2.1 Coating approaches for lPGS on fleece samples

Meltblown polypropylene fleece samples were coated by three different approaches (Figure 2a,b). In the first approach, they were immersed in a solution of lPGS-b-BPh with guanidinium chloride (GHCl) for 10 min and then irradiated with UV light.²⁴ In the other two approaches, they were immersed in a polymerizing solution of either dopamine or MIdPG for 24 h or 1 h, respectively, and then treated with a solution of lPGS-b-NH₂ for 24 h at an elevated temperature of 80°C.

The coating procedures change the hydrophobicity of the fleece materials, as observed in contact angle measurements (Figure 2c). Untreated meltblown PP fleece is hydrophobic and minimizes the contact area between fibers and a water droplet. Each coating procedure changed the fleece samples to hydrophilic and water droplets soak into the fleece.

Scanning electron microscopy (SEM) images were obtained to reveal morphological changes on the fiber surfaces. Since lPGS-b-BPh forms a monolayer of a few nanometers in thickness, no change is observed in that case. Dopamine and MIdPG coatings on the other hand form micrometer large aggregates that deposit on the fibers and are clearly visible in SEM images. Treatment with lPGS-b-NH₂ adds a thin functional layer that does not influence the macroscopic morphology. Further images are shown in Figures S1–S4.

Additional analytical data were obtained by measuring the surface elemental composition with X-ray photoelectron spectroscopy (XPS). The obtained data are summarized in Table 1 (also see Figure S5). Unmodified polypropylene fleece consists of only carbon and hydrogen. XPS does not quantify hydrogen atoms, thus the carbon content is determined to be close to 100%. PDA and MIdPG add oxygen as well as nitrogen to the surface. Small amounts of sulfur may originate from the used buffer, 3-(N-morpholino)propanesulfonic acid. Addition of lPGS-b-NH₂ or lPGS-b-BPh to the surface leads to significant amounts of sulfur, which provides evidence for the presence of the sulfated polymers on the surfaces.

Overall, all of the three tested procedures reliably yield coated fleece materials with lPGS present on the surface. Coating with lPGS-b-BPh potentially is the most promising method because of its easy and fast application. Thus, it may be suitable for possible applications.

2.2 Detection of sulfate groups with methylene blue

Standard techniques for surface characterization, for example, atomic force microscopy, electron microscopy, ellipsometry, or quartz crystal microbalance, cannot be used to directly detect the lPGS coating on a complex surface such as a fleece material. However, the applied coatings add a large amount of negatively charged sulfate groups to the surface. This feature will now be used to detect the polymers by measuring the adsorption of a positively charged dye to the respective surface.

Initially, a sample of lPGS-b-BPh coated fleece was tested in a filtration setup (Figure 3). When a solution of methylene blue in water was filtered through the sample, a portion of the dye was retained on the fleece, turning it blue (step I). Washing with water did not affect the color of the stained fleece (step II), but addition of 2 m sodium chloride immediately removed the dye from the fleece (step III). This demonstrates that the binding of methylene blue to the fleece surface is of electrostatic nature. At low salt concentration, the sodium counterions of the sulfate groups will be replaced by methylene blue ions. Adding an excess of salt causes charge screening, and methylene blue is released. Overall, an equilibrium exchange of the respective counterions is observed.

A qualitative comparison of coated and uncoated fleece samples further proved that lPGS is present on all fully functionalized samples (Figure 4a). In this experiment, fleece samples were incubated in a methylene blue solution (2.5 ml, 30 μM) for 2 h. The methylene blue concentration was determined before and after the incubation by UV/Vis spectroscopy. The difference was used to determine the amount of dye that has been adsorbed to the fleece. Furthermore, the obtained values were normalized to the mass of the respective fleece sample.

As shown in Figure 4a, uncoated samples adsorbed a low amount of methylene blue, which may be attributed to unspecific interactions between the hydrophobic dye and the polypropylene surface. The adhesion layer of polydopamine (PDA) increases the amount of adsorbed dye, likely due to interactions between the PDA surface and the aromatic system of methylene blue. This effect was not observed for MIdPG. All samples with lPGS coating bound substantially more methylene blue than the respective controls. This observation is especially pronounced for lPGS-b-NH₂@MIdPG and lPGS-b-BPh, while lPGS-b-NH₂@PDA adsorbed little more than PDA alone, owing to the high adsorption on PDA.

A quantitative analysis of the methylene blue adsorption was performed exemplarily for lPGS-b-BPh (Figure 4b). The incubation with methylene blue was performed at different dye concentrations. The adsorption of methylene blue to lPGS coated surfaces is an equilibrium exchange of sodium counter ions against methylene blue (MB⁺). Therefore, plotting the residual dye concentration C_R(MB⁺), normalized to the respective starting concentration C₀(MB⁺), over C₀(MB⁺) gives the adsorption isotherm.

The adsorption isotherm of methylene blue on coated meltblown polypropylene fleece allows an estimation of the coating density of lPGS-b-BPh on the fiber surface. The inflection point of this curve at C_I = 57 μM can be taken as the concentration of saturation. Thus, we estimate that at that point every sulfate group has one methylene blue counterion. The sulfate density on the surface is therefore ${\sigma }_{{\mathrm{SO}}_{4-}}=7.5\text{μmol}{\mathrm{g}}^{-1}$, when the concentration reduction of 45% at C_I is taken into account as well as the volume of the dye solution and the mass of the used fleece samples. A detailed description of the calculation is given in the supporting information.

Furthermore, the average surface area per gram of fleece was determined by measuring fiber diameters from SEM images. Together with the known number of sulfate groups per polymer chain, this yields the coating density to be ρ_Polymer ≈ 0.1 nm⁻². This rough estimate represents a sufficiently high coating density and suggests that a polymer brush coating is formed for lPGS-b-BPh.

2.3 Optimizing the coating parameters for lPGS-b-BPh

Evidently, for potential large-scale applications or industrial processing, the coating procedure should be as short as possible. Thus, we utilized the adsorption of methylene blue to test how fast the coating with lPGS-b-BPh can be done without a reduction of the coating density. In that experiment, coating procedures have been performed by either varying the polymer incubation time or the UV irradiation time (Figure S6). The incubation time could be reduced to 1 min without observing significant changes in the adsorption capacity for methylene blue. The irradiation time could be reduced to 2 min before a reduced polymer attachment or less stable coating was observed. In total, the process may be accelerated to be performed within 3 min.

2.4 Protein binding to coated surfaces is charge dependent

Next, the adsorption of proteins from solution was tested (Figure 5). Two protein species were chosen based on their net charge at physiological pH. Lysozyme as a small and positively charged protein, and human serum albumin (HSA) as overall negatively charged protein. The experiment was performed in analogy to the methylene blue adsorption. The virus concentration was determined using a Bradford assay, and the obtained values were normalized to the mass of the tested fleece samples.

Lysozyme exhibits low adsorption rates to uncoated and PDA or MIdPG coated materials. A high adsorption was observed for lPGS coatings, which is in good agreement to the literature.^{24, 35} Furthermore, the samples coated with lPGS-b-BPh adsorbed more lysozyme than the other two lPGS coated samples.

HSA did not exhibit significant adsorption to any of the tested fleece materials. Electrostatic repulsion between negative charges on HSA and sulfate groups on lPGS prevents the attachment of the protein to lPGS-coated surfaces. In contrast to our results, previously reported quartz crystal microbalance (QCM) experiments detected interactions between PGS coated QCM sensors and HSA.^{24, 36} However, QCM is a highly sensitive technique that can detect even weakest interactions between surface and proteins which cannot be observed in our experimental setting. Isothermal titration calorimetry experiments on the other hand did not detect interactions between dPGS and HSA at physiological pH and ionic strength, which is in agreement to our results.³⁷

Overall, the adsorption of proteins to the coated fleece depends on electrostatic interactions between surface and proteins, and thus on the net charge of the protein. This is an important feature of the surface when it comes to the application of binding virus species, which will be tested in the following experiments.

2.5 Virus capture from solution

After it was successfully demonstrated that small molecules as well as proteins bind to the coated fleece samples in a charge dependent manner, next we tested two virus strains that are known to bind to sulfated polymers (Figure 6).^{18, 22}

In this experiment, a suspension of either HSV-1 or SARS-CoV-2 was added to fleece samples and incubated for 1 h. The virus load of the suspension before and after incubation was determined via a plaque assay on Vero cells. Plaque forming units (PFU) per mL of suspension were calculated to yield the relative virus reduction after incubation.

HSV-1 binds efficiently to lPGS coated fleece, while the uncoated control as well as the PDA and MIdPG coated samples did not show a significant inhibition. lPGS-b-NH₂@PDA as well as lPGS-b-BPh achieved a reduction of plaque forming units of about 90%.

Subsequently, SARS-CoV-2 was tested. In this case, the inhibition with lPGS coated surface was lower than for HSV-1. The reduction of the virus titer was about 45% for lPGS-b-BPh on meltblown polypropylene. This observation is in good agreement to the half maximal inhibitory concentrations (IC50) reported for these virus species in literature. The value for lPGS on HSV-1 in solution was below 0.1 nanomolar,¹⁸ while the value on SARS-CoV-2 was in the micromolar range.²² This corresponds to a stronger inhibition of HSV-1 than SARS-CoV-2 by a factor of around 50,000 and explains well why HSV-1 is bound stronger to lPGS functionalized fleece than SARS-CoV-2.

4.1 Materials

All chemicals and solvents obtained from commercial suppliers were used without further purification unless stated otherwise. Meltblown polypropylene fleece was obtained from commercial surgical masks. Dopamine hydrochloride, Lysozyme from chicken egg white and Human Serum Albumin were obtained from Sigma Aldrich (St. Louis, MO, USA). The Coomassie Plus Bradford Assay Kit was obtained from Thermo Scientific (MA, USA). UV irradiation during the coating procedure was done at 366 nm using a Camag laboratory UV lamp (Muttenz, Switzerland) at a constant distance with a power of 2.3 μW cm⁻².

4.2 Synthesis of polymers

The synthesis of lPGS-b-BPh and MIdPG was performed as described in literature.^{24, 38} The novel polymer lPGS-b-NH₂ was synthesized analogous to the respective non-sulfated copolymer lPG-b-NH₂.³⁹ Complete descriptions of the synthetic procedures are given in the Supporting Information.

4.3 General coating procedure with lPGS-b-BPh

Samples of polypropylene fleece were cut into pieces of about 2 cm × 2 cm.²⁴ The fleece was soaked briefly in ethanol to break the surface tension, then washed thoroughly with water and immediately soaked in an aqueous solution of guanidinium chloride (1.9 ml, 1 m) for 5 min. A stock solution of lPGS-b-BPh was added (0.1 ml, 2 mg ml⁻¹) to obtain a final polymer concentration of 0.1 mg ml⁻¹ and a final salt concentration of 0.95 m. The fleece was incubated for 10 min in the dark, then taken from the solution and irradiated with UV light for 5 min on each side. The sample was washed thoroughly with PBS and with water and then dried at 60°C. For a more elaborate explanation of this coating method, the reader is referred to the cited literature.

4.4 General coating procedure for dopamine

Fleece samples were placed in a fresh solution of dopamine hydrochloride (2 mg ml⁻¹) in MOPS buffer (3 ml, 0.1 m, pH = 7.5) and were incubated under constant shaking for 24 h. The fleece was then washed thoroughly with water and dried at 60°C.

4.5 General coating procedure for MIdPG

Fleece samples were placed in a solution of MIdPG (2 mg ml⁻¹) in methanol (2 ml). The polymerization of MIdPG was initiated by adding MOPS buffer (0.7 ml, 0.1 m, pH = 8.5). After 1 h incubation, the fleece samples were washed thoroughly with water and dried at 60°C.

4.6 Postfunctionalization with lPGS-b-NH₂

Fleece samples coated with either dopamine or MIdPG were placed in a solution of lPGS-b-NH₂ (1 mg ml⁻¹) in MOPS buffer (2 ml, 0.1 m, pH = 8.5).³⁰ The mixture was heated to 80°C for 24 h. Subsequently, the fleece samples were washed thoroughly with water and dried at 60°C.

4.7 Contact angle

Contact angle measurements were performed using the contact angle goniometer OCA20 (Dataphysics Instruments, Filderstadt, Germany). A droplet of water (2 μl) was gently placed on the respective surface and allowed to equilibrate for 10 s. The shape of the droplet was recorded and evaluated using the software SCA20 (Dataphysics Instruments, Filderstadt, Germany). Contact angle values were not determined due to the uneven texture of fleece surfaces.

4.8 Scanning electron microscopy

Fleece samples for SEM imaging were pretreated by sputtering with gold to obtain a conductive layer of at least 5 nm, using a compact coating unit CCU-010 by Safematic GmbH (Bad Ragaz, Switzerland). Images were obtained using an SU8030 field-emission scanning electron microscope (FE-SEM) by Hitachi (Tokyo, Japan). The accelerating voltage (Vac) was set to 20 kV, the current to 20 μA, and the working distance was 21.6 cm.

4.9 X-ray photoelectron spectroscopy

X-ray photoelectron spectroscopy (XPS) spectra were recorded on a Kratos (Manchester, UK) Axis Ultra DLD spectrometer, equipped with a monochromatic Al Kα X-ray source. The spectra were measured in normal emission, and a source-to-sample angle of 60° was used. All spectra were recorded utilizing the fixed analyzer transmission (FAT) mode. The binding energy scale of the instrument was calibrated, following a technical procedure provided by Kratos Analytical Ltd (calibration was performed according to ISO 15472). The spectra were recorded utilizing the instrument's slot and hybrid lens modes. An analysis area of approximately 300 μm × 700 μm was investigated; charge neutralization was applied. For quantification, the survey spectra were measured with a pass energy of 80 electron volt (eV), and the spectra were quantified utilizing the empirical sensitivity factors that were provided by KRATOS (the sensitivity factors were corrected with the transmission function of the spectrometer).

4.10 UV/Vis measurements

UV/Vis spectroscopy was used to analyze the concentrations of methylene blue and Coomassie-stained proteins. Methylene blue was measured at its absorption band maximum at 665 nm, while the Coomassie-protein complex showed an absorption maximum at 595 nm. Calibration curves were obtained from serial dilutions of known concentration for the dye and each protein. Absorbance values were only measured in a concentration range of 0–10 μmol L⁻¹ for methylene blue, and 0–10 μg ml⁻¹ for proteins. Samples with higher concentration were diluted to meet the required concentration range. Spectra were recorded using a Cary 8454 spectrometer (Agilent, CA, USA).

4.11 Methylene blue adsorption

Fleece samples were immersed in 2.5 ml of a methylene blue solution in water at room temperature for 2 h. The initial dye concentration was determined by UV/Vis analysis. A sample of the supernatant was taken after incubation, diluted to meet the concentration range required for UV/Vis measurements, and the concentration was determined. The difference of starting concentration and final concentration was used to determine the amount of methylene blue adsorbed on the fleece. The obtained values were normalized to the mass of the respective fleece sample. In case of the adsorption isotherm, where normalizing to the fleece mass is not possible, all fleece samples were weighed to have the same weight of 8.5 mg.

4.12 Protein adsorption

Protein adsorption experiments were performed analogous to the methylene blue adsorption experiments. Proteins were dissolved in PBS (30 μg ml⁻¹ for lysozyme, 10 μg ml⁻¹ for albumin). The Bradford assay was used for quantification by mixing the respective protein solution with a commercial Bradford assay kit in a 1:1 ratio. UV/Vis spectra were measured 5 min after mixing.

4.13 Adsorption of HSV-1

The GFP-tagged HSV-1^{18, 40} (kindly provided by Dr. Yasushi Kawaguchi, University of Tokyo, Japan) was propagated on Vero cells (ATCC CCL-81) for 2 days. The supernatant containing GFP-tagged HSV-1 virus was collected, and the number of infectious virus particles (virus titer) in the supernatant was assessed using a plaque assay on Vero cells. The number of plaques per well was assessed using an epifluorescence microscope (Zeiss AxioVert 100) using the GFP channel and the virus titer of the initially added solution was calculated and is expressed as plaque-forming units (PFU)/ml.

To determine the adsorption of virus to coated fleece materials, samples of equal weight were disinfected with 75% EtOH for 10 min, washed with 0.5 ml DMEM medium for 15 min, and then incubated with 0.5 ml HSV-1 suspension (approx. 5 × 10⁴ PFU ml⁻¹) for 1 h. The virus in the supernatant was then titrated in a plaque assay on Vero cells.

4.14 Adsorption of SARS-CoV-2

SARS-CoV-2 München (SARS-CoV2M; BetaCoV/Germany/BavPat1/2020) was propagated on Vero E6 cells and quantified in a plaque assay.²² Plaques were made visible by fixing the cells with 4% formaldehyde and staining with 0.75% crystal violet (aqueous solution) to make plaques visible. The experiments were performed in a BSL3 laboratory at the Institut für Virologie, Freie Universität Berlin.

The adsorption of SARS-CoV-2 to coated fleece materials was performed in an equal procedure as for HSV-1.