## 3 Results  
### Antitumor Activity of AuFe-MTX Conjugates  
The results from the biological activity analyses include AuFe-MTX obtained with MVS using an acetone dispersion medium (denoted in the figures as AuFe(Ac)-MTX), AuFe-MTX conjugate produced using a toluene dispersion medium (marked in the figures as AuFe(Tol)-MTX) and AuFe nanoparticles (NPs) generated in a toluene organic dispersion medium. Only one BMNP sample was included in the assays and served as a representative unconjugated control. Our previous studies have indicated that NPs obtained with MVS using an acetone or toluene dispersion medium do not significantly differ in their biological activity [33]. Standard in vitro cytotoxicity assays based on the cellular ability for reducing the MTT or NR uptake were performed with three adenocarcinoma cell lines and normal human fibroblasts. Their purpose was to assess the antitumor potential of BMNP conjugates. All cell types tested showed time-dependent and concentration-dependent responses against the treatment with AuFe-MTX conjugates (Figure 2, Table 1). Normal fibroblasts were the least affected by the addition of BMNP-MTX to the culture media, which highlights an antitumor potential of the tested samples. The level of inhibition detected in HEFC cultures did not exceed 25% at the highest tested concentrations of the samples. Furthermore, these negative effects were measured in the early test periods--24 h and 48 h--which indicate the ability of the cells to overcome the cytotoxic influence of BMNP-MTX and highlight the good biocompatibility potential of the samples (Figure 2g,h). The opposite trend is clear from the results obtained with adenocarcinoma cells. More than 50% inhibition of A549 and HeLa tumor cell metabolic activity was detected after 72 h of treatment with nanocomposites (Table 1). The calculated IC\({}_{50}\) concentrations were higher than MTX applied alone, which could be attributed to the potential gradual mode of MTX release by the conjugates. An important activity of BMNP-MTX samples detected with both MTT and NR assays was their higher inhibitory potential when they were applied at low concentrations (Figure 2). Interestingly, a BMNP-MTX concentration of 10 ug/mL induced a significantly higher inhibition of tumor cell metabolic activity and vitality compared to MTX (Figure 2a-f). These effects gradually increased in magnitude during the experiment, reaching a maximum at the 72 or 96 h mark. The HT-29 cell line showed the lowest sensitivity towards the BMNP-MTX treatment among the tested tumor cell lines. In general, AuFe NPs did not show negative effects at low concentrations, except for the longest test period (96 h) with A549 cells (Figure 2a). Thus, the inhibition induced with AuFe-MTX conjugates could be attributed to the effective targeting of the drug and its possible synergistic effects. Figure 2: Longitudinal in vitro cytotoxicity and antitumor potential of BMNPs and BMNP-MTX samples. Data represent \(\pm\) standard error of the mean (\(\pm\)SEM). All graphs display results for A549 (**a,b**), HeLa (**c,d**), HT-29 (**e,f**) and fibroblast (**g,h**) cells treated with 10 \(\upmu\)g/mL AuFe-MTX conjugates, AuFe NPs or methotrexate (MTX). (**a,c,e,g**) MTT assay results; (**b,d,f,h**) NRU assay data. The asterisk sign indicates statistically significant difference between the test sample and MTX control. *, \(p\) < 0.05; **, \(p\) < 0.01; ***, \(p\) < 0.001. MTT and NRU assay analyses were followed by experiments with tumor spheroids formed with HT-29 cells. The aim of these studies was to confirm the detected antitumor potential and prove the efficacy of BMNP-MTX on the level of multicellular tumor structure. HT-29 cells were seeded on polymer-treated ultra-low attachment culture plates.