*3.4. Cytotoxic Activity of Drug-Loaded Dendrimersomes Towards Cancer Cells*

We have tested the performance of triazine-carbosilane dendrimersomes as drug carriers by doing a comparative study of cytotoxic effects of nanoconstructions loaded with doxorubicin and methotrexate towards two leukaemia cell lines, 1301 and K562. 5-Fluorouracil-loaded dendrimersomes were excluded from the study due to the insufficient encapsulation efficiency resulting in the low concentration of the drug in the nanoparticle preparations.

Cell lines 1301 and K562 have been chosen as targets, for they represent leukaemia variants related to different types of progenitor cells: 1301 is the acute T-cell leukaemia cell line; K562 is chronic myelogenous leukaemia cell line derived from the cells of a patient having blast crises [36]. Thus, the use of these cell lines permits to estimate the efficiency of drug-loaded nanoconstructions in the haemoblastosis therapy.

The ability of drug-loaded dendrimersomes to suppress the viability of target cell lines was studied using WST-1 assay. Dose-response cytotoxic effects of nanoconstructions were observed. Doxorubicin-loaded dendrimersomes have been found to be less cytotoxic in comparison with free drug at low concentrations (below 1 μM) and exhibit similar activity at higher concentrations (Figure 5A,B). These differences are likely connected with the slow release of doxorubicin from dendrimersomes, so, being taken at low concentrations, they do not cause a commeasurable cytotoxic effect, which results in higher cell viability after treatment. In the case of methotrexate, free drug and drug-loaded dendrimersomes possess similar cytotoxicity (Figure 5C,D): In most cases, there were no statistically significant differences found between two groups. In general, the cytotoxic effects have been observed

at dendron concentrations below the IC50 (Table 2, Figure S2), which permits to assign them to the encapsulated chemodrugs.

**Figure 5.** Viability profiles of 1301 (**A**,**C**) and K562 (**B**,**D**) cells after incubation with free chemodrugs and drug-loaded dendrimersomes. NT—non-treated cells. Incubation for 72 h, then WST-1 assay, data are presented as Mean <sup>±</sup> S.D. (n <sup>=</sup> 5). \* *<sup>p</sup>* <sup>&</sup>lt; 0.05 vs. NT; # *<sup>p</sup>* <sup>&</sup>lt; 0.05 free drug vs. drug@DS.

**Table 2.** IC50 values of blank dendrimersomes, chemodrugs and drug-loaded dendrimersomes towards 1301 and K562 cells.


<sup>1</sup> DS-dendrimersomes; <sup>2</sup> value predicted from data fitting

The differences observed between effects of doxorubicin- and methotrexate-loaded dendrimersomes on the viability of target cells are likely explained by the distribution of drugs in dendrimersomes. Since doxorubicin is mostly retained in the hydrophobic part of a vesicle, it is released as a result of reorganization (or even dissociation) of the carrier nanoparticle. Meanwhile, methotrexate is retained mostly at the surface of dendrimersomes, so it can be released relatively easier.

To understand the effects of drug-loaded dendrimersomes better, we have quantified cell subpopulations at different stages of cell death after the treatment with blank dendrimersomes, free drug and drug-loaded nanoconstructions. As a model system, we have taken doxorubicin-loaded dendrimersomes and 1301 cells as a cell model.

Both free doxorubicin and doxorubicin-loaded dendrimersomes have been shown to be efficiently accumulated in 1301 cells (Figures S3 and S4). However, treating cells with free doxorubicin (3 μM) resulted in slight increasing of early and late apoptosis cell fractions in comparison with control (ca. 10% each), whereas doxorubicin-loaded dendrimersomes (3 μM doxorubicin) caused a sharp

increase of late apoptosis and necrosis cell fractions (50% and 20%, respectively) (Figure 6 and Figure S5). Blank dendrimersomes do not cause any significant effect on the 1301 cells in comparison to control at the concentration used (5 μM dendron). Certain differences with WST data likely occurred due to methodological features of the techniques.

**Figure 6.** Induction of cell death in 1301 cells by blank dendrimersomes (DS), free doxorubicin (DOX) and doxorubicin-loaded dendrimersomes (DOX@DS). Incubation for 72 h, then FITC-Annexin V/7AAD staining, flow cytometry. Data are presented as mean ± S.D. (n = 3). \* *p* < 0.05 vs. control group; # *p* < 0.05 vs. DS group; § *p* < 0.05 vs. DOX group.

Our findings show that the encapsulation does not improve the rate of accumulation of doxorubicin into tumour cells, however, the cytotoxic effect of the doxorubicin-containing nanoconstruction is visibly higher. This likely occurs due to the switch of the mechanism of drug penetration into a cell. Remarkably, the presence of serum, an important component of the cell culture medium, does not suppress the dendrimersomes' penetration into cells nor their cytostatic activity. The interaction of nanoconstructions with serum components is known to imminently take place during co-incubation with cells. We suppose that the encapsulation of anti-cancer chemodrugs into dendrimersomes can prolongate their persistence in the organism as well as reduce side effects. Such a phenomenon has been reported while using supramolecular associates [37] and macromolecules, in particular, dendrimers [38,39], as carriers for chemodrugs. In respect of novel amphiphilic triazine-carbosilane dendrimersomes reported herein, this hypothesis deserves further validation in animal experiments.
