# 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  
The Human Foreskin Fibroblast (HFF-1 SCRC-1041) cell line originates from human skin fibroblasts and is an appropriate model for the in vitro testing of novel nanomaterials with potential medical applications. These cells possess a normal diploid karyotype, making them particularly valuable for testing the biological activity and potential cytotoxicity of dendrimers. It is well-documented that dendrimers' characteristics--such as generation, size, number of surface groups, and the nature of terminal moieties (anionic, neutral, or cationic)--play a crucial role in modulating their biological effect on target mammalian cells [19; 30]. In the presented study, the zeta potential measurements obtained via DLS showed that both dendrimers possess noticeable positive surface charges (+28 mV for DAB, and +35 mV for DAB-Br). This suggests good colloidal stability of the dispersion, as higher zeta potential values promote repulsion between particles, preventing their aggregation. The observed cationic surface charges of both dendrimers are attributed to the presence of tertiary amines in their internal structures and the terminal amine functional groups [26]. Notably, DAB-Br exhibited a higher zeta potential value compared to the non-halogenic DAB dendrimer due to surface bromination, which enhances the positive charge. Additionally, the hydrodynamic diameters of the dendrimers were determined to be 117 nm for DAB and 234 nm for DAB-Br. These nanoscale dimensions fall within the optimal range for cellular uptake and facilitate effective interactions with plasma membranes, cell organelles (endosomes, mitochondria, and nucleus), and cellular proteins and enzymes [31; 32]. The MTT assay revealed that both tested dendrimers elicited a dose-dependent decrease in cell viability. The cytotoxicity was evident at concentrations exceeding 30 \(\upmu\)g/mL. DAB dendrimers exhibited stronger cytotoxicity (IC\({}_{50}\) = 27 \(\upmu\)g/mL), whereas DAB-Br dendrimers had a much weaker effect (IC\({}_{50}\) = 68 \(\upmu\)g/mL). These findings highlight the influence of dendrimer structure and surface chemistry on cytotoxic outcomes. Cationic dendrimers, while often effective at cellular internalization, are commonly associated with higher cytotoxic potential. In support of this, a previous study has reported that positively charged PPI dendrimers induce pronounced dose-dependent cytotoxicity on target cells [33]. The positive charge is considered a primary contributor to the cytotoxic effects of the dendrimers because it promotes electrostatic interactions with cell components, such as proteins and cell membranes [34]. These interactions can disrupt normal cellular processes and ultimately can lead to cellular impairment and cellular damage. A study conducted by [PERSON] and colleagues has revealed that cationic amino-terminated G2 dendrimers internalize in epidermal and dermal cells, contrary to anionic and uncharged ones [35]. However, the mechanism underlying the cellular uptake of the dendrimers is still debatable and there are various hypotheses regarding their mode of cellular entry. Some authors consider endocytosis as the primary pathway for internalization, while others propose temporary or permanent membrane permeabilization [36; 37]. Based on the results from the TBE test, it can be stated that both DAB and DAB-Br dendrimers enter cells through cell membrane permeabilization, either temporary or permanent, depending on the tested concentrations and duration of exposure. Irreversible membrane permeabilization was observed at cytotoxic concentrations (>30 \(\upmu\)g/mL), indicating strong disruptive interactions with the lipid bilayer. Notably, DAB exerts a stronger and more rapid membrane-disrupting effect compared to the halogenated DAB-Br, consistent with the MTT results.