1 Title: Novel liposome-based and in situ gelling artificial tear formulation for dry eye disease treatment Authors: Duygu Acar1, Irene Teresa Molina-Martínez1,2, Miguel Gómez-Ballesteros1,2, Manuel Guzmán-Navarro3, José Manuel Benítez-del-Castillo2,4, Rocío Herrero-Vanrell1,2 Author affiliations: 1 Department of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, Complutense University of Madrid, Plaza Ramón y Cajal s/n, 28040 Madrid, Spain 2 Pharmaceutical Innovation in Ophthalmology Research Group, Sanitary Research Institute of the San Carlos Clinical Hospital (IdISSC) and the Ocular Pathology National Net (OFTARED) of the Institute of Health Carlos III, Calle Profesor Martín Lagos, s/n, 28040 Madrid, Spain 3 Biomedical Sciences Department, Pharmacy and Pharmaceutical Technology Unit, Faculty of Pharmacy, University of Alcalá, Ctra. de Madrid-Barcelona (Autovía A2) Km. 33,600, 28805 Alcalá de Henares, Madrid, Spain 4 Ocular Surface and Inflammation Unit, San Carlos Clinical Hospital, Calle Profesor Martín Lagos, s/n, 28040 Madrid, Spain Corresponding author: Duygu Acar e-mail: duyacar@ucm.es Tel: (+34) 913941727 Address: Department of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, Complutense University of Madrid, Plaza Ramón y Cajal s/n, 28040 Madrid, Spain mailto:duyacar@ucm.es 2 ABSTRACT Purpose. Artificial tears are widely used in the treatment of dry eye disease, although current formulations do not closely resemble natural tears. The purpose of this study was the design and characterization of a novel in situ gelling artificial tear formulation, containing both lipid and aqueous components, in order to resemble natural tears and replenish the tear film. Methods. Liposomes, containing phosphatidylcholine, cholesterol, vitamins A and E, were prepared by the thin-film hydration method. The aqueous phase of the formulation was comprised of gellan gum, hydroxypropyl methylcellulose, levocarnitine, electrolytes (sodium chloride and potassium chloride), trehalose, and borates. The artificial tear was characterized in terms of liposome size, pH, surface tension, and viscosity. In vitro tolerance studies were performed in a human epithelial carcinoma cell line (HeLa) and a murine macrophage cell line (J774). In vivo tolerance was assessed in rabbits. Results. Liposomes presented a unimodal distribution with a mean size of 200.1 ± 4.4 nm. The resulting surface tension was 53.4 ± 1.1 mN/m (at 33°C) and the pH was 7.6 ± 0.1. The viscosity of the formulation presented a mean value of 4.0 ± 0.1 mPa.s within the shear rate interval of 200-1000 s-1 at 33°C. Cell viability remained higher than 90% in both cell lines. No discomfort or clinical signs were observed in rabbits. Conclusions. The liposome-based and in situ gelling artificial tear formulation presented good tolerance and suitable properties for topical ophthalmic administration. It may be beneficial in the treatment of dry eye disease. Keywords: dry eye, artificial tear, liposome, in situ gel, nanotechnology 3 HIGHLIGHTS ▪ A novel liposome-based, in situ gelling artificial tear formulation was developed. ▪ Physicochemical properties, in vitro and in vivo tolerance were evaluated. ▪ The formulation showed suitable properties for topical ophthalmic administration. ▪ Good in vitro and in vivo tolerance was observed. ▪ The novel artificial tear may be beneficial in the treatment of dry eye disease. 4 INTRODUCTION Dry eye disease (DED) is a highly prevalent health problem affecting approximately 5-35% of the world’s population [1]. It is defined as a multifactorial disease of the tears and ocular surface that results in symptoms of discomfort, visual disturbance and tear film instability [2]. DED is associated with a significant adverse impact on vision-related quality of life, causing difficulties in daily activities [3,4]. It can be caused by various factors such as meibomian gland dysfunction, low blink rate, ocular surface disorders and contact lens wear, among others, resulting in the alteration of the tear film [2]. Artificial tears are widely used for mild to moderate dry eye, and can be prescribed in combination with other therapies for moderate to severe dry eye. Numerous artificial tear formulations with a diverse range of components are currently available in the market; however, most of these formulations do not quite resemble the composition of natural tears [5]. Furthermore, they have a short ocular residence time and require repeated daily administration. An ideal artificial tear formulation should contain both aqueous and lipid components to mimic natural tears, and have certain physicochemical properties such as hypotonicity or isotonicity, neutral to slightly alkaline pH, suitable surface tension and viscosity [6]. A surface tension near the physiological range of natural tears, that is reported to be 40-46 mN/m [7,8], provides a good spreading of the formulation over the ocular surface. For artificial tears that follow Newtonian behavior, the desirable viscosity would be in the physiological range of 1.0-8.3 mPa.s [9,10]; whereas for those that follow non-Newtonian behavior, a dynamic viscosity in the same range would be suitable. Additionally, an ideal artificial tear formulation should be well tolerated on the ocular surface, provide extended ocular residence time, and be preservative-free. This study aimed at designing a novel 5 liposome-based and in situ gelling artificial tear formulation having such properties to replenish the tear film, with potential beneficial effects for the treatment of DED. MATERIALS AND METHODS Materials Phosphatidylcholine obtained from soy lecithin (Phospholipon® 90 G) was purchased from Lipoid GmbH (Ludwigshafen, Germany). Cholesterol, retinyl acetate (vitamin A), DL-α- tocopherol acetate (vitamin E), gellan gum (Gelzan™ CM, Gelrite®), boric acid, sodium tetraborate anhydrous, and benzalkonium chloride were supplied by Sigma-Aldrich Co. (St. Louis, MO, USA). Hydroxypropyl methylcellulose was purchased from Abarán Materias Primas S.L. (Madrid, Spain), levocarnitine from Fagron Ibérica SAU (Barcelona, Spain), and D-(+)-trehalose dihydrate from ThermoFisher (Kandel) GmbH (Karlsruhe, Germany). Sodium chloride and potassium chloride were supplied by Merck KGaA (Darmstadt, Germany), and chloroform (stabilized with ethanol) by Panreac Química SLU (Barcelona, Spain). Animals Six male New Zealand white rabbits (purchased from San Bernardo Farm, Navarra, Spain), weighing between 3.0 to 3.5 kg, were used for this study. The animals were housed individually with free access to food and drinking water, in a room with an alternating 12- hour light/dark cycle. The room was maintained at 22°C with 50% relative humidity. The animal experiments were performed in accordance with the EU Directive 2010/63/EU for animal experiments, U.K. Animals (Scientific Procedures) Act 1986, and ARVO Statement for the Use of Animals in Ophthalmic and Visual Research. The study protocol was approved 6 by the Animal Experimentation Ethics Committee of the Complutense University of Madrid and by the Community of Madrid (approval reference: PROEX 011/16). Preparation of the artificial tear formulation Liposomes were prepared by the thin-film hydration method described by Bangham et al. [11] and modified by Vicario-de-la-Torre et al. [12]. 400 mg of phosphatidylcholine (PC), 50 mg of cholesterol (Chol), 4 mg of vitamin E (vit E), and 400 ng of vitamin A (vit A) were dissolved in 25 mL of chloroform. The solvent was evaporated in a rotary evaporator (Büchi Rotavapor R-205, Massó Analítica S.A., Spain). The resulting dry lipid film was hydrated with 10 mL of an aqueous medium composed of 1.68% trehalose, 0.84% boric acid (H3BO3), and 0.08% sodium tetraborate anhydrous (Na2B4O7). To obtain a homogenous population of lipid vesicles, the liposomal suspension was sonicated (Ultrasons-H, J.P. Selecta S.A., Barcelona, Spain), and extruded (Lipex® Extruder, Transferra Nanosciences Inc., Burnaby, BC, Canada), being forced through polycarbonate filters (Nuclepore™, Whatman International Ltd., Kent, UK) with a pore size of 0.2 μm. The liposomal suspension was diluted with solutions of gellan gum, hydroxypropyl methylcellulose (HPMC), levocarnitine (L-carnitine), sodium chloride (NaCl) and potassium chloride (KCl) to obtain the final artificial tear formulation (Table 1). The preparation was performed under aseptic conditions. The aqueous medium, polymer and electrolyte solutions underwent steam sterilization, whereas the L-carnitine solution was sterilized by filtration. 7 Table 1. Composition of the artificial tear formulation. Formulation component Concentration (% weight/volume) PC (liposomes) 0.5 Gellan gum 0.25 HPMC 0.12 L-carnitine 0.1 Vit E 0.005 Vit A 0.0005 NaCl 0.05 KCl 0.05 Trehalose 1.68 H3BO3 0.84 Na2B4O7 0.08 Characterization of the artificial tear formulation The particle size and the size distribution profile of liposomal nanoparticles were determined at room temperature by dynamic light scattering (Microtrac® Zetatrac™ Particle Size & Zeta Potential Analyzer, Microtrac Europe GmbH, Meerbusch, Germany). The pH of the formulation was determined at room temperature with a calibrated pH meter (GLP 22, Crison Instruments S.A., Barcelona, Spain). The surface tension was measured with a tensiometer (Krüss K-11 Tensiometer, Krüss GmbH, Hamburg, Germany) according to the Wilhelmy plate method. Measurements were performed at 33°C, which corresponds to the temperature of the ocular surface. The viscosity of the artificial tear was determined with a rheometer (HAAKE RheoStress 1, ThermoFisher Scientific), using the parallel plate system (plate diameter 60 mm, separation 0.5 mm) at 33°C. The shear rate was increased in 20 steps from 0 to 1000 s-1. All measurements were performed in triplicate. In vitro tolerance studies The in vitro tolerance of the formulation was assessed by means of cell viability measurements in a human epithelial carcinoma cell line (HeLa) and a murine macrophage 8 cell line (J774). Cellular reduction of the tetrazolium salt MTT to formazan [13] was evaluated after cells were exposed to the formulation for 2 hours. In vivo tolerance studies The in vivo tolerance studies were performed in six male New Zealand white rabbits; 30 μL of the artificial tear was administered in the right eye of each animal every 30 minutes for 6 hours, whereas the contralateral eye received the same volume of isotonic saline solution and served as control. Macroscopic evaluation of the ocular surface and the clinical signs was performed before the first instillation, and at 3, 6, and 24 hours after the first instillation, in compliance with the protocol described by Enríquez de Salamanca et al. [14]. Corneal opacity, conjunctival alterations, discharge, eyelid swelling, and animal discomfort as evident by intense blinking were examined and graded from 0 to 2. RESULTS Characterization of the artificial tear formulation The results obtained in the characterization of the physicochemical properties of the artificial tear are reported in Table 2. Data were expressed as mean ± standard deviation (SD). Table 2. Physicochemical properties of the artificial tear formulation. Property Mean ± SD Liposome size (nm) 200.1 ± 4.4 pH 7.6 ± 0.1 Surface tension (mN/m) 53.4 ± 1.1 Viscositya (mPa.s) 4.0 ± 0.1 aWithin the shear rate interval of 200-1000 s-1. 9 The liposomal nanoparticles of the formulation showed a unimodal size distribution (Figure 1). The rheological properties of the artificial tear followed Newtonian behavior (Figure 2). Figure 1. Size distribution of the liposomal vesicles of the artificial tear formulation. The liposomal nanoparticles showed a unimodal size distribution, determined by dynamic light scattering. Figure 2. Rheogram of the artificial tear formulation. The rheological properties of the artificial tear followed Newtonian behavior within the shear rate interval of 200-1000 s-1. 0 5 10 15 20 1 10 100 1000 F re q u e n c y ( % ) Volume diameter size (nm) 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 0 200 400 600 800 1000 V is c o s it y ( m P a .s ) S h e a r s tr e s s ( P a ) Shear rate (s-1) Shear stress Viscosity 10 In vitro tolerance studies Cell viability resulted in 95.0 ± 4.8% in HeLa cell line and 108.7 ± 10.4% in J774 cell line after exposures of 2 hours (Figure 3). Figure 3. Cell viability of HeLa and J774 cells after a 2-hour exposure to the artificial tear formulation. Untreated cells were used as negative control (100% cell viability), cells treated with a solution of 0.005% benzalkonium chloride (BAK) as positive control (cell death), and cells treated with a solution of 0.9% NaCl as reference. Cell viability was measured by MTT assay and resulted in higher than 90% in both cell lines. In vivo tolerance studies Prior to the experiment, all of the six animals had a normal ocular surface and showed no clinical signs or discomfort. During the experiment, the animals showed neither discomfort nor clinical signs (grade 0) within 24 hours after the first instillation of the artificial tear. 0 20 40 60 80 100 120 Negative control NaCl 0.9% Formulation BAK 0.005% C e ll v ia b il it y ( % ) HeLa J774 11 DISCUSSION DED is associated with alterations and instability of the tear film; therefore, restoring the tear film is fundamental in its management. This study aimed at developing a novel artificial tear formulation that resembles natural tears and replenishes the tear film. Liposomes, consisting of PC and Chol, were employed to replenish the lipids of the tear film. The in situ gelling polymer gellan gum was incorporated because of its unique gel-forming property when in contact with certain ions present in tears [15–18], with the aim of resisting the natural drainage and extending the ocular residence time of the formulation. HPMC was used to provide bioadhesion [19] and increase the ocular residence time. Antioxidants vit A and vit E, and osmoprotectants L-carnitine and trehalose were incorporated to enhance the potential efficacy of the artificial tear in the treatment of DED. When formulating an artificial tear, the key physicochemical parameters such as pH, surface tension, and viscosity should be taken into consideration. Human tears have a pH of 7.0-7.5 [20,21], and a buffering capacity that prevents the ophthalmic formulations within the pH range of 6.6-9.0 from causing discomfort [22–24]. The surface tension of natural tears is reported to be 40-46 mN/m [7,8], and the viscosity 1.0-8.3 mPa.s [9,10]. The surface tension of an ophthalmic formulation affects its interaction with the tear film and spreading ability over the ocular surface [25]. The viscosity, if excessively high, can cause non-uniform mixing of the formulation with natural tears, blurred vision and discomfort. The physicochemical properties of the artificial tear developed in this study, namely the slightly alkaline pH (7.6), a viscosity in the physiological range of natural tears (4.0 mPa.s), and a surface tension close to that of natural tears (53.4 mN/m), all together suggest good spreading of the formulation over the ocular surface without causing tear film instability, blurred vision or discomfort. Furthermore, the liposomal nanoparticles of the artificial tear had a suitable size for a topical ophthalmic formulation. 12 The artificial tear demonstrated absence of toxicity in both HeLa and J774 cell lines. The formulation did not cause any discomfort or clinical signs in rabbits, indicating good in vivo tolerance. In conclusion, the results obtained in this study demonstrated that the liposome-based in situ gelling artificial tear formulation presents suitable properties for topical ophthalmic administration and is well tolerated. Due to its potential of replenishing the tear film lipids, restoring the tear film, and protecting the corneal epithelium, the novel artificial tear developed in this study may be beneficial in the treatment of DED. Acknowledgments This work was supported by the EDEN Project (642760; MSCA-ITN-2014-EJD; Horizon 2020) granted by the European Commission; FEDER-CICYT FIS-PI17/00079 and FIS- PI17/00466; and the Research Group UCM 920415. Conflicts of interest: none. REFERENCES [1] J.A. Smith, J. Albeitz, C. Begley, B. Caffery, K. Nichols, D. Schaumberg, O. Schein, The epidemiology of dry eye disease: Report of the Epidemiology Subcommittee of the International Dry Eye WorkShop (2007), Ocul. Surf. 5 (2007) 93–107. [2] M.A. Lemp, C. Baudouin, J. Baum, M. Dogru, G.N. Foulks, S. Kinoshita, P. Laibson, J. McCulley, J. Murube, S.C. Pflugfelder, M. Rolando, I. Toda, The definition and classification of dry eye disease: Report of the Definition and Classification Subcommittee of the International Dry Eye WorkShop (2007), Ocul. Surf. 5 (2007) 75–92. 13 [3] B. Miljanović, R. Dana, D.A. Sullivan, D.A. Schaumberg, Impact of dry eye syndrome on vision-related quality of life, Am. J. Ophthalmol. 143 (2007) 409–415. [4] J. Benítez-del-Castillo, M. Labetoulle, C. Baudouin, M. Rolando, Y.A. Akova, P. Aragona, G. Geerling, J. Merayo-Lloves, E.M. Messmer, K. Boboridis, Visual acuity and quality of life in dry eye disease: Proceedings of the OCEAN group meeting, Ocul. Surf. 15 (2017) 169–178. [5] S. Pflugfelder, G. Geerling, S. Kinoshita, M.A. Lemp, J. McCulley, D. Nelson, G.N. Novack, J. Shimazaki, C. Wilson, Management and therapy of dry eye disease: Report of the Management and Therapy Subcommittee of the International Dry Eye WorkShop (2007), Ocul. Surf. 5 (2007) 163–178. [6] M. Vicario-de-la Torre, R. Herrero-Vanrell, J.M. Benítez-del-Castillo, I.T. Molina- Martínez, New formulations for dry eye treatment, Arch Soc Esp Oftalmol. 82 (2007) 395– 396. [7] D. Miller, Measurement of the surface tension of tears, Arch Ophthal. 82 (1969) 368– 371. [8] B. Nagyová, J.M. Tiffany, Components responsible for the surface tension of human tears, Curr. Eye Res. 19 (1999) 4–11. [9] J.M. Tiffany, The viscosity of human tears, Int. Ophthalmol. 15 (1991) 371–376. [10] S.M. Gouveia, J.M. Tiffany, Human tear viscosity: An interactive role for proteins and lipids, Biochim. Biophys. Acta. 1753 (2005) 155–163. [11] A.D. Bangham, M.M. Standish, J.C. Watkins, Diffusion of univalent ions across the lamellae of swollen phospholipids, J. Mol. Biol. 13 (1965) 238–252. [12] M. Vicario-de-la-Torre, J.M. Benítez-del-Castillo, E. Vico, M. Guzmán, B. de-las- Heras, R. Herrero-Vanrell, I.T. Molina-Martínez, Design and characterization of an ocular 14 topical liposomal preparation to replenish the lipids of the tear film, Investig. Ophthalmol. Vis. Sci. 55 (2014) 7839–7847. [13] Y. Liu, D.A. Peterson, H. Kimura, D. Schubert, Mechanism of cellular 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, J. Neurochem. 69 (1997) 581–593. [14] A. Enríquez de Salamanca, Y. Diebold, M. Calonge, C. García-Vazquez, S. Callejo, A. Vila, M.J. Alonso, Chitosan nanoparticles as a potential drug delivery system for the ocular surface: Toxicity, uptake mechanism and in vivo tolerance, Invest. Ophthalmol. Vis. Sci. 47 (2006) 1416–1425. [15] M. Milas, X. Shi, M. Rinaudo, On the physicochemical properties of gellan gum, Biopolymers. 30 (1990) 451–464. [16] S. Matsukawa, T. Watanabe, Gelation mechanism and network structure of mixed solution of low- and high-acyl gellan studied by dynamic viscoelasticity, CD and NMR measurements, Food Hydrocoll. 21 (2007) 1355–1361. [17] R. Chandrasekaran, V.G. Thailambal, The influence of calcium ions, acetate and L- glycerate groups on the gellan double-helix, Carbohydr. Polym. 12 (1990) 431–442. [18] E. Miyoshi, T. Takaya, K. Nishinari, Rheological and thermal studies of gel-sol transition in gellan gum aqueous solutions, Carbohydr. Polym. 30 (1996) 109–119. [19] K. Kumar, N. Dhawan, H. Sharma, S. Vaidya, B. Vaidya, Bioadhesive polymers: Novel tool for drug delivery, Artif. Cells, Nanomedicine, Biotechnol. 42 (2014) 274–283. [20] M. Abelson, I. Udell, J. Weston, Normal human tear pH by direct measurement, Arch Ophthalmol. 99 (1981) 301. [21] M. Yamada, H. Mochizuki, M. Kawai, M. Yoshino, Y. Mashima, Fluorophotometric measurement of pH of human tears in vivo, Curr Eye Res. 16 (1997) 482–486. 15 [22] M. Yamada, M. Kawai, H. Mochizuki, Y. Hata, Y. Mashima, Fluorophotometric measurement of the buffering action of human tears in vivo, Curr Eye Res. 17 (1998) 1005– 1009. [23] L.G. Carney, T.F. Mauger, R.M. Hill, Buffering in human tears: pH responses to acid and base challenge, Investig. Ophthalmol. Vis. Sci. 30 (1989) 747–754. [24] R. Hill, L. Carney, Human tear responses to alkali, Invest Ophthalmol Vis Sci. 19 (1980) 207–210. [25] K. Han, O. Woghiren, R. Priefer, Surface tension examination of various liquid oral, nasal, and ophthalmic dosage forms, Chem. Cent. J. 10 (2016) 1–5.