*2.2. Antiaggregant Activity*

The abnormal aggregation of Aβ is one of the main hallmarks of Alzheimer's disease (AD). In the pathological pathway, the amyloid peptide firstly forms soluble and highly toxic oligomers; then, the growing dimensions of the aggregated species lead to the formation of fibrillary and insoluble structures, mainly accumulated into brain plaques [28]. Finding new molecules able to inhibit the formation of amyloid-type aggregates represents an important strategy to prevent the onset of AD or attenuate the development of this devastating disorder. Therefore, we tested the effect of all the PGA polymers on the selfinduced aggregation of Aβ by using a turn-on fluorescent dye Thioflavin T (ThT), sensitive to the formation of fibril species.

The fluorescence data recorded for the amyloid-type aggregation of Aβ fit to a sigmoid curve. The maximum fluorescence gain (*Fmax* − *F*0) is 33 ± 1 and the lag phase lasts 24 ± 2 h. When the compounds of interest are co-incubated with Aβ, the kinetic parameters of the aggregation process could be modified due to non-covalent interactions between Aβ and the PGA polymers. *Fmax* − *F*<sup>0</sup> is proportional to the amount of Aβ fibrils, whereas during the lag phase (*tlag*) only small aggregated species form. As a consequence, the lower *Fmax* − *F*<sup>0</sup> is and/or the higher *tlag* is, the better the antiaggregant activity.

Figure 4 shows the *Fmax* − *F*<sup>0</sup> values obtained by the aggregation of Aβ alone (control (CTRL)) or in the presence of each PGA polymer. Several amounts of the compounds have been tested, the (Aβ)/(Polymer) molar ratio ranging from 1:1 to 1:8.

**Figure 4.** Maximum fluorescence gain values (*Fmax* − *F*<sup>0</sup> ) of the samples containing Aβ1-40 (20 µM) alone (control (CTRL)) or in the presence of the PGA polymers, the (Aβ)/(Polymer) molar ratio ranging from 1:1 to 1:8. (\* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 vs. CTRL, ANOVA test).

The co-incubation of any polymer with Aβ in a 1:1 molar ratio induced little or no effect on the final extent of the aggregation process. Higher amounts of the polymers significantly decrease the *Fmax* − *F*<sup>0</sup> values and the antiaggregant activity is exerted in a dose-dependent manner, as reported in Figure 5 in the case of PGAβCyDArg2.

**Figure 5.** Representative kinetic profiles of the amyloid aggregation due to the co-incubation of Aβ1-40 (20 µM) with PGAβCyDArg2, the (Aβ)/(Polymer) molar ratio ranging from 1:1 to 1:8. Single points represent the experimental data, whereas the straight lines are the fitted curves (adjusted R<sup>2</sup> is 0.9766, 0.9481 and 0.7227 for 1:1, 1:2, 1:8 (Aβ)/(Polymer) molar ratio, respectively).

As for the PGA polymers containing β-CyD (PGAβCyDArg1, PGAβCyDArg2 and PGAβCyDArg4), the antiaggregant activity is very comparable among the polymers, when the Aβ/polymer molar ratio was 1:2. Instead, these β-CyD-containing polymers differently affect the amyloid aggregation extent when the 1:8 Aβ/polymer molar ratio was tested. In particular, it seems that the greater the number of Arg units, the greater the inhibition effect of the amyloid aggregation. Such a trend is also observed when the γ-CyD-containing polymers were taken into account (PGAγCyDArg3 and PGAγCyDArg5). However, the inhibition activity of these γ-CyD polymers is slightly lower than that shown by the corresponding β-CyD polymers (PGAβCyDArg2 and PGAβCyDArg4). Such a difference could be reasonably ascribed to the structural differences between the β- and γ-CyD cavities that, in turn, could affect the non-covalent interaction between the amyloid peptide and the polymers. A similar trend has been reported for single CyDs [14].

The lag phase of the amyloid aggregation was not significantly modified by the polymers (data not shown), meaning that the interaction between the Aβ and these PGA polymers is not altered by the number of CyD and Arg units.

Above all, the antiaggregant activity towards the self-induced formation of amyloid aggregated species exerted by the PGACyD polymers could reasonably be due to the effects of both the CyD cavity and charged units (Arg and PGA). The effect of positive and negative charges of carbohydrate polymers on the on-pathway Aβ aggregation has been recently ascertained [29], thus corroborating these results.
