Design and formulation of nanocarriers for the treatment of diabetic eye diseases

DOCTORAL THESIS DESIGN AND FORMULATION OF NANOCARRIERS FOR THE TREATMENT OF DIABETIC EYE DISEASES Author Axel KATTAR Supervisor: Carmen Alvarez-Lorenzo Tutor: Carmen Alvarez-Lorenzo DOCTORAL PROGRAM IN DRUG RESEARCH AND DEVELOPMENT SANTIAGO DE COMPOSTELA Acknowledgements First and foremost I would like to thank my thesis director, Carmen Alvarez-Lorenzo for giving me the opportunity to work in her research group and to have always been vigilant to make sure that my work was of the highest standard. Thank you for always being available, week in and week out, in Santiago or during the secondments, to correct manuscripts or provide feedback on experimental ideas. Secondly I would like to thank my supervision team, Professor Angel Concheiro, Professor Anuj Chauhan, Professor Hákon Hrafn Sigurðsson, and Dr. Paolo Gasco for the effort put in to advise the course of my thesis and their hospitality during secondments. I would like to extend a special thanks to the Chauhan cluster, with Anuj, Zach, Raj, and Bikram who have made my secondment in Colorado that much more fun. research and innovation program for the financial support through the -Curie Actions grant agreement Nº 813440 (ORBITAL Ocular Research by Integrated Training and Learning). Thank you to Tess and Larry for being an anchor point with anything related to the training program, and all the ESRs for the support over the years. I would like to thank all the people that have made my labwork possible, Ana Filipa, Iago, Diana, Nicola, Maria, Mariana, Paola, Lucia, Xian, Patri, Carol, Alex, Emilio without whom the days would have seemed a lot longer. I would like to extend my thanks to Bebert, Chen, Sami, Lluis, Tici, Ashraf and all the friend I have made in Santiago that have made the town my home. toujours été un modèle pour quotidien. Ik wil mijn moeder bedanken om zo vaak mij te hebben bezocht hier in het zuiden, en altijd positief is gebleven ondanks de omstandigheden die niet altijd makkelijk waren. Bedankt voor je blik die mij heeft laten zien dat ik begrepen kan zijn. Ook wil ik de Adventure Animals bedanken om mij vanaf mijn vertrek uit Enschede nooit hebben laten valllen, altijd in contact te zijn gebleven, en een vriendengroep voor het leven te zijn. vécu mes hauts et mes bas avec moi. Merci pour avoir partagé tout ces moments et les avoir rendu tous plus heureux. INDEX RESUMO ............................................................................................. 1. INTRODUCTION ........................................................................ 15 1.1 DIABETIC EYE DISEASES ...................................................17 1.2 DELIVERY OF DRUGS TO DIABETIC EYE.......................20 1.2.1 Main drug classes and current administration routes ....... 20 1.2.2 Drugs, biologics, and gene therapy in clinical trials.........27 1.3 SELF-ASSEMBLED NANOCARRIERS FOR TOPICAL DIABETIC EYE DRUGS ..............................................................36 1.3.1 The self-assembly process.................................................. 37 1.3.2 Applications to diabetic eye ...............................................41 1.3.2.a Polymeric micelles ..........................................................41 1.3.2.b Liposomes........................................................................47 1.3.2.c Niosomes.......................................................................... 52 1.4 CONCLUSION.........................................................................56 1.5 EXPERT OPINION..................................................................57 1.6 REFERENCES .........................................................................60 2. AIMS .............................................................................................. 8 3. FORMULATION AND CHARACTERIZATION OF EPALRESTAT-LOADED POLYSORBATE 60 CATIONIC NIOSOMES FOR OCULAR DELIVERY..................................... 9 3.1 INTRODUCTION ....................................................................95 3.2 MATERIALS AND METHODS..............................................98 3.3 RESULTS...............................................................................111 3.3.1 Niosome Characterization................................................111 3.3.2 Epalrestat Release ............................................................118 3.3.3 HET-CAM Assay...............................................................120 3.3.4 Gluc-HET Assay ...............................................................121 3.3.5 Zebrafish Embryotoxicity Assay .......................................122 3.3.6 Corneal and Scleral Permeation ......................................123 3.3.7 IR-Raman..........................................................................125 3.4 DISCUSSION .........................................................................129 3.5 CONCLUSIONS.....................................................................135 3.6 REFERENCES........................................................................136 4. STABILITY OF NIOSOMES FOR OPHTALMIC ADMINISTRATION THROUGH LANGMUIR MONOLAYER STUDIES OF NON-IONIC SURFACTANTS AND DOTMA ...149 4.1 INTRODUCTION...................................................................149 4.2 MATERIALS AND METHODS............................................153 4.2.1 Materials...........................................................................153 4.2.2 Monolayer preparation.....................................................153 -A isotherm measurements..............................................154 4.2.4 Compressibility study........................................................155 4.2.5 Brewster Angle Microscopy..............................................156 4.3 RESULTS AND DISCUSSION .............................................157 4.3.1 Effect of monolayer composition ......................................157 4.3.1.1 Single component system...............................................157 4.3.1.2 Binary systems ...............................................................159 4.3.1.3 Tertiary systems.............................................................166 4.3.2 Effect of temperature ........................................................173 4.3.3 Effect of the subphase.......................................................175 4.3.4 Brewster Angle Microscopy ............................................. 176 4.4 CONCLUSION.......................................................................177 4.5 REFERENCES .......................................................................178 5. FORMULATION AND CHARACTERIZATION OF OLEOGELS FOR TOPICAL ADMINISTRATION OF EPALRESTAT ............................................................................... 185 5.1 INTRODUCTION ..................................................................185 5.2 MATERIALS AND METHODS............................................187 5.2.1 Materials. ......................................................................... 187 5.2.2 Oleogel formulation. ........................................................ 187 5.2.3 Oleogel characterization..................................................188 5.2.4 Epalrestat release.............................................................189 5.2.5 HET-CAM......................................................................... 190 5.2.6 Corneal and scleral permeation.......................................190 5.2.7 IR-RAMAN spectroscopy..................................................191 5.3 RESULTS...............................................................................193 5.3.1 Oleogel characterization..................................................193 5.3.2 Epalrestat release.............................................................197 5.3.3 HET-CAM......................................................................... 203 5.3.4 Corneal and scleral permeation.......................................204 5.3.5 IR-RAMAN .......................................................................206 5.4 DISCUSSION.........................................................................207 5.5 CONCLUSION.......................................................................212 5.6 REFERENCES .......................................................................213 6. COMPARISON OF IN VIVO EPALRESTAT OCULAR DISTRIBUTION FROM NIOSOMES, MICELLES AND OLEOGELS ....................................................................................221 6.1 INTRODUCTION...................................................................221 6.2 MATERIALS AND METHODS............................................224 6.2.1 Materials...........................................................................224 6.2.2 Experimental design .........................................................225 6.2.3 Formulations preparation ................................................226 6.2.4 Viscosity............................................................................227 6.2.5 In vivo release...................................................................227 6.2.5.1 Epalrestat quantification in tear fluid ...........................227 6.2.5.2 Epalrestat quantification in the tissues..........................228 6.2.6 Statistical analysis ............................................................229 6.3. RESULTS AND DISCUSSION ............................................229 6.3.1 Characterization of the formulations................................229 6.3.2 Viscosity............................................................................230 6.3.3 In vivo experiment.............................................................232 6.3.3.1 Epalrestat quantification in the lacrimal fluid ..............235 6.3.3.2 Epalrestat in the tissues.................................................237 6.4 CONCLUSION .......................................................................241 6.5 REFERENCES........................................................................242 7. CONCLUSIONS ......................................................................... 25 ANNEXES........................................................................................ 25 RESUMO AXEL KATTAR 6 Resumo 7 AXEL KATTAR 8 Resumo 9 AXEL KATTAR 10 Resumo 11 AXEL KATTAR 12 13 INTRODUCTION The work described in this chapter was published in Diabetic eye: associated diseases, drugs in clinic, and role of self-assembled carriers in topical treatment, Expert Opinion on Drug Delivery, 18(11), 2021, 1589-1607, authored by Axel Kattara, Angel Concheiroa, and Carmen Alvarez-Lorenzoa aDepartamento de Farmacología, Farmacia y Tecnología Farmacéutica, I+D Farma Group (GI-1645), Facultad de Farmacia, Instituto de Materiales (iMATUS) and Health Research Institute of Santiago de Compostela (IDIS), Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain. 15 1. INTRODUCTION Diabetes mellitus currently affects 8.5% people worldwide, and it is expected to impact on the lives of 570 million people in 2025 [1]. There are five different forms of diabetes, with diabetes type 1 (failure in the production of insulin) and type 2 (deficient insulin sensitivity) being the most common. The other three forms are monogenic diabetes, which is hereditary due to a single gene mutation; gestational diabetes, related to pregnancy; and cystic fibrosis-related diabetes, which is linked to scarring of the pancreas that leads to insulin abnormalities. For people with type 1 diabetes, the immune system attacks pancreatic cells responsible for the production of insulin, disrupting their normal function. Both genetic and environmental factors have been identified as causal agents. Type 2 diabetes is indicative of insulin resistance, which may be caused by excess body weight. Diabetes is considered a pandemic disease with an increasing morbidity and the highest rate in years of life lost due to disability in both high-income and lower-middle-income countries [2]. Such a high incidence results in an increase in diseases secondary to diabetes. Diabetes-associated diseases range from cardiovascular problems to diabetic neuropathy including kidney failures and ocular diseases. Indeed, the term diabetic eye disease has a broad meaning as it may encompass multiple illnesses in different parts of the eye, mainly diabetic retinopathy, macular edema, cataracts, glaucoma, and keratopathy [3 6]. AXEL KATTAR 22 Efficient ocular drug delivery is a difficult goal to reach in any case. Depending on the administration procedure and the dosage form, ocular barriers can be either physiological or anatomical, and either static or dynamic [39]. Different barriers located in the anterior and the posterior segments protect the eye against foreign substances coming from outside or inside the body (e.g. the bloodstream). Also, the goal of drug delivery can be different depending on the target cells. If the drug must reach a tissue protected by many barriers, drug permeation through these barriers is critical; prodrugs, penetration enhancers, and encapsulation in nanocarriers may be helpful tools [40,41]. If the aim is to continuously supply the drug to an area with important dynamic turnover of fluids, sustained drug delivery systems may be required [42]. Regarding topical administration, the eye is dynamically protected by the blinking reflex, the tear clearance rate, andthe nasolacrimal drainage. Adhesion to the corneal surface and promotion of the penetration might overcome these barriers. Drugs can follow three routes: corneal, scleral, and conjunctival [43 47]. Major static corneal barriers are the corneal epithelium, which limits the absorption of macromolecules and hydrophilic drugs through tight junctions, and the corneal stroma, which limits the penetration of lipophilic molecules due to its high aqueous content [44]. Thus, midlipophilic drugs are the most suitable candidates for corneal penetration and subsequent diffusion through aqueous humor for intraocular distribution. The iris and the nonpigmented ciliary epithelium further block drugs from passing to the aqueous humor, making make up the blood aqueous barrier [47]. Furthermore, the aqueous humor flow from the ciliary body to the cornea counteracts the diffusion of hydrophilic molecules trying to enter further in the eye. On this blood aqueous barrier and also 1. Introduction 23 on the corneal epithelium, there are efflux pumps that expel the drugs back to the front of the eye [48]. The conjunctiva opposes to drug entry in the eye tissues mainly due to the presence of conjunctival blood capillaries and lymph vessels, which reroute a major fraction of the drug dose to the blood stream. Drug access through the conjunctiva to the posterior segment may occur via passive or active transport [45]. Although there are tight junctions among conjunctival epithelium cells, polar solutes up to 20 kDa can enter through paracellular diffusion across 5-nm pores [49]. Peptides and proteins may find the additional barrier of enzymatic degradation and require co-administration with a protease inhibitor [50]. Lipophilic drugs can still penetrate better via the transcellular route; the surface area is larger than for paracellular pathway, although efflux pumps pose a relevant challenge. Intense active carrier-mediated transport occurs at conjunctiva for some ions and nutrients, which may be exploited for drug and prodrug absorption [46]. Drugs encapsulated in nanocarriers take benefit of the additional pathway of endocytosis, which is feasible both in cornea and conjunctiva [51 53]. Once conjunctiva is crossed, the drug can move through sclera into the uvea and the retinal pigmented epithelium, and then move forward to neural retina and vitreous humor. The sclera performs as a size exclusion barrier, with the permeability decreasing exponentially with molecular radius and lipophilicity [54]. The sclera is negatively charged at physiological pH and therefore electrostatic interactions must be choroid complex traps positively charged lipophilic drugs [55], and sin with age (due to calcification of elastin and crosslinking of collagen) drug diffusion is hindered in elderly patients. AXEL KATTAR 24 Systemic administration of ocular drugs is compromised by the blood-retinal barrier. The inner limiting membrane of the retinal pigment epithelium prevents passage of highmolecular-weight molecules from blood to vitreous and vice versa [56]. Müller cells and astrocytes form tight junctions to regulate the passage of molecules between the outer choroid and the inner retina. Although the information on ocular bioavailability after drug systemic administration in humans is limited, some reports evidenced that for small drugs, such as ciprofloxacin, similar drug levels can be obtained in aqueous humor after topical instillation of the free drug or oral administration. The levels in vitreous humor are commonly higher after oral administration, but at expenses of exposing the whole organism to high drug dose [57]. Also, interestingly, the drug can be found in tear fluid after oral administration but not because of distribution through the eye, as reported for cyclosporine A [58]. The blood-retinal barrier efficiently prevents cyclosporine A diffusion from blood to the anterior segment, except during concomitance of inflammatory processes [59]. Pharmacological treatments intended to stop ocular damage caused by hyperglycemia or at least delay the process rely on (i) reducing IOP (Table 1.2), (ii) blocking the abnormal growth of blood vessels, or (iii) inhibiting negative chemical pathways. Drugs like prostaglandins [60], rho kinase inhibitors [61], nitric oxides [62], or miotic/cholinergic agents [63] drain ocular fluids. Alpha-adrenergic a -blockers, and carbonic anhydrase inhibitors lower the amount of fluid produced in the eye [64]. Both strategies result in a lowering of the IOP and are usually addressed using eye drops (Table 1.2). Additionally, new drug candidates are intended to act on the heme oxygenase 1 (HO-1)/carbon monoxide (CO) physiological pathway that regulates the IOP. The HO1 produces protection against ischemic insult by producing CO, which has anti-inflammatory properties. Incidentally, CO protects retinal 1. Introduction 25 ganglion cells from ischemic/reperfusion injury. Decreased CO levels have been related to increased IOP and, therefore, drugs that release CO may be useful in glaucoma treatment [65]. Table 1.2 . Some active substances of medicines used to reduce the IOP. Data from the European Medicines Agency, https://www.ema.europa.eu/en/medicines Drug class Drug Dosage form Prostaglandin or analog Travoprost Eye drop Bimatoprost Eye drop Latanoprost Eye drop Unoprostone Eye drop Prostaglandin analog nitric oxide Latanoprostene bunod Eye drop Rho kinase inhibitor Netarsudil Eye drop Ripasudil Eye drop Miotic agent Pilocarpine Eye drop Cholinergic agonist Carbachol Eye drop or intraocular injection Alpha-adrenergic agonist Brimonidine Eye drop Apraclonidine Eye drop Beta blocker Betaxolol Eye drop or oral tablet Timolol Eye drop Carteolol Eye drop Carbonic anhydrase inhibitor Methazolamide Oral tablet Acetazolamide Eye drop Brinzolamide Eye drop Dorzolamide Eye drop The growth of abnormal ocular blood vessels can be handled with anti-vascular endothelial growth factor (anti-VEGF) drugs [66]. AXEL KATTAR 26 Bevacizumab and ranibizumab, which are respectively full antibody and antibody fragment that bind VEGF-A, and aflibercept, a recombinant protein that traps VEGF-A and VEGF-B, are the cornerstones for the therapy of diabetes-related macular edema and retinopathy [67]. They require intravitreal injection, which is not absent of complications [68]. Intraocular injections should be used as infrequently as possible, according to pro re nata or treat-and-extend protocols [69]. Biodegradable delivery systems that sustain intraocular release avoiding multiple treatment and maintaining drug stability are under investigation [70,71]. Since each available anti-VEGF agent interacts quite differently with VEGF, characterization of the molecular interactions can improve the design of novel biological drugs potentially useful in clinical practice [72]. Hyperglycemia is also responsible for triggering the polyol pathway. Under normoglycemic conditions, the Embden Meyerhof Parnas catabolism route that transforms glucose into pyruvate, NADH, and ATP becomes saturated. Consequently, the polyol pathway, which commonly transforms 3% glucose, enters into action with the participation of two enzymes: (i) aldose reductase that transforms glucose into sorbitol with the consumption of NADPH and (ii) sorbitol dehydrogenase that slowly converts sorbitol into fructose while consuming NAD+. The polyol pathway, which is very active in retina and lens, metabolizes more than 30% glucose under diabetic conditions [73]. Accumulation of sorbitol causes osmotic stress, triggers leukocyte accumulation, disrupts blood-retinal barrier, favors cells apoptosis, and starts a cascade of oxidative stress-mediated reactions [74]. The excess of fructose acts as precursor of advanced glycation-end products (AGEs). In this context, aldose reductase inhibitors are gaining increased attention, and epalrestat is approved in some countries for oral administration. As an alternative, drugs that accelerate the metabolic 1. Introduction 27 rate of sorbitol dehydrogenase and, thus, decrease the levels of sorbitol are being tested [73]. In the later stages of the disease, laser treatment (mainly for photocoagulation) or surgery (when blood vessel leakage becomes excessive or there is scar tissue) can be proposed [75,76]. Vitreoretinal surgery, for example, involves the removal of part of the vitreous and 1.2.2 Drugs, biologics, and gene therapy in clinical trials Relevance of the morbidity caused by diabetes on eye structures is exemplified by the 868 clinical studies in phases 1 to 4 in February 2021 Refinement of the information to select recruiting, enrolling, active, terminated, or completed trails rendered an outcome of 657 studies, with an ample distribution worldwide (Figure 1.1). Most clinical trials are focused on the efficacy and safety of new molecules or novel administration routes, drug combinations, or delivery systems such as implants, microparticle depot formulations, or biopolymer antibody conjugates. Microneedle patches that can be applied onto cornea or sclera for direct drug delivery in the aqueous or vitreous humor, respectively, are gaining increasing interest, although still in the preclinical phase [78 80]. AXEL KATTAR 28 Figure 1.1. Regional distribution of clinical trials related to diabetic eye. Data source: filters were Recruiting, Active not recruiting, Completed, Enrolling by invitation, and Terminated. Made by the author of this Thesis. Most clinical trials related to diabetic eye refer to the conditions macular edema (Table 1.3) and retinopathy (Table 1.4), and most interventions deal with drugs or biologics, particularly intravitreal injection of antibodies. However, the interest for oral administration as well as topical formulations does not decrease but is gaining attention, spearheaded by the search for novel active substances with improved ocular bioavailability and new therapeutic targets. Intense research on small molecules that perform as anti-inflammatory (e.g. nepafemac, loteprednol etabonate) or as anti-angiogenic/angiolytic (e.g. EXN407, OC-10X) is being carried out. 29 Table 1.3. Pharmacological treatments in clinical trials for diabetes-related macular edema classified as a function of the administration route, drug/biologic active substance, and number of clinical studies Administration route Drug/Biologic class Active substance Number of clinical trials Intravitreal Antibodies or blockers Aflibercept and biosimilars 42 Anti-angiopoietin-2 antibody REGN910 1 REGN910-3 (co-formulation of REGN910 and aflibercept) 1 Anti-erythropoietin LKA651 1 Anti-PlGF recombinant monoclonal antibody 1 Anti-ROBO4 antibody DS-7080a 1 Bevacizumab 16 Bevasiranib 1 Tofacitinib (BI 764,524) 1 Conbercept (KH902) 1 Faricimab 1 Infliximab, anti1 OPT-302, anti-VEGF-C and anti-VEGF-D 1 Pegaptanib 7 Ranibizumab 40 Teprotumumab 1 Small molecules Dexamethasone 31 Fluocinolone acetonide 9 Triamcinolone acetonide 11 Anti-VEGF drugs 7 KVD001 plasma kallikrein inhibitor 2 1. Introduction AXEL KATTAR 30 AR-13,503, small-molecule inhibitor of both Rho kinase and protein kinase C 1 UBX1325, inhibitor of Bcl-xL (antiapoptotic regulatory protein) 1 Peptides AXT107, tyrosine kinase blocking collagen IV derived peptide 1 Luminate (Alg-1001) integrin inhibitor 1 Proteases Ocriplasmin 1 Gene therapy ADVM-022 gene therapy (AAV.7m8aflibercept) 1 iCo-007, a single-stranded antisense that degrades messenger RNA (mRNA) 1 PF-04523655, small-interfering RNA (siRNA) 1 Oral Small molecules GSK2798745, transient receptor potential vanilloid 4 (TRPV4) channel blocker 1 Aliskiren 1 Danazol 1 Fenofibrate/pemafibrate 2 Imatinib mesylate (YD312) 1 Levosulpiride 1 Minocycline 1 MS-533 protein kinase inhibitor 1 Ruboxistaurin 1 Semaglutide 1 Dietary supplements Alzer®, Diamel®, others 2 Topical eyedrops Small molecules Bromfenac 1 Dexamethasone 2 Diclofenac 1 31 EXN407, specific serine/threonine-protein kinase 1 (SRPK1) inhibitor 1 FOV2304, inhibitor of bradykinin B1 receptor 1 Fluocinolone acetonide 1 Ketorolac 3 Nepafenac 5 OC-10X tubulin inhibitor 1 Loteprednol etabonate 1 Mecamylamine nonspecific nACh receptor blocker 1 SF0166 small1 Vitamin E 2 Peptides Elamipretide (MTP-131), mitochondriatargeting peptide 1 Intravenous Small molecules Methotrexate 1 Intramuscular Peptides Octreotide acetate in microspheres 1 Episcleral Small molecules Dexamethasone implant 1 Subconjunctival Antibody Bevacizumab 1 Small molecule Rapamycin 2 Subcutaneous Small molecule Razuprotafib (AKB-9778), inhibitor of VEPTP (vascular endothelial protein tyrosine phosphatase) 2 Sub-macular Antibodies Ranibizumab 1 Suprachoroidal Gene therapy RGX-314 (AAV8 vector containing a transgene for anti-VEGF fab) 1 1. Introduction AXEL KATTAR 38 instability limit their practical use as drug carriers [109]. Micelles may extract relevant components from cells and, if they are made of ionic surfactants, may alter vital cell pathways, compromising the safety of the formulation [110]. Moreover, the self-assembly is a reversible process, and assembled and non-assembled components are in a fragile equilibrium that can be displaced in any direction quite rapidly. Although the volume of liquid at the eye surface is less than in other administration routes, all topically instilled formulations are exposed to relevant tear turnover [111]. Thus, one drop of common micelle formulation in contact with the lachrymal fluid undergoes a rapid decrease in surfactant concentration, and below the CMC, the micelle rapidly disassembles into their individual components [112]. Consequently, few improvements (if any) compared to the instillation of the drug solely solution can be noticed, with the aggravating toxic effects that surfactant molecules may have on eye surface. When searching for more biocompatible and stable selfassembled nanocarriers, two different strategies came up: core-shell polymeric micelles and bioinspired bilayered vesicles. This classification relies on the arrangement of the components, but as explained below, the same component can lead to micelles exhibiting a variety of shapes or to bilayered vesicles depending on its concentration and the presence of certain additives [113]. For the sake of clarity, polymeric micelles are considered here as supramolecular assemblies of amphiphilic polymers (unimers) that have a core formed by apolar segments and a shell formed by polar segments [112]. Thus, a gradient in polarity is observed from inside to outside [113]. Differently, bilayered vesicles are defined as quasispherical structures in which the amphiphilic components are assembled in cell membrane-like bilayers (Figure 1.2). Two or more bilayers can be arranged concentrically being separated from each other by an aqueous compartment. Therefore, bilayered vesicles do not 39 exhibit progressive gradients in polarity, but stepped apolar-polar regions that can respectively encapsulate hydrophobic and hydrophilic compounds [114,115]. Figure 1.2. Dependence of the architecture of the self-assembled nanocarrier on the critical packing parameter (CPP). Made by the author of this Thesis. The amphiphilic component determines the physicochemical and biological properties of the self-assembled nanocarrier. The selfassembly process is regulated by hydrophobic interactions in aqueous environment, and thus the critical packing parameter (CPP) and the hydrophilic/lipophilic balance (HLB) of the amphiphilic component become decisive [116,117]. The hydrophilic heads maximize the contact surface with water, while the hydrophobic tails cluster together to minimize the contact with water. The formed structure arranges into the lower energy configuration, which is spherical or cylindrical depending on the CPP. 1. Introduction AXEL KATTAR 40 The volume of the head group, the volume of the hydrophobic tail (V), the equilibrium area per molecule at the interface surface (A), and the length of the hydrophobic tail (l) are the primary physical characteristics that determine the CPP, as follows [116]: The resulting value, which is unitless, determines the shape of the self-assembled structure. If the CPP is between 0 and 0.33, the selfassembled structure looks like a spherical micelle; between 0.33 and 0.5, the structure has rod-like shape; and only above 0.5 the structure becomes a bilayer, which can form vesicles (Figure 1.2) [118]. The HBL, which is a measure of the balance of the size and strength of hydrophilic to hydrophobic regions, is calculated differently for different classes of amphiphilic substances. The HLB of polyoxyethylene alkyl ethers and polyoxyethylene esters is roughly estimated as the mass percentage of oxyethylene divided by five [119]. The ideal HLB values for vesicle bilayer formation lies between 3 and 8, namely they fall in t Another relevant physical property is the gel liquid transition temperature, which is the temperature at which the amphiphilic components go from closely packed in a gel state to a liquid state where they flow more freely. The aforementioned parameters come into play once the concentration of the amphiphilic component is appropriate for the structure desired, as it is possible to saturate the dispersions and create different aggregates based on the concentration of the surfactant. As an example, in the case of amphiphilic block copolymers, an increase in concentration may drive different unimers and micelles packaging leading to hydrogels and lyotropic liquid crystals [121]. 41 The physical stability of a self-assembled structure depends on thermodynamic and kinetic contributions [112]. Selfassembly is a spontaneous phenomenon and, a priori, does not require solvent exchanges and purification, but it does not mean that occurs quickly and in many cases energy or multistep processes are required to obtain the desired structure. Moreover, an equilibrium between assembled and non-assembled components should be considered. The lower the CMC or the critical aggregation concentration (CAC), the less the ratio of free nonassembled components (unimers). The self-assembled structure is more thermodynamically stable when CMC or CAC are low and, therefore, less prone to disassembly once diluted. The strength of the interactions among the hydrophobic tails also determines the kinetics of the disassembly process. Closely packed components require more time for separation once the formulation is strongly diluted and, in turn, the integrity of the nanocarrier can be maintained for prolonged time [12]. Indeed, physical stability under dilution is an index of the time that the unimers remain aggregate when the concentration is below the CMC and of the capability of the self-assembled carriers to retain the drug inside [91,122]. Strong changes in temperature as those that occur when steam heat sterilization is applied to prepare ophthalmic eye drops may trigger the aggregation or fusion of the self-assembled structure, or an increase in the permeability of the bilayer, which is quite common in the case of liposomes [123]. 1.3.2 Applications to diabetic eye 1.3.2.a Polymeric micelles Polymeric micelles can host a wide variety of lowand midpolarity drugs and provide passive and active targeting [99,124]. 1. Introduction AXEL KATTAR 42 Nevertheless, only a few papers have focused on the design of polymeric micelles for diabetes-related ocular diseases (Table 1.5). Soluplus® (polyvinyl caprolactam-polyvinyl acetatepolyethylene glycol copolymer) micelles (70 80 nm) have shown outstanding capability to solubilize alpha-lipoic acid and to withstand dilution in lachrymal fluid [99]. Alpha-lipoic acid has beneficial effects in dry eye disease and diabetic retinopathy [125], but its solubility and stability in aqueous medium are low. Drug-loaded Soluplus polymeric micelles could be sterilized through membrane filtration, freeze-dried, and reconstituted while maintaining their size and encapsulation efficiency. Corneal permeability (bovine) studies revealed that the micelles facilitate drug accumulation and pass across the tissue, providing alphalipoic acid levels well above those recorded for the commercially available eye drops [125]. Moreover, the in situ gelling performance of Soluplus micelle formulations may provide prolonged retention time on the eye surface. Soluplus micelles also encapsulate progesterone, which has therapeutic potential against retinal degeneration, more efficiently than Pluronic micelles [126]. The hydrophobic core of Soluplus facilitates the assembly at much lower CMC and the micelles are more stable. Studies carried out with cornea and sclera ex vivo from different animal sources (rabbit, pig, cow) revealed the strong influence of the interspecies anatomic differences on the drug permeability results, which may have an impact on the predictions of the performance on human eyes (Figure 1.3) [126]. 43 Figure 1.3. Progesterone (PG) apparent solubility in Soluplus and Pluronic F68 micelles, and permeability coefficients of cornea and sclera recorded for PG encapsulated in Soluplus 20% micelles or Pluronic F68 20% micelles. Reproduced from Alambiaga-Caravaca et al. [126] (Creative Commons Attribution License). 1. Introduction AXEL KATTAR 44 Table 1.5. Recent examples of self-assembled nanocarriers proposed for the topical ocular treatment of diabetic eye Nanocarrier Drug Disease/Outcome Reference Polymeric micelles Alpha-lipoic acid Dry eye and diabetic retinopathy (bovine cornea). Soluplus micelles facilitated alphalipoic acid accumulation and pass across the tissue. [99] Progesterone Retina degeneration (rabbit, pig, cow). Soluplus and Pluronic micelles enhanced drug permeability through cornea with respect to sclera. Important interspecies differences. [128] Dexamethasone, triamcinolone and triamcinolone acetonide Macular edema (cell cultures). Micelles of copolymer of inulin derivatized with ethylenediamine and retinoic acid efficiently encapsulated the drugs, were internalized by different cell lines, and favored transcorneal permeation. [130] Dexamethasone Posterior uveitis (rabbit). Mixed micelles of polyoxyl 40 stearate and polysorbate 80 provided therapeutic levels in retina and choroid after one single instillation. [132] Anti-angiogenic peptide aANGP Diabetic retinopathy and macular edema (cell cultures). aANGP-micelles inhibited tube formation at 1000-fold lower concentration than free peptide. [133] Glycyrrhizin and genistein Wound healing in diabetic eye model (mice). Genipin-loaded glycyrrhizinate micelles down-regulated high mobility group box protein-1 (HMGB1) and its receptors for advanced glycation-end products (RAGEs) and toll-like receptors (TLRs), as well as inflammatory factor interleukin (IL)-6 and ILdiabetic corneal and nerve wound healing. [134] Liposomes Citicoline Diabetic retinopathy (mouse). Topical administration twice a day, for 15 days, prevented glial activation and neural apoptosis. [138] Triamcinolone acetonide Macular edema (patients). Drugloaded QuSomes® instilled every 2 h decreased the central foveal thickness and increased the best-corrected visual acuity. [141] Introduction 45 Triamcinolone acetonide Macular edema (rabbit). Chitosan-coated liposomes instilled five times a day provided efficient delivery to anterior and posterior segments due to enhanced cell uptake. [142] Berberine hydrochloride Macular edema (rabbit). PAMAM G3.0coated liposomes promoted transcorneal permeability. [143] Bevacizumab Macular degeneration (rat and rabbit). Unilamellar vesicles with annexin A5 enhanced uptake and transcytosis through cornea, supplying therapeutic concentrations to the back of the eye. [144] Transforming growth factor- - Macular degeneration (rabbit). Unilamellar vesicles with annexin A5 provided therapeutic levels to the back of the eye. [148] Thrombospondin (TSP)-1-derived peptide Chronic ocular surface inflammation and tear film instability (ex vivo porcine cornea). Liposomes enhanced cornea permeation. [149] Plasmid DNA Gene therapy at retinal pigment epithelium (rat). Transferrin-modified small liposomes (<80 nm) selectively distributed to the retinal pigment epithelium. Larger liposomes could be targeted to choroidal endothelial cells. [151] Niosomes Naltrexone·HCl Diabetic keratopathy (bovine). Span 60 and cholesterol (30 mol%) niosomes sustainedly release the drug while still allowed for high cornea permeability. [167] Flurbiprofen Keratitis (rabbit). Span 60 and cholesterol (50 mol%) niosomes solely or dispersed in Carbopol gel enhanced drug ocular bioavailability. [168] Latanoprost Glaucoma (rabbit). One instillation of Span 60 and cholesterol (50:50 weight ratio) niosomes dispersed in Pluronic F127 gel decreased IOP for more than 48 h. [169] 46 Poly(ethylene glycol)-b-poly(lactic acid) micelles increased 10fold triamcinolone acetonide apparent solubility using a copolymer concentration as low as 0.5 mg/mL. Drug-loaded micelles formulated in chitosan dispersion prolonged drug release for more than 1 week. In vivo (rabbit) evaluation in an inflammatory disease model revealed that twice a day instillation of the micelle solution with or without chitosan was able to recover the normal corneal epithelium [127]. Inulin-based mucoadhesive micelles have been shown suitable for encapsulation of anti-inflammatory drugs adequate for macular edema treatment, such as dexamethasone, triamcinolone, and triamcinolone acetonide, enhancing drug permeability through corneal cells, which could be an alternative to intraocular injections [128]. Also intended for macular degeneration, tacrolimus delivery may benefit from encapsulation in micelles made of PEG-hydrogenated castor oil-40 and octyx-onyl-40, which have low CMC and provide slow drug release. Tacrolimus-loaded micelles showed faster cell internalization than the free drug [129]. Mixed micelles of polyoxyl 40 stearate and polysorbate 80 successfully delivered dexamethasone (0.1%) to the back of the eye, providing therapeutic drug levels in retina and choroid after eye drop instillation [130]. Prevention of abnormal growth of blood vessels in retina can be achieved by means of poly(ethylene glycol)-b-poly (propylene sulfide) micelles decorated with the antiangiogenic peptide aANGP. The aANGP-micelles showed potent angiogenic inhibitory effect at 1000fold lower concentration than the free peptide in cell cultures [131]. Treatment of diabetic keratopathy may notably benefit from the encapsulation of genipin in dipotassium glycyrrhizinate micelles (29.5 nm) [132]. Genipin and glycyrrhizin were shown to synergically block the high mobility group box 1 signaling and, in turn, attenuate the AXEL KATTAR 47 inflammation cascade overexpressed in diabetic eyes. These micelles showed good corneal permeability and favored corneal reepithelialization and nerve regeneration in diabetic mice. 1.3.2.b Liposomes Liposomes are unior multi-bilayer vesicles composed of mainly amphiphilic lipids (phospholipids) and cholesterol. Each bilayer (also known as lamella) resembles the cell membrane, which endows liposomes with high biocompatibility. According to the number of bilayers and the overall size, liposome size may range from 10 to 100 nm (small unilamellar vesicles, SUV) to few microns (large unilamellar vesicles, LUV, and large multilamellar vesicles, LMV). General description of liposomes composition and preparation can be found elsewhere [133]. Liposomes have been widely used as carriers since several decades ago because of their dual capability to simultaneously host hydrophilic and hydrophobic drugs plus the general advantages of passive and active targeting [134,135]. Usefulness of liposomes for topical ophthalmic drug delivery, and especially management of ocular surface diseases, has been recently reviewed [13]. Importantly, the bioinspired structure and composition of liposomes facilitate the fusion with cell membrane at the cornea and conjunctiva surface and drug transfer to cell cytoplasm. Examples related to diabetic eye diseases are summarized in Table 1.5. Regarding diabetic retinopathy, liposomes loaded with citicoline exhibited anti-inflammatory properties [136]. Citicoline or cytidine 5´- diphosphocoline is an endogenous compound involved in the biosynthesis of phospholipids, showing neuroprotective activity. Citicoline oral solution is registered as Food for Special Medical Introduction 54 Niosomes have been shown able to improve oral bioavailability of a variety of treatments for diabetes, including insulin [159], metformin hydrochloride solely or combined with glipizide [160,161], and plant extracts [162]. Niosomes also attract great attention as ocular drug carriers due to their excellent tolerability, prolonged precorneal residence, and enhanced ocular bioavailability [16,151]. Nevertheless, examples of their performance as delivery systems of diabetic eye drugs are still incipient (Table 1.5). Multilayered niosomes (7 been designed to encapsulate naltrexone hydrochloride, which is a potent opioid antagonist that markedly accelerates cornea healing and repairs the signs of diabetic keratopathy. The niosomes prepared with Span 60 and cholesterol using the thin-film hydration method showed high encapsulation efficiency [163]. These niosomes had a gel liquid transition of few degrees above physiological temperature, which is considered as an advantage to avoid dragging by blinking and to control drug release [164]. Ex vivo tests in bovine cornea evidenced that niosomes sustainedly released the drug for several hours and allowed for high permeability [165]. Span 60gel were shown suitable for delivery of the antiinflammatory drug flurbiprofen in the aqueous humor [166]. Once applied onto the cornea (rabbits), flurbiprofen-loaded niosomes either solely or in the Carbopol gel provided drug levels in aqueous humor one order of magnitude above those achieved using a free drug solution. Relative drug bioavailability from the niosome and the niosome-gel formulations was 3.6and 6.2-fold that of the flurbiprofen solution because the gel notably extended the permanence time on the eye surface. The niosome formulations also demonstrated to be therapeutically efficient against keratitis (carrageenan-induced inflammation). The obtained AXEL KATTAR 55 results suggest that once a day instillation may be sufficient to manage ocular inflammatory diseases. Feasibility of using niosomes for glaucoma therapy has been evidenced recently. Niosomes prepared by the reverse-phase evaporation technique covering wide ratios of cholesterol and Span 40 or Span 60 could host latanoprost with an encapsulation efficiency of ~98%. Latanoprost-loaded niosomes (8 Pluronic F127 gel (drug concentration 0.005%) and the effects on IOP -de-sac of rabbit eyes. Latanoprost-loaded niosomes reduced IOP for 3 days, which was remarkably longer than the outcome achieved with the standard latanoprost eye drops (Xalatan®; 0.005%) (Figure 1.6) [167]. Proniosomal gel formulations are promising also for other antiglaucoma agents, including dorzolamide hydrochloride and brimonidine tartrate [168,169]. Introduction 56 Figure 1.6. Reduction in the intraocular pressure (IOP) observed after topical administration to normotensive rabbits of latanoprost-loaded niosome gel or conventional latanoprost eye drops. Error bars represent standard deviation of six replicates. Reproduced from Fathalla et al. [167] with permission from Taylor & Francis. Interestingly, in the last few years, most publications on niosomes refer to the feasibility of forming nioplexes for a variety of therapeutic applications, including gene therapy to the back of the eye [170]. Nevertheless, most reports rely on subretinal or intravitreal injections [171,172]. To the best of our knowledge, the suitability of nioplexes for topical ocular administration is still to be explored. 1.4 CONCLUSION With the upward trend in obesity worldwide, the number of cases of diabetic eye diseases is also bound to increase, making research on the delivery of ocular drugs an active field. This is reflected in the large number of ongoing clinical trials for diabetic eye diseases. The trials are primarily focused on macular edema and retinopathy, with a AXEL KATTAR 57 plethora of active substances to be administered through multiple routes. Although the eye presents challenging anatomical barriers, different self-assembled carriers have been proven to be capable of overcoming them and offer a promising future for drug delivery to the ocular therapeutic sites. Micelles, liposomes, and niosomes offer diverse advantages covering from the increase in drug solubility to the use of additional pathways of penetration into tissues. Preclinical and clinical studies have confirmed their ability to protect the cargo from degradation, target specific areas, enhance permeation through different ocular barriers, and sustain the release, which in turn improves the therapeutic effects both in intensity and in duration. Niosomes made of nonionic surfactants are particularly interesting, and scientific research has grown steadily in the last few years, as an alternative to liposomes in terms of higher stability and lower production costs. Advances in right choosing of the composition and size of the self-assembled carriers may result in more successful management of both anterior and posterior segment eye diseases. 1.5 EXPERT OPINION The diabetic eye is one of the clearest examples of the need to develop adequate drug delivery systems that can improve or reverse the effects of a very relevant chronic disease. Given the pandemic character of diabetes, developing formulations that alleviate or reverse the deleterious effects at ocular level and the subsequent consequences on the quality of life of patients is an unsatisfied clinical demand. Most drugs currently approved or under evaluation in clinical trials require nonviable frequent instillation or intraocular administration using invasive procedures. Since diabetic-eye diseases need chronic Introduction 58 treatments, an adequate balance between patient acceptability and drug ocular bioavailability should be reached. Most clinical trials related to diabetic eye refer to macular edema and retinopathy, although affectation of anterior segment is also considered. Strong research efforts are being made at two levels: (I) to find new drug candidates and to test the therapeutic efficiency of drugs already approved for systemic administration when they are directly administered to the ocular tissues and (II) to find suitable delivery vehicles that can overcome eye barriers and help the drug to get access to the damaged tissue and to remain in it for prolonged time. At the pharmacological level, a myriad of drugs and biologics are being tested, mostly searching for anti-inflammatory and anti-angiogenic activities. Although long sought, the recent approval of gene therapy treatments for inherited eye diseases is undoubtedly driving the development of appropriate approaches for acquired diseases. The feasibility of is closer than ever. Gene therapy relies on a single administration or administrations widely separated in time. One-time treatment may be compatible with invasive maneuvers as the benefit may counteract the risks. Nevertheless, drugs, biologics, and genes may strongly benefit from more patient-friendly approaches. From a pharmaceutical technology level, most therapeutic agents for diabetic eye have poor biopharmaceutic properties because they are poorly soluble or the size is quite large. Thus, self-assembled carriers (polymeric micelles, liposomes, and niosomes) that can facilitate the penetration of small and large molecules deep in the ocular tissues may become valuable tools. Topical management of eye surface and anterior segment diseases has already been shown to improve with selfassembled carriers that show prolonged permanence on ocular surface, AXEL KATTAR 59 facilitate drug permeation through cornea, and diffusion into the aqueous humor. Moreover, the feasibility of the carriers to encapsulate large molecules (including antibodies and genes) pass through sclera while minimizing systemic clearance, and modulate diffusion through vitreous may make topical administration a realistic alternative to intraocular injection. Remarkably, the gain in knowledge about transporters present in each ocular tissue and about the biomarkers that can be found altered due to diabetes may provide interesting clues for the design of drug carriers with greater penetration capacity and targeting ability. This field is still in its infancy, but the intense quest to provide the patients with efficient yet friendly formulations for longterm use will pave the way to clinic of novel topical formulations. Introduction 60 1.6 REFERENCES 1. Lin X, Xu Y, Pan X, et al. Global, regional, and national burden and trend of diabetes in 195 countries and territories: an analysis from 1990 to 2025. Sci Rep.2020;10:14790. 2. 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While different macro sized systems have been explored, the relatively new world of nanometer scale carriers is advancing the field of controlled, targeted and sustained drug delivery at a fast pace. In the context of diabetic eye diseases, and in particular diabetic retinopathy, epalrestat is a drug that has the potential to treat patients with chronic high blood glucose levels, and prevent their loss of vision. However, this molecule is poorly soluble in water. To overcome the hydrophobicity of the epalrestat molecule, different formulations have been investigated. The use of niosomes, micelles and oleogels were explored to encapsulate or solubilize epalrestat with the objective of topical administration. Niosomes and micelles have the advantage of being self-assembled, stable and highly tunable. While micelles encapsulate the drug in their hydrophobic core, niosomes encapsulate the drug in their hydrophobic bilayer. Oleogel for its part dissolves the drug in its midst. All three formulations are then delivered into the eye topically. The main objective of this Thesis was to design reproducible, stable and safe formulations and characterize them in order to validate their potential for topical administration of epalrestat for diabetic eye diseases. This objective was split into four parts that are exposed as follows. 88 1. The design, formulation and characterization of niosomes for ocular delivery of epalrestat Diabetic patients with elevated levels of blood glucose have a metabolic pathway, the polyol pathway, that becomes available which is not active under normal glycemic conditions. The polyol pathway, turns glucose into sorbitol and subsequently sorbitol into fructose. This first step is the rate limiting step, meaning that sorbitol accumulates with time. Sorbitol not being permeable to cell membranes and this first step consuming NADPH, the environment where this reaction takes place is prone to oxidative and osmotic stress, resulting in tissue degradation. The glucose to sorbitol conversion step is catalyzed by aldose reductase, an enzyme present in the retina. Epalrestat is a non-competitive and reversible aldose reductase inhibitor that is used for the treatment of diabetic neuropathy in Japan as an oral formulation. Clinical trials confirmed the efficacy of orally administered epalrestat in the treatment of diabetic retinopathy. However, epalrestat being poorly soluble in water, it needs to be encapsulated to cross the different ocular barriers. Niosomes are self-assembled vesicles that are composed of amphiphilic molecules creating a hydrophobic bilayer capable of accommodating small molecules. They are made by bringing together a non-ionic surfactant and a helper lipid at set ratios in water and use different techniques to tune the size, polydispersity and surface potential of the particle. The first part of the Thesis aimed to elaborate a robust protocol for the preparation of nanometer scale niosomes that are able to encapsulate epalrestat and permeate different ocular tissues. The aim of this section of the Thesis is to assess the reproducibility, stability, safety and AXEL KATTAR 89 compliance with the requirements of ocular drug transport of the niosomes designed and prepared. This allowed for the selection of the best niosomal formulation to be tested in vivo. The zebrafish embryotoxicity experiments for safety characterization of the niosomes were done in collaboration with the laboratory of Professor Laura Sánchez from the Departamento de Zooloxía, Xenética y Antropoloxía Física at the Universidade de Santiago de Compostela in Lugo. 2. The understanding of the underlying physical forces governing molecular interactions of the components of the niosomes Niosomes are self-assembled particles that are composed of a bilayer. The forces governing the molecular arrangement in the bilayer are the ones determining the physical characteristics of the niosomes such as their size or stability. Using a Langmuir-Blodgett through, monolayers made from the molecules present in the niosomal bilayer can be studied under different pressures. This part of the Thesis relies on the hypothesis that the molecular arrangement of a monolayer made from Tween 60, cholesterol and DOTMA in the ratios leading to niosome formation is mainly governed by the Tween 60, is also influenced by interactions with cholesterol and DOTMA and by the temperature and ionic strength of the subphase. The aim of the second chapter of this Thesis was to understand which variables had an influence on the monolayer behavior and to quantify the effects of these variables to ascertain an understanding of the stability of the self-assembly in storage and administration conditions. This was achieved through the study of four effects: (I) the effect of cholesterol on a Tween 60 monolayer, (II) the effect of 2. Aims 90 DOTMA on a Tween 60 monolayer, (III) the effect of cholesterol on a Tween 60/DOTMA binary monolayer, and (IV) the effect of temperature and ionic strength of the subphase on a Tween 60/DOTMA/cholesterol tertiary monolayer. The experiments in this section were performed in collaboration with the Biomemebranes Laboratory of Professor Matilde Casas Parada at the Universidade de Santiago de Compostela in Santiago de Compostela. 3. The formulation and characterization of oleogels for topical ocular delivery of epalrestat Gels are gathering interest in the pharmaceutical industry as a vehicle for drug delivery. They are viscoelastic fluids made of a liquid and a gelator. As the drug to be delivered, epalrestat, is hydrophobic, oleogels were chosen. These oleogels are made up from an oil phase and a gelator, allowing the formulation to tune its viscosity, and homogeneously distribute the drug in the oleogel matrix. The work done in this part of the Thesis was based on the hypothesis that oleogels are suitable carriers to solubilize and release epalrestat, allowing it to permeate through corneal and scleral tissues. The aim of this part of the Thesis was to assess the reproducibility, safety and efficiency in drug release and permeation of the oleogels designed and prepared. This allowed for the selection of the best oleogel formulation to be tested in vivo. Multiple different oleogels were designed and characterized. Soybean oil was chosen as the base, as it possesses a high smoke point and permits higher temperatures during the melting of the gelators. AXEL KATTAR 91 Three gelators were used to prepare single and double gelator oleogels: beeswax, cocoa butter and ethyl cellulose. The oleogel formulation was developed during a research secondment at the Chemical and Biological Engineering Department of the Colorado School of Mines in Golden, Colorado, under the supervision of Professor Anuj Chauhan. 4. The comparison of epalrestat loaded niosomes, micelles and oleogels distribution in vivo Micelles are self-assembled structures that are formed from amphiphilic molecules. These molecules join their hydrophobic moieties to form a core and expose their hydrophilic heads to the aqueous medium. Pluronic® F127 was chosen to prepare micelles as they have already been prepared and characterized in our research group. The best niosomal, micellar and oleogel formulations were selected to be tested in an animal model in the context of in vivo experiments. The aims of the in vivo study were: (I) to assess the safety of the three formulations for topical ocular delivery of epalrestat, (II) to quantify the epalrestat concentration in the lacrimal fluid of the rabbits during the experiment, and (III) to quantify the epalrestat concentration in the different ocular tissues 6 hours after administration of the formulations. The experiments were conducted on rabbits within the 3R principles and with the authorization of both the committee of animal experimentation ethics (CEEA) of University of Santiago de Compostela and the Consellería de Medio Rural of Xunta de Galicia. 2. Aims 93 Chapter 3 The work described in this chapter was published in Formulation and Characterization of Epalrestat-Loaded Polysorbate 60 Cationic Niosomes for Ocular Delivery, Pharmaceutics 15(4), 1247, 2023, authored by: Axel Kattar a, Ana Quelle-Regaldie b, Laura Sánchez b,c, Angel Concheiro a and Carmen Alvarez-Lorenzo a a Departamento de Farmacología, Farmacia y Tecnología Farmacéutica, I+D Farma (GI-1645), Facultad de Farmacia and Health Research Institute of Santiago de Compostela (IDIS), Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain. b Departamento de Zooloxía, Xenética y Antropoloxía Física, Facultade de Veterinaria, Universidade de Santiago de Compostela, 27002 Lugo, Spain c Preclinical Animal Models Group, Health Research Institute of Santiago de Compostela (IDIS), 15706 Santiago de Compostela, Spain 95 3. FORMULATION AND CHARACTERIZATION OF EPALRESTAT-LOADED POLYSORBATE 60 CATIONIC NIOSOMES FOR OCULAR DELIVERY 3.1 INTRODUCTION In 2021 the prevalence of diabetes in adults ranging from 20 to 79 years worldwide was 9.8%, representing 536.6 million patients [1]. Diabetic patients have elevated levels of glucose in their blood, which leads to a range of conditions. A subcategory of complications derived from diabetes is diabetic ocular diseases. Examples include diabetic retinopathy, diabetic keratopathy, or cataracts, which have been linked to the polyol pathway [2]. These different illnesses affect the patient with loss of vision, culminating in blindness if left untreated. The current treatments for diabetic ocular diseases involve intravitreal injections [3], laser treatment [4], or even vitrectomy [5]. The polyol pathway transforms glucose into sorbitol when hexokinases active in the Embden-Meyerhof pathway are saturated, and subsequently, glucose is oxidated to sorbitol and further transformed into fructose. It is important to mention that this pathway is rarely used by the body in a healthy state as it is only activated at high intracellular glucose concentrations [6]. However, in the case of diabetes, the blood glucose levels can be high enough for the reaction balance to favor 102 1 vol% Tween 80 [36] and analyzing the medium with HPLC. The dialysis membrane had a molecular weight cutoff of 12,000 Da and an effective dialysis area of 4.2 cm2. The concentration was confirmed by lysing the niosomes with methanol [34] and measuring the concentration of the drug encapsulated by HPLC. The efficiency was then calculated with Equation 3.1 [35]: , Equation 3.1. Encapsulation efficiency of epalrestat in niosomes. Release study. The release was tested by placing 5 mL of niosomes (0.20 ± 0.01 mg epalrestat/mL) in 14,000 MWCO dialysis tubing (Sigma-Aldrich, St Louis, MO, USA) and using 500 mL ultrapure water with 1% Tween 80 as receptor medium [36]. The medium was left at 20 °C for 20 days or 37 °C for 24 h stirring at 400 rpm with a magnetic stirrer. 1 mL of the medium was taken and replaced with 1 mL of fresh medium every day for 8 days and then every two days until day 21. The concentration of epalrestat in the release medium was quantified with HPLC. The niosomes left in the dialysis bag were lysed, and the remaining epalrestat was quantified with HPLC. HET-CAM. (HET-CAM) assay [28], fertilized eggs (15) were supplied by Coren (Ourense, Spain) and cleaned before incubation in a CCRS 0150 incubator (Ineltec, Tona, Spain) for 9 days at 37 °C and 60% relative humidity. On the day of the experiment, the shell of the eggs was pared off with a circular saw at the location of the air cell. The untouched inner membrane was moistened with a 0.9% NaCl solution, and the eggs were placed back in the incubator for 30 min. The 0.9% NaCl solution was subsequently removed, as well as the inner membrane, while being careful not to damage the blood vessels of the CAM AXEL KATTAR 103 underneath. Any non-viable egg was discarded. The positive control was NaOH 0.1M, and the negative control was 0.9% NaCl. The solutions tested were formulation TCD0, TCD5, and TCD10 in water loaded with 0.2 mg/mL epalrestat, unloaded niosomes of the same molar ratios, and epalrestat dissolved in ethanol: water 10/90 v/v mixture. The 300 µL of the testing solution was then added to the eggs, and the effect on the blood vessels regarding hemorrhage, lysis, and coagulation was recorded. The ocular irritability potential score was calculated with Equation 3.2 [28]: with H= hemorrhage time (s), L= lysis time (s), C= coagulation time (s). Equation 3.2 Membrane assay. Gluc-HET. For the Gluc-HET assay [29], fertilized eggs (15) were supplied by Coren (Ourense, Spain) and cleaned before incubation in a CCRS 0150 incubator (Ineltec, Tona, Spain) for 11 days at 37 °C and 60% relative humidity. On the day of the experiment, the shell of the egg above the air pocket was pierced with a needle, and 300 µL of the testing solution was deposited inside the air compartment. The air compartment of the negative controls was pieced, but no solution was added [29]. The positive control was a solution of 0.002 mg/mL glibenclamide solution in HBSS. The eggshells are then closed off with parafilm. After 2 h incubation, the eggshell above the air compartment was removed, and the chorioallantoic membrane was cut next to a blood vessel with a scalpel. The blood vessel was placed on a flat metal tong and dried with paper. Once no moisture was absorbed anymore by the paper, the vessel was cut, and the blood glucose level was measured 3. Formulation and Characterization of Epalrestat-Loaded Polysorbate 60 Cationic Niosomes for Ocular Delivery 104 with a glucose meter (Contour next, Ascensia Diabetes Care, Basel, Switzerland). Zebrafish embryotoxicity test. Epalrestat-loaded niosome toxicity was assessed using zebrafish embryos (Danio rerio) and the Fish Embryo Acute Aquatic Toxicity (FET) Test. Zebrafish embryos were selected around 3 h post-fertilization (hpf). The test was considered valid if the mortality of fish embryos was at least 30% in the positive control (3,4-dichloroaniline) and lower than 10% in the negative control. In our experiment, the mortality of the negative control was 3.3%, and the mortality of the positive control was 100%. The larvae were grown in autoclaved osmosis water. The experiment was carried out by including 5 or 10 µL of formulation in 200 µL of medium (4.9 µg/mL and 9.5 µg/mL, respectively) and quantifying the mortality at 24, 48, 72, and 96 h. The formulations tested were TCD0, TCD5, and TCD10. The experiments were conducted in triplicate. Corneal permeation. Porcine eyes were supplied by a slaughterhouse and transported to the laboratory in diluted PBS solution at 4 °C in an ice bath. The corneas were dissected with 2 3 mm of surrounding tissue and washed with 0.9% NaCl to remove any attached tissue. The corneas were mounted in Franz diffusion cells with the outer part of the cornea facing up. The area available for permeation was 0.785 cm2. The receiving chamber was filled with 6 mL of Tween 80:water 10:90 v/v solution while making sure no bubbles formed and then agitated with a magnetic stirring rod at 400 rpm. The donor chamber was filled with 2 mL of carbonated buffer (pH 7.2) and closed off with parafilm to prevent evaporation. The system was then left to equilibrate for 1 h at 37 °C. Once the system was balanced, the carbonated buffer in the donor chamber was replaced by 2 mL of either 0.2 mg/mL epalrestat solution in 10 mL of ethanol:water 10:90 v/v or AXEL KATTAR 105 0.2 mg/mL epalrestat encapsulated in the TCD0, TCD5, and TCD10 formulations. After 30 min, at 1 h and then every hour, 1 mL of the solution in the receiving chamber was removed and replaced with 1 mL of fresh Tween 80:water 10:90 v/v solution. After 6 h, the last sample was taken, and the corneas were incubated in ethanol at 37 °C for 24 h. They were then sonicated at 37 °C in an ultrasonic bath for 90 min. The resulting mixture was centrifuged at 1000 rpm at 25 °C for 5 min, and the supernatant was centrifuged at 14,000 rpm at 25 °C for 20 min. After filtration through 0.22 µm pore syringe filters (Scharlab, Barcelona, Spain), all the samples from the receptor chamber as well as the supernatant from the tissue incubation, were analyzed with HPLC according to the protocol described above. All experiments were carried out in triplicate (Figure 3.2) 3. Formulation and Characterization of Epalrestat-Loaded Polysorbate 60 Cationic Niosomes for Ocular Delivery 106 Figure 3.2. Epalrestat permeability data through cornea and sclera recorded for four independent replicates of each niosome formulation (TCD0, TCD5, TCD10) and control AXEL KATTAR 107 Scleral permeation. Scleral permeation was performed the same way as corneal permeation, except the tissue used to permeate through was the porcine sclera instead of the porcine cornea. IR-Raman. Porcine cornea and sclera were permeated with TCD0, TCD5, and TCD10 niosomes for 6 h under the same conditions as the corneal and scleral permeation experiment described above. The IRRaman study was performed by taking a minimum of 3 points and a maximum of 6 points per cornea and per sclera (Figures 3.3 and 3.4) and measuring the Raman scattering of the surface. Furthermore, a line scan was performed in the x-z plane (Figures 3.5-3.7). The excitation wavelength was 532.188 nm, the sample was kept at a temperature of 8 °C for the duration of the experiment, the laser power was 3 mW, and each point was measured with 60 accumulations, with an integration time of 0.3 s and an objective of ×50 (Zeiss LD EC Epiplan-Neofluar Dic 50×/0.55). The measurement was done on the top and bottom part of the tissue, and the absolute height of the peak (Figure 3.8) (CCD cts) was compared between the top and bottom of each tissue. For the line scan, spectra were accumulated by taking 30 spectra per line and 15 lines per image, each spectrum at a distance of 1 µm from the previous point, both in the x and the z direction. 3. Formulation and Characterization of Epalrestat-Loaded Polysorbate 60 Cationic Niosomes for Ocular Delivery 108 Figure 3.3. Image of the cornea sample free drug cornea top under x50 magnification, with the crosses indicating the locations the RAMAN spectrum was taken. Figure 3.4. Image of the cornea sample free drug cornea bottom under x50 magnification, with the crosses indicating the locations the RAMAN spectrum was taken. AXEL KATTAR 109 Figure 3.5. Image of the cornea sample TCD0 cornea bottom under x50 magnification, with the line indicating the locations the RAMAN spectra were taken. Figure 3.6. Image of the cornea sample TCD5 cornea bottom under x50 magnification, with the line indicating the locations the RAMAN spectra were taken. 3. Formulation and Characterization of Epalrestat-Loaded Polysorbate 60 Cationic Niosomes for Ocular Delivery 110 Figure 3.7. Image of the cornea sample TCD10 sclera bottom under x50 magnification, with the line indicating the locations the RAMAN spectra were taken. Figure 3.8. Raman spectrum of sample free drug cornea top at the blue cross in Figure 3.3. AXEL KATTAR 111 Statistical analysis. All conditions in the experiments were carried out in triplicate, and the data were shown as an average with a standard deviation when possible. Statistical analysis was carried out in Origin, making use of a one-way analysis of variance (one-way ANOVA) with statistically significant when the p-value was lower than 0.05. 3.3 RESULTS 3.3.1 Niosome Characterization Cationic niosomes were chosen to allow for efficient encapsulation [37], enhanced stability of the nanoparticles in suspension [38], increased ocular retention time [39], and increased bioavailability [40]. To prepare niosomes, the ratio of surfactant to helper lipids determined the stiffness and curvature of the bilayer. The incorporation of cholesterol changes the assembly of the bilayer as it lodges itself between the hydrophobic tails with the exception of its hydroxyl group [41,42]. In cell membranes, this translates into lowering the membrane permeability to water-soluble molecules, increasing the packing order of the lipids, reducing the bilayer fluidity, and separating the lipid tails to prevent crystallization [43,44,45]. The ratio of cholesterol incorporated in the niosomes affected the physicochemical properties as it intercalated itself within the organic chains of the surfactant in the niosome. The final mol fraction of cholesterol was determined by preparing and characterizing six formulations with different ratios of Tween 60 to cholesterol in terms of size, zetapotential, and polydispersity index (Table 3.2). The shortterm stability of the niosomes was assessed by running the same 3. Formulation and Characterization of Epalrestat-Loaded Polysorbate 60 Cationic Niosomes for Ocular Delivery 118 Table 3.5. The difference in size, PDI, zeta-potential, and drug content of epalrestat loaded Tween 60/cholesterol niosomes with 0, 5, and 10 mol% DOTMA after 7 days in storage at 4 °C and 25 °C. Formulation - Potential (mV) Index Efficiency (%) 4 °C 25 °C 4 °C 25 °C 4 °C 25 °C 4 °C 25 °C TCD0 22.1 26.3 2.3 3.6 0.14 0.13 1.2 0.1 TCD5 33.0 59.1 11.1 7.9 0.27 0.08 1.1 0.2 TCD10 17.9 14.6 21.4 26.3 0.05 0.01 0.7 0.4 3.3.2 Epalrestat Release The release study was carried out in water containing 1% Tween 80 to ensure sink conditions, as epalrestat has a solubility in water of 0.047 mg/mL. With the results from the encapsulation efficiency, the final concentration of epalrestat was between 0.199 and 0.200 mg/mL in the dialysis bag, meaning that 100% epalrestat release corresponds to 1 mg released. The release profile of the niosomes over time is shown in Figure 3.10 In the first 8 days, epalrestat released from the niosomes negatively correlated with the DOTMA percentage of the niosome. However, after 10 days, 0% DOTMA niosomes showed a lower release rate, which may be related to its greater stability. These niosomes were the ones that changed their size during storage (Table 3.4), which indicates that they are less prone to destabilize. AXEL KATTAR 119 Figure 3.10. The release profile of epalrestat encapsulated in niosomes in Tween 80 1% aqueous medium at 20 °C over 20 days. Another release experiment was carried out at 37 °C to mimic inside the back of the human eye. The amounts of released epalrestat over 24 h and non-released epalrestat are summarized in Table 3.6. The mass balance turned out to be about 96% for the niosomes, including DOTMA in their formulation, and 98.6% for niosomes without DOTMA. This finding indicated that niosomes protect epalrestat from degradation, which contrasts with the degradation of unencapsulated epalrestat in an aqueous medium [48]. This finding also confirms that niosomes are stable in the release medium. 3. Formulation and Characterization of Epalrestat-Loaded Polysorbate 60 Cationic Niosomes for Ocular Delivery 120 Table 3.6. Amounts of epalrestat released and remaining in the niosomes after 24 h in Tween 80 1% aqueous medium at 37 °C. Formulation Epalrestat Released after 24 h (%) Epalrestat Remaining in the Niosomes (%) Sum of Released and NonReleased Epalrestat (%) TCD0 12.9 ± 0.50 85.7 ± 0.82 98.6 ± 0.66 TCD5 11.7 ± 0.33 84.3 ± 1.42 96.0 ± 0.87 TCD10 10.3 ± 0.53 86.2 ± 0.97 96.5 ± 0.75 3.3.3 HET-CAM Assay The HET-CAM assay was performed using the three epalrestatloaded niosomal formulations, one unloaded niosome formulation, and epalrestat dissolved in 10/90 v/v ethanol-water solution. The HETCAM assay is not considered an animal experiment under Directive 2010/63/EU [53] as no nervous system is developed before day 11 of the embryo development. The positive (0.1 M NaOH) and negative controls (0.9% NaCl) had ocular irritability potential scores of 19.04 and 0, respectively. Epalrestat solution (0.2 mg/mL) (Figure 3.11A) triggered blood coagulation and had a score of 18.58. Differently, the loaded and unloaded niosomes did not show any noticeable hemorrhage of the blood vessels (Figure 3.11B E) and obtained a score of 0. This indicated that the encapsulation of epalrestat decreases the ocular irritability potential and, therefore, allows for topical administration. Figure 3.11. Pictures of the chorioallantoic membrane after 300 s (A): epalrestat dissolved in 10/90 ethanol/water (0.2 mg/mL), (B): unloaded TCD0 niosomes, (CE): loaded niosomes (0.2 mg/mL) (C): TCD0, (D): TCD5, (E): TCD10. AXEL KATTAR 121 3.3.4 Gluc-HET Assay To measure the effect on the blood glucose level, the gluc-HET [29] test was chosen as it presents a few advantages. It is not considered an animal experiment under Directive 2010/63/EU [53] as no nervous system is developed before day 11 of the embryo development. Furthermore, the embryos exhibit high glucose levels that are susceptible to insulin without interference from naturally produced insulin, which starts on day 12. Both the TCD0 and TCD10 loaded niosomal formulations behaved the same way as the epalrestat in solution (Figure 3.12) in that they reduced the blood glucose levels in a similar fashion to the positive control (glibenclamide). Tests carried out on the effect of the developmental stage on assay performance revealed a significant increase in the sensitivity of the embryos to the glucose-reducing compounds for day 10 and day 11 embryos [29]; therefore, day 11 was chosen to perform the experiment. Formulation TCD5 exhibited blood glucose level reduction but in a lower amount than the positive control. 3. Formulation and Characterization of Epalrestat-Loaded Polysorbate 60 Cationic Niosomes for Ocular Delivery 122 Figure 3.12. Blood glucose level after exposure to glibenclamide, epalrestat in 10/90 ethanol/water solution, TCD0, TCD5, and TCD10 niosomes loaded with epalrestat. * Statistically significant differences (p < 0.05). 3.3.5 Zebrafish Embryotoxicity Assay Zebrafish embryotoxicity tests have been increasing in use for developmental toxicology and ToxCast high-throughput screening of chemicals and nanomaterials [54] due to the high concordance between zebrafish and mammalian studies demonstrating its potential to reduce and refine, if not replace animal studies [30,31,32]. The survival of Danio rerio (zebrafish) embryos was therefore measured after 96 h of exposure to loaded niosomes TCD0, TCD5, and TCD10 (Table 3.7). AXEL KATTAR 123 Table 3.7. Survivability of zebrafish embryos after 96 h of exposure to different loaded niosome formulations. Tested Formulation Survival of Danio rerio (%) 5 µL Solution Exposure 10 µL Solution Exposure Negative control 98.0 96.7 TCD0 98.0 56.7 TCD5 93.0 60.0 TDC10 96.6 43.3 Epalrestat in solution 57.5 40.0 With 5 µL solution, exposure formulation epalrestat-loaded niosomes were all highly compatible with the zebrafish embryos and demonstrated that the niosome encapsulation reduced the toxicity of epalrestat significantly. 3.3.6 Corneal and Scleral Permeation Epalrestat from formulations TCD0, TCD5, and TCD10 permeated at different rates through the porcine cornea and sclera (Figure 3.13). The steady-state flux and lag time were obtained from the slope and xintercept of the linear regressions of the curves in Figure 3.13 (individual plots are shown in Figure 3.1) and used to calculate the permeability coefficient (Table 3.8) [55]. Encapsulated epalrestat permeation through the cornea was lower compared to epalrestat in solution (ANOVA; F3,12 = 9.32; p < 0.05). Differently, no statistically different results were recorded for permeability through the sclera for epalrestat in niosomes compared to free drug. Compared to the free drug solution, formulation in niosomes provided more reproducible data with less variability, and as expected, permeability coefficients through sclera were greater than through cornea, particularly in the case of the most cationic niosomes (TDC10) (Table 3.8). 3. Formulation and Characterization of Epalrestat-Loaded Polysorbate 60 Cationic Niosomes for Ocular Delivery 124 Figure 3.13. Amounts of encapsulated epalrestat permeated through the cornea (left) and sclera (right Table 3.8. Steady-state flux, lag time, and permeability coefficient of different tissues and formulations. Sample Steady State Flux (µg/cm2 × h) Lag Time (min) Permeability Coefficient (×106 cm/s) Cornea Free drug 1.029 (0.356) 152 (66) 1.43 (0.49) TCD0 0.469 (0.204) 163 (12) 0.65 (0.28) TDC5 0.453 (0.295) 138 (36) 0.63 (0.40) TDC10 0.095 (0.008) 33 (32) 0.13 (0.01) Sclera Free drug 0.887 (0.758) 118 (4) 1.23 (1.09) TCD0 0.634 (0.035) 57 (16) 0.88 (0.05) TDC5 0.538 (0.110) 201 (6) 0.75 (0.15) TDC10 0.636 (0.070) 196 (4) 0.88 (0.09) TCD5 and TCD10 niosomes displayed lower accumulation in corneal tissue than TCD0 niosomes and the epalrestat in solution. The niosomes showed lower drug accumulation than the epalrestat in solution in scleral tissue (Figure 3.14). AXEL KATTAR 125 Figure 3.14. Epalrestat retained in corneal (left) and scleral (right) tissue after 6 h permeation experiment in 3.3.7 IR-Raman To confirm the permeation of epalrestat through the different tissues IR-Raman spectroscopy was performed on corneal and scleral cells. The ratio of the Raman spectrum peak for epalrestat from the top part of the tissue (in contact with the donor chamber) to the bottom part of the tissue (in contact with the receiving chamber) was taken as an indication for confirmation of permeation of epalrestat through the tissue (Figure 3.15). Furthermore, pictures were assembled using accumulations of Raman spectra in a plane throughout the different tissues. This allowed for the production of heat maps showing the concentration of epalrestat at different levels of the tissue. Samples of TCD0 cornea, TCD10 cornea, and TCD10 sclera were able to produce readable pictures (Figure 3.16). This can be difficult due to the focus of the laser on the sample changing as the tissue moves with dehydration and burning. White lines were used to mark the tissue delimitation on the heat maps. 3. Formulation and Characterization of Epalrestat-Loaded Polysorbate 60 Cationic Niosomes for Ocular Delivery 126 Figure 3.15. The ratio of the top-to-bottom intensity of Raman peaks indicates the presence AXEL KATTAR 127 Figure 3.16. Cross-section heat map of epalrestat concentration in a 30 × 15 µm plane of the bottom of the cornea or the sclera after 6 h permeation of niosomes loaded with epalrestat (A): TCD0 cornea, (B): TCD10 cornea, (C): TCD10 sclera. Scale bar: 6 µm. 3. Formulation and Characterization of Epalrestat-Loaded Polysorbate 60 Cationic Niosomes for Ocular Delivery 134 typical yellow color of epalrestat was also perceptible by the naked eye. The low value of the ratio for TCD10 sclera (0.92) is unusual but could be explained by interference from gasses liberated during the analysis process if the sample started to burn. Indeed, the peak of epalrestat situated at 1575 cm 1is close to that of carbon (1581 cm 1) [71]. This also would explain the great variance in the results between the four measuring points. The heat maps of the cornea and sclera show the presence of epalrestat inside the tissue, permeating in different channels, adding weight to the results obtained from the Franz cell permeation experiments. While Raman spectroscopy has been used on ocular media for the detection of ganciclovir in ocular media in vivo [72] or for the detection of glutamate in tissues [73], this is, to our knowledge, the first time IR-Raman was used to confirm drug permeation through ocular tissues. Epalrestat is a highly hydrophobic drug, and we found that it can be efficiently encapsulated in niosomes. The niosomes increase the preparation of eyedrops possible. Moreover, niosomes protect from the irritancy that a pure drug solution causes on the ocular surface. According to our results, the niosomes do not release epalrestat during storage but only when subjected to a strong dilution in the presence of other surfactant molecules, which may resemble the conditions on the ocular surface. The formulation TCD5 seemed to be the most balanced, as it was cationic, had sustained release of epalrestat over a long period, was safe for the ocular surface, and permeated both the cornea and the sclera. The capability to sustain drug release in vitro means that most part of the drug dose remains in niosomes when topically placed on the eye, and niosomes have been reported to enhance drug permeability in vivo [24]. Some previous reports on drug-loaded niosomes for ocular delivery have also shown in vitro sustained release in the frame of a few AXEL KATTAR 135 days for hydrophobic drugs such as fluconazole [74] and natamycin [75] or even mid-polarity drugs such as vancomycin [76], doxycycline [77] or naltrexone [78]. In vivo, studies evidenced the success of the niosome approach compared to the free drug solution, indeed probably due to the more sustained release. Niosomes have been shown to remain on the ocular surface longer than the drug solution. Thus, a sustained release may prevent a very rapid washout from the ocular surface while still creating a drug concentration gradient that facilitates the diffusion through eye tissues [24]. 3.5 CONCLUSIONS Our study demonstrated the ability of cationic niosomes to encapsulate epalrestat. Neutral to mild-cationic niosomal formulations showed suitable physicochemical characteristics for topical ocular drug delivery, supported by a low ocular irritability potential, high biocompatibility, sustained release, and permeability through the cornea and scleral tissue. Compared to epalrestat-loaded contact lenses or free drug solutions, niosomes can encapsulate more drugs, increasing apparent solubility, and protect better the drug from premature degradation while promoting the pass towards inner eye tissues. Moreover, as revealed in the HET-CAM and zebrafish embryotoxicity assays, drug encapsulation in niosomes makes the formulation safe. Similar concentrations of epalrestat in solution would be harmful to patients. These findings point out epalrestat-loaded niosomes as suitable for non-invasive drug delivery to inner eye structures. 3. Formulation and Characterization of Epalrestat-Loaded Polysorbate 60 Cationic Niosomes for Ocular Delivery 136 3.6 REFERENCES 1. International Diabetes Foundation. IDF Diabetes Atlas 2021. Available online: www.diabetesatlas.org (accessed on 10 April 2023). 2. Chung, S.S.M.; Ho, E.C.M.; Lam, K.S.L.; Chung, S.K. Contribution of Polyol Pathway to Diabetes-Induced Oxidative Stress. J. Am. Soc. Nephrol.2003, 14, 233 236. 3. Stitt, A.W.; Curtis, T.M.; Chen, M.; Medina, R.J.; McKay, G.J.; Jenkins, A.; Gardiner, T.A.; Lyons, T.J.; Hammes, H.-P.; Simó, R.; et al. The Progress in Understanding and Treatment of Diabetic Retinopathy. Prog. Retin. Eye Res.2016, 51, 156 186. 4. Neubauer, A.S.; Ulbig, M.W. Laser Treatment in Diabetic Retinopathy. Ophthalmologica 2007, 221, 95 102. 5. 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The investigation was carried out in four steps and the conclusions which arise from each chapter are summarized in the following paragraphs. 1. Cationic niosomes were successfully designed with the intent of encapsulating epalrestat. The prepared nanoparticles displayed favorable physicochemical properties for delivering drugs to the ocular surface. These formulations have a low potential for causing eye irritation, are highly biocompatible, offer sustained drug release, and possess the ability to permeate the cornea and scleral tissue. They also provide superior protection against premature drug degradation while facilitating its passage into the inner eye tissues. Furthermore, our research has indicated the safety of drug encapsulation in niosomes, as demonstrated in HET-CAM and zebrafish embryotoxicity assays. In contrast, similar concentrations of epalrestat in solution could be harmful to patients. These findings underscore the suitability of epalrestat-loaded niosomes for non-invasive drug delivery to the inner eye structures. 2. Through experiments involving Langmuir monolayers and the analysis of their mechanical properties, the niosomal arrangement in the bilayer was studied. The results revealed that there was a DOTMA dependent increase in compressibility in all the systems examined. This effect served to impede the formation of tightly packed arrangements, which could have adverse effects on drug loading in the niosomal bilayer. Furthermore, a positive correlation between the concentration of DOTMA and the degree of packing or stability in the films composed of Tween 60, cholesterol, and DOTMA was found. This relationship is expected to mirror what occurs in the bilayer of vesicles. In addition to these findings, the change in niosomes as they transitioned from storage conditions (a water-based formulation at 20°C) to conditions resembling those encountered in the eye during administration (an electrolytic medium at a temperature closer to 30°C) was explored. This chapter showed that the inclusion of DOTMA in the binary system of Tween 60 and cholesterol significantly increased the compression modulus at 30°C and the pressure at higher surface area per molecule, particularly in the air/water interface suggesting that cholesterol holds an important role in the stability of the niosomal bilayer. 3. Successful preparation of olegels capable of incorporating epalrestat was achieved. These oleogels exhibit a significantly greater capacity for loading epalrestat compared to niosomes and contact lenses. Moreover, the release of the drug in simulated lacrimal fluid is ample to saturate the tear film within 20 minutes. Importantly, the loaded oleogels are non-irritating, making them comparable to other ocular formulations that shield the eye's surface from the potential irritations caused by epalrestat in solution. Furthermore, the permeation of epalrestat through the cornea and sclera is akin to the permeation observed when epalrestat is encapsulated in niosomes, and it surpasses the permeation rate seen with epalrestat released from contact lenses. AXEL KATTAR 255 This novel use of oleogels to administer epalrestat topically represents a technologically robust method for delivering the drug to ocular tissues in a non-invasive manner. 4. Micelles formulated from Pluronic® F127 serve as a valuable point of comparison for the in vivo administration of eye drops containing 0.2 mg/mL epalrestat. These micelles outperformed niosomes TCD5 and oleogel C in terms of lacrimal and retinal concentrations. Notably, the micelles maintained high lacrimal concentrations for up to two hours. It is worth mentioning that all three formulations, including TCD5 niosomes, F127 micelles, and oleogel C, effectively delivered epalrestat to various eye tissues in vivo. However, the corneal route may not be the most efficient path to reach the retina, which is the therapeutic target for diabetic retinopathy. On the other hand, the transscleral route appears to offer a viable means for epalrestat, released from each of these formulations, to reach the retina. As a uniform overview of the conclusions put forth in this Doctoral Thesis, the possibilities for topical delivery of epalrestat for the treatment of diabetic retinopathy have been extended. Three drug carriers have been developed from the chemical assembly of the system to the in vivo distribution when applied to a rabbit model. The results obtained during this investigation may pave the way towards less invasive forms of administration for patients suffering from diabetic retinopathy. 7. Conclusions 262 JCR Category: Pharmacology & Pharmacy Category Quartile: (Q1, Pharmacology & Pharmacy); CiteScore (Q1, Pharmaceutical Science). Reproduction permission: The article was published in Open Access. AXEL KATTAR 263 ETHICAL PERMISSIONs FOR ANIMAL EXPERIMENTS 264 AXEL KATTAR 265 266 CHECKLIST FOR THESIS THAT INCLUDE EXPERIMENTAL ANIMALS. EXPERIMENTAL ANIMALS ARRIVE Yes/No/NA page Title Yes Provide as accurate and concise a description of the content of the article as possible. 221 Abstract Yes Provide an accurate summary of the background, research objectives, including details of the species or strain of animal used, key methods, principal findings and conclusions of the study. 10-12 Background Yes Provide an accurate summary of the background, research objectives, including details of the species or strain of animal used, key methods, principal findings and conclusions of the study. 221-249 Yes Explain how and why the animal species and model being used can address the scientific relevance to human biology. 231-240 Objectives Yes Clearly describe the primary and any secondary objectives of the study, or specific hypotheses being tested. 220-223 Methods Ethical statement Yes Indicate the nature of the ethical review permissions, relevant licenses, and national or institutional guidelines for the care and use of animals, that cover the research. 232 Study design AXEL KATTAR 267 Yes Number of experimental and control groups 232 Yes Steps taken to minimize the effects of subjective bias when allocating animals to treatment (e.g. randomization procedure) and when assessing results (e.g. if done, describe who was blinded and when). 232 Yes The experimental unit (e.g. a single animal, group or cage of animals). 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Provide details of any specialist equipment used, including supplier(s). 232 Yes When (e.g. time of day). 232 Yes Where (e.g. home cage, laboratory, water maze). 232 Yes Why (e.g. rationale for choice of specific anesthetic, route of administration, drug dose used). 232 Experimental animals Yes Provide details of the animals used, including species, strain, sex, developmental stage (e.g. mean or median age plus age range) and weight (e.g. mean or median weight plus weight range). 232 Yes Provide further relevant information such as the source of animals, international strain nomenclature, genetic modification status (e.g. knock-out or transgenic), genotype, health/immune status, drug or test naïve, previous procedures, etc. 232 Housing and husbandry Yes Housing (type of facility e.g. specific pathogen free [SPF]; type of cage or housing; bedding 232 268 material; number of cage companions; tank shape and material etc. for fish). Yes Husbandry conditions (e.g. breeding program, light/dark cycle, temperature, quality of water etc for fish, type of food, access to food and water, environmental enrichment). 232 Yes Welfare-related assessments and interventions that were carried out prior to, during, or after the experiment. 232 Sample size Yes Specify the total number of animals used in each experiment, and the number of animals in each experimental group. 232 Yes Explain how the number of animals was arrived at. Provide details of any sample size calculation used. 232 Yes Indicate the number of independent replications of each experiment, if relevant. 232 Allocating animals to experimental groups Yes Indicate the number of independent replications of each experiment, if relevant. 232 Yes Describe the order in which the animals in the different experimental groups were treated and assessed. 232 Experimental outcomes Yes Clearly define the primary and secondary experimental outcomes assessed (e.g. cell death, molecular markers, behavioral changes). 233-240 Statistical methods Yes Provide details of the statistical methods used for each analysis. 229 Yes Specify the unit of analysis for each dataset (e.g. single animal, group of animals, single neuron). 227-229; 232 AXEL KATTAR 269 Yes Describe any methods used to assess whether the data met the assumptions of the statistical approach. 229; 236240 Results and discussion Basal data Yes For each experimental group, report relevant characteristics and health status of animals (e.g. weight, microbiological status, and drug or test naïve) prior to treatment or testing (this information can often be tabulated). 232 Numbers analyzed Yes Report the number of animals in each group included in each analysis. Report absolute numbers (e.g. 10/20, not 50%). 232 Yes If any animals or data were not included in the analysis, explain why. 233 Outcomes and estimation Yes Report the results for each analysis carried out, with a measure of precision (e.g. standard error or confidence interval). 232-240 Adverse events NA Give details of all important adverse events in each experimental group. Yes Describe any modifications to the experimental protocols made to reduce adverse events. 232 Interpretation/scientific implications Yes Interpret the results, taking into account the study objectives and hypotheses, current theory and other relevant studies in the literature. 232-240 Yes Comment on the study limitations including any potential sources of bias, any limitations of the animal model, and the imprecision associated with the results. 232 NA Describe any implications of your experimental methods or findings for the replacement, 270 refinement or reduction (the 3Rs) of the use of animals in research. Generalizability/translation Yes Comment on whether, and how, the findings of this study are likely to translate to other species or systems, including any relevance to human biology. 232-240 Funding Yes List all funding sources (including grant number) and the role of the funder(s) in the study. 232 Based on The ARRIVE guidelines: Animal Research: Reporting of In Vivo Experiments. PhD Student signature AXEL KATTAR u se UNlVERSIDADE DE SANTIAGO DE COMPOSTELA CAMPUS VIDA CAMM º' "'""" ) """""'º"" The eye is a crucial organ that allows us to experience the world visually, and the loss of vision is a fear for many. One significant threat to eye health is diabetic retinopathy, which can result in consequences as severe as blindness if left untreated. Current treatments for diabetic retinopathy involve injections and laser exposure, but they only slow down the degeneration of the retina. Therefore, there exists a need for preventive treatments to protect patients' vision. One potential solution is the use of a small molecule called epalrestat, which can block a metabolic pathway involved in the development of diabetic retinopathy. To enhance the delivery of epalrestat to the eye, drug carriers have been investigated. Niosomes and oleogels were formulated, characterized, and tested in various models including ex vivo, in ovo, and in vivo assays. The self-assembly process of the niosomes was investigated with the help of a monolayer model. These carriers provided a saf e and e:fficient way to help with the transport of epalrestat across the various ocular barriers.