Silver Nanoparticle Based Codelivery of Oseltamivir to Inhibit the

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Silver nanoparticles based co-delivery of Oseltamivir to inhibit the activity of H1N1 influenza virus through ROS-mediated signaling pathways Yinghua Li, Zhengfang Lin, Mingqi Zhao, Tiantian Xu, ChangBing Wang, Liang Hua, Hanzhong Wang, Huimin Xia, and Bing Zhu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b06613 • Publication Date (Web): 02 Sep 2016 Downloaded from http://pubs.acs.org on September 2, 2016

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ACS Applied Materials & Interfaces

Silver nanoparticles based co-delivery of Oseltamivir to inhibit the activity of H1N1 influenza virus through ROS-mediated signaling pathways Yinghua Li †, Zhengfang Lin †, Mingqi Zhao †, Tiantian Xu †, Changbing Wang †, Liang hua †, Hanzhong Wang ‡, Huimin Xia *, † and Bing Zhu *, † †

Center Laboratory, Guangzhou Women and Children’s Medical Center, Guangzhou Medical University Guangzhou, P.R. China

‡

State Key Laboratory of Virology, Chinese Academy of Sciences, Wuhan, P.R. China

ABSTRACT As the therapeutic agent for antiviral, the clinical use of oseltamivir is limited with the appearance of drug-resistant viruses. It is important to explore novel anti-influenza drugs. The antiviral activity of silver nanoparticles (AgNPs) has attracted increasing attention in recent years and was possibility to be employed as biomedical interventions. Herein, we describe the synthesis of surface decoration of AgNPs by using oseltamivir (OTV) with antiviral properties and inhibition of drug resistance. Compared to silver and oseltamivir, oseltamivir modified AgNPs (Ag@OTV) have remarkable inhibition against H1N1 infection and less toxicity was found to MDCK cells by CPE, MTT and TEM. Furthermore, Ag@OTV inhibited the activity of neuraminidase (NA) and hemagglutinin (HA), and then prevented the attachment of H1N1 influenza virus to host cells. The mechanism investigations revealed that Ag@OTV could block H1N1 form infecting MDCK cells and prevent DNA fragmentation, chromatin condensation and activity of caspase-3. Ag@OTV remarkably inhibited the accumulation of reactive oxygen species (ROS) by H1N1 virus and activation of AKT and p53 phosphorylation. Silver nanoparticles based co-delivery of oseltamivir inhibits the

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activity of H1N1 influenza virus through ROS-mediated signaling pathways. These findings demonstrate that Ag@OTV is a novel promising efficient virucide for H1N1. Keywords: Oseltamivir; Silver nanoparticles; Influenza virus; Neuraminidase; Apoptosis

1. Introduction Influenza virus is still a serious health problem around the world and affects millions of people in seasonal epidemics.1, 2 H1N1 which belongs to influenza A type viruses is a highly infectious respiratory disease.3 Influenza H1N1 was discovered and identified in Mexico and the US in 2009, and infected more than 8768 deaths in 207 countries.4 There are many ways to spread H1N1 influenza, for example, sneezes, coughs and the contaminated materials.5 As the antigenic shifts in different species and arising mutation of the genome, the influenza virus show a high variability which may emerge a novel influenza among humans in the future.6 There are several steps were divided in influenza infection cycle: Firstly, the influenza viruses attach to the host cell surface receptor and fuse with the endosomal membrane. Secondly, the uncoating of nucleocapsid and multiplication of the genetic material will be performed. Finally, the influenza protein and new viron is expressed and released.7-9 Hemagglutinin (HA) is a cell-anchoring viral glycoprotein which plays an important role in viral infection by combining sialic acid-containing receptors on host cells and mediating the entry and fusion of virus.10 Neuraminidase (NA) and Hemagglutinin are the most significant glycoprotein on the surface of influenza virus.11, 12 When mature viruses separate from host cell surface, Neuraminidase plays an important role in assisting virus cleave the linkage between sialic acid and Hemagglutinin.13 Vaccination is the common avenue to restraining the spread of influenza infections.14 Nevertheless, the long period


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between the rapid virus evolution and vaccine development, there is an urgent need for the exploration of new antiviral agents to inhibit the spreading of influenza virus.15, 16 Several antiviral drugs were developed in the infection cycle. The classes existing antiviral drugs approved by U.S.FDA are amantadine and Oseltamivir.17 Oseltamivir is a transition state sialic acid analogue with a large hydrophobic group, which is deficiency of sialic acid, developed to against influenza N2 neuraminidase.18 Oseltamivir perform as the primary antiviral drug which block the release of new virion from the cell’s membrane became resistant to influenza A virus.19 At present these synthetic drugs resulted in the emergence of drug-resistant which causes trouble to humans.20 Therefore, the antiviral therapies should promote to control the pandemic influenza A virus. Nanomaterials as new antimicrobials have emerged owing to their unique chemical and physical properties.21-23 Ye et al24 reported that graphene oxide is a promising antiviral agent for the unique property. Polyoxometalate with broad-spectrum may be a new type of antiviral agent by Wang et al.25 PLGA/AgNpPGA/AscH Nanospheres present a promising material for antimicrobial by Stevanovic et al.26 Carbon nanodots have high efficiency of functional as entry inhibitors on the early stage of virus infection by Barras et al.27 Cationic phenylene ethynylene polymers with potential antiviral application was reported by Wang et al.28 Phenylboronic-acid-modified nanoparticles with potential antiviral therapeutics strategy was reported by Khanal et al.29 The functional silica nanoparticles retain binding affinity for HIV-gp120 to by Cheng et al.30 Among of them, silver nanoparticles (AgNPs) attract considerable public attention compared to other pattern of silver source by their unique antimicrobial activities.31 As antibacterial agents, AgNPs have been extensively explored in



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food storage and environmental.32 Furthermore, AgNPs have also been developed to contact with HIV, HSV, HBV and inhibit the virus multiplication inside the host cell.33-35 AgNPs have been considered as a candidate of antimicrobial agent restrain with a widespread of bacteria and viruses.36 In this study, we wish to exposure the novel silver nanoparticles which can interfere with the interaction between H1N1 virus and host cells. Based on the particular properties both of oseltamivir and AgNPs, we look forward to verify that oseltamivir modified AgNPs (Ag@OTV) have excellent antiviral activity against H1N1 virus infection. Reactive oxygen species (ROS)

plays an important role in many physiological processes

and encompass highly reactive molecules.37 Oxidative stress is explicated between consumption of ROS and cellular defense mechanisms.38 The imbalance of redox is contacted with many pathologies, such as cancer, diabetes and other diseases.39 Previous research group36 have described the antimicrobial effects of AgNPs by generating ROS, but litter is reported about the antiviral mechanisms of AgNPs. Therefore, this study was to ascertain how oseltamivir modified AgNPs will antagonize H1N1 influenza virus induced MDCK cells apoptosis.

2. Materials and Methods 2.1 Materials MDCK cells were obtained from ATCC® CCL-34TM. H1N1 influenza virus was provided by Wuhan Institute of Virology. FBS and DMEM were purchased from Gibco. Caspase-3, PARP, p53 and AKT antibodies were purchased form Cell Signaling Technology (CST). AgNO3,

Vitamin

C,

MTT,

JC-1,

propidium

iodide

(PI),

6-coumarin,

4’6-Diamidino-2-phenyindole (DAPI), 2’,7’-dichlorofluorescein diacetate (DCF-DA) were


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purchased from Sigma. Lyso Tracker was obtained from Invitrogen. 2.2 Preparation of Ag@OTV Silver nanoparticles were synthesized as previously described.37 As follow: briefly, 0.1 ml stock solution of Vitamin C was gradually added into 4 ml of stock solution of AgNO3. After that, 80 µl of Oseltamivir (100 nM) was added. The excess Oseltamivir, Vitamin, and AgNO3 were removed by dialysis for overnight. Ag@OTV nanoparticles were sonicated and then filtered through 0.2 µm pore size. The concentration of AgNPs and OTV was measured by ICP-AES. 2.3 Characterization of Ag@OTV The Ag@OTV nanoparticles were characterizes by using multiple methods as follow: The nanoparticles were dispersed onto a holey carbon film on copper grids and micrographs were obtained for TEM (H-7650). Elemental composition of Ag@OTV was analysed by EDX which was carried out on an EX-250 system (Horiba). Zeta potential and Size distribution of Ag@OTV were checked by Zetasizer Nano ZS particle analyzer. 2.4 Cell culture and cell viability by MTT assay The cytotoxicity of Ag@OTV nanoparticles was performed by cell viability as previously described.40 Briefly, after incubation with H1N1 influenza virus for 2 h, the MDCK cells were rinsed to remove influenza virus which were not internalized. The indicated concentrations of Oseltamivir with or without AgNPs were added to MDCK cells for 24 h. After that, 20 µl/well of MTT solution was added and incubated for another 5 h. The formazan crystals were dissolved by adding 150 µl/well DMSO and



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recorded at the absorbance of 570 nm. 2.5 Intracellular Localization of Ag@OTV The intracellular localization of Ag@OTV was performed as previously described.41 MDCK cells were cultured in 6 cm cell culture dishes and incubated with 6-coumarin-load Ag@OTV. The cells were washed with PBS three times and observed under a fluorescence microscope. The localization of 6-coumarin labelled Ag@OTV in MDCK cells was determined by Lyso Tracker and DAPI.42 2.6 Cellular uptake of Ag@OTV Quantitative cellular uptake of Ag@OTV was detected by quantitative analysis as previously described.43 Briefly, MDCK cells were seeded into 96-well plates for 24 h. Then different concentrations of 6-coumarin-loaded Ag@OTV nanoparticles were replaced and incubated in the well for different periods of time. The cellular uptake efficiency of coumarin-6 loaded nanoparticles was determined by fluorescence microplate reader. 2.7 Transmission electron microscopic analysis Ag@OTV-treated H1N1 by TEM Ag@OTV-treated H1N1 influenza virus was negatively stained and morphologically detected by TEM as previously described.16 The H1N1 influenza virus were treated with Ag@OTV at various time points and then were attached to the carbon-coated collodion grid for 10 minutes. The grids were stained with 2% phosphotungstic acid in Sorensen phosphate buffer for 2 minutes. The grids were examined by TEM after rinsing and air-drying the slides 2.8 The Hemagglutinin and Neuraminidase inhibition assay by Ag@OTV


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To determine the effects of Ag@OTV on H1N1 influenza virus proliferation, MDCK cells were infected with H1N1 influenza virus for 2 h. The cell culture supernatants were harvested after 48 h and measured hemagglutinin titer. An equal volume of cell supernatants and 0.5% chicken erythrocytes suspended in phosphate buffered saline was incubated for 1h at room temperature as previously described.16 The NA activity of Influenza virus was determined by quantifying the intensity of fluorescence as previously described.11 2.9 Detection of mitochondrial membrane potential (∆Ψm) The fluorescence intensity from JC-1 monomers was used to estimate the status of ∆Ψm in MDCK cells exposed to Ag@OTV as previously described.44 Cells were trypsinized and resuspended with 10 µg/ml of JC-1. The MDCK cells were then harvested and analyzed by flow cytometery. 2.10 Annexin-V-FLUOS staining assay Plasma membrane alterations in MDCK cells treated with Ag@OTV was detected as previously described.45 Briefly, MDCK cells was treated with Ag@OTV for 24 h and stained with annexin-V-FLUOS, then washed with PBS three times and observed by Flow cytometric analysis. 2.11 Flow cytometric analysis The effect of Ag@OTV on cell cycle distribution was detected through Flow cytometric as previously reported.46 The MDCK cell incubated with Ag@OTV were harvested and fixed with 70% ethanol at -20℃ overnight followed by PI staining. The apoptotic cells were evaluated by detecting the sub-G1 peak. 2.12 TUNEL-DAPI co-staining assay



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The MDCK cells were labeled with TUNEL for 1h and incubated of DAPI for 15 min at 37℃ for nuclear staining. DNA fragmentation was detected with fluorescence staining by the TUNEL apoptosis kit as previously described.47 2.13 Caspase-3 activity The fluorescence intensity of caspase-3 activity was detected under fluorescence microscope with the wavelengths at 380 nm (excitation) and 460 nm (emission), as previously described.48 2.14 TEM image of thin sections of MDCK cells After incubation with H1N1 influenza virus for 2 h, MDCK cells were rinsed with PBS three times and incubated with Ag@OTV for a period time. TEM analysis of MDCK Cells were performed in situ as previous described.49 2.15 Determination of ROS generation ROS accumulation inhibited by Ag@OTV treated MDCK cells were determined as previously described.44 MDCK cells were harvested by centrifugation and suspended in PBS containing 10 mM of DCFH-DA. The ROS level was monitored by analyzing the fluorescence intensity of DCF with excitation (500 nm) and emission (529 nm) wavelengths. 2.16 Western blotting analysis Ag@OTV treated with MDCK cells on the expression of proteins were determined as previously reported.50 The total proteins were obtained after MDCK cells with Ag@OTV treatment for 24 h and incubated with lysis buffer. BCA assay was used to quantify the protein concentration. The bolts were developed with enhanced chemiluminescence reagent using an ECL kit that examines the target proteins on the X-ray film.


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2.17 Statistical analysis All the data are presented as mean ± SD. Differences between two groups were evaluated using two-tailed Student’s t test. One-way analysis of variance (ANOVA) was used in multiple group comparisons. Difference with P

Silver Nanoparticle Based Codelivery of Oseltamivir to Inhibit the

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