*2.11. Drug Internalization Studies*

0.5 <sup>×</sup> 106 1301 cells were cultivated in total volume 100 <sup>μ</sup>L in flat-bottomed 48-well cell culture plates (TPP, Switzerland) for 4 h. Then, water solutions of amphiphilic dendron (5 μM), doxorubicin (3 μM) or doxorubicin-loaded dendrimersomes (doxorubicin content 3 μM) were added. The equal volume of PBS was added to control cells. After cultivation, cells were collected, washed once with PBS, then 50 μL of acidic glycine solution were added, gently pipetted for 30 s and washed again with PBS. Flow cytometric analysis was performed on a FACSCanto II cytometer (BD Biosciences, San Jose, CA, USA). The analysis was made in FITC and PE channels. 15,000 to 50,000 events per sample were acquired and analysed using FACSDiva 6.1.2 software.

1301 cells were incubated with blank dendrimersomes, doxorubicin and doxorubicin-loaded dendrimersomes (6.6 μM DOX) in complete cell culture media for 4 h in humidified atmosphere containing 5% CO2 at 37 ◦C, then fixed in ethanol and glacial acetic acid. Samples were stained with DAPI solution (1.5 μg/mL) with the addition of antifade solution (10 μL) for 20 min, protected from light. The cells were visualized under fluorescence microscopy (Axiopscop 40, Zeiss, Oberkochen, Germany).
