**2. Results and Discussion**

#### **2. Results and Discussion**  *2.1. Particle Size and TEM Data Analysis*

aggregates increase with an increasing CD concentration [30].

*2.1. Particle Size and TEM Data Analysis*  The particle size of 100 mM CD aggregates was measured by Nano Sight and confirmed by Transmission Electron Microscopy (TEM) (Figure 2). CDs tend to form nanosized aggregates when their concentration is increased [29]. Although the aggregation in pure aqueous CD solutions is generally low, the aggregation is frequently enhanced by The particle size of 100 mM CD aggregates was measured by Nano Sight and confirmed by Transmission Electron Microscopy (TEM) (Figure 2). CDs tend to form nano-sized aggregates when their concentration is increased [29]. Although the aggregation in pure aqueous CD solutions is generally low, the aggregation is frequently enhanced by the formation of drug/CD complexes. Furthermore, the aggregation and the size of the aggregates increase with an increasing CD concentration [30].

the formation of drug/CD complexes. Furthermore, the aggregation and the size of the

**Figure 2.** Transmission electron microscopy images of cyclodextrin (CD) aggregates at a magnitude of 30 K. (**a**) 100 mM HPβCD and (**b**) 100 mM RMβCD. The average diameter of RMβCD aggregates appeared to be around two times larger than that of HPβCD. **Figure 2.** Transmission electron microscopy images of cyclodextrin (CD) aggregates at a magnitude of 30 K. (**a**) 100 mM HPβCD and (**b**) 100 mM RMβCD. The average diameter of RMβCD aggregates appeared to be around two times larger than that of HPβCD.

The concentration of CDs used in pharmaceutical formulations depends on the type of CD used, but due to their favorable toxicological and pharmacological profiles, the CD concentrations can be relatively high [31]. However, the limited solubility of the natural CDs, especially βCD, can limit their concentration [1]. Both HPβCD and RMβCD are very soluble in water (Section 3.1), so we used very high concentrations of these CD derivatives to observe their potential toxicity on retinal explants. Aggregates were observed in aqueous HPβCD and RMβCD solutions. The average diameter of the HPβCD aggregate, as determined by Nano Sight, was 148 nm and that of RMβCD was 305 nm. Larger aggregates (diameter > 100 nm) do not have a spherical shape like smaller ones (<100 nm). Instead, they look like clusters of smaller, spherically-shaped aggregates [32]. The aggregate diameters observed with TEM are smaller than those determined by Nano Sight. This can be explained by the TEM sample preparation, where the aggregate size or structure can The concentration of CDs used in pharmaceutical formulations depends on the type of CD used, but due to their favorable toxicological and pharmacological profiles, the CD concentrations can be relatively high [31]. However, the limited solubility of the natural CDs, especially βCD, can limit their concentration [1]. Both HPβCD and RMβCD are very soluble in water (Section 3.1), so we used very high concentrations of these CD derivatives to observe their potential toxicity on retinal explants. Aggregates were observed in aqueous HPβCD and RMβCD solutions. The average diameter of the HPβCD aggregate, as determined by Nano Sight, was 148 nm and that of RMβCD was 305 nm. Larger aggregates (diameter > 100 nm) do not have a spherical shape like smaller ones (<100 nm). Instead, they look like clusters of smaller, spherically-shaped aggregates [32]. The aggregate diameters observed with TEM are smaller than those determined by Nano Sight. This can be explained by the TEM sample preparation, where the aggregate size or structure can change during sample preparation.

change during sample preparation. Generally, it is thought that only the free drug molecules, which have dissociated from the CD complex, are able to permeate cell membranes [1]. However, recent findings have revealed that CD molecules can enter the cells by endocytosis [33] and this may also be true for drug/CD complexes [34]. However, it appears highly unlikely that the large Generally, it is thought that only the free drug molecules, which have dissociated from the CD complex, are able to permeate cell membranes [1]. However, recent findings have revealed that CD molecules can enter the cells by endocytosis [33] and this may also be true for drug/CD complexes [34]. However, it appears highly unlikely that the large and hydrophilic CD aggregates can enter cells.
