Improved release of triamcinolone acetonide from medicated soft contact lenses loaded with drug nanosuspensions

Accepted Manuscript Title: Improved release of triamcinolone acetonide from medicated soft contact lenses loaded with drug nanosuspensions Authors: Eva Garc´ıa-Mill´an, M´onica Quint´ans-Carballo, Francisco Javier Otero-Espinar PII: DOI: Reference:

S0378-5173(17)30269-7 http://dx.doi.org/doi:10.1016/j.ijpharm.2017.03.082 IJP 16554

To appear in:

International Journal of Pharmaceutics

Received date: Revised date: Accepted date:

3-1-2017 27-3-2017 29-3-2017

Please cite this article as: Garc´ıa-Mill´an, Eva, Quint´ans-Carballo, M´onica, OteroEspinar, Francisco Javier, Improved release of triamcinolone acetonide from medicated soft contact lenses loaded with drug nanosuspensions.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2017.03.082 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Improved release of triamcinolone acetonide from medicated soft contact lenses loaded with drug nanosuspensions Eva García-Millána, Mónica Quintáns-Carballoa and Francisco Javier Otero-Espinara,b,* a

Departamento de Farmacología, Farmacia y Tecnología Farmacéutica, Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Vida s/n, 15782-Santiago de Compostela, Spain b

Instituto de Farmacia Industrial, Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Vida s/n, 15782-Santiago de Compostela, Spain *Corresponding author. Facultad de Farmacia, Departamento de Farmacia y Tecnología Farmacéutica, Universidad de Santiago de Compostela, Campus Vida s/n. Tel.: +34 881814878; fax: +34 981547148. E-mail address: [email protected] (F. J. Otero-Espinar)

Garphical abstract

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Abstract Drug nanosuspensions (NSs) show a significant potential to improve loading and release properties of the poorly water soluble drug triamcinolone acetonide (TA) from poly(hydroxyethyl methacrylate) (pHEMA) soft contact lenses. In this work, TA NSs were developed by a controlled precipitation method using a fractional factorial Plackett-Burmann design. Poloxamer 407 (PL) and polyvinyl alcohol (PVA) as stabilizing agents were selected. NSs were characterized in terms of their drug content, particle size and morphology. Results indicate that all studied factors, except homogenization speed and sonication, have significant influence on the drug incorporation yield into NSs. Drug nanoparticles showed an interesting size that may be suitable for their incorporation into topical ocular drug delivery systems, as hydrogels. pHEMA hydrogels and daily-wear Hilafilcon B commercial contact lenses (SCLs) were employed to study TA loading capacity and drug release properties using NSs as loading system. Hydrogels have been synthesised by copolymerization of 2-hydroxyethyl methacrylate (HEMA) with methacrylic acid (MA) in accordance with a previous work (García-Millán et al., 2015). Both synthesised hydrogels and SCLs were characterized in terms of their mechanical and physical properties and TA loading and release properties. Selected TA NS was further characterized by studying its physical-chemical stability during the loading process. Results show that the use of TA NSs as loading medium significantly increases drug loading capacity and release of soft contact lenses in comparison with drug saturated solution. Synthesised pHEMA hydrogels and SCLs lenses have good properties as ophthalmic drug

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delivery systems, but SCLs load higher quantities of drug and release TA in shorter time periods than synthesised pHEMA hydrogel.

Abbreviations. NSs: Nanosuspensions; TA: Triamcinolone acetonide; PL: Poloxamer 407; PVA: Polyvinyl alcohol; HEMA: 2-hydroxyethyl methacrylate; pHEMA: poly(hydroxyethyl methacrylate); SCLs: Commercial soft contact lenses; MA: Methacrylic acid; AIBN: 2,2’-azo-bis(isobutyronitrile); EGDMA: ethyleneglycol dimethacrylate; S/AS: solvent/antisolvent; ALF: Artificial lacrimal fluid; UPLC: Ultra performance liquid chromatography; MS/MS: tandem mass spectrometer.

Keywords: Medicated soft contact lenses, nanosuspensions, ocular, pHEMA hydrogels, triamcinolone acetonide, ocular drug release. Chemical compounds studied in this article: Triamcinolone Acetonide TA (CID: 6436); Polyvinyl alcohol PVA (CID 11199); 2-hydroxyethyl methacrylate HEMA (CID 13360); Methacrylic acid MA (CID: 4093); 2,2’-azo-bis(isobutyronitrile) AIBN (CID 6547); ethyleneglycol dimethacrylate EGDMA (CID:7355) Poloxamer 407.

1. Introduction Topical administration of drugs is widely accepted for the treatment of eye disorders, especially for a localized action. Unfortunately, topical treatments are not often effective due to the efficient protection mechanisms of the human eye. Blinking, baseline, reflex lachrymation and naso-lachrymal drainage remove rapidly foreign substances including drug ophthalmic formulation from the eye surface. Moreover, the anatomy, physiology and barrier function of the cornea compromise the rapid drug absorption. A short pre-corneal contact time of the drug in combination with the low permeability of the cornea itself results in low bioavailability, usually limited to the 1-10% of the total dose administered in conventional

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ocular pharmaceutical dosage forms. Consequently, to achieve the desired therapeutic effect, a frequent and repeated administration of formulations with high concentrations of the drug is

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needed (Alvarez-Lorenzo et al., 2006; Dandagi et al., 2009; Gratieri et al., 2010). Frequent instillation of concentrated solutions can induce significant toxic side effects and cellular damage, as well as promote systemic absorption (Gupta et al., 2010). In order to overcome these problems, new ophthalmic vehicles have been investigated, such as medicated soft contact lenses, primarily to avoid clearance ocular processes. Contact lenses have shown an interesting potential as controlled release systems for drugs in the eye.. Contact lenses are hydrogels, three-dimensional cross-linked networks of water-soluble polymers (Hoare and Koane, 2008). Some of the most popular soft contact lenses are the ones elaborated using poly (hydroxyethyl methacrylate) as polymer base due to their capacity to absorb water, leading to an enhancement of the oxygen permeability and an excellent biocompatibility. Hydrogels of pHEMA are also widely used as drug delivery systems due to their excellent mechanical properties and thermal and chemical stability (Andrade-Vivero et al., 2007; dos Santos et al., 2008). The high water content of pHEMA hydrogel enables the uptake of some drugs by simple immersion in concentrated solution. Once applied loaded lens or hydrogel in the eye, the drug preferentially diffuses towards post-lens lachrymal fluid (i.e. between the lens and the cornea). Since the exchange of this fluid is quite poor, the permanence time of the drug on the corneal surface is significantly increased. Thus, ocular bioavailability could be remarkably enhanced. However, the success of this approach is restricted to few drugs, since most drugs passively diffuse through the aqueous phase of the lens network without interacting effectively. For example, lipophilic drugs in reduced doses may be incorporated, as it is not possible to reach high concentrations in the loading medium. This limits both the amount loaded and the ability to control the release, which is deficient in the absence of mechanisms of drug retention in the

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hydrogel (Alvarez-Lorenzo et al., 2006; Álvarez-Lorenzo and Concheiro-Nine, 2009; dos Santos et al., 2008). Steroidal corticosteroids, as dexamethasone or TA, are lipophilic drugs, which have been assayed to be included in medicated soft contact lenses. The loading and release properties of dexamethasone and TA based on pHEMA soft contact lenses have been studied by Kim and Chauhand (2008) and García-Millán et al. (2015), respectively. For TA, linear isotherms (class C adsorption isotherm), which are characteristic of a partitioning process of the solute onto the hydrogels, were obtained. The favourable drug partition coefficient obtained for both dexamethasone and TA were indicative of the interaction and bound of the drugs and the polymeric network. Nevertheless, the C isotherms obtained for TA are far away from the saturation of adsorption sites indicating that maximum drug-loading dose into hydrogels is strongly limited by the drug concentration in the loading solution, because of its low aqueous solubility. Therefore, the implementation of methods to achieve high drug concentration in the loading medium could be useful to improve the loading process. In order to overcome this limitation, the use of NSs as systems could be increases the incorporation of poorly soluble drugs as TA into the hydrogel structure. NSs are colloidal dispersions of poorly water soluble solids suspended in a dispersion stabilized by surfactants (Ali et al., 2011; Harikumar and Sonia, 2011; Kassem et al., 2007; Kocbek et al., 2006; Lakshmi and Kumar, 2010; Martínez-Pacheco, 2009). Due to the small size drug particles of NSs incorporated or adsorbed into the hydrogels and topically applied, they may enhance the bioavailability. So, the therapeutic efficacy of the drug is increased by prolonged drug residence time in the ocular surface and conjunctival sac, by sustained drug release and/or by reduced precorneal drug loss. Besides, corneal adhesion may be improved and have the ability to penetrate different ocular tissues by circumventing the physiological and anatomical barriers.

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In addition, colloidal drug particles trapped in the matrix structure could act as a reservoir to control and prolong its release over time. Finally, if the dimensions of the particles and crystals in the suspension are suitable and the concentration is not very high, the lenses produced shall retain their optical properties (Álvarez-Lorenzo and Concheiro-Nine, 2009). The drug TA is currently administered by intravitreal injection for a broad spectrum of inflammatory, oedematous and angiogenic ocular diseases (Araújo et al., 2011; Couch and Bakri, 2009; Jermak et al., 2007; Kambhampati et al., 2015; Mansoor et al., 2009). Due to its limited water solubility, TA present major difficulties to be formulated in therapeutic doses. Through particle engineering, the opportunity to improve the efficacy of the drug is offered by reducing size to submicron dimensions (Gupta et al., 2010; Kassem et al., 2007; Kocbek et al., 2006). Increasing the solubility of TA can also lead to increase its bioavailability, permeation through ocular tissue, and intracellular transport (Kambhampati et al., 2015). In this work TA NSs were prepared by controlled precipitation or nanoprecipitation technique. The fundamentals of this method are directly related to the conditions of mixing of the phases, which include intense agitation and temperature. Similarly, the characteristics of the nanoparticles are influenced by the nature of the phases, their relationship and the concentration of components, mainly drug and stabilizing agents (Ali et al., 2010; Kesisoglou et al., 2007; Lakshmi and Kumar, 2010; Mora-Huertas et al., 2010). To prepare and stabilize the NSs, the surfactants PL and PVA were. Both are nontoxic synthetic polymers that have been used to stabilize nanoparticles and their use is authorized for ophthalmic drug formulations. PL is a nonionic surfactant, which belongs to the group of block copolymers and has been used extensively in a variety of pharmaceutical formulations as emulsifier, stabilizers solubilizer, and wetting agent for poor water-soluble drugs (Karolewicz et al., 2014; Wang et al., 2103; Wu et al., 2011). In general, pluronics provide polymer-type

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steric stabilization of NS without modifying the crystal structure of the drug particles (Wang et al., 2103; Wu et al., 2011). PVA is an anionic, biodegradable and hydrophilic polymer used as surfactant to produce stable NSs forming a hydrophilic layer at the nanoparticles surface, which provides stability to the colloidal system (Abrego et al., 2014; Guirguis and Moselhey, 2011; Rowe et al., 2012). The aim of the present work is evaluating the potential of drug NSs as a tool to improve poorly soluble drug loading and controlled release properties of medicated soft contact lenses based on pHEMA hydrogels. The experiments were carried out with synthesised pHEMA hydrogels and SCLs. SCLs were selected owing to similar composition with synthesised pHEMA hydrogel. Both types of soft contact lenses were selected because pHEMA hydrogels have a higher capacity than silicone hydrogels to control load and drug release process. 2. Materials and methods 2.1 Materials The drug TA was supplied by Roig Farma (Spain), poloxamer 407 (PL, from 9840 to 14.600 Da.) and polyvinyl alcohol (PVA; 87-90% hydrolysed, 30.000-70.000 Da.), were purchased from Sigma-Aldrich Chemical (Spain). Ophthalmic grade 2-hydroxyethyl methacrylate (HEMA) monomer and (MA) methacrylic acid were supplied by Merck (Germany); ethylene-glycol di-methacrylate (EGDMA) and 2,2’-azo-bis(isobutyronitrile) (AIBN) by Sigma-Aldrich (Spain). Daily-wear soft commercial contact lenses (SCLs), (Hilafilcon B, Soflens 59 wt.% water content) were purchased from Bauch & Lomb® (Portugal). UPLC LC-MS grade acetonitrile was purchased from Prolabo (France). The ultrapure water used in all the experiments was obtained by reverse osmosis (resistivity >18.2 MΩ cm; MilliQ®, Millipore Spain). All the other chemicals and reagents used in this study were of analytical grade.

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2.2 Optimization of TA nanosuspensions. Factorial design of experiments: Fractional factorial Plackett-Burman design The Plackett- Burmann factorial design for different variables which includes solvent/antisolvent ratio (S/AS) (acetone/water), TA concentration (TA mg/ml), PVA proportion (PVA %), PL proportion (PL%), stirring speed homogenization (ULTX rpm) and ultrasonic homogenization at two levels (Table 1) was used for screening (Plackett and Burman, 1946; Reddy et al., 2008). 12 formulations were studied. Average particle diameter, polydispersity index (PI), TA content ( g/ml) and yield (%) were evaluated were studied as dependent variables. 2.3 Preparation of TA nanosuspensions by controlled precipitation The NSs were prepared by nanoprecipitation technique. Water, PL and PVA 10% (w/v) in water or mixtures thereof were used as an aqueous phase. Moreover, drug solutions were prepared in acetone. The organic phase was slowly added to the aqueous solution at room temperature under stirring using high speed homogenizer Ultra Turrax T25 (Janke & Kunkel, IKA®, Germany) at different speeds (8.000 to 13.500 rpm) (Gupta et al., 2010). In certain formulations, once phases were mixed and homogenized, the suspension recipients were immersed in water-ice bath and sonicated for a period of 90 seconds and 7 cycles of duration using equipment Bandelin Sonopuls HD 2200 (Germany) with an amplitude of 30 % and a probe (SH 213 G) of 40 mm diameter. Finally, the acetone was removed by evaporation at 85 °C in a rotary evaporator (Labo Rota 300, Resona Techins, Switzerland with a Labo Therm SW 200 water-bath) to a final volume of 5 ml (Giannavola et al., 2003). The resulting NSs were stored in a refrigerator at 4 °C for further characterization (Govender et al., 1999).

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2.4 Characterization of nanosuspensions 2.4.1 Determination of the apparent solubility of TA The apparent equilibrium solubility of the drug was determined by adding excess thereof in the solutions studied. 1 mg of TA was weighted and incorporated in test tubes, which contained 5 ml of aqueous solutions of PVA 10% (w/v), PL 10% or their mixtures. Each system was prepared in sextuplicate. Samples were subjected to stirring for 7 days in a horizontal agitation water bath (Selecta® OR UNITRONIC 320, Spain) at a rate of 100 rpm and 25 ± 1°C. The samples were filtered through cellulose acetate filters 0.45

m pore size (Sartorius®, Germany). This filtrate was

suitably diluted with distilled water (1:10). Since samples that incorporate a 20% concentration of surfactants exhibit a high viscosity, which prevents filtration, centrifugation was used to remove slurry. To do this, 1.5 ml samples were taken and centrifuged in Eppendorf tubes at 12.000 rpm (12.235 g) for 30 minutes (Sigma 2-16, Germany), at room temperature for PVA and at 4 ºC for PL to prevent gelation. Finally, 1 ml of the supernatant was taken and diluted with distilled water (1:10) and TA concentration was determined by spectrophotometry (HP 8452 A, Hewlett Packard, Germany) at 242 nm. 2.4.2 TA content determination in nanosuspensions The content of TA was determined on the freshly prepared NSs before and after being filtered through membrane filters of cellulose acetate 0.45 m pore size (Sartorius®, Germany). 1 ml of the sample (filtered and unfiltered) was diluted with distilled water (1:10), determining the concentration of TA by spectrophotometry at 242 nm. TA content was expressed as amount of drug per ml suspension and the relationship was expressed as a percentage of the amount of drug in the suspension and used in its preparation.

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2.4.3 Particle size mean and distribution and zeta potential analysis Mean particle size (expressed in intensity) and PI of all formulations were measured by laser light diffraction (Zetasizer Nano ZS 6.0, Malvern Instruments, UK), and zeta potential (ZP) of the selected formulation 2 (FM2) by Laser Doppler Micro-electrophoresis using the same equipment. Fresh samples were adequately diluted with MilliQ® water to obtain an appropriate obscuration. Selected NS was stored in glass bottles with polypropylene caps at 25º C for 4 days (which correspond to the days of the loading process of NSs into hydrogel) and inter-day variation of particle size and ZP were determined each day. The experiments were performed in triplicate. 2.4.4 Optical Microscopy Optical microscopy images of the suspensions were taken using an Olympus BX60 (New York) microscope connected to a camera DP11. 2.4.5 Electronic Transmission Microscopy (TEM) Qualitative morphological characterization of TA NSs of all fresh samples, and FM2 on the fourth day was performed using the transmission electron microscope Philips CM-12 FEI Company (Netherlands). A drop (5 or 10 µl) of the suspension was placed on a copper grid coated with Formvar film. After 30 seconds, the excess was removed with a filter paper tip and allowed to dry for one hour. After complete drying, a drop of phosphotungstic acid was added, leaving it for 60 seconds. After that, the samples were washed with 10 ml distilled water to remove excess acid. 2.4.6 Chemical stability of TA in solution and nanosuspensions To check the chemical stability of TA in solution and in NSs during the loading and release experiments, Degradation of TA in artificial lachrymal fluid (ALF) and in water was performed

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by UPLC MS/MS. Samples of solutions and NSs stored in the same conditions of release and loading experiments were taken and diluted in MilliQ® water 1:1 and in methanol 1:2. The samples were centrifuged (Eppendorf Centrifuge 5804 R, Germany) at 5000 rpm for 15 min at 25 °C, and the supernatant was assayed for drug content by UPLC MS/MS to determine their possible degradation products. The chromatographic analysis of TA using UPLC-MS/MS analyses were performed on a MS/MS tandem Waters Xevo® TQD detector linked to an Acquity UPLC® H-Class system (Waters®, Czech Republic). Data were collected and processed by chromatographic software TargetLynxTM Application Manager. Chromatographic separation was performed at 40ºC using an Acquity (Waters®, Czech Republic) BEH C18 column (2.1 x 50 mm, 1.7 m particle size). The mobile phase solvents were 20 mM ammonium acetate of pH 5.0 in MilliQ® water (55%, solvent A) and acetonitrile (45%, solvent B). Isocratic conditions were employed at a constant flow rate of 0.45 ml/min. The autosampler was conditioned at 10 ºC and a volume of 10 l of each sample was injected. The total run time including equilibration prior to injection of the next sample was 3 min only. Acquisition of mass spectrometric data was performed in multiple reaction monitoring (MRM) mode via positive electrospray ionization, using the ion transitions of m/z 435.1 > 397.23 (transition qualification/confirmation) and 435.31 > 415.24 (transition quantification) (Liu et al., 2015; Mallet et al., 2013) with desolvation gas flow of 1100 l/h, cone gas of 80 l/h and capillary voltage of 0.55 kV. The desolvation temperature and source temperature were 450 ºC and 146 ºC, respectively.

The optimized mass spectrometric

parameters for TA were cone voltage, 22 v; collision energy for ion product 397.3, 15 v; collision energy for ion product 415.3, 5 v; dwell time, 0.66 min. For Triamcinolone base (Tbase) the parameters were cone voltage, 22 v; collision energy for ion product 375, 10 v; collision energy for ion product 357, 30 v; dwell time, 0.38 min).

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2.5. Synthesis of hydrogels pHEMA-MA200 by thermopolymerization Hydrogels were prepared by radical solution polymerization with thermal initiation. EGDMA crosslinker (80 mM equivalent to 1 mol%) and MA functional monomer (200 mM, 2.5 mol%) were dissolved in HEMA (6 ml, 96.5 mol%). After the addition of AIBN initiator (10mM), the monomer solution was stirred until it was completely dissolved and immediately injected into a mould. These moulds were constituted by two glass plates internally covered with a polypropylene sheet to prevent polymer adhesion on the glass and separated by a silicone frame 1 mm wide. The moulds were then placed in an oven for 12 h at 50 ºC, followed by a 24 h period at 70 °C to complete the polymerization. Each hydrogel was immersed in boiling water for 15 minutes to remove unreactive monomers and facilitate the cut of hydrogel in 10 mm in diameter discs (García-Millán et al., 2015). The discs were immersed in NaCl 10mM solution for 1 week, then in HCl 10mM solution for one day and in ultrapure water for one more day until they were clean. The medium was replaced two times a day. Cleanness was verified by recording UV spectra of the cleaning solution over the range 190 to 800 nm where a complete monomer bands absence was needed. Finally, the clean lenses were dried at 40 °C for 48 h to constant mass. Samples of all hydrogels were characterized as follows in next sections. 2.6. Hydrogels and SCLs characterization 2.6.1. Nitrogen adsorption (BET) Nitrogen adsorption experiments were carried out in a Micromeritics ASAP 2000 (Norcross GA, USA) apparatus, using hydrogel discs that had been degasified by being kept for 8 h at 70 ºC and 10-3 mmHg. Nitrogen adsorption at 77 K was measured over the relative pressure range 0.01–0.98. 2.6.2. Hydrogels of pHEMA-MA and SCLs swelling kinetics in water and water uptake Samples of pHEMA-MA hydrogels and dry SCLs were accurately weighed and

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dimensions were measured (diameter and thickness), and they were placed in a flask with 5 ml of 0.9% NaCl at 25 ºC. Swelling kinetics (Q%) at various times was calculated as relative weight gain; the sample being weighed on each occasion after carefully wiping off its surfaces with a soft tissue (Eq. (1)). This method was repeated until equilibrium water content (EWC), i.e. constant mass, was reached and expressed as final weight gain per gram of dried lenses (Eq. (2)).

Eq. (1)

Eq. (2) where w0 is the weight of the dry lenses and wt its weight at time t and w∞ the final equilibrium weight. Water diffusion coefficients were calculated by fitting the data to Eq. (3):

Eq. (3) where wt (g) is weight gain at time t (h),

∝

(g) the final weight gain, D (cm2s-1) the diffusion

coefficient and h (cm) the lenses thickness. Once the hydrogels reach the swelling equilibrium, they have a diameter of 0.8 and 1.42 cm and a thickness in the middle of the lens of 10-3 cm and 0.257x10-3 cm for pHEMA-MA hydrogels and SCLs, respectively. 2.6.3. Loading of TA from pHEMA-MA hydrogels and SCLs Drug loading experiments were made using two different media, TA saturated in 0.9% NaCl and different concentrations of FM2 nanosuspension, the last ones being assayed to obtain the

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information about the adsorption isotherm. To prepare saturated TA in 0.9% NaCl, drug was added in excess on flasks containing 0.9% NaCl. Flasks were left in a shaking water bath at 25 ºC for two days (Selecta, Unitronic 320 OR, Spain) and the samples were filtered through cellulose acetate filters of 0.45 m pore size (Sartorius, Germany). Concentrations of these filtrates were determined spectrophotometrically at 242 nm. Discs of pHEMA-MA hydrogels and SCLs were placed in TA saturated aqueous solution of 0.9% NaCl (10 ml) or FM2 (5 ml) for four days at room temperature 25 ºC. The amount of drug loaded by pHEMA-MA hydrogels and SCLs was estimated based on release data, determined by High-performance liquid chromatography (HPLC) analysis, following method described in section 2.7. To prove that no TA was remaining in hydrogels after release experiments, an Ultraturrax Tube Drive (IKA®, ULTRA-TURRAX® Tube Disperser, Germany) with a BMT-20-G tube (IKA®, Germany) for grinding with glass balls to 4000 rpm for 5 min was used. Lenses were broken and triturated with acetonitrile (5 ml). Finally, the liquid of extraction was filtered by 0.22 µm Sartolon® polyamide membrane filter (Sartorius®, Germany) and analysed for TA in HPLC. Hydrogels were also analysed by FTIR (See 2.4.7 section for apparatus) to detect TA in the samples. The experiments were carried out in triplicate. Adsorption isotherms were constructed employing loading medium with different TA concentration based on FM2. Data were fitted to Freundlich isotherm, which equation is given by:

Eq. (4)

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Where Qe is the amount of TA adsorbed (mg/g of dry hydrogel) for each equilibrium concentration of adsorbate Ce (mg/l), kf is Freundlich isotherm constant and n the adsorption intensity. 2.6.4. Hydrogels of pHEMA-MA and SCLs optical transparency The samples, SCLs and pHEMA-MA hydrogels, hydrated, loaded with FM2 and saturated solution of TA, were fixed to the inner side of a quartz cell containing MilliQ® water. Then, the transmittance was recorded at 600 nm using UV-Vis spectrophotometry. The experiments were carried out in triplicate. 2.6.5. Release of TA from pHEMA-MA hydrogels and SCLs pHEMA hydrogels and SCLs loaded with TA were rinsed with water to eliminate any TA adsorbed to the surface, placed in 5 ml of artificial lacrimal fluid (ALF; 6.78 g/l NaCl, 2.18 g/l NaHCO3, 1.38 g/l KCl and 0.084 g/l CaCl2·2H2O, pH 8) (García-Millán et al., 2015) and incubated at 37 ºC in an orbital shaker at 100 rpm (Incubator 1000, Unimax 1010, Heidolph, Germany) for four days. The experiments were carried out under sink conditions in sextuplicate. Samples of the solution (1 ml) were withdrawn at regular intervals and replaced with 1 ml of fresh ALF. The amount of drug released was determined by HPLC. The release profiles were fitted to Higuchi square root model (Eq. (5)) (Higuchi, 1962). Eq. (5) where Qt is the total amount of drug released (µg), after time t (h) and K (µg·h-0.5) the rate constant obtained according to Higuchi equation. 2.7. Instrumentation and chromatographic conditions. HPLC systems Chromatographic (HPLC) determination of TA in release studies was performed on a system from Merck Hitachi (Germany) and equipped with a diode array detector (L-4500,

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Merck Hitachi), a compatible pump (L-6200A, Merck Hitachi), an autosampler (AS4000 A, Merck Hitachi) and a chromatographic data processing software (Model D-6500, Hitachi). The column used was Symmetry® C18 5

m particle size 3.9 × 150 mm (Waters®, USA).

Chromatographic analysis was carried out at 40 ºC (Column Thermostat L-5025, Merck). The compounds were eluted using an isocratic system with a mobile phase consisting of acetonitrile:ammonium acetate (pH 5.0, 20 mM) (45:55, v/v). The mobile phase was filtered through a 0.22 µm Sartolon® polyamide membrane filter (Sartorius®, Germany), sonicated for 20 min and delivered at a flow rate of 0.8 ml/min. The effluent was monitored at 242 nm. The injection volume was 100 µl. 2.8. Model Fitting and Statistical analysis Fitting of experimental data to the equations 3, 4 and 5 was made by means of nonlinear regression, using least squares method (GraphPad Prism graphics software 5.0, GraphPad Sofware Inc., CA). To perform the statistical analysis of the Fractional factorial Plackett-Burman design and construct the response surface plots, StatGraphics Plus 5.1 (Statistical Graphics Corp. 19942000) software was used. The analysis of the variance (ANOVA) and T-test used to analyse the loading and release experiments were developed using StatGraphics Plus 5.1 (Statistical Graphics Corp. 19942000), after testing the normal distribution of the variance. 3. Results and discussion Values of the response variables obtained from Plackett-Burman design are summarized in Table 2. A macroscopic evaluation of the appearance of suspensions according to their transparency is also included.

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The drug used in this study presents low aqueous solubility (16.76 g/ml) (García-Millán et al., 2015). In presence of PVA, PL or a mixture of both at a concentration of 10% (44.22 ± 12.76 µg/ml, 176.75 ± 13.33 µg/ml and 89.6 ± 4.27 µg/ml, respectively), aqueous solubility of TA increases. PL has the ability to form polymeric micelles at concentrations above 0.5% (Ravi et al., 2010), which are the responsible structures to increase drug solubility (Nogueiras-Nieto et al., 2009). The slight increases produced by PVA could be associated with a hydrotropic effect at high concentrations of polymer. Concentration of TA in NSs is showed in Table 2. All the conditions assayed give values higher than the one obtained by the solubilization effect of the stabilizers, suggesting the formation of NSs. Statistical analysis of the results did not allow to find any relationship between factors in the design and the amount of drug incorporated into the NS. Apparently, the presence of the stabilizing agents increases the amount of TA in suspensions, especially as both are combined, but no significant effects were found. Nevertheless, all analyzed variables were significant (α< 0.01) in the yield of incorporation of the drug into the NSs, except those related to the conditions of homogenization system (ultraturrax, rpm, and intensity of sonication). The yield parameter is referred to the percentage of TA incorporated to the NS in relation to the total drug used in the preparation. The ANOVA of the experimental design allows us to obtain the polynomial equations used to plot the response surfaces represented in Fig. 1 Complementary (Reddy et al., 2008). Determination coefficient R2, with values of 96.31% and 97%, indicates a good fit of the models to the experimental data for both unfiltered and filtered samples, respectively. Response surface plots show an increase in the yield of TA in NSs when drug concentration in the solvent and ratio solvent/anti-solvent decrease. The yield is also increased using stabilizers, specifically when a mixture of both stabilizers, PL and PVA, is used, which indicates a synergistic effect.

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Both particle size and size distribution of TA in NSs are critical parameters in systems intended for parenteral and ocular administration. As the objective of this work is incorporating TA NSs into contact lenses, the particle size is critical to maintain lens transparency and optical quality. To evaluate particle morphology, optical and transmission electron microscopy (TEM) were used. Most formulations behave as homogeneous transparent systems under optical microscopy (data not shown), indicating submicron particle sizes. Microphotographs of TEM of NSs s are shown in Fig. 1. There was no correlation between the stabilizers and the nanoparticles morphology. All formulations elaborated with polymers show dense structures with spherical shapes and areas of different sizes and electronic density, which suggest the formation of drug particles coated by stabilizers. FM2 numerous, slightly smaller and homogeneous particles (100 nm) embedded in a more dense network are observed. In contrast, nanosuspensions FM8, FM10 and FM12 prepared in absence of stabilizing agents show large aggregates. Additionally, FM10 presents TA crystal with a typical triangular morphology. The high ratio between TA values incorporated to the NSs before and after filtration through filter membranes of 0.45 µm (Table 2) indicates the presence of large particles or aggregates into NSs. The particle size distribution of NSs shows that all formulations have more than one particle size population in the nanometer range, except FM2 and FM11 prepared with mixtures of PVA and PL, and FM8 and FM12 elaborated without stabilizing agent. In general, one population with small size particle, between 10 and 30 nm, which may correspond to the formation of polymeric micelles in PL formulations (Nogueiras-Nieto et al., 2009), together with other larger particles of approximately 200 nm. Note that, in photon correlation, intensity values are proportional to molecular weight squared, larger particles provide much higher intensity values than smaller ones and therefore intensity distributions underestimate the proportion of small particles for the largest. The final mean particle size was not dependent on the nature of the polymer used. Nevertheless, all formulations show a particle size in the

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nanometer range, suitable for ophthalmic applications (Giannavola et al., 2003; Zimmer and Kreuter, 1995). The high polydispersity values confirm that all formulations present very heterogeneous size nanoparticles, except FM2 and FM11, which show lower values, 0.3 and 0.21 respectively (Table 2). The result of this preliminary study allowed us to identify significant variables that affect TA NSs development. To prepare suspensions with a suitable size and high drug content, it is desirable to use a mixture of PVA and PL as stabilizing agents and small amounts of solvent with low concentrations of the drug. Finally, while PI decreases with mechanical stirring, sonication

process

does

not

influence

on

suspensions

properties.

Based on these results and considerations, the FM2 was selected for the posterior studies of TA incorporation into SCLs and pHEMA-MA soft lenses. FM2 nanosuspension shows a monodisperse population with a particle size lower than 150 nm, a low PI of 0.3 and a yield of 80%, with TA concentrations over 600 μg/ml (>500 μg/ml after filtration by 0.45 µm). TA particles precipitation process can produce changes in their crystalline structure, which may be converted to either amorphous or other polymorphic forms. The physical state of drug and polymer in NSs may have an influence on the drug loading process and in vitro release characteristics. Solid state of the drug particles in NSs and interactions polymer-drug were characterized using X-ray diffraction analysis, FT-IR spectroscopy and scanning calorimetry analysis by García-Millán et al. (García-Millán et al. 2017, submitted). Results of this work suggest that an amorphization of TA was produced during the preparation of the NSs, which can promote the drug solubility. The main aim of this work is to evaluate the potential of TA NSs as a tool to improve drug loading and release in medicated soft contact lenses. To evaluate drug loading capacity, the methodology using aqueous solutions of TA, described in a previous work (García-Millán et

21

al., 2015) was used. This method consists in the immersion of the lenses into the drug-loading medium for 4 days. In these conditions, it is necessary to ensure the chemical stability of the TA and physicochemical stability of the NS during the whole loading process. Fig. 2 Complementary shows MS/MS chromatogram of FM2 nanosuspension and a TA saturated solution in water after 4 days of storage at 25 ºC. TA saturated solution chromatograms show a partial loss of the acetonide group in the form of CO2 during the storage. Besides, a signal of triamcinolone base is detected at a retention time of 0.36 min on the fourth day of storage using MS Scan mode, but in a very low proportion compared to the acetonide form. Nevertheless, no triamcinolone base was detected on FM2 nanosuspensions, indicating a better chemical stability of the drug in NSs. Similar results were obtained using ALF as solvent medium during the storage. Fig. 2 shows TA concentration, particle size, surface charge and morphology corresponding to FM2 nanosuspension during four days of storage at room temperature. A decrease in the TA concentration was observed on the first day. Since no chemical degradation of TA is in FM2 systems, the decrease in TA levels could be associated to a process of aggregation (Fig. 2D) and sedimentation of nanoparticles promoted by the low density of surface charges in the NSs, with values of zeta potential between 0 and -2 mv (Fig. 2C). TEM of TA loaded NS (FM2) after four storage days (Fig. 2B) shows that the particles lose their spherical shape compared with the freshly prepared ones, due to particles aggregation. Therefore, these results suggest that NS shows enough chemical and physicochemical stability to be used as hydrogel loading media for four days. To study the drug loading capacity of soft contact lenses from TA NS, two different hydrogels based on pHEMA were used, pHEMA-MA hydrogels prepared by radical solution polymerization as described in a previous work (García-Millán et al., 2015) and daily-wear Hilafilcon B commercial contact lenses.

22

Hilafilcon B are cross-linked polymeric materials that comprise the comonomers HEMA, methacrylic acid, ethyleneglycol dimethacrylate and N-vinylpyrrolidone. Fig. 3 Complementary shows the comparison between internal micro and mesoporosity of the SCLs and the cross-linked polymeric pHEMA-MA hydrogels, characterized by Nitrogen adsorption experiments through the determination of the pores size distribution in the range of 1 to 60 nm. SCLs show low mesoporosity, with internal pores in diameters lower than 4 nm. The pHEMA-MA hydrogels have larger pores with a wide size distribution and lower frequency of small pores than SCLs. The differences found in the internal structure can be critical in the solvent and drug absorption behaviour of SCLs and pHEMA-MA hydrogels. Hydrogels of pHEMA-MA and SCLs swelling were characterized using NaCl 0.9% as medium since it is one of the means, which is normally used as storage solution for soft lenses, and therefore, it is a candidate as charging solution. Water uptake and swelling are extremely fast for dry SCLs, showing a very low EWC value (1.54 ± 0.04), recovering their original shape and size and maintaining its specific dimensions, as its radius of curvature, thickness and diameter. This behaviour is in accordance with the internal porous structure characterized by BET experiments. The presence of internal pores with a very small size causes a very rapid absorption of water promoted by the capillary forces. The absence of large pores that can act as a water reservoir together with the low expansion of the SCLs structure probably caused by a high crosslinking level, give rise to very small EWC values. pHEMA lenses elaborated by radical polymerization swell slowly, being completely swollen in 24 hours, with significantly higher values of EWC (48.0 ± 1.6) and a diffusion coefficient of 8.1x10-8 ± 2.0x10-8 cm2s-1 for water molecules. The high degree of swelling suggests that these hydrogels present lower crosslinking density than SCLs. Moreover, BET

23

structure shows the presence of larger pores, which may act as a water reservoir producing a slow swelling. Synthesised pHEMA-MA hydrogels and SCLs had a clear transparent appearance at the swollen state, gave values above 90 % transmittance. The hydrogels of pHEMA-MA and SCLs were examined to check if the addition of TA NSs induces changes in the transparency. According to the values of transmittance, over 90%, the loading technique maintains adequate optical clarity for lenses and they are compatible with the drug delivery ocular route. Fig. 3 shows the loading ability of the synthesised pHEMA-MA hydrogels and the SCLs from a saturated solution of TA on 0.9% NaCl. Four days in the loading solutions were found to be enough to reach equilibrium. The amount of TA loaded (expressed as mg of drug per g of dry lens; Fig.3A) by SCLs was higher than pHEMA-MA hydrogels (t test, α<0.01). Nevertheless, given the drug loaded per lenses unity (Fig.3B), this behavior is reversed since SCLs load lower amount of TA per lens than pHEMA-MA hydrogel (t test, α<0.01). This is caused by the differences in weight and size between both lenses. SCLs have a mean dry weight of 0.0158 ± 0.0003 g, and a thickness of 0.257 ± 0.067 mm, significantly lower than pHEMAMA hydrogels (0.0619 ± 0.0028g and 1 mm of thickness). In order to improve TA loading and release process based on SCLs and pHEMA-MA hydrogels, FM2 nanosuspensions were used as loading media. Fig. 4 shows the drug loading isotherms developed using FM2 nanosuspensions prepared with different TA concentration. Results show that the loading capacity of hydrogels is significantly increased by the use of NS especially in SCLs. Two-way ANOVA analysis shows an influence of the TA concentration in NS and statistical differences between SCLs and pHEMA-MA with a α<0.001. To characterize the mechanism of drug loading and the affinity and heterogeneity of binding sites of pHEMA-MA hydrogels and SCLs with TA, drug isotherms were fitted to the model of

24

Freundlich (Fig.4) (Voudrias et al., 2002); results are summarized in Table 3. The value of the constant kf and 1/n are the indicators of the adsorption capacity and adsorption intensity, respectively. Values of 1/n = 1 indicate that the adsorption is linear and they are typical when surface sites distribution or interaction adsorbent-adsorbate decreases, increasing surface density. However, values of show a normal physical adsorption process through weak interactions. Values within the range of 1 – 0.1 represent good adsorption (Desta, 2013). Freundlich isotherms of pHEMA-AM show values of 1/ < 1, typical of favourable normal adsorption isotherm where the drug molecules are adsorbed physically on the surface of the hydrogel by weakly interactions (e.g. van der Waals forces) (Zarzycki et al., 2010). SCLs give values of 1/ ≈ 1, which indicates that the partition of TA between the two phases is independent of the concentration. This suggests that adsorption of TA nanoparticles is more favourable in SCLs, which reveals a remarkably high affinity for this drug (Mora-Huertas et al., 2010). The significant increase in the drug loaded capacity with NSs can be likely due to the increase on the activity of the drug in the NS, which increases it diffusion according to Fick's first law, as well as their flow into hydrogels and an adsorption of drug nanoparticles in the lens structures. At the equilibrium, in the loading media drug solid nanoparticles co-exist with the molecules of dissolved drug that are at saturation concentration (Fig.5). Due to the size range and the amorphous state of TA, a high solubility equilibrium concentration is expected. When hydrogels are soaked into the NS, the dissolved drug may penetrate into the network by a diffusion process that depends on the crosslinking density of the matrix and on the drug concentration. Simultaneously, drug nanoparticles can be adsorbed onto the hydrogel surface and if they have the appropriate size, they can diffuse through the pores of the matrix. In accordance with the results obtained from nitrogen adsorption and water uptake analyses, SCLs show lower mesoporosity and higher density of crosslinking than pHEMA-MA. The

25

greater degree of swelling and relaxation of polymer chains of pHEMA-MA may promote the diffusion of the free dissolved molecules of TA inside the network, being efficiently hosted into the hydrogels (Siemoneit et al., 2006). Nevertheless, the higher crosslinking density of SCLs can delay the diffusion of free TA. In relation to the probable diffusion of the TA nanoparticles inside the hydrogel matrix, the low mesoporosity of both lenses precludes their penetration into the hydrogel structure and so the drug nanoparticles may only be adsorbed onto the surface of the hydrogel. The best results obtained in adsorption isotherm from SCLs suggest a more intense interaction between TA nanoparticles in SCLs surface compared with pHEMA-MA. Additional studies are needed to evaluate the interactions implicated on the loading process and to determine if the differences are caused by the lenses composition or by their microstructure. TA release curves from synthesised pHEMA-MA hydrogels and SCLs loaded with an aqueous TA solution of NaCl 0.9% (19 µg/ml) or FM2 nanosuspension are showed in Fig.6. In FTIR-ATR spectra of lenses, the strong band C-F of Ta was not detected and, in addition, the HPLC analysis of the solution used for the drug extraction from hydrogels did not provide any signal at 242 nm. This proved that no drug remained in the lenses after release studies. For all hydrogels, the use of FM2 nanosuspension as loading media produces a significant improvement in the release of TA. Hydrogels loaded with NSs release higher amount and prolonged the time of drug release compared to the lenses loaded with saturated TA solutions. Moreover, as TA concentration in NS increases, the drug loaded and released enhances. Drug release experiments in ALF show differences between pHEMA-MA and SCLs. The pHEMA-MA hydrogels are able to control the TA release for 48h and the drug release is

26

significantly lower than the one obtained using SCLs. Nevertheless, SCLs release the drug content in about 12 hours. The mechanisms of drug retention in the hydrogel described in Fig.5 can likely provide simultaneous diffusion and desorption mechanisms of TA release. As a result, the rate of drug release can be controlled by the interior diffusion of the free drug, by the desorption process of nanoparticles from hydrogel surface and by the dissolution of the desorbed or adsorbed TA NS (Zarzycki et al., 2010). The low values of total drug released and more sustained release profiles of TA for 48 hours in pHEMA-MA suggest that an important proportion of TA loaded into the matrix of the hydrogel is hosted in the aqueous phase or weakly interacting with the polymeric matrix. However, more rapid release profiles of SCLs indicate that the main mechanism of drug release is the simultaneous desorption of nanoparticles from the hydrogels surface, dissolution of the adsorbed and desorbed TA nanoparticles and exterior diffusion processes of the drug (Zarzycki et al., 2010). In all cases, the release profiles fit to the Higuchi model with good correlation coefficients (Table 4), which suggests that diffusion controlled drug release (Dillen et al., 2004). This model is in accordance with the mechanism of interior and exterior drug diffusion and desorption, and higher release rate constants (K) from SCLs compared to pHEMA-AM are indicative of the preponderance of the exterior diffusion process in the marketed lenses. 4. Conclusions The results presented in this study reflect the potential of the NSs by nanoprecipitation method to formulated TA in high concentrations. Of all formulations developed, FM2 was selected in order to use it as a loading media and to obtain medicated soft contact lenses. This formulation is a monodisperse NS in nanometric range, with a mean particle size of 147 nm

27

and a drug load of 0.8 mg/ml, using mixtures of PVA and PL as stabilizers. The good properties obtained with FM2 make it an excellent candidate to be loaded in the hydrogels for ophthalmic application, without blurring the vision. The solid-state characterization of NSs indicates that drug is in amorphous state. This fact and the nanometric size of the TA particles can promote an increase of the solubility of the drug in NSs. Moreover, the formulation of TA as NSs confers chemical stability during all the period of load and release of the lenses. NSs improve the drug loading capacity of soft contact lenses, especially in SCLs. Additionally, the increase of concentration of TA in the NSs improves the drug loaded dose significantly. Finally, the release experiments show controlled TA release profiles for a period of 12 and 48 h for SCLs and pHEMA-MA, respectively. Therefore, NSs could be an efficient clinical approach to load poorly water soluble drugs in hydrogels and treat serious diseases that threaten vision. The results indicate that hydrogels loaded with FM2, particularly SCLs, have a great potential for the fabrication of dailymedicated contact lenses, which would release TA for a day. This practice may improve patient therapeutic compliance in ocular drug delivery. 5. References Abrego, G., Alvarado, H.L., Egea, M.A., Gónzalez-Mira, E., Calpena, A.C., Garcia, M.L., 2014. Design of Nanosuspensions and Freeze-dried PLGA nanoparticles as a novel approach for ophthalmic delivery of pranoprofen, J. Pharm. Sci.103, 3153-3164. Ali, H.S., York, P., Ali, A.M., Blagden, N., 2011. Hydrocortisone nanosuspensions for ophthalmic delivery: a comparative study between microfluidic nanoprecipitation and wet milling. J. Control Release. 149, 175-181. Álvarez-Lorenzo, C., Concheiro-Nine, A., 2008. Lentes de contacto blandas medicadas, Arch. Soc. Esp. Oftalmol. 83, 73-74.

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Fig. 1. TEM images of TA nanosuspensions. 1: 110.000X; 2, 11: 66.000X; 3: 5.600; 4, 8, 12: 53.000X; 5, 7: 40.000X; 6: 31.000X; 9: 88.000X; 10: 19.500X.

Fig. 2. A) Relationship between TA concentration during four days and the initial concentration; B) Image of TA loaded nanosuspension (FM2) the last day of loading process by transmission electron microscopy C) Zeta potential of FM2 nanosuspension (unfiltered), the first day (Day 0) to the four day (Day 4) of storage; D) Evolution of mean diameter of nanosuspension during the four days of storage.

33

Fig. 3. TA loaded, expressed as A) mg/g of dry lens and B) mg per lens, by synthesised pHEMA hydrogels with MA 200 mM (pHEMA-MA) and SCLs using a TA saturated solution on NaCl 0.9% as loading medium.

34

. Fig. 4 TA adsorption isotherms obtained from different concentrations of FM2 employing (A) pHEMA-MA and (B) SCLs expressed in mg/g; (C) TA amount loaded in hydrogels and SCLs obtained from different concentrations of FM2 expressed in mg of drug per hydrogel.

Fig. 5 Schematic illustration of TA nanosuspensions behaviour used as a medium to load into the hydrogels.

35

.

Fig. 6 TA release profiles in artificial lachrymal fluid (ALF) from pHEMA-MA hydrogels (A) and SCLs (B) previously loaded by immersion in a saturated aqueous TA solution of NaCl 0.9% (19 µg/ml) and in FM2 nanosuspensions containing different concentrations of TA: 50 g/ml, 100 g/ml, 200 g/ml or 490 g/ml.

36

.

37

. Table 1 Experimental variables utilised for the production of TA nanosuspensions using fractional factorial Plackett-Burman desing. FM

S/AS (v/v)

TA (mg/ml)

PVA (%)

PL (%)

ULT (rpm)

Ultrasonic homogenization

1 2

5/10 5/10

2 2

10 10

0 10

8.000 13.500

-

3

5/10

1

0

10

8.000

90 s´- 7 cycles

4

5/10

1

0

10

8.000

-

5

5/10

1

10

13.500

90 s´- 7 cycles

6

1/10

2

10

10

8.000

90 s´- 7 cycles

7

1/10

2

0

10

13.500

90 s´- 7 cycles

8

5/10

2

0

0

13.500

90 s´- 7 cycles

9

1/10

1

10

8.000

90 s´- 7 cycles

10

1/10

1

0

0

13.500

-

11

1/10

1

10

10

13.500

-

12

1/10

2

0

0

8.000

-

0

0

38

Table 2 Results obtained in the preliminary study to produce TA nanosuspensions using the experimental Plackett-Burman design. No filtered

Filtered through 0.45 μm Average particle size (nm)

FM contentTA [solvent TA] in Yield (%) index Appearance

content TA [solvent TA] in Yield

Main

Secondary

Polydispersity

(%) ( g/ml)

( g/ml)

( g/ml)

( g/ml)

population

1 120 7.16 12 108 6.44 10.8 184.2 (73.2%) 16.4 (25.1%) 0.51

population

0.11 Transparent 2 769 45.88 76.9 568 33.89 56.8 143.8 (100%) - 0.30

0.01

Translucent 3

287 17.12 Transparent

14.4

238

14.20

11.9

149.4 (50%)

17.8 (50%)

0.70

0.13

4

289 17.24 Transparent

14.5

114

6.77

5.7

6.0 (41.8%)

218.3 (38.4%)

0.63

0.33

5

202 12.05 10.1 138 8.23 6.9 15.8 (64.9%) 102.7(18.2%) 0.33 (10.4%) 0.39

0.05 Transparent 6 449 26.79 100 198 11.81 99.0 285.6 (88.9%) 49.3

0.07 Translucent

7

154 9.19 Transparent

76.5

147

8.75

73.3

76.7 (55.8%)

6.5 (36.6%)

8

102 6.08 Transparent

11.0

97

5.77

9.7

190.9 (100%)

-

9

216 12.89 Transparent

54

226

13.48

56.5

198.9 (81.1%)

15.0 (18.9%)

10

102 6.08 25.5 104 6.18 25.9 236.9 (93.8%) 58.0 (6.2%) 0.34

0.47

0.99

0.36 0.01

0.79

0.21

0.02 Transparent 11 291 17.36 81.5 219 13.07 61.3 365.9 (100%) - 0.21

0.02 Translucent 12 93 5.55 46.3 88 5.23 43.8 205.1 (100%) - 0.83

0.29 Transparent

Table 3 Results of the fitting to the Freundlich model of the values of TA adsorbed by pHEMA hydrogels MA200 and SCLs (mg/g). FM2 kf

1/n

R2

pHEMA-MA

0.109 ± 0.04

0.45 ± 0.04

0.997

SCLs

0.026 ± 0.01

1.07 ± 0.17

0.995

Freundlich

39

Table 4 Results of the fitting of the Higuchi model. Loaded lenses

K (µg·h-0.5)

R2

pHEMA-MA - 50 pHEMA-MA - 100

7.19 ± 0.41 6.86 ± 0.57

0.97 0.93

pHEMA-MA - 200

11.90 ± 0.53

0.98

pHEMA-MA - 490

19.62 ± 0.80

0.98

pHEMA-MA – NaCl 0.9%

5.42 ± 0.19

0.99

SCLs - 50

9.60 ± 1.21

0.91

SCLs - 100

14.15 ± 1.49

0.93

SCLs - 200

35.55 ± 1.86

0.98

SCLs - 490

72.22 ± 3.31

0.98

SCLs – NaCl 0.9%

4.90 ± 0.84

0.85