Enhanced Cellular Uptake of Virus-Like Particles through

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Enhanced Cellular Uptake of Virus-Like Particles through Immobilization on a Sialic Acid-Displaying Solid Surface Noriko Ohtake,† Kenichi Niikura,*,‡ Tadaki Suzuki,§ Keita Nagakawa,† Hirofumi Sawa,§,| and Kuniharu Ijiro‡,⊥ Graduate School of Science, Hokkaido University N21W10, Sapporo 001-0021, Japan, Research Institute for Electronic Science, Hokkaido University N21W10, Sapporo 001-0021, Japan, Department of Molecular Pathobiology, Research Center for Zoonosis Control, Hokkaido University N20W10, Sapporo 001-0020, Japan, 21st Century COE Program for Zoonosis Control, Hokkaido University N20W10, Sapporo 001-0020, Japan, and CREST, JST, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan. Received September 10, 2007; Revised Manuscript Received November 13, 2007

Herein, we present the efficient cellular uptake of immobilized virus-like particles (VLPs) made of recombinant JC virus capsid proteins. VLPs expressed in Escherichia coli were labeled with fluorescein isothiocyanate (FITC). We compared two approaches for the cellular uptake of the FITC-VLPs. In the first approach, FITC-VLPs were immobilized on a polystyrene substrate, and then NIH3T3 cells were cultured on the same substrate. In the second approach, cells were cultured on a polystyrene substrate, and FITC-VLPs were then added to the cell culture medium. Flow cytometric analysis and confocal laser microscopic observation revealed that immobilized VLPs were incorporated into the cells with higher efficiency than were the diffusive VLPs suspended in solution. The cellular uptake of VLPs on the substrate was increased in a VLP density-dependent manner. As a control, disassembling VLPs to form VP1 pentamers abolished incorporation into the cells. Displaying sialic acid on the substrate enhanced VLP density through the specific affinities between the VLPs and sialic acid, resulting in efficient incorporation into the seeded cells. These techniques can be applied to the development of novel drug delivery systems and cell microarrays not only of nucleic acids but also of small molecules and proteins through their encapsulation in VLPs.

INTRODUCTION Recently, much emphasis has been placed on the use of cell microarrays as effective tools for the study of a range of phenomena in mammalian cells, as they provide a rapid and simple method for the high-throughput analysis of multiple gene functions (1–3). Cell microarray technology is based on a “reverse transfection” (4) or “substrate-mediated drug delivery” (5) system, in which preimmobilized plasmid DNAs are transfected directly from the substrate into seeded cells. Compared with the conventional method, in which cells are cultured in a medium containing plasmid DNAs, the reverse transfection method has advantages such as easy handling, low cytotoxicity, and enhanced gene expression even in the presence of serum (6, 7). Several problems, however, remain to be solved with regard to drug-immobilization techniques. For example, in the initial method of reverse transfection established by Sabatini et al. (4), usage was limited to gene delivery, as only DNAs could be coated onto the substrate by mixing with gelatin. Microarrays of small molecules, such as drugs and peptides, have also been constructed through the use of biodegradable polymers or chemolabile linkers as drug reservoirs on the surface of the substrates (8, 9). These methods, however, were limited in that the drugs were only incorporated into cells through a passive (and sometimes time-consuming) process because their release from the substrates depended on free diffusion. To prevent cross-contamination of compounds, immobilization at * Corresponding author. Tel: +81-11-706-9370. Fax: +81-11-7069370. E-mail: [email protected]. † Graduate School of Science, Hokkaido University. ‡ Research Institute for Electronic Science, Hokkaido University. § Research Center for Zoonosis Control, Hokkaido University. | 21st Century COE Program for Zoonosis Control. ⊥ CREST.

Figure 1. Schematic illustration of the substrate-mediated delivery approach using virus-like particles (VLPs). (A) Immobilization of VLPs on a polystyrene surface. (B) Enhanced immobilization of VLPs on a synthetic sialic acid-appended polymer (PV-Sia)-coated surface.

low density and concentration was required, thus affording only a small number of samples spotted in the array and decreased cellular uptake. The future application of cell microarrays requires a high-performance drug carrier that can be uniformly immobilized on the substrates, while providing active and efficient entry of various drugs into a broad variety of cells. Therefore, we propose a new substrate-mediated delivery approach using virus-like particles (VLPs) as potential carriers of various drug components (Figure 1). We focused on the JC virus (10), which belongs to the Polyomavirus family and can enter a broad range of mammalian cells (11). Since it is a

10.1021/bc700348g CCC: $40.75  2008 American Chemical Society Published on Web 01/08/2008

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Figure 2. Characterization of VLPs expressed in E. coli. (A) SDS-PAGE analysis of purified VLPs. The VLPs were subjected to SDS-PAGE, and the resulting gel was stained with Coomassie Brilliant Blue. (B) STEM images of purified VLPs (dark field, negatively stained) at 200 kV. The scale bar is 100 nm. (C) Particle size analysis by dynamic light scattering (DLS), performed at 25 °C.

nonenveloped virus, it is better able to tolerate dry conditions than enveloped viruses; thus, it is suitable for immobilization and preservation on the substrates. The capsid of the JC virus is composed of three capsid proteins: VP1, VP2, and VP3. VP1, the 39 kDa major capsid protein of the JC virus, can be expressed recombinantly in E. coli or insect cells. It is known that recombinant VP1 pentamers self-assemble to form VLPs (12). Since VP1 is known to recognize sialic acid receptors on the cellular surface (13, 14), cellular uptake of VLPs through receptor-mediated endocytosis can be expected. Moreover, VLPs are capable of internalizing not only genes but also proteins, small molecules, and peptides (15, 16). For example, the encapsidation of green fluorescent protein (GFP) and methotrexate (MTX) into Polyoma VLPs has been carried out using VP2 as an anchor molecule (17). In this way, VLPs have a potential as carriers of various drug components to several kinds of mammalian cells. Although some cell microarray systems employing adenoviruses or lentiviruses solely as carriers of DNAs and RNAs have been developed (18, 19), there has been no detailed study of cellular uptake efficiency that compares diffusive viruses in solution with those immobilized on substrates. Herein, as a first step in the fabrication of a cell microarray based on VLPs, we report the efficient cellular uptake of immobilized VLPs on a substrate, compared with that of diffusive VLPs in solution, by means of flow cytometric analyses and confocal laser microscopic observations. We also report the effective immobilization of VLPs using the specific affinity between the VLPs and sugar moieties, which enhanced the cellular incorporation of VLPs.

EXPERIMENTAL PROCEDURES General Methods. Fluorescein isothiocyanate (FITC), fetuin, and asialo fetuin were obtained from Sigma-Aldrich Corp. (USA). Poly(N-p-vinylbenzyl-6-O-carboxymethyl-6′-O-carboxymethyl-Oβ-D-galactopyranosyl-(1f4)-D-gluconamide) (PV-LACOOH) and Poly(N-p-vinylbenzyl-O-2-acetoamide-2-deoxy-β-D-glucopyranosyl-(1f4)-2-acetoamide-2-deoxy-D-gluconamide) (PV-GlcNAc) were purchased from Seikagaku Corp. (Japan). UDP-galactose (UDP-Gal) and CMP-N-acetylneuraminic acid disodium salt (CMP-NANA, 2Na) were obtained from Yamasa Corp. (Japan) and Nacalai Tesque, Inc. (Japan), respectively, and β1,4-

Figure 3. Measurements of the binding affinity between the VLPs and sialo-glycoprotein using surface plasmon resonance (SPR) and hemagglutination (HA) assay. (A) Concentration dependence of maximum response units obtained from the SPR measurements. Fetuin (s) or asialo fetuin (---) were immobilized on the sensor chip. (B) HA assay of (a) original and (b) predissociated VLPs. Twenty-five micoliters of serially diluted aqueous solutions of VLPs were examined. The number represents the reciprocal of the dilution factor.

galactosyltransferase (GalT) and R2,6-sialyltransferase (SiaT) were obtained from Toyobo Co. Ltd. (Japan), and Japan Tobacco, Inc. (Japan), respectively. All samples were prepared with ultrapure water (Milli-Q, Millipore, USA) and used without further purification. Expression and Purification of JC VLPs. JC VLPs were prepared as previously described (11). Briefly, the pET15b plasmid (Novagen, USA) including the JC virus VP1 DNA was transformed into E. coli, BL21 (DE3) pLysS (Stratagene, USA). After overnight colony culture at 37 °C in Terrific broth, the expression of VP1 was induced with 1.0 mM isopropyl-β-Dthiogalactopyranoside (IPTG) for 4 h at 30 °C, and the mixture was collected by centrifugation at 4,000g for 15 min. The pellet was resuspended in reassociation buffer (pH 7.4, 20 mM TrisHCl, 150 mM NaCl, and 1 mM CaCl2) containing 1 mg of lysozyme/mL and kept on ice for 30 min, and then 1% sodium deoxycholate was added. After incubation for 10 min on ice, the sample was treated with DNaseI (100 U/mL) for 30 min at 30 °C and lysed by five cycles of sonication for 30 s. After centrifugation of the lysate at 10,000g for 20 min at 4 °C, the supernatant was centrifuged at 25,000 rpm (SW28 rotor, OptimaL, Beckman Coulter, USA) for 3 h at 4 °C. The white layer in the tube was lysed in a reassociation buffer containing CsCl at a final concentration of 1.29 g/mL and then centrifuged at 32,000 rpm (SW41 rotor, OptimaL, Beckman Coulter, USA) for 16 h at 4 °C. The resultant fractions were dialyzed in reassociation buffer at 4 °C overnight. Dissociation of VLPs/FITC Labeling of VLPs. For the dissociation of particle structures, VLPs were mixed in PBS

Enhanced Cellular Uptake of Immobilized VLPs

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Scheme 1. Cellular Uptake of (a) Immobilized or (b) Diffusive FITC-VLPs through the Two Approaches

containing 3 mM dithiothreitol (DTT) and 10 mM EGTA at 4 °C overnight. For the conjugation of VLPs with FITC, 2.5 mg of VLPs was dissolved in 0.1 M carbonate-bicarbonate buffer (pH 9.0), mixed with 250 µg of FITC, and incubated at room temperature for 2 h. The mixture was dialyzed twice in PBS (pH 7.4) containing 1 mM CaCl2.

Figure 4. Immobilization of FITC-VLPs and their subsequent cellular uptake via the substrate-mediated approach using polystyrene substrates. (A) Measurement of the fluorescence intensity of immobilized FITCVLPs at various concentrations. (B) Flow cytometric analysis of NIH3T3 cells cultured on the FITC-VLP-coated substrates. The histograms of the cellular uptake of FITC-VLPs are shown. The initial concentration of the mounted FITC-VLP solution was (a) 0 nM, (b) 0.16 nM, (c) 0.31 nM, and (d) 0.78 nM.

Electron Microscopy/Dynamic Light Scattering (DLS) Analysis. Before observation, 2 µL of the samples was dropped on carbon-coated TEM grids and then negatively stained with 2% phosphotungstic acid. After desiccation overnight, the morphology of the VLPs was confirmed by scanning transmission electron microscopy (STEM) (HD-2000, Hitachi, Japan). Particle size analysis of the VLPs in reassociation buffer was performed by dynamic light scattering (FDLS-3000, Otsuka Electronics, Japan) with measurements taken at 25 °C. Surface Plasmon Resonance (SPR) Measurement. The measurement of the interactions between the VLPs and glycoproteins were carried out using SPR techniques (Biacore X, Biacore, USA). The sensor chip was equilibrated with a running buffer (pH 7.4, 10 mM Tris-HCl, 150 mM NaCl, and 1 mM CaCl2). Fetuin and asialo fetuin were adsorbed onto an Aucoated sensor chip (SIA Kit Au, Biacore, USA) prior to measurement. The buffer solution containing various VLP concentrations (4-40 nM) was then injected onto the glycoprotein-coated sensor chip, and the signal was recorded. Hemagglutination (HA) Assay. The hemagglutination assay was performed to estimate the functional efficiency of VLPs, as described elsewhere (20). Briefly, 25 µL of serially diluted aqueous solutions of VLPs were prepared in a 96-well V-shaped microplate, using 25 µL of PBS (pH 7.15) containing 0.2% bovine serum albumin (BSA). After incubation at 37 °C for 1 h, 25 µL of 0.5% type-O red blood cells were added to each well. The 96-well plate was kept at 4 °C for 3 h and the end point attained. The HA titer was defined as the reciprocal of the greatest dilution of VLP suspension for which complete hemagglutination was observed. Synthesis of Sugar-Displaying Polymers. Enzymatic synthesis of PV-LacNAc was carried out in 10 mM Hepes-NaOH (pH 7.4), 10 mM MnCl2, 50 mM NaCl, 6.8 mM PV-GlcNAc, 20 mM UDP-Gal, and β1,4-galactosyltransferase (170 mU/mL). The enzymatic reaction mixture was slowly rotated at 30 °C overnight. The prepared crude PV-LacNAc solution was next applied to a glycosyltransferase reaction after simple purification using spin filtration. Then, the sialic acid-displaying polymer (PV-Sia) was synthesized in 20 mM cacodylate buffer (pH 7.5), 3.4 mM PV-LacNAc, 5 mM CMP-NANA, and 50 mU/mL R2,6-sialyltransferase. The enzymatic reaction mixture was slowly rotated at 30 °C overnight. Preparation of FITC-VLP-Coated Substrates. PVLACOOH, PV-LacNAc, and PV-Sia were diluted to 0.17 mM with ultrapure water. After sonication for 1 min, 2 mL of each solution was applied to a 6-well cell culture plate (Nunc, USA). The plate was incubated at room temperature for 1 h and then rinsed twice with PBS. The FITC-VLP solution was diluted with PBS to a final concentration of 0-1.57 nM and added to the plate pretreated with PBS or PV-sugar. The plate was incubated in the dark for 1 h and washed twice with PBS.


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The collected cells were also seeded onto an 8-well chamber slide (Nunc, USA) and cultured for 4 h under the same conditions. After rinsing with PBS, the chambers were removed, and the cells were fixed with 4% paraformaldehyde for 10 min. For staining the cell nuculei, 4′,6′-diamidino-2-phenylindole (DAPI) dihydrochloride solution (Dojindo, Japan) was added to a final concentration of 1 µg/mL. The slides were observed using a confocal laser scanning microscope (FV-300, Olympus, Japan).

RESULTS

Figure 5. Mean fluorescence intensity of incorporated FITC-VLPs in NIH3T3 cells through approaches (a) and (b). The relative fluorescence intensity was estimated by taking the intensity of untreated cells as 1.0. The solid line represents the mean cellular uptake of immobilized FITC-VLPs on the substrates (approach (a)), and the dashed line represents that of diffusive FITC-VLPs in culture medium (approach (b)).

Plate Reader Measurements. The fluorescence intensities of each sample were measured by a plate reader (Ex. 485 nm/ Em. 535 nm, Infinite 2000, Tecan, Switzerland) using an optical bottom plate (Nunc, USA) with a clear bottom and black side walls. The fluorescence intensity derived from the FITC-VLPs was estimated by taking the intensity of PBS as a baseline. Cell Culture. NIH3T3 cells were seeded on the FITC-VLPcoated substrate and grown in Dulbecco’s modified essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin/streptomycin) (SigmaAldrich, USA) in 5% CO2 for 4 h. After rinsing several times with PBS, the cells were treated with trypsin (Gibco, USA) and collected by centrifugation. Flow Cytometry/Confocal Laser Microscopy. The cells were resuspended in PBS and analyzed using a fluorescenceactivated cell sorter (FACS canto, Becton Dickinson, USA). For each analysis, 10,000 events were monitored.

A. Characterization of Virus-Like Particles (VLPs). JC virus VP1 recombinantly expressed in E. coli spontaneously self-assembles into virus-like particles (VLPs) in the presence of calcium ions. Assembled VLPs were purified by CsCl density gradient centrifugation. The purified VLPs were electrophoresized on 5% SDS-polyacrylamide gels (Figure 2A). The main band was observed at around 40 kDa, which is in good agreement with the molecular weight of native JC virus VP1 (39 kDa). The morphology of the VLPs was examined by electron microscopy after negative staining (Figure 2B). A relatively homogeneous population of particles of around 40 nm in diameter was observed. Particle size analysis was also performed by dynamic light scattering (DLS). The mean diameter of the VLPs was estimated to be 41.0 ( 12.1 nm (Figure 2C). These results were similar to those reported in previous literature (21). Although it has been reported that JC VLPs preferentially bind to N-linked glycoproteins containing terminal sialic acid (22), the quantitative binding constant has not yet been determined. Therefore, we measured the binding affinity between the VLPs and sialo-glycoprotein by surface plasmon resonance (SPR) measurements on a Biacore instrument. As a target glycoprotein, we chose fetuin, which is a 48-kDa-protein possessing 8.9 wt% of sialic acid moieties (23, 24). Asialo fetuin, a sialic acid-free protein, was used as a negative control. The two glycoproteins were separately adsorbed onto an Aucoated sensor chip through hydrophobic interactions. Different

Figure 6. Comparision of the localization of FITC-VLPs incorporated into NIH3T3 cells through approaches (a) and (b). (A) Fluorescence images (left), merged fluorescence and nuclear staining images (middle), and differential interference contrast images (right) showing cells after FITC-VLP incorporation through approaches (a) and (b). Images of the negative control using untreated cells are also displayed. The cells were observed by confocal laser scanning microscopy. The scale bars are 20 µm. (B) Relative fluorescence intensities of FITC and DAPI along the dotted lines drawn on the images for approaches (a) and (b) in (A), respectively. (C) Ratio of fluorescence intensities of FITC in the nucleus. The total fluorescence intensity of FITC in the cell was taken as 100%.


Figure 7. Flow cytometric analyses of substrate-mediated cellular uptake of (a) FITC-VLPs and (b) FITC-VLPs disassembled to form VP1 pentamers. The histogram of the negative control using untreated cells is shown as (c).

concentrations of the VLPs were passed over the surface to determine the concentration-dependent maximum response units (∆RU) (Figure 3A). The binding constant of 4.1 × 107 M-1 estimated from the ∆RU curve demonstrates the high selectivity of the VLPs to the sialic acids. This value was similar in order to that of another lectin, SSA (1.4 × 107 M-1) (25). To confirm the ability of our recombinant VLPs to recognize sialic acid, we also performed the hemagglutination (HA) assay (Figure 3B). The HA titer of VLPs obtained from E. coli was 512 HA/ µL, suggesting a relatively high binding affinity between the VLPs and sialic acids present in red blood cells. The samples that were predissociated to VP1 pentamers by treatment with EGTA and DTT did not show any HA activity, probably because they no longer had the ability to bridge blood cells (Figure 3B). B. Introduction of VLPs Deposited on a PolystyreneBased Substrate. In order to quantify cellular uptake, VLPs were conjugated with FITC through a stable covalent linkage with the amine group on the VLPs, and the reaction mixture was then dialyzed to remove the unreacted FITC. The two approaches to the cellular uptake of FITC-labeled VLPs are illustrated in Scheme 1. In our novel approach (a), the FITCVLPs were first immobilized on the substrate and washed with PBS, and then the cells were seeded onto the substrate. In this approach, the immobilized FITC-VLPs were directly introduced from the substrate to the cultured cells. Approach (b) represents a conventional method, in which the cells were cultured on the substrate prior to the addition of FITC-VLPs into the culture medium. First, we investigated the immobilization of FITC-VLPs and their subsequent cellular uptake in approach (a). We employed a tissue culture polystyrene plate to immobilize the FITC-VLPs for cell culture. An aqueous solution of FITC-VLPs was mounted on a polystyrene substrate and incubated for 1 h, and then the substrate was rinsed well with PBS. The fluorescence intensities of the substrate before and after rinsing with PBS were measured using a fluorescence plate reader. Approximately 1/10 of FITC-VLPs in the mounted solution were deposited on the substrate even after rinsing with PBS (data not shown).


Various concentrations of FITC-VLP in aqueous solutions were also mounted on the substrate, followed by washing with PBS and measurement of fluorescence intensities (Figure 4A). The fluorescence intensity curve shows that the number of FITCVLPs immobilized on the substrate increased exponentially with increases in the initial concentration of FITC-VLPs in the aqueous solution, up to the saturation of the FITC-VLP binding sites on the polystyrene substrate. NIH3T3 cells were cultured on the FITC-VLP-coated substrate in DMEM containing 10% FBS for 4 h and then removed from the substrate by treatment with trypsin. After collection, the cellular uptake of FITC-VLPs was analyzed by flow cytometry. The histogram shown in Figure 4B indicates the introduction of FITC-VLPs into the cells cultured on the FITCVLP-coated substrates. The cellular uptake of FITC-VLPs increased with increases in the initial concentration of FITCVLPs because of an increase in the number of VLPs immobilized on the substrate. We next compared the efficiency of cellular uptake of FITCVLP through approaches (a) and (b). Mean cellular uptake using various quantities of FITC-VLPs (0-0.32 pmol) is plotted in Figure 5. The number of FITC-VLPs used in the two approaches was calculated as follows. Aqueous solutions of FITC-VLPs at identical concentrations were first mounted in both approaches. In approach (a), as the substrate was rinsed with PBS, some of the FITC-VLPs in the mounted solution were washed out. Therefore, the number of FITC-VLPs immobilized on the substrate was estimated from the relative fluorescence intensity at each concentration as previously determined by plate reader measurement. However, in approach (b), the number of FITCVLPs was estimated only by culture volume of the mounted solution. We used 2 mL of FITC-VLP-containing DMEM for approach (b), which is the minimum volume required for cell culture, and the number of FITC-VLPs in solution was considered to be sufficient compared with that in previous reports (26). Surprisingly, the introduction of FITC-VLPs through approach (a) was possible at a much lower quantity than was possible through approach (b). While approach (b) requires more than 0.1 pmol of FITC-VLPs for the detection of cellular uptake, less than 0.1 pmol of FITC-VLPs was sufficient for high cellular uptake in approach (a). This result suggests that immobilization on the substrate leads to efficient cellular uptake of FITC-VLPs. Cellular uptake of FITC-VLPs was further confirmed by confocal laser scanning microscopy (Figure 6A). To eliminate the fluorescence derived from FITC-VLPs retained on the substrate, the cells were removed from the substrate by trypsin treatment and cultured again on new glass slides for a further 4 h. The observation of cells prepared by approaches (a) and (b) showed that FITC-VLPs were internalized into the cells, not just attached to the cell surface. Interestingly, the VLPs from the substrate were more dispersed into the nucleus than were those from the solution (Figure 6B and C). As a control, FITC-VLPs were disassembled to VP1 pentamers by the addition of DTT and EGTA prior to immobilization on the substrate (Figure 7). In this case, there was little incorporation of the disassembled sample from the substrate as compared to that of the original VLPs, suggesting that VLP formation is necessary for cellular uptake from the substrate. C. Increased Cellular Uptake Using Sialic-Acid Displaying Substrate. On the basis of our results using polystyrenebased substrates, deposition of a large number of VLPs on the substrate was significant enough to increase the cellular uptake of the immobilized VLPs. Therefore, we sought to enhance cellular uptake through increasing the number of VLPs immobilized on the substrate using their specificity to sialic acid (22).


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Figure 8. Immobilization of FITC-VLPs and subsequent cellular uptake via the substrate-mediated approach using sugar-displaying substrates. (A) Fluorescence intensities of FITC-VLPs attached to the substrate precoated with 0.5 mg/mL fetuin and asialo fetuin. (B) Comparison of the fluorescence intensities of FITC-VLPs immobilized on the polystyrene (PS) and sugar-displaying substrates. (C) Fluorescence images of NIH3T3 cells cultured on the FITC-VLP-coated sugar-displaying substrates. The cells were observed by confocal laser scanning microscopy. The scale bars are 50 µm. (D) Comparison of the NIH3T3 cellular uptake of FITC-VLPs attached to the sugar-displaying substrates. Whole fluorescence intensities of the confocal images were estimated using image analysis, and the mean values obtained from three images are shown. The relative fluorescence intensity was estimated by taking the intensity of PS as 1.0.

Using fetuin and asialo fetuin, we initially confirmed that displaying sialic acid from the polystyrene substrate could increase the number of immobilized FITC-VLPs (Figure 8A). The result shows that the FITC-VLPs were more effectively immobilized onto the fetuin-coated substrate than that coated with asialo fetuin, which is in good agreement with the results of the SPR measurement. This suggests that displaying sialic acid from a polystyrene substrate is effective in increasing FITCVLP immobilization. For the preparation of a sugar-displaying substrate more appropriate for cell culture, we next used PV-sugars, which are synthetic sugars grafted to polystyrene (27, 28). PV-sugars are reported to dissolve in water, forming a characteristic bottlebrush conformation composed of a polystyrene backbone covered with sugar brushes (29). Mounting aqueous solutions of PV-sugars onto substrates can provide a uniform glycopolymer-coated surface, which has good biocompatibility for cell culture (30, 31).

The synthesis of PV-sugar containing sialic acid (PV-Sia) was performed by two-step glycosyltransferase reaction (Scheme 2). In the first stage, an aqueous solution of PV-GlcNAc was treated with β1,4-galactosyltransferase (GalT) in the presence of UDP-Gal, providing the formation of PV-LacNAc. The product was further treated with R2,6-sialyltransferase (SiaT) in the presence of CMP-NANA, generating the final product PV-Sia. Since the polymers formed a micelle structure, they were not identifiable by NMR. Therefore, glycosylation was confirmed both by the specific binding of lectins for galactose and by resorcinol assay sensitive to sialic acid. FITC-labeled RCA120, which is a lectin that specifically associates with Galβ1-4GlcNAc (32, 33), did not bind to the PV-GlcNAc-coated substrate but strongly bound after the galactosyltransferase reaction (see Supporting Information). Subsequent sialylation of the polymer by sialyltransferase caused a large decrease in RCA120 binding since the lectin only slightly associates with



Scheme 2. Enzymatic Syntheses of Sialic Acid-Displaying PV-Sugars

sialic acid (34). The content of sialic acid in the polymer was then determined by colorimetry using the resorcinol assay (35). The sialic acids released from glycosidic linkage by heating in a strong aqueous acid solution were quantified by absorption at 580 nm. The ratio of side chains possessing a sialic acid in PV-Sia was estimated to be around 19% (see Supporting Information). These experiments showed that carbohydrates were transferred step by step, and that almost 20% of sialic acids were finally displayed on the polymer chain. We otherwise used PV-LACOOH as a control for the electrostatic interaction of PV-Sia. Ellipsometric measurements were carried out in order to detect the film thickness of each polymer (see Supporting Information). Glass slides were spin-coated with PV-sugars and washed well with water. After drying overnight, the thickness of the films was examined. The thicknesses of the PVLACOOH, PV-LacNAc, and PV-Sia films were 79.3 ( 3.7, 77.7 ( 4.3, and 79.6 ( 3.7 nm, respectively, indicating that the polymers were tightly fixed to the surface of the substrates. Plate reader measurements after the binding of the FITCVLPs showed that the fluorescence intensity obtained from the PV-Sia-coated substrate was 2-fold higher than that obtained from the polystyrene substrate (Figure 8B). The difference in fluorescence intensity between the PV-Sia-coated and the polystyrene substrates was in good agreement with that between the substrates coated with fetuin and asialo fetuin (Figure 8A). The PV-LacNAc-coated substrate provided a moderate increase in FITC-VLP immobilization. FITC-VLP immobilization onto the PV-LACOOH-coated substrate was approximately equivalent to that onto the polystyrene substrate. These results indicate that FITC-VLPs were effectively immobilized on the PV-Siadisplaying substrate because of their specific interaction with sialic acid, not because of electrostatic interaction. Finally, the introduction of FITC-VLPs from sugar-displaying substrates was investigated using confocal laser scanning microscopy as described above. Fluorescence images of NIH3T3 cells cultured on the FITC-VLP-coated sugar-displaying substrates are shown in Figure 8C. Seeded cells adhered well to all sugar-displaying substrates showing no cytotoxicity (data not shown). The cellular uptake of FITC-VLPs was also observed on all sugar-displaying substrates. The digitalized intensities of the fluorescence images demonstrate that the cells cultured on the PV-Sia-displaying substrate provided the significantly highest fluorescence intensity (Figure 8D). Our

results show that coating the substrate with sialic acid-displaying biomaterials leads to more effective cellular uptake of VLPs.

DISCUSSION JC virus VP1 recombinantly expressed in E. coli spontaneously self-assembles into virus-like particles (VLPs), which can be isolated by CsCl density gradient centrifugation. Surface plasmon resonance (SPR) measurements indicated that the VLPs have specific affinity to the sialic acid moiety of glycoprotein. We investigated the immobilization of VLPs and their subsequent cellular uptake in approach (a) using a tissue culture polystyrene plate. A comparison of this approach with the control, approach (b), revealed that immobilized VLPs were incorporated into the cells with higher efficiency than were the diffusive VLPs suspended in solution. When VLPs are added to a solution, a limited number of VLPs can access the cell surface because of their Brownian movements. However, when introduced from a substrate, a large number of VLPs can spontaneously come into contact with the cellular surface, leading to more effective uptake. Another possible explanation of this enhanced cellular uptake from the substrate is cell adhesion. There have been some reports on reverse transfection showing that increased cell adhesiveness through the addition of an extracellular matrix (ECM) enhances gene expression (36, 37). In our study, cell adhesion to the VLP-coated substrates was actually increased in comparison to that to the untreated substrates (data not shown). This indicates that VLPs themselves acted as an ECM and strengthened cell adhesion, resulting in the enhanced cellular uptake of the VLPs. Subsequent confocal laser microscopic observation indicated that VLPs introduced from the substrate were more dispersed into the nucleus than were those from the solution (Figure 6). In this study, we used JC virus-derived VLPs, containing a nuclear localizing signal at the C-terminus, known to localize in the nucleus after cellular uptake (26). We speculated that FITC-VLPs from the substrate could be localized into the nucleus faster than those from the solution since the cellular uptake from the substrate occurred more rapidly. Previously, Shea et al. demonstrated that the expression of immobilized DNAs leveled up to 100-fold greater than bulk delivery of DNAs (38). Our results showing rapid cellular uptake of VLPs from the substrate and subsequent dispersion into the nucleus could


explain the fact that substrate-mediated transfection induces efficient and enhanced gene expression. We next proved that VLP formation was necessary for cellular uptake from the substrate. Even if VP1 pentamers bind to the receptors on the cell surface, they might be incapable of inducing structural change in the receptor, thus they cannot induce endocytosis. In addition, size effect is also important for the incorporation of VLPs. According to the report of Aoyama et al., the optimal size of nanoparticles for endocytosis is around 50 nm (39), which is almost the same size as that of the VLPs in this study. Finally, PV-Sia was enzymatically synthesized and used for the immobilization of FITC-VLPs, resulting in enhancement of the cellular uptake of FITC-VLPs. Two factors are thought to be mainly responsible for this enhancement using PV-Sia: increased FITCVLP immobilization and strengthened adhesion of seeded cells. Akaike et al. reported that PV-LA, a PV-sugar, acts as an ECM and enables progressive hepatocyte adhesion (40). This function of PV-sugar as an ECM might be crucial to the enhancement of cell adhesion. Though various factors are thought to contribute to the efficiency of cellular uptake, we demonstrated that increased VLP immobilization using sialic acid significantly increased the incorporation of VLPs from the substrate. In conclusion, we report the efficient cellular uptake of immobilized VLPs. VLPs expressed in E. coli were labeled with FITC and immobilized on the substrate. Flow cytometric analysis and confocal laser microscopic observation revealed that immobilized VLPs were incorporated into the cells with higher efficiency than were diffusive VLPs suspended in solution. Our approach was extended to sialic acid-displaying surfaces aimed at increasing the number of FITC-VLPs immobilized on the substrate via the specific affinity between VLPs and sialic acid. These techniques have potential applications to the fabrication of cell microarray systems using VLPs as multiple carriers of nucleic acids, small molecules, and proteins.

ACKNOWLEDGMENT We appreciate Associate Professor Yoshiko Miura of JAIST for kindly providing a PV-sugar sample. We also thank Professor Kohei Uosaki and Assistant Professor Satoru Takakusagi of Hokkaido University for their help with ellipsometric measurements. Noriko Ohtake and Tadaki Suzuki appreciate the financial support provided by JSPS. This study was supported in part by grants from the Program of Founding Research Centers for Emerging and Reemerging Infectious Diseases, MEXT, Japan. Supporting Information Available: Details of experimental procedures and results for the characterization of synthetic polymers. This material is available free of charge via the Internet at http://pubs.acs.org.

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