Silver Coordination Polymers Driven by Adamantoid Blocks for Advanced Antiviral and Antibacterial Biomaterials

The development of sustainable biomaterials and surfaces to prevent the accumulation and proliferation of viruses and bacteria is highly demanded in healthcare areas. This study describes the assembly and full characterization of two new bioactive silver(I) coordination polymers (CPs) formulated as [Ag(aca)(μ-PTA)]n·5nH2O (1) and [Ag2(μ-ada)(μ3-PTA)2]n·4nH2O (2). These products were generated by exploiting a heteroleptic approach based on the use of two different adamantoid building blocks, namely 1,3,5-triaza-7-phosphaadamantane (PTA) and 1-adamantanecarboxylic (Haca) or 1,3-adamantanedicarboxylic (H2ada) acids, resulting in the assembly of 1D (1) and 3D (2). Antiviral, antibacterial, and antifungal properties of the obtained compounds were investigated in detail, followed by their incorporation as bioactive dopants (1 wt %) into hybrid biopolymers based on acid-hydrolyzed starch polymer (AHSP). The resulting materials, formulated as 1@AHSP and 2@AHSP, also featured (i) an exceptional antiviral activity against herpes simplex virus type 1 and human adenovirus (HAd-5) and (ii) a remarkable antibacterial activity against Gram-negative bacteria. Docking experiments, interaction with human serum albumin, mass spectrometry, and antioxidation studies provided insights into the mechanism of antimicrobial action. By reporting these new silver CPs driven by adamantoid building blocks and the derived starch-based materials, this study endows a facile approach to access biopolymers and interfaces capable of preventing and reducing the proliferation of a broad spectrum of different microorganisms, including bacteria, fungi, and viruses.


Supporting Tables
Binding and thermodynamic parameters for the interaction of HSA with the studied compounds at different temperatures S-25 Table S2.The percentage of HSA secondary structure in the absence and presence of 1 and 2 calculated with the CDPro software S-26 Table S3.Selected bond lengths for 1 S-28 Table S4.Selected bond angles for 1 S-28 Table S5.Hydrogen bonding parameters for 1 S-28 Table S6.Selected bond lengths for 2 S-29 Table S7.Selected bond angles for 2 S-29 Table S8.Hydrogen bonding parameters for 2 S-30 Table S9.Physicochemical parameters of tested biomaterials S-31

S14
Despite washing procedures, a residual concentration of Cl -ions is probably present in polymer matrix.It is known that Ag + ions in pure solvent tend to adopt tetrahedral geometry.S1 However, this tendency changes in the presence of heteroleptic ligands due to s-d hybridization, and the formation of very stable [AgL2] + species occurs.S2 Based on these results, we can conclude that a trace amount of chloride anion plays an important role in the solution speciation of these compounds.This particular ion in the solution can be strictly related to the antimicrobial activity of these compounds and their composite materials.Probably, in a cell medium, the formation of [Ag2(PTA)2Cl] + species also occurs and plays a significant role in the mechanism of antimicrobial action.However, more detailed studies are required to confirm the role of this species in the antimicrobial action of compounds 1 and 2. Coordination of chloride ligand can significantly increase lipophilicity, enhance permeation through the bacterial lipid layer, and cause death by bacteria.to the ion source via a 20 µL loop at the beginning of each analysis, using a six-port valve.
Calibration was then performed using a high-precision calibration mode (HPC).The full scan mass spectra were acquired over a mass range of 100-1000 m/z at a spectra rate of 1 Hz.Data was processed using the Data Analysis 5.2 software.shifts are relative to an external H3PO4 (85% aqueous solution).The IR spectra were measured on a BIO-RAD FTS3000MX spectrophotometer in the range from 400 to 4000 cm -1 (KBr discs).The ICP-OES measurements were made on an iCAP 7400 ICP-OES emission spectrometer (model DUO, Thermo Scientific, manufactured in 2018), with sample excitation at the temperature of inductively coupled argon plasma (approx.10,000K).The spectrometer is equipped with an ASX 280 autosampler (Teledyne), a classic pneumatic concentric nebulizer was used for nebulization.Solutions containing Ag + in the range of 0.01 -1.0 mg/L were used for calibration.The emission was recorded for the 328.068 nm and 338.289 nm lines in the axial plasma observation mode, three times for each solution.The instrument limits of detection (LoD) are: LoD (328.068nm)= 0.0026mg/L and LoD (338.289nm)= 0.0022mg/L M2.Thickness and Flatness Measurements of 1@AHSP and 2@AHSP.Biofilms thickness was measured by a digital caliper (Absolute Digimatic) using the square cut samples of 1@AHSP and 2@AHSP.The samples were divided into three different sections and each section was measured (L1) by caliper and cut.Each strap was then measured again (L2).The percent of constriction of both materials was calculated according to the following equation: According to the standard, a disinfectant is considered as having virucidal effectiveness if within the recommended exposure time the titer is reduced by ≥4 log10 steps, corresponding to ≥ 99.99% inactivation).
Calculation of the infective dose TCID50/mL was calculated by the Spearman and Karber method using the following formula: log10TCID50 = x0 -0.5 + Σ r/n wherein x0 = log 10 of the lowest dilution with 100% positive reaction, r = number of positive determinations of lowest dilution step with 100% positive and all higher positive dilution steps, and n = number of determinations for each dilution step, TCID50 = tissue culture infectious dose.
The polymer biofilms doped with 1 and 2 were also tested using the same viruses and medium as above, according to the standard ISO 21702 [Measurement of the antiviral activity on plastics and other non-porous surfaces; ISO, Switzerland, Geneva, 2019].This document specifies proper protocols for measuring antiviral activity on plastics and other non-porous surfaces of antiviral-treated products against specified viruses.For this purpose, a sample of the test material treated with SCDLP (soybean casein digest broth with lecithin and polyoxyethylene sorbitan monooleate) neutralizer was placed in a sterile Petri dish.On the sample prepared in this way, 400 µL of the test virus was applied and covered with a 2×3 cm layer of thermoplastic.
The sample was incubated at 25 °C for 24 h at 90% humidity.After this time, the virus was collected by pipette.The study was conducted in 3 replicates.In parallel, as a control, the whole procedure was repeated using a control disc as a non-virucidal material of the same dimensions as the test sample, also in triplicate.In addition, immediately after virus inoculation onto the control disc, virus titers were tested (3 replicates).
Given that ISO 21702 permits the use of the TCID50 method described in EN 14476 instead of the plaque test, serial dilutions up to 10 -12 of the viruses collected from each disc were prepared and inoculated into 96-wells polystyrene plate.They were observed for the development of viral cytopathic effect (CPE) that is an indicator of virus survival.
M5. Antimicrobial Activity.Determination of MICs for 1 and 2 against bacteria and yeasts was performed as described before.S4 Briefly, the method of serial dilutions in the antibiotic broth (AB) described by Grove and Randall S5   60, 50, 40, 30, 20, 10, 9, 8, 7,     6, 5, 4, 3, 2, and 1 μg mL -1 .Broth sterility and growth controls were also performed.Inoculated plates were then incubated at 37 °C for 24 h.This procedure was carried out three times and with two technical replicates each.The minimum inhibitory concentration (MIC, μg mL -1 ) was defined as the lowest concentration of the compound that fully inhibited the growth of microorganisms.For comparison, the MIC values were normalized for the molar content of silver and are presented in a nmol mL -1 scale.Additionally, activities of AgNO3 and the ligands (PTA, Haca, and H2adc) were also tested.1mL of saline).The obtained suspensions were diluted 1:100 in tryptic soy broth (TSB), and 0.5 mL aliquots of the dilution were placed onto the surface of square-shaped starch polymers (3×3 cm, 1 mm thick).Four squares were used for each strain and compound: two blank ones (T0 and T24, corresponding to 0 and 24 h, respectively), and two (duplicate) compound-loaded squares, intended for a 24 h incubation (T24).Immediately after inoculation, the squares assigned to the T0 analysis were transferred into Falcon tubes containing 40 mL of D/E

M6. Surface Activity of Starch Polymers
Neutralizing Broth and vortexed for 3 min.The obtained suspensions of microorganisms were then serially diluted in saline, and inoculated onto plates containing tryptic soy agar (TSA).The plates were incubated overnight, and the number of colony forming units (CFUs) on each plate was estimated.Based on the number of CFUs, the number of living bacterial cells detached from each polymer square was calculated.The procedure was carried out in duplicate, and the results were expressed as an arithmetic mean of colony forming units (CFUs) grown on a given medium, which, in turn, were converted into the logarithm or percentage of the microbial reduction.The T24 squares were incubated for 24 h at 37 o C in a humid chamber.After the incubation period, the squares were proceeded as described for T0.
M7. Autoxidation Measurements.The antioxidant behavior of 1, 2, and free ligands (Haca and H2ada) was measured by monitoring the rate of peroxidation in a heterogeneous model system (micelles, Triton X-100).The uptake of dissolved oxygen in micelles during peroxidation was carried out at 37 °C by using a Biological Oxygen Monitor equipped with a Clark-type oxygen electrode.To a glass chamber containing a magnetic stirrer, 5 mL of micelles (pH 7.0) was added and then saturated with oxygen.Next, the electrodes were placed inside the chambers and a peroxidation was initiated by injection of the AAPH solution (final concentration: 10 mM).After 10% of oxygen consumption, 10 µL of the solution of studied compounds was added (final concentration was 1 µM).The influence of tested compounds on the peroxidation was determined graphically.Due to the neutrality of 1 to peroxidation of micelles or weak retardation activity of 1, Haca, and H2ada, the kinetic parameters were not determined.

M8. Silver Release and Stability
Studies.Silver concentration and ion release process from the 1@AHSP and 2@AHSP materials were monitored for 24 h in a medium representing simulated physiological conditions (phosphate buffered saline (PBS), pH 7.4, 10 mL).The concentration of silver release from 1@AHSP and 2@AHSP was established by ICP-OES.
Stock solution samples were stored during 24 h at room temperature.Small aliquots (20 µL) of samples were withdrawn every 4 h and replaced with the same volume of PBS.After the analysis, the polymer samples were dried, weighted, and analyzed by IR spectroscopy.M9.

M9. Interaction of 1 and 2 with Human Serum Albumin. Human serum albumin was
purchased from Sigma-Aldrich Co. (Catalog No. A3782, ≥99% purity).The protein stock solution with a concentration 2 μM was prepared in PBS buffer (pH=7.40).Metal complexes stock solutions with a concentration 833 μM were prepared by dissolution in double distilled water.Human serum albumin concentration was determined spectrophotometrically using ε280 = 36600M -1 cm -1S6 .Fluorescence measurements were performed on Jasco 8200 spectrofluorometer using a 1.0 cm quartz cell.The excitation/emission slits were set at 5.0 nm.Spectra were recorded in the range of 300 -500 nm at the excitation wavelength of 280 nm.
PBS buffer was taken as a blank.The fluorescence studies were done by titration of 2 ml HSA with 10 μl appropriate silver complex.The samples were measured after 5 mins incubation at 298K and 310K.The final spectra are an average of 3 scans.Circular dichroism measurements were performed on Jasco J-1500 CD spectrometer using 0.1 cm quartz cell.A bandwidth of 1 nm, a response time of 2 s and scanning speed at 50 nm/min were fixed to obtain final spectra as an average of 3 scans.The final concentration of the protein was 2 μM and molar ratio [HSA]:[complex] = 1:0, 1:10 and 1:20.Spectra were recorded after 24h incubation at 310K in the range of 200-250nm.
Interaction of 1 and 2 with Human Serum Albumin.Fluorescence spectroscopy is one of the most effective methods to investigate the interaction of biomolecules with small particles and determine the structural changes and binding parameters between them.We used this technique to determine the quenching mechanism of HSA fluorescence by 1 and 2 and calculate the binding and thermodynamic parameters of the systems.Human serum albumin shows a strong fluorescence signal at 335.5 nm after excitation at 280 nm.Under tested conditions, the analysed compounds do not show any fluorescence signal.Titration with 1 and 2 causes a slight quenching of the protein fluorescence and blueshift of the emission peak (2 nm).This behavior indicates the interaction between human serum albumin and metal compounds and some conformational changes of the protein.
To determine the quenching parameters of the systems, number of binding sites and association constant between HSA and the ligands, the equations 1-3 were used.All data are collected in Table S1.From the data collected in Table S1 and Figures S25-S26 it is clear that the binding constants (Ka) decrease with temperature raising when the protein interacts with both compounds.Such relationships indicate that 1 and 2 form unstable complexes that undergo partial decomposition at higher temperatures.The n values approximate close to 1 in both systems indicate that only one binding site in protein is accessible for the complex.The binding constants of the systems are in the order of 10 2 -10 3 M − 1 and are comparable with the value of HSAcisplatin (Ka = 8.52×10 2 M − 1 ).S10 For comparison, the association constants of the ruthenium complex tested in clinical trials KP1339 is Ka = 4.57×10 4 M − 1 .S11 Using the Equations 4-6 and the Ross & Subramanian theory the thermodynamic parameters and acting forces between 1 or 2 and HSA were determined: Eq. 4 In the equations, ∆H 0 , ∆S 0 , ∆G 0 are the enthalpy, free energy and entropy, respectively, while Ka is the binding constant at the corresponding temperature, T stands for the experimental temperature [K], and R is the gas constant.
In general, four types of bonding forces between small molecules and biological macromolecules may be present in the interaction.These include electrostatic (Coulomb) forces, hydrophobic and ionic interactions, as well as Van der Waals forces and hydrogen bonds.Furthermore, contribution of metal ions in the interaction is also possible in the case of coordination compounds.The obtained data show that Van der Waals interactions and hydrogen bonds as the main forces in the HSA-1 and HSA-2 systems because of the both negative ∆H 0 and ∆S 0 .Additionally, negative ∆G 0 values prove spontaneous interaction between molecules.
Table S1.Binding and thermodynamic parameters for the interaction of HSA with the studied compounds at different temperatures Circular dichroism (CD) spectroscopy is a widely used technique for studying protein conformations.We used the CD technique to determine the effect of 1 and 2 on the HSA secondary structure.The CD spectrum in the far UV CD region (190−250 nm) is characterised by negative bands at 209 and 221 nm, assigned to its α-helical structure and π→π* and n→π* transitions.S12 Under the experimental conditions, 1 and 2 do not generate a CD signal.Human serum albumin is mostly an α-helical protein.The α-helical content of free HSA at 209 nm was found to be 66.5%.A decrease in α-helicity to 64.4/64.3%without changes in bands position was observed after addition of 1 or 2 at the molar ratio of [HSA]/[Ag] = 1:20.These changes arise from the interaction between serum albumin and silver compounds.The obtained results were analysed by the CDPro software and are summarized in Table S2.CD spectra are shown in Figure S27, SI.HSA-2 1:20 64.3 (±0.12) 2.1 (±0.12) 11.6 (±0.12) 21.3 (±0.12)M10.Computational Studies.Computational docking of diverse moieties originating from the speciation of the investigated coordination polymers was carried out according to the following procedure.Structures of ligands (1,3,5-triaza-7-phosphaadamantane, 1adamantanecarboxylic acid) and fragments of the coordination network were generated from the experimental X-ray data of 1 using a CCDC Mercury 2020.1 program.The compounds of the AgL2 type were linearized to represent the s-d hybridization of the silver atom.The structures of two macromolecules chosen for this study, namely the M2 proton channel of influenza A virus and the p7 cationic channel of hepatitis C virus, were taken from the Protein Data Bank S13 (deposits 6BKK and 2M6X,respectively).The protein and tentative ligand structures were processed using AutoDock Tools component of the MGLTools 1.5.7 suite S14 polar hydrogen atoms were added, Gasteiger atomic charges and AutoDock4 atom types were assigned.Detection of rotatable bonds was carried out for the ligands.The docking was performed with the AutoDock Vina 1.1.2program S15 requesting up to 12 binding modes to be generated.The search exhaustiveness parameter was set to 18.The search boxes were centered on the experimental coordinates of the amantadine ligand in case of 6BKK, and on the barycenter of the protein in case of 2M6X.The respective cubic box sizes were 30 and 60 Å.
The results were analyzed with the help of the AutoDock Tools program.Additional docking runs used amantadine as the ligand, so that the docking accuracy could be assessed against the experimental location of the adamantane-like binding site in 6BKK.
Then the squares were transferred to a desiccator filled with a 100 mL saturated solution of KCl.The potassium chloride was used to provide ca.84% humidity.The biofilms were kept under this condition in a desiccator until the constant weight was observed.Moisture uptake was calculated as follows: %moisture uptake = [(W2 -W1)/W1]×100%.S16

Figure S1 .
Figure S1.PXRD patterns of compound 1: calculated from the single-crystal X-ray data (blue), experimental for bulk product (red).

Figure S2 .
Figure S2.PXRD patterns of compound 2: calculated from the single-crystal X-ray data (blue), experimental for bulk product (red).

Figure S7 .
Figure S7.Low resolution full scan mass spectrum acquired in the ESI positive mode for 1 in aqueous medium.

Figure S8 .
Figure S8.Low resolution full scan mass spectrum acquired in the ESI positive mode for 2 in aqueous medium.

Figure S12 .
Figure S12.FTIR spectrum of the stock corn starch.

Figure S16 .
Figure S16.SEM images of 2@AHSP showing the morphology of porous flat surface (a,c) with some visible recesses (e) as well as distribution of compound 2 (b,d,f).

Figure S19 .
Figure S19.HR full scan mass spectra obtained in the ESI positive mode for solutions after immersing biocomposites for 1 h in water: a) 1@AHSP; b) 2@AHSP.The group of peaks centered at m/z 564.9316 display an isotopic distribution pattern that agrees well with that calculated for [Ag2(C6H12N3P)2Cl] + as shown in c).

Figure S20 .
Figure S20.TGA data of the starting AHSP.

Figure S24 .
Figure S24.Oxygen uptake for autoxidation of micellar system initiated with AAPH at 37 °C, pH 7.0.Autoxidation in the absence (AAPH, blank) or presence of tested compounds (1 µM): Haca, H2ada, 1, 2. Final concentration: LinMe 2.74 mM, Triton X-100 8 mM.Final results are determined based on four series of results obtained in two independent experiments (each experiment provided two series of results).Presented data are the most similar to the arithmetic average of 4 series of measurement data.The standard deviation was calculated and is 5-15%; however, the error bars are not included in the chart for clarity.
Lines and Media.A549 -human lung carcinoma (ATCC, No CCL-185 TM ) and HeLa -human cervix carcinoma (ATCC, No CCL-2 TM ) cell lines from American Type Culture Collection-ATCC (Rockville, MD, USA) were used for determination of virucidal properties.Dulbecco's Modified Eagle's Medium -DMEM (Lonza, Basel, Switzerland) served as substrate and was supplemented with fetal bovine serum (FBS, 10%) and L-glutamine (4 mM) (Biological Industries, Kibbutz Beit-Haemek, Israel), 100 U/mL of penicillin, and 100 µg/L of streptomycin (Sigma-Aldrich, Munich, Germany).M4.Antiviral Assays.The solutions of 1 and 2 in 0.3 mg/mL were tested using herpes simplex virus type 1 (HSV-1 -ATCC ® VR-1493 ™ ) and human adenovirus 5 (HAdv-5, virus-strain Adenoid 75, ATCC VR-5 TM ).The assays were performed according to the standard EN 14476 [Chemical Disinfectants and Antiseptics − Quantitative Suspension Test for the Evaluation of Virucidal Activity in the Medical Area − Test Method and Requirements (Phase2/Step 1); European Committee for Standardization: Brussels, Belgium, 2013].This standard describes a quantitative suspension test for the evaluation of virucidal activity in the medical area (Phase 2/Step 1).One part by volume of test virus suspension (0.1 mL of 1×10 12 TCID50 HAd-36 virusor HSV-1), one part by volume of interfering substance (0.1 mL of PBS), and eight parts by volume of disinfectant (investigated substances in a concentration 0.3 mg/mL) were mixed.At specified contact times (60 min), aliquots were taken, and serial dilutions from 10 -1 to 10 -12 of each mixture were prepared.Then, 50 µL of each dilution was added to the microtiter plate containing a monolayer of confluent A549 or HeLa cells.Each dilution was tested in eight repeats.The plates were observed daily for up to 4 days for the development of viral cytopathic effect (CPE), using an inverted microscope (Olympus Corp., Hamburg, Germany; Axio Observer, Carl Zeiss MicroImaging GmbH).Then, a residual infectivity was determined.
and F are the relative fluorescence intensities of protein in the absence and presence of the quencher, respectively, Ksv is the Stern-Volmer quenching constant, Kq is the quenching rate constant, τ refers to an average lifetime of the protein (10 ns), Ka is the binding constant, n is the number of binding sites, and [Q] stands for the quencher concentration.The calculated Stern-Volmer quenching constants (Ksv) are directly correlated with temperature.The values are 3.09×10 3 and 4.04×10 3 M − 1 for 1 and 2.91×10 3 and 3.54×10 3 M − 1 for 2 at 298 and 310 K, respectively.These point at dynamic quenching mechanisms of the protein which involve fluorophore and a quencher collisions during transient existence of the excited state.But taking into account the maximum scatter collision quenching constant of various quenchers with the biopolymer (Kq = 2.0×10 10 M − 1 s − 1 ) this indicates the contribution of static quenching mechanism of the protein fluorescence by the compounds and refers to the formation of HSA-1 or HSA-2 complexes (appropriate 1 and 2 values of 3.09, 4.04 and 2.91, 3.54 are in the order of 10 11 M − 1 s -1 ).S9

Table S2 .
The percentage of HSA secondary structure in the absence and presence of 1 and 2

Table S9 .
Physicochemical parameters of tested biomaterials.