Glutathione-stabilized fluorescent gold nanoclusters vary in their

†State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University,. Wuhan 430070, P.R. China. §College of Science, Huazhong Agri...
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Cite This: ACS Appl. Nano Mater. 2018, 1, 969−976

Glutathione-Stabilized Fluorescent Gold Nanoclusters Vary in Their Influences on the Proliferation of Pseudorabies Virus and Porcine Reproductive and Respiratory Syndrome Virus Yanli Bai,†,‡,⊥ Yanrong Zhou,†,§,⊥ Huabing Liu,†,‡ Liurong Fang,†,§ Jiangong Liang,*,†,‡ and Shaobo Xiao*,†,§ State Key Laboratory of Agricultural Microbiology, ‡College of Science, and §College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, P. R. China

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S Supporting Information *

ABSTRACT: Gold nanoclusters (Au NCs) are widely used in biological imaging and antitumor treatment because of their excellent cell membrane permeability, good fluorescence properties, and high biocompatibility. However, their effects on viruses are still largely unknown. Here, pseudorabies virus (PRV) and porcine reproductive and respiratory syndrome virus (PRRSV) were used respectively as the models of DNA virus and RNA virus to investigate the influences of glutathione-stabilized fluorescent Au NCs on viruses by plaque assay, indirect immunofluorescence assay, quantitative realtime polymerase chain reaction assay, and Western blot assay. The experimental data indicated that Au NCs selectively inhibited proliferation and protein expression of PRRSV but not that of PRV. Mechanistically, Au NCs directly inactivated PRRSV and blocked viral adsorption but showed no effect on viral genome replication. These findings prompt the possibility of developing Au NCs as an effective specific antiviral nanomaterial against RNA virus infection in the future. KEYWORDS: gold nanoclusters, porcine reproductive and respiratory syndrome virus, pseudorabies virus, proliferation, selective inhibitory effect imaging.25,26 For instance, Au NCs acted as drug carriers for the therapy of cancer.20 Chen et al. established a dual targeting function luminescent gold nanocluster (AuNC-cRGD-Apt) for tumor imaging and deep tissue therapy.22 However, to our knowledge, the influence of Au NCs on viruses has not yet been studied. Many RNA viruses, such as Ebola virus, hepatitis C virus, and influenza virus, pose great threats to human health and the livestock industry,27−29 suggesting the necessity to develop selective inhibitors of RNA virus.30,31 In this study, both porcine reproductive and respiratory syndrome virus (PRRSV), a single-stranded RNA virus of the Arteriviridae family,32 and pseudorabies virus (PRV), a double-stranded DNA virus of the Herpesvirinae family that causes porcine Aujeszky’s disease,33−35 are important pathogens that have caused serious losses to the pig industry worldwide36,37 and were selected as models of RNA or DNA virus to investigate the effect of glutathione (GSH)-stabilized Au NCs on RNA virus (PRRSV) and DNA virus (PRV). Mechanistic studies revealed a selective antiviral effect of Au NCs on PRRSV proliferation by the direct

1. INTRODUCTION Functional nanoparticles (NPs) have attracted increasing attention in the field of nanomedicine.1 Many kinds of NPs with good antiviral activities have been found, such as quantum dots (QDs),2 carbon dots (CDs),3 silver nanoparticles (Ag NPs),4−8 gold nanoparticles (Au NPs),9−11 glycofullerenes,12 and graphene oxide.13 Deokar et al. reported that sulfonated magnetic NPs functionalized with reduced graphene oxide is an effective and rapid antiviral agent for herpes simplex virus type 1 (HSV-1).14 Illescas et al. demonstrated that multivalent glycosylated fullerene significantly prevents Ebola virus infection in the subnanomolar range.15 However, there are two common drawbacks for current antiviral NPs: (i) the majority of nanoparticles release heavy metals (besides gold) with cytotoxicity; (ii) the antiviral properties of most NPs are of no selectivity and specificity. For example, Ag NPs inhibit the propagation of both RNA viruses (human immunodeficiency virus and influenza virus)4,7 and DNA viruses (adenoviruses, HSV, and monkey pox virus).5,6,8 Gold nanoclusters (Au NCs) are widely used as a novel type of fluorescent nanomaterial in biology for their good biocompatibility and biosecurity.16 Previous studies have revealed their morphological properties17−19 and antitumor effects,20−24 as well as their potential application in cell © 2018 American Chemical Society

Received: December 23, 2017 Accepted: January 29, 2018 Published: January 29, 2018 969

DOI: 10.1021/acsanm.7b00386 ACS Appl. Nano Mater. 2018, 1, 969−976

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ACS Applied Nano Materials

Figure 1. (A) UV−vis absorption and fluorescence spectra of Au NCs. (B) Hydrodynamic diameter distribution of Au NCs from DLS. The fluorescent excitation wavelength was 425 nm. NCs (450 μM) for another 2 h. The effect of Au NCs on the PRV infection was further evaluated using confocal microscopic analysis. Similarly, PRRSV (MOI = 0.1) was treated with Au NCs (450 μM) and infected MARC-145 cells as described above, and the influence of Au NCs on PRRSV proliferation was further observed with indirect immunofluorescence assay and confocal microscopic analysis at 36 hpi. 2.5. Indirect Immunofluorescence Assay and Confocal Microscopic Analysis. The PRRSV in MARC-145 cells was observed using indirect immunofluorescence assay, as reported in our previous work.3 In brief, MARC-145 cells were treated as described in “antiviral activity assay” and fixed at 36 hpi. Primary antibody against nucleocapsid (N) protein of PRRSV and Alexa Fluor 594-conjugated donkey antimouse IgG were used to detect fluorescence signals of the infected cells. As for GFP-PRV, PK-15 cells were treated as described in “antiviral activity assay” and fixed at 24 hpi. A confocal microscope was used to observe the fluorescence signals of GFP-PRV-infected cells. 2.6. Plaque Assay. PRV (MOI = 0.1) were treated as described in “virus pretreatment assay” and collected separately at 6, 9, 12, 18, and 24 hpi. The virus titers of PRV were detected by plaque assay, as reported by Luo et al.43 Briefly, PK-15 cells were cultured in 6-well plates for 24 h until 95% confluence. Next, virus samples infected the PK-15 cells for 1 h. Then, the cells were covered with a mixture of lowmelting-point agarose and 2 × DMEM with 1% penicillin− streptomycin and 3% FBS.44 The results were obtained from three parallel experiments after 2−3 days postinfection at 37 °C. Likewise, virus titers of PRRSV were obtained as described above. 2.7. Western Blot Assay. PRRSV (MOI = 0.1) were treated as described in “virus pretreatment assay” and then harvested at 36 hpi. The expression of PRRSV N protein was analyzed with Western blot assay by using mouse anti-N antibody.45 2.8. RT-qPCR Assay. MARC-145 cells were treated with Au NCs (450 μM) after infection with 0.1 MOI PRRSV for 6 h. The negativesense PRRSV RNA at 7, 8, 9, and 10 h after Au NC incubation was detected by the reported methods.46 The primer specific to the 5′terminal region of a PRRSV genomic was as follows: 5′GACGTATAGGTGTTGGCTC-3′. The primers used in qPCR assay were sense 5′-GCATTTGTATTGTCAGGAGC-3′ and antisense 5′-AGCAGTGCAACTCCGGAAG-3′. The gene expression was analyzed as previously described.47,48 2.9. Statistical Analysis. Cytotoxicity and antiviral activity of Au NCs were analyzed with one-way analysis of the variance. Results were shown as the mean ± standard deviation (SD) of three independent experiments. P < 0.05 or P < 0.001 stand for statistical significance.

inactivation of viruses and interference with viral adsorption but not that of PRV. These data have paved the way for further research on Au NCs as a specific inhibitor against RNA virus infection.

2. EXPERIMENTAL SECTION 2.1. Synthesis of Au NCs. Reagents used in this study are listed in Table S1. All glassware was immersed in a freshly prepared aqua regia solution for 12 h and then washed thoroughly with ultrapure water. Au NCs were prepared as reported by Zhang et al.38 with some modifications. Briefly, 15.0 mL of a histidine (His) solution (0.10 M) and 5.0 mL of a HAuCl4 solution (0.010 M) were added to a small glass bottle and reacted at 25 °C in the dark for 2 h. After that, 5.0 mL of a GSH solution (0.12 M) was mixed with the above Au NC precursor solution, and Au NCs were obtained after reaction at 25 °C in the dark for 12 h. The concentration of AuNCs was calculated according to the Au-atom concentration of the Au NC solution, which was detected according to the method from Corpuz et al.39 2.2. Cytotoxicity Assay. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect the cytotoxicity of Au NCs as reported previously.40 Briefly, PK-15 or MARC-145 cells were incubated with Au NCs (0, 56, 112, 225, 450, and 900 μM) in 96-well plates. After incubation for 2 h, the supernatant was replaced with fresh Dulbecco’s modified Eagle medium [DMEM; 2% fetal bovine serum (FBS)], followed by incubation separately for 24 or 48 h. Cells were then treated with MTT and maintained for 4 h. After removal of the MTT reagent, the formazan precipitate was dissolved with dimethyl sulfoxide, followed by gentle shaking of the plates for 10 min. Finally, the viability was calculated by a comparison with control cells without exposure to Au NCs according to absorbance at 570 nm. 2.3. Virus Propagation. GFP-PRV, a kind of recombinant PRV that expresses green fluorescent protein, was propagated in PK-15 cells for 24 h postinfection (hpi). Similarly, PRRSV were propagated in MARC-145 cells for 48 hpi. Next, the cells were frozen at −80 °C. The progeny viruses were released and kept at −80 °C for further use.41 2.4. Antiviral Activity Assay. The main processes of the virus replication cycle include attachment, entry, genome replication, protein synthesis, and exocytosis. To explore the exact step(s) that Au NCs target for the antiviral effect, antiviral activity assays with four different treatment conditions were performed by changing the incubation sequence of Au NCs and viruses with the cells using a previously reported method.42 (1) Virus pretreatment assay: 0.1 multiplicity of infection (MOI) GFP-PRV was mixed with Au NCs (450 μM) for 1 h, and then PK-15 cells were infected with the mixture for another 2 h. (2) Cotreatment assay: 0.1 MOI GFP-PRV was mixed with Au NCs (450 μM) and immediately infected PK-15 cells for 2 h. (3) Cell pretreatment assay: PK-15 cells were pretreated with Au NCs (450 μM) for 1 h, followed by infection with 0.1 MOI GFP-PRV. (4) Cell post-treatment assay: PK-15 cells were infected with 0.1 MOI GFP-PRV for 2 h in the absence of Au NCs and then treated with Au

3. RESULTS AND DISCUSSION 3.1. Characteristics of Au NCs. The apparatuses used to characterize Au NCs in our study are listed in Table S2. The UV−vis absorption and fluorescent spectra of Au NCs are shown in Figure 1A. The absorption spectra were detected between 230 and 650 nm, with a small peak observed near 402 970

DOI: 10.1021/acsanm.7b00386 ACS Appl. Nano Mater. 2018, 1, 969−976

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Figure 2. FT-IR (A) and XPS spectra of Au NCs (B), Au 4f (C), S 2p (D), C 1s (E), N 1s (F), and O 1s(G).

indicating discrete excitation−emission bands because of interband transitions.39,49 The fluorescence lifetimes of Au NCs were also indicated in Figure S1, which was calculated to be 5.20 ± 0.05 ns. The quantum yield of Au NCs was 6.8%

nm. It can be seen that no obvious absorption spectra were detected at >550 nm, which is consistent with previously reported Au NCs.39 In addition, a strong fluorescent emission peak was observed at 502 nm, with a strong green fluorescence 971

DOI: 10.1021/acsanm.7b00386 ACS Appl. Nano Mater. 2018, 1, 969−976

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ACS Applied Nano Materials according to a reported procedure.39 The transmission electron microscopy (TEM) image (Figure S2) of Au NCs indicated that the average diameter was 1.5 ± 0.5 nm. The results of dynamic light scattering (DLS; Figure 1B) revealed good dispersity of the Au NCs, and the hydrodynamic diameter distribution was 2.5 ± 0.7 nm, which is consistent with the results of TEM. Figure 2A shows the Fourier transform infrared (FT-IR) spectroscopy of the as-prepared Au NCs. Stretch vibrations of COH at 3418 cm−1, C−H at 2945 cm−1, C−NH at 1506 cm−1 and CO at 1631 cm−1 represented the characteristic absorption bands of GSH and His,9,10,49,50 and the stretch vibration of S−H at 2620 cm−1 showed the characteristic peaks of Au−S. The full-range X-ray photoelectron spectrometry (XPS) analysis (Figure 2B) showed five evident peaks at 84.0, 163.0, 285.0, 400.1, and 530.6 eV, which could be attributed to Au 4f, S 2p, C 1s, N 1s, and O 1s, respectively. The Au 4f core-level spectra are shown in Figure 2C, with the peaks at 84.0 and 87.6 eV corresponding to Au 4f7/2 and Au 4f5/2, respectively.51 The spectrum of S 2p (Figure 2D) revealed the existence of S22−, S2−, and S0/Sn2−,52 demonstrating the Au−S bond on the surface of Au NCs.39 The peaks of C 1s (Figure 2E) at 284.4, 285.1, 285.8, and 287.5 eV were assigned to C−C, C−N, C−O, and OC−N, respectively.53−55 N−(C)3, C−N−C, and C N were confirmed according to the three fitted peaks at 400.6, 399.3, and 398.1 eV in the N 1s spectrum (Figure 2F) .53,56 C− O (531.6 eV) and CO (530.6 eV) were also proven by the XPS spectrum of O 1s (Figure 2G).57 The presence of C−NH and Au−S from FT-IR and XPS suggested that His and GSH are on the surface of the Au NCs and COOH and NH2 are the main functional groups of Au NCs. 3.2. Cytotoxicity of Au NCs. In previous reports, Au NCs have been found to have low cytotoxicity on various types of cells.58−60 For instance, Zhang et al. observed that ATII and A549 cells still have high viability under the treatment of GSHstabilized Au NCs even at a concentration of 1000 μM.39 To assess the potential cytotoxicity of Au NCs, the MTT assays were conducted in both PK-15 and MARC-145 cells. As shown in Figure 3, after incubation with different concentrations (0, 56, 112, 225, 450, and 900 μM) of Au NCs, the relative viabilities of the two types of cells were both more than 95% when treated with 450 μM Au NCs and close to 94 and 91% at 900 μM. These results indicated low toxicity of Au NCs and

good biocompatibility with different types of cells. Hence, 450 μM was selected for the following studies. 3.3. Influence of Au NCs on Viral Proliferation. To evaluate the effect of Au NCs on the propagation of DNA or RNA virus, PRV or PRRSV was treated with Au NCs, as described in the Antiviral Activity Assay section, and the results were analyzed using indirect immunofluorescence assay and confocal microscopic analysis. As depicted in Figure 4A, Au NCs showed no effect on the proliferation of PRV under all four treatment conditions. However, Au NCs significantly prevented PRRSV infection when PRRSV was preincubated with Au NCs before infection (Figures 4B, third line, and S3) or when MARC-145 cells were cotreated with PRRSV and Au NCs (Figure 4B, fourth line) but did not affect the proliferation of PRRSV when MARC-145 cells were preincubated with Au NCs before PRRSV infection (Figure 4B, fifth line) or when PRRSV infected MARC-145 cells prior to Au NC incubation (Figure 4B, sixth line). These results demonstrated that Au NCs selectively inhibit the infection of PRRSV (a model of RNA virus) through direct inactivation of viruses and interference with viral adsorption but not that of PRV (a model of DNA virus). 3.4. Effect of Au NCs on Viral Infection Measured with Plaque Assay. To further confirm the inactivation effect of Au NCs on viral infection, plaque assay, a classic method to investigate viral titers, was used here as described in the Experimental Section. As displayed in Figure 5A, the virus titer of Au NCs-treated PRV (Au NCs-PRV) resembled that of control PRV, indicating that Au NCs have no obvious influence on the propagation of PRV. In addition, we found that the titers of both Au NCs-PRV and control PRV could reach approximately 108 pfu/mL at 18 hpi, suggesting that the infectivity of PRV was well preserved under the treatment of 450 μM Au NCs, which was extremely vital for the potential application of Au NCs in the intracellular trafficking of labeled virus. On the contrary, as shown in Figure 5B, the relative titers of Au NCs-PRRSV showed a remarkable reduction relative to the PRRSV control. Interestingly, the antiviral effect of Au NCs on the later stage (36 and 48 hpi) of virus multiplication was more significant than that on the early stage (6, 12, and 24 hpi). The results from plaque assay further proved the effective antiviral effect of Au NCs on PRRSV, rather than PRV, which were in accordance with the data form confocal microscopic analysis. 3.5. Effect of Au NCs on PRRSV N Protein. PRRSV N protein, as an abundant viral protein during infection, has been found to be associated with the pathogenicity of PRRSV.61 The expression levels of N protein were detected through Western blot assay, as described in the Experimental Section to further validate the direct inactivation activity of Au NCs on PRRSV. As depicted in Figure 6, Au NCs were found to significantly inhibit the N protein synthesis of PRRSV. Collectively, the results of indirect immunofluorescence assay, plaque assay, and Western blot assay verified that Au NCs can significantly inhibit the proliferation of RNA virus (PRRSV) but have no significant influence on the progeny virus infection of PRV in PK-15 cells. 3.6. Preliminary Exploration of the Potential Mechanism for the Selective Antiviral Effect of Au NCs. Currently, numerous NPs have been found to play antiviral roles by targeting various stage(s) of virus infection. For example, Barras et al. reported that boronic acid-modified carbon nanodots can inhibit HSV-1 infection by interfering with the virus entry,62 while mercaptoethanesulfonate-modified

Figure 3. Cytotoxicity of Au NCs analyzed by MTT assay. 972

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Figure 4. Antiviral activity assay of Au NCs against PRV infection (A) or PRRSV infection (B) with indirect immunofluorescence assay and confocal microscopic analysis: (first row) cells (PK-15 and MARC-145 cells) without any treatment; (second row) virus-infected cells; (third row) virus incubated with Au NCs for 1 h before infecting the cells; (fourth row) cells cotreated with virus and Au NCs; (fifth row) Au NCs added after virus infection; (sixth row) Au NCs added before virus infection.

Figure 5. Propagation of PRV (A) and PRRSV (B) with or without 450 μM Au NCs.

To investigate whether the virus-infected cell type is related to the discrepancy in antiviral properties, we tested the effects of Au NCs on PRV propagation in MARC-145 cells, as described in the Experimental Section using plaque assay. As presented in Figure S4, Au NCs also had no obvious influence on the PRV proliferation in MARC-145 cells. These data eliminated the involvement of the cell type in the different antiviral effects of Au NCs on PRV and PRRSV propagation. Considering that the replication process of DNA viruses (PRV) mainly happens in the nucleus while that of RNA viruses (PRRSV) mostly occurs in the cytoplasm,64 cellular imaging of Au NCs was performed for a preliminary investigation of whether the localization of Au NCs is similar to that of PRRSV. PK-15 and MARC-145 cells were incubated separately with 450 μM Au NCs for 2 h, and then the cells were observed with an excitation wavelength of 405 nm. From Figure S5, it can be seen that Au NCs are strictly localized to the cytoplasm, rather than the nucleus, of both PK-15 and MARC145 cells. Therefore, we speculate that Au NCs might specifically inhibit the proliferation of RNA viruses based on the similar localization.

Figure 6. Western blot analysis of PRRSV N protein with or without the treatment of Au NCs.

Au NPs could act as an effective anti-HSV-1 agent via a cellsurface-receptor heparan sulfate-dependent cell entry mechanism.9 In addition, Ziem et al. also found that the polyvalent 2D entry inhibitor, which was based on polyglycerol sulfatefunctionalized graphene sheets, can act as efficient entry inhibitors for enveloped viruses, such as PRV and African swine fever virus.63 In the present study, Au NCs were found to have a specific antiviral effect on PRRSV infection by the direct inactivation of viruses and interference with viral adsorption. However, the detailed mechanism for the selective antiviral activity of Au NCs remains to be elucidated in future work. 973

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ACKNOWLEDGMENTS We gratefully acknowledge financial support from the National Key Research and Development Program (2016YFD0500105) and National Natural Science Foundation of China (Grants 31490602 and 31372439).

To verify the hypothesis, the effect of Au NCs on PRRSV genome replication was tested as described in a previous paper.46 As shown in Figure S6, no significant differences were observed in PRRSV negative RNA levels before and after the treatment of Au NCs, suggesting that Au NCs have no noticeable effect against the genome replication process of PRRSV. Meanwhile, the results imply that the localization of Au NCs to the cytoplasm might not be the predominant factor for the selective antiviral activity of Au NCs on RNA viruses. Additionally, we notice that the concentration of Au NCs used in our study is beyond the general bioavailability of tissues and may not be suitable for in vivo application. However, PRRSV was reported to spread through close contact with fomites, such as feces and urine from infected pigs, and aerial transmission,65 suggesting the potential of Au NCs as a novel PRRSV inhibitor via the inactivation of viruses in the environment. Furthermore, coupling virus-specific agents, such as antibodies and aptamers, on the surface of NPs is an effective strategy to improve the antiviral activity and reduce the working concentration of NPs, enabling Au NCs to be applied in the future as a safe antiviral nanomaterial in vivo.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsanm.7b00386. Supplementary data of Au NCs, including apparatuses and reagents used in our study, fluorescence decay curves, a TEM image, inactivation activity on PRRSV infection using indirect immunofluorescence assay, the effect on PRV infection in MARC-145 cells, fluorescence microscopy images, and the effect on PRRSV genome replication (PDF)

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REFERENCES

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4. CONCLUSIONS In summary, we systematically investigated the antiviral activities of Au NCs using PRRSV and PRV as RNA and DNA virus models. Through indirect immunofluorescence assay, Au NCs were found to have a selective antagonistic effect against the propagation of RNA virus (PRRSV) by the direct inactivation of PRRSV and interference with viral adsorption but not that of DNA virus (PRV) at a noncytotoxic concentration. The results were further confirmed by Western blot assay, plaque assay, and RT-qPCR assay, which means Au NCs may play an important role in inhibiting RAN viruses as a novel antiviral agent, and facilitate a better understanding of the relationship between Au NCs and viruses.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (J.L.). *E-mail: [email protected] (S.X.). ORCID

Jiangong Liang: 0000-0002-8210-0210 Shaobo Xiao: 0000-0003-0023-9188 Author Contributions ⊥

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. These authors contributed equally. Notes

The authors declare no competing financial interest. 974

DOI: 10.1021/acsanm.7b00386 ACS Appl. Nano Mater. 2018, 1, 969−976

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DOI: 10.1021/acsanm.7b00386 ACS Appl. Nano Mater. 2018, 1, 969−976