Graphene Sheets with Defined Dual Functionalities for the Strong SARS‐CoV‐2 Interactions

Abstract Search of new strategies for the inhibition of respiratory viruses is one of the urgent health challenges worldwide, as most of the current therapeutic agents and treatments are inefficient. Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has caused a pandemic and has taken lives of approximately two million people to date. Even though various vaccines are currently under development, virus, and especially its spike glycoprotein can mutate, which highlights a need for a broad‐spectrum inhibitor. In this work, inhibition of SARS‐CoV‐2 by graphene platforms with precise dual sulfate/alkyl functionalities is investigated. A series of graphene derivatives with different lengths of aliphatic chains is synthesized and is investigated for their ability to inhibit SARS‐CoV‐2 and feline coronavirus. Graphene derivatives with long alkyl chains (>C9) inhibit coronavirus replication by virtue of disrupting viral envelope. The ability of these graphene platforms to rupture viruses is visualized by atomic force microscopy and cryogenic electron microscopy. A large concentration window (10 to 100‐fold) where graphene platforms display strongly antiviral activity against native SARS‐CoV‐2 without significant toxicity against human cells is found. In this concentration range, the synthesized graphene platforms inhibit the infection of enveloped viruses efficiently, opening new therapeutic and metaphylactic avenues against SARS‐CoV‐2.


Zeta-potential measurements. Zeta-potential experiments were performed on a Malvern
Zetasizer nano machine at 25 °C. Millipore water was used in all experiments. Measurements were performed with a Malvern-folded capillary zeta cell in automatic mode. (v/v) during ultrasonication at room temperature for 10 min. Then they were washed with the DI water 5 times and with acetone 2 times. After drying overnight, the studied compounds were dissolved in methanol and evenly distributed dropwise across the surface of gold substrates. XPS spectra were recorded using a Kratos Axis Ultra DLD spectrometer equipped with a monochromatized Al Kα X-ray source (1486.69 eV) using an analyzer pass energy of 80 eV for survey spectra that were used for quantification. High-resolution, core-level O1s, C1s, and N1s spectra were recorded in FAT (fixed analyzer transmission) mode at a pass energy of 20 eV. Both the electron emission angle and the source-to-analyzer angle were 60 °.
The binding energy scale of the instrument was calibrated following a Kratos Analytical Ltd procedure that used ISO 15472 binding energy data. Spectra were recorded by setting the instrument to the hybrid lens mode and the slot mode, which provided approximately a 300 x 700 µm 2 analysis area and using charge neutralization. All XPS spectra were processed with the UNIFIT program (version 2017). A Gaussian/Lorentzian product function peak shape model GL (30) was used in combination with a Shirley background. If not otherwise denoted, the L-G mixing for component peaks in all spectra were constrained to the value of 0.39. Peak fitting of C1s spectra was performed by using an asymmetric peak shape model for the graphene C1s' component peak and a symmetric peak shape model for all other component peaks. After peak fitting of the C1s spectra, all the binding energies were calibrated in reference to the graphene C1s component at a binding energy of 284.6 eV.
Atomic Force Microscopy (AFM). Samples were prepared by spin-casting dispersions of graphene materials (0.1 mg/mL) in deionized water onto freshly cleaved mica substrates at 50 rps for 60 seconds. The mica substrates were mounted onto Ø12 mm stainless steel discs with double-sided tape. Measurements were performed using a Bruker Multimode 8, Nanoscope 5 with a J-type scanner, operated in tapping mode with Nanosensors PPP-NCLR cantilever tips with a typical resonant frequency of 190 kHz, a spring constant of 48 N m -1 , and a tip radius of < 10 nm. Images were recorded at a minimum resolution of 1024 x 1024 and a scan speed of 0.8 Hz or lower. All experiments were conducted under ambient conditions, and results were analyzed using the Bruker NanoScope Analysis 1.8 software, along with Gwyddion.
Images were line-flattened using a first order (linear) fit. Statistical analysis. All viability and plaque reduction assays were repeated three independent times. Statistical evaluation was performed using GraphPad Prism 5 (GraphPad software).
One-way and two-way ANOVA was used to test for significance. Bonferroni adjustment was applied for multiple comparisons. Data represent mean values; standard deviations are indicated by error bars. P values less than 0.05 are considered significant. Trz). Synthesis of G-Trz was conducted according to our recently reported method (yield 78 %). [1] Synthesis of polyglycerol with a few amino groups (PG-NH 2 5 %). First, polyglycerol was synthesized according to a reported procedure (yield 81 %). [2] M n = 13.1 kDa. Polyglycerol with 5 % amino functional groups (PG-NH 2(5 %) ) was synthesized according to the procedure reported in the literature. [3] The hyperbranched polyglycerol (PG, M n = 13.1 kDa) was initially mesylated and subsequently azidated to convert the hydroxyl to azide functional groups with further reduction to the amino functional groups forming amino-functionalized PG (yield 60 %). Polyglycerol-mesylate. 1

Synthesis of graphene sheets with polyglycerol coverage (G-PG). Dispersion of G-Trz (0.2 g)
in NMP (50 mL) was sonicated for 2 h and further stirred in an ice bath. Solution of PG-NH 2(5 %) (0.96 g, 0.07 mmol) in NMP (10 mL) was added to G-Trz dispersion at 0 °C and stirred at same temperature for 3 h. TEA (21 µL, 0.15 mmol) was further added to the mixture and stirred for 1 h at 0 °C. The temperature of the mixture was increased to 25 °C and stirred for 2 days. Later, the mixture was dialyzed by a dialysis bag (cutoff MWCO 20 kDa) against water for 5 days and freeze-dried (yield 74 %). (G-PGS). G-PG was sulfated according to the reported procedures in the literature. 10-12 Shortly G-PG (0.6 g) was dispersed in dry DMF (20 mL). Afterwards, pyridine sulfur trioxide (2.5 g, 15.7 mmol) in same solvent (20 mL) was added to the G-PG and stirred for other 24 h at 60 °C. Further, deionized water (20 mL) was added and the pH of the reaction mixture was adjusted to pH 9 using NaOH (20 % w/v), and the mixture was dialyzed using a dialysis bag (cutoff MWCO 20 kDa) against 1 M NaCl solution for 5 days, against deionized water for 7 days and in the end freeze-dried (yield 76 %).

Synthesis of G-PGS-Cx.
Synthesis of G-PGS-Cx was performed according to our recently reported procedure (yield 78-82%). [4] Briefly, G-PGS (0.3 g) was dispersed in DMF (25 mL) and sonicated at room temperature for 30 minutes. Alkyl amines with different aliphatic chains (C 6 H 13 -NH 2 , C 9 H 19 -NH 2 , C 10 H 21 -NH 2 , C 11 H 23 -NH 2 , C 12 H 25 -NH 2 ) (42 μmol) were dissolved in DMF (15 mL) and added to the G-PGS dispersion at 25 °C. After stirring for 30 minutes at this temperature, triethylamine (5.6 μL, 0.042 mmol) was added to the reaction flask, mixture was heated till 60 °C, and stirred for 48 h. The product was dialyzed (MWCO 20 kDa) against water/isopropanol 1/1 mixture for 5 days. The solvent was evaporated, and the product was dried by lyophilization (yields 75-82%).   Table S1. Table S1. Relative ratios of C=C/C-O components obtained by quantification of the XPS spectra displayed in Figure S1a.