Cellular Binding and Internalization of Functionalized Silicon

Letter pubs.acs.org/NanoLett

Cellular Binding and Internalization of Functionalized Silicon Nanowires Weixia Zhang,† Ling Tong,† and Chen Yang*,†,‡ †

Department of Chemistry, and ‡Department of Physics, Purdue University, West Lafayette, Indiana 47907, United States S Supporting Information *

ABSTRACT: Nanostructures with precise control of sizes and shapes, intrinsic read-out signals for tracking, and flexible surface chemistry for bioconjugation can offer an excellent system to study interaction between nanomaterials and cells. In this paper, a new nanobio system based on functionalized silicon nanowires (SiNWs) was developed. Using the intensive and intrinsic nonlinear optical signal of SiNWs, we visualized the interaction between the folate and amine group functionalized SiNWs and cells by monitoring the cellular binding and uptake of SiNWs in real time. We demonstrated that the strong specific ligand−receptor interaction between folate on NWs and folate receptors on CHO-β cell membranes expedited agglomeration of folate modified SiNWs on cells and internalization of NWs. Such specific targeting was further confirmed through control experiments done with normal CHO cell without folate receptors. Weaker nonspecific charge−charge attraction led to longer time required for amino group modified SiNWs to be bound on cell membrane. No effective accumulation was noticed for unmodified SiNW with native oxidized surface layer. In addition, we also observed the binding was independent of length for NWs ranging between 2.5 and 8.0 μm. Uptake of NWs highly depended on length and NWs longer than 5 μm were difficult to be internalized. Our results provided an insight of cellular interaction with 1-dimensional nanomaterials. KEYWORDS: Cellular interaction, silicon nanowire, surface functionalization

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from the objects to be studied. Radioisotopes used for radioactive labeling are hazardous and must be handled with extreme care, and some radioisotopes with short half-lives are not suitable for long-term monitoring. Intrinsic signals of specific 0D or 1D nanostructures have been utilized to study cellular interactions. Strong luminescence of gold nanoparticles allowed researchers to investigate the effects of shape and surfactant coating on cellular uptake and cytotoxicity;20−22 nevertheless, photothermal effect of gold gives rise to lethal damage to cells. Although near-infrared fluorescence23 and spontaneous Raman scattering24 were used to track carbon nanotubes and to study uptake of carbon nanotubes by cells, difficulty in controlling aspect ratio could limit the usage of carbon nanotubes for systematic cellular studies. Intrinsically fluorescent ZnO nanowires were recently successfully adopted for imaging of cancer cells, but low penetration of UV excitation for fluorescence made it not suitable for future in vivo studies.25 Previously, our study demonstrated that SiNWs emit an intense, intrinsic nonlinear optical (NLO) signal. Its deep penetration enabled by near-infrared or infrared pump beams and high 3D spatial resolution can be useful for in vivo

ith the rapid development of nanotechnology, a wide range of nanomaterials, such as liposomes,1 polymeric micelles,2 quantum dots,3 carbon nanotubes,4 gold nanostructures5,6 (e.g., nanospheres, nanoshells, nanocages, and nanorods), and nanowires7−9 have shown exciting potential as imaging and sensing probes, drug and gene delivery carriers, and therapeutic agents. In spite of much progress in this nanobio direction, to advance the translation of nanomedicine based on these nanostructures to a clinical setting, it requires a fundamental understanding of the interactions between nanomaterials and cells. To establish such an essential understanding, tremendous efforts have been made using various 0D and 1D nanostructures. For example, after being fluorescently or radioactively labeled, liposomes were used to study the effect of ligand density on the binding efficiency.10,11 The size effect on cellular uptake efficiency of nanoparticles, including labeled organic polymers and inorganic silica, were reported,12−14 and the significant roles of particle shape in endocytosis and phagocytosis have been revealed.15,16 Recently, magnetic nanowires were utilized to exploit their interaction with living cells in which cells were labeled with fluorescence probes.17 However, fluorescence probes may bring in toxicity and interference with normal biological processes and might suffer from photobleaching.18,19 The interpretation of fluorescence data could be further complicated due to dissociation of probes © 2012 American Chemical Society

Received: November 23, 2011 Revised: December 21, 2011 Published: January 23, 2012 1002

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application.26 Such a NLO signal can be used for label-free and noninvasive monitoring of the dynamic behavior of the NWs in a biological environment in real time. Additionally, SiNWs with desirable dimensions and aspect ratios could be fully controlled via chemical vapor deposition (CVD) method.27 In this report, we suggest that the SiNW system provides an excellent system to study the cellular response to 1D nanomaterials when implementing its unparalleled capability in size and shape control, its intensive intrinsic nonlinear optical signal for imaging, and the flexible chemistry on the silicon surface. Functionalization of nanomaterials, especially for the inorganic nanostructures, not only prevents agglomeration28,29 and renders them compatible with other phases or systems25,30,31 but also provides them with functional groups for targeting, essential for cellular interaction and regulation. Specifically, SiNWs with a native oxide layer on its surface could be easily functionalized based on the well-studied silica surface chemistry.32 Functional molecules conjugated onto SiNW surface will enable specific targeting and thus cellular regulation. In this work, we choose folate conjugation on SiNW surface since primarily malignant cells often express large numbers of folate receptors and folate conjugation has been conceivably proved useful in the targeting of certain types of cancer cells. Successful folate conjugation on SiNW surface is demonstrated in this work. We visualized cellular interaction of 1D nanomaterials, specifically binding and internalization, using functionalized single crystalline SiNWs. Controlled Synthesis and Characterization of SiNW. SiNWs were synthesized on the Al2O3 substrates by CVD method with silane as the precursor and 40 nm Au nanoparticles as catalysts. Growth pressure and growth time were optimized to produce SiNWs with lengths of 2.5 ± 0.8, 5 ± 1.1, and 8 ± 2.0 μm, respectively. As-grown SiNWs were characterized by scanning electron microscopy (SEM) and NLO imaging system26 (Figure 1a,b).

first treated with oxygen plasma to obtain clean and oxidized surfaces for further modification. The cleaned SiNWs were treated with 1% (v/v) 3-aminopropylmethoxysilianes (APTMS) in pure ethanol for about 24 h. After rinsing with ethanol, the substrate was transferred into 80 °C oven and was allowed to stand for 2 h to stabilize the functionalized groups. The amino modified samples were washed with methanol, and dried under nitrogen.33 On the substrates with the resulting products (1) (denoted as “SiNW-NH2”) triethylamine (TEA) and N-[β-maleimidopropyloxy] succinimide ester (BMPS, 3 mg/mL in anhydrous DMSO) were dropped and stood for 30 min. The unreacted NH2 groups were blocked by adding excess acetic anhydride. Following rinsing and drying, folate-cysteine (1 mM in MES buffer, pH 6.5) was added on the substrates and reacted for 2−3 h.34 Excess folate-cysteine was washed away with phosphate-buffered saline (PBS). This product (2) was denoted as “SiNW-Folate”, representing folate functionalized surface. After the modification, functionalized SiNWs were removed from the substrates into ethanol through sonication. Suspensions obtained were washed several times by the centrifuge method. At last, functionalized SiNWs were dispersed into PBS for future use. The coverage of amino groups on our SiNW surface was estimated to be 1.7 molec/nm2 based on the total covalently bonded APTES coverage on silica.35 The maximum coverage of folate was estimated to be 1.2 molec/nm2, considering the yield of reaction between amino groups and succinimidyl ester of BMPS (83%)36 and the yield of addition reaction between thiol group of folate-cysteine and maleimide group of BMPS (85%).37 Additionally, note that the length of folate-cysBMPS-APTMS was about 2.6 nm obtained from Chem3D using MM2 minimization; therefore, the maximum surface area required for each folate-cys-BMPS-APTMS was approximately 21 square nanometer.38 Thus we estimated the minimum coverage of folate to be approximately 0.05 molec/nm2. Surface functionalization of SiNW was confirmed by monitoring the changes in zeta potential after each modification, with a Malvern Zeta Sizer Nano-ZS90 (Malvern Instruments). For the zeta potential measurement, SiNWs was washed with pure ethanol and water and finally suspended in ultrapure water to form a well-dispersed suspension. Untreated SiNW had a zeta potential of −31.8 ± 16.7 mV, due to the oxide layer on the SiNW surface. The zeta potentials of SiNWNH2 and SiNW-Folate were 24.2 ± 16.5 and −20.9 ± 9.72 mV, respectively. Since the pKa of alkyl-aminium and carboxylic groups are about 10.6 and 4.0, respectively, under neutral condition (the pH of pure water is 7), amino groups of SiNWNH2 were protonated, and the SiNW-NH2 surface was positively charged, whereas the folate ligand features both a positive amino group (protonated) and two negative carboxylic groups (deprotonated), resulting in an overall negative charge. These zeta potential results suggested successful surface functionalization of SiNWs. Cellular Interaction Study. We used both immortalized Chinese hamster ovary (CHO) cells stably transfected with folate receptor β (CHO-β) and normal CHO cells in our cellular interaction studies. Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2. In a typical experiment, a 1 mL suspension of CHO cells (105/ml) was plated onto a glass-bottomed Petri dish, then treated with 100 μg/mL SiNWs suspension and incubated at 37 °C with periodic monitoring.

Figure 1. Characterization of as-synthesized SiNWs. (a) SEM images of SiNWs on the Al2O3 substrate, inset: 90° tilted SEM image of the same sample. Scale bars, 4 μm. (b) FWM image and spectrum of SiNWs. The pump and Stokes laser power at the sample were 0.8 and 1.2 mW, respectively. Scale bar, 2 μm.

SiNWs with uniform diameters were grown with random growth orientations on the amorphous Al2O3 substrate (Figure 1a). Notably, NWs were free-standing on the substrate, which enables effective surface modification later. SiNWs emitted intensive four wave mixing (FWM) signals at 645 nm when excited by the pump and Stokes beams at 790 and 1018 nm, respectively (Figure 1b). Functionalization of SiNWs. Amino modified SiNWs (SiNW-NH2) and folate groups modified SiNWs (SiNWFolate) were obtained through surface modification of SiNWs, as illustrated in Scheme 1. As-synthesized SiNWs samples were 1003

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Scheme 1. Schematic Illustration of SiNW Surface Functionalization

(Figure 2b). For SiNW-folate, despite of the smaller folate coverage, the rapid conjugation of folate with receptors on the cell membranes was finished within the initial 1 h of incubation (Figure 2c), which was comparable with the result previously reported for folate derivatives.11 Further incubation until 4 h, almost all SiNW-Folate were internalized by cells and accumulated locally inside the cells likely in the perinuclear area, where microtubule organization center locates39 (Figure 2d). Internalization of NWs by cells was confirmed using FWM imaging superimposed with fluorescence imaging, in which CHO-β cells were labeled with folate-FITC after internalization of SiNWs (Figure 2f). Internalization can also be visualized by 3D reconstruction of z-scan images (see the video in the Supporting Information). Quantitative analysis of binding is shown in Figure 2g. Each data point was taken based on the analysis of 5−7 cell images. The average number of bound NWs per cell before the onset of internalization was seen to increase over incubation time for both functionalized SiNWs. SiNW-Folate showed considerably higher binding rate than that of SiNW-NH2. In contrast, no obvious binding or internalization was observed in the cells treated with the same concentration of unmodified SiNWs even after 11 h incubation (Figure 2e). Differences in incubation duration required for binding could be explained by the effects of surface properties on NW−cell interactions. It has been shown that compared to the positive surface, neutral or negative surface of nanomaterials is not preferred in the cellular interaction.40−43 No effective binding and thus no uptake of unmodified SiNWs is consistent with these observations. Cationic nanoparticles should be favorable to bind to negatively charged cell surface because of attractive electrostatic interaction.44 Such interaction facilitated the adsorption of SiNW-NH2 to cell membrane. The overall negatively charged folate should have led to, from the perspective of charge−charge interaction, unfavorable interaction between the SiNW-Folate and the negatively charged cell membrane.43 Our result indicated that the strong ligand (folate)-receptor (FR) interaction with the strength of about 1 nN45 could overcome this repulsive electrostatic force. In fact, it is reported that when cultured with Fe3O4 nanoparticles conjugated with folate, cell lines significantly overexpressing the FR showed considerably faster uptake than those cultured with folate-free Fe3O4 nanoparticles.46 Additionally, irreversible folate-FR binding interaction prevents release of NWs from

SiNW-NH2 was used to study the nonspecific charge−charge interaction between cells and SiNWs, since its surface was positively charged under the pH of the cell culture medium of 7.4. SiNW-folate was used to investigate the specific interaction between folate on SiNWs and FR on the CHO-β cell membrane. Binding and internalization of SiNW-NH2, SiNW-Folate, and unmodified SiNWs with 2.5 μm in length in CHO-β cells were monitored using FWM combined with optical transmission imaging. After incubation with SiNW-NH2 for 3 h, accumulation was clearly observed on the cell membrane (Figure 2a), and a portion of SiNW-NH2 were internalized by cells at 10 h

Figure 2. Binding and internalization of SiNWs (red) in CHO-β cells. Overlay of FWM and transmission images of (a and b) SiNW-NH2 after incubation for 3 and 10 h, respectively, (c and d) SiNW-Folate after incubation for 1 and 4 h, respectively, and (e) untreated SiNWs after incubation for 11 h. (f) Overlay of FWM and fluorescence image of a CHO-β cell after internalization of SiNWs. Green, fluorescence from folate-FITC labeled cell membrane. (g) Average number of bound NWs per cell before the onset of internalization as a function of incubation time. Scale bars in a−f, 10 μm. 1004

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cell membranes. Since a cell membrane carries negative charge of approximately 8.3 × 10−9 C per square cm,47 the strength of interaction between amino groups and the cell could be approximately estimated as about 4 × 10−11 N by using simple single point charge model assuming the diameter of the cell to be about 10 μm. Thus, the specific ligand−receptor binding is able to overcome the nonspecific electrostatic interaction, leading to an efficient uptake of the SiNW-Folate into CHO-β. To further confirm our explanation, a control experiment was conducted with normal CHO cells without β FRs. Differences of binding and internalization of these three groups of SiNWs were again examined (Figure 3). After 1 h incubation, no obvious binding was observed for all samples (Figure 3a,d,g).

Figure 4. Surface binding and internalization of SiNW-NH2 with length of (a) 2.5 ± 0.8 μm, (b) 5 ± 1.1 μm, and (c) 8 ± 2.0 μm after incubation with CHO-β cell for 3, 6, 10, and 23 h, respectively. Scale bars, 10 μm.

contact area between straight and rigid NWs and curved cell surface was very limited. The interface utilized for interaction was very small, so that longer NWs with larger surface area were not helpful for binding. As for internalization, however, the length played an important role. For the SiNW-NH2 of 2.5 μm in length, further incubation after binding led to uptake of NWs, and at 10 h SiNWs were internalized with a high density inside cells (Figure 4a). For SiNWs with the length of 5 and 8 μm, although binding yields were comparable to that of 2 μm NWs, internalization rarely occurred. Instead, many NWs were found on the cell membranes in the form of bundles (Figure 4b,c). Our results clearly demonstrate the length effect on the cellar responses, specifically internalization. The study of internalization mechanism is ongoing in our lab.

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CONCLUSION In conclusion, we demonstrated a new nanobio system based on functionalized SiNW to study 1D nanomaterial and cell interactions. The strong intrinsic NLO signal of SiNW is utilized for real-time monitoring. Functionalizations of Si surface with folate is demonstrated for the first time and used for regulating cellular response to SiNWs. The results showed that compared to weak dipole-charge and moderate charge− charge interaction, the strong ligand-specific targeting dominated in binding and uptake of SiNW-Folate by CHO-β cell. The length of NWs was found to affect internalization but not binding rate due to the restriction of geometry. Our pilot study not only provides better understanding of cellular interaction with 1D nanostructures, but also offers new opportunities for the study of nanobio interaction with intrinsic optical signals and controllable structures. Insights on the mechanism of cellular uptake, factors (e.g., ligand density) affecting nanobio interactions, cytotoxicity, and potential applications to diagnosis and drug delivery will be investigated.

Figure 3. Surface binding and internalization of SiNWs (red) with CHO cells. Overlay of FWM (red) and transmission (gray) images of (a−c) SiNW-NH2, (d−f) SiNW-Folate after incubation for 1, 3, and 6 h, and (g−i) untreated SiNWs after incubation for 1, 3, and 7 h, respectively. Scale bars, 10 μm.

Accumulation of SiNW-NH2 on cells membrane was clearly observed at 3 h (Figure 3b), consistent with incubation time when SiNW-NH2 binding with CHO-β cells occurred. The internalization of SiNW-NH2 by normal CHO cells was slightly faster than that by CHO-β cells, occurring at 6 h (Figure 3c). In contrast to interaction with CHO-β cells, when incubating with normal CHO cells, all SiNW-Folate behaved similarly to unmodified SiNWs, present in the culture media instead of accumulation to cell membranes or internalization, after 3 h incubation (Figure 3e,h) and even until incubation for 6 and 7 h, respectively (Figure 3f,i). The results of this control experiment clearly confirmed that the specific interaction between folate and folate receptor attributed to the binding and internalization of SiNW-Folate by CHO-β cells. Effect of lengths on NW-Cell interaction was investigated using SiNW-NH2 with three different lengths of approximately 2.5, 5, and 8 μm, at the same concentrations of 100 μg/mL. As shown in Figure 4, after incubation for 3 h, bindings were clearly observed in all three samples, which indicated that the binding rates could be independent of the length of SiNWs. The result could be explained by geometric restriction. The

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ASSOCIATED CONTENT

S Supporting Information *

Estimation of surface coverage and 3D animation of z-scan mode of optical microscopy confirmed internalization (Supporting video). This material is available free of charge via the Internet at http://pubs.acs.org. 1005

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected].

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ACKNOWLEDGMENTS Authors gratefully acknowledge Dr. J. X. Cheng for insightful discussions and access to measurement facility and Dr. P. S. Low for helpful discussion. C.Y. acknowledges Purdue University and the National Science Foundation grant 103796 for financial assistance.

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