3.1. Chemical Structure of Cu-Modified Polymers
A series of copper-modified polymers with different DA:
molar ratios were prepared (
Figure 1). This was done to find the optimal concentration of copper on the surface of Cu-modified polymers. Previously it was established that catecholamines, and in particular pDA, form stable metal-pDA complexes [
32] and the molar ratio of dopamine to metal salt in the solution should be between 2:1 and 3:1 to ensure full incorporation of metal into the pDA [
33]. A higher ratio of copper ions in the incubation solution leads to aggregation of pDa in solution. The polymer samples with different DA:
molar ratios were prepared (
Figure 1).
The results of FTIR analysis of Cu-modified samples are shown in
Figure 2. Spectra of unmodified PVC and PU are typical of these polymers as reported in literature. Their assignation is given in the legend to
Figure 2. In the spectrum of PVC/pDA at 3333 cm
−1 and in the region between 1500–1600 cm
−1 new peaks at 1600 cm
−1 and 1515 cm
−1 appeared, as shown in
Figure 2a. These peaks can be attributed to pDA as reported in [
31]. Although the chemical structure of pDA is still under discussion, it is generally accepted that it contains indole and/or indoline units [
29]. Accordingly, these peaks were assigned to the overlapping C=C stretching vibrations from indole ring and, by different authors, to N–H scissoring vibrations or C=N vibrations thus presenting evidence of pDA formation [
34]. A peak at 333 cm
−1 in the spectrum of Cu-modified PVC was attributed to N–H vibrations. The peaks at 2867 cm
−1, 2850 cm
−1 and 2817 cm
−1 in the FTIR spectrum of non-modified PVC were assigned to C–H stretching vibrations (
Figure 2a, line 1).
The appearance of these peaks confirmed the presence of pDA film on the PVC surface. In the spectrum of Cu-modified PVC (
Figure 2a) the band at 1600 cm
−1 shifted to higher wavenumbers appearing at 1674 cm
−1 and its intensity was decreased. The band at 1515 cm
−1 split into two bands at 1493 cm
−1 and 1562 cm
−1. Such split indicates metal binding with polydopamine [
35]. The FTIR spectra of Cu-modified PU are shown in
Figure 2b. The peaks centred at 3333 cm
−1, 2955 cm
−1, 2874 cm
−1, 1728 cm
−1, 1701 cm
−1, 1531 cm
−1 are peaks originating from the PU. Peaks at 3333 cm
−1, 2954 cm
−1, 2874 cm
−1 are from N–H and C–H groups; the band at 1728 cm
−1 is due to the carbonyl groups in urethane bonds (C=O); the band centred at 1701 cm
−1 is attributed to carbonyl groups in urea bond; the 1531 cm
−1 band was assigned to secondary amide (RCONHR’). The FTIR spectra of PU modified by PDA and Cu did not provide clear evidence of the presence of PDA and copper on the modified PU surface because PU matrix bands overlapped with PDA peaks which have absorbance in the same region.
The characteristic peaks of pDA appeared in the FTIR spectra after modification confirms the successful modification by pDA. The shift peak at 1600 cm
−1 to higher wavenumbers and split of the band at 1515 cm
−1 into two bands at 1491 cm
−1 and 1562 cm
−1 indicates the formation of a Cu-PDA complex as observed previously for Fe-PDA and Zn-PDA complexes [
8,
36].
It was reported that the amount of 13 nmol of
immobilized on a modified PU was sufficient to produce physiologically relevant levels of NO in 2 mL of 10 µM solution of GSNO/GSH in PBS [
15]. The amount of copper in the coatings was estimated using ICP-OES. The results are shown in
Table 1. The highest copper content was found in the samples obtained at DA:
molar ratio 3.5:1, being 3.86 nmol cm
−2 and 6.04 nmol cm
−2 for Cu-modified PVC and PU, respectively. Subsequently, the samples obtained at DA:
molar ratio 3.5:1 were chosen for further investigation.
XPS analysis was performed to detect copper in the coating along with finding the valence states of individual elements to better understand about their chemical surroundings and the mechanism of attachment to the polymer surface. In
Figure 3, the narrow scan spectra of Cu2p, O1s, C1s and N1s of Cu-modified PVC surface are shown. The two peaks observed for the binding energy (BE) of copper with maxima at 955 eV and 935 eV can be attributed to Cu 2p
1/2 orbital and Cu 2p
3/2 orbitals respectively, and peaks around 943 and 964 eV are the satellite peaks, which are common to Cu2p XPS data. The position of the main Cu 2p
3/2 peak ca. 933–934 eV and the satellite peak ca. 942–943 eV is typical of Cu(II) catecholate complexes [
37]. Deconvoluted Cu2p
3/2 narrow scan data for four representative systems are shown in
Figure S1. Cu is present in both of its chemical states of Cu
+ and Cu
+2, with Cu
2O, CuO and Cu(OH)
2. All peaks are assigned with references under NIST database. The XPS data suggest that Cu-pDA was successfully immobilized on the surface of PVC and PU, which are consistent with the results of FTIR and ICP-OES studies. XPS of C1s and N1s also confirm the presence of C–N bonds, i.e., pDA and XPS of O1s indicate the presence of oxygen atoms in the state similar to that in Cu–O bonds. The presence of Cu(I) species in the surface layer is likely to be the result of a redox reaction between pDA and Cu(II).
The atomic fraction of copper element in the surface coating of Cu-modified PVC is 1.5% (
Table 2), which is close to the reported 1.4% on polysulfone and 1.36% on polyvinydienefluoride ultrafiltration membranes [
38]. In case of Cu modified PU, there is much less Cu detected than expected from other experimental results. It could be due to various reasons, but most possible reason could be that majority of the Cu is located below 10 nm of top surface layer, making it relatively unquantifiable using XPS and the other reason could be possible adventitious organic layer on the top.
Using XPS, we evaluated the leaching of copper from Cu-modified polymers to 100 µM GSNO/GSH solution during 1 h (
Table 2). Copper content of PVC/pDA/Cu and PU/pDA/Cu decreased from 1.5 to 1.1 atomic % and from 0.54 to 0.44 atomic %, respectively, after incubation in GSNO/GSH solution. A decrease in surface carbon, nitrogen and oxygen contents should be observed in case of the detachment of PDA. The XPS results show that the carbon content, O/C and N/C atomic ratio of initial PVC/pDA/Cu are 72.4, 0.22 and 0.08, respectively, whereas the carbon content after incubation in the 100 µM GSNO/GSH solution reduced to 67.8 atomic %, and the atomic ratios of O/C and N/C of PVC/pDA/Cu increased to 0.27 and 0.10, which indicated that chemical reactions took place during incubation of the sample in GSNO/GSH. The same trend similar to PVC/pDA/Cu is observed for PU/pDA/Cu. Despite the widespread application of pDA coatings for biomaterials development, its structure is still under discussion. It is known that GSNO exhibits oxidizing properties, so the increase in the O/C ratio could be explained by the surface oxidation, but it is difficult to determine which groups were oxidized. Similarly, the reduction in carbon content could be explained by the surface oxidation, however it requires further investigation. On the other hand, the pDA-coated material used in the catalytic oxidation in the reaction medium containing 30% aqueous hydrogen peroxide exhibited good stability [
39]. Moreover, the Michael addition reaction between pDA and glutathione (GSH) can occur due to the ability of pDA to react with amine- and thiol-containing molecules, which could also explain the increase in O/C and N/C ratios.
Leaching of Cu from Cu-modified samples to PBS during 5 days was evaluated. Cumulative leaching from Cu-modified PVC for 5 days was 1.51 nmol·cm
−2, for Cu-modified PU was equal to 1.41 nmol·cm
−2 (
Figure 4). That is about 39% of Cu content on Cu-modified PVC and 23% on Cu-modified PU.
The normal level of free copper in the human blood serum is 1.6–2.4 μM or 100–150 ppb [
40]. The highest measured Cu leaching level from samples was recorded at 50.7 ppb in five days (
Table 3), which is well below cytotoxic concentrations for mammalian cells. It was reported that the viability of mammalian cells was not affected until the copper concentration reached 1000 ppb [
41]. Our results suggest that copper leaching from the samples should pose no risk to health.
3.2. Measurement of NO Generation Catalyzed by Modified Materials
According to XPS data copper on the surface of Cu-modified polymers exists in the form of
and
. The actual catalyst of GSNO decomposition is
. However, the catalytic mechanism includes Cu redox-cycle between the
and
. The NO generating mechanism involves the decomposition of GSNO by
ions, which partially exist in the sample surface and also form by reduction of
ions by glutathione (GSH) (
Figure 5) [
35]. The traces amounts of
affect the catalysis. These reactions proceeds until GSNO depletion and its conversion to disulfide.
NO generation ability of Cu-containing samples was studied using GSNO as the nitric oxide donor, because GSNO is a compound relatively stable to self-decomposition and has long half-life. The NO release from
S-nitrosoglutathione catalyzed by Cu-polymer samples was found to be within the range (0.43 − 1.4) × 10
−10 (
Figure 6). These data are within the physiological level of NO release by endothelial cells, which is (0.5 − 4) × 10
−10 [
4].
It was reported that GSNO is also able to release NO in the presence of GSH without Cu [
42], but both endogenous and synthetic RSNOs are prone to catalytic decomposition by copper ions [
17,
32]. We have shown that Cu-pDA modified PVC and PU can catalyse NO generation in the presence of GSNO. The highest NO generation rate was exhibited by the Cu-modified PU which correlated with higher copper content in the polymer in comparison with Cu-modified PVC according to ICP-OES analysis.
According to the kinetics of NO generation, about 35% of GSNO was catalytically decomposed by PVC/pDA/Cu and 25% by PU/pDA/Cu within the first hour (
Figure 7).