# Cellular Entry, Cytotoxicityicity, and Antifungal Activity of Newly Synthesized Dendrimers
## 4 Discussion
### Cellular Entry, Cytotoxicity, and Interactions of DAB and DAB-Br with HFF-1 Cells
No changes in cellular morphology were detected, implying that at high concentrations, both dendrimersmay function similarly to chemical fixatives, preserving cell structure despite inducing membrane permeabilization. At sub-toxic concentrations (<30 \(\mathrm{\SIUnitSymbolMicro g}\)/mL) both dendrimers accumulated in the cells by temporary and reversible membrane permeabilization. The integrity of the cell membranes remained intact, indicating an occurrence of reversible processes within the cell membranes, likely occurring after transient pore formation. The nanoscale size and cationic surface charge of both dendrimers facilitate their permeation through the lipid bilayer by promoting the formation of nanoscale pores, particularly within the more fluid membrane regions. These pores lead to a significant increase in membrane permeability. At higher concentrations, this enhanced permeability culminates in membrane disruption [36; 38; 39]. To exert cytotoxicity and membrane permeabilization, dendrimers interact with cell membranes mainly through electrostatic forces. Cationic dendrimers are attracted to the slightly negatively charged phospholipids in the membrane bilayer, facilitating their binding, adhesion, and accumulation on the membrane surface [40; 41]. Our previous studies on the molecular mechanisms of interaction between DAB, DAB-Br, and POPC model membranes (monolayers and liposomes) demonstrated that dendrimers interact with hydrophilic head groups of phospholipids, leading to their incorporation into the lipid bilayers [42]. Due to their cationic surface charge and nanoscale dimensions, dendrimers are electrostatically attracted to the surfaces of cell membranes. This attraction causes them to adhere to the membrane and penetrate the bilayer to some extent, substituting water molecules around the glycerol backbone of the lipids, and consequently, this leads to an increase in the lipid order [41]. Laurdan fluorescence spectroscopy provided valuable insight into the interaction of the tested dendrimers with cell membranes by assessing changes in the lipid order [43], an important parameter influencing membrane fluidity, permeability, and functions. A blue shift in the Laurdan emission spectra (420-440 nm) was observed at 3 \(\mathrm{\SIUnitSymbolMicro g}\)/mL for DAB and DAB-Br, indicating increased membrane lipid order, likely due to dendrimer incorporation into the bilayer. This effect was more pronounced for DAB, which also exhibited higher generalized polarization values, suggesting a tighter lipid packing and reduced hydration at the membrane interface. This was further supported by experiments with model vesicles (POPC), where DAB induced greater ordering than DAB-Br, pointing to structural differences in membrane interaction [42]. Interestingly, the greater ordering effect of DAB may be associated with its smaller particle size (118 nm vs. 221 nm for DAB-Br), which may allow more efficient insertion into the lipid bilayer. In contrast, DAB-Br, despite its higher positive \(\zeta\)-potential (+35.2 mV vs. +28.7 mV for DAB), induced a less marked increase in the lipid order. While a higher surface charge typically enhances electrostatic interactions with negatively charged membranes, the larger size of DAB-Br may limit its ability to integrate into the membrane as effectively as DAB. This suggests that particle size, not just surface charge, plays a critical role in determining how dendrimers modulate membrane structure. At the higher concentration of 30 \(\mathrm{\SIUnitSymbolMicro g}\)/mL, both dendrimers caused a decrease in the lipid order, consistent with Laurdan's sensitivity to water penetration and indicative of increased membrane permeability. This threshold concentration also corresponded with the results from the TBE assay, where both dendrimers caused irreversible membrane permeabilization. Overall, the data point to a concentration-dependent dual effect: membrane stabilization at lower concentrations and increased permeability at higher dendrimer doses.