Micelles of Progesterone for Topical Eye Administration: Interspecies and Intertissues Differences in Ex Vivo Ocular Permeability

pharmaceutics Article Micelles of Progesterone for Topical Eye Administration: Interspecies and Intertissues Differences in Ex Vivo Ocular Permeability Adrián M. Alambiaga-Caravaca 1, María Aracely Calatayud-Pascual 1, Vicent Rodilla 1, Angel Concheiro 2, Alicia López-Castellano 1,* and Carmen Alvarez-Lorenzo 2,* 1Departamento de Farmacia, Facultad de Ciencias de la Salud, Instituto de Ciencias Biomédicas, Universidad Cardenal Herrera-CEU, CEU Universities, C/Santiago Ram ó n y Cajal, s/n., Alfara del Patriarca, 46115 Valencia, Spain; [email protected] (A.M.A.-C.); [email protected] (M.A.C.-P.); [email protected] (V.R.) 2Departamento de Farmacología, Farmacia y Tecnología Farmacéutica, I+D Farma Group, Facultad de Farmacia, and Health Research Institute of Santiago de Compostela (IDIS), Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain; [email protected] *Correspondence: [email protected] (A.L.-C.); carmen.alvarez.lor[email protected] (C.A.-L.); Tel.: +34-961-369-000 (ext. 64906) (A.L.-C.); +34-881-815-239 (C.A.-L.) Received: 17 June 2020; Accepted: 23 July 2020; Published: 26 July 2020   Abstract: Progesterone (PG) may provide protection to the retina during retinitis pigmentosa, but its topical ocular supply is hampered by PG poor aqueous solubility and low ocular bioavailability. The development of efficient topical ocular forms must face up to two relevant challenges: Protective barriers of the eyes and lack of validated ex vivo tests to predict drug permeability. The aims of this study were: (i) To design micelles using Pluronic F68 and Soluplus copolymers to overcome PG solubility and permeability; and (ii) to compare drug diffusion through the cornea and sclera of three animal species (rabbit, porcine, and bovine) to investigate interspecies differences. Micelles of Pluronic F68 (3–4 nm) and Soluplus (52–59 nm) increased PG solubility by one and two orders of magnitude, respectively and exhibited nearly a 100% encapsulation efficiency. Soluplus systems showed in situ gelling capability in contrast to the low viscosity Pluronic F68 micelles. The formulations successfully passed the hen’s egg-chorioallantoic membrane test (HET-CAM) test. PG penetration through rabbit cornea and sclera was faster than through porcine or bovine cornea, although the differences were also formulation-dependent. Porcine tissues showed intermediate permeability between rabbit and bovine. Soluplus micelles allowed greater PG accumulation in cornea and sclera whereas Pluronic F68 promoted a faster penetration of lower PG doses. Keywords: progesterone; retinitis pigmentosa; interspecies ocular permeability differences; polymeric micelles; Soluplus; Pluronic; ocular drug delivery; solubility; hen’s egg-chorioallantoic membrane test (HET-CAM) assay 1. Introduction Retinitis pigmentosa (RP) is a degenerative disease that involves heterogeneous inherited photoreceptors apoptosis affecting the peripheral retina. RP is the most common hereditary retinal degeneration, causing blindness [ 1 ]. One of the concerns in RP is that a single mutation can cause a variety of different clinical phenotypes, so there is a large clinical and genetic heterogeneity; also, different mutations can cause the same syndrome [ 2 ]. In the early stages of the disease, rod photoreceptors are affected causing difficulties with dark adaptation, night blindness, and loss of mid-peripheral visual field in adolescence or young adulthood. As the RP advances and cones Pharmaceutics 2020,12, 702; doi:10.3390/pharmaceutics12080702 www.mdpi.com/journal/pharmaceutics Pharmaceutics 2020,12, 702 2 of 18 die, the central vision is lost. Nevertheless, RP is a highly variable disorder; some patients develop symptomatic visual loss in childhood whereas others remain asymptomatic until mid-adulthood [ 1 , 3 , 4 ]. Different therapeutic approaches have been adopted depending on the stage of the disease. In the early stages, the aim is to halt the degeneration using gene therapy or pharmacological treatments (neurotrophic growth factors or anti-apoptotic factors), reducing the production of retino-toxic molecules, and limiting oxidative damage. In advanced stages, when there are barely any functional photoreceptors left, retinal transplantation or electronic retinal implants may be needed [ 4 ]. In the early stages when rods die and oxygen consumption in the outer retina is reduced, the supply of oxygen is not decreased because there is no self-regulation of choroidal blood vessels. This leads to increased levels of oxygen in the external retina. It has been suggested that long-term protection against oxidative damage can delay the death of cones in patients with RP regardless of the causative mutation [5]. Estrogens exert neuroprotection on the retina and optic nerve. Progesterone (PG; a C-21 steroid hormone, Figure 1a-A) and its derivatives may promote the formation of new myelin and reduce the extent of myelin sheath loss, showing therapeutic potential against retinal degeneration [ 6 – 10 ]. The retina is considered a true steroidogenic central nervous system (CNS) structure [ 9 ]. PG has been reported to promote cell survival and proliferation in non-neuronal tissues [ 10 ] and to provide protection to the retina during retinitis pigmentosa [ 11 , 12 ]. Recently, Hernandez-Rabaza et al. [ 13 ] studied the effects of PG on RP highlighting benefits such as reduction of the typical gliosis, decrease in the concentration of glutamate in the retina and increase of the concentration of glutathione. Similar results were found when PG administration was performed in the RP mouse model rd10. However, topical ocular delivery of PG is hindered by its poor aqueous solubility and low ocular bioavailability [ 13 ]. PG dosage forms for eye administration have barely been investigated and, to the best of our knowledge, there is only a report on PG encapsulation into nanocarriers [ 14 ]. That previous study revealed that the high affinity of PG for polybutylcianoacrylate nanoparticles causes that PG levels in ocular tissues were even lower than when applied as a solution, highlighting the need of an adequate design of the nanocarrier [14]. The development of safe and effective topical ocular formulations remains a challenging task due to the inherent anatomical and physiological barriers of the eye [ 15 – 18 ]. Topical administration is a painless, non-invasive, and simple practice method, but corneal tissue is a formidable barrier for drug penetration [ 5 , 6 ]. Corneal epithelium (lipophilic) limits the permeation of hydrophilic molecules, while the stroma, i.e., the layer below, is composed of a highly hydrated extracellular matrix that limits the permeation of lipophilic substances [ 1 ]. Moreover, instillation of eye drops activates defense mechanisms, such as lacrimation, which together with tear turnover, dilute the drug and clear it from the ocular surface. The excess volume is drained via the nasolacrimal duct into systemic circulation [ 3 , 5 , 6 ]. Overall, ocular drug bioavailability is less than 5% [ 19 – 21 ]. Polymeric micelles are an attractive option for ocular delivery due to their capability to encapsulate hydrophobic drugs, facilitating the development of clear aqueous solutions that avoid the sticky sensation and blurred vision of semisolid formulations. The amphiphilic excipients used in micelles can facilitate the permanence of the formulation on the eye surface and inhibit efflux pumps on corneal tissue [ 22 ]. Moreover, micelles can facilitate drug penetration through both the cornea and sclera, providing therapeutic drug levels to the back of the eye [23,24]. The preclinical assessment of ocular drug delivery systems commonly involves ex vivo permeability studies using non-human cornea or sclera. The availability of each animal species is different according to the region [ 25 ] and thus ex vivo tests of human formulations have been reported in the literature for a variety of animal tissues, mainly rabbit, porcine, and bovine [ 26 ]. These tests in animal tissues are mainly intended to predict human drug permeability assuming that the test renders similar results disregarding the species. Rabbit cornea and sclera have been preferred in the majority of ex vivo permeation studies in order to correlate with in vivo preclinical tests of safety and efficacy carried out also in rabbits, although now these in vivo tests are being restricted by most Animal Ethical Committees over the world [ 4 ]. Porcine eye tissues seem to be the most similar to the human ones: Pharmaceutics 2020,12, 702 3 of 18 The eyeballs have similar weight and size, and also the volumes of aqueous humor and the vitreous body are comparable [ 27 ]. The permeability of certain active substances through the porcine cornea is estimated to be similar or slightly lower than through the human cornea since the porcine corneal epithelial layer is a little thicker. In parallel, the permeability of bovine corneas may be lower because bovine epithelium has almost twice as many cell layers as the human corneal epithelium [ 27 ]. Despite differences between human and bovine corneas, the Bovine Cornea Opacity/Permeability test (BCOP) is one of the currently approved alternatives to in vivo ocular irritation assay in rabbits [ 28 ]. There is only one study that reports on the interspecies (porcine, rabbit, and bovine) differences in the apparent permeation coefficient, P app , of ciprofloxacin hydrochloride, lidocaine hydrochloride, timolol maleate, and dexamethasone dissolved in a buffer solution. P app values were recorded for each drug through sclera, cornea, conjunctiva, choroid and retina, and the combination of the different tissue layers. As expected, disregarding the drug and the animal, P app was higher for conjunctiva and sclera and lower for cornea. The values recorded for rabbits were in general higher and also showed greater variability than those recorded for porcine tissues. Moreover, tissue dissimilarities were proved not to be the only cause of the P app differences. Drug lipophilia (log P between 1 and 3) seems to facilitate the penetration in all tissues investigated [ 21 , 26 ]. The cornea has a molecular weight cut-offof 400–600 Da while sclera allows the pass of 70 kDa molecules [29]. Pharmaceutics 2020, 12, x 3 of 18 Figure 1. (a) Structure of progesterone (PG) (A), Soluplus ® (B), and Pluronic ® (C); (b) appearance of micelle dispersions (A) 16% Soluplus + PG, (B) 20% Soluplus + PG, (C) 16% Soluplus, (D) 20% Soluplus, (E) 16% Pluronic + PG, (F) 20% Pluronic + PG, (G) 16% Pluronic, and (H) 20% Pluronic; (c) PG solubility in Soluplus and Pluronic F68 micelle dispersions formulated in a phosphate saline buffer (PBS) pH 7.4. Error bars represent the standard deviations (n = 3). The preclinical assessment of ocular drug delivery systems commonly involves ex vivo permeability studies using non-human cornea or sclera. The availability of each animal species is different according to the region [25] and thus ex vivo tests of human formulations have been reported in the literature for a variety of animal tissues, mainly rabbit, porcine, and bovine [26]. These tests in animal tissues are mainly intended to predict human drug permeability assuming that the test renders similar results disregarding the species. Rabbit cornea and sclera have been preferred in the majority of ex vivo permeation studies in order to correlate with in vivo preclinical tests of safety and efficacy carried out also in rabbits, although now these in vivo tests are being restricted by most Animal Ethical Committees over the world [4]. Porcine eye tissues seem to be the most similar to the human ones: The eyeballs have similar weight and size, and also the volumes of aqueous humor and the vitreous body are comparable [27]. The permeability of certain active substances through the porcine cornea is estimated to be similar or slightly lower than through the human cornea since the porcine corneal epithelial layer is a little thicker. In parallel, the permeability of bovine corneas may be lower because bovine epithelium has almost twice as many cell layers as the human corneal epithelium [27]. Despite differences between human and bovine corneas, the Bovine Cornea Opacity/Permeability test (BCOP) is one of the currently approved alternatives to in vivo ocular irritation assay in rabbits [28]. There is only one study that reports on the interspecies (porcine, rabbit, and bovine) differences in the apparent permeation coefficient, P app , of ciprofloxacin hydrochloride, lidocaine hydrochloride, timolol maleate, and dexamethasone dissolved in a buffer solution. P app values were recorded for each drug through sclera, cornea, conjunctiva, choroid and retina, and the combination of the different tissue layers. As expected, disregarding the drug and the animal, P app Figure 1. ( a ) Structure of progesterone (PG) (A), Soluplus ® (B), and Pluronic ® (C); ( b ) appearance of micelle dispersions (A) 16% Soluplus +PG, (B) 20% Soluplus +PG, (C) 16% Soluplus, (D) 20% Soluplus, (E) 16% Pluronic +PG, (F) 20% Pluronic +PG, (G) 16% Pluronic, and (H) 20% Pluronic; ( c ) PG solubility in Soluplus and Pluronic F68 micelle dispersions formulated in a phosphate saline buffer (PBS) pH 7.4. Error bars represent the standard deviations (n=3). Pharmaceutics 2020,12, 702 4 of 18 The aim of the present study was two-fold: (i) To design micelle-based formulations of PG that can overcome the drug solubility and permeability limitations when intended for topical treatment of RP; and (ii) to compare drug diffusion through cornea and sclera of three animal species, namely, rabbit, porcine, and bovine, in order to investigate interspecies differences. Specifically, two polymers, Soluplus and Pluronic F68, were selected to prepare the micelles. Soluplus is a polyvinyl caprolactam—polyvinyl acetate—polyethylene glycol graft copolymer (PCL-PVAc-PEG) (Figure 1a-B). Soluplus has a very low critical micelle concentration (CMC) value (6.6 × 10 −5 mM) and its micelles are highly stable against dilution and may also undergo in situ gelling on the ocular surface, enhancing penetration into ocular structures [ 23 , 30 ]. The hydrophobic core of Soluplus micelles allows the encapsulation of poorly soluble drugs, notably enhancing their apparent solubility [ 31 ]. Pluronic F68 is a linear triblock poly(ethylene oxide)—poly(propylene oxide)—poly(ethylene oxide) (PEO-PPO-PEO) copolymer (Figure 1a-C) that, in addition to the self-assembling properties, exhibits the capability to inhibit P-glycoprotein efflux pumps at the eye surface and to undergo sol-gel transitions upon heating [ 30 ]. To carry out the study, first the capability of micelles prepared covering a wide range of copolymers concentrations to solubilize PG was investigated. After a screening of ocular compatibility using the HET-CAM test, ex vivo drug permeability through the cornea and sclera from the three selected animal species was investigated. 2. Materials and Methods 2.1. Materials Progesterone (PG, 314.46 g/mol), Pluronic ® F68 (8350 g/mol), and PG: Methylβ -cyclodextrin complex (85.2 mg PG/g; Progresterone-water soluble) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA), Soluplus ® (115,000 g/mol) from BASF (Ludwigshafen, Germany), acetonitrile was provided from Scharlab SL (Barcelona, Spain), ethanol absolute from VWR (Fontenay-sous-Bois, France), propylene glycol from Guinama (La Pobla de Vallbona, Spain). Ultrapure water was obtained by reverse osmosis (Milli-Q, Millipore Ib é rica, Madrid, Spain). All other chemicals used were of analytical reagent grade and used as received. 2.2. Micelles Preparation and Characterization Micelles were prepared by dispersing Soluplus or Pluronic F68 at different concentrations (4%, 8%, 12%, 16%, and 20% w/w) in triplicate in a phosphate saline buffer (PBS) pH 7.4. The dispersions were kept under magnetic stirring for 24 h at room temperature. The size, zeta potential, and polydispersion index (PDI) of the micelles (filtered through 0.22 µ m membranes; Acrodisc ® Syringe Filter, GHP Minispike, Waters, Milford, MA, USA) were measured in triplicate in a Zetasizer ® 3000HS (Malvern Instruments, Worcestershire, UK). The pH was measured using a calibrated GLP22 pH meter (Crison Instruments, L’Hospitalet de Llobregat, Spain) [24]. 2.3. Solubility of Progesterone (PG) in Micelle Dispersions Aliquots (10 mL) of Soluplus and Pluronic F68 dispersions prepared as above were placed in tubes containing PG (2.6–3.0 mg) and kept under constant magnetic agitation (Unitronic, JP Selecta, Barcelona, Spain) at 300 rpm and room temperature [ 24 , 30 ]. The PBS pH 7.4 medium without a copolymer was used as the control. After 96 h of stirring, the dispersions were centrifuged (centrifuge model 5804R, Eppendorf AG, Hamburg, Germany) at 5000 rpm for 30 min to separate the non-solubilized PG. The supernatants were collected gently and diluted with an ethanol:water (30:70 v/v) mixture to determine the apparent solubility of PG. The experiments were carried out in triplicate. The PG concentration was measured in a HPLC (AS-4140 autosampler, PU-4180 pump, LC-NetII/ADC interface box, CO-4060 column oven, MD-4010 photodiode array detector) from Jasco (Tokyo, Japan) fitted with a C18 column (LiquidPurple ODS C18, 5 µ m, 4.6 × 150 mm) and operated using the ChromNAV software (ver. 2, Jasco, Tokyo, Japan). The mobile phase was acetonitrile:water (20:80 Pharmaceutics 2020,12, 702 5 of 18 v/v) at 1 mL/min and 25 ◦ C. The injection volume was 50 µ L. PG was quantified at 245 nm (retention time 5.1 min). Standard solutions of PG (0.01–5 µg/mL) in ethanol/water (30:70 v/v) were prepared. The solubilization capability was characterized taking into account PG solubility in the medium without (S W ) and with micelles (S tot ), the concentration of the copolymer used (C copol ), the CMC of the copolymer, and the universal constant of gases (R), as follows [ 24 , 30 ]. The molar solubilization capacity (moles of drug solubilized per mol of copolymer forming micelles): X=Stot −Sw Ccopol−CMC (1) The micelle-water partition coefficient (ratio between the drug concentration in the micelle and the aqueous phase): P=Stot −Sw Sw(2) The molar micelle-water partition coefficient, assigning a default copolymer concentration of 1 M: PM =X(1−CMC) Sw(3) Gibbs standard-free energy of solubilization was estimated from the molar micelle/water partition coefficient (PM) and the micelle-water partition coefficient (P), as: ∆Gs =−RT ×ln(PM)(4) ∆Gs =−RT ×ln(P)(5) The proportion of drug molecules encapsulated in the micelles: mf =Stot −Sw Stot (6) 2.4. Rheological Analysis The dependence on temperature of storage (G 0 ) and loss (G 00 ) moduli of Soluplus and Pluronic F68 dispersions was evaluated in a Rheolyst AR-1000N rheometer (TA Instruments, Newcastle, UK) equipped with an AR-1000N data analyzer, a Peltier plate, and cone geometry (59 mm diameter, 2.1 ◦ ). The angular frequency was fixed at 5 rad/s and the temperature increased from 15 to 40 ◦ C with a ramp rate of 2 ◦C/min, while the oscillation stress was kept at 0.1 Pa. 2.5. Hen’s Egg Test Chorioallantoic Membrane (HET-CAM) Fertilized hen’s eggs from the Coren Technological Incubation Center (San Cibrao das Viñas, Spain) were incubated at 37 ◦C and 60% RH (Ineltec, model CC SR 0150, Barcelona, Spain). Each of the eggs was manually rotated 180 ◦ every 8 h to ensure the correct embryo development. After nine days of incubation, a circular cut (1 cm) was made in the air chamber of the eggshell. The inner membrane was moistened with 0.9% NaCl for 30 min and then carefully removed to expose the chorioallantoic membrane (CAM). Dispersions of Soluplus and Pluronic F68 micelles loaded with PG (200 µ L) were deposited in the CAM of different eggs. Solutions of 0.1 N NaOH and 0.9% NaCl were used as positive and negative controls, respectively (300 µ L). The eggs were monitored for bleeding, vascular lysis, and coagulation of the CAM vessels for 300 s. The irritation score (IS) was calculated as: IS =(301 −tH)×5 300 +(301 −tL)×7 300 +(301 −tC)×9 300 (7) Pharmaceutics 2020,12, 702 6 of 18 In this equation, t H represents haemorrhage time (s), t L is lysis time (s) and t C is coagulation time (s) [ 30 ]. According to the IS values, the substances are classified as non-irritant (IS <1), mildly irritant (1 ≤IS <5), moderately irritating (5 ≤IS <10), or severely irritant (IS >10) [32]. 2.6. Ex Vivo Corneal and Sclera Permeability Assay Bovine, porcine, and rabbit eyes were supplied by a local slaughterhouse and transported immersed in a PBS medium without antibiotics in an ice bath. Corneas and scleras were carefully isolated, washed with PBS, and used to separate donor and receptor compartments in vertical diffusion Franz cells [33–37]. The receptor chamber was filled with propyleneglycol:water (40:60% w/w) pH 7.4 (6 mL). The donor chamber (2 mL) was filled with the same solution and the tissue was allowed to balance for 1 h. The solubility of PG in the receptor medium has already been established [ 38 ] and ensured sink conditions. The receptors were kept at 35 ◦ C (temperature controlled bath), and gentle magnetic stirring was applied for 1 h. After 1 h, the solution at the donor chamber was completely removed using a Pasteur pipette and replaced by the micelles formulations (1 mL) prepared as described above in a PBS pH 7.4. The donor compartments (0.785 cm 2 permeation available area) were covered with parafilm. Samples (1 mL) were taken manually from the receptor chamber at 30, 60, 90, 120, 180, and 240 min, and replaced with a fresh medium avoiding bubbles. All the experiments were carried out in quadruplicate. As a control, a solution of PG (343.04 µ g/mL) was prepared by dispersion of the commercially available PG: Methylβ -cyclodextrin complex powder (85.2 mg PG/g) (Sigma-Aldrich Co.; St. Louis, MO, USA) in a buffer pH 7.4 and evaluated in parallel. PG permeated was analyzed by HPLC and the cumulative amount of the drug ( µ g/cm 2 ) collected in the receptor medium was plotted against time to estimate the apparent permeability coefficients (Papp, cm/s), as follows: Papp =∆Q ∆t×(A×Co ×60)−1(8) In this equation, ∆ Q/ ∆ t( µ g/min) is the steady state flow (J) across corneal or scleral tissue, Ais the exposed surface area of ocular tissue (0.785 cm 2 in bovine and porcine tissues; 0.567 cm 2 in rabbit), C 0 is the initial PG concentration ( µ g/mL) in the donor compartment, and 60 is taken as the factor to convert minute into second [30,39]. Each experiment wasperformed in triplicate, andtheresults were reportedas themean value ± standard deviation. The steady state flow (J) was calculated from the slope as well as the lag time (T 0 ) by intersecting with the x-axis of the linear regression [ 31 ]. The drug concentration in the donor chamber was also quantified at the end of the test (4 h). All corneas and scleras were visually inspected after the test to verify that all of them were in good conditions, without cracks or changes in their appearance. Corneas and scleras were placed in Falcon tubes containing acetonitrile (2 mL). The tubes were kept under mild stirring at 37 ◦ C for 24 h, immersed in an ultrasound bath for 99 min at 37 ◦ C, and centrifuged (1000 rpm, 5 min, 25 ◦ C). The supernatants were filtered (Acrodisc ® Syringe Filter, 0.22 µ m GHP Minispike, Waters), centrifuged (14,000 rpm, 20 min, 25 ◦C), and filtered to be analyzed in HPLC. 2.7. Statistical Analysis Data analysis was performed using the Mann-Whitney or the Kruskal-Wallis tests as appropriate. Post-hoc multiple comparisons were carried out using the Man-Whitney test applying Bonferroni’s correction for significance. Statistical analysis was carried out using SPSS 24.0. 3. Results and Discussion 3.1. Micelles Preparation and PG Solubilization Both Pluronic and Soluplus copolymers (Figure 1a-B,a-C) are attractive excipients for ocular drug delivery owing to their self-assembling properties, which enable forming micelles and also in situ Pharmaceutics 2020,12, 702 7 of 18 gelling systems at moderate concentrations. Compared to Pluronic, the literature on the applications of Soluplus to formulate eye drops is more limited. Soluplus has the advantage of its extremely low CMC (6.6 × 10 −5 mM vs. 4.0 × 10 −2 mM for Pluronic F68) [ 40 ] because of the higher hydrophobicity of the caprolactam moieties, and thus Soluplus micelles are more stable against dilution [ 41 ]. Moreover, differently, Soluplus solutions in water are slightly acid (pH 5.6) while Pluronic F68 solutions are neutral (pH 7.4). Although these pH values are well tolerated by the eye surface, for comparison purposes all micelle dispersions were prepared in a buffered medium. Accordingly, Soluplus and Pluronic F68 micelles were prepared in a PBS pH 7.4 covering a wide range of copolymer concentrations (4, 8, 12, 16, and 20% w/w) (Table 1). The concentrations of each copolymer chosen for the study were well above the CMC values, and ranged between 0.35 and 1.74 mM for Soluplus, and 4.79–23.95 mM for Pluronic F68 (corresponding to 4–20% w/wconcentration for both copolymers). Table 1. pH, particle size (number distribution), polydispersion index (PDI), and Z-potential (mV) of Soluplus and Pluronic (12%) micelles in a PBS pH 7.4 at 25 ◦ C before and after being loaded with progesterone (PG). Mean values ±standard deviations; n=3. Formulation pH Particle Size (nm) PDI Z-Potential (mV) Soluplus 12% 6.46 52.32 ±10.13 0.24 ±0.01 −1.17 ±0.38 Soluplus 12% +PG 6.38 59.19 ±0.41 0.24 ±0.02 −1.67 ±0.62 Pluronic F68 12% 7.39 3.70 ±1.01 0.74 ±0.08 −1.76 ±1.50 Pluronic F68 12% +PG 7.30 3.52 ±0.15 0.73 ±0.07 −0.46 ±0.50 Unimodal number-based size distributions were observed for all micelle dispersions (Figure S1). Soluplus micelles had an average size of 52.32 ± 10.13 nm and a polydispersity index of 0.24 ± 0.01, while Pluronic F68 micelles were much smaller showing an average size of 3.74 ± 1.13 and a polydispersity index of 0.53 ± 0.08 (Table S1 in Supplementary Material). The PG load did not cause a relevant change in the size of the micelles (Table 1). The surface charge of all polymeric micelles was slightly negative or near zero according to the composition of the two copolymers. The initial transparent (Pluronic F68) or opalescent (Soluplus) dispersions kept the same appearance when adding PG (Figure 1b). Pluronic produced clearer formulations due to its high hydrophilic-lipophilic balance (HLB =29), while Soluplus dispersions opalescence is related to that the aggregates are larger and less hydrophilic (HLB =16). The transmittance of 20% Soluplus dispersion in a PBS pH 7.4 measured using a 10 mm light path cuvette is shown in Figure S2 (Supplementary Materials). Dispersions including PG behaved similarly. The transmittance of a liquid layer of Soluplus/PG system of about 10 microns, which corresponds to the thickness of the human tear film [ 42 ], can be estimated to be close to 100% in the visible light range. Therefore, no change in visual acuity is expected after instillation. Once the micelles were prepared, their capability to encapsulate PG was investigated. PG solubility in a PBS at 25 ◦ C was 4.4 µ g/mL, which was lower than the solubility in water reported in the literature, i.e., 7.0 µ g/mL [ 43 ]. In the presence of the copolymers, PG solubility experienced a remarkable increase (Figure 1c). Pluronic F68 micelles (20% in PBS) increased the apparent solubility up to 46.8 ± 0.2 µ g/mL. In the case of Soluplus (20% in PBS) the increase was even more remarkable; an apparent solubility of 258.2 ± 17.6 µ g/mL was recorded and the remaining solid PG was not detected. This means that Soluplus 20% micelles dispersions could even encapsulate larger amounts of PG. Parameters used to quantify the solubilization efficiency are summarized in Tables 2and 3. Micelle-water partition coefficients, expressed as log P-values, ranged between 3 and 4 for Pluronic micelles and between 4 and 5 for Soluplus micelles. These values clearly indicated that the proportion of PG encapsulated inside the micelles is to 3-to-5 orders of magnitude higher than the free drug remaining in the aqueous medium. Indeed, an increase in the copolymer concentration favored the encapsulation of more PG molecules, and according to the mf values (Table 2), more than 99.9% of drug was hosted inside the micelles for the higher Soluplus concentrations tested. Therefore, the outstanding encapsulation Pharmaceutics 2020,12, 702 8 of 18 efficiency of ca. 100% means that almost no free drug is outside the micelles (when solubilization equilibrium was reached and excess of solid drug was removed). The huge range of PG amounts that can be solubilized using Pluronic and Soluplus micelles may offer an enormous versatility to formulate the dose required for each patient. Table 2. PG solubilization capacity of Soluplus dispersions in a PBS pH 7.4, calculated using Equations (1)–(6). SP: Soluplus; PG: Progesterone; χ : Molar solubilization capacity; log P: Logarithm of the partition coefficient; PM: Molar partition coefficient; ∆ G: Gibbs free standard solubilization energy; mf: Molar fraction of the encapsulated drug within the micelle. Copolymer (% w/w)SP (M) PG (M) PG (µg/mL) χlog P PM ∆G for PM (KJ/mol) mf 43.47 ×10−42.74 ×10−486.25 7.89 ×10−14.29 56,163,941.1 −44,211.8 0.99995 86.95 ×10−44.74 ×10−4149.12 6.82 ×10−14.53 48,546,060.2 −43,850.6 0.99997 12 1.04 ×10−36.88 ×10−4216.50 6.60 ×10−14.69 46,987,265.0 −43,769.7 0.99998 16 1.39 ×10−38.13 ×10−4255.77 5.85 ×10−14.76 41,631,784.1 −43,469.9 0.99998 20 1.73 ×10−38.21 ×10−4258.18 4.72 ×10−14.77 33,619,107.1 −42,940.3 0.99998 Table 3. PG solubilization capacity of Pluronic F68 dispersions in a PBS pH 7.4, calculated using Equations (1)–(6). PL F68: Pluronic F68; PG: Progesterone; χ : Molar solubilization capacity; log P: Respect of the partition coefficient; PM: Molar partition coefficient; ∆ G: Gibbs free standard solubilization energy; mf: Molar fraction of the encapsulated drug within the micelle. Copolymer (% w/w)PL F68 (M) PG(M) PG (µg/mL) χlog P PM ∆G for PM (KJ/mol) mf 44.79 ×10−33.07 ×10−59.67 6.47 ×10−33.34 460,565.1 −32,309.9 0.99954 89.58 ×10−33.52 ×10−511.08 3.69 ×10−33.40 262,942.2 −30,921.1 0.99960 12 1.44 ×10−25.47 ×10−517.21 3.82 ×10−33.59 271,837.0 −31,003.5 0.99974 16 1.92 ×10−29.60 ×10−530.18 5.02 ×10−33.83 357,388.1 −31,681.5 0.99985 20 2.39 ×10−21.49 ×10−546.81 6.23 ×10−34.02 443,288.4 −32,215.2 0.99991 The free energy of solubilization was negative in all cases; probably the encapsulation process was thermodynamically driven by hydrophobic interactions with the micelle cores. So far the largest increases in PG solubility have been reported for cyclodextrins in a PEG:water medium, attaining values close to 1 mg/mL [ 43 ]. The remarkable advantages of PG encapsulation in the prepared polymeric micelles compared to the inclusion complex formation with cyclodextrins rely on: (i) The outstanding 100% encapsulation efficiency in micelles, which is never reached with cyclodextrins; (ii) the higher thermodynamic and kinetic stability of the micelles, which are less prone to release the encapsulated drug than the cyclodextrins [44,45], and (iii) that organic cosolvents are not needed. 3.2. Rheological Properties of the Formulations The rheological properties of a topical ocular formulation notably determine the time of permanence on the ocular surface and, in turn, the drug ocular bioavailability [ 46 ]. The use of thickening agents is frequent, but their concentration requires a fine balance between the required flowability of the liquid formulation during dosing (viscosity under shearing conditions below 25 × 10 −3 Pa · s) and the consistency demanded to remain on the eye (shear viscosity ≥ 10 × 10 −3 Pa · s) [ 47 ]. The upper limit of viscosity could be expanded by means of polymers that undergo in situ gelling phenomena [ 48 ]. Thus, the next step was to investigate whether the presence of the micelles can increase by themselves the viscosity of the formulation and to determine the effect of temperature on the rheological properties. Viscoelastic behavior of copolymer dispersions with and without PG is depicted in Figure 2. Pluronic F68 dispersions (4–20% w/w) with and without PG showed a viscous behavior with negligible storage modulus (G 0 ) in the 15–40 ◦ C range. The G 00 values were constant in the temperature interval evaluated and no sol-gel transition was observed. The presence of PG caused minor changes in the loss modulus of Pluronic F68. Differently, Soluplus blank dispersions exhibited the typical sol-gel transition showing a remarkable increase in G’ when a certain temperature was reached (Figure S3 Pharmaceutics 2020,12, 702 9 of 18 in Supplementary Materials). The transition temperature decreased as the copolymer concentration increased: 4%, 37.5 ◦ C; 8%, 35.6 ◦ C; 12%, 35.7 ◦ C; 16%, 34.6 ◦ C, and 20%, 32.7 ◦ C. For the PG-loaded micelle dispersions the transition temperature was: 4%, 37.4 ◦ C; 8%, 34.9 ◦ C; 12%, 33.5 ◦ C; 16%, 36.3 ◦ C, and 20%, 35.6 ◦ C. Nearby and above the transition temperature both G 0 and G 00 showed a progressive increase in their values. This temperature-dependence pattern agreed well with other reports on Soluplus dispersions [ 26 ] and was quite different to the typical in situ gelling behavior reported for other temperature-responsive copolymers that exhibit brusque transitions. Interestingly, at 35 ◦ C Soluplus 12–20% micelle dispersions could transform into soft gels on the eye surface prolonging the retention time and the drug release. The complex viscosity values (i.e., the frequency-dependent viscosity) recorded at 35 ◦ C for Soluplus 16% and 20% in a PBS with PG were 4.08 and 4.63 Pa · s, respectively, while for Pluronic 16% and 20% in a PBS with PG, the values of complex viscosities were 9.87 × 10 −3 and 14.21 × 10 −3 Pa · s, respectively. These findings indicate that Pluronic micelles communicate shear viscosity above the critical level to maintain precorneal residence in man [47]. Pharmaceutics 2020, 12, x 9 of 18 negligible storage modulus (G′) in the 15–40 °C range. The G′′ values were constant in the temperature interval evaluated and no sol-gel transition was observed. The presence of PG caused minor changes in the loss modulus of Pluronic F68. Differently, Soluplus blank dispersions exhibited the typical solgel transition showing a remarkable increase in G’ when a certain temperature was reached (Figure S3 in Supplementary Materials). The transition temperature decreased as the copolymer concentration increased: 4%, 37.5 °C; 8%, 35.6 °C; 12%, 35.7 °C; 16%, 34.6 °C, and 20%, 32.7 °C. For the PG-loaded micelle dispersions the transition temperature was: 4%, 37.4 °C; 8%, 34.9 °C; 12%, 33.5 °C; 16%, 36.3 °C, and 20%, 35.6 °C. Nearby and above the transition temperature both G′ and G′′ showed a progressive increase in their values. This temperature-dependence pattern agreed well with other reports on Soluplus dispersions [26] and was quite different to the typical in situ gelling behavior reported for other temperature-responsive copolymers that exhibit brusque transitions. Interestingly, at 35 °C Soluplus 12–20% micelle dispersions could transform into soft gels on the eye surface prolonging the retention time and the drug release. The complex viscosity values (i.e., the frequencydependent viscosity) recorded at 35 °C for Soluplus 16% and 20% in a PBS with PG were 4.08 and 4.63 Pa·s, respectively, while for Pluronic 16% and 20% in a PBS with PG, the values of complex viscosities were 9.87 × 10 −3 and 14.21 × 10 −3 Pa·s, respectively. These findings indicate that Pluronic micelles communicate shear viscosity above the critical level to maintain precorneal residence in man [47]. Figure 2. Evolution of the storage (G′) and the loss (G′′) moduli as a function of temperature of (A) PG-loaded Soluplus micelle dispersions, (B) blank Soluplus dispersions, (C) PG-loaded Pluronic F68 micelle dispersion, (D) blank Pluronic dispersions. Total copolymer concentration was 4, 8, 12, 16, and 20% w/w in a PBS pH 7.4 buffer (n = 4). 3.3. HET-CAM Test A first screening of ocular compatibility was carried out placing the formulation in contact with the hen´s egg chorioallantoic membrane (HET-CAM). This test is an alternative to the in vivo Draize Figure 2. Evolution of the storage (G 0 ) and the loss (G 00 ) moduli as a function of temperature of ( A ) PG-loaded Soluplus micelle dispersions, ( B ) blank Soluplus dispersions, ( C ) PG-loaded Pluronic F68 micelle dispersion, ( D ) blank Pluronic dispersions. Total copolymer concentration was 4, 8, 12, 16, and 20% w/win a PBS pH 7.4 buffer (n=4). 3.3. HET-CAM Test A first screening of ocular compatibility was carried out placing the formulation in contact with the hen ´ s egg chorioallantoic membrane (HET-CAM). This test is an alternative to the in vivo Draize test to evaluate the irritancy that ocular formulations may cause [ 30 , 31 , 49 ]. CAM is a fetal membrane that is not innervated but highly vascularized and responds to injury similarly to the rabbit conjunctiva made of fused chorion and allantois [ 50 ]. The effects induced by the substance to be tested on the small Pharmaceutics 2020,12, 702 16 of 18 8. Doonan, F.; O’Driscoll, C.; Kenna, P.; Cotter, T.G. Enhancing survival of photoreceptor cells in vivo using the synthetic progestin Norgestrel. J. Neurochem. 2011,118, 915–927. [CrossRef] 9. Hern á ndez-Rabaza, V.; L ó pez-Pedrajas, R.; Almansa, I. Progesterone, lipoic acid, and sulforaphane as promising antioxidants for retinal diseases: A review. Antioxidants 2019,8, 53. [CrossRef] 10. S á nchez-Vallejo, V.; Benlloch-Navarro, S.; L ó pez-Pedrajas, R.; Romero, F.J.; Miranda, M. Neuroprotective actions of progesterone in an in vivo model of retinitis pigmentosa. Pharmacol. Res. 2015 ,99, 276–288. [CrossRef] 11. Nuzzi, R.; Scalabrin, S.; Becco, A.; Panzica, G. Gonadal hormones and retinal disorders: A review. Front. Endocrinol. 2018,9, 1–15. [CrossRef] [PubMed] 12. Allen, R.S.; Olsen, T.W.; Sayeed, I.; Cale, H.A.; Morrison, K.C.; Oumarbaeva, Y.; Lucaciu, I.; Boatright, J.H.; Pardue, M.T.; Stein, D.G. Progesterone treatment in two rat models of ocular ischemia. Investig. Ophthalmol. Vis. Sci. 2015,56, 2880–2891. [CrossRef] [PubMed] 13. Hassan, A.S.; Soliman, G.M.; El-Mahdy, M.M.; El-Gindy, G.E.A. Solubilization and enhancement of ex vivo vaginal delivery of progesterone using solid dispersions, inclusion complexes and micellar solubilization. Curr. Drug Deliv. 2017,15, 110–121. [CrossRef] [PubMed] 14. Li, V.H.K.; Wood, R.W.; Kreuter, J.; Harmia, T.; Robinson, J.R. Ocular drug delivery of progesterone using nanoparticles. J. Microencapsul. 1986,3, 213–218. [CrossRef] [PubMed] 15. Vadlapudi, A.D.; Cholkar, K.; Vadlapatla, R.K.; Mitra, A.K. Aqueous nano micellar formulation for topical delivery of biotinylated lipid prodrug of acyclovir: Formulation development and ocular biocompatibility. J. Ocul. Pharmacol. Ther. 2014,30, 49–58. [CrossRef] [PubMed] 16. Barar, J.; Aghanejad, A.; Fathi, M.; Omidi, Y. Advanced drug delivery and targeting technologies for the ocular diseases. BioImpacts 2016,6, 49–67. [CrossRef] 17. Kang-Mieler, J.J.; Dosmar, E.; Liu, W.; Mieler, W.F. Extended ocular drug delivery systems for the anterior and posterior segments: Biomaterial options and applications. Expert Opin. Drug Deliv. 2017 ,14, 611–620. [CrossRef] 18. Gote, V.; Sikder, S.; Sicotte, J.; Pal, D. Ocular drug delivery: Present innovations and future challenges. J. Pharmacol. Exp. Ther. 2019,370, 602–624. [CrossRef] 19. Soliman, O.A.E.-A.; Mohamed, E.A.; Khatera, N.A.A. Enhanced ocular bioavailability of fluconazole from niosomal gels and microemulsions: Formulation, optimization, and in vitro – in vivo evaluation. Pharm. Dev. Technol. 2019,24, 48–62. [CrossRef] 20. Bachu, R.; Chowdhury, P.; Al-Saedi, Z.; Karla, P.; Boddu, S. Ocular drug delivery barriers—Role of nanocarriers in the treatment of anterior segment ocular diseases. Pharmaceutics 2018,10, 28. [CrossRef] 21. Ramsay, E.; del Amo, E.M.; Toropainen, E.; Tengvall-Unadike, U.; Ranta, V.P.; Urtti, A.; Ruponen, M. Corneal and conjunctival drug permeability: Systematic comparison and pharmacokinetic impact in the eye. Eur. J. Pharm Sci. 2018,119, 83–89. [CrossRef] [PubMed] 22. Weng, Y.H.; Ma, X.W.; Che, J.; Li, C.; Liu, J.; Chen, S.Z.; Wang, Y.Q.; Gan, Y.L.; Chen, H.; Hu, Z.B.; et al. Nanomicelle-assisted targeted ocular delivery with enhanced anti-inflammatory efficacy in vivo .Adv. Sci. 2018,5, 1700455. [CrossRef] [PubMed] 23. Grimaudo, M.A.; Pescina, S.; Padula, C.; Santi, P.; Concheiro, A.; Alvarez-Lorenzo, C.; Nicoli, S. Topical application of polymeric nanomicelles in ophthalmology: A review on research efforts for the non-invasive delivery of ocular therapeutics. Expert Opin. Drug Deliv. 2019,16, 397–413. [CrossRef] [PubMed] 24. Varela-Garcia, A.; Concheiro, A.; Alvarez-Lorenzo, C. Surplus micelles for acyclovir ocular delivery: Formulation and cornea and sclera permeability. Int. J. Pharm. 2018,552, 39–47. [CrossRef] 25. Agarwal, P.; Rupenthal, I.D. In vitro and ex vivo corneal penetration and absorption models. Drug Deliv. Transl. Res. 2016,6, 634–647. [CrossRef] 26. Loch, C.; Zakelj, S.; Kristl, A.; Nagel, S.; Guthoff, R.; Weitschies, W.; Seidlitz, A. Determination of permeability coefficients of ophthalmic drugs through different layers of porcine, rabbit and bovine eyes. Eur. J. Pharm. Sci. 2012,47, 131–138. [CrossRef] 27. Moiseev, R.V.; Morrison, P.W.J.; Steele, F.; Khutoryanskiy, V.V. Penetration enhancers in ocular drug delivery. Pharmaceutics 2019,11, 321. [CrossRef] 28. OECD Guideline for Testing of Chemicals. Bovine Corneal Opacity and Permeability Test Method for Identifying I) Chemicals Inducing Serious Eye Damage and II) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage TG 437; OECD: Paris, France, 2015; pp. 1–27. Pharmaceutics 2020,12, 702 17 of 18 29. Ambati, J.; Canakis, C.S.; Miller, J.W.; Gragoudas, E.S.; Edwards, A.; Weissgold DJKim, I.; Delori, F.C.; Adamis, A.P. Diffusion of high molecular weight compounds through sclera. Invest. Ophthalmol. Vis. Sci. 2000,41, 1181–1185. 30. Lorenzo-Veiga, B.; Sigurdsson, H.H.; Loftsson, T.; Alvarez-Lorenzo, C. Cyclodextrin–amphiphilic copolymer supramolecular assemblies for the ocular delivery of natamycin. Nanomaterials 2019,9, 745. [CrossRef] 31. Alvarez-Rivera, F.; Fern á ndez-Villanueva, D.; Concheiro, A.; Alvarez-Lorenzo, C. α -Lipoic acid in soluplus ® polymeric nanomicelles for ocular treatment of diabetes-associated corneal diseases. J. Pharm. Sci. 2016 ,105, 2855–2863. [CrossRef] 32. In Vitro Ocular Evaluation Report (ICCVAM). Test Method Evaluation Report: Current Validation Status of In Vitro Test Methods Proposed for Identifying Eye Injury Hazard Potential of Chemicals and Products; National Toxicology Program: Research Triangle Park, NC, USA, 2010; pp. 10–755. 33. Sebasti á n-Morell ó , M.; Calatayud-Pascual, M.A.; Rodilla, V.; Balaguer-Fern á ndez, C.; L ó pez-Castellano, A. Ex vivo rabbit cornea diffusion studies with a soluble insert of moxifloxacin. Drug Deliv. Transl. Res. 2018 ,8, 132–139. [CrossRef] [PubMed] 34. Rao, P.C.M.; Nappinnai, M.; Raju, S.; Rao, U.M.V.; Reddy, V.B. Fluconazole ocular inserts: Formulation and in-vitro evaluation. J. Pharm. Sci. Res. 2010,2, 693–699. 35. Franca, J.R.; Foureaux, G.; Fuscaldi, L.L.; Ribeiro, T.G.; Rodrigues, L.B.; Bravo, R.; Castilho, R.O.; Yoshida, M.I.; Cardoso, V.N.; Fernandes, S.O.; et al. Bimatoprost-loaded ocular inserts as sustained release drug delivery systems for glaucoma treatment: In vitro and in vivo evaluation. PLoS ONE 2014 ,9, 1–11. [CrossRef] [PubMed] 36. Grimaudo, M.A.; Nicoli, S.; Santi, P.; Concheiro, A.; Alvarez-Lorenzo, C. Cyclosporine-loaded cross-linked inserts of sodium hyaluronan and hydroxypropylβ -cyclodextrin for ocular administration. Carbohydr. Polym. 2018,201, 308–316. [CrossRef] 37. Dave, V.; Paliwal, S.; Yadav, S.; Sharma, S. Effect of in vitro transcorneal approach of aceclofenac eye drops through excised goat, sheep, and buffalo corneas. Sci. World J. 2015,2015, 432376. [CrossRef] [PubMed] 38. Klang, V.; Matsko, N.; Zimmermann, A.M.; Vojnikovic, E.; Valenta, C. Enhancement of stability and skin permeation by sucrose stearate and cyclodextrins in progesterone nanoemulsions. Int. J. Pharm. 2010 ,393, 153–161. [CrossRef] [PubMed] 39. Ahuja, M.; Dhake, A.S.; Majumdar, D.K. Effect of formulation factors on in-vitro permeation of diclofenac from experimental and marketed aqueous eye drops through excised goat cornea. J. Pharm. Soc. Jpn. 2006 , 126, 1369–1375. [CrossRef] [PubMed] 40. BASF. Technical Information Soluplus; BASF: Ludwigshafen am Rhein, Germany, 2010; pp. 1–8. 41. Daxer, A.; Blumthaler, M.; Schreder, J.; Ettl, A. Effectiveness of eye drops protective against ultraviolet radiation. Ophthalmic Res. 1998,30, 286–290. [CrossRef] 42. Lu, Y.; Zhang, E.; Yang, J.; Cao, Z. Strategies to improve micelle stability for drug delivery. Nano Res. 2018 , 11, 4985–4998. [CrossRef] 43. Nandi, I.; Bateson, M.; Bari, M.; Joshi, H.N. Synergistic effect of PEG-400 and cyclodextrin to enhance solubility of progesterone. AAPS Pharm. Sci. Tech. 2004,4, 1–5. [CrossRef] 44. Gidwani, B.; Vyas, A. A comprehensive review on cyclodextrin-based carriers for delivery of chemotherapeutic cytotoxic anticancer drugs. Biomed. Res. Int. 2015, 198268. [CrossRef] [PubMed] 45. Lu, Y.; Park, K. Polymeric micelles and alternative nanonized delivery vehicles for poorly soluble drugs. Int. J. Pharm. 2013,453, 198–214. [CrossRef] 46. Awwad, S.; Mohamed Ahmed, A.H.A.; Sharma, G.; Heng, J.S.; Khaw, P.T.; Brocchini, S.; Lockwood, A. Principles of pharmacology in the eye. Br. J. Pharmacol. 2017,174, 4205–4223. [CrossRef] [PubMed] 47. Rahman, M.Q.; Chuah, K.S.; MacDonald, E.C.A.; Trusler, J.P.M.; Ramaesh, K. The effect of pH, dilution, and temperature on the viscosity of ocular lubricants-shift in rheological parameters and potential clinical significance. Eye 2012,26, 1579–1584. [CrossRef] [PubMed] 48. Jumelle, C.; Gholizadeh, S.; Annabi, N.; Dana, R. Advances and limitations of drug delivery systems formulated as eye drops. J. Control. Release 2020,321, 1–22. [CrossRef] [PubMed] 49. Butt, U.; ElShaer, A.; Snyder, L.; Al-Kinani, A.; Le Gresley, A.; Alany, R. Fatty acid based microemulsions to combat ophthalmia neonatorum caused by Neisseria gonorrhoeae and Staphylococcus aureus.Nanomaterials 2018,8, 51. [CrossRef] Pharmaceutics 2020,12, 702 18 of 18 50. McKenzie, B.; Kay, G.; Matthews, K.H.; Knott, R.M.; Cairns, D. The hen’s egg chorioallantoic membrane (HET-CAM) test to predict the ophthalmic irritation potential of a cysteamine-containing gel: Quantification using Photoshop®and ImageJ. Int. J. Pharm. 2015,490, 1–8. [CrossRef] 51. Fathalla, Z.M.A.; Vangala, A.; Longman, M.; Khaled, K.A.; Hussein, A.K.; El-Garhy, O.H.; Alany, R.G. Poloxamer-based thermoresponsive ketorolac tromethamine in situ gel preparations: Design, characterisation, toxicity and transcorneal permeation studies. Eur. J. Pharm. Biopharm. 2017,114, 119–134. [CrossRef] 52. Nicoli, S.; Ferrari, G.; Quarta, M.; Macaluso, C.; Govoni, P.; Dallatana, D.; Santi, P. Porcine sclera as a model of human sclera for in vitro transport experiments: Histology, SEM, and comparative permeability. Mol. Vis. 2009,15, 259–266. 53. Souza, J.G.; Dias, K.; Pereira, T.A.; Bernardi, D.S.; Lopez, R.F.V. Topical delivery of ocular therapeutics: Carrier systems and physical methods. J. Pharm. Pharmacol. 2014,66, 507–530. [CrossRef] 54. Savla, R.; Browne, J.; Plassat, V.; Wasan, K.M.; Wasan, E.K. Review and analysis of FDA approved drugs using lipid-based formulations. Drug Dev. Ind. Pharm. 2017,43, 1743–1758. [CrossRef] [PubMed] 55. Mandal, A.; Bisht, R.; Rupenthal, I.D.; Mitra, A.K. Polymeric micelles for ocular drug delivery: From structural frameworks to recent preclinical studies. J. Control. Release 2017 ,248, 96–116. [CrossRef] [PubMed] 56. del Amo, E.M.; Rimpelä, A.K.; Heikkinen, E.; Kari, O.K.; Ramsay, E.; Lajunen, T.; Schmitt, M.; Pelkonen, L.; Bhattacharya, M.; Richardson, D.; et al. Pharmacokinetic aspects of retinal drug delivery. Prog. Retin. Eye Res. 2017,57, 134–185. [CrossRef] [PubMed] 57. Bachu, R.D.; Stepanski, M.; Alzhrani, R.M.; Jung, R.; Boddu, S.H.S. Development and evaluation of a novel microemulsion of dexamethasone and tobramycin for topical ocular administration. J. Ocul. Pharmacol. Ther. 2018,34, 312–324. [CrossRef] [PubMed] 58. Alvarez-Rivera, F.; Concheiro, A.; Alvarez-Lorenzo, C. Epalrestat-loaded silicone hydrogels as contact lenses to address diabetic-eye complications. Eur. J. Pharm. Biopharm. 2018,122, 126–136. [CrossRef] [PubMed] 59. Desai, A.R.; Maulvi, F.A.; Pandya, M.M.; Ranch, K.M.; Vyas, B.A.; Shah, S.A.; Shah, D.O. Co-delivery of timolol and hyaluronic acid from semi-circular ring-implanted contact lenses for the treatment of glaucoma: In vitro and in vivo evaluation. Biomater. Sci. 2018,6, 1580–1591. [CrossRef] 60. Ghosn, M.G.; Tuchin, V.V.; Larin, K.V. Nondestructive quantification of analyte diffusion in cornea and sclera using optical coherence tomography. Investig. Ophthalmol. Vis. Sci. 2007,48, 2726–2733. [CrossRef] 61. Aburahma, M.H.; Mahmoud, A.A. Biodegradable ocular inserts for sustained delivery of brimonidine tartarate: Preparation and in vitro / in vivo evaluation. AAPS Pharm. Sci. Tech. 2011 ,12, 1335–1347. [CrossRef] © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).