**1. Introduction**

Cyclodextrins (CDs) are cyclic oligosaccharides consisting of (α-1,4-)-linked α-Dglucopyranose units with a hydrophobic central cavity and a hydrophilic outer surface. They can form water-soluble inclusion complexes with numerous lipophilic drugs provided that their structure (or part of it) fits in the CD cavity. No covalent bonds are formed or broken during the complexation and drug molecules in the complex are in rapid equilibria with free molecules in the aqueous complexation media [1]. The complexation affects many physicochemical properties of drugs, such as their aqueous solubility and chemical stability [2]. Natural CDs (i.e., αCD, βCD, and γCD) have a limited solubility in water and, thus, CD derivatives with an improved solubility have been synthesized and are currently used as solubilizing complexing agents in various marketed pharmaceutical products, particularly 2-hydroxypropyl-β-cyclodextrin (HPβCD) and sulfobutylether-β-cyclodextrin (SBEβCD), as well as randomly methylated β-cyclodextrin (RMβCD), although to a lesser extent [1]. CDs have also undergone extensive safety studies and have been approved by both the European Medicines Agency (EMA) and the Food and Drug Administration (FDA) for pharmaceutical use and in dietary supplements [3,4]. CDs have been the subject of numerous review publications [5–13].

**Citation:** Prajapati, M.; Christensen, G.; Paquet-Durand, F.; Loftsson, T. Cytotoxicity of β-Cyclodextrins in Retinal Explants for Intravitreal Drug Formulations. *Molecules* **2021**, *26*, 1492. https://doi.org/10.3390/ molecules26051492

Academic Editor: Marina Isidori

Received: 15 February 2021 Accepted: 3 March 2021 Published: 9 March 2021

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Drugs for the treatment of retinal diseases are most often delivered via intravitreal injections or implants, where the drug is administered directly into the vitreous humor, which is the hydrogel-type fluid that occupies the space between the lens and the retina. The vitreous mainly consists (99%) of water in a network of collagen and hyaluronic acid. In humans, the volume of the vitreous humor is about 4 mL [14]. Drug molecules must be dissolved in the aqueous vitreous to permeate into the retinal tissue. After an intravitreal injection, hydrophilic and high molecular weight drugs (e.g., proteins and peptides) are known to be excreted via an anterior route to the aqueous humor, while small lipophilic drugs easily pass the retina and are removed via the posterior choroidal flow [15]. The half-life of a dissolved drug in the vitreous humor is typically less than 10 to 24 h, where small molecules have a shorter half-life than biomolecules such as proteins [16,17]. It is expected that the hydrophilic CD molecules (molecular weight between about 1000 and 2000 Da) are readily removed from the vitreous humor after an intravitreal injection. CDs might be able to enhance the retinal delivery of poorly soluble lipophilic drugs after intravitreal administration.

The ability of CDs to complex biomolecules depends upon the molecular structure and the CD binding constants and generally follows the order carbohydrates << nucleic acids << proteins < lipids [18]. Therefore, most biological effects of CDs are based on their interaction with membranes rich in lipids and their ability to extract lipids (e.g., phospholipids and cholesterol) from the plasma membrane [18]. In ophthalmic drug delivery, CDs have mainly been applied topically to the eye, with little or no reports on their intravitreal administration [19–21]. Although hydrophilic CDs, such as HPβCD and SBEβCD, have been shown to be well-tolerated when applied topically to the eye, with no detectable side effects, their parent βCD and RMβCD are known to extract cholesterol from cell membranes and form cholesterol/CD complexes [22–24]. Furthermore, Nociari et al. observed that βCD extracted lipofuscin bisretinoids from the retinal pigment epithelium (RPE) [25]. Based on these observations, it was proposed that CDs can be used to develop therapeutic candidates for many retinal degenerative diseases, such as Stargardt disease, which is an inherited form of macular degeneration causing central vision loss and sometimes referred to as juvenile macular degeneration. The cause of Stargardt disease is characterized by an abnormal accumulation of lipofuscin in the retina [26]. Similarly, the topical administration of HPβCD as eye drops over 3 months has shown a significant efficacy in reducing amyloid-beta and inflammation in aged mouse retina, consequently improving the retinal function by elevating retinal pigment epithelium-specific protein 65 (RPE 65), which is a key molecule in the visual cycle [27]. Even oral HPβCD treatment in mice resulted in a reduction in the retinal cholesterol content and changes in the retinal sterol, gene, and protein levels [28].

Our study aimed to examine the cytotoxicity of CDs in mouse retinal explants using βCD derivatives to explore their applicability for future ophthalmic formulations. Additionally, fluorophore-conjugation to the same CDs was used to trace the uptake and localization within the retina (Figure 1).

**Figure 1.** Chemical structures of (**a**) 2-hydroxypropyl-β-cyclodextrin (HPβCD), (**b**) randomly methylated β-cyclodextrin (RMβCD), (**c**) 6-deoxy-6-[(5/6)-fluoresceinylthioureido]-HPβCD (FITC-RMβCD), (**d**) 6-deoxy-6-[(5/6)-fluoresceinylthioureido]-RMβCD(FITC-HPβCD), and (**e**) 6-deoxy-6- [(5/6)-rhodaminylthioureido]-HPβCD (RBITC-HPβCD). **Figure 1.** Chemical structures of (**a**) 2-hydroxypropyl-β-cyclodextrin (HPβCD), (**b**) randomly methylated βcyclodextrin (RMβCD), (**c**) 6-deoxy-6-[(5/6)-fluoresceinylthioureido]-HPβCD (FITC-RMβCD), (**d**) 6-deoxy-6-[(5/6) fluoresceinylthioureido]-RMβCD(FITC-HPβCD), and (**e**) 6-deoxy-6-[(5/6)-rhodaminylthioureido]-HPβCD (RBITC-HPβCD).
