## 4 Discussion
Another perspective for our further investigations includes the analyses of the potential of MVS-produced AuFe-MTX samples to serve as hyperthermia or radiosensitizing agents providing stringent site-specific drug release under the influence of external magnetic or X-ray signals. Recent findings demonstrate such ability of drug-modified AuFe NPs. [PERSON] et al. have shown a controlled drug release via hyperthermia mediated using MTX-conjugated iron-gold alloy nanoparticles [23]. [PERSON] et al. have reported efficient tumor chemoradiation therapy in a murine model of breast cancer using curcumin and folic acid-loaded Fe\({}_{3}\)O\({}_{4}\)-Au bimetallic heteronanostructures [43]. An important biological activity to investigate is antibacterial potential and, eventually, activity against antibiotic-resistant species. The reason to investigate the bactericidal effects of BMNP-MTX is that both components of this conjugate have been shown to possess such abilities. The antibacterial properties of MTX have been described [44]. The present data indicate moderate antibacterial activity for the studied AuFe NPs. Taken together, in a joint agent, MVS-generated AuFe NPs and MTX could exhibit a stronger antibacterial activity. However, our data do not confirm such enhanced properties. The emerging role of nanomaterials concerns their immunomodulatory potential. Gold, iron and iron oxide nanoparticles have demonstrated intrinsic immune-regulatory and anti-inflammatory properties [39]. In this study, we have analyzed the levels of two main cytokines--II-2 and IFN-\(\gamma\). Although they are generally considered as pro-inflammatory, IL-2 and IFN-\(\gamma\) play complex roles to support functional immune responses. IFN-\(\gamma\) is necessary for macrophage activation to increase phagocytosis and enhance antitumor properties, and is used for the destruction of intracellular pathogens [45]. IL-2 is essential for the maintenance of a peripheral tolerance, T-lymphocyte growth and differentiation [46]. Therefore, the increase caused by the AuFe-MTX treatment IL-2 and IFN-\(\gamma\) production and secretion should not be solely attributed to the exacerbation of pro-inflammatory responses. In fact, it should be considered as a complex immuno-stimulatory effect with a prospective potential that will be further analyzed in our future studies. In conclusion, this study describes the generation and characterization of biologically active bimetallic nanoparticles and their conjugation with MTX based on the environmentally safe method of metal-vapor synthesis, which enables an effective production of bimetallic drug nanocarriers, and is a simple approach for drug conjugation that does not involve additional organic reactions, improving the sustainability of the process and possible future manufacture. The obtained modified nanomaterials were subjected to physicochemical analyses and multilateral biological activity evaluations. BMNP-MTX demonstrated prominent antitumor activity even after the treatment of tumor cells with low sample concentrations and inhibition of multicellular 3D tumor aggregates' growth and vitality. This indicates that they could serve as efficient drug carriers of antitumor agents and could act synergistically. In addition, AuFe-MTX demonstrated an ability to stimulate cytokine production with peripheral blood leukocytes, proving to have complex biological effects and a vast potential for biomedical applications. The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app132312894/s1](https://www.mdpi.com/article/10.3390/app132312894/s1)]([https://www.mdpi.com/article/10.3390/app132312894/s1](https://www.mdpi.com/article/10.3390/app132312894/s1)), Figure S1: Survey of XPS spectra of the AuFe and AuFe-MTX sample; Methods [47; 48; 49]. Conceptualization, T.B. and A.V. ([PERSON]); methodology, T.B., A.V. ([PERSON]) and B.D. ; formal analysis, T.B., A.V. ([PERSON]), A.V. ([PERSON]), A.N., D.M., I.T. and B.D. ; biological evaluations, T.B., D.M., I.T. and B.D.