*2.3. Fluorescent Microscopy of Fluorescently-Labeled Cyclodextrin Derivates to Study Cellular Uptake in Retinal Cultures*

Following this, we investigated the overall distribution of fluorescently-labeled CDs in the retina. To this end, RBITC-HPβCD, FITC-HPβCD, and FITC-RMβCD were used and the fluorescent intensity in the inner and outer retina was quantified (Figure 5) and compared to the control specimen to account for auto-fluorescence coming from the tissue itself. No difference between FITC-labeled CDs was observed for the inner and outer retina, indicating that the HPβCD and RMβCD distribute similarly within the retina. Therefore, the difference in cytotoxicity between the two compounds was likely due to their respective effect on the cells, as discussed above, and not because of differences in the overall tissue distribution.

Some subtle differences between FITC-labeled CDs were observed. For FITC-HPβCD, a string-like structure was seen, spanning across the inner and outer retina. This was not as prominent in cultures where FITC-RMβCD was applied (Figure 5a). This type of staining suggests that the CDs had partly been taken up by Müller glial cells.

When using a rhodamine-labeled CD, we observed an elevated signal in the retina. The signal-to-noise ratio was higher for RBITC-HPβCD than for FITC-HPβCD (Figure 5b,c). This allowed us to detect specific uptake in photoreceptors, which supported the result from the cytotoxicity analysis, where HPβCD mainly killed cells in the outer retina.

However, these results should be taken with a grain of salt as the dye labelling could also have affected cell uptake. First of all, we did not observe the same Müller cell uptake in retinas with RBITC-HPβCD, compared with retinas with FITC-HPβCD. Since the rhodamine molecule is positively charged, the rhodamine-conjugated CDs might bind to negatively charged cell membranes and extracellular matrix elements, possibly facilitating cellular uptake. FITC and RBITC derivatives behave differently and are internalized by different processes. The labelling increases the molecular weight and may alter the properties of the parental CDs, while these still retain high water solubility and cannot cross the cell membrane by passive diffusion. However, there are reports suggesting that endocytosis was observed in fluorescent CDs as well [34]. Müller cells have the capacity to assemble and secrete lipoproteins which can be utilized by photoreceptors or inner retinal neurons, serving as an intraretinal source of cholesterol [52]. This could explain the Müller cell uptake of HPβCD.

While the fluorescent CDs do not behave exactly as their non-fluorescent form, these studies enhance the understanding of the behavior of labelled CD derivatives at the tissue level [53]. Together with the toxicity analysis, these results may be employed in further the development of CDs as drugs or drug carriers for the treatment of retinal diseases.

**Figure 5.** Uptake of fluorescently-labeled CDs in the retina explant culture. (**a**) Overview of tissue sections from wild type (WT) mice explant cultures to which three different fluorescently-labeled CDs were added to the side facing down in the image. (**b**) Close-up images of the sections were taken for an analysis of the fluorescent signal. Images from control sections (without added CDs) were used to measure the intensity of the red and green auto-fluorescence (AF) coming from the tissue itself. For this analysis, the outer retina is defined as the area from the outer nuclear layer (ONL) to the retinal pigment epithelium (RPE), while the inner retina encompasses the area from the ganglion cell layer (GCL) to the inner nuclear layer (INL). Seg. = segments of the photoreceptors (inner and outer segment). \* membrane on which the retinal explant was cultured. (**c**) Analysis of the average fluorescent intensity coming from either the inner or outer retina. For both inner and outer retina, a high signal was detected from RBITC-labeled CDs, while the signal was lower and similar for FITC-labeled CDs. Results represent the mean ± SD for *n* = 3, where *n* is the number of animals. **Figure 5.** Uptake of fluorescently-labeled CDs in the retina explant culture. (**a**) Overview of tissue sections from wild type (WT) mice explant cultures to which three different fluorescently-labeled CDs were added to the side facing down in the image. (**b**) Close-up images of the sections were taken for an analysis of the fluorescent signal. Images from control sections (without added CDs) were used to measure the intensity of the red and green auto-fluorescence (AF) coming from the tissue itself. For this analysis, the outer retina is defined as the area from the outer nuclear layer (ONL) to the retinal pigment epithelium (RPE), while the inner retina encompasses the area from the ganglion cell layer (GCL) to the inner nuclear layer (INL). Seg. = segments of the photoreceptors (inner and outer segment). \* membrane on which the retinal explant was cultured. (**c**) Analysis of the average fluorescent intensity coming from either the inner or outer retina. For both inner and outer retina, a high signal was detected from RBITC-labeled CDs, while the signal was lower and similar for FITC-labeled CDs. Results represent the mean ± SD for *n* = 3, where *n* is the number of animals.

#### Some subtle differences between FITC-labeled CDs were observed. For FITC-**3. Materials and Methods**

#### HPβCD, a string-like structure was seen, spanning across the inner and outer retina. This *3.1. Materials*

was not as prominent in cultures where FITC-RMβCD was applied (Figure 5a). This type of staining suggests that the CDs had partly been taken up by Müller glial cells. When using a rhodamine-labeled CD, we observed an elevated signal in the retina. The signal-to-noise ratio was higher for RBITC-HPβCD than for FITC-HPβCD (Figure Randomly methylated β-cyclodextrin (RMβCD, also referred to as RAMEB) with a degree of substitution (DS) of 12.6 (molecular weight, MW 1312) was purchased from Wacker Chemie (Munich, Germany), and cholesterol from Sigma Chemical Co. (St. Louis, MO, USA), while 2-hydroxypropyl-β-cyclodextrin (HPβCD) with DS 4.2 (MW 1380) was

kindly provided by Janssen Pharmaceutica, Belgium. The solubility of HPβCD in water is >600 mg/mL, while that of RMβCD is >500 mg/mL [1].

6-deoxy-6-[(5/6)-fluoresceinylthioureido]-RMβCD, 6-deoxy-6-[(5/6)-fluoresceinyl thioureido]-HPβCD and 6-deoxy-6-[(5/6)-rhodaminylthioureido]-HPβCD were kindly provided by CycloLab, Budapest, Hungary. Milli-Q water and sterile saline solution were used for the preparation of CD solution and all other chemicals were commercially available products (Sigma-Aldrich, Saint Louis, MO, USA) of special reagent grade.
