# Cellular Entry, Cytotoxicityicity, and Antifungal Activity of Newly Synthesized Dendrimers
## 3 Results
### Permeability of Cell Membranes of HFF-1 Cells and Changes in Cell Morphology  
The evaluation of cell membrane integrity by the TBE test revealed that both dendrimers induced permeabilization of the cell membrane in a dose-dependent manner (Figure 4). Insignificant permeabilization was observed at the lowest tested concentration of 3 \(\mathrm{\SIUnitSymbolMicro\SIUnitSymbolMicro m}\) (2-3%) at the 2 nd, 24 th, and 48 th hour after treatment with both dispersions of dendrimers. However, a noticeable effect on permeabilization was observed at the highest tested concentrations. At the 2 nd hour, permeabilization reached 100% at a concentration of 60 \(\mathrm{\SIUnitSymbolMicro\SIUnitSymbolMicro m}\)/mL for both dendrimers (Figure 4a). At the 24 th hour, DAB exhibited a significant effect, reaching 100% permeabilization at 30 \(\mathrm{\SIUnitSymbolMicro\SIUnitSymbolMicro m}\)/mL (Figure 4b), while for DAB-Br this parameter at the same concentration was detected at the 48 th hour (Figure 4c). A noticeable dynamic in cell permeabilization was observed at lower concentrations, 7.5 and 15 \(\mathrm{\SIUnitSymbolMicro\SIUnitSymbolMicro m}\)/mL, suggesting the occurrence of reverse processes in the cell membranes. In both cases, a stronger permeabilization was noted by DAB: at 15 \(\mathrm{\SIUnitSymbolMicro\SIUnitSymbolMicro m}\)/mL at the 2 nd hour, permeabilization was increased to 60% (Figure 4a). However, at the same concentration at the 24 th hour, permeabilization dropped to 40% and further decreased to 20% by the 48 th hour (Figure 4b,c). A similar effect was observed with DAB-Br, with a significant decrease in membrane permeabilization after the 24 th hour post treatment.  
Figure 4: Comparative quantitative analysis of cell membrane permeabilization following treatment of HFF-1 cells with DAB and DAB-Br at the (**a**) 2 nd hour, (**b**) 24 th hour, and (**c**) 48 th hour. Statistical significance is denoted by *** (\(p<0.001\)) when comparing effects of both dendrimers.  
Following the evaluation of cell membrane permeabilization, alterations in the cell morphology were monitored, as these changes indicate adaptation and stress responses. The morphology of the untreated adherent cells reveals spindle-shaped and highly elongated forms, typical for fibroblasts, which were closely spaced one next to the other (Figures 5a and 6a). At the 24 th and 48 th hour, the fibroblasts spread onto the Petri dish and formed a thick layer due to extracellular interactions (Figure 5d,g and Figure 6d,g). However, after treatment with DAB and DAB-Br, noticeable morphological changes occurred. At the lower concentration of 7.5 ug/mL, both dendrimers induced similar cell morphological changes at the 24 th and 48 th hour post treatment (Figure 5e,h and Figure 6e,h). The cells appeared more rounded, with accumulated brown particles (internalized dendrimers) evident in the cytoplasm. However, cell viability was not affected. The cells continued to spread and formed a thick layer (Figures 5h and 6h).  
At the higher concentration of 30 ug/mL 2 h post treatment, all recorded cells appeared blue under the microscope, indicating compromised membrane integrity and dead or dying cells (Figures 5c and 6c). At the 24 th and 48 th hour, brown clusters of dendrimers in the medium surrounding the cells were observed (Figure 5f,i and Figure 6f,i). Presumably, the dendrimers passed through the cell membrane when cell death occurred. However, apart from the coloring, the cells did not show alterations in their morphology.  
Figure 5: Changes in cell morphology and cell permeabilization with trypan blue staining at the 2 nd, 24 th, and 48 th hour post treatment with DAB at concentrations of 7.5 ug/mL and 30 ug/mL. For photo shooting, random fields were selected. Controls (untreated cells, **a**,**d**,**g**); cells treated with DAB at a concentration of 7.5 ug/mL (**b**,**e**,**h**) and 30 ug/mL. (**c**,**f**,**i**). Magnification 200\(\times\).