Cu(II) complexes using acylhydrazones or cyclen for biocidal antifouling coatings

Synthesis

A water-soluble polyacylhydrazone (DiLevDEG/ADH) was synthesised from dilevulinoyl diethylene glycol (DiLevDEG) and adipic acid dihydrazide (ADH) as described previously [12]. Simple acyldihydrazone test compounds were synthesised following the procedure of Zha and You [13] as described previously [12].

Copper chelation

The polyacylhydrazone copper complex (Cu(II)-DiLevDEG/ADH) was prepared by adding an aliquot of DiLevDEG/ADH solution (∼50 % w/v; 56 µL) to Cu(NO₃)₂·3H₂O (32 mg, 0.13 mmol) dissolved in water (1 mL). The UV–Vis spectra of DiLevDEG/ADH, the Cu(II)-DiLevDEG/ADH complex, and Cu(NO₃)₂ (50 mM, aq) were recorded as dilute solutions.

Paint/coating formulation

A 50 % w/v aqueous solution of DiLevDEG/ADH was formulated in a commercial acrylic paint preparation as described in Daines et al. [12]: 35 % w/w of the resin was substituted by DiLevDEG/ADH to give DAp/acr. For comparison, standard acrylic paint was prepared using a conventional recipe [12]. Following formulation and drying on a surface the paints were exposed to Cu(II) and tested for their Cu(II)-retention as well as impact on bacterial growth.

Antibacterial testing

The effects of the DAp/acr and acrylic paints (±Cu(II)) on the growth of E. coli NZRM 3647 were determined. To wells in a 24-well plate (JET BIOFIL^®), 250 µL of DAp/acr paint, acrylic paint, or a commercial marine paint (Altex Yacht and Boat Paint, Ablative Antifouling No. 5; 40–50 % w/w Cu₂O) was added, and the plate was agitated while drying, to coat the well bottoms evenly. The paints were cured at 25 °C while shaking gently for 72 h to dry, then washed with water (3 × 500 µL) and air-dried. An aqueous solution of Cu(NO₃)₂ (50 mM; 500 µL/well) was added to a row of wells coated with the acrylic paint (“Acrylic + Cu(II)”) and a row of wells coated with the DAp/acr paint (“DAp/acr + Cu(II)”). Water (500 µL/well) was added to a row of uncoated wells and to rows of wells coated with the DAp/acr, acrylic and marine paints. After 24 h, all wells were washed with water (3 × 500 µL), air-dried, and sterilised with UV light for 20 min. The approximate surface area of the paint in each well was measured. An culture of E. coli NZRM 3647 was prepared overnight by inoculating nutrient broth (3 mL) with a colony from a nutrient agar plate and incubating for 16 h at 37 °C with shaking. This culture was diluted 1:1 v/v with nutrient broth, and an aliquot of the diluted culture (500 µL; 1.3 × 10⁸ CFU/mL) was added to each well. The plate was incubated at 37 °C, and the OD600 of the culture in each well was measured at t = 0, 2, 4, 6, 8, 10, and 24 h to generate growth curves. The experiment was repeated independently three times.

Quantification of Cu(II) leaching

The wells in a 24-well plate were coated with DAp/acr paint, acrylic paint, or a commercial marine paint as described above. Nutrient broth (500 µL) was added to each well and the plate was incubated at 37 °C for 24 h. The supernatant was removed from each well, diluted 10,000× with nitric acid (3 % v/v aq), and analysed by ICP-MS to quantify the copper that had leached into the nutrient broth.

Assessment of DiLevDEG/ADH polyacylhydrazone leaching

The bottom of a well in a 24-well plate was coated evenly with 260 mg of the DAp/acr paint and was cured at 50 °C for 24 h. The cured paint was washed with water (3×), air-dried and deuterium oxide (500 µL) was added and left for 24 h. The ¹³C NMR spectrum of the resulting deuterium oxide solution was recorded and compared to the spectrum of DiLevDEG/ADH polyacylhydrazone.

Functionalisation of silica with cyclen

Two methods were utilised for the functionalisation of silica with cyclen (1,4,7,10-tetraazacyclododecane), as shown in Scheme 1. The difference in these two methods is the order in which the particle-ligand system is built, but is ultimately intended to produce identical products. Detailed methodology is provided in the Supplementary Information.

Scheme 1:

Two silica functionalisation methods. Method 1: reaction between GLYMO and the silanol groups of silica (SiO₂) to produce GLYMO-functionalised silica (GmSiO₂). Then, the reaction between GmSiO₂ and cyclen to produce cyclen-GLYMO-silica (CnGmSiO₂). Method 2: GLYMO and cyclen react first to produce cyclen-GLYMO (CnGm), and then CnGm reacts with silica to produce CnGmSiO₂.

For Method 1, four procedures were trialled for synthesis of GLYMO-silica (CnGmSiO₂) as summarised in Table 1. Subsequent functionalisation with cyclen was modelled on the procedure of Gros et al. [14]. Briefly, GmSiO₂ was end-capped by suspending in trimethylchlorosilane (TMCS; 92 mmol) and heating at reflux temperature for 4.25 h under argon. The silica product was vacuum filtered, washed with water to neutral pH, and dried for 18 h under high vacuum. Next, an aqueous solution of cyclen (59 mL, 3.3 mmol) in water was added to the end-capped GmSiO₂ and the suspension heated at 80 °C for 48 h with occasional gentle swirling. The resulting product was filtered to remove excess cyclen and was washed with dilute hydrochloric acid (0.1 M; 2 × 10 mL), then water (until the filtrate no longer turned a dark blue colour upon the addition of 50 mM CuSO₄), and hot methanol.

For Method 2, six procedures were trialled for the synthesis of cyclen-GLYMO (Cn(Gm)_n) as summarised in Table 2. Immobilisation of Cn(Gm)_n on to silica was done in the presence or absence of Cu(NO₃)₂ to produce preloaded Cu(II)-CnGmSiO₂ and free, uncoordinated CnGmSiO₂ (Table 2). A solution of Cu(NO₃)₂ in methanol was added to the crude Cn(Gm)_n for copper incorporation into the ligand. Then, methoxysilane groups were hydrolysed following the procedure of Lu [15]. Briefly, methanol, aqueous ammonium hydroxide (28 % v/v) and silica (26.68 g) were added to the reaction mixture, and stirred and heated for 1 h at 60 °C. The mixture was concentrated under vacuum and the royal blue Cu(II)-CnGmSiO₂ product was washed by Soxhlet extraction with water for 24 h, then dried. Uncoordinated CnGmSiO₂ was prepared in the same way, but without addition of copper(II) nitrate.

Incorporation of CnGmSiO₂ into coatings

A two-pack epoxy was prepared by mixing a commercial hardener (Ancamine^® 2459 Curing Agent; Air Products and Chemicals, Inc., PA, USA) and a commercial epoxy resin (Epikote™ 235; Resolution Performance Products, Columbus, OH, USA) in accordance with the manufacturer specifications: 0.563 g Ancamine^® 2459/g Epikote™ 235. Immediately following manual mixing of the two components, one coat of the viscous, dark yellow epoxy resin was applied to a single side of a black vinyl chloride/acetate copolymer (black scrub test panel, Leneta). An inverted funnel was then used to add silica to a defined surface area of the still-uncured resin so that, at minimum, one layer of silica covered the resin. The coating was allowed to cure at room temperature overnight, and, the following day, the procedure was repeated for the second side. After allowing the second side to cure at room temperature overnight, the silica/epoxy (SiO₂/epx)-type samples were brushed, washed with water, and dried with compressed air, and squares (23 mm × 23 mm) coated on both sides were cut from the material. These squares were tested immediately for bacterial adherence. The four silica samples prepared for testing were as follows: 1) CnGmSiO₂ (free ligand), 2) CnGmSiO₂ + Cu(II) (post-loaded), 3) Cu(II)-CnGmSiO₂ (pre-loaded), and 4) unfunctionalised SiO₂. To post-load CnGmSiO₂, the squares coated on both sides with CnGmSiO₂ were submerged in an aqueous solution of an excess of Cu(NO₃)₂ (50 mM; 16.5 mL) for 24 h, washed with water, dried with compressed air, and tested immediately for bacterial adherence. Images and the elemental composition of the SiO₂/epx-type coatings were obtained by SEM-EDS.

Bacterial adherence assay

The ability of the coatings to deter the adherence of bacteria was assessed by determining the attachment of E. coli NZRM 3647. An uncoated sample square and sample squares coated with the commercial epoxy resin and marine paint were also tested. An overnight culture was prepared by inoculating nutrient broth (3 mL) with a colony of E. coli NZRM 3647 from a nutrient agar plate and incubating for 16 h at 37 °C with shaking. The squares were sterilised with UV light for 20 min per side in a laminar flow hood, and then each of the sterilised squares were submerged in overnight culture diluted with M63 minimal medium to OD600 0.05 (final volume 16.5 mL; 5 ± 2 (×10⁷) CFU/mL). The diluted cultures were incubated 24 h at 37 °C without shaking. Following incubation, the OD600 of each culture was measured, and the squares were transferred to sterile saline (20 mL) for 5 min to remove planktonic bacteria. A pipet was used to rinse both sides of the squares with sterile saline (500 µL/side) before transferring the squares to tubes containing sterile solutions of 0.05 % v/v Tween 20 in saline (final volume 20.0401 mL). These solutions were vortexed 2 min at maximum speed and sonicated 8 s at 40 % amplitude to detach bacteria from the squares. Serial dilutions (10×) of the solutions were performed to count colonies by the drop plate method. The experiment was independently repeated three times.

Measurement of copper leaching

The concentrations of copper leachate from CnGmSiO₂ + Cu(II)/epx, Cu(II)-CnGmSiO₂/epx, and the marine paint were determined by incubating the test squares in M63 minimal medium statically for 24 h at 37 °C. This experiment was conducted with and without E. coli in order to determine if the presence of bacteria affected copper-leaching. After 24 h incubation of the samples with E. coli, each culture was filter-sterilised and centrifuged, and the supernatant was isolated for analysis. All solutions were diluted, as needed, with dilute nitric acid for copper quantification by ICP-MS.

Cyclen modified silica

Silica is a low-cost, common additive and extender that can enhance multiple properties of coatings and is found in several coating types, including antifouling coatings. Mesoporous silica nanoparticles functionalised with quaternary ammonium salts and formulated into a coating, have been found to reduce biofouling coverage from 39 % to below 10 % [29]. These results suggested that modified silica particles are rich with potential to serve as functional fillers in antifouling coatings. The known positive influence of silica on the physical characteristics of the coatings, together with their existing demonstrated antifouling characteristics, led us to consider this material as a solid support for the cyclen ligands and/or Cu-complexes. The incorporation of cyclen in a coating through the modification of silica, was considered as a flexible strategy since copper loading could, in theory, be undertaken before or after formulation into a surface coating. In addition, the inclusion of aza-macrocycles into surface coatings using silica particles should reduce leaching of the loaded copper into seawater (due to the macrocycle’s high copper affinity), while still presenting copper ions at the surface of the coating at its coating-liquid interface [30].

Two methods were trialled for functionalisation of silica with the cyclen ligand; method 1 in which the reaction between GLYMO and the silanol groups of silica gives GmSiO₂, and then the reaction between GmSiO₂ and cyclen produces CnGmSiO₂; method 2 where GLYMO and cyclen react first to produce CnGm, and then CnGm reacts with silica to produce CnGmSiO₂. All attempts to synthesise CnGmSiO₂ via method 1 were unsuccessful. Several procedures for the synthesis of GmSiO₂ were evaluated and the outcomes are summarised in Table 4. Procedure 1 yielded mostly unreacted GLYMO with almost no functionalisation of the silica. The other procedures yielded GmSiO₂ with varying degrees of functionalisation, but only procedure 4 showed evidence of silica modification (appearance of the epoxy ring C–O stretch peak at 908–920 cm⁻¹ in the IR spectrum; Fig. S1, Table S6). Unfortunately, attempts to end-cap GmSiO₂ and subsequent synthesis of CnGmSiO₂ were unsuccessful, as determined by IR spectroscopy.

Turning to the synthesis of CnGmSiO₂ via method 2, several procedures were trialled to synthesise Cn(Gm)_n (Scheme 2, Table 2). Most of the procedures trialled were unsuccessful and only the reaction of GLYMO with cyclen (1.1 eq) in anhydrous chloroform at room temperature under dry conditions and an argon atmosphere (Table 2, procedure 6), showed the reaction go to completion. This was evident in the ¹H NMR spectra of the recovered product (Fig. 6) from the disappearance of the resonance associate with the H2 proton of the epoxide at δ _H 3.14. The presence of the CnGm product was confirmed by HRMS (m/z calcd for C₁₇H₄₁N₄O₅Si [CnGm + H]⁺ 409.2846; found 409.2843). The regioselectivity of the ring-opening reaction was determined through 2-D NMR experiments (HSQC; (¹H, ¹³C)- and (¹H, ¹⁵N)-HMBC; COSY) (Figs. S2 and S3).

Scheme 2:

Synthesis of Cn(Gm)_n.

Fig. 6:

¹H NMR spectra (CDCl₃, δ _H 4.00–3.00 ppm) of GLYMO and the samples at 3, 5, 6, and 10 days from the Cn(Gm)_n procedure 6 reaction mixture, showing the change in the spectrum with the progression of the ring-opening reaction. H is the proton responsible for the indicated peak.

The CnGm product was then taken for synthesis of CnGmSiO₂ and pre-loaded Cu(II)-CnGmSiO₂ (Scheme 3). To create the preloaded Cu(II)-CnGmSiO_2, first excess of copper(II) nitrate (aq, light blue solution) was added to the CnGm reaction mixture in chloroform and the complexation was evidenced by the colour change of the reaction mixture from colourless to dark blue. Initial attempts to synthesise Cu(II)-CnGmSiO₂ using 0.05 % v/v aqueous ammonium hydroxide at room temperature to hydrolyse the GLYMO methoxy groups followed by addition of silica, concentration of the reaction mixture under vacuum while heating, and Soxhlet extraction yielded a royal blue Cu(II)-CnGmSiO₂ product. CHN analysis of the product revealed a percent functionalisation of the silica was only 32 %. We hypothesised that this low degree of functionalisation was due, at least partly, to incomplete hydrolysis of the GLYMO methoxy groups which would preclude subsequent silica modification. Monitoring the hydrolysis in 0.05 % v/v aqueous ammonium hydroxide at room temperature showed that while 78 % of the methoxy groups were hydrolysed within 0.2 h, hydrolysis was still incomplete after 4.7 h (Fig. 7).

Scheme 3:

In order to synthesise Cu(II)-CnGmSiO₂, Cu(II) may be added first to produce the Cu(II)-CnGm complex (1). Then, hydrolysis of the methoxysilane groups (2) is followed by the addition of silica (3) and heating and concentration (4) to drive the dehydration/condensation reaction (5). Finally, the ±Cu(II)-CnGmSiO₂ product is washed via Soxhlet extraction (6).

Fig. 7:

¹H NMR spectra (CDCl₃) of GLYMO (δ _H 3.61–3.43 ppm), following methoxy group hydrolysis over time.

Increasing the percentage of ammonium hydroxide to 1 % v/v (CD₃OD, 3.7 % v/v H₂O, 11 % v/v CHCl₃) and heating at 60 °C gave almost complete hydrolysis of the methoxy groups in 1 h. Subsequent addition of silica and concentration of the mixture under vacuum promoted the dehydration/condensation reactions, and Soxhlet extraction of the product yielded dark blue Cu(II)-CnGmSiO₂. The degree of functionalisation of this silica was 73 %. A similar degree of functionalisation was observed for the two batches of CnGmSiO₂, 75 and 88 % functionalisation respectively.

Measurement of the copper content of the two pre-loaded batches of Cu(II)-CnGmSiO₂ products showed that the one with higher degree of functionalisation had 2.60 % copper, compared with 0.48 % for the one with a lower degree of functionalisation. After exposure of the CnGmSiO₂ products to aqueous Cu(NO₃)₂ solutions for 24 h (post-loading with Cu(II)) the measured copper contents were 0.96 % w/w and 1.02 % w/w, respectively. Previous studies found that harsh conditions (2–3 h refluxing in ethanol with an excess of Cu(II)) were necessary for complete metallation of cyclam-modified silica [32], [33], [34], which may account for the lower loading obtained for post-loading with Cu(II) compared with pre-loaded Cu(II)-CnGmSiO₂.

These functionalised silica products were incorporated into coatings using a two-pack epoxy system, composed of an epoxy resin and a commercial hardener. Resins containing CnGmSiO₂ were post-loaded with Cu(II) by submerging in an aqueous solution containing an excess of Cu(NO₃)₂ for 24 h, washed with water and dried. Magnified images of the SiO₂/epx-type coatings scanning electron microscopy revealed consistent, but inhomogeneous, surface coverage with the functionalised silica products and photographs of the prepared squares are shown in the top left-hand corner (Fig. 8). Analysis of the elemental compositions of the coatings obtained via scanning electron microscopy/energy dispersive X-ray spectroscopy (SEM-EDS) showed the presence of the copper, although the results varied compared to the ICP-MS analysis of silica products due to the inhomogeneity of the surfaces: (1.02 % w/w (ICP-MS) vs. 1.0 % (SEM-EDS) for CnGmSiO₂ + Cu(II)/epx and 2.60 % w/w (ICP-MS) vs. 1.4 % w/w (SEM-EDS) for Cu(II)-CnGmSiO₂/epx copper (Tables S7 and S8).

Fig. 8:

SEM secondary electron images at 200× magnification of the SiO₂/epx-type squares to be tested for bacterial adherence (HV 20 kV, spot size 4, WD 10 mm, LFD). The coatings include SiO₂/epx, Cu(II)-CnGmSiO₂/epx (preloaded), CnGmSiO₂/epx, and CnGmSiO₂ + Cu(II)/epx (postloaded), and pictures of these coatings are in the top left corner of each SEM image.

The ability of these coatings to deter the adherence of bacteria was assessed by determining the attachment of E. coli (NZRM 3647). The results of the assay showed that there was no deterrence of bacterial adhesion on any of the functionalised silica coatings (Fig. 9), including the biocide-containing CnGmSiO₂ + Cu(II)/epx and Cu(II)-CnGmSiO₂/epx coatings. Interestingly, Cu(II)-CnGmSiO₂/epx had the greatest number of viable, adherent bacteria, and this result was statistically different from the adherence results for all of the other coatings but SiO₂/epx. The number of bacteria attached to Cu(II)-CnGmSiO₂/epx was approximately six-fold higher than the number for CnGmSiO₂/epx and CnGmSiO₂ + Cu(II)/epx and approximately twice the number for SiO₂/epx. This was unexpected, given that Cu(II)-CnGmSiO₂ had the highest % w/w copper (2.6 %) out of all of the SiO₂/epx-type coatings. The marine paint gave the lowest CFU/cm² (±SD), at the LOD of the assay, and none of the other coatings had comparable activity.

Fig. 9:

Results of E. coli NZRM 3647 CFU/cm² on the SiO₂/epx-type coatings (pictured right), as well as on uncoated squares, the epoxy resin, and the marine paint. The bars represent the mean (biological replicates = 6 for “Uncoated”, biological replicates = 3 for all others) ± SD. To determine if the means from the data sets were significantly different from each other, a one-way ANOVA with Tukey’s post hoc test was conducted, and the difference was considered to be significant when the p-value was <0.05 (*p < 0.05, **p < 0.01, ***p < 0.001; LOD = 106 CFU/cm²).

In order to investigate possible factors impacting the adherence results, the amount of copper leaching from each copper-containing coating into the medium during the 24 h incubation was measured, both in the presence and absence of E. coli (Table 5). Although the copper concentrations in the marine paint (40–50 % w/w) and Cu(II)-CnGmSiO₂ (2.6 ± 0.3 % w/w) were vastly different, the concentrations of copper leachate in the absence of E. coli were statistically equivalent: 3.3 ± 0.5 ppm (52 ± 8 µM) and 3.6 ± 0.3 ppm (57 ± 5 µM). The concentration of copper leached from CnGmSiO₂ + Cu(II)/epx was slightly lower (2.3 ± 0.1 ppm, 36 ± 2 µM) than the other coatings when bacteria were absent. Interestingly, the presence of bacteria resulted in increased copper-leaching for all samples, and CnGmSiO₂ + Cu(II)/epx leached the most copper (12 ± 2 ppm, 190 ± 30 µM), while the marine paint (7.3 ± 0.4 ppm, 115 ± 6 µM) and Cu(II)-CnGmSiO₂/epx (6.0 ± 0.4 ppm, 94 ± 6 µM) leached similar, lower amounts. This increase in copper-leaching in the presence of E. coli could be due to the uptake of Cu(II) from the surface by bacteria, since cations are attracted to the negatively charged lipopolysaccharide (LPS) in the outer membrane [35, 36].

Copper leachate concentrations were measured because they were thought to be relevant to the bacterial adherence results, but there was not a straightforward relationship between the two. The marine paint and Cu(II)-CnGmSiO₂/epx leached the same amount of copper (94–115 µM, Table 5), but there was an almost 3 Log₁₀ reduction in the number of viable, adherent bacteria when the test square was coated with the marine paint instead of Cu(II)-CnGmSiO₂/epx (Fig. 9).

The mechanisms of the contact killing of bacteria on a copper surface are not fully understood, but there is a consensus in the literature that copper ions released from the surface play a role [9, 37, 38]. It is proposed that, following damage to the bacterial cell membrane, the influx of copper ions into the cell causes oxidative damage by ROS and enzyme inhibition, leading to cell death and DNA degradation [37,39]. In culture, copper toxicity is due to the displacement of iron by copper in iron-sulfur cluster dehydratases, which are essential for the branched-chain biosynthesis of amino acids [40].

The unexpected results of the bacterial adhesion assays may be attributed to the relationship that exists between surface roughness and bacterial colonisation. Rough surfaces have a greater surface area for bacterial adhesion and can provide shelter from shear forces that would otherwise remove the bacteria [41]. However, it was observed that at the appropriate biocide concentration, even if the surface is rough, bacterial adhesion will be deterred. Kozlovsky et al. [42] assessed antibacterial activity (against Streptococcus mutans) of chlorohexidine (CHX) on titanium disks that were either sand-blasted, acid-etched or machine-smoothed. Greater surface area of the rough disk provided more sites for adsorption of CHX resulting in improved antibacterial activity compared to smooth surface [42]. Therefore, in future work we aim to achieve a high enough copper concentration at the silica surface to overcome the positive effect of the rough surface on bacterial growth.