Nano-Decoration of the Hemagglutinating Virus of Japan Envelope

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Nano-Decoration of the Hemagglutinating Virus of Japan Envelope (HVJ-E) Using a Layer-by-Layer Assembly Technique Takaharu Okada,†,‡ Koichiro Uto,‡ Masao Sasai,§ Chun Man Lee,§ Mitsuhiro Ebara,‡ and Takao Aoyagi*,†,‡ †

Graduate School of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577, Japan Biomaterials Unit, International Center for Materials Nanoarchitectonics (WPI-MANA), National Institute for Materials Science (NIMS), 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan § Medical Center for Translational Research, Osaka University Hospital, 2-15 Yamadaoka, Suita, Osaka 565-0871, Japan ‡

S Supporting Information *

ABSTRACT: In this study, we created a nanoscale layer of hyaluronic acid (HA) on the inactivated Hemagglutinating Virus of Japan envelope (HVJ-E) via a layer-by-layer (LbL) assembly technique for CD-44 targeted delivery. HVJ-E was selected as the template virus because it has shown a tumor-suppressing ability by eliciting inflammatory cytokine production in dendritic cells. Although it has been required to increase the tumor-targeting ability and reduce nonspecific binding because HVJ-E fuses with virtually all cells and induces hemagglutination in the bloodstream, complete modifications of single-envelope-type viruses with HA have been difficult. Therefore, we studied the surface ζ potential of HVJ-E at different pH values and carefully examined the deposition conditions for the first layer using three cationic polymers: poly-L-lysine (PLL), chitosan (CH), and glycol chitosan (GC). GC-coated HVJ-E particles showed the highest disperse ability under physiological pH and salt conditions without aggregation. An HA layer was then prepared via alternating deposition of HA and GC. The successive decoration of multilayers on HVJ-E has been confirmed by dynamic light scattering (DLS), ζ potentials, and transmission electron microscopy (TEM). An enzymatic degradation assay revealed that only the outermost HA layer was selectively degraded by hyaluronidase. However, entire layers were destabilized at lower pH. Therefore, the HA/GC-coated HVJ-E describe here can be thought of as a potential bomb for cancer immunotherapy because of the ability of targeting CD44 as well as the explosion of nanodecorated HA/GC layers at endosomal pH while preventing nonspecific binding at physiological pH and salt conditions such as in the bloodstream or normal tissues.

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INTRODUCTION The surface modification of cells, viruses, or bacteria by conjugating with bioactive molecules or synthetic polymers has been a versatile way to add new value, advanced features, and unique properties to inserted ones. Creating a nanoscale layer on them significantly improves or even completely changes their biological properties and introduces new, unique properties such as chemical functionality,1−3 imaging,4,5 immune camouflaging,6,7 maintenance of viability,8 and control of stability in the body.9,10 The ability to visualize mammalian cell surfaces in both in vitro and in vivo environments, for example, is essential to gaining further insight into the function of specific molecules or the entire entity. In addition to mammalian cells, myriad viruses and viruslike particles have been genetically and chemically reprogrammed to function as drug- and gene-delivery vehicles11 and nanomaterials.12 Genedelivery vectors based on adenoviral (Ad) vectors, for example, have enormous potential for the treatment of both hereditary and acquired diseases.13 However, many of the therapeutically relevant target cells for gene therapy are refractory to Ad © 2013 American Chemical Society

transduction because of the low expression of primary receptors. The chemical modification of the Ad capsid is one of the most direct approaches to modifying vector tropism. Surface-modified Ads with a multivalent reactive poly[N-(2hydroxypropyl)methacrylamide] (PHPMA)-based copolymer successfully shielded them from recognition by antibodies.14 The direct attachment of ligands such as fibroblast growth factor-2 (FGF-2) through bifunctional poly(ethylene glycol) (PEG) has been shown to augment coxsackie and adenovirus receptor (CAR)-independent gene transfer.15 The reaction to PEG has also been shown to improve the in vivo pharmacokinetics of the vector by increasing the vector persistence in the blood, preventing antibody neutralization.16 PEG coating also prolonged transgene expression and allowed partial readministration with a native virus.17 Special Issue: Interfacial Nanoarchitectonics Received: November 15, 2012 Revised: February 25, 2013 Published: February 26, 2013 7384

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an envelope-type virus being successfully decorated with bioactive polymer multilayers by the LbL method. Therefore, here we describe an original approach to decorating single HVJE particles with HA layers that are programmed to be degraded enzymatically by hyaluronidase secreted by growing tumors or to be destabilized at lower pH in endosomes or a tumor’s periphery while stabilizing HVJ-E at physiological pH and salt conditions such as those in the bloodstream or normal tissues. First, we examined three cationic polymers, poly-L-lysine (PLL), chitosan (CH) and glycol chitosan (GC), as the first layer and explored the adsorption condition to avoid the undesirable aggregation of HVJ-E during LbL processes. Then, the HA layer was prepared via alternating deposition of HA and cationic polymers. The successive decoration of multilayers on HVJ-E has been studied in detail by dynamic light scattering (DLS), ζ potentials, and transmission electron microscopy (TEM). We further demonstrated the degradation of the decorated layers on HVJ-E by enzymatic degradation and lowpH treatment.

In this study, we create a nanoscale layer of hyaluronic acid (HA) on inactivated Hemagglutinating Virus of Japan envelope (HVJ-E) for CD-44 targeted delivery (Figure 1). HVJ-E is a

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MATERIALS AND METHODS

Materials. GenomeONE-CF (Hemagglutinating Virus of Japan envelope, HVJ-E) was purchased from Ishihara Sangyo Kaisha, Ltd. (Osaka, Japan). Glycol chitosan (GC, Mw = 68 000 g/mol), chitosan (CH, Mw = 70 000 g/mol), poly-L-lysine (PLL, Mw = 15 000−30 000 g/mol), hyaluronidase from bovine testes, albumin from bovine serum, 12 tungsten(VI) phosphoric acid n-hydrate, and fluorescein isothiocyanate (FITC) were purchased from Sigma-Aldrich (St. Louis, MO). Hyaluronic acid (HA; Mw = 35 000 g/mol) was purchased from R&D Systems (Minneapolis, MN). Dulbecco’s phosphate-buffered saline (PBS, pH 7.4, 150 mM) was purchased from Lonza (Basel, Switzerland). Chicken preserved blood was purchased from Nippon Bio-Test Laboratories (Tokyo, Japan). FITC-labeled glycol chitosan (FITC-GC) was prepared as follow: 200 mg of GC was dissolved in 20 mL of distilled water (1% w/v), and then the solution pH was adjusted to 6.0. FITC was added to the solution and stirred for 24 h at room temperature. The reaction mixture was transferred to a dialysis membrane (cutoff Mw = 3500 g/ mol, Spectrum Laboratories Inc., Piscataway, NJ). FITC-GC was obtained after freeze dehydration of the dialyzed solution. Surface Coating of HVJ-E with a Polycation Layer. Three different types of polycations, GC, CH, and PLL, were examined in this study (Scheme S1, Supporting Information). HVJ-E (0.3 mg) was first dissolved in 1 mL of PBS and centrifuged in a 1.5 mL tube for 15 min at 15 000 rpm at 4 °C. The supernatant was then removed from each tube and replaced with 500 μL of PBS. The HVJ-E solution was added dropwise to 500 μL of a solution of PBS containing 0.25, 0.5, 1.0, and 2.0 mg/mL of GC, CH, and PLL, respectively. The reaction mixtures were allowed to agitate slowly for 15 min at 4 °C. The solutions were then centrifuged for 15 min at 15 000 rpm at 4 °C, and the supernatant was removed. This centrifugation/redispersion process was repeated three times to obtain cationic polymer-coated HVJ-E particles. Surface Coating of HVJ-E with One, Two, and Three Pairs of HA/GC Layers. Obtained cationic polymer-coated HVJ-E (0.24 mg) was redispersed in 500 μL of PBS. The total number of HVJ-E particles was determined by UV−vis absorbance at 280 nm. The solution was then added dropwise to 500 μL portions of PBS solutions containing 0.25, 0.5, 1.0, or 2.0 mg/mL HA. The reaction mixtures were allowed to agitate slowly for 15 min at 4 °C. The solutions were then centrifuged for 15 min at 15000 rpm at 4 °C, and the supernatant was removed. The precipitate of HA/GC-coated HVJ-E particles was redispersed in 1 mL of PBS. This centrifugation/redispersion process was repeated three times to obtain purified HA/GC-coated HVJ-E. This procedure was followed in cycles to obtain six layers. Characterization. The multilayer growth was monitored by dynamic light scattering (DLS) (DLS-8000, Otsuka Electronics Co.,

Figure 1. Design concept for CD-44 targeted delivery of nanodecorated HVJ-E with HA layers. The HA layer is expected to prevent HVJ-E from immune recognition, fusion with normal tissue, and hemagglutination of erythrocytes but allows binding to the tumor cells through the CD44 surface receptor. The nanofilms can be degraded by hyaluronidase or can destabilize at acidic pH. Thus, HVJ-E can locally induce tumor-specific antitumor immunity in the tumor tissue.

purified product prepared through the complete inactivation of the genome in HVJ by UV irradiation. HVJ-E was originally developed as a novel vector for plasmid DNAs,18 peptides,19 and drugs20 because fusion proteins of the HVJ such as HN and F are retained after inactivation. Recently, Kaneda and coworkers have reported the tumor-suppressing ability of the inactivated, replication-defective HVJ-E itself.21 Although many virus vectors have shown the ability to stimulate host immune responses against cancers,22 HVJ-E alone induced tumorspecific antitumor immunity by eliciting IL-6 production in dendritic cells (DCs) and eradicated 60 to 80% of tumors growing in mice without exogenous gene expression.21 Thus, HVJ-E has recently attracted much attention as a new type of therapeutic material for cancer immunotherapy. However, because HVJ-E fuses with virtually all cells and induces hemagglutination in the bloodstream, it has been required to increase the tumor-targeting ability while reducing nonspecific binding. From these perspectives, we focus on HA, which is one of the major components of the extracellular matrix glycosaminoglycan composed of disaccharides of N-acetylglucosamine and glucuronic acid. HA is nonadhesive toward most proteins, binding only through specific sites to some epitopes of the CD44 cell surface receptor,23−25 and a number of HAs expressed by tumor cells have been correlated with metastasis and proliferation. We employ a layer-by-layer (LbL) technique to encapsulate a single HVJ-E with HA. The LbL technique has quickly become one of the most popular and well-established methods for the preparation of multifunctional thin films because of its versatility, simplicity, and robustness.26−29 The LbL technique has shown broad biomedical applications.30−33 Although studies on the surface modification of capsid-type viruses have been already reported, there are few examples of 7385

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Figure 2. Effects of polymer concentrations on the first cationic layer deposition on HVJ-E. Both the hydrodynamic diameter and ζ potential of the HVJ-E are plotted against the polymer concentrations of (a) PLL, (b) CH, and (c) GC. (Reaction conditions: 4 °C, pH 7.4. 150 mM PBS, reaction time 15 min.). Osaka Japan) at 25 °C. LbL-coated HVJ-E samples were dissolved in 1 mL of PBS (0.3 mg), and the analysis was conducted with a lightscattering apparatus equipped with a 75 mW Ar laser at a scattering angle of 90°. The ζ potential of LbL-coated HVJ-E particles was measured with electrophoretic light scattering (ELS) equipped with a laser Doppler system (ELS-6000, Otsuka Electronics Co., Osaka, Japan). LbL assembly on the HVJ-E was traced by measuring the changes in ζ potential after each layer deposition. All of the samples were suspended in PBS (pH 7.0, 10 mM), and the measurements were performed at 25 °C. Transmission electron microscopy (TEM) measurements were carried out on a JEM-1010 microscope operated at an accelerating voltage of ∼100 kV (Jeol, Tokyo, Japan). The TEM samples were prepared by dropping the HVJ-E suspension onto a carbon-coated grid. The samples were then negatively stained with 2 wt % 12 tungsten(VI) phosphoric acid n-hydrate. TEM images were randomly taken at magnifications of 2000×, 5000×, and 10 000× in a 0.7 cm spaced grid. A quartz crystal analyzer (QCA917, Seiko EG&G, Tokyo, Japan) was employed for the characterization of planar multilayers of GC and HA on the solid substrates. QA-A9M-AU goldcoated quartz crystals (0.2 cm2) from Seiko were used as substrates. The deposition was traced by changes in the frequency of the quartz crystal (Figure S2 in the Supporting Information). Enzymatic Degradation Assay. First, 1000 units of hyaluronidase was dissolved in 1 mL of enzyme diluent (pH 7.0, 20 mM sodium phosphate, 77 mM sodium chloride, 0.01% bovine albumin). A 250 μL portion of the enzyme diluent was mixed with a 200 μL portion of a sodium phosphate buffer solution (pH 5.35, 300 mM) and placed in a 48-well plate. Then 50 μL of PBS containing bare and LbLcoated HVJ-E (0.04 mg) was added to the 48-well plate to allow an enzymatic reaction at 37 °C. After 24 h, the solutions in each well were centrifuged for 15 min at 15 000 rpm at 4 °C to collect the HVJ-E particles. To confirm the existence of the GC layer, FITC-labeled GC was coated onto HVJ-E. After the enzymatic reaction at 37 °C, the UV absorbance of the solution was observed. Effects of Solution pH on the Stability of Multilayers. The two, four, and six layers of (HA/GC)-coated HVJ-E (0.04 mg) were dissolved in 1 mL of PBS (pH 7.4, 150 mM). The solution pH was then adjusted to 4.3 and 6.0 by adding a 0.1 M HCl solution. The solutions were incubated at room temperature for 24 h. The diameters

of the coated HVJ-E in pH 4.3, 6.0, and 7.4 solutions were measured by DLS. Hemolysis Assay of Bare and LbL-Coated HVJ-E. A 3 mL portion of chicken preserved blood was suspended in PBS and purified by centrifugation three times (2500 rpm, 3 min). The bottom solution of red blood cells (RBCs) was dissolved in PBS and adjusted to a concentration of 2% (v/v). Blood solutions (100 μL each) were added to 100 μL each of bare and one-, two-, three-, four-, five-, and six-layercoated HVJ-E solutions (0.1 mg/mL). The mixture was held at 37 °C for 2 h. After the incubation, the reaction solution was centrifuged at 2500 rpm for 3 min. The supernatant’s measured absorbance at 541 nm was compared to that of control sample Triton-X 100-treated RBCs. The percent hemolysis was calculated with the following equation:

%hemolysis =

(absorbance of sample) − (absorbance of blank) (absorbance of control)

× 100

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RESULTS AND DISCUSSION Adsorption of the First Polycation Layer. Of the many biomaterials, capsid-type viruses are of particular interest to the nanotechnology field because of their highly uniform structures, small size, and ability to self-assemble. Recent reports have demonstrated the addition of new functionality to the surfaces of viruses such as cowpea mosaic virus (CPMV)34 and tobacco mosaic virus (TMV),35 yielding efficient routes to spherical materials. Although several approaches have been tried for the surface modification of capsid-type viruses, complete modifications of single-envelope-type viruses with bioactive polyelectrolytes such as HA have been extremely difficult. Wang et al. developed a facile encapsulation method for singleenvelope-type virus human yellow fever vaccine 17D (YF-17D) by the LbL assembly of synthetic polymers poly(allylamine hydrochloride) (PAH) and poly(styrenesulfonate) (PSS).36 Because the combination of PAH and PSS has been known to 7386

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Figure 3. (a−d) TEM micrographs of (a) bare, (b) PLL-, (c) CH-, and (d) GC-coated HVJ-E (polymer concentration 1.0 mg/mL, scale bar 200 nm). (e−h) Size distribution histograms for (e) bare, (f) PLL-, (g) CH-, and (h) GC-coated HVJ-E obtained from DLS measurement (polymer concentration 1.0 mg/mL).

between interparticle attractive and repulsive forces, namely, the electrostatic and hydrophobic interactions. Significant aggregation was not observed for the samples at pH above 5.9 and below 3.9, which has been verified by DLS. This behavior also depends on the salt concentration. When a higher salt concentration was used (150 mM), HVJ-E aggregated even in pH 6.0 solutions (data not shown). Therefore, we employed the following experiments under physiological conditions (PBS, pH 7.4, 150 mM). First, we explored the stable, complete adsorption method of the first polycation layer to avoid the undesirable aggregation of HVJ-E during LbL processes. Figure 2 shows the particle sizes and ζ potentials of coated HVJ-E with different polycations in PBS as a function of the polymer concentrations. Initially, the surface ζ potential of bare HVJ-E (0.3 mg/mL) was negatively charged with a value of approximately −20 mV. Although the ζ potential increases with increasing polycation concentration, it reaches a plateau at an excess of 0.5 mg/mL, around −10 mV for PLL, +5 mV for CH, and −10 mV for GC, respectively. Because the measurements were carried out in PBS, the HVJ-E was still negatively charged even after polycation adsorption. DLS demonstrated that the hydrodynamic diameter of bare HVJ-E was around 250 nm. With different polycations as the first layer, the HVJ-E particles show different stabilities in PBS. When PLL was added, the diameter dramatically increased up to around 1000 nm (Figure 2a). This result indicates that HVJE particles aggregated upon interaction with PLL and thereby induced the increase in the hydrodynamic diameter, even though PLL is one of the most conventional polycations for

produce one of the most stable polyelectrolyte layers, the encapsulated YF-17D viruses were stably shielded by PAH/PSS layers even under denatured conditions. The main purpose of this study is to create stable and complete layers of HA on HVJE under physiological conditions. The major restrictions in single-envelope-type virus encapsulation with HA by the LbL technique are (1) the soft and flexible nature of the lipid bilayer with a viral envelope37 and (2) the surface curvature of objects smaller than a few micrometers or nanometers. In addition, (3) the poor stability of weak polyelectrolyte layers makes the LbL assembly of HA more difficult under the physiological salt condition (150 mM).38 Furthermore, (4) the isoelectric point (pI) of the virus and (5) the membrane fusion pH also limit the LbL deposition of HA. Because there are a few reports on the physical properties of HVJ-E, the pI of bare HVJ-E was measured by ELS. Figure S1a (Supporting Information) plots the ζ potential of HVJ-E measured as a function of pH. The pI is defined as the point of zero ζ potential and is at approximately pH 4.0− 4.5. Therefore, HA can be deposited directly onto the surface of HVJ-E at a pH between the pKa of HA (pH 2.9) and the pI of HVJ-E (pH 4.0−4.5). However, because the viral membrane fusion pH for HVJ-E is around 6.0, where the conformational change in the membrane fusion proteins occurs, single HVJ-E particles cannot exist stably without aggregation below the fusion pH.39 Figure S1b (Supporting Information) shows the time-dependent aggregation of bare HVJ-E particles in solutions of different pH (10 mM). Generally, the stabilization or aggregation of particles depends on the net potential 7387

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LbL deposition on solid surfaces or rigid particles.40 This can be explained by the higher pKa value of PLL (pKa = 9.041), which caused bridging between particles during the centrifugation process. The strong interaction between positively charged PLL and the envelope may also cause domain construction and a disruption of the soft, flexible envelopes at pH 7.4.42 The undesirable aggregation was also observed when CH was added, most likely because of the weak polyelectrolyte and relatively stronger hydrophobicity (poor water solubility) of CH at pH 7.4 (above pKa = 6.543) (Figure 2b). The GCcoated HVJ-E, however, exhibited excellent suspension stability in PBS (Figure 2c). The hydrodynamic diameter remains almost constant at around 250 nm over the concentration range of GC from 0.125 to 1.0 mg/mL, and the ζ potential increases from −20 to −10 mV. TEM was also utilized further to characterize the size, morphology, and structure of the HVJ-E. As seen in Figure 3d, the GC-HVJ-E particles have a single spherical shape with a diameter of 250 nm corresponding to the size obtained from DLS. These results were similar to those for unmodified HVJ-E (Figure 3a). In contrast, images of individual HVJ-E particles were not obtained for PLL- or CH-HVJ-E (Figure 3b,c). Figure 3e−h shows the size distribution histograms of modified HVJ-E with PLL, CH, and GC as determined by DLS. The successful encapsulation of HVJ-E with GC is attributed to the ethylene glycol moiety of GC, leading to better water solubility. Shutava et al. have reported that poly(ethylene glycol)-grafted cationic polymer containing multilayered nanoparticles exhibited excellent suspension stability in PBS because the flexible PEG chains outside can repel particle aggregation.44 Because of the greater solubility of GC compared to that of CH, single GC-HVJ-E particles showed a higher dispersion ability in PBS without aggregation. Therefore, we used GC for the following multilayer formation experiments. Multilayer Formation on HVJ-E. Next, the multilayer growth was studied as a function of the assembly cycles. In general, the LbL assembly of HA has been shown to be unstable at physiological salt concentration because highmolecular-weight weak polyelectrolytes tend to form soluble complexes between the solvated polymer and the previous layer.45 In addition, HA molecules form a double-helical structure because of hydrophobic interactions that may also contribute to the difficulty of obtaining a stable LbL assembly.46 Figure S2 (Supporting Information) shows the QCM signal obtained for HA/GC films plotted as a function of the number of layers. The growing HA/GC film could be built in 1 mM PBS as seen in Figure S2a. The HA/GC film, however, became unstable with the addition of 150 mM PBS. Next, the HA/GC multilayer growth was examined in 150 mM PBS with stirring. A linear increase in the frequency upon HA/GC adsorption was successfully observed even at a high salt concentration by stirring (closed symbols in Figure S2b). This was not observed without stirring (open symbols in Figure S2b). These observations were attributed to the slower growth of HA/GC films resulting from the weak interaction at high ionic strength and pH 7.4. It may also be attributed to the hydrophilic glycol chains of GC, which repel particle aggregation. The vigorous stirring during the LbL buildup processes can increase the reaction rate and shear stress. Therefore, we used the same conditions for the deposition of the second HA layer on GCHVJ-E. Figure 4 shows the effect of HA concentration on the particle sizes and ζ potentials of GC-HVJ-E. Although the increase in the hydrodynamic diameter was small, the ζ

Figure 4. Effects of polymer concentration on the second HA layer deposition on GC-HVJ-E. Both the hydrodynamic diameter and ζ potential of the coated HVJ-E are plotted against the polymer concentrations (reaction conditions: 4 °C, pH 7.4, 150 mM PBS, reaction time 15 min.).

potential decreased from around −10 to −20 mV. This result suggests that the HA coating was successfully employed on single HVJ-E particles without the aggregation of the particles or the solvation of the layers. Figure 5a shows size changes in (HA/GC)-HVJ-E with an increasing number of layers. DLS experiments show the increase in diameter during coating. For each single polyelectrolyte layer, the hydrodynamic diameter increases by approximately 13 nm (i.e., each layer has a thickness of approximately 6 to 7 nm). The size distribution histograms for (HA/GC)-HVJ-E are shown in Figure S3 (Supporting Information). All samples showed the monophasic distribution of the diameter, indicating the successful coating of single HVJE particles. To verify the charge inversion of the outermost layer during LbL deposition, ζ-potential measurements were carried out after each adsorbed layer (Figure 5b). As expected, the surface ζ potential changed alternately with different polyelectrolytes as the top layer. TEM micrographs of individual coated HVJ-E molecules are shown in Figure 6. Although an increase in diameter during coating was observed, mean distances between the surface of the HVJ-E core and the polymer layer could not be determined because no clear image of the surrounding thin polymer layers was obtained. However, the images show that all of the particles had a relatively regular spherical shape with diameters of approximately 244 ± 32, 266 ± 19, 302 ± 40, 306 ± 36, 346 ± 73, and 355 ± 45 nm for zero and two−six layers, respectively. These values consisted of the sizes determined by DLS. In addition, many of small objects can be observed, especially in the TEM image of bare HVJ-E. Presumably, these may be inclusions of HVJ-E or components of the membrane. These results suggested that the HVJ-E was successfully encapsulated in the HA/GC layers not only to stabilize and functionalize it but also to prevent from rapture of the envelope during drying process. Degradation of the HA Layer by Hyaluronidase. Hyaluronidases are a family of enzymes involved in extracellular 7388

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Figure 5. (a) Hydrodynamic diameter and (b) ζ-potential of the LbL coated HVJ-E with GC/HA as a function of layer number. Alternating deposition of 1 mg/mL of GC and HA under pH 7.4, 150 mM PBS for 15 min.

Figure 6. TEM micrographs of HA/GC-coated HVJ-E with zero, two, three, four, five or six layers. Scale bars in the top and bottom images are 1 μm and 200 nm, respectively. All of the TEM samples were negatively stained with 2 wt % tungsten(VI) phosphoric acid nhydrate.

matrix remodeling during normal processes such as developmental cell migration and mammalian fertilization.47 However, the dysregulated expression of specific hyaluronidases within the tumor extracellular matrix has been shown to accompany invasive cancer progression.48,49 Therefore, if HAcoated HVJ-E is successfully delivered to the tumor periphery, then the selective degradation of HA layers can be possible. Figure 7a shows the ζ potentials and the hydrodynamic diameters for (HA/GC)-HVJ-E particles after incubation with hyaluronidase at 37 °C for 24 h. Before the enzyme reaction, the surface ζ potential changed alternately with different electrolytes as the top layer (Figure 5b). After degradation, however, the surface ζ-potential values were observed at around −5 to −8 mV, independent of the outermost layer. Unexpectedly, both the ζ-potential value and the size of bare HVJ-E also increased from −20 to −6 mV and from 240 to 280 nm after the enzymatic reaction, respectively. A similar trend has been observed in all of the samples tested. One of the plausible reasons for these results is that hyaluronidase adsorbed on the surface of HVJ-E because of the higher pI value of 8.0 to 9.0.50 The hyaluronidase may also be able to adsorb onto the GC-coated HVJ-E electrostatically because the ζ potential of GC-HVJ-E particles had negative values as shown in Figure 5b. To confirm the hyaluronidase adsorption on HVJE, the absorbance at 280 nm was measured before and after the enzymatic reaction (Figure S4 in the Supporting Information). After the enzymatic reaction, increased absorbance at 280 nm was clearly observed. These results raise the question, did

Figure 7. (a) Effects of enzymatic treatment on the HA/GC layers on HVJ-E particles. Both hydrodynamic diameter and ζ-potential are plotted against the layer numbers. (b) Schematic illustration of degradation of the outermost surface of HA on the HVJ-E.

hyaluronidase degrade only the outermost HA layer or entire (HA/GC) layers? To assess this, we compared the hydrodynamic diameters for each (HA/GC)-HVJ-E before and after enzymatic degradation. Before the enzyme reaction, the diameters were linearly increased with the assembly steps (Figure 5a). After the degradation however, nonlinear increases in diameter were observed. Interestingly, the particles with five and six layers have similar diameters of around 350 nm. This can also be observed for particles with three and four layers (around 325 nm). Although no significant differences in diameter were observed for zero-, one-, and two-layer samples, these results clearly suggested that hyaluronidase selectively degraded only the outermost HA layer. The existence of a GC layer was also confirmed using FITC-GC-coated HJV-E. As shown in Figure S5 (Supporting Information), fluorescence was still observed after the enzymatic reaction with UV light illumination. 7389

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pH Stability of the Decorated Layers on the Surface of HVJ-E. Finally, to explore the impact of the low pH on the release of decorated layers from HVJ-E, we examined the stability of the decorated HA/GC layers under different pH conditions. In general, the human body exhibits variations of pH along the gastrointestinal tract, tumor areas, inflamed or infected tissues, and the endosomal lumen.51 The endosomal pH, for example, is acidic (5.0 to 6.0) compared to physiological pH (6.0 to 7.4).52 If the nanodecorated film on HVJ-E can be triggered to be released from the surface in an acidic environment, then HVJ-E can efficiently merge with the host endosomal membranes. Several studies have already demonstrated the explosion of LbL multilayers on the particles by external stimuli such as pH and IR laser and ultrasonic treatments.53−55 We examined the explosion of the decorated films at physiological pH (7.4), the membrane fusion pH (6.0), and the pI of HVJ-E (4.3). Figure 8a shows the hydrodynamic

because LbL-coated HVJ-E did not aggregate in pH 6.0 solution as shown in Figure 8. Holmes et al. have reported that the LbL multilayer composed of glycol chitosan and hyaluronic acid on the substrate showed the unique surface feature that was an island structure with high surface roughness.56,57 This result indicates that the homogeneous (flat) coating of GC/HA is difficult and the F protein on the surface of HVJ-E may be partially exposed. The incomplete charge inversion of the zeta potential (Figures 2 and 5b) may be attributed to the exposure of F proteins (Figures 2 and 5b). These results indicate that the formed GC/HA multilayer on HVJ-E successfully improved the stability of HVJ-E particles in aqueous media, whereas the fusion ability originating from the F protein of HVJ-E against RBCs was preserved. At pH 4.3, both bare and LbL-coated HVJ-E aggregated into particles that were greater than 7 μm in diameter (not detected by DLS). It is known that weak polyelectrolytes are easily destabilized by changes in environmental conditions such as the solution pH or salt concentration.58,59 In addition, the surface net charge of HVJE at pH 4.3 may also be an influence because the ζ potential becomes zero at this pH. Interestingly, the increased diameters of the LbL-coated HVJ-E particles were observed at pH 5.0, which is near the endosomal pH (Figure S7 in the Supporting Information). This result indicates that the interactions between GC and HA or between GC and HVJ-E were destabilized by the changes in solution pH between 5.0 and 6.0, although the surface net charge of HVJ-E becomes zero below pH 4.5 (Figure S1). Although it is difficult to identify the disruption mechanism of LbL-coated HVJ-E without obtaining further results, the HA/GC-coated HVJ-E describe here can be thought of as a potential bomb for anticancer therapy because of the ability to target CD44 as well as the explosion of nanodecorated HA/GC layers at endosomal pH while preventing nonspecific binding.

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CONCLUSIONS An LbL assembly has been employed under physiological salt and pH conditions for the decoration of envelope-type HVJ-E with HA. We have first explored a stable, complete adsorption method of the first polycation layer to avoid the undesirable aggregation of HVJ-E during LbL processes. Because of the hydrophilic ethylene glycol side chain of GC, GC-coated HVJ-E exhibited excellent suspension stability in PBS, having a single spherical shape with a diameter of approximately 250 nm. Next, the optimal conditions for the second HA layer assembly were explored. Although the LbL assembly of HA has been difficult in a high-salt environment, vigorous stirring and the complete removal of unmodified polyelectrolytes from the solution by centrifugation enabled us to decorate single GC-HVJ-E particles with HA without aggregation or solvation of the layers. By utilizing this method, we succeeded in forming HA/ GC multilayers on HVJ-E for up to six assembly cycles. DLS experiments show the increase in diameter during coating and the monodisperse size distributions. The ζ-potential measurements were also carried out to verify the charge inversion of the outermost layer during LbL deposition. Monodispersion was confirmed by TEM observation. Furthermore, the outermost HA layer could be selectively degraded by hyaluronidase. However, entire layers were destabilized under the endosomal pH condition. Because HA is nonadhesive toward most proteins, binding only through specific sites to some epitopes of the CD44 cell surface receptor that are overexpressed on tumor cell membranes, the new characteristics of HA-decorated

Figure 8. (a) Effects of solution pH on the stability of HA/GC layers on HVJ-E particles. All of the samples were incubated in solutions of different pH at room temperature for 24 h (7.4, 150 mM). The diameters were measured by DLS. (b) TEM micrographs of bare and HA/GC-coated HVJ-E with four layers at different pH values (scale bars 1 μm).

diameters for bare, two-, four-, and six-layer-coated HVJ-E particles after 24 h of incubation at room temperature in solutions of different pH (150 mM). At pH 6.0, the LbL-coated HVJ-E particles stably dispersed in the solution whereas bare HVJ-E aggregated, triggered by the conformational changes in fusion proteins (F proteins). This result implies that the LbL layers successfully prevented F proteins from interactions via electrostatic and/or steric repulsion. The TEM images also confirm the stability of LbL-coated HVJ-E at pH 6.0 where bare HVJ-E aggregated (Figure 8b). A hemolysis assay was also conducted to confirm whether the fusion ability of HVJ-E was maintained after six layers of LbL. Although the hemolytic activity of HVJ-E was slightly decreased with increasing layer numbers, more than 85% of hemolysis was observed for LbLcoated HVJ-E with six layers. This is an interesting result 7390

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HVJ-E will significantly handle the main shortcomings of bare HVJ-E. In addition, coated HVJ-E still possessed a viral fusion ability. This simple and versatile technique may expand the application of HVJ-E with relatively low cost, easy separation, convenient handling, and improved biological safety on a large scale.

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ASSOCIATED CONTENT

S Supporting Information *

Chemical structures of polyelectrolytes used in this study. Quartz crystal analysis of planar multilayers on QCM substrates. Isoelectric point of HVJ-E. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful for a challenging exploratory research grant (23650295) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.

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