Dissertation
Ocular drug delivery systems for preserving corneal functions
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Summer 2023
DOI: 10.25820/etd.007127
Abstract
Fuchs endothelial corneal dystrophy (FECD), a progressive polygenic disease that causes degeneration of the corneal endothelium, is the leading indication for corneal transplantation in the U.S. Characteristically, loss of corneal endothelial cells and formation of degenerative extracellular “guttae” result in failure to maintain appropriate corneal hydration through active ion pumping to counter the passive leakage of aqueous humor. Defective cellular metabolism, impaired mitochondrial function, and increased cell death are major problems that adversely affect donor tissues during hypothermic preservation prior to transplantation. We have recently discovered that ubiquinol – the reduced and active form of coenzyme Q10 and a powerful antioxidant – significantly enhances mitochondrial function and reduces apoptosis in human donor corneal endothelial cells. However, ubiquinol is highly lipophilic, underscoring the need for an aqueous-based formulation of this molecule. Herein, we report a highly dispersible and stable formulation comprising a complex of ubiquinol and gamma cyclodextrin (γ-CD) for use in aqueous-phase ophthalmic products. Complexed ubiquinol, also referred to as solubilized ubiquinol, showed significantly higher stability compared to free ubiquinol in different aqueous ophthalmic products. Complexed ubiquinol was more effective at lowering reactive oxygen species (ROS) compared to free ubiquinol. Complexed ubiquinol inhibited lipid peroxidation and protected human corneal endothelial cells (HCECs) against erastin-induced ferroptosis. No evidence of cellular toxicity was detected in HCEC-B4G12 cells after treatment with complexed ubiquinol. Topically applied complexed ubiquinol penetrated the entire thickness of human donor corneas with markedly greater ubiquinol retention in the endothelium compared to free ubiquinol.
In the second study, FECD pathogenesis was studied to find therapeutics to prevent FECD progression, which will reduce the demand for corneal transplantation. FECD pathogenesis is linked to impaired response to oxidative stress and environmental ultraviolet A (UVA) exposure. Although UVA is known to cause nonapoptotic oxidative cell death resulting from iron-mediated lipid peroxidation, ferroptosis has not been characterized in FECD. Herein, we investigated the roles of genetic background and UVA exposure in causing HCEC degeneration in FECD. Using ungenotyped FECD patient surgical samples, we found increased levels of cytosolic ferrous iron (Fe2+) and lipid peroxidation in end-stage diseased tissues compared with healthy controls. Using immortalized and primary cell cultures modeling the TCF4 intronic trinucleotide repeat expansion genotype, we found altered gene and protein expression involved in ferroptosis compared to controls including elevated levels of Fe2+, basal lipid peroxidation, and the ferroptosis-specific marker transferrin receptor 1. Increased cytosolic Fe2+ iron levels were detected after physiologically relevant doses of UVA exposure, indicating a role for ferroptosis in FECD disease progression. Finally, FECD cultured cells were more prone to RSL3 induced ferroptosis than healthy controls with deferoxamine chelation indicating increased susceptibility in FECD cells, and cell death from experimentally induced ferroptosis was preventable using complexed ubiquinol indicating a role for anti-ferroptosis therapies in FECD. This investigation demonstrates that genetic background and UVA exposure contribute to iron-mediated lipid peroxidation and cell death in FECD, and provides the basis for future investigations of ferroptosis-mediated disease progression in FECD.
In the third study, transferrin-targeted, ripasudil-loaded liposomal drug delivery systems (LNPs) were developed for treating FECD. Ripasudil is known to have a stimulatory effect on the proliferation and migration of HCECs. In an earlier study, ripasudil has shown the capability to repair damaged corneas and is being used as an adjuvant therapy in corneal transplantation. Ripasudil therapy comes with potential side effects, and the current dosage regimen is not patient-friendly. To solve these issues, a lipid-based targeted drug delivery system was designed. The ammonium sulfate salt gradient active loading method was found to be superior in loading ripasudil into LNPs. The LNPs were characterized for their size, polydispersity, surface charge, morphology, drug loading, drug encapsulation efficiency, and transferrin conjugation efficiency. LNPs were stable at both 4 and 25°C without showing any sign of rapid change in size and did not show drug leakage during storage. LNPs were efficient in crossing the full thickness of the mouse cornea and delivering cargo to CECs. LNPs were well tolerated and did not show any local or systemic side effects in the Col8A2Q455K FECD mouse model. The efficacy of LNPs in preventing FECD progression is under ongoing investigation.
In the fourth study, integrin-targeted, ubiquinol-loaded PLGA polymeric nanoparticles (UBNPs) were developed as a superior solubility enabling formulation for ubiquinol. UBNPs were prepared by the nanoprecipitation method and characterized for their size, polydispersity, surface charge, morphology, drug loading, and encapsulation efficiency. UBNPs protected ubiquinol from oxidation and dramatically improved the efficacy of ubiquinol in preventing RSL3-induced ferroptosis in the FECD cell cultures. Integrin-targeted UBNPs decorated with cRGD were superior to non-targeted UBNPs in inhibiting ferroptosis and significantly outperformed complexed ubiquinol. In a pilot study, UBNPs showed a decrease in average guttae development in the Col8A2Q455K FECD mouse model. UBNPs also increased the percent hexagonality of CECs in the mouse model. UBNPs were well tolerated and did not show any signs of side effects.
In summary, we have successfully developed two solubility enabling formulations for ubiquinol to prevent FECD progression. In another therapeutic approach, we have developed a lipid-based drug delivery system for ripasudil to repair damaged corneas in FECD. This dissertation opens the path to develop a first-line therapy in preventing FECD progression.
Details
- Title: Subtitle
- Ocular drug delivery systems for preserving corneal functions
- Creators
- Sanjib Saha
- Contributors
- Aliasger K. Salem (Advisor)Mark A. Greiner (Committee Member)Lewis L. Stevens (Committee Member)Reza Nejadnik (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Pharmacy
- Date degree season
- Summer 2023
- DOI
- 10.25820/etd.007127
- Publisher
- University of Iowa
- Number of pages
- xxvii, 198 pages
- Copyright
- Copyright 2023 Sanjib Saha
- Language
- English
- Date submitted
- 05/17/2023
- Description illustrations
- Illustrations, tables, graphs, charts
- Description bibliographic
- Includes bibliographical references (pages 183-198).
- Public Abstract (ETD)
The cornea is the clear outer layer of the eye, which works as a structural barrier and window to control the entry of light into the eye and helps in focusing it on the retina. Fuchs endothelial corneal dystrophy (FECD) is a genetic disorder that affects the cornea and makes it cloudy. Currently, around 6.1 million people in the USA are affected by FECD, and there is no first-line therapy available. Corneal transplantation is the only way to cure this disease in its advanced stages. Beside genetic factors, oxidative stress, UVA irradiation, and ferroptosis have been known to play crucial roles in the pathogenesis of FECD. Previously, we have seen that ubiquinol is effective in preserving corneal function to prevent FECD progression. Ubiquinol is very hydrophobic and unstable, and comes with multiple formulation-related challenges, resulting in poor aqueous solubility and bioavailability in its conventional form. To solve these formulation challenges, we have developed two solubility-enabling formulations for ubiquinol, including guest-host physical complexation with -CD (UBCD) and integrin receptor-targeted PLGA (poly(lactic-co-glycolic acid)) polymeric nanoparticles (UBNP). In another therapeutic approach, we have developed transferrin receptor-targeted liposomal drug delivery systems (LNPs) for ripasudil to repair damaged corneas in FECD by increasing cell proliferation and migration
In Chapter 2, complexed ubiquinol was developed by the physical complexation of ubiquinol with -CD, and was found to be well dispersed in aqueous phase ophthalmic products. It protected ubiquinol from oxidation and increased its efficacy in preserving corneal function by scavenging free radicals and preventing ferroptosis. This formulation was efficient in delivering ubiquinol to the corneal endothelial cells (HCECs) in human donor corneas.
In Chapter 3, ferroptosis susceptibility in FECD was systematically investigated. We have shown that iron-mediated lipid peroxidation, known as ferroptosis, plays a crucial role in the pathogenesis of FECD. We have found evidence of the hallmarks of ferroptosis in FECD, including upregulation of a specific ferroptosis marker (transferrin receptor 1), higher toxic iron accumulation, upregulation of lipid peroxidation, and downregulation of key antioxidant defense mechanisms. UVA irradiation was also found to be causing toxic iron release, resulting in higher ferroptosis susceptibility. We have also shown that solubilized ubiquinol was efficient in preventing lipid peroxidation and ferroptosis in FECD cell cultures.
In Chapter 4, lipid-based drug delivery systems (LNPs) were developed for ripasudil with transferrin receptor-targeted delivery functionality. This delivery system was designed to increase the efficacy of ripasudil in preventing FECD progression by increasing cell proliferation and migration. It was also designed to mitigate off-target side effects and increase patient compliance. LNPs had desirable properties and the capability to cross the full thickness of the cornea to deliver cargo to CECs in mouse. LNPs were well tolerated in a preclinical mouse study. The efficacy of LNPs in preventing FECD progression is under ongoing investigation.
In Chapter 5, PLGA polymeric nanoparticle-based drug delivery systems (UBNPs) were designed for ubiquinol with integrin receptor targeting capability. UBNPs protected ubiquinol from oxidation and improved its efficacy in preventing ferroptosis in FECD. It also outperformed complexed ubiquinol. UBNPs remarkably decreased disease progression in the FECD mouse model and were well tolerated.
In summary, we have developed two drug delivery systems for ubiquinol to protect it from oxidation and increase its bioavailability and efficacy in preventing FECD progression. In addition, we developed a liposomal drug delivery system for ripasudil to improve its efficacy and mitigate side effects while treating FECD.
- Academic Unit
- Pharmacy; Craniofacial Anomalies Research Center
- Record Identifier
- 9984454644502771
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