# Cellular Entry, Cytotoxicityicity, and Antifungal Activity of Newly Synthesized Dendrimers
## 5 Conclusions  
The biological activities of newly synthesized dendrimers, 4-dimethylamino-1,8-naphthalimide (DAB) and its halogenated analog 3-bromo-4-dimethylamino-1,8-naphthalimide (DAB-Br), specifically their cytotoxicity, mechanisms of cellular entry, and antifungal properties were investigated on eukaryotic cells. Dynamic light scattering measurements revealed that both dendrimers carried strong positive surface charges and different nanoscale hydrodynamic diameters. These size dimensions, along with their cationic nature, facilitate effective interactions with cellular membranes and promote internalization into target cells. The treatment of the HFF-1 cell line and five fungal strains with the dendrimers tested revealed that DAB dendrimers were more cytotoxic to human cells, whereas DAB-Br exhibited greater antifungal potency. The MTT assay using the HFF-1 cell line showed that DAB had a lower IC\({}_{50}\) value, indicating stronger cytotoxicity, while DAB-Br was less toxic. In contrast, antifungal testing using the resazurin method showed that DAB-Br induced complete growth inhibition of all tested fungal strains at a lower minimum inhibitory concentration than DAB. Furthermore, DAB-Br maintained its antifungal activity for up to 96 h at lower concentration, whereas DAB required a twofold higher concentration to produce a comparable effect. The TBE assay indicated that both DAB and DAB-Br dendrimers enter HFF-1 cells via temporary or permanent membrane permeabilization. At the threshold concentration of 30 \(\upmu\)g/mL, both dendrimers induced irreversible, time-dependent membrane permeabilization, as higher concentrations accelerated this effect. Microscopic observations and Laurdan fluorescence spectroscopy confirmed compromised membrane integrity, as treated cells exhibited a predominantly blue fluorescence and a decreased lipid order, which is indicative of disrupted membrane integrity and permeability. At non-toxic concentrations below 30 \(\upmu\)g/mL, dendrimers crossed the cell membrane via temporary permeabilization. Treated cells showed dendrimer accumulation in the cytoplasm, while membrane integrity remained intact, suggesting the formation of transient nanoholes and reversible membrane processes under these conditions. **Supplementary Materials:** The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app15147764/s1](https://www.mdpi.com/article/10.3390/app15147764/s1)]([https://www.mdpi.com/article/10.3390/app15147764/s1](https://www.mdpi.com/article/10.3390/app15147764/s1)), Supplement S1. Cell morphology after treatment with dendrimers (a) at low concentration of 7.5 \(\mathrm{\SIUnitSymbolMicro g}\)/mL and (b) high concentration of 30 \(\mathrm{\SIUnitSymbolMicro g}\)/mL. The arrows point the dendrimers in cell periphery and the triangles point the dendrimers outside of the cells. The cells appeared more rounded, with accumulated brown particles (internalized dendrimers) evident in the cytoplasm (a, arrows). However, cell viability was not affected. The cells continued to spread and formed a thick layer. At the higher concentration of 30 \(\mathrm{\SIUnitSymbolMicro g}\)/mL 2 h post-treatment, all recorded cells appeared blue under the microscope, indicating compromised membrane integrity and dead or dying cells, and brown clusters of dendrimers in the medium surrounding the cells were observed (b). **Author Contributions:** Conceptualization, A.K., T.T.-H., A.T. and A.J. ; methodology, A.K., T.T.-H., G.S. and I.G. ; validation, T.T.-H., M.M. and G.S. ; formal analysis, D.B. ; investigation, A.K., E.G., T.T.-H., D.B., G.S., E.K., R.H., M.M. and I.G. ; data curation, A.K. ; writing--original draft preparation, A.K., T.T.-H., D.B., E.K. and M.M. ; writing--review and editing, A.T. and A.J. ; visualization, D.B. and A.T.; supervision, A.J. ; project administration, A.J. ; funding acquisition, A.J. All authors have read and agreed to the published version of the manuscript.