*2.4. Antiproliferative Activity (MTT Assay)*

New polymers were studied as drug delivery systems for the topoisomerase inhibitor DOX. We performed cell proliferation assays on A2780, A549 and MDA-MB-231 cancer cell lines. Complexes polymer/DOX (1:10 molar ratio) were prepared and assayed. Data obtained are reported in Table 2. Polymers alone did not show toxicity for cells (data not shown).

**Table 2.** Half maximal inhibitory concentration (IC50) values (nM) of doxorubicin (DOX) in the presence of CyD polymers in human tumor cells.


<sup>a</sup> *p* = 0.0003, as detected by ANOVA; <sup>b</sup> *p* = 0.0657 vs. DOX; <sup>c</sup> *p* = 0.0001 vs. PGAβCyDArg1; <sup>d</sup> *p* = 0.0016 vs. PGAβCyDArg1; <sup>e</sup> *p* = 0.0007 vs. PGAβCyDArg1, all calculated by Bonferroni/Dunn post-hoc analysis of data.

> Overall, the data show that the polymers did not change the antiproliferative activity of DOX significantly. In fact, in A549 and MDA-MB-231 cell lines, the half maximal inhibitory concentration (IC50) values do not change significantly depending on the type of functionalisation. Conversaly, in A2780 cells the complexes with PGAγCyDArg3 (*p* = 0.0028), and PGAβCyDArg5 (trend, *p* = 0.0738) showed higher IC<sup>50</sup> values compared to free DOX.

> In the case of the polymers based on γ-CyD, the higher affinity for DOX suggested by solubility data may explain the effect on the cytotoxicity, as reported for similar systems studied by us [26]. The reduction in the antiproliferative effect was observed for many systems and only in vivo studies may provide information on the potential of the drug carriers [31].

> Only PGAβCyDArg1/DOX showed a slight trend towards a higher antiproliferative activity (*p* = 0.0657) than free DOX and a significant difference compared to PGAγCyDArg3, PGAγCyDArg4, and PGAβCyDArg5 (Table 2).
