High Efficiency of Functional Carbon Nanodots as Entry Inhibitors of

Research Article www.acsami.org

High Efficiency of Functional Carbon Nanodots as Entry Inhibitors of Herpes Simplex Virus Type 1 Alexandre Barras,† Quentin Pagneux,† Famara Sane,‡ Qi Wang,§ Rabah Boukherroub,† Didier Hober,‡,* and Sabine Szunerits*,† †

Institute of Electronics, Microelectronics, and Nanotechnology (IEMN, UMR CNRS 8520), Université Lille 1, Cité Scientifique, Avenue Poincaré, BP60069, 59652 Villeneuve d’Ascq, France ‡ Laboratoire de Virologie EA3610, Université Lille 2 et CHU Lille, Batiment P Boulanger Hôpital A Calmette CHRU de Lille, Boulevard du Professeur Jules Leclerc, 59037 Lille, France § Key Laboratory for Liquid−Solid Structural Evolution and Processing of Materials, Ministry of Education, School of Materials Science and Engineering, Shandong University, Jinan 250061, P. R. China ABSTRACT: Nanostructures have been lately identified as an efficient therapeutic strategy to modulate viral attachment and entry. The high concentrations of ligands present on nanostructures can considerably enhance affinities toward biological receptors. We demonstrate here the potential of carbon nanodots (C-dots) surfacefunctionalized with boronic acid or amine functions to interfere with the entry of herpes simplex virus type 1 (HSV-1). C-dots formed from 4-aminophenylboronic acid hydrochloride (4-AB/C-dots) using a modified hydrothermal carbonization are shown to prevent HSV-1 infection in the nanograms per milliliter concentration range (EC50 = 80 and 145 ng mL−1 on Vero and A549 cells, respectively), whereas the corresponding C-dots formed from phenylboronic acid (B/C-dots) have no effects even at high concentrations. Some of the presented results also suggest that C-dots are specifically acting on the early stage of virus entry through an interaction with the virus and probably the cells at the same time. KEYWORDS: carbon nanodots, herpes simplex virus 1, inhibition, viral entry, 4-aminophenylboronic acid

1. INTRODUCTION For several virus-based diseases, therapies are not readily available. In contrast to bacterial infections, viruses, hiding inside cells, are difficult to reach with antibodies, and other approaches are needed to eradicate them. The mechanism of action of conventional antiviral agents can vary, ranging from targeting viral and cellular proteins to strengthening the immune response to the viral infection. Nanoparticles are considered to be potentially effective in terms of interfering with viral infection.1−3 Like other biologically-based interactions, the attachment and entry of viruses into the host cells are based on multivalent interactions between the surface of the virus with receptors present in the cell membrane.4 The multivalent character of nanostructures makes them welladapted to interfere with viral attachment and blocking viral entry into cells. We have explored lately boronic acid modified nanostructures as therapeutic agents for inhibiting the entry of highly glycosylated enveloped hepatitis C viruses (HCV).2,3 Motivated by these encouraging results, we were intrigued if boronic acid modified nanostructures are appropriate for blocking the attachment and entry of other enveloped viruses or if these structures specifically target HCV.2,3 A common human pathogen possessing an envelope like HCV is the herpes simplex virus type 1 (HSV-1).5 The central core of HSV-1, containing the linear double-stranded DNA © 2016 American Chemical Society

genome, is enclosed by an icosahedral capsid, further surrounded by a lipid-bilayer envelope accommodating viral glycoprotein spikes with variable packing densities.5 Viral envelope glycoproteins as well as host-cell membrane receptors are believed to be involved in the HSV-1 infection of cells.6 The strong affinity between the viral envelope glycoproteins gC and heparin sulfate receptors of the cell membrane, with a Ka ≈ 10−8 M, makes this interaction one of the most important binding interaction during attachment. It was shown that in the absence of gC, a strong reduction in viral binding to the cell surface occurs.7,8 Here, we investigated the potential of functional carbon nanodots (Figure 1) as HSV-1 viral entry inhibitors. Currently, no antiherpes drugs licensed for HSV treatment are targeted against viral entry inhibition because viral penetration is a very complex process.9 Nevertheless, some negatively charged dendrimers,10,11 polymers,12 and nanostructures13−16 interacting directly with the viral envelope glycoproteins have been proposed over the last years. Silver and gold nanoparticles modified with sulfonate functions, mimicking heparan sulfate (HS), have been proposed as HSV-1 inhibitors.13,14 Tannic acid Received: February 8, 2016 Accepted: March 25, 2016 Published: March 25, 2016 9004

DOI: 10.1021/acsami.6b01681 ACS Appl. Mater. Interfaces 2016, 8, 9004−9013

Research Article

ACS Applied Materials & Interfaces

Figure 1. (A) Formation of boronic acid (B/C-dots) and amine−boronic acid functionalized C-dots (3-AB/C-dots, 4-AB/C-dots) based on the hydrothermal carbonization of phenylboronic acid (PBA), 3-aminophenylboronic acid (3-APBA), or 4-aminophenylboronic acid hydrochloride (4APBA·HCl, Sigma-Aldrich) upon heating in a Teflon-lined autoclave chamber for 8 h at 160 °C. (B) UV−vis absorption of C-dots (5 μg mL−1) and fluorescence spectra at pH 7 upon excitation at 320 nm. (C) Fluorescence spectra of C-dots (5 μg mL−1) in phosphate buffer at pH 8 upon different excitations (from 240 to 420 nm). (D) TEM images of different B/C-dots, 3-AB/C-dots, and 4-AB/C-dots. (E) XRD spectrum of 4-AB/C-dots: inset is the corresponding HRTEM image. 9005


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ACS Applied Materials & Interfaces modified silver nanoparticles have been proposed lately.17 The high affinity of tannins toward glycoproteins is believed to prevent viral attachment and entry. Herein, we describe the first example of specifically engineered carbon nanodots (C-dots) to behave as HSV entry inhibitors.18,19 The good dispersibility of C-dots in aqueous solution and biological media, their low toxicity19 and favorable optical properties (together with their reliable fabrication on a larger scale)19−21 make functional C-dots ideal nanostructures for biomedical applications. Traditional Cdots were employed for optical imaging.20,22 In this work, the focal point is the use of C-dots for viral inhibition. A modified hydrothermal carbonization method, as described by Shen et al.,23 was selected for the formation of the nanostructures. We show that some of the structures were able to reduce considerably viral entry of cell-culture-derived HSV into monkey kidney cancer cells and human lung cancer cells using in vitro assays.

X-ray photoelectron spectroscopy (XPS) spectra were recorded on a ESCALAB 220 XL spectrometer. Fourier transform infrared (FTIR) spectra were recorded using a ThermoScientific FTIR instrument (Nicolet 8700) with a resolution of 4 cm−1. Dried nanomaterials (1 mg) were mixed with KBr powder (100 mg) in an agate mortar. The mixture was pressed into a pellet under 10 tons of load for 2−4 min, and the spectrum was recorded immediately. A total of 16 accumulative scans were collected. The signal from a pure KBr pellet was subtracted as a background. Raman spectroscopy measurements were performed with a Horiba Jobin Yvon LabRam HR Microraman system with a 473 nm laser diode excitation source. Visible light was focused by a 100× objective. The scattered light was collected by the same objective in backscattering configuration, dispersed by a monochromator (1800 mm focal length) and detected by a CCD. The average hydrodynamic diameter, the polydispersity index (PDI), and the ζ potential were recorded on a Zetasizer Nano ZS (Malvern Instruments S.A., Worcestershire, UK). All of the batches were diluted to 100 μg mL−1 in water and analyzed in triplicate at 25 °C. The ζ potentials of HSV-1, Vero cells, and mixtures in diluted DMEM (by 10) were measured at 37 °C using a folded ζ capillary cell. Experiments consisted of 25 runs per measurement with an applied voltage of 50 V. Cell Culture and Toxicity Assay. Monkey kidney cancer cells (Vero) [ATCC CCL-81, ECACC, Sigma-Aldrich, France] and human lung cancer cell (A549) [ATCC CCL-185] were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, 100 U mL−1 penicillin, and 100 μg mL−1 streptomycin in a humidified atmosphere of 5% CO2 at 37 °C. The cytotoxicity was evaluated by the Uptiblue (Interchim) method based on absorption measurements at 570 and 595 nm following the manufacturer’s protocol. Vero and A549 cells were plated in 96-well plates (6 × 103 cells/well), grown for 24 h, and then various concentrations of functional C-dots were added to the wells and the plates incubated for 2 h. Afterward, the cell layers were washed four times with cold PBS, and then the plates were incubated. At 72 h postincubation, the cell morphology was evaluated under an inverted microscope (Olympus CKX41, France). The cell viability was assessed by using Uptiblue. Briefly, the cell layers were washed, 100 μL of Uptiblue diluted at 10% in DMEM with 10% FBS were added per well and incubated at 37 °C during 4 h. The absorbance values were then measured at 570 and 595 nm. The results were expressed as percentage compared with controls. Virus. Herpes simplex virus 1 (HSV-1) [ATCC VR-260] was produced in Vero cell culture as previously reported24 and adapted to A549 cells. The virus was incubated with cells until the complete destruction of cell monolayers. Afterward, the supernatant was harvested, centrifuged at 3000 rpm during 10 min, and stored at −80 °C. Virus Titration. The virus titers of supernatants of Vero and A549 cell cultures were determined. Corresponding cells (A549 or Vero) were plated 24 h in 96-well plates (6 × 103 cells/well) before test. Serial 6-fold dilution of supernatants were distributed in the wells in DMEM supplemented with 10% FBS, 1% antibiotics, and 1% Lglutamine. The plates were incubated for 5 days in atmosphere with 5% CO2 at 37 °C. Afterward, the plates were examined using an inverted microscope (Olympus CKX41, France) to evaluate the extent of the virus-induced cytopathic effect in cell culture. Calculation of estimated virus concentration M was carried out by the SpearmanKarber method25 and expressed as log10 TCID50/mL (50% tissue culture infectious dose). Antiviral Assay. Cells were plated 24 h in 96-well plates (6 × 103 cells/well) before testing. Functional C-dots were diluted in the appropriate solution (DMEM without FBS) at different concentrations and incubated for 30 min in atmosphere with 5% CO2 at 37 °C with HSV-1 at 103 virus mL−1. This mixture was further incubated with Vero or A549 cells during 2 h at 37 °C. Afterward, they were washed 4 times with PBS, and the plates were incubated. At 72 h postincubation, the cell morphology was evaluated under an inverted

2. EXPERIMENTAL SECTION Synthesis of Functional Carbon Nanodots. Boronic acid- and amine-functionalized C-dots were prepared from phenylboronic acid derivatives (phenylboronic acid (PBA), 3-aminophenylboronic acid (3-APBA), or 4-aminophenylboronic acid hydrochloride (4-APBA· HCl, Sigma-Aldrich) using a modified hydrothermal carbonization method, as described by Shen et al.23 In brief, phenylboronic acid derivatives (200 mg) were dissolved in water (20 mL) and the pH adjusted to 9.0 by adding NaOH (0.5 M). The solution was degassed with nitrogen gas during 1 h to remove dissolved oxygen and heated in a Teflon-lined autoclave chamber (125 mL acid digestion vessel no. 4748, Parr, France) for 8 h at 160 °C. After cooling to room temperature, solutions were centrifuged at 10 000 rpm during 30 min to remove large precipitates and were dialyzed against water for 24 h, with the water being changed every 6 h (SpectraPor 1, pore size: 1000 Da). The formed functional C-dots solutions (200 μL) were dried and then weighed with a Sartorius microbalance (TG 209 F3 Tarsus, Netzsch) to estimate the exact mass concentration. Stock solutions were adjusted to 500 μg mL−1 with DI water and were conserved at 4 °C. Characterization of Functional Carbon Nanodots. UV−vis measurements were performed with a PerkinElmer lambda 950 dualbeam spectrophotometer (resolution of 1 nm) with a diluted aqueous solution of nanomaterials at 5 μg mL−1. Emission fluorescence spectra were recorded between 340 and 800 nm using a Cary Eclipse spectrometer (Agilent, France). Solutions were excited at 320 nm and at different wavelengths (excitation and emission slit 10 nm, scan rate 600 nm/min). Fluorescence measurements were performed with diluted phosphate buffer (pH 8.0) aqueous solution of nanomaterials at 5 μg mL−1. The quantum yields (Φ) of the C-dots was calculated by comparing their fluorescence intensities (excitation at 350 nm) and absorbance values at 350 nm with those of quinine sulfate with the following equation: Φ = ΦREF(I/IREF)(AREF/A)(η2/η2REF), where Φ is the quantum yield, A is the optical density, I is the measured integrated emission fluorescence intensity, and η is the refractive index. Quinine sulfate (quantum yield of 0.54 at 350 nm) was chosen as standard and dissolved in 0.1 M H2SO4 (refractive index: 1.33), and the C-dots were dissolved in water (refractive index: 1.33). Absorbance values of the C-dot solutions were kept under 0.05 at the excitation wavelength to minimize reabsorption effects. Transmission electron microscopy (TEM) images were recorded on a JEOL JEM-2100 electron microscope operated at an accelerating voltage of 200 kV. The X-ray diffraction (XRD) patterns of the C-dot powders were recorded in the range of 10−90° on a BrukerAXS D8-advance X-ray diffractometer using Cu Kα radiation (λ = 1.54 Å) at 40 kV and 40 mA. 9006


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ACS Applied Materials & Interfaces microscope (Olympus CKX41, France). The cell viability was assessed by using Uptiblue, as described above. Competitive Assay with Fructose. C-dots (from 2 to 50 μg mL−1) were mixed with a large excess of fructose (520 μg mL−1) and incubated at 37 °C for 1 h before adding the virus. Subsequently, further dilutions of sugar-coated nanomaterials were mixed with HSV1 virus and incubated for 30 min at 37 °C. These mixtures were mixed with 24 h-old Vero and A549 cell monolayers. After 2 h of incubation, cell layers were washed 4 times with cold PBS before cultured in fresh media. The cell layers were examined 72 h later under an inverted microscope (Olympus CKX41, France) at the objective lens (10×). Cell viability assays were performed using UptiBlue.

Table 1. Quantum Yield of C-Dots

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of Functional CDots. We opted here for the synthesis of C-dots modified with boronic acid and amine groups (Figure 1A). Hydrothermal decomposition of low-temperature-melting molecular precursors such as phenylboronic acid (PBA, mp. 216 °C), 3aminophenylboronic acid (3-APBA, mp. 94−96 °C), or 4aminophenylboronic acid hydrochloride (4-APBA, mp 62−66 °C) in a Teflon-lined autoclave chamber was employed and resulted in different functional C-dots. This synthetic approach is highly attractive as it directly leads to surface-modified Cdots, simply by the careful selection of the initial carbon source. PBA was used because boronic acid groups are known for their ability of recognizing cis-diol containing molecules such as saccharides by forming five- or six-membered cyclic esters.26 Boronic acid modified C-dots (B/C-dots) are expected to interact strongly with glycan receptors such as heparin sulfates of the host-cell membrane and might interfere with viral attachment.27 Alongside B/C-dots, two different boronic acidamine structures, 3-AB/C-dots and 4-AB/C-dots were synthesized with the understanding that the generation of efficient receptors for heparin, a glycosaminoglycan with high anionic charge density, requires a number of complementary cationic groups.27 The UV−visible spectra of B/C-dots, 3-AB/C-dots, and 4AB/C-dots in water are displayed in Figure 1B. All structures show strong optical absorption in the UV region below 205 nm, attributed to the π−π* transition of the CC structures, with a tail extending to the visible range. The absorption shoulders at 250−300 nm represent a typical absorption of an aromatic π − system in accordance with the literature.28 When the C-dots are excited at 320 nm, emission bands centered at 414 nm (B/C-dots) and 430 nm (3-AB/C-dots and 4-AB/C-dots) are observed, indicating a blue fluorescence (Figure 1C). Because the initial phenylboronic acid derivatives are nonfluorescent in the visible region, the bright fluorescence is indicative of the formation of functional C-dots. Figure 1C shows, in addition, that the fluorescence maximum and intensity is dependent on the used excitation wavelength (Figure 1C). Increasing the excitation wavelength from 240 to 420 nm red-shifts the emission gradually, a behavior specific to C-dots.19,29 The fluorescent quantum yields of the different Cdots varied between 0.027% (B/C-dots), 0.018 (3-AB/C-dots) and 0.005 (4-AB/C-dots) using quinine sulfate as reference with a quantum yield of 0.54 (Table 1).30 The emission efficienty is moderate for B/C-dots and 3-AB/C-dots, and low for 4-AB/C-dots. Transmission (TEM) images of B/C-dots, 3-AB/C-dots and 4-AB/C-dot are shown in Figure 1D, demonstrating that the Cdots are geometrically uniform (inset in Figure 1D) with an average diameter of 14 ± 4 nm (B/C-dots), 24 ± 6 nm (3-AB/

C-dots), and 22 ± 7 nm (4-AB/C-dots). The average diameter is larger in comparison to the C-dots reported by Shen and Xia, being 4.5 nm for phenylboronic acid modified C-dots.29 This is most likely due to the different chemical composition of our Cdots, as shown by XPS and FTIR, where oxygen functions are, in addition, present on the particles. Furthermore, the lack of any perceptible lattice fringes from the HRTEM images (data not shown) suggests an amorphous nature of all C-dots. This result was further confirmed by XRD analysis of 4-AB/C-dots (Figure 1E). The XRD pattern displays a broad (002) diffraction peak at around 21.3° (0.42 nm), corresponding to Bragg’s reflection plane of graphitic carbon and revealing the existence of amorphous structure. The surface composition of the prepared functional C-dots was investigated by X-ray photoelectron spectroscopy (XPS) (Figure 2A) and FTIR analysis (Figure 2B). XPS survey spectra display bands due to C1s (285 eV), O1s (540 eV), B1s (188 eV), N1s (400 eV), and Cl2p (199 eV) depending on the C-dots. The percentage of B1S integrated into the C-dots varies from 1.75 at % for B/C-dots to only 0.40 and 0.10 at % for 3-AB/C-dots and 4-AB/C-dots, respectively (Table 2). Most of the functional groups in 3-AB/C-dots and 4-AB/C-dots are N1s functions. In the case of 4-AB/C-dots, the presence of Cl2p is additionally detected. The high-resolution XPS spectra of the C1s region of the three particles are seen in Figure 2A. In the case of B/Cdots, three peaks at 283.9, 285.0, and 287.5 eV attributed to the C−C (sp2)/C−B, C−C/C−H, and CO functions, respectively are observed. The 3-AB/C-dots and 4-AB/C-dots show next to the bands at 283.9 eV (C−C (sp2)/C−B), 285.0 eV (C−C/C−H) and 287.3 eV (CO/C−N), a contribution at 290.3 eV due to O−CO functions. Indeed, some oxygen functions are present on the C-dots, albeit mainly only CO functions in the case of B/C-dots as well as O−CO functions for AB/C-dots and 4-AB/C-dots. Indeed, the C/O ratio is in all three cases somehow larger than the theoretical one predicted of 3 (6C/2O) from the initial organic precursors, being 3.5 (B/ C-dots), 3.1(3-AB/C-dots), and 3.3 (3-AB/C-dots), respectively. This is different for phenylboronic acid modified C-dots reported by Shen and Xia, where a C/O ratio of 2.9 was determined.29 This indicates that some of the phenylboronic acid groups are carbonized, which is in line with the rather low content of boronic acid functions obtained in our procedure. The band at 399.6 eV in the N 1s high-resolution spectra of 3AB/C-dots and 4-AB/C-dots demonstrates the presence of amine groups.31 FT-IR spectra of the different C-dots (Figure 2C) exhibit distinct bands at 3465, 1618, 1333, and 1090 cm−1 that are attributed to adsorbed water molecules, graphitic CC, B−O stretching, and the deformation vibration modes of the boroxol bond of the boronic acid moieties.29 The bands observed at 2855 and 2924 cm−1 are due to CH2 stretching bands, and the

samples quinine sulfate B/C-dots 3-AB/Cdots 4-AB/Cdots

9007

integrated fluorescence intensity (I)

absorbance at 350 nm (A)

refractive index (η)

quantum yield (Φ)

46866

0.0153

1.33

0.5430

204 1131

0.0013 0.0111

1.33 1.33

0.027 0.018

550

0.0179

1.33

0.005


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Figure 2. (A) XPS-survey spectra; (B) C1s and N1s high-resolution survey spectra; (C) FT-IR spectra; (D) Raman spectra.

characteristic peak of NH2 bending vibration is observed at 1631 cm−1. The ζ potential of all three C-dots show a strongly negative potential (Table 2) in water and in biological medium such as Dulbecco’s Modified Eagle Medium (DMEM). This is most

indicative for the absence of protonated amines and the presence of predominantly negatively charged oxygen functions, which is in line with XPS analysis (Figure 2A). Raman studies show contributions from both the G band at 1570 cm−1, related to the in-plane vibration of sp2 carbon, and 9008


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3.3. Antiviral Assay. The first event of virus-cell interactions often involves the attachment and adsorption of viral particles to receptors of the host, followed by penetration and viral replication. The attachment of HSV-1 to cells is mediated by the viral envelope. Interfering with this adsorption process is thus considered as a promising strategy to inhibit the infection of the cell.33 The antiviral activity of the different C-dots was evaluated on A549 and Vero cell monolayers, infected with HSV-1 in the absence and the presence of C-dots. The addition of B/C-dots for 2 h and incubation for additional 72 h at 37 °C show an important loss of cell viability for both cell lines, indicating no blocking effect on viral entry. This contrasts to the use of 3-AB/ C-dots and 4-AB/C-dots, where concentration-dependent virus inactivation is observed. The dose−response curves are seen in panels A and B of Figure 4 for the two cell lines. The effective concentration to achieve 50% inhibition (EC50) against HSV-1 infections was 424 ± 19 ng mL−1 (A549) and 469 ± 22 ng mL−1 (Vero) for 3-AB/C-dots and 145 ± 12 ng mL−1 (A549) and 80 ± 13 ng mL−1 (Vero) for 4-AB/C-dots, respectively. At concentrations of 3-AB/C-dots or 4-AB/C-dots higher than 5 μg mL−1, no infection occurs, and 100% of cell viability is observed. For comparison, Table 3 summarizes EC50 values of other reported anti-HSV compounds such as dextrane sulfate, poly-L-lysine, acyclovir, polyanionic dendrimers, and tannic acid modified silver particles structures.10,17,34 In comparison to the reported structures, the 3-AB/C-dots and 4-AB/C-dots display high levels of antiviral activity. The morphologies of cells before and after infection with HSV-1 with and without 3-AB/C-dots or 4-AB/C-dots were investigated. From the images shown in Figure 4C, it becomes clear that in the absence of C-dots, important morphological changes are observed upon infection. In contrast, in the presence of nanostructures, the morphology of cells remained unchanged comparable with controls. Altogether, these data indicate that 3-AB/C-dots and 4-AB/C-dots can inhibit the cytopathic effect induced by HSV-1. 3.4. Mechanistic Considerations. Polyanionic compounds such as dextrane sulfates34 and sulfonate-modified metallic nanoparticles13,14 as well as dendrimers10 have shown the potential to inhibit the infection of cells with HSV-1. Almost all cell types in the body express negatively charged heparan sulfate (HS) on their surface, which interacts with a number of proteins mainly through ionic binding. The potential of these agents is based on the principle that they mimic heparan sulfate (HS) and compete for the binding of the virion to the cell membrane. It was shown that heparan sulfate and 3O-sulfated heparan sulfate (3-OS HS) generate fusion receptors for HSV entry and spread.35,36 Preincubation of cells with HS and 3-OS HS recognizing peptides significantly reduced the viral entry and spread in human corneal fibroblasts.35−37 Indeed, Vero cells show negative ζ-potentials at physiological pH (7.4) (Figure 5), determined to be approximately −10 ± 4 mV. The ζ-potentials of the different C-dots are also negative, being −19 ± 5 mV for B/C-dots and −23 ± 7 mV or −25 ± 5 mV for 3-AB/C-dots or 4-AB/C-dots, respectively. The incubation of cells with B/C-dots did not alter the ζ potential. These observations indicate that B/C-dots were not bound to Vero cells within this concentration range and that the boronic acid moieties present on the C-dots do not mediate high affinity with HS of the host-cell membrane. The interaction of the C-dots with HS or 3-OS HS is consequently ruled out. The same was observed upon incubation of B/C-dots with HSV-1,

Table 2. Physicochemical Properties of Boronic Acid and Amine−Boronic Acid Functionalized C-Dots B/C-dots hydrodynamic size/ nm polydispersity index (PDI) ζ-potential/mV (water) ζ-potential/mV (DMEM) C 1s O 1s N 1s B 1s Cl 2p

3-AB/C-dots

4-AB/C-dots

332 ± 60

55 ± 1

96 ± 1

0.314 ± 0.055

0.248 ± 0.007

0.171 ± 0.006

−27.0 ± 6

−37 ± 1

−41 ± 1

−19 ± 5

−23 ± 7

−25 ± 5

76.6 21.7 0 1.7 0

70.6 22.7 6.3 0.4 0

70.4 21.2 7.1 0.1 1.2

the D band at 1350 cm−1, related to the presence of sp3 defects (Figure 2D). The intensity ratio of the D and G band (ID/IG) is a measure of the disorder extent and the ratio of sp3/sp2 carbon and is 1.24 ± 0.1 for these C-dots.32 In addition, a broad peak centered at ∼2920 cm−1, corresponding to the 2D component of graphitic structures, was found. 3.2. Cytotoxicity Assay. To determine the concentration zone where the functional C-dots can be used without any significant side effects, we investigated the cytotoxicity toward two cell lines, A549 and Vero cells. Vero cells are used for many purposes, in particular as host cell models for growing viruses. The cytotoxicity was assessed using the Uptiblue cell-viability assay. Figure 3 shows the results obtained. All nanostructures

Figure 3. Viability of cells treated with C-dots: A549 (A) and Vero (B) cells were grown in 96-well plates (6 × 103 cells per well) with 10% FBS in DMEM. C-dots were added to cell cultures at various concentrations; the plates were incubated for 2 h, and afterward, the cell layers were washed and the plates incubated for 72 h. Next, the cell layers were washed, and the viability was evaluated by using Uptiblue. The results, expressed as percentage of viability, are the means of two independent experiments with each treatment performed in triplicate. Negative control: without C-dots.

were found to be noncytotoxic on A549 at concentrations up to 300 μg mL−1. For Vero cells, 3-AB/C-dots showed moderate cytotoxicity, with a cell viability drop to 67% at 300 μg mL−1, and neither B/C-dots nor 4-AB/C-dots showed any significant cytotoxicity. The cell viability at 100 μg mL−1 was found to be higher than 95% for A549 and 92% for Vero for all C-dots, comparable to the absence of cytotoxicity of mercaptoethanesulfate-capped gold13 or silver14 nanoparticles toward Vero cells. 9009


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Figure 4. C-dots inhibit the cytopathic effect of HSV-1: HSV-1 (103 TCID50 mL−1) was mixed with C-dots in DMEM or with DMEM at various concentrations for 30 min (in atmosphere with 5% CO2 at 37 °C), and then the mixtures were incubated with A549 (A) and Vero (B) cells (6 × 103 cells per well) in medium without FBS for 2 h. Afterward, the cell layers were washed and grown in complete medium for 72 h. Then, after washings, the cell viability was assessed by using Uptiblue. The results, expressed as percentage of viability, are the means of two independent experiments with each treatment performed in triplicate. Symbols: * represents significant differences with p ≤ 0.05, while ** represents p ≤ 0.001. (C) A549 cells and Vero cell layers were observed under an inverted microscope (×10) 24 h after inoculation with HSV-1 or HSV-1 mixed with 4-AB/C-dots (10 μg mL−1); the bar length indicates 200 μm.

Table 3. Anti-HSV Activity of Different Compounds in Cellsa compounds

EC50 / ng mL−1

cell line

reference

dextran sulfate poly-L-lysine acyclovir SPL7013 tannic acid modified Ag NPs 3-AB/C-dots 4-AB/C-dots

2000−5000 8000−145000 1090 2000 1990−2530 469 ± 22 80 ± 13

Vero cells Vero cells Vero cells Vero cells 291.03C Vero cells Vero cells

34 34 39 10 17 current study current study

a

PCL: polycaprolactone; SPL7013: amide dendrimer. Figure 5. ζ potential of HSV-1 (103 TCID50 mL−1), Vero cells (60 000 cells mL−1), and C-dots (2 μg mL−1) in DMEM at 37 °C.

where no significant alteration on ζ potentials was observed. The negatively charged B/C-dots are therefore also not interacting by ionic interactions with positively charged viral envelope glycoproteins, as observed in the case of dextrane sulfate,34 or partially negatively charged ZnO micronano filopodia-like structures.38 These results suggest that B/Cdots are not blocking the interaction of HSV-1 with cells and

viral attachment and penetration occurs even in the presence of B/C-dots (Figure 4). The incubation of Vero cell with 3-AB/C-dots or 4-AB/Cdots results in a significant change of ζ potential (Figure 5) due to the believed binding of C-dots to the cell surface. It is not clear to which cellular receptor the particles are binding. The 9010


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Figure 6. Effect of fructose-saturated C-dots on the infection with HSV-1: various concentrations of functional C-dots (from 5 to 50 μg mL−1) were first mixed with a large excess of fructose (520 μg mL−1) and incubated at 37 °C for 1 h. Subsequently, HSV-1 (103 TCID50 mL−1) was added and 30 min later incubated with A549 (A) and Vero (B) cells (6 × 103 cells/well) in medium without FBS for 2 h. Afterward, the cell layers were washed and grown in complete medium for 72 h. After the washing steps, Uptiblue assay was performed. The results, expressed as a percentage of viability, are the means of two independent experiments with each treatment performed in triplicate. Symbols: ** represents significant differences with p ≤ 0.001.

Figure 7. C-dots interact in the early step of HSV-1 infection of cells: schematic representation of C-dots treatment. (A) Cells were treated with Cdots before inoculating with HSV-1 (a); cells were inoculated with HSV-1 mixed with C-dots (b); cells were inoculated with HSV-1 afterward C-dots were added (c). Viability of A549 cells (B) and Vero cells (C) (6 × 103 cells per well) infected with HSV-1 (103 TCID50 mL−1) treated with 10 μg mL−1 C-dots as described in (A). The cell viability on day 72 post-infection was evaluated by using Uptiblue assay. The results, expressed as a percentage of viability, are the means of two independent experiments with each treatment performed in triplicate. Symbols: ** represents significant differences with p ≤ 0.001.

NH2 groups present in 3-AB/C-dots or 4-AB/C-dots might bind electrostatically to heparan or 3-OS heparan receptors:

however, binding to proteins such as nectin-1 cannot be excluded at this stage. However, again no change in ζ potentials 9011


Research Article


interactions between virus and cell receptors. In addition, future studies should address the effect of such nanostructures on different clinical relevant cell lines to demonstrate that C-dots are equally good in human skin fibroblasts, genital epithelial, or cornea cells as well. On the same token, in addition to HSV-1 strains, clinically relevant HSV-1 isolates will be tested.

upon the incubation of 3-AB/C-dots or 4-AB/C-dots with HSV-1 was observed (Figure 5), as was the case of B/C-dots. This makes us believe that the particles are not interacting with the HSV-1 viron but mainly with cellular receptors. The interaction with Vero cells receptors seems in addition not to be governed by surface-charge effects of the particles (all three C-dots show negative ζ potentials, but only 3-AB/C-dots or 4AB/C-dots interact with Vero cells) but either by size or chemical functions such as NH2 and COOH groups present on the surface of the particles (Figure 2 and Table 2). Indeed, the hydrodynamic size of B/C-dots is 3−6 times larger than 3-AB/ C-dots or 4-AB/C-dots, respectively (Table 2). To validate the hypothesis of a boronic acid independent inhibition mechanism, we mixed 3-AB/C-dots and 4-AB/Cdots with a large excess of fructose (520 μg mL−1) resulting in the formation of five-membered cyclic esters.26 Figure 6 shows that the effects of C-dots and fructose-saturated C-dots are similar. C-dots and fructose-saturated C-dots can as well inhibit the cytopathic effect of HSV-1. This furthermore confirms that boronic acid functions on C-dots are not involved in the inhibition of HSV-1-induced cytopathic effect obtained with Cdots. To determine whether C-dots specifically act on the early stage of HSV-1 virus entry, we added 3-AB/C-dots and 4-AB/ C-dots (10 μg mL−1) at different time points before, during, and after the infection of A549 and Vero cells with HSV-1 (Figure 7A). The results presented in panels B and C of Figure 7 for A549 and Vero cells, respectively, indicate that preincubation of both cell lines with 3-AB/C-dots or 4-AB/ C-dots before infection does not inhibit the cytopathic effect of HSV-1. This might indicate that the C-dots are only very weakly bound, and any formed bond is cleaved upon rinsing. The addition of C-dots after infection also has no effect because HSV-1 induced a cytopathic effect. Co-incubation of HSV-1 virions with C-dots for 30 min before adding the mixture to the cell cultures exhibits a remarkable inhibition of the HSV-1induced cytopathic effect because the cell viability was 92%. These results suggest that C-dots are specifically or preferentially acting on the early stage of virus entry. Altogether, our data suggest that the interaction of 3-AB/Cdots or 4-AB/C-dots is stronger with Vero cells than with viral particles, which can interfere in the early step of interaction of HSV-1 with the cell resulting in an inhibition of the infection.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] *E-mail: [email protected] Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Support from the Centre National de Recherche Scientifique (CNRS), the Université Lille 1, the Nord Pas de Calais region, and the Institut Universitaire de France (IUF) is acknowledged. We thank A. Addad (Unité Matériaux et Transformations, Lille 1) and L. Brunet (Centre Commun de Mesures Imagerie Cellulaire, Lille 1) for technical support with TEM characterizations. This study was supported by Ministère de l’Education Nationale de la Recherche et de la Technologie, Université Lille 2 (Equipe d’Accueil 3610), France, and Centre Hospitalier Régional et Universitaire de Lille.

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REFERENCES

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4. CONCLUSIONS The purpose of HSV-1 treatments is to reduce the replication and spreading of the virus. In primary infection or in reactivation, viral particles are produced. We present here the interest of functional carbon nanodots (C-dots), formed by hydrothermal carbonization of aminophenylboronic acid derivatives, as an antiviral agent. Although it cannot be excluded that inhibitors like C-dots are able to limit the spreading of the virus from cell to cell, it could be shown that 4AB/C-dots interfere with HSV-1 infection in a concentrationdependent manner with EC 50 = 80 ng mL −1 . The corresponding C-dots formed from phenylboronic acid (B/Cdots) show no effect even at high particle concentrations. In contrast to HCV, boronic acid functions are not involved in the viral entry inhibition mechanism of HSV-1. Classical in vitro models of HSV-1 infection have been used in this work. Although the mechanism of action is still not completely understood, the results suggest that C-dots are specifically acting on the early stage of virus infection in the course of 9012


Research Article

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High Efficiency of Functional Carbon Nanodots as Entry Inhibitors of

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