Journal of Controlled Release xxx (2011) xxxxxx

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Journal of Controlled Release

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / j c o n r e l

Development and characterization of Cyclosporine A loaded nanoparticles for ocular drug delivery: Cellular toxicity, uptake, and kinetic studies
Pelin Aksungur a,, Murat Demirbilek b, Emir B. Denkba b, Jo Vandervoort c, Annick Ludwig c, Nuren nl a
a b c

Faculty of Pharmacy, Department of Pharmaceutical Technology, Hacettepe University, 06100 Ankara, Turkey Nanotechnology Nanomedicine Division, Chemistry Department - Biochemistry Division, Beytepe, Hacettepe University, 06800 Ankara, Turkey Laboratory of Pharmaceutical Technology and Biopharmacy, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, 2610 Antwerp, Belgium

a r t i c l e

i n f o

a b s t r a c t
Dry eye syndrome is a common disorder of the tear lm caused by decreased tear production or increased evaporation. The objective of this study was to evaluate the potential effectiveness of Cyclosporine A (CsA) nanoparticles (NPs) for the treatment of inammation of the eye surface. Topical CsA is currently the only and safe pharmacologic treatment of severe dry eye symptoms. The NPs were prepared using either poly-lactideco-glycolide (PLGA) or a mixture of PLGA with EudragitRL or were coated with Carbopol. The mean size of CsA loaded NPs was within the range from 148 to 219 nm, except for the Carbopol coated NPs (393 nm). The drug entrapment efciency was very high (from 83 to 95%) and production yield was found between 75 and 92% in all preparations. The zeta potential of the Eudragit RL containing NPs was positive (1925 mV). The NPs formulations exhibited a biphasic drug release with initial burst followed by a very slow drug release and total cumulative release within 24 h ranged from 75 to 90%. Kinetically, the release proles of CsA from NPs appeared to t best with the Weibull model. The viability of L929 cells was decreased by increasing the concentration of the various NPs examined as well as the incubation time. The amount of NPs uptake was related to the polymer type used. The highest degree of cellular uptake (52.2%), tear lm concentration of the drug (366.3 ng/g) and AUC0 24 (972.6 ng h/g) value were obtained from PLGA: Eudragit RL (75:25)-CsA NPs formulations. The change of surface characteristics of NPs represents a useful approach for improvement of ocular retention and drug availability. 2011 Elsevier B.V. All rights reserved.

Article history: Received 29 September 2010 Accepted 9 January 2011 Available online xxxx Keywords: Nanoparticles Cyclosporin A PLGA EudragitRL Carbopol Dry eye

1. Introduction Keratoconjunctivitis sicca (KCS) is a common disease characterized by unstable tear lm associated with abnormality of the lipid, protein, and mucin proles. Changes in tear composition also promote inammation on the ocular surface. For untreated patients, the risk of ocular infection increases at considerable level and clinical course of the disease may proceed up to infection, corneal ulcer and blindness [13]. CsA, which has a selective immunosuppressive effect, is used for several autoimmune and contingent eye diseases because it specically inhibits the T-cell-dependent immune reactions at high rates [46]. In order to overcome the poor solubility of CsA in water, preparations in different oils (castor, olive, and peanut, etc.) or emulsions were developed, but side-effects such as itching, redness, epithelial keratitis, burning, and toxic effects on the cornea has limited their use [7,8]. The increase of both the residence time of the drug in the precorneal area or cul-de-sac and the absorption would improve

Corresponding author. E-mail address: pelinaks@yahoo.com (P. Aksungur). 0168-3659/$ see front matter 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2011.01.010

its therapeutic effect. One of the signicant efforts towards this aim has been the use of colloidal drug delivery systems such as liposomes, micro- or nanoparticles. Positively charged liposomes increased the corneal penetration of drugs when compared to neutral or negatively charged counterparts [9,10], but the potential of liposomes as a topical delivery system remains limited. Nanoparticle systems are able to encapsulate and protect the drug, improve tolerance, penetration efciency and increase corneal uptake [7]. Calvo et al. prepared poly-caprolactone nanocapsules in order to improve the ocular penetration of CsA [11]. After topical administration, these nanocapsules were taken up by corneal epithelial cells and achieved corneal levels of CsA that were ve times higher than a CsA oily solution, but this formulation failed to prolong corneal graft survival in an experimental rat model [12]. In rabbits, topical administration of chitosan NPs achieved and maintained high CsA levels in external ocular tissues for an extended period of time [13]. Based on these considerations, various strategies have been employed to modify NPs properties, including the use of cationic or mucoadhesive polymers. We have evaluated the particle formation process of the NPs prepared from PLGA polymers with the benet of positively charged polymers, EudragitRL in different ratios, which could interact with the anionic mucins present in the mucus layer at

Please cite this article as: P. Aksungur, et al., Development and characterization of Cyclosporine A loaded nanoparticles for ocular drug delivery: Cellular toxicity, uptake, and kinetic studies, J. Control. Release (2011), doi:10.1016/j.jconrel.2011.01.010

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the surface of the eye [1316]. Also, PLGA NPs were coated with hydrophilic mucoadhesive Carbopol that possesses carboxylic acid groups available for hydrogen bonding with mucin glycoproteins [17 19]. This kind of NPs formulations offer the prospects of prolonging the residence time of NPs ensuring optimal contact between the formulation and the mucosa resulting in adequate drug concentration in external ocular tissues. The aim of present study was to load the hydrophobic peptide CsA in NPs and to investigate the new vehicles for delivering the drug to the outer ocular structures. Thus, we developed and characterized CsA NPs as an attempt for improving its corneal uptake and efciency to treat ocular surface disease. Meanwhile, the impacts of both nanoparticle concentration and exposure time on the cell viability were investigated. To facilitate further research on the feasibility of NPs, we also compared NPs with widely applied and commercially available RestasisA emulsion. 2. Materials and methods 2.1. Materials The PLGA polymer Resomer RG 503 was obtained from Boehringer Ingelheim (Ingelheim am Rhein, Germany) and EudragitRL100 from Rhm Pharma (Darmstadt, Germany). CsA was a gift from Novartis (Turkey). Poly(vinylalcohol) (PVA) (MW 30,000 70,000) was supplied by Sigma Chemicals Co. (St. Louis, USA), Dmannitol by Riedel de Han (Germany), and Carbopol 940NF by BF Goodrich Chemical Co. (Ohio, USA) Dichloromethane was purchased from Sigma (Steinheim, Germany) and acetonitrile (HPLC grade) from Merck (Darmstadt, Germany). Puried MilliQ water (Millipore Simplicity 185, USA). MTT (3-(4-5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) was obtained from Sigma (St. Louis, MO, USA). Fetal bovine serum, penicilin G and streptomycin were from Biochrom ( Germany). L-glutamine was from Biological Industries (Beit Haemek, Israel). 2.2. Preparation of CsA loaded NPs The polymeric NPs were prepared with EudragitRL, PLGA or mixtures hereof (75:25, 50:50, 25:75% w/w) (Table 1). The preparation was achieved by o/w emulsication solvent evaporation followed by lyophilization. Briey, PLGA and/or EudragitRL (50 mg), was dissolved in 3 mL dichloromethane with or without CsA (10 mg). This organic mixture was then emulsied in 10 mL of an aqueous PVA solution (1% w/v) by sonication (amplitude 20%, 60 s) using an ultrasound probe (Bandelin D-12207, electronic GmbH & Co. KG Germany) in an ice bath. This emulsion was then diluted in 40 mL of PVA stabilizer solution (0.36% w/v). The organic solvent was allowed to evaporate at room temperature under magnetic stirrer (700 rpm), for 4 h at room temperature (Variomag Telesystem 15.07, Thermo Electron Corporation, USA). The nanosuspensions were then centri-

fuged at 14,000 rpm for 30 min. Collected NPs were washed twice with ultra puried water (UPW) and resuspended in mannitol solution (5% w/v). The resulting nanosuspension was subsequently cooled down to 20 C and freeze-dried (Heto PowerDry PL 3000, Denmark). Carbopol coated NPs were prepared applying the same protocol. Carbopol (0.05%w/v) was dissolved in both 1% w/v and 0.36%w/v PVA solutions. A 1N NaOH solution was used to adjust the pH to a value of 7.4 0.1. Nile red (NR) labeled NPs were prepared for uptake studies as described above. A 500 L amount of the dye solution (0.04% w/v) was added to the polymer solution. 2.3. In vitro characterization of NPs 2.3.1. Evaluation of entrapment efciency and yield of NPs The drug content in NPs was determined directly by measuring the encapsulated CsA amount in NPs. A known weight of freeze-dried powder was dissolved in acetonitrile: water (7:3) solution and put for 1 h in an ultrasonic bath. After centrifugation for 30 min at 14,000 rpm, CsA content of the samples was determined by HPLC (Waters Instrument 2695, USA). A Phenomenex-Luna C18 (2) column (250 4.60 mm) was used. The mobile phase consisted of an 80:20 (v/v) mixture of acetonitrile: water. The detector wavelength, ow rate, and column temperature were 210 nm, 1 mL/min and 65 C respectively. The HPLC method was validated with respect to linearity, repeatability and the limit of quantitation and limit of detection. CsA entrapment efciency of the CsA (Eq. (1)) and the yield of NPs (Eq. (2)) were calculated three times as indicated below:
EE = Weight of drug determined mg = Weight of drug added mgx100

1
Yield = The total weight of obtained NPs g = drug+ polymer weight g 100

2 Nile red content in NPs was determined with Phenomenex-Luna C18 (2) column (250 4.60 mm), mobile phase, acetonitrile; ow rate, 1 mL/min; excitation and emission wavelength, 550 and 600 nm. Chromatographic analyses were performed at room temperature (25 C). 2.3.2. NPs size and zeta potential analysis NPs size was determined using Photon Correlation Spectroscopy with a Zetasizer 3000 (Malvern Instruments, Malvern, UK). The analysis was performed at a scattering angle of 90 and a temperature of 25 C. The mean particle size of each sample was determined three times and the average values were calculated. The zeta potential of the particles was determined by electrophoretic light scattering using the same instrument. NPs were suspended either in simulated lacrimal uid (SLF) or UPW. The composition of SLF was 8.3 g of NaCl, 0.084 g of CaCl2.2H2O, 1.4 g of KCl in 1 L of UPW [20]. The average values of three replicates were calculated. 2.3.3. Morphology of NPs The morphology of the CsA loaded NPs were investigated by scanning electron microscopy (SEM) with Jeol-SEM 7000S (Jeol, Japan) at an accelerating voltage of 20 kV. Prior to examination, samples were prepared on aluminum stubs and coated with gold under argon atmosphere by means of a sputter coater. 2.4. In vitro release studies The freeze-dried NPs containing 50 g CsA were suspended in vials containing 15 mL SLF and incubated in a water bath at 32 C which is

Table 1 CsA entrapment efciency and yield of NPs prepared different polymer types (mean S.D., n = 3). Polymer PLGA PLGA and Carbopol PLGA, Eudragit RL (75:25) PLGA, Eudragit RL (50:50) PLGA, Eudragit RL (25:75) EudragitRL PLGA, NR PLGA, Eudragit RL (75:25), NR PLGA, Carbopol, NR Formulation code P-CsA P:C-CsA P:E (75:25)-CsA P:E (50:50)-CsA P:E (25:75)-CsA E-CsA NR-P-CsA NR-P:E (75:25)-CsA NL-P:C-CsA % CsA 93.4 2.4 83.0 2.2 89.4 1.3 86.5 1.7 91.3 0.9 85.5 0.5 94.9 1.7 88.2 0.8 84.5 2.3 % Yield 90.5 2.5 77.1 1.3 91.7 2.1 89.2 0.9 90.0 2.0 91.6 0.5 89.5 1.9 92.2 2.0 74.8 0.8

Please cite this article as: P. Aksungur, et al., Development and characterization of Cyclosporine A loaded nanoparticles for ocular drug delivery: Cellular toxicity, uptake, and kinetic studies, J. Control. Release (2011), doi:10.1016/j.jconrel.2011.01.010

P. Aksungur et al. / Journal of Controlled Release xxx (2011) xxxxxx

the temperature of the eye surface, using continuous magnetic stirrer at 200 rpm. At given time intervals 1 mL samples were withdrawn, centrifuged for 30 min at 14,000 rpm and the amount of CsA in the samples was determined by HPLC method as described before. Various mathematical equations were applied to dene the kinetics of the drug release. The best curve t of the release data was tested with the mathematical models of zero and rst order, HixsonCrowell, Higuchi, and Weibull (RRSBW) kinetics [21]. Similarity factor f2 has been used to compare in vitro dissolution proles in order to investigate the effect of different type of polymers used. The P-CsA formulation was regarded as the reference formulation. 2.5. In vitro permeation studies: Franz diffusion cells The permeation studies were performed using Franz diffusion cells with a diffusion area of 2.26 cm2. Sigma dialysis membrane having a molecular weight cut-off of 12,400 Da was used. Membranes were soaked in UPW for 24 h before mounting in Franz diffusion cell. The SLF in the thermostated (32 C) receptor chamber was stirred at 200 rpm. An amount of NPs containing 50 g CsA or RestasisA were dispersed in 5 mL UPW and gently placed into the donor chamber. At specied time intervals, 1 mL samples were withdrawn for HPLC determination and replaced immediately with an equal volume of SLF solution. 2.6. Cell culture studies L929 (mouse broblast) cells (ATCC) (Manassas, VERSAmax, USA) were cultured in 75 cm2 culture asks containing RPMI 1640 medium (Biological Industries, Israel) supplemented with 10% fetal bovine serum (Biochrom, Germany), 1% L-glutamine (Biological Industries, Israel), 50 U mL 1 penicilin G and 50 U mL 1 streptomicin (Biochrom, Germany) at 37 C in a humidied incubator (Heraus, Germany) containing 5% CO2 air. Conuent cell monolayers were trypsinized and cells in the exponentially growing phase were used in the cytotoxicity experiments. For in vitro cytotoxicity and cellular uptake studies; UPW, rubber materials (caps) were sterilized in autoclave at 115 C for 30 min. All metal and glass materials, magnetic stirrers were sterilized using dry heat (Nve FN055 drying oven, Turkey) at 170 C for 2 h. The NPs preparation process was carried out in an aseptic room under aseptic condition. 2.6.1. Analysis of in vitro cytotoxicity Cell viability was measured by the MTT assay [22] L929 cells were seeded on 96-well tissue culture plate at the initial density of 15 103 cells/well. After a 24 h stabilization of the cells, fresh medium containing either different concentrations of CsA (0.5, 5, 20, 50, 125 and 250 g/mL), blank or loaded NPs (0.125, 0.25 and 0.5 mg/mL) was added. Incubation with the different CsA solutions was performed at 37 C for 24 h. In the case of blank or loaded NPs cells were incubated at 37 C for 24 h and 48 h in order to compare the cytotoxicity of different concentrations and incubation times on L929 cells. After the incubation, media was removed and 100 L fresh medium and 13 L MTT solution (5 g/mL, diluted with RPMI 1640 without phenol red) were added to each well. Incubation was allowed for another 4 h in darkness at 37 C. Since living cells metabolize the MTT and form blue formazan crystals, 100 L/well isopropanol-HCl (absolute isopropanol containing 0.08M HCI) solution was added to dissolve the formazan crystals. Absorbance values were measured by reading the plates at 570 nm on an ELISA plate reader (ASYS Hiteck GmbH, Austria), and percentage of viability was calculated. The viability of the treated cell cultures was expressed as a percentage of control untreated cell cultures assumed to be 100%.

2.7. Cellular NPs uptake studies L929 cells (25 103) were seeded in 6-well plates, incubated overnight at 37 C with 5% CO2 in a humidied atmosphere. Nile red labeled NPs {NR-P-CsA, NR-P:E (75:25)-CsA and NR-P:C-CsA} were diluted separately with cell culture medium in order to obtain a NPs concentration of 0.5 mg/mL and were incubated at 37 C for 6 h approximately. The experiment was stopped by washing the cells three times with sterile PBS to eliminate excess NPs which were not entrapped by the cells. The cells were diluted in 300 L acetonitrile, vortexed for 2 min and centrifuged for 30 min at 14,000 rpm. The uptake was calculated directly by measuring the % of Nile red taken up. The L929 cells were photographed with Leica DFC320 digital Camera (Leica Microsystems Imaging Solutions Ltd, UK). 2.8. Cyclosporine A determination in rabbit tear The use and the treatment of rabbits in this study was conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The test protocol was carried out on six male New Zealand white rabbits with a single instillation of 50 L of the NPs formulation (25 g CsA). The untreated contralateral eye was used as control and 0.9% (w/v) NaCl solution was instilled into the control eye. All protocols were approved by the Hacettepe University Animal Ethics Committee (Project number: 2008/51). Tear uid samples were collected with Schirmer lter paper. Schirmer papers were weighed before use and gently inserted into the inferolateral cul-de-sac of the eye, close to the middle of the eye, during 30 s. Immediately after collection; papers were removed, placed into the eppendorf tube and weighed again. Papers were then placed in 300 L acetonitrile and vortexed for 3 min. After centrifugation for 15 min at 14,000 rpm, CsA content of the samples was determined. 2.9. Statistical analysis Data of particle size, zeta potential, entrapment efciency, cell culture and in vivo studies were tested statistically by one-way analysis of variance (ANOVA) followed by Tukey HSD test. Differences were considered to be statistically signicant at a level of p 0.05. 3. Results and discussion 3.1. In vitro characterization studies 3.1.1. Evaluation of entrapment efciency and yield of NPs The validation assay of the HPLC procedure used showed good linearity (R2 = 0.999). Limit of detection and limit of quantication were 0.04 g/mL, 0.1 g/mL respectively. The CsA loading efciency was approximately 88% and the production yields was over 89% except for P:C-CsA NPs (77%) (Table 1). The reason of low production yield of Carbopol coated NPs was attributed to the possible loss of water soluble coating polymer from the surface during the washing procedure. Due to the hydrophobic character of CsA, high entrapped efciency and production yield was obtained in PLGA and Eudragit RL NPs. Because of CsA is a very poorly water soluble drug, it was partitioned in the organic phase of the initial emulsion and consequently, very little amount of drug was lost to the aqueous phase [23]. The CsA loading capacity of the Nile red labelled NPs was found above 84% for all preparations. Incorporation of Nile red into the NPs did not show any signicant effect on the CsA entrapment efciency. Nile red entrapment efciency was found between 72 and 78% in all preparations with no signicant difference (p N 0.05).

Please cite this article as: P. Aksungur, et al., Development and characterization of Cyclosporine A loaded nanoparticles for ocular drug delivery: Cellular toxicity, uptake, and kinetic studies, J. Control. Release (2011), doi:10.1016/j.jconrel.2011.01.010

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The homogenous distribution and the nearly spherical structure of NPs are shown in Fig. 1. 3.1.2. Particle size and zeta potential Particle size distributions of all preparations are homogenous and well suited for ocular use. The particle size for ophthalmic applications should not exceed 10 m to prevent scratching and foreign body sensation [24]. The size of NPs was affected by the type of polymer used (Fig. 2). The NPs sizes were distributed mainly in a narrow range of 148 219 nm, while NPs coated with Carbopol showed the largest size among the preparations (393 nm, p b 0.05). The larger size is due to the presence of carbomer polymer chains at the surface of the NPs [19]. Very good polydispersity index values (PI 0.2) were obtained for all preparations.

Fig. 2. Particle size distribution of CsA loaded NPs before and after lyophilization (mean S.D., n = 3).

The smallest NPs were obtained with Eudragit RL and the NPs size was decreased with increasing Eudragit RL concentration in NPs, due to the physicochemical properties of the polymer. The size of P:E (50:50)-CsA and P:E (75:25)-CsA NPs were signicantly larger in the case of NPs prepared by either Eudragit RL or in combination with PLGA in ratio 25:75. Particle size decrease can be attributed to the fact that the viscosity of the internal phase of the emulsion may also be changed regarding to the polymer type used. An increase in viscosity of internal phase leads to an increase in size of the particles under a constant sonication input. During the formation of NPs prepared by EudragitRL and a mixture of PLGA-EudragitRL (25:75), the viscosity of the internal phase of the emulsion might be lower than other polymer compositions. A viscosity decrease of organic phase during emulsication process would decrease the dropsize of the internal phase and afterwards facilitate the diffusion of organic solvent into external water phase. Therefore smaller NPs would be formed during precipitation. The results obtained correlated well with previous studies investigated the inuence of Eudragit and PLGA polymers on NPs size [14,15,25]. No inuence of drug loading on particle size was observed (p N 0.05) (data not shown). These results are in accordance with the study of Zhang et al. [26] reporting that the size of blank or CsA loaded particles was not affected by the drug present. It has been reported that the particle size was a key factor on cellular uptake of NPs and the uptake increased with decreasing particle diameter [27]. The size of the blank or CsA loaded Nile red labelled NPs used in cellular uptake studies was examined (Fig. 3). Incorporation of Nile red had no effect on particle size compared to the blank (p N 0.05). Also, no difference in size was observed between blank and CsA loaded Nile red labeled NPs. Freeze drying of CsA loaded NPs induced minor increase in particle size (p b 0.05). Similarly, there are many studies reporting an increase in particle size after lyophilization [14,28,29]. Moreover, the PI values which provides information about the homogeneity of particle size, was lower than 0.2 for all preparations. Zeta potential measurement is an adequate method in order to evaluate NPs surface properties and to detect any eventual modication after freeze-drying. The zeta potential values were found dependent on the polymer type used (Table 2). P-CsA and Carbopol coated PLGA NPs were negatively charged. Carbomer coated NPs

Fig. 1. SEM images of the CsA loaded NPs: a) P-CsA, b) P:E (75:25)-CsA, and c) P:C-CsA.

Fig. 3. Particle size distribution of blank and CsA loaded Nile red labeled NPs (mean S.D., n = 3).

Please cite this article as: P. Aksungur, et al., Development and characterization of Cyclosporine A loaded nanoparticles for ocular drug delivery: Cellular toxicity, uptake, and kinetic studies, J. Control. Release (2011), doi:10.1016/j.jconrel.2011.01.010

P. Aksungur et al. / Journal of Controlled Release xxx (2011) xxxxxx Table 2 Zeta potential values (mean SD) of CsA loaded NPs, measured in UPW or SLF (n = 3). Formulation code UPW Before FD (mV) P-CsA P:E (75:25)-CsA P:E (50:50)-CsA P:E (25:75)-CsA E-CsA P:C-CsA NR-P-CsA NR-P:E (75:25)-CsA NL-P:C-CsA 21.1 1.8 + 25.2 1.4 + 26.2 0.4 + 26.8 1.9 + 24.3 1.2 45.2 0.8 18.7 0.9 + 19.1 1.4 43.4 2.1 After FD (mV) 25.1 3.4 + 21.4 2.5 + 19.2 1.7 + 22.2 0.8 + 19.3 2.2 49.0 1.3 20.3 0.7 + 20.5 2.5 41.2 1.1 SLF After FD (mV) 4.3 1.4 + 5.6 1.6 + 4.2 1.0 + 4.1 2.1 + 3.4 1.7 5.6 1.7 5.4 1.6 + 4.4 1.3 7.2 1.3

exhibited the highest zeta potential values (p b 0.05). Presence of carbomer polymer chains at the particle surface allowed to form carboxylates and consequently negatively charged polymer chains in neutral or alkaline media. The surface charges of the NPs prepared by using EudragitRL and PLGA-EudragitRL mixtures were positive. The charge ratio was not related to EudragitRL ratio (p N 0.05), because only a small amount of amphiphilic Eudragit molecules are needed to obtain a positive charge at the particle surface. Same results were obtained in the studies of Vandervoort et al. [19] and Dillen et al. [14]. It is important to use positively charged NPs since it will prolong the residence time of the formulation in the precorneal area, because of interactions with negatively charged mucins. The zeta potential of the NPs was measured in both SLF and UPW. The absolute zeta potential values of the NPs were reduced in SLF compared to puried water (p b 0.05). The charges on the surface of the particles were neutralized by the salts present in SLF, and resulted in a decrease of zeta potential values. No signicant difference on surface charge of blank and CsA loaded particles was measured (data not shown) because of the neutral structure of the drug. Also lyophilization did not inuence the zeta potential of the different NPs examined (p N 0.05). On the contrary Dillen et al. [14] have reported that freeze drying of NPs caused a signicant decrease in zeta potential. Nile red incorporation did not inuence the surface charge of the NPs as can be deduced from zeta potential measurements. Scott et al. also reported that the particles' surface charge of Nile red labelled PLGA NPs was not affected by the uorescent dye [30]. 3.2. In vitro release studies Biphasic release pattern characterized by an initial burst release of CsA was observed (Fig. 4). This might be a result of the internal structure of NPs gained by freeze drying and lyophilization procedures as reported by other authors [31,32]. In our study, another reason of high burst might be the result of crystal structure of CsA being transformed to an amorphous structure after the lyophilization process [33]. Formation of amorphous structure from crystal may lead to an increase in CsA solubility. During preparation of NPs, CsA could precipitate as amorphous substance in the PLGA matrix. After the initial burst effect, the drug was released slowly. The release rate from Eudragit RL and PLGA- Eudragit RL NPs was lower and their surface area was larger than those of the particles prepared with PLGA, but no signicant difference was observed in release proles. This nding is in agreement with the results of Dillen et al. [14] who have reported that ciprooxacin loaded PLGA, Eudragit RS and PLGA-EudragitRS NPs did not show a signicantly different drug release. All NPs of the present study showed similar release behaviours (f2 N 50). Total cumulative release within 24 h was found between 75 and 90% for all formulations. The f2 values indicate relatively good similarity in dissolution proles compared with the reference formulation's (P-CsA) release pattern.

Fig. 4. Release proles of CsA from NPs prepared with different type of polymers (n = 6).

Kinetic models are useful to understand the dissolution characteristics of the dosage form. According to the correlation coefcients at the end of 24 h measurements, the release patterns best tted to the RRSBW model (Table 3). All values were between 0.147 and 0.221 mentioning b 1. This means a fast release at the beginning and it is followed by slow drug release. Adipkia et al. [34] also have reported that among the kinetic models employed, the Weibull's distribution model showed a better t compared to the other models within the all piroxicam loaded Eudragit RS nanoparticle formulations studied. 3.3. In vitro permeation studies: Franz diffusion cell This study aimed to obtain the pre-information about in vivo studies by using cellulose membrane simulating human cornea. No signicant difference in release and diffusion through the membrane was observed between the developed NPs formulations and RestasisA (Fig. 5). The cumulative percentage of CsA in receptor compartment was between 41 and 47% for all formulations at the end of 24 h. Taken into account the MW of CsA, the rate limiting step is probably the diffusion of CsA and not the release out of the NPs or emulsion droplets. 3.4. Cell culture studies In vitro cell culture assays are commonly used for cytotoxicity evaluations. The advantages of L929 mouse broblasts cell cultures include relatively well-controlled variables and quantitative results in short time periods. Finally, they are considered a very sensitive means of cytotoxicity testing. 3.4.1. Cytotoxicity of CsA Cell viability was decreased in a dose-depending manner with increasing concentrations of CsA (Fig. 6). CsA showed a toxic effect on the L929 cells in higher concentration levels. The survival rate of L929 mouse broblast cells at 5 g/mL and 0.5 g/mL CsA concentrations
Table 3 shape factor, and correlation coefcients (R2) calculated after tting the release proles for different mathematical models. Formulations P-CsA P:E (25:75)- P:E (50:50)- P:E (75:25)- E-CsA P:CCsA CsA CsA CsA RRSBW T%63.2 94.38 0.159 r2 0.917 23.76 0.221 0.972 41.58 0.205 0.950 34.98 0.147 0.761 71.40 0.185 0.936 42.24 0.149 0.930

Please cite this article as: P. Aksungur, et al., Development and characterization of Cyclosporine A loaded nanoparticles for ocular drug delivery: Cellular toxicity, uptake, and kinetic studies, J. Control. Release (2011), doi:10.1016/j.jconrel.2011.01.010

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Fig. 5. Franz diffusion cell: CsA concentration in receptor compartment (n = 6).

was signicantly higher (p b 0.05) than the other concentrations of CsA (20, 50, 125 and 250 g/mL). 3.4.2. Cytotoxicity of NPs A promising nanoparticle system intended for ocular use must be capable of delivering sufcient levels of the active agent without compromising the viability of the host cells. Cationic polymers are known to exhibit cytotoxic effects by inducing cell membrane damage. EudragitRL, like most cationic macromolecules, could interact with anionic components of the glycoproteins on the surface of epithelia cells, causing cytotoxic effects. For this reason, it was interesting to study the effect of different NPs on the viability of L929 cell lines. The survival rate of L929 cells was decreased with increasing incubation time (p b 0.05) (Figs. 7 and 8). Survival rate of L929 cells was also decreased with increasing the NPs concentration applied. A signicant difference in survival rate for the cells treated with 0.125 mg/mL and 0.5 mg/mL NPs concentrations was observed (p b 0.05). However, no signicant difference was found for CsA loaded or blank NPs at the end of the 24 h and 48 h experiments. Although the survival percentage of the blank or loaded NPs was high, a low percentage cell death was thought to be related to the polymer or residue of organic solvent remaining from the preparation procedure. 3.5. Cellular uptake studies The presence of Nile red labelled NPs in L929 mouse broblasts cells was observed microscopially at each time interval and for each kind of NPs preparation (Fig. 9). Percentage amount of cellular uptake varied regarding to the polymer type used. An obvious time-dependent increase in uptake

Fig. 7. Survival rate of L929 cells after incubation with blank NPs (n = 8); a) 24 h and b) 48 h.

was observed from 0.5 to 6 h (Fig. 10). These results supported the study of Zhang et al. [35]. At given time points, P:E (75:25)-CsA NPs showed signicantly higher uptake reaching to 52.1% after 6 h. It was conceivable that the positively charged particle surface facilitated ionic interaction with the negatively charged L929 cells membrane. Besides, it was assumed that as the P:E (75:25)-CsA NPs are smaller than the others, the cellular uptake may increase accordingly [27]. The uptake of Carbopol coated NPs (40%) was much higher than in case of the uncoated NPs (33.2% after 6 h) (p b 0.05). Although P:CCsA NPs, which has gained adhesive properties by coating with carbomer, had larger particle size and more negative surface charge, their uptake was increased signicantly compared to P-CsA particles. Carbopol was suggested to endow bioadhesive properties to NPs, which facilitate adhesion on the cell surface, thus improving the uptake. Particle size and surface charge are considered the most important parameters governing tissue uptake of NPs. It could be concluded that

Fig. 6. Cytotoxicity of CsA in L929 cells (n = 8).

Fig. 8. Survival rate of L929 cells after incubation with CsA loaded NPs (n = 8); a) 24 h and b) 48 h.

Please cite this article as: P. Aksungur, et al., Development and characterization of Cyclosporine A loaded nanoparticles for ocular drug delivery: Cellular toxicity, uptake, and kinetic studies, J. Control. Release (2011), doi:10.1016/j.jconrel.2011.01.010

P. Aksungur et al. / Journal of Controlled Release xxx (2011) xxxxxx

Fig. 9. Time-dependent NPs uptake by L929 cells. Photographs of representative series of cells exposed to 0.5 mg/mL NPs for 30, 120 and 360 min. Original magnication 400.

P-CsA, P:E (75:25) and P:C-CsA particles are the formulations of choice for uptake studies according to their negative, cationic and adhesive properties respectively. 3.6. Determination of the amount of cyclosporine in rabbit tear lm After applying CsA loaded NPs and emulsion (RestasisA) to rabbit eye, drainage and elimination processes started. In the rst few minutes, the CsA tear lm concentration was high for all formulations instilled. But a rapid decrease in concentration was observed after 10th min (Fig. 11). After 30 min, the elimination of CsA from the precorneal area was slowed down. The fast decrease of CsA concentration could be explained by the mechanical elimination of the excess of instilled volume from the cul-de-sac. When the tearlm volume returned to normal, the elimination of CsA proceeds depending on normal tear turn-over. The drug concentration obtained after instillation of P:E (75:25)-CsA particles was higher than for other

formulations at all time intervals. The cationic characteristics of Eudragit RL and positive charges of P:E (75:25)-CsA NPs cause ionically interaction with the negatively charged mucus layer at the eye surface. Positive charged surface properties offer the prospects of prolonging the residence time of NPs in the cul-de-sac and consequently ensuring optimal contact between the formulation and the eye. Second higher CsA concentration in tearlm was measured after application of the emulsion (RestasisA) in the rst few minutes, but afterwards the CsA level also decreased rapidly. Even though the size of P:C-CsA NPs are larger and they have higher zeta potential than P-CsA NPs, CsA concentrations measured after instillation of these particles were higher than in the case of P-CsA NPs. Carbopol chains could entangle with the mucus layer and moreover they could make hydrogen bond with the mucin glycoproteins resulting in a strengthened gel network, which allows the

Fig. 10. Cellular uptake efciency (%) of Nile red labelled NPs.

Fig. 11. CsA tear lm concentration after administration of a single topical dose to rabbits in the cul-de-sac (n = 6).

Please cite this article as: P. Aksungur, et al., Development and characterization of Cyclosporine A loaded nanoparticles for ocular drug delivery: Cellular toxicity, uptake, and kinetic studies, J. Control. Release (2011), doi:10.1016/j.jconrel.2011.01.010

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Table 4 Pharmacokinetic parameters for tear after the application of CsA loaded NPs suspension or drug emulsion (RestasisA) (n = 6). Formulation code P-CsA P:E-CsA (75:25) P:C-CsA Restasis A
a b a

Acknowledgements This project was supported by NOVARTIS Research Grant. Authors are grateful to Novartis, Turkey for their generous gift of Cyclosporine A. References
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AUC

0 24h

(ng h/g)

C max (ng/g)

490.42 972.59 776.57 514.24

126.12 366.30 211.08 299.02

Area under the concentrationtime curve between 0 and 24 h. Peak drug concentration (ng CsA/g tear).

mucoadhesive system to remain adhesive for an extended period of time [36]. In the case of ocular application, it is difcult to maintain the therapeutic effects for a signicant period of time because the liquid dosage forms are very easily removed from the eye. As a result of corneal uptake of the NPs, cornea acts like a reservoir and CsA is being released in a controlled manner. Besides, the burst release would ensure a sufcient drug level just after instillation. It could be expected that CsA would precipitated within the limited volume of lacrymal uid present in the precorneal environment because of burst release and accumulate in the cul-de-sac [37]. In that case, the precipitated CsA would also act like a reservoir and redissolve slowly in the lacrymal uid, hence leading to a constant release of CsA. The long-term release of the CsA would lead to an increase in efciency of the treatment. Kinetic parameters were calculated in order to examine the elimination of CsA from the precorneal area (Table 4). The AUC0 24 value was affected by the nature of polymers used. The AUC0 24 value for P:E (75:25)-CsA formulation was the highest (p b 0.05). These results are in accordance with data of CsA determination in the lacrymal uid and cellular uptake studies. Positively charged NPs interact with the negatively charged mucus layer at the eye surface and the prolonged residence increases the CsA concentration in the tear lm. AUC0 24 value obtained from P:C-CsA NPs was signicantly higher compared to those of P-CsA and emulsion formulations. Since P:C-CsA NPs possess adhesive properties they adhere to the mucus layer and increase the residence time in the cul-de-sac. For P-CsA formulation, which showed the lowest AUC0 24 value, area under curve was found very close to the AUC value of RestasisA.

4. Conclusion The present study reports the preparation and physicochemical characterization of NPs which combine the PLGA with the positively charged properties of EudragitRL or coated with the mucoadhesive Carbopol. The results indicate that both the mean diameter and the surface charge on NPs were markedly affected by the polymer type. The P:ECsA (75:25) NPs showed small particle size and positive surface charge, which makes them suitable for ocular use. The cytotoxic effects of NPs formulations were found to be time and concentration dependent. The P:E-CsA (75:25) NPs formulation also showed signicantly higher degree of cellular uptake, tear lm concentration of the CsA and AUC0 24 value in comparison with the other NPs formulations. In conclusion, we have demonstrated that NPs with different properties could modulate the drug release in the cul-de sac or in the ocular tissues after uptake by epithelial cells. The system interacts with eye surface, ensuring optimal contact between the formulation and the mucosa. It provides sustained drug release in the cul-de-sac for an extended period of time. These results indicate that CsA loaded NPs have great potential as drug delivery systems and are promising formulations in the management of external inammatory/autoimmune ocular diseases.

Please cite this article as: P. Aksungur, et al., Development and characterization of Cyclosporine A loaded nanoparticles for ocular drug delivery: Cellular toxicity, uptake, and kinetic studies, J. Control. Release (2011), doi:10.1016/j.jconrel.2011.01.010

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