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Development and evaluation of fast forming nano-composite hydrogel for ocular delivery of diclofenac
International Journal of Pharmaceutics 448 (2013) 96–100
Contents lists available at SciVerse ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Development and evaluation of fast forming nano-composite hydrogel for ocular delivery of diclofenac XingYi Li, Zhaoliang Zhang, Hao Chen ∗ Institute of Biomedical Engineering, School of Ophthalmology & Optometry and Eye hospital, Wenzhou Medical College, 270 Xueyuan Road, Wenzhou 325027, China
a r t i c l e
i n f o
Article history: Received 22 January 2013 Accepted 13 March 2013 Available online xxx Keywords: Nano-composite hydrogel Ocular drug delivery Micelles
a b s t r a c t In this paper, a fast forming nano-composite hydrogel was developed for potential application in ocular drug delivery. The optical transmission (OT) as well as rheological properties of nano-composite hydrogel was characterized. The developed nano-composite hydrogel given a high diclofenac micelles loading and provided a sustained release manner of diclofenac within 6 h. The developed nano-composite hydrogel formulation was administrated into the eye as flowable solution, quickly forming a hydrogel that is able to resist of the blinking and flushing of tear, yet resulting in the prolonged residence time of precorneal. In vivo eye irritation test suggested that the developed nano-composite hydrogel was none-eye irritation might be suitable for various ocular applications. In vivo pharmacokinetic study indicated that the developed nano-composite hydrogel could significantly increase the bioavailability of diclofenac and maintain the concentration of diclofenac in aqueous humor above MEC at least 24 h after administration as compared with that of the commercial diclofenac sodium eye drops, which might be able to reduce the frequency of administration for patients. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Eye drops has been widely used to the treatment of ocular diseases, but for traditional formulation only less than 5% of the administrated drugs could penetrate the cornea to reach the desired intraocular tissue due to the various anatomical and physicochemical barriers such as lacrimation, tear dilution, etc. (Diebold et al., 2007; Baba et al., 2011; Li et al., 2012). As a result, a frequency of instillation of eye drops was needed to achieve therapeutic efficacy. However, frequently administration of drugs might cause the undesirable side effects, resulting from the systemic absorption of drugs through the nasolacrimal duct (Asasutjarit et al., 2011; Mahmoud et al., 2011). In order to overcome these shortcomings and increase ocular drug bioavailability, several strategies including viscosity solution, micro/nanoparticles, hydrogel and, etc. were developed and investigated (El-Kamel, 2002; Sultana et al., 2006; Diebold et al., 2007; Asasutjarit et al., 2011; Gupta et al., 2011; Casolaro et al., 2012). In the case of viscous solution, numerous studies have been demonstrated that viscous solutions did not have enough mechanical strength to resist ocular clearance mechanism and only offered a transient improvement in ocular residence time (Davies et al., 1991). Alternative strategy for increasing ocular drug
∗ Corresponding author. Tel.: +86 577 88833806; fax: +86 577 88833806. E-mail address: [email protected] (H. Chen). 0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2013.03.024
penetration is using nanoparticles. Even though the hydrophobic drugs itself did not dissolved into water solution, it can be regarded as showing a great enhancement of water solubility by means of forming nanoparticles in aqueous solution (Rafie et al., 2010; Gupta et al., 2011; Yoncheva et al., 2011; Li et al., 2012; Stella et al., 2012). Furthermore, previous studies also demonstrated that the drug penetration capability across the cornea could be significantly improved by means of the decreasing particle size of nanoparticles (Rafie et al., 2010; Gupta et al., 2011; Li et al., 2012). However, nanoparticles with smaller particle size were not always ideal for increasing the ocular penetration of drugs, because it might lead to toxicity and quick elimination associated with nanoparticles. Our recent studies also showed that ocular instillation of diclofenac micelles could greatly improve the bioavailability of drug, whereas it was also suffered from the quick elimination of drug after 8 h of administration (Li et al., 2012). Nowadays, a great progress in development of ophthalmic formulations has been performed by the ophthalmic hydrogel technology (Anumolu et al., 2009; Casolaro et al., 2012). Hydrogel was a three-dimensional structure made from hydrophilic polymer that has the ability to adsorb large amounts of water solution. As the ocular drug delivery system, it could prolong the corneal contact time and reduce pre-corneal drug loss. The viscoelastic characterization of hydrogels assumed for sufficient mechanical strength to resist clearance due to the blinking, resulting the improvement of bioavailability of drugs (Chen et al., 2011). Recently,
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thermosensitive hydrogel with a sol–gel transition behavior at physiological temperature has gained considerable attention due to its convenience for instillation (Hsiue et al., 2002; Jeong et al., 2002). One of well-known polymer types possessing thermosensitive behavior was poly(ethylene oxide)–poly(propylene oxide) (PEO–PPO–PEO) block copolymers, so called Poloxamers. A number of literatures on the application of Poloxamer hydrogel for ocular drug delivery system were viewed in past three decade. However, a major shortcoming of Poloxamer hydrogel for ocular application was its low mucoadhesive activity on the surface of cornea (El-Kamel, 2002; Ma et al., 2008; Asasutjarit et al., 2011). In present study, we developed a fast forming nano-composite system composed of diclofenac micelles and F-127 hydrogel for topical ocular drug delivery. We assumed that the combination of diclofenac micelles and F-127 hydrogel could efficiently prolong the drug residence time at pre-corneal by hydrogel property, while increase the drug penetration across cornea barrier by micellization of drug, yet resulting in the improvement of bioavailability of drugs.
2.5. In vitro release study
2. Materials and methods
2.6. In vivo eye irradiation test
2.1. Materials
The potential eye irritancy and/or damaging effects of either nano-composite hydrogel (containing 0.1% diclofenac) or blank hydrogel were carefully evaluated by Draize test using a slit-lamp microscope. Six male mature New Zealand albino rabbits weighing 1.8–2.2 kg and free of any signs of ocular inflammation or gross abnormality were employed to evaluate the eye irritancy. All these tests were compiled with the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, and were approved by the Institutional Animal Care and Use Committee of Wenzhou medical college. Twenty microliters of either nanocomposite hydrogel or blank hydrogel was instilled into the lower conjunctival sca of the rabbit’s right eye, while the left eye was instilled with saline solution as the reference. Observation of ocular tissue condition was performed after 1 h, 6 h, 24 h, 48 h, and 72 h’s treatment. The degree of eye irritation was scored by an empirical doctor.
Diclofenac was purchased from Wenzhou Ding Sheng Co. Ltd (China). MPEG/PCL copolymer (Mn = 4000) was synthesized by our previous study. Diclofenac sodium eye drop (DiFei® ) was purchased from the Eye hospital of Zhejiang Province. F-127 was purchased from Sigma–Aldrich (USA). All the materials used in the study were analytic reagent degree. 2.2. Preparation of nano-composite hydrogel The nano-composite hydrogel was developed by a simple mixing of diclofenac micelles and F-127 solution. Briefly, 10 mg/ml of diclofenac micelles were first successfully synthesized by a thin-film evaporation method with our previous study (Li et al., 2012). Thereafter, a series of amounts of diclofenac micelles was added to the F-127 solution (25%, w/v) at 4 ◦ C with continuous stirring to form homogeneous mixture with final drug concentrations of 0.1%, 0.2% and 0.3% (w/v), respectively. Finally, the mixture was incubated at 37 ◦ C for gelation. 2.3. Optical transmission measurement According to the previous study, the optical transmission (OT) of hydrogel was performed in a quartz cuvette containing distilled water followed by measurement of its transmission at 480 nm (Anumolu et al., 2009). The quartz cuvette containing distilled water solution was used as reference. Eight hydrogel samples were used for measurement of OT. And the results were expressed as mean value ± S.D. 2.4. Rheological study Rheological measurement was performed with a rheometer (DHR-3, TA Instruments, USA). Hydrogel samples were prepared from 0.1% diclofenac loading F-127 hydrogel. The hydrogel was placed on a parallel-plate of 40 mm diameter with a gap of 31 mm for measurement. Rheological test parameters, storage moduli (G ) and loss moduli (G ) were monitored as a function of temperature at a 1 Hz frequency. All rheological studies were performed in triplicate and results were expressed as mean value ± S.D.
In vitro release behavior of diclofenac from hydrogel was investigated at 37 ◦ C in PBS solution. Briefly, 1 ml of nano-composite hydrogel containing various concentrations of diclofenac (0.1%, 0.2%, 0.3%; w/v) were prepared in a 10 ml glass bottle followed by the addition of 5 ml PBS solution for release medium. Thereafter, the samples were incubated at air bath (37 ◦ C; 100 rpm/min) for periodical study. At pre-determined time points, 1 ml of aliquot release medium was withdrawn for detection of drug concentrations by High Performance Liquid Chromatography (HPLC, Aglilent 1200 series, USA) and the resident release medium was discarded and another freshly prepared PBS solution was added into glass bottle for continuous study. HPLC analysis was carried out on a reversed phase C18 column (4.6 × 150 mm, 5 mm, ZORBAX Eclipse XDB-C18) at room temperature. The mobile phase was composed of acetonitrile and 0.1% triethylamine/phosphate buffer solution (65/35; v/v), and filtered through a 0.22 m Millipore filter and degassed prior to use. The flow rate was 1.0 ml/min and the eluent was detected by a DAD detector at 276 nm.
2.7. Pre-corneal resident time measurement The fluorescein sodium and its hydrophobic form-fluorescein were employed to monitor the resident time of nano-composite hydrogel on the corneal surface. Briefly, a certain amount of fluorescein micelles (5 mg/ml) was incorporated into F-127 solution to form fluorescein loaded nano-composite hydrogel (0.1%, w/v), and then instilled into the lower conjunctival sca of the rabbit’s eye at dosage of 20 l/eye. At specific time points, the intensity of fluorescein was observed by a slit-lamp microscope with exciting by blue light. The rabbit eye with instillation of 0.1% (w/v) fluorescein sodium was used a reference. 2.8. Pharmacokinetic study Twelve mature male New Zealand albino rabbits (1.8–3.0 kg) were adopted for the test and divided into two groups, six rabbit for each group. Each group was treated with either nano-composite hydrogel (containing 0.1% diclofenac) or the commercial diclofenac eye drops (0.1%, w/v) by instillation 50 l of each test formulation into the lower conjunctival sac of rabbit’s right eye. At specific time points (15 min, 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h and 24 h) after instillation of each formulation, 20 l aqueous humor samples were collected using an insulin syringe fitted with a 29 G needle followed by mixing with 80 l of HPLC mobile phase to remove the protein by centrifugation at 13,000 rpm/min for 10 min. Finally, the
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Fig. 1. Sol–gel transition behavior of nano-composite hydrogel system.
concentration of drug in humor aqueous solution was detected by HPCL method as described in Section 2.5. 2.9. Statistical analysis Data were analyzed using the software program Origin Pro 7.5. Statistical comparison between various groups were determined by using one-way ANOVA using SPSS software (p ≤ 0.05). 3. Results and discussions
Fig. 2. Optical transmission of nano-composite hydrogel with various diclofenac concentrations (0.1%, 0.2% and 0.3%; w/v).
3.1. Development of nano-composite hydrogel In past three decade, numerous thermosensitive hydrogels based on natural and synthesized polymer were developed and evaluated for ophthalmic applications (El-Kamel, 2002; Casolaro et al., 2012). Among these hydrogels, poloxamer hydrogel has gained great attentions due to its non-cytotoxic and prolonged residence time of drug on the surface of cornea (Jeong et al., 2002). However, it also suffered from its low mucoadhesive activity as well as none-enhancement of drug penetration. Our previous study has been demonstrated that micelles/nanoparticles formulation could greatly increase the drug across the corneal barrier, yet resulting in the enhancement of bioavailability. However, the major shortcoming for micelles/nanoparticles formulation was its quick clearance from ocular tissue (Li et al., 2012). Therefore, in this paper, we assumed that the combined with using nanoparticles and thermosensitive hydrogel could significantly improve the bioavailability of drug by the improvement of retention time of drug on ocular tissue and enhanced penetration of drug across the cornea. As depicted in Fig. 1, it was clearly observed that the system was flowable liquid at lower temperature (4 ◦ C), while turned into the unflowable gel at physiological temperature (37 ◦ C) suitable for ocular applications. As the drug delivery system for ocular application, it should ideally be transparent, yet would not interfere vision of patient. Since human eye was almost responsive around 480 nm, the optical transmission (OT) of hydrogel was monitored at this wavelength. Hydrogel with OT > 90% was considered as transparent, while less than 90% but greater than 10% was consider as translucent. And the hydrogel with OT < 10% was opaque. As presented in Fig. 2, we could find that the OT of blank F-127 hydrogel was about 95%, which was transparent suitable for various ocular applications. With the increase of diclofenac micelles content in system, the OT of hydrogel decreased gradually from 95% to 85%, indicating that incorporation of diclofenac micelles had slightly influenced on the optical property of hydrogel. The similar result was also observed by Anumolu et al. (2009), whom found that an increase in polymeric concentration in system would decrease the transparency of the hydrogel. Therefore, these results suggested that nano-composite hydrogel with excellent optical properties might be suitable as ocular drug delivery system.
to predict its retention behavior and physical integrity in vivo. As presented in Fig. 3, both storage moduli (G ) and loss moduli (G ) were very low with G smaller than G as the temperature was lower than 23 ◦ C, corresponding to the sol state of system. As the temperature was reached to 23 ◦ C, a great transition of G and G was observed with G greater than G , suggesting that the system was more elastic than viscous. The elastic property of nano-composite hydrogel was expected to maintain its integrity and help to prevent drug loss from blinking, yet resulting in the prolonged residence time on the surface of cornea (Davies et al., 1991). 3.3. In vitro release study In vitro release of diclofenac from nano-composite hydrogel was performed in PBS (pH = 7.4) at 37 ◦ C. The percentage of diclofenac released from hydrogel was plotted as a function with time. As depicted in Fig. 4, it was clearly observed that all diclofenac were completely released from hydrogel within 6 h, which might be induced by the collapse of F-127 hydrogel in PBS solution. As well known to us, F-127 hydrogel was formed mainly by the physical aggregation of micelles, which was very easily collapse as placed in
3.2. Rheological analysis The mechanical strength and viscoelastic property of the nanocomposite hydrogel was detected by using a rheological instrument
Fig. 3. Rheological property of nano-composite hydrogel as a function of temperature.
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Fig. 4. In vitro release profile of diclofenac from nano-composite hydrogel.
PBS solution within a very short period of time. However, with the collapse of F-127 hydrogel, the encapsulated diclofenac micelles was released, which might be able to serve as the binary sustained release system for diclofenac (El-Kamel, 2002; Ma et al., 2008). 3.4. In vivo eye irritation test To evaluate the potency irritation of nano-composite hydrogel prepared as ophthalmic drug delivery systems, an eye irritation experiments on the rabbit was evaluated by the modified Draize test. Ocular tolerability results showed that no clinical signs or changes (corneal opacity, conjunctival redness, abnormality of the iris, etc.) were observed in rabbit eyes after the instillation of either blank nano-composite hydrogel or nano-composite hydrogel (containing 0.1% diclofenac) at any time point. Furthermore, fluorescein staining did not suggest any corneal or conjunctival epithelial defects. Thus, our developed nano-composite hydrogel with none-eye irritation might be suitable for various ocular applications. 3.5. Pre-corneal residence time measurement In order to estimate the pre-corneal residence time of the nanocomposite hydrogel as the ocular drug delivery application, sodium fluorescein and its hydrophobic form fluorescein were employed to measure the residence time of drugs at different time points. Fig. 5 depicts the pre-corneal distribution of fluorescein following by instillation of either 0.1% (w/v) sodium fluorescein or 0.1% (w/v) fluorescein nano-composite hydrogel. It was clearly observed that the sodium fluorescein was quickly eliminated after 5 min of instillation, which was in agreement with the previous study that the hydrophilic eye drops had short residence time on the surface of cornea. For the fluorescein nano-composite hydrogel group, it was clearly observed that the nano-composite hydrogel was immediately dispersed on the surface of cornea after the instillation. With the result of blinking and flushing of tear, the nano-fluorescein was slowly released from hydrogel to achieve the greatest fluorescence intensity after 10–20 min of instillation. Even 30 min later, the apparent fluorescence was observed in saccus conjunctivae, suggesting that nano-composite hydrogel could significantly increase the pre-corneal residence time of drug as compared with that of common eye drops.
Fig. 5. Pre-corneal distribution of instillation of sodium fluorescein and nanofluorescein/F-127 hydrogel (20 l) at different time point.
3.6. Pharmacokinetics study Fig. 6 depicts the concentration of diclofenac in rabbit’s aqueous humor at various time points after instillation of 50 l of a commercial diclofenac sodium eye drops (DiFei® ) and diclofenac nano-composite hydrogel into the rabbit’s eye. The pharmacokinetic parameters of diclofenac in aqueous humor after instillation of these two formulations were presented in Table 1. As showed in Table 1 Pharmacokinetic parameters of instillation of sodium diclofenac eye drops and diclofenac nano-composite hydrogel (50 l). Key parameters
Diclofenac nano-composite hydrogel
Sodium diclofenac eye drops
Volume of instillation AUC0–24h (mg/l/h) Cmax (mg/l) Tmax (h) MRT (h)
50 l 20.37 ± 7.45 1.49 ± 0.26 1 11.62 ± 0.53
50 l 2.25 ± 1.58 0.23 ± 0.15 1 5.74 ± 0.64
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flushing of tear, yet resulting in the prolonged residence time of pre-corneal. In vivo eye irritation test suggested that the developed nano-composite hydrogel was none-eye irritation might be suitable for various ocular applications. In vivo pharmacokinetic study indicated that the developed nano-composite hydrogel could significantly increase the bioavailability of diclofenac and maintain the concentration of diclofenac in aqueous humor above MEC at least 24 h after administration as compared with that of the commercial diclofenac sodium eye drops, which might be able to reduce the frequency of administration for patients. Acknowledgments This research was supported by the Fund for Key Scientific and Technological Innovation in Zhejiang Province (2009R50039), Talent project of Zhejiang province (2012R413040) and Science the Technology Bureau of wenzhou city (Y20120196). Fig. 6. Concentration of diclofenac in rabbits’ aqueous humor at various times after instillation of a commercial diclofenac sodium eye drop (DiFei® ) and diclofenac nano-composite hydrogel (n = 3, mean ± SD).
Fig. 6 and Table 1, the maximum concentration (Cmax ) of diclofenac in rabbit’s aqueous humor was achieved at 1 h after instillation of diclofenac nano-composite hydrogel (Cmax = 1.49 ± 0.26 mg/l), which was about 6.5-fold higher than that of instillation of diclofenac sodium eye drops at 1 h (Cmax = 0.23 ± 0.15 mg/l). Furthermore, detectable drug concentrations in aqueous humor were measured up to 24 h after instillation of diclofenac nano-composite hydrogel with concentration of 0.31 ± 0.14 mg/l. It was 2-fold higher than the upper value of in vivo minimum effective concentration (MEC) of diclofenac, which was in rang of 14–158.2 ng/ml. Conversely, the diclofenac concentration in aqueous humor could not be detected after 8 h of instillation of commercial diclofenac sodium eye drops (DiFei® ), indicating the quick elimination of diclofenac sodium. As shown in Table 1, the area under the curve (AUC0–24h ) of diclofenac nano-composite hydrogel was ∼10-fold increase as compared with that of the commercial diclofenac sodium eye drops, suggesting that the better ophthalmic bioavailability could be obtained by administration of diclofenac nano-composite hydrogel. This might be explained by the enhancement of penetration of diclofenac micelles and the prolonged residence time of F-127 hydrogel (Li et al., 2012). Thus, all these results indicated that the developed diclofenac nano-composite hydrogel could increase the bioavailability of diclofenac and maintain the concentration of diclofenac in aqueous humor above MEC at least 24 h after administration, which might be able to reduce the frequency of administration for patients (Baba et al., 2011; Yoncheva et al., 2011; Stella et al., 2012). 4. Conclusion In this paper, the fast forming nano-composite hydrogel was developed and its optical transmission (OT) as well as the rheological properties was evaluated. The developed nano-composite hydrogel give a high diclofenac micelles loading and provide sustained diclofenac release within 6 h. This nano-composite hydrogel formulation was administrated into the eye as flowable solution, quickly forming a hydrogel that is able to resist of the blinking and
References Anumolu, S.N.S., Singh, Y., Gao, D., Stein, S., Sinko, P.J., 2009. Design and evaluation of novel fast forming pilocarpine-loaded ocular hydrogels for sustained pharmacological response. J. Control. Release 137, 152–159. Asasutjarit, R., Thanasanchokpibull, S., Fuongfuchat, A., Veeranodha, S., 2011. Optimization and evaluation of thermoresponsive diclofenac sodium ophthalmic in situ gels. Int. J. Pharm. 411, 128–135. Baba, K., Tanaka, Y., Kubota, A., Kasai, H., Yokokura, S., Nakanishi, H., Nishida, K., 2011. A method for enhancing the ocular penetration of eye drops using nanoparticles of hydrolysable dye. J. Control. Release 153, 278–287. Casolaro, M., Casolaro, I., Lamponi, S., 2012. Stimuli-responsive hydrogels for controlled pilocarpine ocular delivery. Eur. J. Pharm. Biopharm. 80, 553–561. Chen, X., Li, X., Zhou, Y., Wang, X., Zhang, Y., Fan, Y., Huang, Y., Liu, Y., 2011. Chitosanbased thermosensitive hydrogel as a promising ocular drug delivery system: preparation, characterization, and in vivo evaluation. J. Biomater. Sci. Polym. E, doi:0885328211406563. Davies, N.M., Fair, S.J., Hadgraft, J., Kellaway, I.W., 1991. Evaluation of mucoadhesive polymers in ocular drug delivery. I. Viscous solutions. J. Pharm. Sci. 8, 1039–1043. Diebold, Y., Jarrin, M., Saez, V., Carvalho, E.L.S., Orea, M., Calonge, M., Seijo, B., Alonso, M.J., 2007. Ocular drug delivery by liposome-chitosan nanoparticle complexes (LCS-NP). Biomaterials 28, 1553–1564. El-Kamel, A.H., 2002. In vitro and in vivo evaluation of Pluronic F127-based ocular delivery system for timolol maleate. Int. J. Pharm. 241, 47–55. Gupta, H., Aqil, M., Khar, R.K., Ali, A., Bhatnagar, A., Mittal, G., 2011. Biodegradable levofloxacin nanoparticles for sustained ocular drug delivery. J. Drug Target. 19, 409–417. Hsiue, G.H., Hsu, S., Yang, C.C., Lee, S.H., Yang, I., 2002. Preparation of controlled release ophthalmic drops, for glaucoma therapy using thermosensitive poly-Nisopropylacrylamide. Biomaterials 23, 457–462. Jeong, B., Kim, S.W., Bae, Y.H., 2002. Thermosensitive sol–gel reversible hydrogels. Adv. Drug Deliv. Rev. 54, 37–51. Li, X., Zhang, Z., Li, J., Sun, S., Weng, Y., Chen, H., 2012. Diclofenac/biodegradable polymer micelles for ocular applications. Nanoscale 4, 4667–4673. Ma, W.D., Xu, H., Wang, C., Nie, S.F., Pan, W.S., 2008. Pluronic F127-g-poly (acrylic acid) copolymers as in situ gelling vehicle for ophthalmic drug delivery system. Int. J. Pharm. 350, 247–256. A.A., El-Feky, G.S., Kamel, R., Awad, G.E.A., 2011. Mahmoud, Chitosan/sulfobutylether-[beta]-cyclodextrin nanoparticles as a potential approach for ocular drug delivery. Int. J. Pharm. 413, 229–236. Rafie, F., Javadzadeh, Y., Javadzadeh, A.R., Ghavidel, L.A., Jafari, B., Moogooee, M., Davaran, S., 2010. In vivo evaluation of novel nanoparticles containing dexamethasone for ocular drug delivery on rabbit eye. Curr. Eye Res. 35, 1081–1089. Stella, B., Arpicco, S., Rocco, F., Burgalassi, S., Nicosia, N., Tampucci, S., Chetoni, P., Cattel, L., 2012. Nonpolymeric nanoassemblies for ocular administration of acyclovir: pharmacokinetic evaluation in rabbits. Eur. J. Pharm. Biopharm. 80, 39–45. Sultana, Y., Aqil, M., Ali, A., 2006. Ion-activated, Gelrite® -based in situ ophthalmic gels of pefloxacin mesylate: comparison with conventional eye drops. Drug Deliv. 13, 215–219. Yoncheva, K., Vandervoort, J., Ludwig, A., 2011. Development of mucoadhesive poly (lactide-co-glycolide) nanoparticles for ocular application. Pharm. Dev. Technol. 16, 29–35.
Contents lists available at SciVerse ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Development and evaluation of fast forming nano-composite hydrogel for ocular delivery of diclofenac XingYi Li, Zhaoliang Zhang, Hao Chen ∗ Institute of Biomedical Engineering, School of Ophthalmology & Optometry and Eye hospital, Wenzhou Medical College, 270 Xueyuan Road, Wenzhou 325027, China
a r t i c l e
i n f o
Article history: Received 22 January 2013 Accepted 13 March 2013 Available online xxx Keywords: Nano-composite hydrogel Ocular drug delivery Micelles
a b s t r a c t In this paper, a fast forming nano-composite hydrogel was developed for potential application in ocular drug delivery. The optical transmission (OT) as well as rheological properties of nano-composite hydrogel was characterized. The developed nano-composite hydrogel given a high diclofenac micelles loading and provided a sustained release manner of diclofenac within 6 h. The developed nano-composite hydrogel formulation was administrated into the eye as flowable solution, quickly forming a hydrogel that is able to resist of the blinking and flushing of tear, yet resulting in the prolonged residence time of precorneal. In vivo eye irritation test suggested that the developed nano-composite hydrogel was none-eye irritation might be suitable for various ocular applications. In vivo pharmacokinetic study indicated that the developed nano-composite hydrogel could significantly increase the bioavailability of diclofenac and maintain the concentration of diclofenac in aqueous humor above MEC at least 24 h after administration as compared with that of the commercial diclofenac sodium eye drops, which might be able to reduce the frequency of administration for patients. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Eye drops has been widely used to the treatment of ocular diseases, but for traditional formulation only less than 5% of the administrated drugs could penetrate the cornea to reach the desired intraocular tissue due to the various anatomical and physicochemical barriers such as lacrimation, tear dilution, etc. (Diebold et al., 2007; Baba et al., 2011; Li et al., 2012). As a result, a frequency of instillation of eye drops was needed to achieve therapeutic efficacy. However, frequently administration of drugs might cause the undesirable side effects, resulting from the systemic absorption of drugs through the nasolacrimal duct (Asasutjarit et al., 2011; Mahmoud et al., 2011). In order to overcome these shortcomings and increase ocular drug bioavailability, several strategies including viscosity solution, micro/nanoparticles, hydrogel and, etc. were developed and investigated (El-Kamel, 2002; Sultana et al., 2006; Diebold et al., 2007; Asasutjarit et al., 2011; Gupta et al., 2011; Casolaro et al., 2012). In the case of viscous solution, numerous studies have been demonstrated that viscous solutions did not have enough mechanical strength to resist ocular clearance mechanism and only offered a transient improvement in ocular residence time (Davies et al., 1991). Alternative strategy for increasing ocular drug
∗ Corresponding author. Tel.: +86 577 88833806; fax: +86 577 88833806. E-mail address: [email protected] (H. Chen). 0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2013.03.024
penetration is using nanoparticles. Even though the hydrophobic drugs itself did not dissolved into water solution, it can be regarded as showing a great enhancement of water solubility by means of forming nanoparticles in aqueous solution (Rafie et al., 2010; Gupta et al., 2011; Yoncheva et al., 2011; Li et al., 2012; Stella et al., 2012). Furthermore, previous studies also demonstrated that the drug penetration capability across the cornea could be significantly improved by means of the decreasing particle size of nanoparticles (Rafie et al., 2010; Gupta et al., 2011; Li et al., 2012). However, nanoparticles with smaller particle size were not always ideal for increasing the ocular penetration of drugs, because it might lead to toxicity and quick elimination associated with nanoparticles. Our recent studies also showed that ocular instillation of diclofenac micelles could greatly improve the bioavailability of drug, whereas it was also suffered from the quick elimination of drug after 8 h of administration (Li et al., 2012). Nowadays, a great progress in development of ophthalmic formulations has been performed by the ophthalmic hydrogel technology (Anumolu et al., 2009; Casolaro et al., 2012). Hydrogel was a three-dimensional structure made from hydrophilic polymer that has the ability to adsorb large amounts of water solution. As the ocular drug delivery system, it could prolong the corneal contact time and reduce pre-corneal drug loss. The viscoelastic characterization of hydrogels assumed for sufficient mechanical strength to resist clearance due to the blinking, resulting the improvement of bioavailability of drugs (Chen et al., 2011). Recently,
X. Li et al. / International Journal of Pharmaceutics 448 (2013) 96–100
97
thermosensitive hydrogel with a sol–gel transition behavior at physiological temperature has gained considerable attention due to its convenience for instillation (Hsiue et al., 2002; Jeong et al., 2002). One of well-known polymer types possessing thermosensitive behavior was poly(ethylene oxide)–poly(propylene oxide) (PEO–PPO–PEO) block copolymers, so called Poloxamers. A number of literatures on the application of Poloxamer hydrogel for ocular drug delivery system were viewed in past three decade. However, a major shortcoming of Poloxamer hydrogel for ocular application was its low mucoadhesive activity on the surface of cornea (El-Kamel, 2002; Ma et al., 2008; Asasutjarit et al., 2011). In present study, we developed a fast forming nano-composite system composed of diclofenac micelles and F-127 hydrogel for topical ocular drug delivery. We assumed that the combination of diclofenac micelles and F-127 hydrogel could efficiently prolong the drug residence time at pre-corneal by hydrogel property, while increase the drug penetration across cornea barrier by micellization of drug, yet resulting in the improvement of bioavailability of drugs.
2.5. In vitro release study
2. Materials and methods
2.6. In vivo eye irradiation test
2.1. Materials
The potential eye irritancy and/or damaging effects of either nano-composite hydrogel (containing 0.1% diclofenac) or blank hydrogel were carefully evaluated by Draize test using a slit-lamp microscope. Six male mature New Zealand albino rabbits weighing 1.8–2.2 kg and free of any signs of ocular inflammation or gross abnormality were employed to evaluate the eye irritancy. All these tests were compiled with the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, and were approved by the Institutional Animal Care and Use Committee of Wenzhou medical college. Twenty microliters of either nanocomposite hydrogel or blank hydrogel was instilled into the lower conjunctival sca of the rabbit’s right eye, while the left eye was instilled with saline solution as the reference. Observation of ocular tissue condition was performed after 1 h, 6 h, 24 h, 48 h, and 72 h’s treatment. The degree of eye irritation was scored by an empirical doctor.
Diclofenac was purchased from Wenzhou Ding Sheng Co. Ltd (China). MPEG/PCL copolymer (Mn = 4000) was synthesized by our previous study. Diclofenac sodium eye drop (DiFei® ) was purchased from the Eye hospital of Zhejiang Province. F-127 was purchased from Sigma–Aldrich (USA). All the materials used in the study were analytic reagent degree. 2.2. Preparation of nano-composite hydrogel The nano-composite hydrogel was developed by a simple mixing of diclofenac micelles and F-127 solution. Briefly, 10 mg/ml of diclofenac micelles were first successfully synthesized by a thin-film evaporation method with our previous study (Li et al., 2012). Thereafter, a series of amounts of diclofenac micelles was added to the F-127 solution (25%, w/v) at 4 ◦ C with continuous stirring to form homogeneous mixture with final drug concentrations of 0.1%, 0.2% and 0.3% (w/v), respectively. Finally, the mixture was incubated at 37 ◦ C for gelation. 2.3. Optical transmission measurement According to the previous study, the optical transmission (OT) of hydrogel was performed in a quartz cuvette containing distilled water followed by measurement of its transmission at 480 nm (Anumolu et al., 2009). The quartz cuvette containing distilled water solution was used as reference. Eight hydrogel samples were used for measurement of OT. And the results were expressed as mean value ± S.D. 2.4. Rheological study Rheological measurement was performed with a rheometer (DHR-3, TA Instruments, USA). Hydrogel samples were prepared from 0.1% diclofenac loading F-127 hydrogel. The hydrogel was placed on a parallel-plate of 40 mm diameter with a gap of 31 mm for measurement. Rheological test parameters, storage moduli (G ) and loss moduli (G ) were monitored as a function of temperature at a 1 Hz frequency. All rheological studies were performed in triplicate and results were expressed as mean value ± S.D.
In vitro release behavior of diclofenac from hydrogel was investigated at 37 ◦ C in PBS solution. Briefly, 1 ml of nano-composite hydrogel containing various concentrations of diclofenac (0.1%, 0.2%, 0.3%; w/v) were prepared in a 10 ml glass bottle followed by the addition of 5 ml PBS solution for release medium. Thereafter, the samples were incubated at air bath (37 ◦ C; 100 rpm/min) for periodical study. At pre-determined time points, 1 ml of aliquot release medium was withdrawn for detection of drug concentrations by High Performance Liquid Chromatography (HPLC, Aglilent 1200 series, USA) and the resident release medium was discarded and another freshly prepared PBS solution was added into glass bottle for continuous study. HPLC analysis was carried out on a reversed phase C18 column (4.6 × 150 mm, 5 mm, ZORBAX Eclipse XDB-C18) at room temperature. The mobile phase was composed of acetonitrile and 0.1% triethylamine/phosphate buffer solution (65/35; v/v), and filtered through a 0.22 m Millipore filter and degassed prior to use. The flow rate was 1.0 ml/min and the eluent was detected by a DAD detector at 276 nm.
2.7. Pre-corneal resident time measurement The fluorescein sodium and its hydrophobic form-fluorescein were employed to monitor the resident time of nano-composite hydrogel on the corneal surface. Briefly, a certain amount of fluorescein micelles (5 mg/ml) was incorporated into F-127 solution to form fluorescein loaded nano-composite hydrogel (0.1%, w/v), and then instilled into the lower conjunctival sca of the rabbit’s eye at dosage of 20 l/eye. At specific time points, the intensity of fluorescein was observed by a slit-lamp microscope with exciting by blue light. The rabbit eye with instillation of 0.1% (w/v) fluorescein sodium was used a reference. 2.8. Pharmacokinetic study Twelve mature male New Zealand albino rabbits (1.8–3.0 kg) were adopted for the test and divided into two groups, six rabbit for each group. Each group was treated with either nano-composite hydrogel (containing 0.1% diclofenac) or the commercial diclofenac eye drops (0.1%, w/v) by instillation 50 l of each test formulation into the lower conjunctival sac of rabbit’s right eye. At specific time points (15 min, 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h and 24 h) after instillation of each formulation, 20 l aqueous humor samples were collected using an insulin syringe fitted with a 29 G needle followed by mixing with 80 l of HPLC mobile phase to remove the protein by centrifugation at 13,000 rpm/min for 10 min. Finally, the
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Fig. 1. Sol–gel transition behavior of nano-composite hydrogel system.
concentration of drug in humor aqueous solution was detected by HPCL method as described in Section 2.5. 2.9. Statistical analysis Data were analyzed using the software program Origin Pro 7.5. Statistical comparison between various groups were determined by using one-way ANOVA using SPSS software (p ≤ 0.05). 3. Results and discussions
Fig. 2. Optical transmission of nano-composite hydrogel with various diclofenac concentrations (0.1%, 0.2% and 0.3%; w/v).
3.1. Development of nano-composite hydrogel In past three decade, numerous thermosensitive hydrogels based on natural and synthesized polymer were developed and evaluated for ophthalmic applications (El-Kamel, 2002; Casolaro et al., 2012). Among these hydrogels, poloxamer hydrogel has gained great attentions due to its non-cytotoxic and prolonged residence time of drug on the surface of cornea (Jeong et al., 2002). However, it also suffered from its low mucoadhesive activity as well as none-enhancement of drug penetration. Our previous study has been demonstrated that micelles/nanoparticles formulation could greatly increase the drug across the corneal barrier, yet resulting in the enhancement of bioavailability. However, the major shortcoming for micelles/nanoparticles formulation was its quick clearance from ocular tissue (Li et al., 2012). Therefore, in this paper, we assumed that the combined with using nanoparticles and thermosensitive hydrogel could significantly improve the bioavailability of drug by the improvement of retention time of drug on ocular tissue and enhanced penetration of drug across the cornea. As depicted in Fig. 1, it was clearly observed that the system was flowable liquid at lower temperature (4 ◦ C), while turned into the unflowable gel at physiological temperature (37 ◦ C) suitable for ocular applications. As the drug delivery system for ocular application, it should ideally be transparent, yet would not interfere vision of patient. Since human eye was almost responsive around 480 nm, the optical transmission (OT) of hydrogel was monitored at this wavelength. Hydrogel with OT > 90% was considered as transparent, while less than 90% but greater than 10% was consider as translucent. And the hydrogel with OT < 10% was opaque. As presented in Fig. 2, we could find that the OT of blank F-127 hydrogel was about 95%, which was transparent suitable for various ocular applications. With the increase of diclofenac micelles content in system, the OT of hydrogel decreased gradually from 95% to 85%, indicating that incorporation of diclofenac micelles had slightly influenced on the optical property of hydrogel. The similar result was also observed by Anumolu et al. (2009), whom found that an increase in polymeric concentration in system would decrease the transparency of the hydrogel. Therefore, these results suggested that nano-composite hydrogel with excellent optical properties might be suitable as ocular drug delivery system.
to predict its retention behavior and physical integrity in vivo. As presented in Fig. 3, both storage moduli (G ) and loss moduli (G ) were very low with G smaller than G as the temperature was lower than 23 ◦ C, corresponding to the sol state of system. As the temperature was reached to 23 ◦ C, a great transition of G and G was observed with G greater than G , suggesting that the system was more elastic than viscous. The elastic property of nano-composite hydrogel was expected to maintain its integrity and help to prevent drug loss from blinking, yet resulting in the prolonged residence time on the surface of cornea (Davies et al., 1991). 3.3. In vitro release study In vitro release of diclofenac from nano-composite hydrogel was performed in PBS (pH = 7.4) at 37 ◦ C. The percentage of diclofenac released from hydrogel was plotted as a function with time. As depicted in Fig. 4, it was clearly observed that all diclofenac were completely released from hydrogel within 6 h, which might be induced by the collapse of F-127 hydrogel in PBS solution. As well known to us, F-127 hydrogel was formed mainly by the physical aggregation of micelles, which was very easily collapse as placed in
3.2. Rheological analysis The mechanical strength and viscoelastic property of the nanocomposite hydrogel was detected by using a rheological instrument
Fig. 3. Rheological property of nano-composite hydrogel as a function of temperature.
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Fig. 4. In vitro release profile of diclofenac from nano-composite hydrogel.
PBS solution within a very short period of time. However, with the collapse of F-127 hydrogel, the encapsulated diclofenac micelles was released, which might be able to serve as the binary sustained release system for diclofenac (El-Kamel, 2002; Ma et al., 2008). 3.4. In vivo eye irritation test To evaluate the potency irritation of nano-composite hydrogel prepared as ophthalmic drug delivery systems, an eye irritation experiments on the rabbit was evaluated by the modified Draize test. Ocular tolerability results showed that no clinical signs or changes (corneal opacity, conjunctival redness, abnormality of the iris, etc.) were observed in rabbit eyes after the instillation of either blank nano-composite hydrogel or nano-composite hydrogel (containing 0.1% diclofenac) at any time point. Furthermore, fluorescein staining did not suggest any corneal or conjunctival epithelial defects. Thus, our developed nano-composite hydrogel with none-eye irritation might be suitable for various ocular applications. 3.5. Pre-corneal residence time measurement In order to estimate the pre-corneal residence time of the nanocomposite hydrogel as the ocular drug delivery application, sodium fluorescein and its hydrophobic form fluorescein were employed to measure the residence time of drugs at different time points. Fig. 5 depicts the pre-corneal distribution of fluorescein following by instillation of either 0.1% (w/v) sodium fluorescein or 0.1% (w/v) fluorescein nano-composite hydrogel. It was clearly observed that the sodium fluorescein was quickly eliminated after 5 min of instillation, which was in agreement with the previous study that the hydrophilic eye drops had short residence time on the surface of cornea. For the fluorescein nano-composite hydrogel group, it was clearly observed that the nano-composite hydrogel was immediately dispersed on the surface of cornea after the instillation. With the result of blinking and flushing of tear, the nano-fluorescein was slowly released from hydrogel to achieve the greatest fluorescence intensity after 10–20 min of instillation. Even 30 min later, the apparent fluorescence was observed in saccus conjunctivae, suggesting that nano-composite hydrogel could significantly increase the pre-corneal residence time of drug as compared with that of common eye drops.
Fig. 5. Pre-corneal distribution of instillation of sodium fluorescein and nanofluorescein/F-127 hydrogel (20 l) at different time point.
3.6. Pharmacokinetics study Fig. 6 depicts the concentration of diclofenac in rabbit’s aqueous humor at various time points after instillation of 50 l of a commercial diclofenac sodium eye drops (DiFei® ) and diclofenac nano-composite hydrogel into the rabbit’s eye. The pharmacokinetic parameters of diclofenac in aqueous humor after instillation of these two formulations were presented in Table 1. As showed in Table 1 Pharmacokinetic parameters of instillation of sodium diclofenac eye drops and diclofenac nano-composite hydrogel (50 l). Key parameters
Diclofenac nano-composite hydrogel
Sodium diclofenac eye drops
Volume of instillation AUC0–24h (mg/l/h) Cmax (mg/l) Tmax (h) MRT (h)
50 l 20.37 ± 7.45 1.49 ± 0.26 1 11.62 ± 0.53
50 l 2.25 ± 1.58 0.23 ± 0.15 1 5.74 ± 0.64
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flushing of tear, yet resulting in the prolonged residence time of pre-corneal. In vivo eye irritation test suggested that the developed nano-composite hydrogel was none-eye irritation might be suitable for various ocular applications. In vivo pharmacokinetic study indicated that the developed nano-composite hydrogel could significantly increase the bioavailability of diclofenac and maintain the concentration of diclofenac in aqueous humor above MEC at least 24 h after administration as compared with that of the commercial diclofenac sodium eye drops, which might be able to reduce the frequency of administration for patients. Acknowledgments This research was supported by the Fund for Key Scientific and Technological Innovation in Zhejiang Province (2009R50039), Talent project of Zhejiang province (2012R413040) and Science the Technology Bureau of wenzhou city (Y20120196). Fig. 6. Concentration of diclofenac in rabbits’ aqueous humor at various times after instillation of a commercial diclofenac sodium eye drop (DiFei® ) and diclofenac nano-composite hydrogel (n = 3, mean ± SD).
Fig. 6 and Table 1, the maximum concentration (Cmax ) of diclofenac in rabbit’s aqueous humor was achieved at 1 h after instillation of diclofenac nano-composite hydrogel (Cmax = 1.49 ± 0.26 mg/l), which was about 6.5-fold higher than that of instillation of diclofenac sodium eye drops at 1 h (Cmax = 0.23 ± 0.15 mg/l). Furthermore, detectable drug concentrations in aqueous humor were measured up to 24 h after instillation of diclofenac nano-composite hydrogel with concentration of 0.31 ± 0.14 mg/l. It was 2-fold higher than the upper value of in vivo minimum effective concentration (MEC) of diclofenac, which was in rang of 14–158.2 ng/ml. Conversely, the diclofenac concentration in aqueous humor could not be detected after 8 h of instillation of commercial diclofenac sodium eye drops (DiFei® ), indicating the quick elimination of diclofenac sodium. As shown in Table 1, the area under the curve (AUC0–24h ) of diclofenac nano-composite hydrogel was ∼10-fold increase as compared with that of the commercial diclofenac sodium eye drops, suggesting that the better ophthalmic bioavailability could be obtained by administration of diclofenac nano-composite hydrogel. This might be explained by the enhancement of penetration of diclofenac micelles and the prolonged residence time of F-127 hydrogel (Li et al., 2012). Thus, all these results indicated that the developed diclofenac nano-composite hydrogel could increase the bioavailability of diclofenac and maintain the concentration of diclofenac in aqueous humor above MEC at least 24 h after administration, which might be able to reduce the frequency of administration for patients (Baba et al., 2011; Yoncheva et al., 2011; Stella et al., 2012). 4. Conclusion In this paper, the fast forming nano-composite hydrogel was developed and its optical transmission (OT) as well as the rheological properties was evaluated. The developed nano-composite hydrogel give a high diclofenac micelles loading and provide sustained diclofenac release within 6 h. This nano-composite hydrogel formulation was administrated into the eye as flowable solution, quickly forming a hydrogel that is able to resist of the blinking and
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