Reversal of H1N1 influenza virus-induced apoptosis by silver nanoparticles functionalized with amantadine

Abstract

Amantadine is an antiviral agent, but its clinical use against influenza viruses is limited because of the emergence of drug-resistant viruses. Thus, there is a need for novel anti-influenza agents. The antiviral activity of silver nanoparticles (AgNPs) has attracted increasing attention, with such nanoparticles being employed in biomedical interventions in recent years. Herein, we describe a simple method for surface decoration of AgNPs using amantadine. Co-delivery of AgNPs and amantadine was designed to overcome drug resistance. Compared with AgNPs and AM, amantadine-modified AgNPs (Ag@AM) were shown to inhibit H1N1 infection by CPE, MTT and TEM. Ag@AM also inhibited the activity of hemagglutinin (HA) and neuraminidase (NA). Mechanism investigations revealed that Ag@AM can block H1N1 from infecting host cells and prevent DNA fragmentation, chromatin condensation and activity of caspase-3. Ag@AM inhibited accumulation of reactive oxygen species (ROS) and reversed virus-induced apoptosis by H1N1 virus. Taken together, these findings suggest that Ag@AM is a novel promising efficient virucide for H1N1.

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1 Introduction

Influenza virus remains one of the most burdensome and widespread health problems globally, affecting millions of people in seasonal epidemics.¹ H1N1, an influenza A type virus, is a highly infectious respiratory disease.² Influenza H1N1 was discovered and identified in Mexico in March 2009.^3,4 H1N1 influenza can be spread by, for example, sneezes, coughs and contaminated materials.⁵ Antigenic shifts in different species and mutation of the genome mean that the influenza virus has high variability, which may result in novel future influenza among humans.⁶

The influenza infection cycle comprises several steps: first, the influenza viruses attach to the host cell surface receptor and fuse with the endosomal membrane; second, uncoating of nucleocapsid and multiplication of the genetic material occurs; finally, the influenza protein and new viron is expressed and released.^7,8 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.⁹ Neuraminidase (NA) and hemagglutinin are the most significant glycoproteins on the surface of influenza virus.^10,11 There are four different domains in neuraminidase glycoproteins: a cytoplasmic domain, transmembrane domain, a stalk and the globular head. The stalk region of neuraminidase, which is between the globular head and anchored domains, relates to increased virulence and replication of virus.¹² When mature viruses separate from the host cell surface, neuraminidase assists the virus in cleaving the linkage between sialic acid and hemagglutinin.¹³ Vaccination is the most common method of attempting to restrict the spread of influenza infections.¹⁴ However, the long period between rapid virus evolution and development of vaccines means that there is an urgent need for new antiviral agents to inhibit the spread of influenza virus.¹⁵

Existing antiviral drugs approved by the U.S. FDA are neuraminidase inhibitors such as oseltamivir/zanamivir and M2 ion channel inhibitors such as rimantadine/amantadine.¹⁶ The M2 proton channel is a critical factor in viral replication; the replication cycle is arrested and infection of the host is halted when proton transport through the channel is inhibited.^17,18 The M2 protein is a 97-residue integral membrane protein with a transmembrane (TM) domain of 19 residues and a 54-residue cytoplasmic tail, with several point mutations of pore-lining residues of the A/M2 TM domain resulting in resistance to amantadine.^19,20 The emergence of such drug resistance is problematic for humans.^21,22 Therefore, the antiviral therapies must be promoted in an attempt to control the pandemic influenza A virus.

Nanomaterials have emerged as potential new antimicrobials owing to their unique chemical and physical properties.^23–25 Among these, silver nanoparticles (AgNPs) have attracted considerable interest compared with other sources of silver because of their unique antimicrobial activities.^26–28 As antibacterial agents, AgNPs have been explored extensively in food storage and environment.²⁹ Furthermore, AgNPs have also been developed to contact with HIV, HSV, HBV, Ad3 and inhibit virus multiplication inside host cells.^30–33 AgNPs have been considered as potential antimicrobial agents for a wide range of bacteria and viruses.³⁴ In this study, we examine whether novel silver nanoparticles can interfere with interaction between H1N1 virus and host cells. Based on the particular properties of amantadine and AgNPs, we suggest that amantadine-modified AgNPs (Ag@AM) will have excellent antiviral activity against H1N1 virus infection.

Reactive oxygen species (ROS) play an important role in many physiological processes and encompass highly reactive molecules.³⁵ Oxidative stress is explicated between consumption of ROS and cellular defense mechanisms.³⁶ The imbalance of redox occurs in many pathologies, such as cancer, diabetes and other diseases.³⁷ Previous research groups have described the antimicrobial effects of AgNPs by generating ROS, but little has been reported on the antiviral mechanisms of AgNPs. Therefore, this study aimed at ascertaining how amantadine-modified AgNPs antagonize H1N1 influenza virus-induced MDCK cell apoptosis.

2 Experimental

2.1 Materials

MDCK cells were obtained from ATCC® CCL-34™. H1N1 influenza virus was provided by Wuhan Institute of Virology (State Key Laboratory of Virology, Chinese Academy of Sciences, Wuhan, PR China). DMEM and FBS were purchased from Gibco. Caspase-3, antibody was obtained from Cell Signaling Technology. AgNO₃, Vitamin C, MTT, JC-1, propidium iodide (PI), 4′6-diamidino-2-phenyindole (DAPI) and 2′,7′-dichlorofluorescein diacetate (DCF-DA) were purchased from Sigma.

2.2 Preparation and characterization of Ag@AM

Silver nanoparticles were synthesized as previously described.³⁵ Briefly, 0.1 ml stock solution of Vitamin C was gradually added to 4 ml of stock solution of AgNO₃. After that, 0.8 ml of amantadine (1 μM) was added. The excess VC, AgNO₃ and amantadine were removed by dialysis overnight. The Ag@AM nanoparticles were sonicated and then passed through filters of 0.2 μm pore size. Ag@AM concentration was detected by ICP-AES.

The Ag@AM nanoparticles were characterized using multiple methods: the nanoparticles were dispersed onto a holey carbon film on copper grids and micrographs were obtained for TEM (H-7650). Elemental composition of Ag@AM was analyzed by EDX, which was carried out on an EX-250 system (Horiba). Zeta potential and size distribution of Ag@AM were checked using a Zetasizer Nano ZS particle analyzer.

2.3 Cell viability assay

The cytotoxicity of Ag@AM nanoparticles was determined using a cell viability assay as previously described.³⁸ Briefly, after incubation with H1N1 influenza virus for 2 h, MDCK cells were washed with PBS to remove influenza virus that was not internalized. The indicated concentrations of amantadine, AgNPs, or Ag@AM were added to MDCK cells for 24 h. After that, 20 μl per well of MTT solution was added and incubated for another 5 h. The formazan crystals were dissolved by adding 150 μl per well DMSO and recorded at an absorbance of 570 nm.

2.4 Hemagglutinin and neuraminidase inhibition assay by Ag@AM

To determine the effects of Ag@AM 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 hemagglutinin titer was measured. An equal volume of cell supernatants and 0.5% chicken erythrocytes suspended in phosphate buffered saline was incubated for 1 h at room temperature as previously described.¹⁵ The NA activity of influenza virus was determined by quantifying the intensity of fluorescence as previously described.¹¹

2.5 Detection of mitochondrial membrane potential (ΔΨ_m)

The fluorescence intensity from JC-1 monomers was used to estimate the status of ΔΨ_m in MDCK cells exposed to Ag@AM as previously described.³⁹ Cells were trypsinized and resuspended with 10 μg ml⁻¹ of JC-1. The MDCK cells were then harvested and analyzed by flow cytometry.

2.6 Annexin-V-FITC staining assay

Translocation of phosphatidylserine in MDCK cells treated with Ag@AM was detected using an Annexin-V-FITC staining kit as previously described.⁴⁰ Briefly, the cells were treated with Ag@AM for 24 h and stained with Annexin-V-FITC solution for 15 min. The cells were then rinsed with PBS and analyzed by flow cytometry.

2.7 Flow cytometric analysis

The effect of Ag@AM on cell cycle distribution was detected through flow cytometry as previously reported.⁴¹ Cells incubated with Ag@AM were collected and centrifuged at a speed of 1500 rpm for 10 min. The harvested cells were fixed with 70% ethanol at −20 °C overnight followed by PI staining.

2.8 TUNEL-DAPI co-staining assay

The MDCK cells were labeled with TUNEL for 1 h and incubated with DAPI for 15 min at 37° for nuclear staining. DNA fragmentation was detected with fluorescence staining by the TUNEL apoptosis kit as previously described.⁴²

2.9 Caspase-3 activity

The fluorescence intensity of caspase-3 activity was detected under a fluorescence microscope with the wavelengths at 380 nm (excitation) and 460 nm (emission), as previously described.⁴³

2.10 Determination of reactive oxygen species (ROS)

ROS accumulation inhibited by Ag@AM-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 the fluorescence intensity of DCF with excitation (500 nm) and emission (529 nm) wavelengths.

2.11 Western blotting analysis

The effect of Ag@AM treatment on expression of proteins in MDCK cells was determined as previously reported.⁴⁵ The total proteins were obtained after MDCK cells were treated with Ag@AM 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.

2.12 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. A difference of P < 0.05 (*) or P < 0.01 (**) was considered to be statistically significant.

3 Results and discussion

3.1 Synthesis and characterization of Ag@AM

The morphology of as-prepared Ag@AM was first characterized by TEM. TEM images of AgNPs and Ag@AM show that Ag@AM presented uniform and monodisperse spherical particles (Fig. 1A). EDX showed the presence of a signal from C atoms (22%), resulting from the amantadine (Fig. 1B). Average particle size distribution of different nanoparticles is shown in Fig. 1C and D, with Ag@AM decreasing from 3 nm to 2 nm facilitating easier entry into cells. The zeta potential of Ag@AM was lower than that of AgNPs after amantadine surface decoration, explaining the higher stability of Ag@AM than AgNPs (Fig. 1E). Furthermore, from Fig. 1F, the results revealed that Ag@AM was stable for at least 28 days. The small size of Ag@AM with low zeta potential contributed to highly stable nanostructures, able to cross the cell membrane.

Fig. 1 Characterization of AgNPs and Ag@AM. (A) TEM images of AgNPs and Ag@AM scale bar 2 nm. (B) EDX analysis of Ag@AM. (C) and (D) Size distribution of AgNPs and Ag@AM. (E) Zeta potentials of AgNPs and Ag@AM. (F) Stability of Ag@AM in aqueous solutions.

3.2 In vitro antiviral effects of Ag@AM

Negatively stained H1N1 virus was used to explain the interaction between H1N1 and Ag@AM. The H1N1 virus control (approximately 20 nm) showed typical elliptical or spherically shaped normal H1N1 virus containing virus matrix and capsid (Fig. 2A). After interaction of Ag@AM with H1N1 virus for 30 minutes, parts of the H1N1 viral edges were lost and the viral morphology was destroyed. The results demonstrate that Ag@AM directly interacted with virus particles, leading to disruption of viral function. The cytotoxic effects of H1N1 influenza virus on MDCK cells and protective effects of Ag@AM were detected using an MTT assay. MDCK cells treated with H1N1 influenza virus showed cell viability of 39%. Amantadine and AgNPs increased the cell viability to 56% and 65%, respectively (Fig. 2B); however, the cell viability was increased to 90% by Ag@AM. This suggests that the antiviral effects of AgNPs were amplified by amantadine on the surface of the nanoparticles. Cells treated with H1N1 influenza virus showed cytoplasmic shrinkage, loss of cell-to-cell contact and reduction in cell numbers (Fig. 2C). The MDCK cell morphology was slightly changed by co-treatment with Ag@AM, but the cells appeared healthy with regularity in shape. These results suggest that Ag@AM effectively inhibited the proliferation of H1N1 influenza virus.

Fig. 2 Effects of Ag@AM on H1N1 infection of MDCK cells by MTT assay. (A) Morphologic abnormalities in Ag@AM-treated H1N1 (2Aa: H1N1 virus control, 2Ab: H1N1 virus was interacted with Ag@AM). (B) Antiviral activity of Ag@AM was measured by MTT assay. 0.1 μM of AM was loaded onto the AgNPs. The free AM was 0.4 μM, the AgNPs was 2.5 μg ml⁻¹. (C) Morphological changes in H1N1 infection of MDCK cells observed by phase-contrast microscopy.

3.3 HA and NA as the potential targets of Ag@AM

A hemagglutination inhibition assay was used to examine the potential interaction of the envelope HA protein with Ag@AM. Compared with AM and AgNPs, Ag@AM nanoparticles showed effective capability to inhibit influenza virus-induced aggregation of chicken erythrocytes (Fig. 3A). This suggests that Ag@AM nanoparticles prevent interactions of viruses with MDCK cells. Untreated or Ag@AM-treated influenza H1N1 virus was used to detect NA enzymatic activity. Ag@AM-treated influenza H1N1 virus had enzymatic activity, indicating that the Ag@AM nanoparticles had an effect on NA activity (Fig. 3B). The proposed mechanism for Ag@AM-mediated anti-influenza activity is that Ag@AM nanoparticles bind tightly to the HA and NA, thus preventing attachment of H1N1 viruses to the MDCK cells.

Fig. 3 HA and NA as the potential targets of Ag@AM. (A) Comparisons of the behaviors of AgNPs, amantadine and Ag@AM inhibition of influenza virus-induced aggregation of chicken erythrocytes. (B) The NA inhibition assay was performed by quantifying the fluorescence. 0.1 μM of AM was loaded onto the AgNPs. The free AM was 0.4 μM, the AgNPs was 2.5 μg ml⁻¹.

3.4 Depletion of mitochondrial membrane potential (ΔΨ_m) and translocation of phosphatidylserine induced by Ag@AM

MDCK cells were treated with the mitochondrial-selective JC-1 dye. This was used to show that H1N1 influenza virus caused increased mitochondrial depolarization and dysfunction in MDCK cells (Fig. 4A). Compared with AM and AgNPs, when MDCK cells were exposed to Ag@AM, the percentage of mitochondrial membrane potential was increased. These results demonstrate that Ag@AM inhibited H1N1 influenza virus by apoptosis in MDCK cells by inducing mitochondrial dysfunction. As shown in Fig. 4B, treatment with Ag@AM resulted in decreased cell number of MDCK cells. The Ag@AM restrained the H1N1 virus infection MDCK cells mainly by inhibiting apoptosis.

Fig. 4 Depletion of mitochondrial membrane potential and translocation of phosphatidylserine induced by AgNPs, AM and Ag@AM. (A) Mitochondrial membrane potential of H1N1 infection of MDCK cells exposed to AgNPs, AM and Ag@AM. (B) Translocation of phosphatidylserine induced by AgNPs, AM and Ag@AM in H1N1 infection of MDCK cells. 0.1 μM of AM was loaded onto the AgNPs. The free AM was 0.4 μM, the AgNPs was 2.5 μg ml⁻¹.

3.5 Inhibition of H1N1 influenza virus infection of MDCK cells by Ag@AM

Flow cytometry and TUNEL-DAPI were used to examine the potential anticancer mechanisms of Ag@AM. As shown in Fig. 5A, the cell population of sub-G1, which represents apoptotic cells, was increased as a result of H1N1 infection. However, Ag@AM decreased the apoptotic cell population to 13.5%. As shown in Fig. 5B, H1N1 influenza virus caused the MDCK cells to exhibit typical apoptotic features of DNA fragmentation and nuclear condensation. However, treatment with Ag@AM prevented such H1N1 influenza virus-induced changes in nuclear morphology. These results indicate that Ag@AM rescues MDCK cells from H1N1 influenza virus-induced apoptosis.

Fig. 5 Ag@AM induced apoptosis in H1N1 infection of MDCK cells. (A) The cell cycle distribution after different treatments was analyzed by quantifying DNA content using flow cytometric analysis. (B) DNA fragmentation and nuclear condensation as detected by TUNEL-DAPI co-staining assay. 0.1 μM of AM was loaded onto the AgNPs. The free AM was 0.4 μM, the AgNPs was 2.5 μg ml⁻¹. All results are representative of three independent experiments.

3.6 Inhibition of caspase-3 activation by Ag@AM

Caspase-3 is an important mediator of cell apoptosis. As shown in Fig. 6A, H1N1 influenza virus increased the activity of caspase-3 in MDCK cells, but in cells treated with Ag@AM caspase-3 activity was decreased (Fig. 6B). These results show that the synthetic nanosystem inhibits H1N1 influenza virus activity by preventing cell apoptosis.

Fig. 6 Inhibition of caspase-3 activity by Ag@AM in H1N1 infection of cells. (A) Cells were treated with Ag@AM and caspase-3 activity was detected by synthetic fluorogenic substrate. (B) Protein expression of caspase-3 by western blot, β-actin was used as loading control. 0.1 μM of AM was loaded onto the AgNPs. The free AM was 0.4 μM, the AgNPs was 2.5 μg ml⁻¹.

3.7 Inhibition of ROS generation by Ag@AM

Generation of ROS was monitored by DCF assay to indicate the action mechanisms of Ag@AM. The H1N1 influenza virus increased ROS generation in MDCK cells (Fig. 7A). Amantadine and AgNPs, separately, slightly inhibited ROS generation. However, Ag@AM decreased intracellular ROS generation. The stronger fluorescent intensity of DCF was also found in MDCK cells by H1N1 influenza virus (Fig. 7B). The fluorescent intensity of DCF in MDCK cells treated with H1N1 influenza virus was much stronger than in those treated with Ag@AM. These results indicate the involvement of ROS in the antiviral action of Ag@AM.

Fig. 7 ROS overproduction induced by Ag@AM in H1N1 infection of MDCK cells. (A) ROS levels were detected by DCF fluorescence intensity. (B) H1N1 infected MDCK cells were pre-incubated with 10 μM DCF for 30 min and then treated with Ag@AM. 0.1 μM of AM was loaded onto the AgNPs. The free AM was 0.4 μM, the AgNPs was 2.5 μg ml⁻¹.

4 Conclusions

In conclusion, this study described a simple chemical method for preparation of amantadine-surface decorated silver nanoparticles. The Ag@AM exhibits superior abilities to enhance cellular uptake and block H1N1 influenza virus infection. Ag@AM decreased the apoptotic cell population infected by the H1N1 influenza virus. The underlying molecular mechanisms of the study indicated that the main pattern of cell death reduced by Ag@AM was apoptosis. Ag@AM inhibited caspase-3 mediated apoptosis through ROS generation. The proposed mechanism for Ag@AM-mediated anti-influenza activity is that the Ag@AM nanoparticles bind tightly to HA and NA, thus preventing attachment of the H1N1 virus to the MDCK cells. Therefore, the nanosystem of Ag@AM offers a prospective silver species with antiviral properties.

Acknowledgements

This work was supported by the China Postdoctoral Science Foundation (2015M582366), the Technology Planning Project of Guangdong Province (2014A020212697), and the Technology Planning Project of Guangdong (201607010120).

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