain) using the luminescence from the respective controls without siRNA. To analyze the solution mediated delivery into cells, PEI (Wako Pure Chem. Ind., Japan, MW 10 000) was complexed with siRNA as mentioned elsewhere.51 After formation of a complex mixture with different concentrations of siRNA in solution, the solution was dropped into wells containing cells. The luciferase activity was measured after 48 h. Confocal FRAP Analyses for Membrane Fluidity. All FRAP experiments were carried out with an FV1200 Laser Scanning Confocal microscope (Olympus, Japan) equipped with a laser light stimulation scanner and 60× objective lens (water immersion, NA 1.2). To record and analyze FRAP data, we used FLUOVIEW FV1200 software provided by Olympus. To carry out FRAP in A549-Luc cells, Bodipy FL C12 (Invitrogen) was loaded into the cell membrane at a concentration of 4 μM, which was found to sufficiently stain without affecting cell viability. For bleaching, one cell was selected, the zoom factor was set to 4, and the region of interest in the cell was simultaneously adjusted to 4 μm in diameter. The bleaching was carried out for 1000 ms. After an interval of 3 s, recovery of fluorescence was recorded as images and text files. FRAP was compared in A549 cells cultured on PD-Plain and PD-SiNW surfaces. The FRAP data were analyzed by FLUOVIEW FV1200 and diffusion measurement package software to calculate the diffusion coefficient (D) in each experimental condition. The lateral diffusion constant of lipids in the membrane was calculated by curve fitting into Axelrods̀ equation that is shown below:47 −1 ∞ ⎡ (− K )″ ⎤ ⎡ ⎛ 2t ⎞⎟⎤ F(t ) = C0 ∑ ⎢ ⎥ ⎢1 + n⎝⎜1 + ⎥ ⎣ n ⎦⎣ τD ⎠⎦ n=0

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*E-mail: [email protected] (Y.I.). Author Contributions

Y.I. conceived and designed the experiments. B.N. and K.H. performed the experiments. B.N. and Y.I. analyzed the data. M.U., H.Y., and H.R.T. contributed reagents/materials/analysis tools. B.N. and Y.I. wrote the paper. All authors have given approval to the final version of the manuscript. Funding

RIKEN’s programs for Junior Scientists (SPDR), 100547201300019130; Japan Society for the Promotion of Science (JSPS), 22220009; and KAKENHI grant 15H01810. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by the RIKEN Programs for Junior Scientists. We acknowledge the Support Unit for Biomaterial Analysis, RIKEN BSI for molecular imaging facility and BSIOlympus Collaboration Center (BOCC) for confocal imaging. We are grateful for preliminary trials of PD treatment presented by Dr. B. Joddar and cell culture support by Ms. Akiko Yumoto, Nano Medical Engineering Laboratory, RIKEN.

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(1)

(2)

(3)

The extracted lipid coefficients of the two conditions were compared and analyzed for the membrane fluidity and membrane penetration caused by SiNWs.

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REFERENCES

(1) Yan, L.; Zhang, J.; Lee, C. S.; Chen, X. Micro- and Nanotechnologies for Intracellular Delivery. Small 2014, 10, 4487− 4504. (2) Stephens, D. J.; Pepperkok, R. The Many Ways to Cross the Plasma Membrane. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 4295−4298. (3) Singhal, R.; Orynbayeva, Z.; Sundaram, R. V. K.; Niu, J. J.; Bhattacharyya, S.; Vitol, E. A.; Schrlau, M. G.; Papazoglou, E. S.; Friedman, G.; Gogotsi, Y. Multifunctional Carbon-Nanotube Cellular Endoscopes. Nat. Nanotechnol. 2011, 6, 57−64. (4) Caffrey, D. R.; Zhao, J.; Song, Z. L.; Schaffer, M. E.; Haney, S. A.; Subramanian, R. R.; Seymour, A. B.; Hughes, J. D. Sirna Off-Target Effects Can Be Reduced at Concentrations That Match Their Individual Potency. PLoS One 2011, 6, e2150310.1371/journal.pone.0021503 (5) Adolfsson, K.; Persson, H.; Wallentin, J.; Oredsson, S.; Samuelson, L.; Tegenfeldt, J. O.; Borgström, M. T.; Prinz, C. N. Fluorescent Nanowire Heterostructures as a Versatile Tool for Biology Applications. Nano Lett. 2013, 13, 4728−4732. (6) Xie, C.; Hanson, L.; Cui, Y.; Cui, B. X. Vertical Nanopillars for Highly Localized Fluorescence Imaging. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 3894−3899. (7) Kim, W.; Ng, J. K.; Kunitake, M. E.; Conklin, B. R.; Yang, P. D. Interfacing Silicon Nanowires with Mammalian Cells. J. Am. Chem. Soc. 2007, 129, 7228−7229. (8) Wong, I. Y.; Bhatia, S. N.; Toner, M. Nanotechnology: Emerging Tools for Biology and Medicine. Genes Dev. 2013, 27, 2397−2408. (9) Mendes, P. M. Cellular Nanotechnology: Making Biological Interfaces Smarter. Chem. Soc. Rev. 2013, 42, 9207−9218. (10) Park, Y.-S.; Yoon, S. Y.; Park, J. S.; Lee, J. S. Deflection Induced Cellular Focal Adhesion and Anisotropic Growth on Vertically Aligned Silicon Nanowires with Differing Elasticity. NPG Asia Mater. 2016, 8, e249.

Finally, the lateral diffusion of lipids in membrane of cells grown on PD-SiNWs and PD-plain surface were calculated based on the following equation.

D = w 2/4τD

AUTHOR INFORMATION

Corresponding Author

where F(t) is the fluorescence intensity as a function of time, C0 is the maximum recovery intensity, K is the bleach constant, w is bleach radius, and τD is the diffusion time. K and w were calculated based on the pure two-dimensional diffusion by curve fitting with the formula for the Gaussian intensity profile and uniform disc profile for a laser beam on a fixed cell sample.

C(r ) = exp[− K (exp(− 2r 2/w 2)]

Quantification of siRNA deposited on SiNWs; quantitative evaluations of adhesion and spreading based on the basic cell morphology; and fluorescent microscope images of A549 luc cells grown on a bare SiNW substrate (PDF)

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b04913. F

DOI: 10.1021/acsami.6b04913 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

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DOI: 10.1021/acsami.6b04913 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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DOI: 10.1021/acsami.6b04913 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX