Study of biocompatibility and antitumor cytotoxic activity in vitro of Zn – 1 %Mg and Zn – 1 %Mg – 0.1 %Ca alloys strengthened by equal angular pressing

Introduction. The biological activity of potential biodegradable zinc-based alloys that are promising for oncoorthopedics was studied in this work. The alloys were processed by equal-channel angular pressing, which made it possible to increase their strength due to microstructure refinement and the ability to provide the functionality of osteosynthesis, fixed due to the metal structure developed on their basis.

Aim. Investigation of effect of equal-channel angular pressing (ECAP) treatment on strength, ductility, degradation rate, biocompatibility in vitro and cytotoxicity against SKOV-3 tumor cells of the Zn – 1 %Mg and Zn – 1 %Mg – 0.1 %Ca alloys.

Materials and methods. The Zn – 1 %Mg and Zn – 1 %Mg – 0.1 %Ca alloys in the initial state and after ECAP were used as objects of study, and blood cells of CBA mice were used as model systems. To assess the hemolytic activity, the samples were incubated with red blood cells for 4 and 24 hours at 37 °C, assessing the relative increase in the level of extracellular hemoglobin compared to the intact control. The cytotoxicity of the alloys was assessed by the change in the level of extracellular lactate dehydrogenase (LDH) activity after 24 hours of incubation with mononuclear white blood cells. The study of antitumor cytotoxic activity was carried out on human ovarian cancer cells of the SKOV-3 line in vitro, assessing their survival after 48 hours of incubation with alloy samples using the LDH test.

Results. As a result of the studies, it was concluded that the studied alloys after ECAP treatment retained their biocompatibility, since there were no signs of hemolysis and cytotoxicity with respect to blood cells. However, contact with samples of all studied alloys in vitro induced a significant inhibition of the metabolic activity of the ovarian cancer cell culture in comparison with the control. Incubation with alloys samples leads to a decrease in cellular activity by an average of 49 % and 59 % for Zn – 1 %Mg and Zn – 1 %Mg – 0.1 %Ca alloys, respectively. The addition of calcium to the composition of the alloy Zn – 1 %Mg contributed to the growth of antitumor cytotoxic activity.

Conclusion. Thus, based on the results of assessing the hemolytic activity and cytotoxicity of the samples, we can conclude that the studied alloys are biocompatible. It was also found that Zn – 1 %Mg and Zn – 1 %Mg – 0.1 %Ca alloys had a pronounced cytotoxic effect on SKOV-3 tumor cells. The obtained data indicate the prospects for the development of a new type of medical devices based on the studied alloys, promising, in particular, for oncoorthopedics: a metal structure developed on their basis can ensure the strength of osteosynthesis, reduce the risk of local recurrence of oncological disease and does not require a second operation to remove the device.

1. Staiger M.P., Pietak A.M., Huadmai J. et al. Magnesium and its alloys as orthopedic biomaterials: A review. Biomaterials 2006;27(9):1728–34. DOI: 10.1016/j.biomaterials.2005.10.003

2. Zhuo X., Wu Y., Ju J. et al. Recent progress of novel biodegradable zinc alloys: from the perspective of strengthening and toughening. J Mater Res Technol 2022;17:244–69. DOI: 10.1016/j.jmrt.2022.01.004

3. Han H.-S., Loffredo S., Jun I. et al. Current status and outlook on the clinical translation of biodegradable metals. Materials Today 2019;23:57–71. DOI: 10.1016/j.mattod.2018.05.018

4. Zhao B., Qiu X., Wang D. et al. Application of bioabsorbable screw fixation for anterior cervical decompression and bone grafting. Clinics 2016;71:320–4. DOI: 10.6061/clinics/2016(06)06

5. Tsakiris V., Tardei C., Clicinschi F.M. Biodegradable Mg alloys for orthopedic implants – A review. J Magnes Alloy 2021;9(6):1884–905. DOI: 10.1016/j.jma.2021.06.024

6. Li S., Ren J., Wang X. et al. Dilemmas and countermeasures of Fe-based biomaterials for next-generation bone implants. J Mater Res Technol 2022;20:2034–50. DOI: 10.1016/j.jmrt.2022.07.089

7. Zhang S., Zhang X., Zhao C. et al. Research on an Mg–Zn alloy as a degradable biomaterial. Acta Biomater 2010;6:626–40. DOI: 10.1016/j.actbio.2009.06.028

8. Witte F., Fischer J., Nellesen J. et al. In vitro and in vivo corrosion measurements of magnesium alloys. Biomaterials 2006;27:1013–8. DOI: 10.1016/j.biomaterials.2005.07.037

9. Zheng Y.F., Gu X.N., Witte F. Biodegradable metals. Mater Sci Eng R Rep 2014;77:1–34. DOI: 10.1016/j.mser.2014.01.001

10. Frederickson C.J., Koh J.-Y., Bush A.I. The neurobiology of zinc in health and disease. Nat Rev Neurosci 2005;6:449–62. DOI: 10.1038/nrn1671

11. Vallee B.L., Falchuk K.H. The biochemical basis of zinc physiology. Physiol Rev 1993;73:79–118. DOI: 10.1152/physrev.1993.73.1.79

12. Fosmire G.J. Zinc toxicity. Am J Clin Nutr 1990;51:225–7. DOI: 10.1093/ajcn/51.2.22

13. Kabir H., Munir K., Wen C., Li Y. Recent research and progress of biodegradable zinc alloys and composites for biomedical applications: Biomechanical and biocorrosion perspectives. Bioact Mater 2021;6(3):836–79. DOI: 10.1016/j.bioactmat.2020.09.013

14. Vojtěch D., Kubásek J., Serák J., Novák P. Mechanical and corrosion properties of newly developed biodegradable Zn-based alloys for bone fixation. Acta Biomater 2011;7:3515–22. DOI: 10.1016/j.actbio.2011.05.008

15. Bowen P.K., Drelich J., Goldman J. Zinc exhibits ideal physiological corrosion behavior for bioabsorbable stents. Adv Mater 2013;25:2577–82. DOI: 10.1002/adma.201300226

16. Bowen P.K., Guillory R.J., Shearier E.R. et al. Metallic zinc exhibits optimal biocompatibility for bioabsorbable endovascular stents. Mater Sci Eng C Mater Biol Appl 2015;56:467–72. DOI: 10.1016/j.msec.2015.07.022

17. Du S., Shen Y., Zheng Y. et al. Systematic in vitro and in vivo study on biodegradable binary Zn–0.2 at% Rare Earth alloys (Zn–RE: Sc, Y, La–Nd, Sm–Lu). Bioact Mater 2023; 24:507–23. DOI: 10.1016/j.bioactmat.2023.01.004

18. Jia B., Yang H., Zhang Z. et al. Biodegradable Zn–Sr alloy for bone regeneration in rat femoral condyle defect model: In vitro and in vivo studies. Bioact Mater 2021;6(6):1588–604. DOI: 10.1016/j.bioactmat.2020.11.007

19. Guo P., Zhu X., Yang L. et al. Ultrafineand uniform-grained biodegradable Zn–0.5Mn alloy: Grain refinement mechanism, corrosion behavior, and biocompatibility in vivo. Mater Sci Eng C Mater Biol Appl 2021;118:111391. DOI: 10.1016/j.msec.2020.111391

20. Guruviah K., Annamalai S.K., Ramaswamy A. et al. Comparative antimicrobial and anticancer activity of biologically and chemically synthesized zinc oxide nanoparticles toward breast cancer cells. Nanomed J 2020;7(4):272–83. DOI: 10.22038/nmj.2020.07.00003

21. Al-Enazi N.M., Alsamhary K., Kha M. et al. In vitro anticancer and antibacterial performance of biosynthesized Ag and Ce codoped ZnO NPs. Bioprocess Biosyst Eng 2023;46:89–103.

22. Feyerabend F., Fischer J., Holtz J. et al. Evaluation of short-term effects of rare earth and other elements used in magnesium alloys on primary cells and cell lines. Acta Biomater 2010;6(5):1834–42. DOI: 10.1016/j.actbio.2009.09.024

23. Liu S.S., Lu D., Miao L.F. et al. Effects of lanthanum chloride on proliferation and migration of human cervical cancer cell line HeLa cells. Zhonghua Fu Chan Ke Za Zhi 2010;45(8):609–13. DOI: 10.3760/cma.j.issn.0529-567x.2010.08.012

24. Feng C., Gan Q., Liu X. et al. Synthesis, antitumor and apoptosis inducing activities of novel 5-fluorouracil derivatives of rare earth (Sm, Eu) substituted polyoxometalates. Chin J Chem Eng 2012;30(7):5–8. DOI: 10.1002/cjoc.201100744

25. Li T., Xu W., Liu C. et al. Anticancer effect of biodegradable magnesium on hepatobiliary carcinoma: an in vitro and in vivo study. ACS Biomater Sci Eng 2021;7(6):2774–82. DOI: 10.1021/acsbiomaterials.1c00288

26. Wei X., Tang Z., Wu H. et al. Biofunctional magnesium-coated Ti6Al4V scaffolds promote autophagy-dependent apoptosis in osteosarcoma by activating the AMPK/mTOR/ULK1 signaling pathway. Mater Today Bio 2021;12:100147. DOI: 10.1016/j.mtbio.2021.100147

27. Dou J.-P., Wu Q., Fu C.-H. et al. Amplified intracellular Ca2+ for synergistic anti-tumor therapy of microwave ablation and chemotherapy. J Nanobiotechnology 2019;17(1):118. DOI: 10.1186/s12951-019-0549-0

28. Valiev R.Z., Langdon T.G. Principles of equal-channel angular pressing as a processing tool for grain refinement. Prog Mater Sci 2006;51(7):881–981. DOI: 10.1016/j.pmatsci.2006.02.003

29. ASTM G1-03-E. Standard practice for preparing, cleaning, and evaluating corrosion test specimens. West Conshohocken, PA: ASTM International, 2011.

30. Choudhary R., Venkatraman S.K., Bulygina I. et al. Biomineralization, dissolution and cellular studies of silicate bioceramics prepared from eggshell and rice husk. Mater Sci Eng C Mater Biol Appl 2021;118:111456. DOI: 10.1016/j.msec.2020.111456

31. Zhuo X., Gao W., Zhao L. et al. A bimodal grain structured Zn–0.4Mg–0.02Mn alloy with superior strength-ductility synergy. Materials Science and Engineering: A 2023;862:144514. DOI: 10.1016/j.msea.2022.144514

32. Huang H., Liu H., Ren K. et al. Improvement of ductility and work hardening ability in a high strength Zn–Mg–Y alloy via micron-sized and submicron-sized YZn12 particles. J Alloys Compd 2021;877:160268. DOI: 10.1016/j.jallcom.2021.160268

33. Niu K., Zhang D., Qi F. et al. The effects of Cu and Mn on the microstructure, mechanical, corrosion properties and biocompatibility of Zn–4Ag alloy. J Mater Res Technology 2022;21:4969–81. DOI: 10.1016/j.jmrt.2022.11.083

34. Wątroba M., Mech K., Bednarczyk W. et al. Long-term in vitro corrosion behavior of Zn–3Ag and Zn–3Ag–0.5Mg alloys considered for biodegradable implant applications. Mater Des 2022;213:110289. DOI: 10.1016/j.matdes.2021.110289

35. Venezuela J., Dargusch M.S. The influence of alloying and fabrication techniques on the mechanical properties, biodegradability and biocompatibility of zinc: A comprehensive review. Acta Biomater 2019;87:1–40. DOI: 10.1016/j.actbio.2019.01.035

36. Duan J., Li L., Liu C. et al. Novel Zn–2Cu–0.2Mn–xLi (x = 0, 0.1 and 0.38) alloys developed for potential biodegradable implant applications. J Alloys Compd 2022;916:165478. DOI: 10.1016/j.jallcom.2022.165478

37. Yin Y.-X., Zhou C., Shi Y.-P. et al. Hemocompatibility of biodegradable Zn–0.8 wt% (Cu, Mn, Li) alloys. Mater Sci Eng C Mater Biol Appl 2019;104:109896. DOI: 10.1016/j.msec.2019.109896

38. Rybalchenko O.V., Anisimova N.Yu., Kiselevsky M.V. et al. Effect of equal-channel angular pressing on structure and properties of Fe–Mn–С alloys for biomedical applications. Mater Today Commun 2022;30:103048. DOI: 10.1016/j.mtcomm.2021.103048

39. Martynenko N., Lukyanova E., Anisimova N. et al. Improving the property profile of a bioresorbable Mg–Y–Nd–Zr alloy by deformation treatments. Materialia 2020;13(2):100841. DOI: 10.1016/j.mtla.2020.100841

40. Martynenko N., Anisimova N., Rybalchenko O. et al. Effect of high pressure torsion on microstructure, mechanical and operational properties of Zn–1%Mg–0.1%Ca alloy. Metals 2022;12(10):1681. DOI: 10.3390/met12101681

41. Martynenko N., Anisimova N., Rybalchenko O. et al. Structure, biodegradation and in vitro bioactivity of Zn–1%Mg alloy strengthened by high pressure torsion. Materials 2022;15(24):9073. DOI: 10.3390/ma15249073

42. Tong X., Zhang D., Lin J. et al. Development of biodegradable Zn–1Mg–0.1RE (RE = Er, Dy, and Ho) alloys for biomedical applications. Acta Biomater 2020;117:384–99. DOI: 10.1016/j.actbio.2020.09.036

43. Su Y., Fu J., Du S. Biodegradable Zn–Sr alloys with enhanced mechanical and biocompatibility for biomedical applications. Smart Mat Med 2021;3:117–27. DOI: 10.1016/j.smaim.2021.12.004

44. Martynenko N., Anisimova N., Kiselevskiy M. et al. Structure, mechanical characteristics, biodegradation, and in vitro cytotoxicity of magnesium alloy ZX11 processed by rotary swaging. J Magnes Alloy 2020;8(4):1038–46. DOI: 10.1016/j.jma.2020.08.008

45. Ma Z., Cao Y., Li Q. et al. Synthesis, characterization, solid-state photo-luminescence and anti-tumor activity of zinc(II) 4′-phenyl-terpyridine compounds. J Inorg Biochem 2010;104(7):704–11. DOI: 10.1016/j.jinorgbio.2010.03.002

46. Magda D., Lecane P., Wang Z. et al. Synthesis and anticancer properties of water-soluble zinc ionophores. Cancer Res 2008;68:5318–25. DOI: 10.1158/0008-5472.can-08-0601

47. Feng P., Li T.L., Guan Z.X. et al. Effect of zinc on prostatic tumorigenicity in nude mice. Ann N Y Acad Sci 2003;1010: 316–20. DOI: 10.1196/annals.1299.056

48. Liguori P.F., Valentini A., Palma M. et al. Non-classical anticancer agents: synthesis and biological evaluation of zinc(II) heteroleptic complexes. Dalton Trans 2010;39:4205–12. DOI: 10.1039/b922101h