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Proniosome-derived niosomes for tacrolimus topical ocular delivery: In vitro cornea permeation, ocular irritation, and in vivo anti-allograft rejection
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Qi Li a,1, Zhanrong Li b,1, Weidong Zeng a, Shumin Ge a, Haoyang Lu a, Chuanbin Wu a, Li Ge a, Dan Liang b, Yuehong Xu a,⇑ a b
School of Pharmaceutical Science, Sun Yat-sen University, Guangzhou 510006, China State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060, China
a r t i c l e
i n f o
Article history: Received 4 February 2014 Received in revised form 23 March 2014 Accepted 26 May 2014 Available online xxxx Keywords: Tacrolimus Proniosome Niosome Ocular delivery Allograft rejection
a b s t r a c t The objective of this study was to develop proniosome-derived niosomes for topical ophthalmic delivery of Tacrolimus (FK506). The FK506 loaded proniosomes containing poloxamer 188 and lecithin as surfactants, cholesterol as a stabilizer, and minimal amount of ethanol and trace water reconstituted to niosomes prior to use. The stability of FK506 loaded proniosomes was assessed, and the morphology, size, zeta potential, surface tension, and entrapment efficiency of the derived niosomes were characterized, indicating they were feasible for instillation in the eyes. The in vitro permeation of FK506 through the freshly excised rabbit cornea, the cumulative permeation amount of FK506 from niosomes, and the drug retention in the cornea all exhibited significant increase as compared to 0.1% FK506 commercial ointments. The in vivo ocular irritation test of 0.1% FK506 loaded niosomes instilled 4 times per day in rat eyes for 21 consecutive days showed no irritation and good biocompatibility with cornea. The in vivo anti-allograft rejection assessment was performed in a Sprague–Dawley (SD) rat corneal xenotransplantation model. The results showed treatment with 0.1% FK506 loaded niosomes delayed the occurrence of corneal allograft rejection and significantly prolonged the median survival time of corneal allografts to13.86 ± 0.80 days as compared with those treated with 1% Cyclosporine (CsA) eye drops, drug-free niosomes, or untreated. In conclusion, the proniosome-derived niosomes may be a promising vehicle for effective ocular drug delivery of FK506. Ó 2014 Published by Elsevier B.V.
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1. Introduction Corneal allograft is clinically performed to improve the vision of patients who suffer from damaged or diseased corneas. However, corneal graft failure is commonly caused by allograft rejection (Coster et al., 2005). Although steroids are normally used to suppress the rejection reaction, the occurrence of allograft rejection still keeps high as 10–30% in the patients, and commonly followed with the secondary complications such as glaucoma, cataract, and systemic infection (Lindstrom, 1986). Tacrolimus (FK506), an immunosuppressive agent, can inhibit the action of enzyme calcineurin phosphatase and the transcription of IL-2 gene, resulting in suppression of T-lymphocyte response (Thomson et al., 1995). It can efficiently prevent post-transplant rejection in patients who are resistant to steroids and cyclosporine A (Scott et al., 2003). ⇑ Corresponding author. Tel./fax: +86 20 39943119. 1
E-mail address: [email protected] (Y. Xu). The authors contributed equally to the present work.
By far, FK506 has been topically administered to prevent the rejection of penetrating keratoplasty (Fei et al., 2008; Hikita et al., 1997; Reinhard et al., 2005; Sloper et al., 2001) and treat the intractable allergic conjunctivitis (Attas-Fox et al., 2008) and refractory inflammatory ocular surface diseases with immunologic causes (Lee et al., 2013). At present, FK506 ointment (0.1% and 0.03%, w/w), which is known as ProtopicÒ (Astellas Toyama Co., Ltd., Toyama Plant, Japan), has been approved by the US Food and Drug Administration (FDA) for treating moderate to severe atopic dermatitis (Ruzicka et al., 1997). But no ophthalmic preparation of FK506 is commercially available, and the dermatological FK506 ointment was clinically used instead of ophthalmic preparation in some reports (AttasFox et al., 2008; Lee et al., 2013; Miyazaki et al., 2008). Therefore, it is urgent to develop an ophthalmic delivery system for FK506 for its application in preventing and treating eye diseases. Owing to its highly hydrophobic characteristic and high molecular weight (822.5 D), FK506 had difficulties in penetrating the cornea and reaching the effective therapeutic intraocular level.
http://dx.doi.org/10.1016/j.ejps.2014.05.020 0928-0987/Ó 2014 Published by Elsevier B.V.
Please cite this article in press as: Li, Q., et al. Proniosome-derived niosomes for tacrolimus topical ocular delivery: In vitro cornea permeation, ocular irritation, and in vivo anti-allograft rejection. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.05.020
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Moreover, its poor water stability hinders formulating it with massive aqueous medium into eye drops. Therefore, the ideal delivery system for FK506 should not only enhance drug penetration through the cornea but also maintain its stability and reduce its undesirable side effects such as alterations in kidney function and glucose metabolism, neurotoxicity and susceptibility to infection or malignancy with systemic administration (Fung et al., 1991). Some delivery systems including liposomes (Pleyer et al., 1993; Zhang et al., 2010; Dai et al., 2013), nanospheric suspensions (Fei et al., 2008), in situ nanosuspensions (Luschmann et al., 2013), nanoparticles (Nagarwal et al., 2012) and nanoemulsions (Garg et al., 2013) have been investigated for potential ocular application. Niosomes which are made of nonionic surfactants and lipids can load either hydrophilic or hydrophobic drugs. They also possess many advantages over other vesicular drug delivery systems, such as low production cost, low toxicity, and ease of formulation without unacceptable solvents (Choi and Maibach, 2005; Sankar et al., 2010). Moreover, the nonionic surfactants used in niosomes can increase the bioavailability of poor water soluble drugs by enhancing the solubility and permeability through the biological membrane. It was reported that niosomes could provide a prolonged and controlled action at the corneal surface and prevent the drug metabolism by enzymes present at the tear/corneal surface (Abdelbary and El-Gendy, 2008). Several studies have demonstrated the successful use of niosomes as a potential ocular drug delivery system for drugs such as cyclopentolate, acetazolamide, Q2 timolol maleate, gentamicin, and naltrexone (Abdelbary and Elgendy, 2008; Abdelkader et al., 2012; Aggarwal and Kaur, 2005; Guinedi et al., 2005). However, niosomes also exhibit physical and chemical instability during storage, such as aggregation, sedimentation, fusion and leakage or hydrolysis of encapsulated drugs, Q3 which may affect the shelf life of dispersion (Hu and Rhodes, 2000). Therefore, the latest approach in vesicular delivery system is to take the provesicular approach to format ‘‘proniosomes’’ (Ammar et al., 2011; Sankar et al., 2010), which are liquid crystalline-compact niosomal hybrids and will convert into niosomes upon hydration with water prior to administration. Proniosomes are generally present in a stable semisolid gel structure, which can prevent the hydrolysis of encapsulated drug during storage and transport. Many studies have shown that proniosomes might be a promising drug delivery system via transdermal route (Ei-Laithy et al., 2011; Fang et al., 2001; Jukanti et al., 2011; Vora et al., 1998). Proniosomes can be hydrated by water from the skin to form niosomes, and both phospholipids and non-ionic surfactants in proniosomes can act as penetration enhancers (Ammar et al., 2011; Sankar et al., 2010). These properties may also make proniosomes potential vehicles for topical ophthalmic drug delivery. Therefore, the aim of the present work was to develop a proniosome system to reconstitute niosomes for ophthalmic delivery of FK506. The stability of FK506 loaded proniosomes was evaluated, and the niosomes were characterized in terms of morphology, vesicle size and size distribution, zeta potential, surface tension, and entrapment efficiency. Permeation of the niosomes through rabbit cornea was evaluated in vitro and ocular irritation in rabbits was assessed in vivo. Furthermore, the in vivo anti-allograft rejection of the FK506 loaded niosomes was evaluated in a mode of corneal xenotransplantation.
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2. Materials and methods
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2.1. Chemicals
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FK506 was purchased from Teva Czech Industries (S.R.O. Ostravska 29305, 74770 Opava-Komarov, Czech Republic). Soybean phosphatidylcholine (SPC, Lipoid S100, purity >98%) was obtained
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from Lipoid GmbH (Ludwigshafen, Germany). Cholesterol (CHOL, Tian Ma Chemical Plant, Guangzhou), Poloxamer 188 (BASF, Ludwigshafen, Germany), 0.1% FK506 commercial ointment (ProtopicÒ, Astellas Toyama Co., Ltd. Toyama Plant, Japan), and 1% Cyclosporine (CsA) eye drops (Zhongshan Ophthalmic Center, Guangzhou) were used in the study. Other reagents were of analytical grade.
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2.2. Animals
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New Zealand albino rabbits (male and female, weighing 2–3 kg) were provided by the Animal Experimental Center of Zhongshan Ophthalmology Center in Sun Yat-sen University. Wistar (female, weighing 200–220 g, 6–8 weeks old) and Sprague–Dawley (SD) rats (female, weighing 200–220 g, 6–8 weeks old) were obtained from Guangzhou Animal Testing Center. The animals were housed under standard condition (temperature of 25–28 °C, humidity of 40–60% RH, and light/dark cycle of 12/12 h) prior to operation and fed with a standard pellet diet and water at free. The protocols for animal use and care were approved by the Institutional Animal Care and Use Committee of Zhongshan Ophthalmic Center, Sun Yat-sen University.
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2.3. Preparation of FK506 loaded proniosomes and reconstruction of niosomes
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Proniosomes were prepared according to a modified literature Q4 method (Vora et al., 1998). Briefly, poloxamer 188, lecithin, and cholesterol in a ratio of 9:9:1 (w/w/w) were mixed with the required amount of ethanol in a glass tube. Then the required amount of FK506 was added in and the tube was warmed in a water bath at 65 ± 2 °C for 10 min. Afterwards, the phosphate buffer saline (pH 7.4) was dropped into the tube which was kept in the water bath for about 5 min until a clear solution formed. The solution was cooled down at room temperature until the proniosomal gel formed. Blank proniosomes were prepared similarly but without addition of FK506. Prior to application, 0.1% FK506 loaded niosomes were reconstituted by adding 10 ml of normal saline solution to 0.2 g of proniosomes and shaking for 3 min.
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2.4. Characterization of FK506 loaded niosomes
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2.4.1. Transmission electron microscopy (TEM) The morphological characteristics of the proniosome-derived niosomes were examined using transmission electron miroscropy (JEM1400, Tokyo, Japan). Briefly, a drop of niosomes was applied onto a collodion coated 300 mesh copper grid and remained for 1 min to allow the niosomes to adhere to the collodion. Then, a drop of 2% uranyl acetate solution was applied for staining, and the grid was air dried at room temperature and examined by TEM (Abd-Elbary et al., 2008).
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2.4.2. Particle size, size distribution and zeta potential The particle size, polydispersity index (PDI) and zeta potential of niosomes were determined at 25 °C by photon correlation spectroscopy at the scattering angle of 90° equipped with a Zetasizer (Malvern Instruments Nano ZS90, Worcestershire, UK). Each sample was measured in triplicate.
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2.4.3. Surface tension The surface tension of FK506 loaded niosomes was measured at room temperature using an interfacial tension meter (Hengping Instrument, Shanghai, China), and the FK506 aqueous suspension and normal saline solution were used as the controls. All measurements were carried out in triplicate.
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Please cite this article in press as: Li, Q., et al. Proniosome-derived niosomes for tacrolimus topical ocular delivery: In vitro cornea permeation, ocular irritation, and in vivo anti-allograft rejection. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.05.020
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2.4.4. Entrapment efficiency Free FK506 was separated from FK506 loaded niosomes by centrifugation at a speed of 14000 rpm for 30 min at 4 °C (Ultra centrifuge, Thermo, USA) (Hu and Rhodes, 1999). The amount of free FK506 in the supernatant was determined by High Performance Liquid Chromatography (HPLC). The entrapment efficiency (EE) of FK506 in niosomes was calculated as follows (Rajera et al., 2011a):
EE ¼ ðC t C f Þ=C t 100%
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where Ct and Cf was the concentration of total FK506 and free FK506, respectively.
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2.5. Stability
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The variations of drug loading and entrapment efficiency during storage were monitored to determine the stability of the FK506 loaded proniosomes, which may be affected by aggregation, fusion or leakage of the vesicles, and storage temperature. The prepared FK506 loaded proniosomes were stored in glass vials and kept at 4 ± 2 °C/75 ± 5%RH; 25 ± 2 °C/60 ± 5%RH; 40 ± 2 °C/75 ± 5%RH, respectively as per ICH guidelines. The drug loading and entrapment efficiency were determined after preparation, 1, 2, and 3 months of storage. Moreover, the 1 day for 24 h storage stability of reconstituted FK506 loaded niosomes at 37 ± 0.5 °C has been measured because the niosomes were kept for 24 h for administration or in vitro permeation.
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2.6. In vitro permeation through excised rabbit cornea
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In vitro corneal permeation study was carried out using a modified Franz diffusion cell suitable for cornea with an effective permeation area of 50.24 mm2. Rabbits were sacrificed by overdose of anesthesia. The fresh corneas were excised from the globes and carefully mounted onto the receptor cell with the outside of cornea facing the donor cell. Based on the preliminary experiment (data not shown), 5 ml of 25% ethanol/artificial tears (v/v) were used as receptor medium to maintain a sink condition for the release of FK506. The medium was continuously stirred at 250 rpm with a magnetic bar and maintained at 37 ± 0.5 °C with a circulating water bath. Niosomes and the commercial ointment (ProtopicÒ) containing equal amount of FK506 were carefully spread onto the corneal surface, respectively. At 0.5, 1, 2, 3, 4, 6, 12, 24 h, 1 ml of the receiving medium was withdrawn and replaced with equal volume of fresh receiving medium. The experiments were performed in triplicate and all the samples were analyzed with HPLC to determine the content of FK506.
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2.7. FK 506 content
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HPLC was employed to determine FK506 content in all the samples. The HPLC system (Agilent 1100 series) with a C8 column (GraceÒ, 250 mm 4.6 mm, 5 lm) was equipped with a UV detector (G1314A VWD) and data processing software (Agilent Chem Station for LC systems) for FK506 analysis at wavelength of 220 nm. The mobile phase was water/isopropyl alcohol/tetrahydrofuran (5:2:2, v/v/v) at a flow rate of 0.8 ml/min at 55 °C.
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2.8. Ocular irritation evaluation
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Six New Zealand albino rabbits were used to evaluate the ocular irritation by the Draize eye test (Wilhelmus, 2001) and histological examination. Before drug administration, bilateral eyes of the animals were inspected with 2% fluorescein sodium to make sure free of irritation, corneal defects, or conjunctival damage. Then, the left eye of each rabbit was administered with 100 ll of 0.1% FK506 loaded niosomes 4 times a day for a period of
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21 days, while the right eye was administered with normal saline solution 4 times a day as the untreated control. Ocular response of a single dose was observed at 1, 2, and 4 h after the first instillation. The cumulative ocular response was observed every 24 h after four administrations daily for a consecutive 21 days, as well as 1, 2, 4, and 24 h after the last instillation. The rabbit eyes were inspected with a slit lamp microscope (SL 120; Carl Zeiss AG, Oberkochen, Germany) for local ocular reaction. Irritation was scored using the method of Draize, and the responses were evaluated according to Table 1 (Gettings et al., 1996) and reported as the maximum average score. Upon completion of eye irritation study, all the rabbits were sacrificed with an overdose injection of ketamine. The eye balls were immediately enucleated and fixed in 10% formalin solution. Sections were made from the paraffin blocks at a thickness of 5 lm, and stained by hematoxylin and eosin. Histologic examination was performed with a light microscopy (T8-100, Nikon, Japan) for the changes of cornea, iris, and retina components of the tissue.
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2.9. In vivo assessment of anti-allograft rejection
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2.9.1. Corneal transplantation Wistar and Sprague–Dawley (SD) rats were used for corneal transplant, 32 female Wistar rats served as donors and 32 female SD rats were recipients. Full thickness corneal transplants were performed as previously reported (Holland et al., 1991b). Briefly, the donor rats were sacrificed by an overdose of chlorpromazine, while the recipient rats were deeply anesthetized with ketamine (25 mg/kg) and chlorpromazine (25 mg/kg) by intra peritoneal injection before surgical procedure. Compound tropicamide eye drops (Shenyang Sinqi Pharma. Co., Ltd.) were topically applied to dilate the pupil of the recipient right eye, where only the right cornea was transplanted. The corneal button was excised from the donor rat with a 3.5-mm trephine and Vannas scissors, and the graft was placed into chilled PBS until use. The donor cornea was secured by 8 stitches of interrupted 10–0 nylon suture (Alcon Laboratories, Fort Worth, TX) into the 3.0 mm recipient bed. The suture knot was not buried and kept in place postoperatively in order to stimulate the corneal neovascularization. Antibiotic ointment (Ofloxacin, Floxal™, Mann Pharma, Germany) was applied immediately to the eye after transplantation. The corneal suture was not removed during the entire duration of the experiment. The transplanted rats were randomly divided into four treatment groups (eight rats per group) as follows: control group treated with a drop of normal saline; blank group treated with blank proniosomed-derived niosomes without FK506; Cyclosporine (CsA) group treated with a drop of 1% CsA eye drops, and FK506 group treated with 0.1% FK506 loaded niosome eye drops. The frequency of administration in each group was four times a day after transplantation.
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2.9.2. Clinical assessment of graft survival After surgery, the transplants were examined daily for occurrence of graft rejection under the slit lamp microscopy until the rejection occurred as previously reported (Fei et al., 2008). The grafts were evaluated according to the Holland’s scoring system
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Table 1 Evaluation criteria for eye irritation reaction. Scores
Evaluation
0–3 4–8 9–12 13–16
Nonirritant Slightly irritant Moderate irritant Severe irritant
Please cite this article in press as: Li, Q., et al. Proniosome-derived niosomes for tacrolimus topical ocular delivery: In vitro cornea permeation, ocular irritation, and in vivo anti-allograft rejection. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.05.020
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described previously (Holland et al., 1991; Pan et al., 2003), in which the corneal opacity, edema, and neovascularization were taken into account (Table 2). Corneal grafts which showed technical complications such as infection, cataract, and intraocular hemorrhage were excluded from the study. Corneas with combined scores of 6 or higher were considered as rejected.
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2.10. Statistical analysis
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All experiments were performed at least in triplicate. Comparison between two groups was analyzed by Student’s t-test using IBM SPSS Statistic 19 software (SPSS Inc., Chicago, IL, USA). Data were reported as mean ± SD, and P < 0.05 was considered as statistically significant difference.
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3. Results and discussion
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3.1. Preparation of proniosomes and reconstitution of niosomes
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Excipients suitable for ophthalmic application were used to prepare FK506 loaded proniosomes for topical ophthalmic drug delivery. Poloxamer 188 and lecithin with excellent ocular compatibility were chosen as the surfactants (Jiao, 2008). Cholesterol was the important component of the cell membrane and its incorporation in the vesicle is known to improve the stability and permeability (Fang et al., 2001a). The surfactants and cholesterol were mixed with alcohol and aqueous phase to form the FK506 loaded semisolid proniosomal gel. The proniosomes can spontaneously convert to a niosomal dispersion upon dilution with excess aqueous phase under gentle shaking. The mechanism of proniosome formation may be as following: lamellar liquid crystal will form from lecithin and the non-ionic surfactant at krafft temperature in the presence of alcohol, and the crystalline phase will convert to dispersion of niosomes with excess water (Bai and Abbott, 2011). The organization of lipid/ethanol/water mixture in the lamellar structure can be conveniently utilized for ophthalmic delivery of drugs. This method avoids the use of pharmaceutically unacceptable solvents and is easy to scale up (Vora et al., 1998). The proniosome gel product can be hydrated immediately before use so that it may avoid the stability problems associated with aqueous niosome dispersions such as fusion, aggregation, and leakage.
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3.2. Characterization of FK506 loaded niosomes
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As observed from TEM images (Fig. 1), the niosomes derived from proniosomes were dispersed, spherical particles in the diameter of about 1 lm. Consistently, the niosome mean size of 1.33 ± 0.32 lm with polydispersity index (PDI) of 0.21 ± 0.03 were measured by the dynamic light scattering particle size analyzer (Table 3). The niosomes showed a negative zeta potential of about 8 mV because of the application of nonionic surfactants. The surface tension of the FK506 loaded niosomes (39.13 ± 0.35 dynes/cm) was significantly lower than those of the FK506 aqueous suspension (56.50 ± 0.26 dynes/cm) and normal saline solution (72.13 ± 0.06 dynes/cm) (p < 0.001). The lower surface tension made the niosomes easily wet the hydrophobic surface of the corneal epithelium and the lipid layer of the precorneal tear film, which may contribute to a better spreading ability and enhance the permeation of the formulation (Pawar and Majumdar, 2006). The amount of FK506 loaded in the niosomes was 1.00 ± 0.17 mg/g (w/w). The entrapment efficiency of FK506 in the proniosome-derived niosomes was 95.34 ± 0.02% (Table 3), which indicated that FK506 was effectively entrapped in the niosomes. The high entrapment efficiency may be because the highly lipophilic drug tends to intercalate almost completely within the lipid bilayer of niosomes (Gulati et al., 1998). Also, lecithin as a penetration enhancer in the formulation could increase the entrapment efficiency due to its high phase transition temperature preventing drug leakage from the vesicles. Cholesterol as a stabilizing surfactant also enhanced the stability of bilayer membrane by increasing gel liquid transition temperature and preventing drug leakage from the bilayer (Hsieh et al., 2013).
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3.3. Stability studies
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Physical stability studies were carried out to investigate the leakage of drug from proniosomes during storage under different conditions. After 3 months of storage at 4 °C, 25 °C, and 40 °C, the proniosomes remained visually unchanged and the loading of FK506 in derived niosomes changed from initial 1.00 ± 0.17 mg/g to 0.97 ± 0.06, 0.94 ± 0.08, and 0.57 ± 0.04 mg/g, respectively (Table 4). It indicates that 4 °C is the favorable storing condition for FK506 loaded proniosomes. Additionally, the entrapment efficiency of FK506 in niosomes only slightly dropped from initial
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Table 2 Clinical scoring system for the corneal transplantation rejection. Score
Clinical finding
Opacity 0 1 2 3 4
Clear cornea Slight haze, details of iris clearly visible Increased haze, some details of iris no longer visible Advanced haze, pupil still recognizable Opaque cornea without view of anterior chamber
Edema 0 1 2 3 4
No edema Mild stromal thickness Diffuse stromal edema Pronounced edema with small bleb of epithelium Bullous keratopathy
Neovascularization 0 1 2 3 4
No neovascularization Neovascularization of peripheral cornea Neovascularization appearing in the graft periphery Neovascularization extending deeper Neovascularization extending to the entire graft
a
The sum of scores of opacity, edema and neovascularization was termed as rejection index (RI). With RI of 6 or greater, the cornea was considered as rejected.
Fig. 1. Representative TEM images of proniosome-derived niosomes.
Please cite this article in press as: Li, Q., et al. Proniosome-derived niosomes for tacrolimus topical ocular delivery: In vitro cornea permeation, ocular irritation, and in vivo anti-allograft rejection. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.05.020
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Q. Li et al. / European Journal of Pharmaceutical Sciences xxx (2014) xxx–xxx Table 3 Characterization of FK506 loaded niosomes. Data represent mean ± SD (n = 3). Appearance
Size (lm)
Polydispersity index
Zeta potential (mV)
Entrapment efficiency (%)
Drug content (mg/ml)
Clear solution
1.33 ± 0.32
0.21 ± 0.03
8.25 ± 0.16
95.34 ± 0.02%
1.00 ± 0.17
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95% to 93% after 3 month storing at 4 °C. The ability of proniosomes-derived niosomes to retain the entrapment efficiency is in accordance with the previous report (Ammar et al., 2011). For 1 day of 24 h storage stability of FK506 loaded noisome (Table 5), the changes of drug loading and the entrapment efficiency of FK506 indicated that it was stable when being administered for 1 day or in vitro permeation study.
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3.4. In vitro permeation through rabbit corneal
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The corneal and conjunctival epithelial barriers cover the ocular surface and limit the access of drug to the targets. In vitro transcorneal permeation was conducted through the isolated rabbit corneal and the efficiency of the niosomes to enhance the corneal penetration of FK506 was assessed by measuring the transcorneal flux and the cumulative permeation amount. The reasons that the dermal preparation ProtopicÒ was chosen to be the control were as follows: First, dermal preparation ProtopicÒ was the only topical delivery formulation approved by US FDA and there are many reports that the dermatological FK506 ointment was topically used in clinic for treating different eye diseases (Attas-Fox et al., 2008; Joseph et al., 2005; Lee et al., 2013; Miyazaki et al., 2008; Ryu et al., 2012; Virtanen et al., 2006). We chose it as the control of a commercial preparation. Second, owing to highly hydrophobic characteristic of tacrolimus, it is very difficult to dispense it in aqueous medium to form a relatively uniform suspension without adding surfactant or organic solvent. However, if adding surfactant or organic solvent to medium enhances the dispensing, the permeation of tacrolimus will be affected by the surfactant or organic solvent. Taken together, the dermal preparation ProtopicÒ was chosen to be the control. The cumulative amount of FK506 permeated from 0.1% FK506 loaded niosomes through the cornea was significantly greater than that from 0.1% (w/w) FK506 commercial ointment ProtopicÒ throughout the entire experiment (P < 0.05, Fig. 2). After 24 h permeation, the cumulative permeation of FK506 from niosomes (117.36 ± 8.38 lg/cm2) was significantly greater than that from ProtopicÒ (70.88 ± 5.78 lg/cm2), which demonstrated that niosomes significantly enhanced the corneal penetration of FK506. The transcorneal flux of niosomes and ointment was 3.84 and 2.30 lg/cm2/h, respectively, confirming the permeation enhancement of niosomes. In present study, the size of FK506 loaded niosome is about 1.33 lm, the relatively large size
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makes it difficult permeate through excised rabbit cornea in intact form, the mechanism of FK506 permeation into corneal tissue is mainly anticipated that the interaction of noisome-cornea enhances free FK506 to permeate through corneal tissue. Moreover, the transcorneal penetration enhancement of niosomes may be due to several mechanisms. First, both alcohol and surfactants poloxamer 188 and lecithin in the formulation acted as penetration enhancers (Fang et al., 2001), the penetration enhancement of surfactants was proved as removing the mucus layer and breaking junctional complexes (Abdelbary and El-Gendy, 2008). Second, the surfactants can significantly improve the solubility of FK506 in the preparation. According to our preliminary experiment, when poloxamer 188 was added in the water-glycerin system, the solubility of FK506 was increased 2742%. The solubilized FK506 was more ready for transport through the cornea. Third, owing to the low surface tension, niosomes had better wetting and spreading properties, which can promote an intimate contact with the lipophilic corneal epithelium, expand the contact surface area, and enhance corneal permeation (Abdelkader et al., 2012). Additionally, the drug retention in the cornea after 24 h administration of FK506 loaded niosomes and ointment was measured, and the niosomes showed significantly higher drug retention (7.31 ± 0.22 lg/ cm2) than ointment (3.71 ± 0.43 lg/cm2) (P < 0.05). The enhancement of niosomes on FK506 permeation would achieve effective therapeutic drug concentration for eye diseases through normal topical administration.
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3.5. Ocular irritation studies
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Ocular irritancy test was carried out on the proniosomederived niosomes to evaluate their acute ocular tolerance and biocompatibility.
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3.5.1. General observation and visual inspection During the experiment, no premature deaths related to the treatment of FK506 loaded niosomes occurred, and no seizures, manifested changes in animal behavior were observed. The scores of irritation reactions of cornea, conjunctiva, and iris were recorded, and the grade of corneal and conjunctival staining was described (data not shown). All the observed scores met the requirements of no irritation, and there was no statistical difference between niosomes treated group and the saline control group.
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Table 4 The loading and entrapment efficiency (%) of FK506 in proniosomal formulations during 3 month storage under different conditions. T (°C)
4 25 40
Drug loading (mg/g)
Entrapment efficiency (%)
Month 0
Month 1
Month 2
Month 3
Month 0
Month 1
Month 2
Month 3
1.00 ± 0.17 1.00 ± 0.17 1.00 ± 0.17
1.08 ± 0.21 0.97 ± 0.18 0.76 ± 0.16
1.00 ± 0.14 0.93 ± 0.16 0.58 ± 0.07
0.97 ± 0.06 0.94 ± 0.08 0.57 ± 0.04
95.34 ± 0.02 95.34 ± 0.02 95.34 ± 0.02
93.21 ± 0.08 93.17 ± 0.03 90.21 ± 0.04
93.37 ± 0.04 92.48 ± 0.07 85.28 ± 0.01
93.37 ± 0.01 93.47 ± 0.02 84.13 ± 0.01
Table 5 The loading and entrapment efficiency (%) of FK506 niosomes during 24 h storage at 37 ± 0.5 °C. T (°C)
37
Drug loading (mg/g)
Entrapment efficiency (%)
0h
8h
16 h
24 h
0h
8h
16 h
24 h
1.00 ± 0.17
1.05 ± 0.18
1.03 ± 0.16
1.06 ± 0.21
95.34 ± 0.02
95.78 ± 0.07
96.12 ± 0.06
95.96 ± 0.03
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Time (h) Fig. 2. In vitro transcorneal permeation of FK506 from proniosome-derived niosomes in comparison with commercial FK506 ointments. Each point represents the mean ± SD from 6 determinations.
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Sodium fluorescein is used by ophthalmologists for initial detection of breaks in the continuity of the epithelium of the cornea. As observed under the slip lamp microscope (Fig. 3), there was no damage to the cornea when FK506 loaded niosomes were continuously administered for 21 days.
3.5.2. Histological inspection To investigate the influence of FK506 loaded niosomes on the corneal cell structure and tissue integrity, cross-sections of cornea, iris, and retina after 21 day of administration were inspected. Compared to the saline control group, the epithelium and stroma structure treated with FK506 loaded niosomes was normal, the corneal structure and integrity were almost visibly unaffected, and no
obvious irritation reaction was observed in the iris and retina (Fig. 4). Together with the general observation, the results proved the good corneal biocompatibility of FK506 loaded niosomes and the formulation was nonirritant for short and long term treatment. Meanwhile, Poloxamer 188 as a polyoxyethylated nonionic surfactant has been widely used in the topical delivery of ophthalmic drugs for the treatment of various ocular disorders (Jiao, 2008). Other components such as lecithin and cholesterol have been demonstrated well biocompatible and safe to the eye tissues with chronic exposure (Gan et al., 2013).
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3.6. In vivo studies
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3.6.1. Evaluation of the rat corneal transplantation survival In the present study, the same inbred strains of rats as previously reported (Fei et al., 2008) were used to examine the immunosuppressive effect of topically applied FK506 loaded niosomes. To determine whether treatment with niosomes could prolong corneal graft survival in rats, 0.1% FK506 loaded niosomes, 1% CsA eye drops, saline, and blank niosomes were respectively administrated on each day after surgery. The opacity, edema, and neovascularization were observed and scored regularly under slim lamp microscope till 16 days after the surgery. On the postoperative day 8, almost all grafts in groups of control and blank niosomes showed rejection (Fig. 5A and B) with severe edema, transparency decline, and corneal angiogenesis penetrating the graft buttons. However, in groups of CsA and FK506 loaded niosomes, almost all grafts exhibited no immune-mediated rejection and remained transparent (Fig. 5C and D). On the postoperative day 16, the corneal neovascularization in groups of control and blank niosomes reached the center of graft buttons, and the grafts in groups of FK506 loaded niosomes and CsA exhibited immunemediated rejection. However, treatment with FK506 loaded
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Fig. 3. Local ocular reaction observed under slit lamp microscope after in vivo instillation of normal saline (control) or 0.1% FK506 loaded niosomes for 21 days.(A) and (B) observed with visible light; (C) and (D) observed with Cobalt blue light after a drop of fluorescein sodium was instilled in the eyes. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 4. Histological cross-sections of rabbit cornea, iris and retina after in vivo instillation of normal saline (control; A and C) or 0.1% FK506 loaded niosomes (B and D) for 21 days (original magnification, 100).
Fig. 5. Rat corneas on day 8 after transplantation observed under the slim lamp microscope. The control groups of normal saline (A) and blank niosomes (B) showed rejection with severe edema, opacity decline, and neovascularization penetrating the transplanted graft; the groups of 1% CsA (C) and 0.1% FK506 loaded niosomes (D) exhibited no rejection, transparent corneal graft, and less neovascularization.
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niosomes apparently delayed the occurrence of graft rejection with less edema in comparison with the controls. Table 6 summarized the mean survival time (MST) of the corneas transplanted in four groups. The grafts in control group underwent clinical rejection on a median of 5–8 days after corneal transplantation with the MST of 6.28 ± 0.42 days. Whilst the MST was 6.25 ± 0.59 days for the blank niosomes group, and 0.1% FK506 loaded niosomes and 1% CsA significantly prolonged the
survival of corneal graft to MST of 13.86 ± 0.80 and 10.57 ± 0.65 days (p < 0.05) respectively. Meanwhile, the survival curves of corneal transplantation treated with FK506 loaded niosomes or CsA groups are significantly different, and both are significantly different from that of the control group (p < 0.05, Fig. 6). The mean clinical scores for all four groups after corneal transplantation were graphed in Fig. 7. Corneal allografts in rats treated with FK506 loaded niosomes appeared much less edematous,
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Table 6 Mean survival time (MST) of rat corneal transplantation prolonged in each group.
a
Groups
Survival time (days)
MST ± SD
Median (max/min, day)
No. of rats
Control Blank niosomes CsA FK506 loaded niosomes
5, 5, 6, 6, 7, 8, 7 4, 5, 5, 6, 6, 7, 8,9 8, 9, 10, 11, 11, 12, 13 10, 12, 14, 15, 15, 15, 16
6.29 ± 0.42 6.25 ± 0.59 10.57 ± 0.65 13.86 ± 0.80
6 (5/8) 6 (4/9) 11 (8/13) 15 (10/16)
7 8 7 7
One rat was excluded from the study for each of the control, CsA, and FK506 loaded niosomes groups due to technical complications.
Fig. 6. Survival curves of SD rats taken corneal transplantation with graft buttons from wistar rats. Treatment of 1% CsA or 0.1% FK506 loaded niosomes significantly prolonged the survival of corneal grafts as compared with the control (P < 0.05). The prolongation effect of FK506 loaded niosomes was even significantly better than that of CsA (P < 0.05).
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opaque, and neovascularized at all experimental time points after transplantation. All grafts in groups of control and blank niosomes exhibited rejection after 9 days. In comparison with the control,
the mean clinical scores for grafts in groups of 0.1% FK506 loaded niosomes and 1% CsA were significant lower till postoperative day 16 (p < 0.05, Fig. 7A). And, the opacity and edema diminished to significant lower levels for grafts in groups of FK506 loaded niosomes and CsA till postoperative day 20 (p < 0.05, Fig. 7B and C). The neovascularization score for grafts in group of FK506 loaded niosomes showed a significant decrease till postoperative day 7 (p < 0.05, Fig. 7D).
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4. Conclusions
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Stable proniosomal formulation containing FK506 was prepared and successfully reconstituted to niosomes with drug entrapment efficiency up to 95.34% (w/w). The nonirritant niosomes enhanced precorneal permeation and retention for FK506 in vitro, and prolonged the corneal graft survival and showed practical anti-allograft rejection efficacy in vivo on corneal transplantation rats. Therefore, the proniosomes-derived niosomes may be a promising vehicle for effective ocular drug delivery of FK506. Further works, such as the mechanism of FK506 permeation into corneal tissue and in vivo topical ocular distribution of FK506, need to be carried out in the near future.
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Fig. 7. Average rejection score for four groups. There were significant differences in the mean total scores (A), opacity (B), edema (C), and neovascularization (D) between the control and 0.1% FK506 loaded niosomes groups (P < 0.05).
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Proniosome-derived niosomes for tacrolimus topical ocular delivery: In vitro cornea permeation, ocular irritation, and in vivo anti-allograft rejection
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Qi Li a,1, Zhanrong Li b,1, Weidong Zeng a, Shumin Ge a, Haoyang Lu a, Chuanbin Wu a, Li Ge a, Dan Liang b, Yuehong Xu a,⇑ a b
School of Pharmaceutical Science, Sun Yat-sen University, Guangzhou 510006, China State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060, China
a r t i c l e
i n f o
Article history: Received 4 February 2014 Received in revised form 23 March 2014 Accepted 26 May 2014 Available online xxxx Keywords: Tacrolimus Proniosome Niosome Ocular delivery Allograft rejection
a b s t r a c t The objective of this study was to develop proniosome-derived niosomes for topical ophthalmic delivery of Tacrolimus (FK506). The FK506 loaded proniosomes containing poloxamer 188 and lecithin as surfactants, cholesterol as a stabilizer, and minimal amount of ethanol and trace water reconstituted to niosomes prior to use. The stability of FK506 loaded proniosomes was assessed, and the morphology, size, zeta potential, surface tension, and entrapment efficiency of the derived niosomes were characterized, indicating they were feasible for instillation in the eyes. The in vitro permeation of FK506 through the freshly excised rabbit cornea, the cumulative permeation amount of FK506 from niosomes, and the drug retention in the cornea all exhibited significant increase as compared to 0.1% FK506 commercial ointments. The in vivo ocular irritation test of 0.1% FK506 loaded niosomes instilled 4 times per day in rat eyes for 21 consecutive days showed no irritation and good biocompatibility with cornea. The in vivo anti-allograft rejection assessment was performed in a Sprague–Dawley (SD) rat corneal xenotransplantation model. The results showed treatment with 0.1% FK506 loaded niosomes delayed the occurrence of corneal allograft rejection and significantly prolonged the median survival time of corneal allografts to13.86 ± 0.80 days as compared with those treated with 1% Cyclosporine (CsA) eye drops, drug-free niosomes, or untreated. In conclusion, the proniosome-derived niosomes may be a promising vehicle for effective ocular drug delivery of FK506. Ó 2014 Published by Elsevier B.V.
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1. Introduction Corneal allograft is clinically performed to improve the vision of patients who suffer from damaged or diseased corneas. However, corneal graft failure is commonly caused by allograft rejection (Coster et al., 2005). Although steroids are normally used to suppress the rejection reaction, the occurrence of allograft rejection still keeps high as 10–30% in the patients, and commonly followed with the secondary complications such as glaucoma, cataract, and systemic infection (Lindstrom, 1986). Tacrolimus (FK506), an immunosuppressive agent, can inhibit the action of enzyme calcineurin phosphatase and the transcription of IL-2 gene, resulting in suppression of T-lymphocyte response (Thomson et al., 1995). It can efficiently prevent post-transplant rejection in patients who are resistant to steroids and cyclosporine A (Scott et al., 2003). ⇑ Corresponding author. Tel./fax: +86 20 39943119. 1
E-mail address: [email protected] (Y. Xu). The authors contributed equally to the present work.
By far, FK506 has been topically administered to prevent the rejection of penetrating keratoplasty (Fei et al., 2008; Hikita et al., 1997; Reinhard et al., 2005; Sloper et al., 2001) and treat the intractable allergic conjunctivitis (Attas-Fox et al., 2008) and refractory inflammatory ocular surface diseases with immunologic causes (Lee et al., 2013). At present, FK506 ointment (0.1% and 0.03%, w/w), which is known as ProtopicÒ (Astellas Toyama Co., Ltd., Toyama Plant, Japan), has been approved by the US Food and Drug Administration (FDA) for treating moderate to severe atopic dermatitis (Ruzicka et al., 1997). But no ophthalmic preparation of FK506 is commercially available, and the dermatological FK506 ointment was clinically used instead of ophthalmic preparation in some reports (AttasFox et al., 2008; Lee et al., 2013; Miyazaki et al., 2008). Therefore, it is urgent to develop an ophthalmic delivery system for FK506 for its application in preventing and treating eye diseases. Owing to its highly hydrophobic characteristic and high molecular weight (822.5 D), FK506 had difficulties in penetrating the cornea and reaching the effective therapeutic intraocular level.
http://dx.doi.org/10.1016/j.ejps.2014.05.020 0928-0987/Ó 2014 Published by Elsevier B.V.
Please cite this article in press as: Li, Q., et al. Proniosome-derived niosomes for tacrolimus topical ocular delivery: In vitro cornea permeation, ocular irritation, and in vivo anti-allograft rejection. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.05.020
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Moreover, its poor water stability hinders formulating it with massive aqueous medium into eye drops. Therefore, the ideal delivery system for FK506 should not only enhance drug penetration through the cornea but also maintain its stability and reduce its undesirable side effects such as alterations in kidney function and glucose metabolism, neurotoxicity and susceptibility to infection or malignancy with systemic administration (Fung et al., 1991). Some delivery systems including liposomes (Pleyer et al., 1993; Zhang et al., 2010; Dai et al., 2013), nanospheric suspensions (Fei et al., 2008), in situ nanosuspensions (Luschmann et al., 2013), nanoparticles (Nagarwal et al., 2012) and nanoemulsions (Garg et al., 2013) have been investigated for potential ocular application. Niosomes which are made of nonionic surfactants and lipids can load either hydrophilic or hydrophobic drugs. They also possess many advantages over other vesicular drug delivery systems, such as low production cost, low toxicity, and ease of formulation without unacceptable solvents (Choi and Maibach, 2005; Sankar et al., 2010). Moreover, the nonionic surfactants used in niosomes can increase the bioavailability of poor water soluble drugs by enhancing the solubility and permeability through the biological membrane. It was reported that niosomes could provide a prolonged and controlled action at the corneal surface and prevent the drug metabolism by enzymes present at the tear/corneal surface (Abdelbary and El-Gendy, 2008). Several studies have demonstrated the successful use of niosomes as a potential ocular drug delivery system for drugs such as cyclopentolate, acetazolamide, Q2 timolol maleate, gentamicin, and naltrexone (Abdelbary and Elgendy, 2008; Abdelkader et al., 2012; Aggarwal and Kaur, 2005; Guinedi et al., 2005). However, niosomes also exhibit physical and chemical instability during storage, such as aggregation, sedimentation, fusion and leakage or hydrolysis of encapsulated drugs, Q3 which may affect the shelf life of dispersion (Hu and Rhodes, 2000). Therefore, the latest approach in vesicular delivery system is to take the provesicular approach to format ‘‘proniosomes’’ (Ammar et al., 2011; Sankar et al., 2010), which are liquid crystalline-compact niosomal hybrids and will convert into niosomes upon hydration with water prior to administration. Proniosomes are generally present in a stable semisolid gel structure, which can prevent the hydrolysis of encapsulated drug during storage and transport. Many studies have shown that proniosomes might be a promising drug delivery system via transdermal route (Ei-Laithy et al., 2011; Fang et al., 2001; Jukanti et al., 2011; Vora et al., 1998). Proniosomes can be hydrated by water from the skin to form niosomes, and both phospholipids and non-ionic surfactants in proniosomes can act as penetration enhancers (Ammar et al., 2011; Sankar et al., 2010). These properties may also make proniosomes potential vehicles for topical ophthalmic drug delivery. Therefore, the aim of the present work was to develop a proniosome system to reconstitute niosomes for ophthalmic delivery of FK506. The stability of FK506 loaded proniosomes was evaluated, and the niosomes were characterized in terms of morphology, vesicle size and size distribution, zeta potential, surface tension, and entrapment efficiency. Permeation of the niosomes through rabbit cornea was evaluated in vitro and ocular irritation in rabbits was assessed in vivo. Furthermore, the in vivo anti-allograft rejection of the FK506 loaded niosomes was evaluated in a mode of corneal xenotransplantation.
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2. Materials and methods
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2.1. Chemicals
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FK506 was purchased from Teva Czech Industries (S.R.O. Ostravska 29305, 74770 Opava-Komarov, Czech Republic). Soybean phosphatidylcholine (SPC, Lipoid S100, purity >98%) was obtained
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from Lipoid GmbH (Ludwigshafen, Germany). Cholesterol (CHOL, Tian Ma Chemical Plant, Guangzhou), Poloxamer 188 (BASF, Ludwigshafen, Germany), 0.1% FK506 commercial ointment (ProtopicÒ, Astellas Toyama Co., Ltd. Toyama Plant, Japan), and 1% Cyclosporine (CsA) eye drops (Zhongshan Ophthalmic Center, Guangzhou) were used in the study. Other reagents were of analytical grade.
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2.2. Animals
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New Zealand albino rabbits (male and female, weighing 2–3 kg) were provided by the Animal Experimental Center of Zhongshan Ophthalmology Center in Sun Yat-sen University. Wistar (female, weighing 200–220 g, 6–8 weeks old) and Sprague–Dawley (SD) rats (female, weighing 200–220 g, 6–8 weeks old) were obtained from Guangzhou Animal Testing Center. The animals were housed under standard condition (temperature of 25–28 °C, humidity of 40–60% RH, and light/dark cycle of 12/12 h) prior to operation and fed with a standard pellet diet and water at free. The protocols for animal use and care were approved by the Institutional Animal Care and Use Committee of Zhongshan Ophthalmic Center, Sun Yat-sen University.
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2.3. Preparation of FK506 loaded proniosomes and reconstruction of niosomes
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Proniosomes were prepared according to a modified literature Q4 method (Vora et al., 1998). Briefly, poloxamer 188, lecithin, and cholesterol in a ratio of 9:9:1 (w/w/w) were mixed with the required amount of ethanol in a glass tube. Then the required amount of FK506 was added in and the tube was warmed in a water bath at 65 ± 2 °C for 10 min. Afterwards, the phosphate buffer saline (pH 7.4) was dropped into the tube which was kept in the water bath for about 5 min until a clear solution formed. The solution was cooled down at room temperature until the proniosomal gel formed. Blank proniosomes were prepared similarly but without addition of FK506. Prior to application, 0.1% FK506 loaded niosomes were reconstituted by adding 10 ml of normal saline solution to 0.2 g of proniosomes and shaking for 3 min.
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2.4. Characterization of FK506 loaded niosomes
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2.4.1. Transmission electron microscopy (TEM) The morphological characteristics of the proniosome-derived niosomes were examined using transmission electron miroscropy (JEM1400, Tokyo, Japan). Briefly, a drop of niosomes was applied onto a collodion coated 300 mesh copper grid and remained for 1 min to allow the niosomes to adhere to the collodion. Then, a drop of 2% uranyl acetate solution was applied for staining, and the grid was air dried at room temperature and examined by TEM (Abd-Elbary et al., 2008).
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2.4.2. Particle size, size distribution and zeta potential The particle size, polydispersity index (PDI) and zeta potential of niosomes were determined at 25 °C by photon correlation spectroscopy at the scattering angle of 90° equipped with a Zetasizer (Malvern Instruments Nano ZS90, Worcestershire, UK). Each sample was measured in triplicate.
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2.4.3. Surface tension The surface tension of FK506 loaded niosomes was measured at room temperature using an interfacial tension meter (Hengping Instrument, Shanghai, China), and the FK506 aqueous suspension and normal saline solution were used as the controls. All measurements were carried out in triplicate.
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2.4.4. Entrapment efficiency Free FK506 was separated from FK506 loaded niosomes by centrifugation at a speed of 14000 rpm for 30 min at 4 °C (Ultra centrifuge, Thermo, USA) (Hu and Rhodes, 1999). The amount of free FK506 in the supernatant was determined by High Performance Liquid Chromatography (HPLC). The entrapment efficiency (EE) of FK506 in niosomes was calculated as follows (Rajera et al., 2011a):
EE ¼ ðC t C f Þ=C t 100%
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where Ct and Cf was the concentration of total FK506 and free FK506, respectively.
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2.5. Stability
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The variations of drug loading and entrapment efficiency during storage were monitored to determine the stability of the FK506 loaded proniosomes, which may be affected by aggregation, fusion or leakage of the vesicles, and storage temperature. The prepared FK506 loaded proniosomes were stored in glass vials and kept at 4 ± 2 °C/75 ± 5%RH; 25 ± 2 °C/60 ± 5%RH; 40 ± 2 °C/75 ± 5%RH, respectively as per ICH guidelines. The drug loading and entrapment efficiency were determined after preparation, 1, 2, and 3 months of storage. Moreover, the 1 day for 24 h storage stability of reconstituted FK506 loaded niosomes at 37 ± 0.5 °C has been measured because the niosomes were kept for 24 h for administration or in vitro permeation.
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2.6. In vitro permeation through excised rabbit cornea
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In vitro corneal permeation study was carried out using a modified Franz diffusion cell suitable for cornea with an effective permeation area of 50.24 mm2. Rabbits were sacrificed by overdose of anesthesia. The fresh corneas were excised from the globes and carefully mounted onto the receptor cell with the outside of cornea facing the donor cell. Based on the preliminary experiment (data not shown), 5 ml of 25% ethanol/artificial tears (v/v) were used as receptor medium to maintain a sink condition for the release of FK506. The medium was continuously stirred at 250 rpm with a magnetic bar and maintained at 37 ± 0.5 °C with a circulating water bath. Niosomes and the commercial ointment (ProtopicÒ) containing equal amount of FK506 were carefully spread onto the corneal surface, respectively. At 0.5, 1, 2, 3, 4, 6, 12, 24 h, 1 ml of the receiving medium was withdrawn and replaced with equal volume of fresh receiving medium. The experiments were performed in triplicate and all the samples were analyzed with HPLC to determine the content of FK506.
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2.7. FK 506 content
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HPLC was employed to determine FK506 content in all the samples. The HPLC system (Agilent 1100 series) with a C8 column (GraceÒ, 250 mm 4.6 mm, 5 lm) was equipped with a UV detector (G1314A VWD) and data processing software (Agilent Chem Station for LC systems) for FK506 analysis at wavelength of 220 nm. The mobile phase was water/isopropyl alcohol/tetrahydrofuran (5:2:2, v/v/v) at a flow rate of 0.8 ml/min at 55 °C.
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2.8. Ocular irritation evaluation
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Six New Zealand albino rabbits were used to evaluate the ocular irritation by the Draize eye test (Wilhelmus, 2001) and histological examination. Before drug administration, bilateral eyes of the animals were inspected with 2% fluorescein sodium to make sure free of irritation, corneal defects, or conjunctival damage. Then, the left eye of each rabbit was administered with 100 ll of 0.1% FK506 loaded niosomes 4 times a day for a period of
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21 days, while the right eye was administered with normal saline solution 4 times a day as the untreated control. Ocular response of a single dose was observed at 1, 2, and 4 h after the first instillation. The cumulative ocular response was observed every 24 h after four administrations daily for a consecutive 21 days, as well as 1, 2, 4, and 24 h after the last instillation. The rabbit eyes were inspected with a slit lamp microscope (SL 120; Carl Zeiss AG, Oberkochen, Germany) for local ocular reaction. Irritation was scored using the method of Draize, and the responses were evaluated according to Table 1 (Gettings et al., 1996) and reported as the maximum average score. Upon completion of eye irritation study, all the rabbits were sacrificed with an overdose injection of ketamine. The eye balls were immediately enucleated and fixed in 10% formalin solution. Sections were made from the paraffin blocks at a thickness of 5 lm, and stained by hematoxylin and eosin. Histologic examination was performed with a light microscopy (T8-100, Nikon, Japan) for the changes of cornea, iris, and retina components of the tissue.
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2.9. In vivo assessment of anti-allograft rejection
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2.9.1. Corneal transplantation Wistar and Sprague–Dawley (SD) rats were used for corneal transplant, 32 female Wistar rats served as donors and 32 female SD rats were recipients. Full thickness corneal transplants were performed as previously reported (Holland et al., 1991b). Briefly, the donor rats were sacrificed by an overdose of chlorpromazine, while the recipient rats were deeply anesthetized with ketamine (25 mg/kg) and chlorpromazine (25 mg/kg) by intra peritoneal injection before surgical procedure. Compound tropicamide eye drops (Shenyang Sinqi Pharma. Co., Ltd.) were topically applied to dilate the pupil of the recipient right eye, where only the right cornea was transplanted. The corneal button was excised from the donor rat with a 3.5-mm trephine and Vannas scissors, and the graft was placed into chilled PBS until use. The donor cornea was secured by 8 stitches of interrupted 10–0 nylon suture (Alcon Laboratories, Fort Worth, TX) into the 3.0 mm recipient bed. The suture knot was not buried and kept in place postoperatively in order to stimulate the corneal neovascularization. Antibiotic ointment (Ofloxacin, Floxal™, Mann Pharma, Germany) was applied immediately to the eye after transplantation. The corneal suture was not removed during the entire duration of the experiment. The transplanted rats were randomly divided into four treatment groups (eight rats per group) as follows: control group treated with a drop of normal saline; blank group treated with blank proniosomed-derived niosomes without FK506; Cyclosporine (CsA) group treated with a drop of 1% CsA eye drops, and FK506 group treated with 0.1% FK506 loaded niosome eye drops. The frequency of administration in each group was four times a day after transplantation.
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2.9.2. Clinical assessment of graft survival After surgery, the transplants were examined daily for occurrence of graft rejection under the slit lamp microscopy until the rejection occurred as previously reported (Fei et al., 2008). The grafts were evaluated according to the Holland’s scoring system
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Table 1 Evaluation criteria for eye irritation reaction. Scores
Evaluation
0–3 4–8 9–12 13–16
Nonirritant Slightly irritant Moderate irritant Severe irritant
Please cite this article in press as: Li, Q., et al. Proniosome-derived niosomes for tacrolimus topical ocular delivery: In vitro cornea permeation, ocular irritation, and in vivo anti-allograft rejection. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.05.020
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described previously (Holland et al., 1991; Pan et al., 2003), in which the corneal opacity, edema, and neovascularization were taken into account (Table 2). Corneal grafts which showed technical complications such as infection, cataract, and intraocular hemorrhage were excluded from the study. Corneas with combined scores of 6 or higher were considered as rejected.
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2.10. Statistical analysis
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All experiments were performed at least in triplicate. Comparison between two groups was analyzed by Student’s t-test using IBM SPSS Statistic 19 software (SPSS Inc., Chicago, IL, USA). Data were reported as mean ± SD, and P < 0.05 was considered as statistically significant difference.
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3. Results and discussion
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3.1. Preparation of proniosomes and reconstitution of niosomes
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Excipients suitable for ophthalmic application were used to prepare FK506 loaded proniosomes for topical ophthalmic drug delivery. Poloxamer 188 and lecithin with excellent ocular compatibility were chosen as the surfactants (Jiao, 2008). Cholesterol was the important component of the cell membrane and its incorporation in the vesicle is known to improve the stability and permeability (Fang et al., 2001a). The surfactants and cholesterol were mixed with alcohol and aqueous phase to form the FK506 loaded semisolid proniosomal gel. The proniosomes can spontaneously convert to a niosomal dispersion upon dilution with excess aqueous phase under gentle shaking. The mechanism of proniosome formation may be as following: lamellar liquid crystal will form from lecithin and the non-ionic surfactant at krafft temperature in the presence of alcohol, and the crystalline phase will convert to dispersion of niosomes with excess water (Bai and Abbott, 2011). The organization of lipid/ethanol/water mixture in the lamellar structure can be conveniently utilized for ophthalmic delivery of drugs. This method avoids the use of pharmaceutically unacceptable solvents and is easy to scale up (Vora et al., 1998). The proniosome gel product can be hydrated immediately before use so that it may avoid the stability problems associated with aqueous niosome dispersions such as fusion, aggregation, and leakage.
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3.2. Characterization of FK506 loaded niosomes
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As observed from TEM images (Fig. 1), the niosomes derived from proniosomes were dispersed, spherical particles in the diameter of about 1 lm. Consistently, the niosome mean size of 1.33 ± 0.32 lm with polydispersity index (PDI) of 0.21 ± 0.03 were measured by the dynamic light scattering particle size analyzer (Table 3). The niosomes showed a negative zeta potential of about 8 mV because of the application of nonionic surfactants. The surface tension of the FK506 loaded niosomes (39.13 ± 0.35 dynes/cm) was significantly lower than those of the FK506 aqueous suspension (56.50 ± 0.26 dynes/cm) and normal saline solution (72.13 ± 0.06 dynes/cm) (p < 0.001). The lower surface tension made the niosomes easily wet the hydrophobic surface of the corneal epithelium and the lipid layer of the precorneal tear film, which may contribute to a better spreading ability and enhance the permeation of the formulation (Pawar and Majumdar, 2006). The amount of FK506 loaded in the niosomes was 1.00 ± 0.17 mg/g (w/w). The entrapment efficiency of FK506 in the proniosome-derived niosomes was 95.34 ± 0.02% (Table 3), which indicated that FK506 was effectively entrapped in the niosomes. The high entrapment efficiency may be because the highly lipophilic drug tends to intercalate almost completely within the lipid bilayer of niosomes (Gulati et al., 1998). Also, lecithin as a penetration enhancer in the formulation could increase the entrapment efficiency due to its high phase transition temperature preventing drug leakage from the vesicles. Cholesterol as a stabilizing surfactant also enhanced the stability of bilayer membrane by increasing gel liquid transition temperature and preventing drug leakage from the bilayer (Hsieh et al., 2013).
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3.3. Stability studies
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Physical stability studies were carried out to investigate the leakage of drug from proniosomes during storage under different conditions. After 3 months of storage at 4 °C, 25 °C, and 40 °C, the proniosomes remained visually unchanged and the loading of FK506 in derived niosomes changed from initial 1.00 ± 0.17 mg/g to 0.97 ± 0.06, 0.94 ± 0.08, and 0.57 ± 0.04 mg/g, respectively (Table 4). It indicates that 4 °C is the favorable storing condition for FK506 loaded proniosomes. Additionally, the entrapment efficiency of FK506 in niosomes only slightly dropped from initial
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Table 2 Clinical scoring system for the corneal transplantation rejection. Score
Clinical finding
Opacity 0 1 2 3 4
Clear cornea Slight haze, details of iris clearly visible Increased haze, some details of iris no longer visible Advanced haze, pupil still recognizable Opaque cornea without view of anterior chamber
Edema 0 1 2 3 4
No edema Mild stromal thickness Diffuse stromal edema Pronounced edema with small bleb of epithelium Bullous keratopathy
Neovascularization 0 1 2 3 4
No neovascularization Neovascularization of peripheral cornea Neovascularization appearing in the graft periphery Neovascularization extending deeper Neovascularization extending to the entire graft
a
The sum of scores of opacity, edema and neovascularization was termed as rejection index (RI). With RI of 6 or greater, the cornea was considered as rejected.
Fig. 1. Representative TEM images of proniosome-derived niosomes.
Please cite this article in press as: Li, Q., et al. Proniosome-derived niosomes for tacrolimus topical ocular delivery: In vitro cornea permeation, ocular irritation, and in vivo anti-allograft rejection. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.05.020
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Q. Li et al. / European Journal of Pharmaceutical Sciences xxx (2014) xxx–xxx Table 3 Characterization of FK506 loaded niosomes. Data represent mean ± SD (n = 3). Appearance
Size (lm)
Polydispersity index
Zeta potential (mV)
Entrapment efficiency (%)
Drug content (mg/ml)
Clear solution
1.33 ± 0.32
0.21 ± 0.03
8.25 ± 0.16
95.34 ± 0.02%
1.00 ± 0.17
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95% to 93% after 3 month storing at 4 °C. The ability of proniosomes-derived niosomes to retain the entrapment efficiency is in accordance with the previous report (Ammar et al., 2011). For 1 day of 24 h storage stability of FK506 loaded noisome (Table 5), the changes of drug loading and the entrapment efficiency of FK506 indicated that it was stable when being administered for 1 day or in vitro permeation study.
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3.4. In vitro permeation through rabbit corneal
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The corneal and conjunctival epithelial barriers cover the ocular surface and limit the access of drug to the targets. In vitro transcorneal permeation was conducted through the isolated rabbit corneal and the efficiency of the niosomes to enhance the corneal penetration of FK506 was assessed by measuring the transcorneal flux and the cumulative permeation amount. The reasons that the dermal preparation ProtopicÒ was chosen to be the control were as follows: First, dermal preparation ProtopicÒ was the only topical delivery formulation approved by US FDA and there are many reports that the dermatological FK506 ointment was topically used in clinic for treating different eye diseases (Attas-Fox et al., 2008; Joseph et al., 2005; Lee et al., 2013; Miyazaki et al., 2008; Ryu et al., 2012; Virtanen et al., 2006). We chose it as the control of a commercial preparation. Second, owing to highly hydrophobic characteristic of tacrolimus, it is very difficult to dispense it in aqueous medium to form a relatively uniform suspension without adding surfactant or organic solvent. However, if adding surfactant or organic solvent to medium enhances the dispensing, the permeation of tacrolimus will be affected by the surfactant or organic solvent. Taken together, the dermal preparation ProtopicÒ was chosen to be the control. The cumulative amount of FK506 permeated from 0.1% FK506 loaded niosomes through the cornea was significantly greater than that from 0.1% (w/w) FK506 commercial ointment ProtopicÒ throughout the entire experiment (P < 0.05, Fig. 2). After 24 h permeation, the cumulative permeation of FK506 from niosomes (117.36 ± 8.38 lg/cm2) was significantly greater than that from ProtopicÒ (70.88 ± 5.78 lg/cm2), which demonstrated that niosomes significantly enhanced the corneal penetration of FK506. The transcorneal flux of niosomes and ointment was 3.84 and 2.30 lg/cm2/h, respectively, confirming the permeation enhancement of niosomes. In present study, the size of FK506 loaded niosome is about 1.33 lm, the relatively large size
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makes it difficult permeate through excised rabbit cornea in intact form, the mechanism of FK506 permeation into corneal tissue is mainly anticipated that the interaction of noisome-cornea enhances free FK506 to permeate through corneal tissue. Moreover, the transcorneal penetration enhancement of niosomes may be due to several mechanisms. First, both alcohol and surfactants poloxamer 188 and lecithin in the formulation acted as penetration enhancers (Fang et al., 2001), the penetration enhancement of surfactants was proved as removing the mucus layer and breaking junctional complexes (Abdelbary and El-Gendy, 2008). Second, the surfactants can significantly improve the solubility of FK506 in the preparation. According to our preliminary experiment, when poloxamer 188 was added in the water-glycerin system, the solubility of FK506 was increased 2742%. The solubilized FK506 was more ready for transport through the cornea. Third, owing to the low surface tension, niosomes had better wetting and spreading properties, which can promote an intimate contact with the lipophilic corneal epithelium, expand the contact surface area, and enhance corneal permeation (Abdelkader et al., 2012). Additionally, the drug retention in the cornea after 24 h administration of FK506 loaded niosomes and ointment was measured, and the niosomes showed significantly higher drug retention (7.31 ± 0.22 lg/ cm2) than ointment (3.71 ± 0.43 lg/cm2) (P < 0.05). The enhancement of niosomes on FK506 permeation would achieve effective therapeutic drug concentration for eye diseases through normal topical administration.
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3.5. Ocular irritation studies
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Ocular irritancy test was carried out on the proniosomederived niosomes to evaluate their acute ocular tolerance and biocompatibility.
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3.5.1. General observation and visual inspection During the experiment, no premature deaths related to the treatment of FK506 loaded niosomes occurred, and no seizures, manifested changes in animal behavior were observed. The scores of irritation reactions of cornea, conjunctiva, and iris were recorded, and the grade of corneal and conjunctival staining was described (data not shown). All the observed scores met the requirements of no irritation, and there was no statistical difference between niosomes treated group and the saline control group.
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Table 4 The loading and entrapment efficiency (%) of FK506 in proniosomal formulations during 3 month storage under different conditions. T (°C)
4 25 40
Drug loading (mg/g)
Entrapment efficiency (%)
Month 0
Month 1
Month 2
Month 3
Month 0
Month 1
Month 2
Month 3
1.00 ± 0.17 1.00 ± 0.17 1.00 ± 0.17
1.08 ± 0.21 0.97 ± 0.18 0.76 ± 0.16
1.00 ± 0.14 0.93 ± 0.16 0.58 ± 0.07
0.97 ± 0.06 0.94 ± 0.08 0.57 ± 0.04
95.34 ± 0.02 95.34 ± 0.02 95.34 ± 0.02
93.21 ± 0.08 93.17 ± 0.03 90.21 ± 0.04
93.37 ± 0.04 92.48 ± 0.07 85.28 ± 0.01
93.37 ± 0.01 93.47 ± 0.02 84.13 ± 0.01
Table 5 The loading and entrapment efficiency (%) of FK506 niosomes during 24 h storage at 37 ± 0.5 °C. T (°C)
37
Drug loading (mg/g)
Entrapment efficiency (%)
0h
8h
16 h
24 h
0h
8h
16 h
24 h
1.00 ± 0.17
1.05 ± 0.18
1.03 ± 0.16
1.06 ± 0.21
95.34 ± 0.02
95.78 ± 0.07
96.12 ± 0.06
95.96 ± 0.03
Please cite this article in press as: Li, Q., et al. Proniosome-derived niosomes for tacrolimus topical ocular delivery: In vitro cornea permeation, ocular irritation, and in vivo anti-allograft rejection. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.05.020
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Cumulative permeated amount (µg/cm2)
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Time (h) Fig. 2. In vitro transcorneal permeation of FK506 from proniosome-derived niosomes in comparison with commercial FK506 ointments. Each point represents the mean ± SD from 6 determinations.
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Sodium fluorescein is used by ophthalmologists for initial detection of breaks in the continuity of the epithelium of the cornea. As observed under the slip lamp microscope (Fig. 3), there was no damage to the cornea when FK506 loaded niosomes were continuously administered for 21 days.
3.5.2. Histological inspection To investigate the influence of FK506 loaded niosomes on the corneal cell structure and tissue integrity, cross-sections of cornea, iris, and retina after 21 day of administration were inspected. Compared to the saline control group, the epithelium and stroma structure treated with FK506 loaded niosomes was normal, the corneal structure and integrity were almost visibly unaffected, and no
obvious irritation reaction was observed in the iris and retina (Fig. 4). Together with the general observation, the results proved the good corneal biocompatibility of FK506 loaded niosomes and the formulation was nonirritant for short and long term treatment. Meanwhile, Poloxamer 188 as a polyoxyethylated nonionic surfactant has been widely used in the topical delivery of ophthalmic drugs for the treatment of various ocular disorders (Jiao, 2008). Other components such as lecithin and cholesterol have been demonstrated well biocompatible and safe to the eye tissues with chronic exposure (Gan et al., 2013).
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3.6. In vivo studies
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3.6.1. Evaluation of the rat corneal transplantation survival In the present study, the same inbred strains of rats as previously reported (Fei et al., 2008) were used to examine the immunosuppressive effect of topically applied FK506 loaded niosomes. To determine whether treatment with niosomes could prolong corneal graft survival in rats, 0.1% FK506 loaded niosomes, 1% CsA eye drops, saline, and blank niosomes were respectively administrated on each day after surgery. The opacity, edema, and neovascularization were observed and scored regularly under slim lamp microscope till 16 days after the surgery. On the postoperative day 8, almost all grafts in groups of control and blank niosomes showed rejection (Fig. 5A and B) with severe edema, transparency decline, and corneal angiogenesis penetrating the graft buttons. However, in groups of CsA and FK506 loaded niosomes, almost all grafts exhibited no immune-mediated rejection and remained transparent (Fig. 5C and D). On the postoperative day 16, the corneal neovascularization in groups of control and blank niosomes reached the center of graft buttons, and the grafts in groups of FK506 loaded niosomes and CsA exhibited immunemediated rejection. However, treatment with FK506 loaded
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Fig. 3. Local ocular reaction observed under slit lamp microscope after in vivo instillation of normal saline (control) or 0.1% FK506 loaded niosomes for 21 days.(A) and (B) observed with visible light; (C) and (D) observed with Cobalt blue light after a drop of fluorescein sodium was instilled in the eyes. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article in press as: Li, Q., et al. Proniosome-derived niosomes for tacrolimus topical ocular delivery: In vitro cornea permeation, ocular irritation, and in vivo anti-allograft rejection. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.05.020
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Fig. 4. Histological cross-sections of rabbit cornea, iris and retina after in vivo instillation of normal saline (control; A and C) or 0.1% FK506 loaded niosomes (B and D) for 21 days (original magnification, 100).
Fig. 5. Rat corneas on day 8 after transplantation observed under the slim lamp microscope. The control groups of normal saline (A) and blank niosomes (B) showed rejection with severe edema, opacity decline, and neovascularization penetrating the transplanted graft; the groups of 1% CsA (C) and 0.1% FK506 loaded niosomes (D) exhibited no rejection, transparent corneal graft, and less neovascularization.
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niosomes apparently delayed the occurrence of graft rejection with less edema in comparison with the controls. Table 6 summarized the mean survival time (MST) of the corneas transplanted in four groups. The grafts in control group underwent clinical rejection on a median of 5–8 days after corneal transplantation with the MST of 6.28 ± 0.42 days. Whilst the MST was 6.25 ± 0.59 days for the blank niosomes group, and 0.1% FK506 loaded niosomes and 1% CsA significantly prolonged the
survival of corneal graft to MST of 13.86 ± 0.80 and 10.57 ± 0.65 days (p < 0.05) respectively. Meanwhile, the survival curves of corneal transplantation treated with FK506 loaded niosomes or CsA groups are significantly different, and both are significantly different from that of the control group (p < 0.05, Fig. 6). The mean clinical scores for all four groups after corneal transplantation were graphed in Fig. 7. Corneal allografts in rats treated with FK506 loaded niosomes appeared much less edematous,
Please cite this article in press as: Li, Q., et al. Proniosome-derived niosomes for tacrolimus topical ocular delivery: In vitro cornea permeation, ocular irritation, and in vivo anti-allograft rejection. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.05.020
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Table 6 Mean survival time (MST) of rat corneal transplantation prolonged in each group.
a
Groups
Survival time (days)
MST ± SD
Median (max/min, day)
No. of rats
Control Blank niosomes CsA FK506 loaded niosomes
5, 5, 6, 6, 7, 8, 7 4, 5, 5, 6, 6, 7, 8,9 8, 9, 10, 11, 11, 12, 13 10, 12, 14, 15, 15, 15, 16
6.29 ± 0.42 6.25 ± 0.59 10.57 ± 0.65 13.86 ± 0.80
6 (5/8) 6 (4/9) 11 (8/13) 15 (10/16)
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One rat was excluded from the study for each of the control, CsA, and FK506 loaded niosomes groups due to technical complications.
Fig. 6. Survival curves of SD rats taken corneal transplantation with graft buttons from wistar rats. Treatment of 1% CsA or 0.1% FK506 loaded niosomes significantly prolonged the survival of corneal grafts as compared with the control (P < 0.05). The prolongation effect of FK506 loaded niosomes was even significantly better than that of CsA (P < 0.05).
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opaque, and neovascularized at all experimental time points after transplantation. All grafts in groups of control and blank niosomes exhibited rejection after 9 days. In comparison with the control,
the mean clinical scores for grafts in groups of 0.1% FK506 loaded niosomes and 1% CsA were significant lower till postoperative day 16 (p < 0.05, Fig. 7A). And, the opacity and edema diminished to significant lower levels for grafts in groups of FK506 loaded niosomes and CsA till postoperative day 20 (p < 0.05, Fig. 7B and C). The neovascularization score for grafts in group of FK506 loaded niosomes showed a significant decrease till postoperative day 7 (p < 0.05, Fig. 7D).
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4. Conclusions
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Stable proniosomal formulation containing FK506 was prepared and successfully reconstituted to niosomes with drug entrapment efficiency up to 95.34% (w/w). The nonirritant niosomes enhanced precorneal permeation and retention for FK506 in vitro, and prolonged the corneal graft survival and showed practical anti-allograft rejection efficacy in vivo on corneal transplantation rats. Therefore, the proniosomes-derived niosomes may be a promising vehicle for effective ocular drug delivery of FK506. Further works, such as the mechanism of FK506 permeation into corneal tissue and in vivo topical ocular distribution of FK506, need to be carried out in the near future.
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Fig. 7. Average rejection score for four groups. There were significant differences in the mean total scores (A), opacity (B), edema (C), and neovascularization (D) between the control and 0.1% FK506 loaded niosomes groups (P < 0.05).
Please cite this article in press as: Li, Q., et al. Proniosome-derived niosomes for tacrolimus topical ocular delivery: In vitro cornea permeation, ocular irritation, and in vivo anti-allograft rejection. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.05.020
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Please cite this article in press as: Li, Q., et al. Proniosome-derived niosomes for tacrolimus topical ocular delivery: In vitro cornea permeation, ocular irritation, and in vivo anti-allograft rejection. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.05.020
