Prednisolone-loaded nanocapsules as ocular drug delivery system: development, in vitro drug release and eye toxicity.

http://informahealthcare.com/mnc ISSN: 0265-2048 (print), 1464-5246 (electronic) J Microencapsul, Early Online: 1–10 ! 2014 Informa UK Ltd. DOI: 10.3109/02652048.2013.879930

RESEARCH ARTICLE

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Prednisolone-loaded nanocapsules as ocular drug delivery system: development, in vitro drug release and eye toxicity Tatiele Katzer1, Paula Chaves2, Andressa Bernardi1, Adriana Pohlmann3,4, Silvia S. Guterres1,4, and Ruy Carlos Ruver Beck1,4 1

Programa de Po´s-Graduaçaõ em Cieˆncias Farmaceûticas, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil, 2Faculdade de Farmaćia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil, 3Departamento de Quı´mica Orgaˆnica, Instituto de Quı´mica, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil, and 4Programa de Po´s-Graduaçaõ em Nanotecnologia Farmaceûtica, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil Abstract

Keywords

Objective: To develop non-toxic aqueous ocular drug delivery systems containing prednisolone by means of its nanoencapsulation. Materials and methods: Nanocapsules were prepared by interfacial deposition of preformed polymer [poly("-caprolactone) or EudragitÕ RS100]. Particle size distribution was determined by laser diffractometry, photon correlation spectroscopy and nanoparticle tracking analysis. Ocular irritation and cytotoxicity were evaluated in vitro on the chorioallantoic membrane (CAM) and rabbit corneal epithelial cell line, respectively. Results and discussion: Nanocapsules showed mean particle sizes between 100 and 300 nm and prednisolone encapsulation efficiency of around 50%. Controlled release of prednisolone occurred for 5 h for both formulations according to the biexponential model. Both formulations were found to be non-irritant in the CAM test and non-cytotoxic toward rabbit corneal epithelial cells. Conclusions: Encapsulation of prednisolone in nanocapsules was reported for the first time, being suitable for producing eye drops for the treatment of ocular inflammatory and no eye toxicity was indicated.

Chorioallantoic membrane, cytotoxicity, eye drops, eye toxicity, nanoparticles, ocular

Introduction The excess tear production caused by the instillation of an eye drop into the conjunctival sac, increased drainage and reflex palpebral blinking (Bonferoni et al., 2004; Vandervoort and Ludwig, 2004) lead to the elimination of considerable amounts of an administered drug (Urtti and Salminen, 1993; Jarvinen et al., 1995). Hence, the frequent instillation of eye drops is necessary to maintain a therapeutic drug level in the tear film or at the site of action (Salminen, 1990; Arici et al., 2000). However, this may induce cellular damage at the ocular surface, as well as toxic side effects (Mello Filho et al., 2010). New non-invasive strategies have been studied to overcome the low residence time of drugs in the precorneal area, thereby reducing wastage and minimising side effects. In this context, nanoparticles have become the object of increasing scientific interest (Pignatello et al., 2006; Nagarwal et al., 2009; Gupta et al., 2010). Drug-loaded nanoparticles can reportedly facilitate the application of drugs in eye drops (Le Bourlais et al., 1998), affording therapeutic action using lower doses and causing fewer systemic and ocular side effects (Vandamme, 2002; Cunha Juńior et al., 2003; Alany et al., 2006). These advantages, which make treatments more patient friendly, are due to less frequent application and longer retention in the extraocular region (Nagarwal et al., 2009; Zarbin et al., 2010). Another important

Address for correspondence: Dr Ruy Carlos Ruver Beck, Faculdade de Farmaćia – Universidade Federal do Rio Grande do Sul, Av. Ipiranga, 2752 – Sala 404, CEP 90610-000 Porto Alegre, RS – Brazil. Tel: +55 (0)51 3308-5411. Fax: +55 (0)51 3308-5277. E-mail: [email protected]

History Received 14 July 2013 Revised 22 November 2013 Accepted 17 December 2013 Published online 1 April 2014

factor is the potential of these drug delivery systems to adhere preferentially to inflamed eyes (Fiscella, 2008). Polymeric nanocapsules are composed of oily nanodroplets surrounded by a polymeric wall. Polymers used to prepare these nanocapsules should be biocompatible and preferentially biodegradable for biomedical applications, an example being poly("-caprolactone) (Reús et al., 2009). Polymers which are biocompatible but are not biodegradable, such as EudragitÕ RS100, a co-polymer of poly(ethylacrylate, methyl–methacrylate), have shown good ocular tolerability (Pignatello et al., 2002a). The polymer EudragitÕ RS100 is commonly used in controlled-release preparations for drugs, since it is insoluble in physiological media and capable of swelling, thus representing an appropriate material for this purpose (Pignatello et al., 2001, 2002b). Since it is positively charged, due to the presence of quaternarium ammonium groups (Domingues et al., 2008; Evonik, 2011), particles prepared with this polymer can have a prolonged residence time on the corneal surface due to the interactions with anionic mucins present in the tear film (Dillen et al., 2006; Nagarwal et al., 2011). In this context, in this study we developed prednisolone-loaded nanocapsules to produce controlled-delivery eye drops for the treatment of ocular diseases. Prednisolone is a corticosteroid drug widely used in the treatment of various inflammatory eye diseases (Arici et al., 2000; Finamor et al., 2002; Fialho et al., 2003), in the form of eye drops (solution or suspension) or ointment. The design of the study was based on the physicochemical characterisation of the nanodevices, their in vitro drug release behaviour and in vitro ocular irritation. To the best of our knowledge, this is the first report on the encapsulation of


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J Microencapsul, Early Online: 1–10

prednisolone in nanocapsules for the treatment of ocular inflammatory diseases as well as the first study to evaluate the eye toxicity of such nanocapsules using two complimentary in vitro methods. The Draize rabbit eye test is the standard test for determining the eye toxicity of a formulation (Kishore et al., 2008) and it is currently still in use (Guo et al., 2012; Cariello et al., 2013); however, since it may cause pain and distress to the animals, alternative methods have been developed. Thus, the in vivo Draize test should be performed solely to assure the lack of toxicity of the potential formulation or substance after a combination of two or more in vitro tests have been carried out. In this study, two in vitro alternatives to the in vivo Draize test were performed to evaluate the ocular compatibility of the formulations developed.

magnetic stirring (10 min) to an aqueous solution containing the high hydrophilic-lipophilic balance surfactant (polysorbate 80). Thus, acetone was removed and the aqueous phase concentrated by evaporation under reduced pressure (Rotavapor R-114, Bu¨chi, Switerzland) to a final volume of 10 mL of the formulation. Blank formulations were prepared (n ¼ 3) by the same method, without prednisolone. The qualitative and quantitative compositions of the formulations are shown in Table 1. The formulations are referred to herein as: prednisoloneloaded nanocapsules prepared with EudragitÕ RS100 (PD-EUD-NC) and its corresponding blank formulation (B-EUD-NC); and prednisolone-loaded nanocapsules prepared with poly("-caprolactone) (PD-PCL-NC) and its corresponding blank formulation (B-PCL-NC). Polymeric film swelling/dissolution test

Materials and methods Materials Prednisolone, poly("-caprolactone) (PCL) Mw ¼ 80 000 gmol1 (glass transition temperature around 60 C) and sorbitan monostearate were purchased from Sigma-Aldrich (Saõ Paulo, Brazil). Castor oil and mineral oil were obtained from Delaware (Porto Alegre, Brazil) and Alpha-Quı´mica (Porto Alegre, Brazil), respectively. EudragitÕ RS100 (glass transition temperature around 65 C) was obtained from Degussa (Saõ Paulo, Brazil), polysorbate 80 from DEG (Saõ Paulo, Brazil) and SoflensÕ 66 contact lenses from Bausch & Lomb (Porto Alegre, Brazil). Fertilised chicken eggs were kindly donated by Avia´rio de Ensino e Pesquisa (Departamento de Zootecnia, UFRGS, Porto Alegre, Brazil). Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F12, L-glutamine, sodium bicarbonate and ([3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide]) (MTT) were obtained from Sigma Aldrich (St. Louis, MO). Foetal bovine serum (FBS), FungizoneÕ (Amphotericin B) and 0.5% trypsin/ EDTA solution were obtained from Gibco BRL (Carlsbad, CA). Gentamicin was obtained from Schering do Brazil (Rio de Janeiro, Brazil). The 96-well and 24-well culture plates were obtained from TPP (Tissue culture test plates-TPP, Trasadingen, Switzerland). All other chemicals and solvents used were of analytical or pharmaceutical grade. Preparation of nanoparticles Prednisolone-loaded nanocapsules were prepared (n ¼ 3) by interfacial deposition of the preformed polymer (Fessi et al., 1988). The organic solution consisted of the drug (prednisolone), oils (castor and mineral oil), a low hydrophilic-lipophilic balance surfactant (sorbitan monostearate, except in the formulations prepared with EudragitÕ RS100), a polymer [EudragitÕ RS100 or poly("-caprolactone)] and acetone. After the solubilisation of all components, this organic phase was added under moderate

Since the use of castor oil and mineral oil to prepare polymeric nanocapsules has not been previously reported, the possible swelling or dissolution of the polymers used in this study in the presence of these oils was evaluated. Polymeric films of EudragitÕ RS100 or poly("-caprolactone) were obtained by their complete dissolution in chloroform, followed by the evaporation of the organic solvent (Guterres et al., 2008). Fragments of these films (n ¼ 3) were accurately weighed and then immersed separately in glass containers with the oil (castor or mineral oil) and stored protected from light at room temperature. The films were withdrawn from the vials, dried with smooth paper and weighed using an analytical balance (Denver APX-200, Brazil, readability 0.1 mg, linearity ± 0.2 mg) after predetermined periods (2, 5, 15, 30 and 60 d) of contact with the oils. Characterisation of formulations Particle size distribution, mean diameter and polydispersity index (PDI) The particle size distribution was determined by laser light 2000, Malvern Instruments, diffraction (MastersizerÕ Worcestershire, England). The mean diameter over the volume distribution (d4.3) was used as a particle size distribution parameter and the span values were considered as a measure of the width of the distribution. The measurements were performed in distilled water with no previous treatment of the samples. For a more specific determination of the particle size, and also to determine the PDI, the formulations were analysed by photon correlation spectroscopy (PCS) (ZetasizerÕ Nanoseries ZEN3600, Malvern Instruments, Worcestershire, England) after adequate dilution (1:500, v/v) of an aliquot of the suspension in filtered water (0.45 mm, Millipore, Bedford, MA). The formulations were also analysed by nanoparticle tracking analysis based on laserilluminated optical microscopy (NanosightÕ LM 10, Amesbury, England), a method which considers individual particles and, for

Table 1. Qualitative and quantitative composition of nanocapsule formulations (n ¼ 3). Organic phase* Formulation

Prednisolone (g)

Castor oil (ml)

Mineral oil (ml)

PD-EUD-NC B-EUD-NC PD-PCL-NC B-PCL-NC

0.005 – 0.005 –

0.165 0.165 0.165 0.165

0.165 0.165 0.165 0.165

Notes: PD, prednisolone; NC, nanocapsules; B is blank formulation. *acetone (27 ml). ypurified water (53 ml). zEudragitÕ RS100. ôpoly("-caprolactone). ‘‘–’’ means the absence of such component.

Aqueous phasey Polymer (g) 0.1, 0.1, 0.1, 0.1,

EUDz EUDz PCLô PCLô

Sorbitan monostearate (g)

Polysorbate 80 (g)

– – 0.0385 0.0385

0.077 0.077 0.077 0.077

DOI: 10.3109/02652048.2013.879930

Prednisolone-loaded nanocapsules as ocular drug delivery system

this reason, may provide a higher resolution when multimodal samples are used. The samples were diluted with purified water (1:5000, v/v) before analysis (Le et al., 2008).

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hydroalcoholic solution (7:3, v/v) at the same prednisolone concentration (0.5 mgmL1). Determination of the presence of drug crystals

Zeta potential The zeta potential (z-potential) (ZetasizerÕ Nanoseries ZEN3600, Malvern Instruments, Worcestershire, England) measurements were performed at 25 C according to the electrophoretic mobility principle after diluting (1:500, v/v) the samples with a filtered (0.45 mm, Millipore, Bedford, MA) 10 mM NaCl aqueous solution.


pH measurements The pH values were determined in the undiluted suspensions using a calibrated potentiometer (Denver, UB-10, Santo Andre´, Brazil). Prednisolone content and encapsulation efficiency

The encapsulation of hydrophobic drugs in nanocapsules can lead to the simultaneous formation of drug nanocrystals (Pohlmann et al., 2008). Since the ultrafiltration–centrifugation technique is unable to segregate nanocapsules from nanocrystals, it is essential to determine whether there are drug crystals in the formulation. Equal volumes of each prednisolone-loaded nanocapsule suspension (PD-EUD-NC or PD-PCL-NC) were separated into two different flasks (A and B), kept at room temperature (25 ± 2 C) and protected from light. The samples from flask A were not shaken before HPLC–UV analysis, and the aliquots were taken from the top, while the samples from flasks B were shaken before sampling for HPLC–UV analysis. The drug contents of formulations were analysed as a function of time during 15 d of storage. Rheological profile

The concentration of prednisolone and its encapsulation efficiency were determined by high performance liquid chromatography with ultraviolet detection (HPLC–UV). The HPLC system consisted of a Perkin Elmer Series 200 (S-200) pump, UV-VIS detector, autosampler and vacuum degasser. A C18 Gemini reverse ˚ pore phase column (150 mm 4.6 mm, 5 mm particle size, 110 A diameter) (Phenomenex, Torrance, CA) was used. The mobile phase consisted of acetonitrile–water (40:60, v/v) with an isocratic flow rate of 1.0 mLmin1. The volume injected was 20 mL. Prednisolone was detected at 241 nm with a retention time of 3.2 min. Before the injections, samples were filtered through a 0.45 mm membrane (Millipore, Bedford, MA). The method was validated according to the following characteristics: linearity (y ¼ 43960 x 30657, n ¼ 3, r ¼ 0.9999, range between 5 and 40.0 mgmL1), precision (repeatability: RSD 1.08%; intermediate precision: RSD 1.75%) and specificity (blank formulations), which demonstrated that the nanocapsule adjuvants did not alter the prednisolone assay. Data analysis by ANOVA showed a linear regression with no deviation from linearity. The limits of detection (LD) and quantification (LQ) of the method were calculated using the following Equations (1) and (2), respectively:

The rheological profiles for the formulations were obtained using a rotary viscosimeter (BrookfieldÕ LV-DV-II + Pro, Middleboro, MA) with an ultra low adapter (ULA). The results express the viscosity as a function of the shear rate. Morphology The morphology of the particles for all formulations was analysed by transmission electron microscopy – TEM (Jeol, JEM 1200ExII, Tokyo, Japan). The samples were diluted in ultrapure water (1:10, v/v) and placed on grids (400 mesh). Uranyl acetate (2%) was used as the contrast substance. These grids were kept in desiccators for 24 h before analysis by TEM, operating at a voltage of 80 kV. Analyses were carried out at the university Electron Microscopy Center (Centro de Microscopia Eletroˆnica, UFRGS, Brazil). Preliminary stability studies

LD ¼ 3:3 S1

ð1Þ

The tendency toward the physical instability of the formulations was studied by multiple light scattering by means of evaluating the coalescence, creaming or flocculation (Turbiscan LAbÕ , Formulaction, L’Union, France). The analysis was performed with undiluted samples for 1 h (one scan every 5 min) at 25 C.

LQ ¼ 10 S1

ð2Þ

In vitro drug release study

where is the standard deviation of the response (peak areas) and S is the slope of the calibration curve (ICH, 2005). The LD and LQ were 0.622 and 1.884 mgmL1, respectively. The drug contents immediately after preparation and after 30 d of storage at room temperature were determined by diluting the formulations (0.5 mgmL1) with acetonitrile to a concentration of 20 mgmL1. The resulting solutions were filtered through a 0.45 mm membrane (Millipore, Bedford, MA) before injection in the HPLC–UV system. Prednisolone entrapped in the nanocapsules was determined by means of an ultrafiltrationcentrifugation method (MicroconÕ 100 00 Da, Millipore, Bedford, MA), which allows the separation of the nonencapsulated drug, which in turn was able to pass through the filter during centrifugation (Eppendorf 5417 R, Hamburg, Germany) (10 min, 2150 g). The encapsulation efficiency was calculated through the difference between the total drug content in the formulation and in the non-encapsulated drug. To assure that the drug does not interact with the ultrafilter membrane, which could lead to a misinterpretation of the results, the same ultrafiltration–centrifugation protocol was carried out with a

The profiles for the prednisolone release from the nanocapsules (n ¼ 3) were obtained by means of the direct dialysis method. A control representing the non-encapsulated drug was prepared as a hydroalcoholic solution (ethanol:water, 3:7, v/v) at the same prednisolone concentration (0.5 mgmL1). Each dialysis bag containing 1 mL of formulation was placed into a closed glass flask containing 200 mL of the release medium (ethanol:water, 10:90, v/v) under mixing in a water bath (37 ± 1 C). The prednisolone saturation in the release medium was evaluated to ensure the maintenance of sink conditions. After predetermined time intervals, 1 mL samples were withdrawn from the flasks and replaced with fresh medium. The samples were analysed by HPLC–UV and then the cumulative drug released was calculated. The HPLC–UV method had been previously validated (Katzer et al., 2013). However, the injection volume was increased from 20 mL to 100 mL and the range was narrowed down to allow the detection of smaller amounts of prednisolone. Good linearity (y ¼ 213 365 x + 8157, n ¼ 3, r ¼ 1, range 0.5–5.0 mgmL1) and precision (repeatability: RSD 1.39%; intermediate precision: RSD 1.38%) were observed. Data analysis by ANOVA showed a linear

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regression with no deviation from linearity. The limits of detection and quantification were 0.102 and 0.311 mgmL1, respectively. Mathematical monoexponential [Equation (3)] and biexponential [Equation (4)] models (ScientistÕ software, MicroMathÕ , St. Louis, MO) were used to better understand the influence of the nanostructure type on the prednisolone release behaviour, C ¼ C0 ekt

ð3Þ

C ¼ A ek1t þ B ek2t

ð4Þ

where C (dimensionless) is the amount of prednisolone released at time t (h), C0 (dimensionless) is the initial total drug content (in percentage terms) and k, k1 and k2 (h1) are the observed kinetics rate constants. A and B represent the concentrations of prednisolone associated with the burst and sustained release phases, respectively. The fits were evaluated considering the best graphic adjustment, the correlation between experimental points and theoretical profiles and the highest value for the model selection criteria (MSC) (Contri et al., 2010). In vitro study of ocular irritation The eye irritation potential of prednisolone-loaded and unloaded nanocapsules was examined by means of the hen’s egg test (HET), which uses the chorioallantoic membrane (CAM), a foetal vascularised respiratory tissue, to mimic the conjunctiva as an in vitro alternative to the in vivo Draize test. Fertilised chicken (Cobb 500) eggs on the tenth day of incubation (37.5 C, 60% relative humidity) were used. The assay is based on the observation of irritation (vasoconstriction, haemorrhage and coagulation), which results in a scale that considers the observed phenomena (Spielmann et al., 1993; Luepke, 1995). Egg shells were opened at the air cell, the white membrane was removed and 300 mL of the formulation was applied to the CAM (n ¼ 6). Considering the opacity of nanocapsule suspensions, the CAM was rinsed with physiological solution 20 s after the application and the time (in seconds) of the first occurrence of irritant endpoints was monitored for 300 s. Positive controls (0.1 M NaOH and 0.1% sodium lauryl sulfate – SLS, n ¼ 3) and a negative control (0.9% NaCl, n ¼ 3) were performed. The irritation score (IS) was determined according to Equation (5): IS ¼

5 ð301 haemorrhage timeÞ 300 7 ð301 vasoconstriction timeÞ þ 300 9 ð301 coagulation timeÞ : þ 300

ð5Þ

The lesions were classified by means of IS, as non-irritant (0–0.9), slightly irritant (1–4.9), moderately irritant (5–8.9) and extremely irritant (9–21) (Luepke, 1995). Assessment of in vitro cytotoxicity Maintenance of cell line The rabbit corneal epithelial cell line (SIRC) was donated by Fundaçaõ Oswaldo Cruz (FIOCRUZ, Departamento de Imunologia – Laborato´rio de Vacinas Virais e Cultura de Ce´lulas, Rio de Janeiro, Brazil). SIRC was cultured as previously described (Mediero et al., 2010). Cells at 415–430 passages were grown and maintained in Dulbecco’s modified Eagle medium/ Ham’s nutrient mixture F12 (DMEM HAM F12), containing

3 mM L-glutamine, 13.8 mM NaHCO3, 0.1% FungizoneÕ and 0.1 mgmL1 gentamicin, supplemented with 10% (v/v) FBS. Cells were kept at 37 C, with a relative humidity of 95%, and an atmosphere of 5% CO2 in air. SIRC viability determined by MTT (3-[4,5-dimethylthiazol-2-yl]2,5 diphenyl tetrazolium bromide) assay SIRC cells were seeded in a 96-well plate (1 103 cells per well). After reaching semi-confluence, the cultures were treated with prednisolone hydroalcoholic solution (PD-solution) or prednisolone-loaded nanocapsules (PD-EUD-NC or PD-PCL-NC) at different concentrations of prednisolone (0.06, 0.12, 0.25, 0.31 and 0.37 mM) for 48 or 72 h. The intermediate dose (0.25 mM) was based on the recommended dosage of commercially available eye drops (10 mgmL1 of prednisolone acetate, 402.5 g/mol) (Pred FortÕ , Allergan, Irvine, CA), which is 1–2 drops (40–80 mL) 2–4 times daily. Assuming an application of two drops (80 mL), the dosage of prednisolone base (360.4 g/mol) is 0.72 mg. However, only 12.5% (90 mg, i.e. 0.25 mM) of this amount will actually be present in the pre-corneal area within 1–2 min (Chrai et al., 1973) considering that the capacity of the corneal surface is 10 mL (Reddy and Ganesan, 1999; Ansel et al., 2011). Additionally, according to Tomlinson and Khanal (2005), as cited by Alvarez-Lorenzo and co-workers (2006), the normal human tear turnover, 10–20% per min, facilitates the removal of the remaining formulation from the pre-corneal area. Two higher and lower concentrations were selected to construct a curve. The control cultures (untreated cells) were maintained with culture medium only. After 48 h or 72 h of treatment, each culture medium containing the formulation was removed and the cells were washed twice with 100 mL of pH 7.4 phosphate buffered saline (PBS). After removing the PBS, 90 mL of culture medium and 10 mL of MTT were added to each of the wells, to assess the cell viability. Incubation was maintained for 3 h. After, the solution was removed from the precipitate. In order to solubilise the formazan precipitate, 100 mL of DMSO was added to the wells. Absorbance levels were read using an ELISA plate reader (Zenty Microplate reader, Anthos, Cambridge, UK) at 490 nm. A linear regression was achieved between the plotting absorbance as a function of the number of live cells with active mitochondria. Cell viability was calculated using the following Equation (6): ðAbss =Abscontrol Þ 100

ð6Þ

where Abss is the absorbance of cells treated with different formulations and Abscontrol is the absorbance of control cells. Cytotoxicity was expressed as the percentage of live cells relative to the control against the concentration. All experiments were repeated four times in duplicate. Trypan blue exclusion method of SIRC cell viability SIRC cells were seeded at 5 103 cell per well in the same medium described above in 24-well plates and allowed to grow until reaching semi-confluence. The SIRC cells were then treated (n ¼ 3) with the intermediate concentration (0.25 mM) of each formulation for 48 or 72 h. Control cultures were maintained with culture medium only. At the end of the treatment, the medium was removed. Cells were washed with pH 7.4 PBS and 100 mL of 0.25% trypsin/EDTA solution was added to detach the cells; 200 mL of DMEM/HAM F12 was added to neutralise the medium. The cells were then incubated with 0.4% (v/v) trypan blue solution and counted immediately in a haemocytometer. Cytotoxicity was expressed as the percentage of live cells relative to the control.


DOI: 10.3109/02652048.2013.879930

Statistical analysis Data are expressed as mean ± standard deviation. Statistical analysis was carried out by one-way analysis of variance (ANOVA) followed by post-hoc analysis for multiple comparisons (Tukey test) using BioEstat 5.0. Differences were considered significant when p50.05.

Results


Swelling/dissolution test The weight of poly("-caprolactone) films immersed in both oils increased slightly (56%) over 60 d (Figure 1). Similar findings have been reported in an evaluation of the interaction of poly("-caprolactone) with tea tree oil (Flores et al., 2011). EudragitÕ RS100 films had a mass loss of 510%, in both oils, after 2 d, which remained constant within the period of the experiment. Physicochemical characterisation of nanocapsules Regardless of the technique used to evaluate the particle size distribution, all nanocapsule formulations showed a unimodal particle population within the nanoscale, with a narrow size distribution, represented by span values of 5 2 and PDI50.20 (Table 2). Mean particle sizes were between 134 and 274 nm, which is often observed for the method of interfacial deposition of a preformed polymer (Schaffazick et al., 2003; Poletto et al., 2011). Morphological analysis by TEM showed spherical particles (Figure 2) whose diameters were in agreement with those determined by other techniques. The values for the other physicochemical properties of the formulations are reported in Table 3. The pH values showed that

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the formulations were slightly acidic. The statistical analysis of the results (mean diameter, PDI, pH and zeta potential) did not show any influence of the presence of prednisolone, as the results were similar for loaded and unloaded formulations. The drug contents of prednisolone-loaded formulations were close to the theoretical value (0.5 mgmL1). In the retention test, the total amount of prednisolone added to an ultrafilter device was recovered in the ultrafiltrate after centrifugation, indicating that there was no interaction with the membrane. Hence, the encapsulation efficiency of nanocapsules prepared with poly("-caprolactone) or EudragitÕ RS100 were 51.2 ± 2.9% and 45.9 ± 2.7%, respectively, without significant statistical differences. However, in the presence of drug nanocrystals, nanocapsule formulations can present a decrease in the total drug content under storage due to the drug precipitation/sedimentation after the agglomeration of nanocrystals (Guterres et al., 1995). Furthermore, erratic results for the encapsulation efficiency may be obtained. The drug content values remained constant after 15 d of storage for both immobile and shaken samples during the evaluation of the concomitant presence of drug nanocrystals. Regarding the rheological profile, the formulations presented low viscosity (around 1.2 mPa.s1) and a Newtonian behaviour (data not shown), which is often observed for these systems (Hussein et al., 2009; Shakeel et al., 2009). Predictive physical stability studies To predict the physical stability of the dispersed system, formulations were analysed by multiple light scattering. As the formulations showed transmittance below 0.02%, the backscattering profile was studied. Relative backscattering as a function of time (Delta backscattering) presented variations of 55% at the top of the cuvette, indicating that the tendency toward creaming of the largest nanocapsules is not expressive. The central part of the graph shows a very low tendency toward aggregation or flocculation within 1 h of analysis, since the backscattering variation was 5 1% (data not shown). The presence of prednisolone did not influence the physical stability of these suspensions. In vitro drug release

Figure 1. Swelling/dissolution experiment: polymeric (EudragitÕ RS100 or poly["-caprolactone]) film weight after different times of contact with castor or mineral oil (n ¼ 3).

The profiles for the in vitro prednisolone release from nanocapsules were obtained as a percentage of the drug released as a function of time (Figure 3). Prednisolone showed a fast diffusion through the dialysis bag from the control (hydroalcoholic solution), showing 99.91 ± 0.68% of drug in the release medium after 2 h, while within the same time the values for the PD-PCL-NC and PD-EUD-NC release were 83.16 ± 0.54% and 80.8 ± 3.6%, respectively. The total release of prednisolone (approximately 100%) from the nanocapsule formulations was reached only after 5 h for both PD-PCL-NC (99.88 ± 0.91%) and PD-EUD-NC (101.5 ± 3.2%).

Table 2. Particle size distribution, mean particle size and width of distribution (Span and PDI values) of the formulations by different techniques: laser diffraction, nanoparticle tracking analysis (NTA) and photon correlation spectroscopy (PCS) (n ¼ 3). Laser diffraction* Formulation

D[4,3] (nm)

Span

NTA Mean size (nm)y


149 ± 5 137 ± 3 234 ± 16 240 ± 34

1.33 ± 0.09 1.24 ± 0.08 1.77 ± 0.09 1.85 ± 0.08

176 210 210 213

PCS Mean size (nm)

PDI

173 ± 6 165 ± 6 216 ± 11 224 ± 9

0.17 ± 0.02 0.11 ± 0.02 0.12 ± 0.02 0.14 ± 0.02

Notes: Mean ± standard deviation. *Values obtained by volume distribution measurement; refractive index: 1.38 and 1.59 for EudragitÕ RS 100 and poly("caprolactone) nanocapsules, respectively. yn ¼ 1.

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Figure 2. Transmission electron microscopy images of (A) B-EUD-NC, (B) PD-EUD-NC, (C) B-PCL-NC and (D) PD-PCL-NC (bar ¼ 200 nm [100 000 ]).

Table 3. Results of the physicochemical parameters of the formulations: pH, zeta potential, drug content and encapsulation efficiency (n ¼ 3). Formulation

pH

z-potential (mV)

Drug content (mgmL1)

Encapsulation efficiency (%)


5.04 ± 0.13 5.16 ± 0.25 5.60 ± 0.16 6.16 ± 0.32

+ 10.3 ± 1.3 + 9.16 ± 0.45 5.76 ± 0.23 3.83 ± 0.34

0.500 ± 0.007 – 0.485 ± 0.018 –

51.2 ± 2.9 – 45.9 ± 2.7 –

Figure 3. Profiles for in vitro release of prednisolone from PD-EUD-NC and PD-PCL-NC as well as the diffusion profile for the prednisolone ethanolic solution (control). The lines correspond to the fitting to the biexponential (PD-EUD-NC and PD-PCL-NC) or monoexponential (ethanolic solution) models.

The results for the drug release (%) were statistically analysed. At all points the amount of drug released from the nanoformulations (NC) differed in relation to the control (hydroalcoholic solution). No differences were found between the amounts of prednisolone released (%) from PD-PCL-NC and PD-EUD-NC for any of the time periods considered (p40.05). To elucidate the influence of the nanoencapsulation on the prednisolone release behavior, mathematical modeling was carried out. The kinetic rate constants (k), correlation coefficients (r) and MSC are shown in Table 4. The model that best described the release profile for the nanocapsule formulations was the biexponential model. This profile characterises a biphasic release, i.e. a burst release (k1) at an early stage followed by a sustained phase (k2). Rate constants for the burst phase (k1) were 2.73 ± 0.26 h1 (PD-PCL-NC) and 2.7 ± 1.0 h1 (PD-EUD-NC) and for the sustained phase the rate constants (k2) were 0.709 ± 0.081 (PD-PCL-NC) and 0.49 ± 0.20 h1 (PD-EUDNC). For the burst phase, the rate constant for PD-PCL-NC was not significantly different to that for PD-EUD-NC (p40.05), while in the controlled phase the rate of prednisolone release from PD-EUD-NC was lower (p 0.05). The percentages of prednisolone related to the burst phase (coefficient A, Equation (4)) were


DOI: 10.3109/02652048.2013.879930

Table 4. Observed rate constants k, coefficients A and B, correlation coefficients (r) and model selection criteria (MSC) obtained by fitting prednisolone release profiles from formulations to monoexponential and biexponencial equations.


Parameters

Ethanolic solution

PD-PCL-NC

PD-EUD-NC

1.40 ± 0.11 0.994 ± 0.004 3.87 ± 0.47

1.04 ± 0.04 0.996 ± 0.001 4.46 ± 0.29

0.96 ± 0.06 0.992 ± 0.006 3.70 ± 0.74

– – – – – –

2.73 ± 0.26 0.71 ± 0.08 0.467 ± 0.070 0.534 ± 0.069 0.999 ± 0.001 5.14 ± 0.32

2.7 ± 1.00 0.49 ± 0.20 0.346 ± 0.067 0.655 ± 0.067 0.998 ± 0.001 4.49 ± 0.68

Monoexponential k (h1) r MSC Biexponential k1 (h1) k2 (h1) A B r MSC

46.7 ± 7.0% and 34.6 ± 6.7%, while the percentages related to the sustained phase (coefficient B, Equation (4)) were 53.4 ± 6.9% and 65.5 ± 6.7% for PD-PCL-NC and PD-EUD-NC, respectively. In vitro study of ocular irritation (CAM assay) The CAM bioassay was used as a tool for this evaluation. The following were applied directly to the egg’s chorioallantoic membrane: 0.3 mL of the positive (0.1 N NaOH and 1% SLS) and negative (physiological solution) controls, and the prednisoloneloaded and the blank nanocapsules. Positive controls were classified as extremely irritant (1% SLS IS ¼ 11.6 ± 1.6 and 0.1 N NaOH IS ¼ 12.95 ± 0.73), which validated our experiment. On the other hand, the negative control and all of the formulations (independently of drug presence and type of polymer) did not show any of the possible reactions (haemorrhage, coagulation and vasoconstriction). Cytotoxicity assays Analysis of the MTT results showed that, after 48 or 72 h of treatment, except for the higher concentration, nanocapsules did not cause a significant decrease in cell viability, when compared to control cells. A slight decrease (510%) in cell viability after 48 h of treatment with B-PCL-NC and PD-EUD-NC at the highest concentration was observed. After 72 h of treatment, except for the hydroalcoholic solution, all formulations (prednisolone-loaded and unloaded) caused a slight decrease in cell viability (510%). Regarding the trypan blue exclusion assay, no significant differences were found between the cell viability values for treated and control cells, after 48 and 72 h of treatment (Figure 4).

Discussion At the beginning of this study, the swelling and dissolution of the polymers (PCL and EUD) in the presence of castor or mineral oil was evaluated, as the interaction between these components had not been previously reported. Since no significant changes in the weight of the polymeric slices were observed after contact with the oils the feasibility of preparing the nanocapsules with castor and mineral oil without the risk of affecting the integrity of the polymeric wall was veified. Afterwards, three batches of poly("-caprolactone) or EudragitÕ RS100 nanocapsules containing both oils were prepared and characterised. Three different techniques were used to evaluate the particle size distribution and mean size of the nanocapsule suspensions. All results confirmed their unimodal particle size distribution in the nanoscale range, with mean particle sizes of between 100 and 300 nm.

7

As expected, the zeta potential was positive for EudragitÕ RS100 nanocapsules due to the cationic character of the polymer, which has a low content of quaternary ammonium groups (Evonik, 2011), as previously reported (Dillen et al., 2006; Domingues et al., 2008). This cationic charge may contribute to a higher retention time in the precorneal area after topical administration due to mucoadhesion. Poly("-caprolactone) nanocapsules had slightly negative zeta potential values, probably as a consequence of the particle coating with polysorbate 80, which presents a negative surface charge due to the presence of oxygen atoms in its molecules (Fontana et al., 2009). In addition, for poly("-caprolactone) nanocapsules these values can be explained by the presence of ester groups in the polymer structure (Mu¨ller et al., 2001). This slightly negative zeta potential at the surface of the PCL-NC, as well as nanoparticles prepared with other nonionic or anionic polymers, such as poly(D,L-lactide-co-glycolide) (PLGA), does not make these particles mucoadhesive in nature, but due to their small size they can be retained on the eye (Gupta et al., 2010). The encapsulation efficiency of prednisolone of around 50% can be explained by the low partition coefficient (log P ¼ 1.6) of prednisolone. A previous report demonstrated similar efficiencies for melatonin encapsulation (log P ¼ 1.62) in poly("-caprolactone) or EudragitÕ RS100 nanocapsules, using caprylic/capric triglyceride as the oily core (Schaffazick et al., 2006). On the other hand, Kshirsagar and coworkers (2012) produced EudragitÕ S100 nanocapsules in which the encapsulation efficiency of prednisolone was close to 90%. The simultaneous presence of nanocrystals in the suspensions was discarded by the assaying of the drug in the formulations (immobile and shaken samples) as a function of time. The control in this respect is very important, since the presence of these nanocrystals could contribute negatively to the physical stability of these systems, as the sedimentation of nanocrystals can lead to a lack of homogeneity (Pohlmann et al., 2008). Furthermore, the predictive physical stability of the suspensions, characterised as Newtonian fluids (in the rheological analysis) was also confirmed by the low variation in the backscattering at the top and bottom of the cuvette. Multiple light scattering is currently used to study the physical stability of colloidal systems, nanoparticle suspensions and emulsions. Its main advantage is the ability to detect instability phenomena like coalescence, flocculation, creaming or sedimentation much faster than observation with the naked eye (Lemarchand et al., 2003). From the physicochemical point of view, these particle size, drug content and rheology results suggest the suitability of these suspensions as ocular drug delivery systems. Regarding the in vitro drug release studies, although the amount of prednisolone encapsulated was around 50%, the amount of drug released from the nanocapsules differed from the control (hydroalcoholic solution), showing their ability to prolong the drug release time. The slower release from nanocapsules can be explained by the presence of the polymeric wall around the oily nanodroplets. This property, along with the property of a longer retention time in the eye, makes these formulations an innovative alternative for the treatment of inflammatory ocular diseases (Nagarwal et al., 2009; Zarbin et al., 2010). Furthermore, these release profiles fit the biexponential release model, regardless of the type of polymeric material. The percentage of drug released in the burst phase can be explained by the amount of free drug (around 50%) present in these formulations, assessed using the ultrafiltration– centrifugation technique, as previously discussed. Recently, Ibrahim and coworkers (2013) published a study on the use of nanoparticle-based topical ophthalmic formulations (eye drops, in situ gelling system and gel) for the sustained release of an


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Figure 4. Cell viability of rabbit cornea cell line (SIRC) after 48 h (top graph) and 72 h (bottom graph) treatment with formulations by MTT assay; control: SIRC not exposed to nanoparticles. Data represent the means ± standard deviation of four experiments carried out in duplicate. *Significantly different from the control group (p50.05).

anti-inflammatory drug (celecoxib). They also suggested that the high encapsulation efficiency positively affects the release profile. Gupta and coworkers (2010) developed sparfloxacin-loaded PLGA nanoparticles for ocular delivery. The particle size was similar to those reported in the present study (180–190 nm), with negative zeta potential, and the nanoparticles were evaluated in terms of retention on the precorneal area of male New Zealand albino rabbits after topical instillation. A marketed drug formulation used for comparison was cleared very rapidly from the corneal region and reached the systemic circulation through the nasolacrimal drainage system, as significant radioactivity was recorded in the kidney and bladder after 6 h of ocular administration. On the other hand, the nanosuspension prepared cleared at a very slow rate and remained on the corneal surface for a longer duration, as no radioactivity was observed in the systemic circulation, demonstrating that nanoparticles per se can improve the precorneal residence time and possibly ocular penetration. In order to propose the developed formulations as suitable ophthalmic drug release systems, it was necessary to evaluate their potential irritation to the eyes. According to the data from CAM assay, the formulations were classified as non-irritant. It is important to mention that this test does not replace the in vivo Draize test. On the other hand, HET-CAM has shown potential

to refine and reduce animal use in eye irritation testing (Barile, 2010). In order to confirm this result and to further assess the suitability of these formulations for ocular use, other alternative in vitro tests were also carried out. To investigate whether or not prednisolone-loaded and blank nanocapsules (data not shown) affect the viability of SIRC cells, we used MTT and trypan blue exclusion assays, which measure mitochondrial activity and cell membrane integrity (Strober, 2001), respectively, as indicators of cell viability. Cultured SIRC cells were treated with different concentrations (0.06, 0.12, 0.25, 0.31 and 0.37 mM) of prednisolone for the MTT assay and with 0.25 mM of prednisolone for the trypan blue assay, for 48 or 72 h. The percentage of cell viability was determined by comparison with cells which were not exposed to nanocapsules, i.e. the control group. The results showed no significant loss of viability after the treatment with the formulations developed, although a slight decrease was observed in cell viability at the highest dose of prednisolone used in the MTT assay. It should be noted that, due to a lack of studies in the literature on the evaluation of the in vitro cytotoxicity of corticosteroids such as prednisolone, we stipulated the doses based on the recommend dosage of a commercially available prednisolone eye drop formulation. Hence, a plausible explanation for this slight decrease is that the volume of formulation added


DOI: 10.3109/02652048.2013.879930


9

Figure 5. Cell viability of rabbit cornea cell line (SIRC) after 48 h (left bar) and 72 h (right bar) of treatment with the formulations (0.25 mM of prednisolone) by trypan blue assay; control: SIRC not exposed to nanoparticles; B-solution: hydroalcoholic solution (7:3, v/v) without prednisolone. Data represent the means ± standard deviation of 4 experiments carried out in duplicate.

to the well, given the content of prednisolone in the formulations, was high (around 25% of the well capacity) and probably affected the osmolarity of the culture medium. Thus, under the experimental conditions of this study, our results indicate that these nanocapsules are not cytotoxic to the SIRC cell line.

Conclusions This article reports for the first time the encapsulation of prednisolone in nanocapsules for the treatment of ocular inflammatory disorders, as innovative eye drops, presenting low viscosity and particles exclusively on the nanoscale (130– 260 nm). These formulations were able to control the prednisolone release, regardless of the type of polymeric wall. Their potential use as suitable ocular nanodevices was demonstrated by the lack of ocular irritancy or ocular cytotoxicity.

Declaration of interests The authors thank FAPERGS, CNPq, INCT_if, PRONEX-FAPERGS/ CNPq and Rede Nanocosme´ticos/CNPq for the financial support. T. Katzer is grateful for the scholarship from CPNq/Brazil. The authors have no declarations of conflicting interests to report.

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Notice of Correction: The version of this article published online ahead of print on 3rd April 2014 contained an error on page 2. The sentence ‘‘. . . and it is currently still in use (Gratieri et al., 2011; Rodriguez-Aller et al., 2012); however, since it may cause pain and distress to the animals, alternative methods have been developed.’’ should have read ‘‘. . . and it is currently still in use (Guo et al., 2012; Cariello et al., 2013); however, since it may cause pain and distress to the animals, alternative methods have been developed.’’. The references have been updated and the error has been corrected for this version.

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