Based on the biological functions of copper, an increasing number of researchers in the field of biomaterials have focused on developing novel copper-containing biomaterials, which exhibit unique properties in protecting the cardiovascular system, promoting endothelialization, angiogenesis, and tissue regeneration, and exerting antibacterial effects.13,2113. P. Wang, Y. Yuan, K. Xu, H. Zhong, Y. Yang, S. Jin, K. Yang, and X. Qi, Bioact. Mater. 6, 916 (2021). https://doi.org/10.1016/j.bioactmat.2020.09.01721. J. Hua, H. You, X. Li, R. You, and L. Ma, Int. J. Biol. Macromol. 158, 275 (2020). https://doi.org/10.1016/j.ijbiomac.2020.04.094 The biological metal coordination interactions between proteins and common transition metal ions (e.g., Cu, Fe, Zn, and Ni) have been mostly studied.2222. L. A. Finney and T. V. O'halloran, Science 300, 931 (2003). https://doi.org/10.1126/science.1085049 Protein–metal interactions—traditionally regarded for roles in metabolic processes—are now known to enhance the performance of certain biogenic materials.2323. E. Degtyar, M. J. Harrington, Y. Politi, and P. Fratzl, Angew. Chem. Int. Ed. 53, 12026 (2014). https://doi.org/10.1002/anie.201404272 SF is a fibrous protein produced by Bombyx mori silkworm. The coordination between Cu(II) and SF has been shown to occur in the dilute SF solution and film utilizing spectrophotometry and electronic paramagnetic resonance.1919. L. Zhou, X. Chen, W. Dai, and Z. Shao, Biopolymers 82, 144 (2006). https://doi.org/10.1002/bip.20472 A previous study showed that the formation of SF/Cu(II) complex may be achieved by the coordination of Cu(II) to an SF macromolecular chain, in which Cu(II) coordinates with both N and O atoms in the SF.19,2119. L. Zhou, X. Chen, W. Dai, and Z. Shao, Biopolymers 82, 144 (2006). https://doi.org/10.1002/bip.2047221. J. Hua, H. You, X. Li, R. You, and L. Ma, Int. J. Biol. Macromol. 158, 275 (2020). https://doi.org/10.1016/j.ijbiomac.2020.04.094In this study, copper ions were loaded to the SF film by a simple immersion processing using the coordination interactions between silk protein and copper ions (Fig. 1). As shown in Fig. 2(a), the content of copper ions on the surface of SF films was determined by EDX. The copper ion content in the SF films was expressed by x-ray fluorescence intensity. The x-ray fluorescence intensity gradually increased with increasing immersion time. After 1 h of adsorption, the amount of copper ions reached the maximum, indicating that the adsorption reached an equilibrium state. Furthermore, the effect of copper sulfate concentration on the kinetics of copper ion adsorption was studied. The results show that the final adsorption amount of copper ions did not increase significantly as the concentration increased. However, the content of copper ions began to decrease after 1 h in the high-concentration copper sulfate solution. Copper sulfate is an acid salt, and the increased concentration of copper sulfate will corrode and hydrolyze the surface of the SF films so that the content of adsorbed copper ions will decrease after a long time of soaking. According to the results of adsorption kinetics, one hour of adsorption was used for subsequent experiments.To examine the copper ions on the surface of the SF films, the elemental and chemical composition of the films were determined by performing the XPS analysis. Figure 3 shows the deconvoluted spectra of each region before and after the adsorption of Cu(II) ions. Besides the peaks of C 1s, N 1s, and O 1s, two other peaks corresponding to Cu 2p1/2 and Cu 2p3/2 occur. It is found that an increased Cu(II)/SF ratio causes both N/C (from 0.271 to 0.084) and O/C (from 0.193 to 0.169) to decrease. The decrease in N/C and O/C ratios reflect a “shielding” of N and O by other atoms. The shielding effect should be caused by the Cu present in the films interacting with N and/or O. Correspondingly, the copper content significantly increased (from 0.36% to 0.56%) as the increase in adsorption time from 2 to 60 min, which is consistent with the EDX results. The binding energies of Cu 2p spectra were fitted with two components, which are Cu0 + Cu1 (2p3/2, 932.4 eV; 2p1/2, 952.3 eV, indistinguishable) and CuII (2p3/2, 934.2 eV; 2p1/2, 953.9 eV), respectively.24,2524. Y. Feng et al., Green Chem. 21, 4319 (2019). https://doi.org/10.1039/C9GC01331H25. R. Feng, Q. Zhu, M. Chu, S. Jia, J. Zhai, H. Wu, P. Wu, and B. Han, Green Chem. 22, 7560 (2020). https://doi.org/10.1039/D0GC03051A There are 18 types of amino acid residues in each SF protein molecule, leading to greater capping and reducing capabilities. The amino acids in the SF protein could reduce most of the Cu(II) ions to Cu(I), leading to the formation of Cu(I) complexes.2626. G. Zhang et al., J. Mater. Chem. C 4, 3540 (2016). https://doi.org/10.1039/C6TC00314A The presence of binding energies at 2p3/2 (932.4 eV, 934.2 eV) and 2p1/2 [952.3 eV, 953.9 eV indicate that Cu(II) and Cu(I) may coexist on the surface of SF films [Fig. 3(b)]. It is known that the catalytic species of copper to decompose RSNOs are either Cu(I) or Cu(II). Cu(I) would be the direct ion to decompose RSNO to generate NO, and Cu(II) and Cu(0) might also make sense to change their state to Cu(I) because of the convenience via redox or corrosion, respectively2727. R. Luo, Y. Liu, H. Yao, L. Jiang, L. Wang, A. Zhao, Y. Weng, and N. Huang, ACS Biomater. Sci. Eng. 1, 771 (2015). https://doi.org/10.1021/acsbiomaterials.5b00131 As mentioned before, Cu(I) or Cu(II) (in the presence of reductant l-GSH) can catalyze the decomposition of S-nitrosothiols. Synthetic RSNOs, such as SNAP, have been extensively studied as in vitro donors and are often used to replicate the physiological environment.88. C. W. Mccarthy, R. J. Guillory, J. Goldman, and M. C. Frost, ACS Appl. Mater. Interfaces 8, 10128 (2016). https://doi.org/10.1021/acsami.6b00145 In this work, the in vitro catalytic degradation of SNAP was evaluated using the Griess reagent. The NO release content from SNAP at different time points is presented in Fig. 4(a) over a 60 min period. It could be seen that the copper-loaded SF films have a considerable NO-generating catalytic activity. In the SF films without copper ions, the amount of NO released is 1.5 μM after five minutes, which is due to the spontaneous decomposition of SNAP, because NO release from RSNOs can also be triggered by their exposure to heat and light.2828. S. Ghalei, A. Mondal, S. Hopkins, P. Singha, R. Devine, and H. Handa, ACS Appl. Mater. Interfaces 12, 53615 (2020). https://doi.org/10.1021/acsami.0c13813 The copper-incorporated samples catalytically generate NO with a content of about 2.3 μM. The relative content of Cu(I)/Cu(II) might directly reflect the effect of NO decomposing from SNAP. The amount of copper ions on the surface of the SF film is related to the adsorption time. To test the effect of the amount of copper ions on the catalytic efficiency, the SF films with different adsorption times were used to catalyze the release of NO, respectively [Fig. 4(b)]. It can be seen from Fig. 2 that the relative copper ion contents on the SF films are 0.22% and 0.36% after 2 min and 60 min of adsorption, respectively. In the presence of SNAP, when the content of copper ions increases from 0.22% to 0.36%, the amount of NO released increases from 1.8 to 2.3 μM. The results further confirmed the NO release catalyzed by copper ions and also provided a strategy for tunable NO release. Furthermore, the effect of the concentration of donor SNAP was detected (Fig. 5). As the concentration of SNAP increases from 13 μM to 65 to 130 μM, the presence of copper increases the amount of NO released. From the point of view of the efficiency of promoting the release of NO, the catalysis is higher at low concentrations of SNAP, which is mainly due to the higher concentration of NO produced by a spontaneous decomposition under high concentrations of SNAP. In the body, normal blood contains μM levels of NO precursors in the form of nitrite and S-nitrosothiols (RSNOs) [e.g., S-nitrosoglutathione (GSNO), S-nitrosocysteine (CysNO), etc.].66. B. K. Oh and M. E. Meyerhoff, J. Am. Chem. Soc. 125, 9552 (2003). https://doi.org/10.1021/ja035775x Therefore, the SNAP concentration with 13 μM was used for subsequent experiments.To detect the effect of degradation on the release of copper ions and the catalytic release of NO, the SF films with copper ions were degraded in the PBS solution and collagenase IA solution. Figure 6(a) shows the degradation rate of the SF films in PBS and collagenase IA solution. Previous studies have shown that SF can be degraded by collagenase IA. After 7 days, the degradation ratio of the SF films in PBS and collagenase was 3% and 5%, respectively, indicating that the presence of collagenase IA promoted the degradation rate of the SF films. However, the release of copper ions was not accelerated by the presence of collagenase IA. During the 21-day degradation time, there was no significant difference in the release of copper ions between the PBS solution and the collagenase IA solution [Fig. 6(b)], which may be due to the strong chelation between copper ions and proteins. During the degradation process, copper ions are slowly released from the surface of the SF film. Although the release rate is faster within 7 days, it still has 0.15% relative content after 7 days in the enzyme solution. Even after 21 days, there is still a small amount of copper ions. The NO catalytic ability of copper ions after being released for different periods was tested. As shown in Fig. 6(c), with the extension of the degradation time, the content of copper ions gradually decreases, and the catalyzed NO gradually decreases. However, there is still a release of 1.7 μM of NO after 7 days of degradation, which is higher than the catalytic ability of the SF film without copper ions (∼1.5 μM). The slow release of copper ions from the SF film surface provides the possibility for sustainable catalytic release for NO.SF has been widely used in biomaterial applications, including tissue engineering and drug release. In particular, SF is a promising candidate for the coating of implants due to low inflammation, excellent mechanical properties, tunable degradation rate, and outstanding drug encapsulation ability.29–3229. X. Wang, H. J. Kim, P. Xu, A. Matsumoto, and D. L. Kaplan, Langmuir 21, 11335 (2005). https://doi.org/10.1021/la051862m30. X. Wang, H. Xiao, A. Daley, O. Rabotyagova, P. Cebe, and D. L. Kaplan, J. Controlled Release 121, 190 (2007). https://doi.org/10.1016/j.jconrel.2007.06.00631. B. Marelli, M. A. Brenckle, D. L. Kaplan, and F. G. Omenetto, Sci. Rep. 6, 1 (2016). https://doi.org/10.1038/srep2526332. O. Bayraktar, Ö Malay, Y. Özgarip, and A. Batıgün, Eur. J. Pharm. Biopharm. 60, 373 (2005). https://doi.org/10.1016/j.ejpb.2005.02.002 Copper ion is an important metal ion in the body and plays an important role in many biological functions. For example, copper ions play important roles in endothelialization and angiogenesis during tissue regeneration.1313. P. Wang, Y. Yuan, K. Xu, H. Zhong, Y. Yang, S. Jin, K. Yang, and X. Qi, Bioact. Mater. 6, 916 (2021). https://doi.org/10.1016/j.bioactmat.2020.09.017 Copper ion has been immobilized on cardiovascular stent surfaces to form in situ NO-generating coatings, showing excellent biocompatibility and efficient prevention of stent thrombosis and restenosis.9,339. F. Zhang, Q. Zhang, X. Li, N. Huang, X. Zha, and Z. Yang, Biomaterials 194, 117 (2019). https://doi.org/10.1016/j.biomaterials.2018.12.02033. X. Li, J. Liu, T. Yang, H. Qiu, L. Lu, Q. Tu, K. Xiong, N. Huang, and Z. Yang, Biomaterials 241, 119904 (2020). https://doi.org/10.1016/j.biomaterials.2020.119904 Copper ions are effectively bound to the surface of SF biomaterials based on protein–metal coordination, which provides a new idea for promoting the bioactivity of SF biomaterials. For example, recent studies have confirmed that SF can be used as a coating for drug-eluting stents.16–1816. W. Xu, K. Yagoshi, T. Asakura, M. Sasaki, and T. Niidome, ACS Appl. Bio Mater. 3, 531 (2020). https://doi.org/10.1021/acsabm.9b0095717. X. Cheng, D. Deng, L. Chen, J. A. Jansen, S. G. C. Leeuwenburgh, and F. Yang, ACS Appl. Mater. Interfaces 12, 12018 (2020). https://doi.org/10.1021/acsami.9b2180818. C. Wang, H. Fang, X. Qi, C. Hang, Y. Sun, Z. Peng, W. Wei, and Y. Wang, Acta Biomater. 91, 99 (2019). https://doi.org/10.1016/j.actbio.2019.04.048 The catalytic release of NO regulated by copper ions provides the feasibility for the construction of bioactive SF coatings. Therefore, copper ion loading provides an option for the construction of bioactive SF biomaterials by virtue of the biological effects of copper ions and the ability to catalyze the release of NO from RSNOs.