Surfactant-modified nanoclay exhibits an antiviral activity with high potency 1 and broad spectrum

44 Pages·2014·1.73 MB·English

JVI Accepts, published online ahead of print on 29 January 2014
J. Virol. doi:10.1128/JVI.03256-13
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
1 Surfactant-modified nanoclay exhibits an antiviral activity with high potency
2 and broad spectrum
3 Jian-Jong Liang,a Jiun-Chiou Wei,b Yi-Ling Lee,a Shan-hui Hsu,b# Jiang-Jen Lin, b
4 and Yi-Ling Lina,c#
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5 Institute of Biomedical Sciences,a and Genomics Research Center,c Academia Sinica, o
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6 Taipei, Taiwan; and Institute of Polymer Science and Engineering, National Taiwan a
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7 University, Taipei, Taiwan b f
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8 Running title: antiviral activity of surfactant-modified nanoclay h
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9 #To whom correspondence should be addressed. //jv
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10 Yi-Ling Lin, PhD, Institute of Biomedical Sciences, Academia Sinica, No. 128, Sec. 2, m
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11 Academia Road, Taipei 11529, Taiwan, Phone: [phone], E-mail: /

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12 [email protected]
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13 Shan-hui Hsu, PhD, Institute of Polymer Science and Engineering, National Taiwan ,
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14 University, No. 1, Sec. 4, Roosevelt Road, Taipei, 10617, Taiwan, Phone:
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15 886-2-3366-5313, E-mail: [email protected] u
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16 Manuscript information: 199 words in abstract and 7 figures.
17 Keywords: nanoscale silicate platelet; surfactant; electrostatic interaction; antiviral
18 activity; Japanese encephalitis virus; dengue virus; influenza A virus
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19 Abstract
20 Nanomaterials have the characteristic of high surface-to-volume ratio and have
21 been explored for their antiviral activity. Despite some success, cytotoxicity has been
22 an issue of nanomaterial-based antiviral strategy. We previously developed a novel
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23 method to fully exfoliate montmorillonite clay to generate the most fundamental units o
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24 of nanoscale silicate platelet (NSP). We further modified NSP by capping with a
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25 various surfactants and found the surfactant-modified NSP (NSQ) was less cytotoxic. f
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26 In this study, we tested the antiviral potential of a series of nature clay-derived h
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27 nanomaterials. Among the derivatives, NSP modified with anionic sodium dodecyl /jv
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28 sulfate (NSQc), but not the pristine clay, unmodified NSP, silver nanoparticle-NSP m
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29 hybrid, NSP modified with cationic n-octadecanylamine hydrochloride salt, or NSP g
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30 modified with nonionic Triton X-100, significantly suppressed the plaque-forming A
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31 ability of Japanese encephalitis virus (JEV) at noncytotoxic concentrations. NSQc 1
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32 also blocked the infection with dengue virus (DEN) and influenza A virus. Regarding
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33 the antiviral mechanism, NSQc interfered with viral binding through electrostatic g
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34 interaction, since its antiviral activity can be neutralized by polybrene, a cationic t
35 polymer. Furthermore, NSQc reduced the lethality of JEV and DEN infection in
36 mouse challenge models. Thus, the surfactant-modified exfoliated nanoclay NSQc
37 may be a novel nanomaterial with broad and potent antiviral activity.
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38 Importance
39 Nanomaterials have being investigated as antimicrobial agents, yet their antiviral
40 potential is overshadowed by the cytotoxicity. By using a novel method, we fully
41 exfoliate montmorillonite clay to generate the most fundamental units of nanoscale
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42 silicate platelet (NSP). Here, we show that the surfactant-modified NSP (NSQ) is less o
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43 cytotoxic and NSQc (NSP modified with sodium dodecyl sulfate) could potently a
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44 block infection of dengue virus (DEN), Japanese encephalitis virus (JEV) and f
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45 influenza A virus at noncytotoxic concentrations. For the antiviral mechanism, we h
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46 find that the electrostatic interaction between the negative-charged NSQc and the /jv
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47 positive-charged virus particles blocks viral binding. Furthermore, we used mouse m
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48 challenge models of JEV and DEN to demonstrate the in vivo antiviral potential of g
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49 NSQc. Thus, NSQc may function as a potent and safe antiviral nanohybrid against A
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50 several viruses and our success in synthesizing surfactant-modified NSP with antiviral 1
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51 activity may shed some light on future antiviral development.
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52 Introduction
53 Emerging viral infections have been threatening public health constantly, for
54 example the SARS coronavirus outbreak ~10 years ago and the recent H7N9 avian
55 influenza A virus infection (1). Mosquito-borne flaviviruses, such as dengue virus
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56 (DEN) and Japanese encephalitis virus (JEV) are reemerging and affecting humans o
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57 living in tropical and sub-tropical areas. DEN infection in humans causes a wide a
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58 spectrum of illnesses ranging from mild dengue fever to severe complications such as f
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59 dengue hemorrhagic fever and dengue shock syndrome (2, 3). JEV is the most h
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60 important agent of viral encephalitis and causes acute encephalitis with high mortality /jv
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61 in Asia (4). Inactivated and attenuated vaccines are available for JEV, but no vaccine m
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62 exists for DEN, in part because of the complexity of 4 serotypes of DEN and the g
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63 potential involvement of antibody-dependent enhancement in severe dengue diseases. A
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64 So far, no specific antiviral therapeutics are available for treating JEV and DEN 1
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65 infection (5, 6), thus there is a great need to explore novel technology such as
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66 nanomaterials for their antiviral potentials against these viruses. g
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67 Nanomaterials have the characteristics of high surface-to-volume ratios and t
68 generally exhibit unique properties not occurring in the micrometer-size analogs. The
69 novel properties due to miniaturization of bulk materials have been intensively
70 exploited in many research fields, and the practical applications are numerous (7). The
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71 concept of creating nanosized pharmaceuticals has been explored for treating and
72 preventing human diseases (8, 9). For example, high potency of silver nanoparticle
73 (AgNP) for antibacterial and antifungal activities has been well demonstrated (10).
74 Continuing research on incorporating AgNP into a wide range of medical devices
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75 such as bone cement, surgical instruments and wound dressings are actively pursued. o
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76 Recently, the antiviral effects of AgNP have been demonstrated against several a
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77 viruses (10) such as HIV (11), herpes simplex virus (12), hepatitis B virus (13), f
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78 respiratory syncytial virus (14), and influenza virus (15). However, the adverse effects h
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79 of using nanoparticles such as AgNP found highly cytotoxic to many mammalian /jv
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80 cells (10, 16, 17) are a concern and applications of nanomaterials as antiviral agents m
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81 have lagged behind similar antibacterial studies. g
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82 Naturally occurring clays such as montmorillonite (MMT) are conventionally A
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83 used for catalysts and adsorbent agents and have been used as natural medicine 1
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84 (18-20). We developed a novel method to fully exfoliate MMT layered silicate clay to
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85 generate the most fundamental units of nanoscale silicate platelet (NSP) (21) (Fig. g
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86 1A), which possess higher antibacterial activity than the parental MMT in stack t
87 structure (22). The high surface-to-volume ratio and polyvalent anionic charges on a
88 single platelet render intense forces for 2-dimensional non-covalent bonding attraction
89 and provide an extensive reacting surface for hybridizing AgNP on 1-nm-thick NSP.
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90 Synthesized AgNP/NSP nanohybrids inhibited the growth of several bacterial
91 pathogens and even Ag-resistant E. coli and drug-resistant Staphylococcus aureus (23,
92 24). Recently, we also found that AgNP/NSP protected chicks against salmonella
93 infection (25). However, NSP with polyvalent ions could directly interact with the cell
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94 membrane, thus leading to some cytotoxic effects. To reduce the cytotoxicity, NSP o
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95 was modified by capping with surfactants, because surfactant-capped nanomaterials a
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96 are known to have lower cytotoxicity in general (26). NSP was modified by the f
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97 cationic n-octadecanylamine hydrochloride salt (Qa), nonionic Triton X-100 (Qb), or h
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98 anionic sodium dodecyl sulfate (SDS, Qc) (27) (Fig. 1B). The surfactant-modified /jv
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99 NSP such as NSQa, NSQb and NSQc, commonly showed less cytotoxicity and m
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100 enhanced antimicrobial activities, probably due to improvement of dispersibility in g
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101 water (27). A
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102 In this study, we evaluated the antiviral potential of a series of clay 1
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103 MMT-derived nanomaterials and found that NSQc, but not the pristine clay,
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104 unmodified NSP, AgNP/NSP, NSQa, or NSQb, potently blocked the plaque-forming g
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105 ability of JEV and also suppressed infection with DEN serotype 2 (DEN-2) and t
106 influenza A virus. The antiviral potency of NSQc depends on the composition of NSP
107 and the surfactant SDS, because NSQc(A30) with a higher ratio of surfactant to NSP
108 (70/30 weight ratio) showed stronger antiviral activity than NSQc(A50) (50/50 weight
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109 ratio). For the antiviral mechanism, we demonstrated an electrostatic interaction
110 between the negative-charged NSQc and the positive-charged virus particles as the
111 predominant factor in antiviral action. Furthermore, we used mouse models of JEV
112 and DEN infection to demonstrate the in vivo antiviral potential of NSQc. Thus, the
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113 complexes of NSP and negative-charged surfactant may be potent and safe antiviral o
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114 nanomaterials against several pathogenic viruses. a
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115 MATERIALS AND METHODS
116 Cell lines and viruses. Baby hamster kidney fibroblast BHK-21 cells, human
117 neuroblastoma SK-N-SH cells (ATCC HTB-11) and human lung carcinoma A549
118 cells were cultured as described (28, 29). The propagation and plaque formation of
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119 JEV RP-9 strain and DEN-2 PL046 and NGC-N strains have been described o
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120 previously (28, 29). Influenza A virus (H1N1, WSN) and MDCK (Madin-Darby a
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121 canine kidney) cells were kindly provided by Dr. Michael Lai (Institute of Molecular f
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122 Biology, Academia Sinica, Taipei, Taiwan). h
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123 Chemicals and antibodies. Hexadimethrine bromide (polybrene) was from /jv
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124 Sigma-Aldrich. Mouse antibodies against actin (Novus Biologicals), influenza A virus m
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125 nucleoprotein (Abcam, ab20343), JEV NS3 and DEN-2 NS3 proteins (30) were used. g
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126 NSQ preparation and characterization. NSP was isolated by one-step A
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127 exfoliation of natural sodium MMT clay and toluene/aqueous NaOH extraction (21, 1
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128 22). Three types of NSQ (NSQa, NSQb, and NSQc) were created by use of different
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129 surfactants with NSP as described (27). To remove the free SDS, NSQc was dialyzed g
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130 against double distilled water by use of a dialysis membrane (3500 MWCO, t
131 Membrane Filtration Products, Inc.) for three days. The mass percentages of NSP and
132 SDS were obtained with a thermogravimetric analyzer (TGA 7, PerkinElmer, USA) at
133 a heating rate of 10 °C per min from 100 to 750 °C. Nanomaterials were examined by
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134 using a transmission electron microscope (Hitachi H-7100) operated at 100 kV. The
135 zeta potentials of nanomaterials in water were determined by laser light scattering
136 using a zeta potential and submicrometer particle analyzer (Delsa Nano S; Beckman
137 Coulter). For gel electrophoresis, the nanomaterials were mixed with loading dye (5%
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138 glycerol, 0.05% bromophenol blue, 0.05% xylene cyanol and 0.05% neutral red) and o
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139 separated on a 1% agarose gel in 0.5x TBE buffer for 10-15 min at a constant voltage a
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140 of 100 V. Gel was stained with ethidium bromide and photographed under UV light. f
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141 Cytotoxicity test. Cytotoxicity was assessed by use of the Cell Proliferation Kit h
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142 II (XTT) (Roche). Briefly, BHK-21 cells (1 x 104 /well) were seeded in 96-well plates //jv
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143 and incubated with the indicated compound (0-80 μg/ml) for 2 h, washed and then sm
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144 cultured in fresh medium for 24 h. XTT labeling mixture was added for 1-2 h at 37 °C g
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145 before measurement for spectrophotometrical absorbance with a microplate reader. A
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146 Antiviral tests in cells. For plaque reduction assay, BHK-21 cells seeded in 1
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147 6-well plates were adsorbed with JEV [~200 plaque forming unit (PFU)] mixed with
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148 the indicated compounds at room temperature for 1 h. After 2 h of viral adsorption, g
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149 the virus and compounds were washed away and cells were overlaid with t
150 agarose-containing medium and incubated for 4 days. The plaques were fixed and
151 stained with crystal violet solution (28). To determine the step of viral infection
152 affected by NSQc, cells were treated with NSQc before virus adsorption for 2 h
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153 (before), during virus adsorption for 2 h (during), after virus adsorption for 22 h (post),
154 or during all of these times for 26 h (all). After viral adsorption with a multiplicity of
155 infection (MOI) of 5, cells were washed and incubated for 22 h at 37 °C. Culture
156 supernatants were collected for virus titration by plaque-forming assay and cell lysates
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157 were harvested for western blot analysis of viral protein expression (28). For virus o
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158 binding assay, JEV (MOI 1) was preincubated with 1 or 10 μg/ml of the indicated a
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159 nanocompounds or PBS for 1 h at room temperature. Cells were adsorbed with the f
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160 virus mixture for 2 h at 4 °C and then were washed three times with cold serum-free h
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161 medium. Total RNA was extracted with RNeasy kit (Qiagen) and analyzed by /jv
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162 real-time RT−PCR as previously described (31). sm
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163 Mouse challenge assays. Stat1–deficient mice (Stat1-/-) (32) and wild-type g
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164 C57BL/6 mice were bred in the animal facility of Institute of Biomedical Sciences, A
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165 Academia Sinica (Taipei, Taiwan). The mouse experiments were approved and 1
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166 performed in accordance with the guidelines of the Academia Sinica Institutional
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167 Animal Care and Utilization Committee. To test whether NSQc-treatment abolished g
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168 the in vivo viral infectivity, JEV (RP-9: 2 x 105 PFU/mouse) and DEN-2 (NGC-N: 1 x t
169 105 PFU/mouse) were incubated with NSQc (10 and 20 μg/ml) or PBS at 37 °C for 2
170 h. Groups of 5-week-old mice were intraperitoneally (IP) inoculated with virus and
171 intracerebrally (IC) injected with 30 μl of PBS (IP plus IC route) (33, 34). For the
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