## 3 Results
### Physicochemical Analyses  
Biological evaluations highlighted the AuFe-MTX sample generated in the acetone organic dispersion medium as a nanocarrier with better properties compared to AuFe(Tol)-MTX. Therefore, the physicochemical evaluations were concentrated on this sample. Figure 6 shows the EDX spectra of bimetallic AuFe nanoparticles obtained in the reaction with acetone and their conjugates with MTX. Along with the presence of metals in the AuFe system, a significant amount of carbon material has been registered, which may indicate the chemisorption of acetone or its fragments on the metal surface. Figure 4: Longitudinal evaluations of the effects of BMNP-MTX on bacterial growth and viability. (**a**) Gram-negative bacteria (_E. coli_); (**b**) Gram-positive bacteria (_B. cereus_). All samples were analyzed in triplicates. The graphs display mean results (\(\pm\)SEM). \({}^{*}\), \(p<0.05\). Figure 5: LL-2 (**a**) and IFN-\(\gamma\) (**b**) production with peripheral blood leukocytes treated ex vivo with 100 \(\upmu\)g/mL BMNP-MTX. Asterisks indicate statistically significant differences between the test sample and the untreated control. \({}^{***}\), \(p<0.001\). Figure 6: Energy dispersive X-ray spectra of AuFe (**a**) and AuFe-MTX (**b**). The composition of the surface plays a key role in the biological activity, catalysis and other properties of metal nanoparticles. AuFe nanoparticles and their conjugates with MTX were studied with X-ray photoelectron spectroscopy, which is the leading analytical method used for the characterization of various chemical/physical forms of elements on the surface of materials. The relative atomic concentrations of the elements were determined from the survey spectra of the samples (see Supplementary Materials, Figure S1) (Table 3). The Au 4f photoelectron spectra of the AuFe (1) and AuFe-MTX (2) samples, shown in Figure 7, are described by three spin-orbit doublets 4f\({}_{7/2}\)-4f\({}_{5/2}\) preserving the 4f\({}_{7/2}\)/4f\({}_{5/2}\) branching ratio of 4/3. The characteristics of the photoelectronic peaks and their interpretations are presented in Table 4.  
The binding energy of the Au 4f\({}_{7/2}\) peak in the spectrum of AuFe nanoparticles equal to 84.1 eV is 0.1 eV higher than the value for pure Au and can be attributed to the formation of a Au-Fe bond. The value of 84.8 eV corresponds to the Au\({}^{+}\) state. As for the binding energy of 86.2 eV, it corresponds to the interaction of gold atoms with hydroxyl groups of the shell with the formation of the Au\({}^{3+}\) state [35]. \begin{table}
\begin{tabular}{c c c c c c c} \hline \hline \multirow{2}{*}{**Sample**} & **State** & **Au\({}^{0}\)** & \multicolumn{2}{c}{**Au\({}^{+}\)**} & \multicolumn{2}{c}{**Au\({}^{3+}\)**} \\ \cline{2-7}  & **Shell** & **4f\({}_{7/2}\)**, eV** & **4f\({}_{5/2}\)**, eV** & **4f\({}_{7/2}\)**, eV** & **4f\({}_{5/2}\)**, eV** & **4f\({}_{7/2}\)**, eV** & **4f\({}_{5/2}\)**, eV** \\ \hline \multirow{3}{*}{AuFe} & E\({}_{\text{b}}\), eV & 84.1 & 87.8 & 84.8 & 88.5 & 86.2 & 89.8 \\  & W, eV & 0.81 & 0.81 & 0.99 & 0.96 & 1.6 & 1.7 \\  & I\({}_{\text{rel}}\) & 0.94 & 0.725 & 0.2 & 0.15 & 0.145 & 0.11 \\ \hline \multirow{3}{*}{AuFe-MTX} & E\({}_{\text{b}}\), eV & 84.2 & 87.9 & 84.7 & 88.4 & 86.7 & 89.3 \\  & W, eV & 0.8 & 0.77 & 0.92 & 0.9 & 1.55 & 1.48 \\ \cline{1-1}  & I\({}_{\text{rel}}\) & 0.58 & 0.435 & 0.64 & 0.495 & 0.29 & 0.218 \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Characteristics of photoelectron spectra Au 4f (E\({}_{\text{b}}\)—binding energy; W—peak width; I\({}_{\text{rel}}\)—relative intensity). \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Sample** & **Au** & **Fe** & **O** & **C** & **N** \\ \hline MTX & - & - & 13.3 & 69.3 & 17.4 \\ AuFe & 12.0 & 14.5 & 25.8 & 47.8 & - \\ AuFe-MTX & 25.9 & 8.1 & 21.7 & 65.4 & 3.7 \\ \hline \hline \end{tabular}
\end{table} 
Table 3: X-ray photoelectron spectroscopy (XPS) quantification data (at. %) of samples comprising MTX, AuFe and AuFe-MTX.