## 4 Discussion  
Nowadays, the development of nanomaterials for specialized biomedical applications is a leading research branch that opens numerous perspectives. Nanoparticles exhibit superior properties compared to the bulk material, providing them with unique abilities and numerous beneficial applications [39]. Bimetallic nanoparticles, in particular, contain two nano-sized metals, which leads to structural changes introduced by an additional degree of freedom [5]. Synergistic effects induced by the combination of different metals could enhance the physical, chemical and biological properties of the nanomaterial, resulting in improved optical, magnetic, catalytic, thermal, antibacterial, antitumor, anti-inflammatory or immunomodulatory properties [5; 10; 39], as well as new specific activities. In our previous studies, we have proven that biologically active gold and iron nanoparticles can be generated by the environmentally safe method of metal-vapor synthesis [33]. Both types of metal nanoparticles have been shown to selectively accumulate in tumor and inflamed tissues [39]. Their further modification with drugs could allow them to achieve a better therapeutic efficacy, a reduction in negative side effects and prolonged circulation time in blood. Improvement of solubility, tissue availability and the penetration profile together with the diminution of adverse effects is particularly necessary for MTX--a well-known antitumor and anti-inflammatory agent used for the treatment of certain tumors and inflammatory conditions (rheumatoid arthritis, psoriasis). The benefits of MTX conjugation with nanoparticles, particularly metal nanoparticles, have already been reported [40; 41]. Our previous studies also contribute to this field, demonstrating antitumor properties of Au and Fe monometallic MTX conjugates produced with MVS [29; 42]. Interestingly, Au NPs-MTX and Fe NPs-MTX generated using the MVS method showed highest inhibitory potential against HT-29 rectal adenocarcinoma cells in our previous study [29], while the data in this study showed a better efficacy against lung A549 and cervical HeLa adenocarcinoma cells. This difference could be due to the use of bimetallic nanoparticles and their specific physicochemical properties manifested in combination with the modification using MTX. Another interesting finding in this study is the high effectiveness of the BMNP-MTX nanomaterial at low concentrations. Treatment with the same concentration of MTX or AuFe NPs did not induce significant inhibition of tumor cell metabolic activity and viability. On the other hand, we could not detect stronger than MTX-induced inhibition during the treatment with higher concentrations of BMNP-MTX. A possible reason for this could be the saturation effect of the sample's concentration and the inactive state of BMNPs-bound MTX. [PERSON] et al. have proven that MTX exerts its activity only after its release from the conjugate with AuFe alloy nanoparticles [23]. In the present research scenario, we suggest that AuFe-MTX samples used in higher concentrations (100-200 \(\upmu\)g/mL) could prevent effective cellular uptake and drug release, while lower concentrations of the conjugate favor synergistic effects together with efficient interactions with cells and drug release. Further experiments need to be performed to confirm this hypothesis. Our results on the antitumor activity of AuFe-MTX nanocarriers are exclusively based on in vitro experiments, which could be considered as a partial limitation of our study. However, an important step in the process of the validation of a certain substance or nanomaterial as an antitumor agent includes in vitro analyses. Moreover, our experiments with 3D spheroids demonstrate the efficacy of BMNP-MTX on the level of multicellular tumor structures. The results precede and motivate future in vivo evaluations of AuFe-MTX, for which effective concentration ranges of the test agent as well as tumor cell type specific effects were defined.