Accepted Manuscript Sustained Delivery of Latanoprost by Thermosensitive Chitosan-Gelatin-based Hydrogel for Controlling Ocular Hypertension Yung-Hsin Cheng, Kuo-Hsuan Hung, Tung-Hu Tsai, Chia-Jung Lee, Ruy-Yu Ku, Allen Wen-hsiang Chiu, Shih-Hwa Chiou, Catherine Jui-lin Liu PII: DOI: Reference:
S1742-7061(14)00243-8 http://dx.doi.org/10.1016/j.actbio.2014.05.031 ACTBIO 3253
To appear in:
Acta Biomaterialia
Received Date: Revised Date: Accepted Date:
1 February 2014 14 May 2014 27 May 2014
Please cite this article as: Cheng, Y-H., Hung, K-H., Tsai, T-H., Lee, C-J., Ku, R-Y., Chiu, A.W-h., Chiou, S-H., Liu, C.J-l., Sustained Delivery of Latanoprost by Thermosensitive Chitosan-Gelatin-based Hydrogel for Controlling Ocular Hypertension, Acta Biomaterialia (2014), doi: http://dx.doi.org/10.1016/j.actbio.2014.05.031
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Sustained Delivery of Latanoprost by Thermosensitive Chitosan-Gelatin-based Hydrogel for Controlling Ocular Hypertension Yung-Hsin Chenga,b Kuo-Hsuan Hungc,d,e, Tung-Hu Tsaib,f, Chia-Jung Leeb,f, Ruy-Yu Kud, Allen Wen-hsiang Chiu d, Shih-Hwa Chioua,f,*, Catherine Jui-lin Liud,g,* a
Department and Institute of Pharmacology, National Yang-Ming University, Taipei, Taiwan b c
Department of Education and Research, Taipei City Hospital, Taipei, Taiwan Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan d
e
f g
National Yang-Ming University School of Medicine, Taipei ,Taiwan Division of Ophthalmology, National Yang-Ming University Hospital, I-Lan,
Taiwan Institute of Traditional Medicine, National Yang-Ming University, Taipei, Taiwan Department of Ophthalmology, Taipei Veterans General Hospital, Taipei, Taiwan
YUNG-HSIN CHENG: Ph.D. Assistant Professor Institute of Pharmacology, School of Medicine, National Yang-Ming University, No.155, Sec.2, Linong Street, Taipei, 112, Taiwan (R.O.C.) Adjunct Assistant Research Fellow Department of Education and Research, Taipei City Hospital, No.145, Zhengzhou Rd., Datong Dist., Taipei, 103, Taiwan (R.O.C.) Tel: 886-2-28712121 ext. 8431 Fax: 886-2-28720959 E-mail: [email protected]; [email protected]
KUO-HSUAN HUNG: M.D. Institute of Clinical Medicine, National Yang-Ming University, No.155, Sec.2, Linong Street, Taipei, 112, Taiwan (R.O.C.) Division of Ophthalmology, National Yang-Ming University Hospital, No.152, Xinmin Rd., Yilan City, 260, Taiwan (R.O.C.) National Yang-Ming University School of Medicine, No.155, Sec.2, Linong Street, Taipei, 112, Taiwan (R.O.C.) Tel: 886- 3-905-1688 Fax: 886-2-28720959 E-mail: [email protected]
TUNG-HU TSAI: Ph.D. Professor Institute of Traditional Medicine, School of Medicine, National Yang-Ming University, No.155, Sec.2, Linong Street, Taipei, 112, Taiwan (R.O.C.) Director Department of Education and Research, Taipei City Hospital, No.145, Zhengzhou Rd., Datong Dist., Taipei, 103, Taiwan (R.O.C.) Tel: 886-2-28267115 Fax: 886-2-28225044 E-mail: [email protected]
CHIA-JUNG LEE: Ph.D. Assistant Professor Institute of Traditional Medicine, School of Medicine, National Yang-Ming University, No.155, Sec.2, Linong Street, Taipei, 112, Taiwan (R.O.C.) Adjunct Assistant Research Fellow Department of Education and Research, Taipei City Hospital, No.145, Zhengzhou Rd., Datong Dist., Taipei, 103, Taiwan (R.O.C.) Tel: 886-2-28267115 Fax: 886-2-28225044 E-mail: [email protected]
RUY-YU KU: MS Institute of Pharmacology, School of Medicine, National Yang-Ming University, No.155, Sec.2, Linong Street, Taipei, 112, Taiwan (R.O.C.) Tel: 886-2-28712121 ext. 8453 Fax: 886-2-28720959 E-mail: [email protected]
1
ALLEN WEN-HSIANG CHIU: M.D., Ph.D. Dean National Yang-Ming University School of Medicine, No.155, Sec.2, Linong Street, Taipei, 112, Taiwan (R.O.C.) Professor National Yang-Ming University School of Medicine, No.155, Sec.2, Linong Street, Taipei, 112, Taiwan (R.O.C.) Tel: 886-2-28211699 Fax: 886-2-28202190 E-mail: [email protected]
SHIH-HWA CHIOU*: M.D., Ph.D. (Corresponding author) Professor Institute of Pharmacology, School of Medicine, National Yang-Ming University, No.155, Sec.2, Linong Street, Taipei, 112, Taiwan (R.O.C.) Tel: 886-2-28211699 Fax: 886-2-28202190 E-mail: [email protected]
CATHERINE JUI-LIN LIU*: M.D., Ph.D. (Corresponding author) Professor National Yang-Ming University School of Medicine, No.155, Sec.2, Linong Street, Taipei, 112, Taiwan (R.O.C.) Director Glaucoma Service, Department of Ophthalmology, Taipei Veterans General Hospital, No.201, Sec. 2, Shipai Rd., Beitou District, Taipei, 112, Taiwan (R.O.C.) Tel: 886-2-82757325 Fax: 886-2-28761351 E-mail: [email protected]
2
Abstract Glaucoma is an irreversible ocular disease that may lead to progressive visual field loss and eventually to blindness with inadequately controlled intraocular pressure (IOP). Latanoprost is one of the most potent ocular hypotensive compounds, the current first-line therapy in glaucoma. However, the daily instillation required for efficacy and undesirable side effects are major causes of treatment adherence failure and persistence in glaucoma therapy. In the present study, we developed an injectable thermosensitive chitosan/gelatin/glycerol phosphate (C/G/GP) hydrogel as a sustained-release
system
of
latanoprost
for
glaucoma
treatment.
The
latanoprost-loaded C/G/GP hydrogel can gel within 1 minute at 37°C. The results show a sustained release of latanoprost from C/G/GP hydrogel in-vitro and in-vivo. The latanoprost-loaded C/G/GP hydrogel showed a good in-vitro and in-vivo biocompatibility. A rabbit model of glaucoma was established by intravitreal injection of
triamcinolone
acetonide.
After
a
single
subconjunctival
injection
of
latanoprost-loaded C/G/GP hydrogel, IOP was significantly decreased within 8 days and then remained at a normal level. The results of the study suggest that latanoprost-loaded C/G/GP hydrogel may have a potential application in glaucoma therapy.
3
1. Introduction Glaucoma is an irreversible optic neuropathy that may lead to progressive visual field loss and blindness if intraocular pressure (IOP) is inadequately controlled. There are an estimated 67 million people worldwide with glaucoma, which is the second leading cause of irreversible blindness [1-2]. To date, IOP is still the only amendable therapeutic strategy target, regardless of diagnosed glaucoma type; thus, IOP-lowering medications play a key role in long-term treatment [3]. Various anti-glaucoma agents, including beta-blockers, prostaglandin analogs, a carbonic anhydrases inhibitor, an α-2 agonist and several fixed combination therapies, have been developed in different formulations for greater potency, longer release and less systemic or local side effects. Latanoprost belongs to the prostaglandins family and is one of the most potent ocular hypotensive compounds that reduces IOP by increasing uveoscleral outflow [4]. However, daily instillations of latanoprost eye drops are required to maintain drug efficacy, which is accompanied by undesirable local side effects, such as conjunctival hyperemia. Moreover, latanoprost eye drops contain an antimicrobial preservative that may cause ocular discomfort and a temporary burning sensation. These side effects and for those who are unable to apply eye drops by themselves due to physical limitations or vision impairment are the major causes of failure of adherence and persistence in glaucoma therapy [5-8]. 4
Several novel drug delivery systems of ocular hypertension agents are under investigation with the hope of reduced side effects and improved patient adherence to anti-glaucoma medications [9-12]. Compared with topically administered eye drops, injectable drug delivery systems, which have attracted considerable attention in recent years, can provide the sustained release of anti-glaucoma medication [13-15]. Thermosensitive hydrogel formation by simple sol-gel transition and without the use of organic crosslinking agents is attracting increased interest for a wide range of biomedical and pharmaceutical applications [16-18]. Chenite et al. [16] developed thermosensitive chitosan/β-glycerophosphate (C/GP) hydrogel, which has been used widely in the drug delivery of both hydrophobic and hydrophilic compounds [18, 19]. In
a
previous
study,
we
developed
a
thermosensitive
injectable
chitosan/gelatin/β-glycerophosphate (C/G/GP) system that is liquid at room temperature but gels at body temperature. The C/G/GP hydrogel showed better gel strength and optimal gelation time than those of C/GP hydrogel [20]. The C/G/GP hydrogel showed promising gelation properties and great biocompatibility that may be more suitable for clinical applications [20, 21]. In the present study, we aimed to evaluate the feasibility of using the thermosensitive C/G/GP hydrogel as a sustained release system of latanoprost to control ocular hypertension. The gelation temperature and time of latanoprost-loaded 5
C/G/GP hydrogel was evaluated using a rheometer. The release of latanoprost from C/G/GP hydrogel was analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). The biocompatibility of latanoprost-loaded C/G/GP hydrogel was analyzed by cell viability, Draize eye irritancy test and histology analysis. A rabbit model of glaucoma was established by intravitreal injection of triamcinolone acetonide (TA). After ocular hypertension had been successfully established, latanoprost-loaded C/G/GP hydrogel was injected subconjunctivally through a 30-gauge needle. The possible therapeutic effects of latanoprost-loaded C/G/GP hydrogel were evaluated by IOP measurement.
6
2. Materials and methods 2.1 Preparation of latanoprost-loaded thermosensitive chitosan/gelatin/β-glycerol phosphate (C/G/GP) hydrogel A combination of 2.5% chitosan (degree of deacetylation > 95%, viscosity ≒ 581 mPa·s, Xing Cheng Biochemical Factory Nantong, China) with 1% gelatin (G2500, Sigma, USA) was dissolved in 0.1 M acetic acid (242853, Sigma, USA) and sterilized by autoclave. A 44.4% (w/v) glycerol 2-phosphate disodium salt hydrate (GP, G5422, Sigma, USA) solution was sterilized by filtration (0.22 µm; Millex-GV, Millipore, USA). The GP solution was added dropwise into the C/G solution under stirring, and the pH value was adjusted to 7.4. The C/G/GP solution was stored at 4 °C and utilized as a controlled release system of latanoprost. Latanoprost (L1167, Sigma, USA) was added into the C/G/GP solution under stirring and cooling in an ice-water bath for 30 minutes. Then, 500 µg/ml of latanoprost-containing C/G/GP hydrogel was prepared under a laminar flow hood and stored at 4 °C until further use.
2.2 Rheological characterization of latanoprost-loaded C/G/GP hydrogel Rheological measurements were performed using a TA Instruments HR-1 rheometer equipped with a Peltier plate (Aluminum, 20-mm plate) in oscillatory mode (TA Instruments, New Castle, DE). The elastic modulus (G’) and viscous modulus (G’’) were measured at a gap of 1 mm and a fixed frequency of 1.0 Hz. In the gelation 7
temperature analysis, the samples were measured with a temperature range from 15 °C to 45 °C at a rate of 1 °C per minute. All samples were prepared and tested in triplicate.
2.3 In-vitro drug release Latanoprost-loaded C/G/GP solution (500 µg/ml) was added to the transwell (100 µl/well) mounted on 24-well plates, and 1.5 ml of PBS was added into each well and then incubated at 37 °C. The 1.5 ml of PBS was collected on days 1, 7, 14 and 28, and 1.5 ml of fresh PBS was replenished after each collection. The amount of latanoprost was analyzed by a LC-MS/MS system equipped with an electrospray ionization source. The LC-MS/MS consisted of a Sciex API 3000 tandem mass spectrometer and an Agilent 1100 series LC system. The separation of latanoprost was achieved using a Phenomenex Luna C18 column (particle size 5 µm, 50 mm×4.6 mm i.d.). The mobile phase consisted of water containing 0.1% formic acid (mobile phase A) and acetonitrile with 0.1% formic acid (mobile phase B) (A:B = 7:3, v/v) and delivered at 0.3 ml per minute. The mass spectrometer was operated in positive mode using multiple-reaction monitoring. The precursor to produce an ion transition of m/z 374.2→195.2 was monitored.
2.4 Cytotoxicity evaluation 8
The cytotoxicity of C/G/GP hydrogel on human corneal epithelial cells (HCEC) was evaluated by crystal violet assay. Hydrogel (0.1 g) was immersed in 1 ml of Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham (DMEM-F12, D8900, Sigma, USA) containing 5% fetal bovine serum (FBS, AXB30114, Hyclone, USA), 10 ng/ml epidermal growth factor (AF-100-15, PeproTech, USA) and 1% penicillin-streptomycin (15140-122, Gibco, USA) in a 48-well plate at 37°C, 5% carbon dioxide and 95% relative humidity for 72 hours to prepare the extraction medium for the cytotoxicity test. HCECs were seeded in the 96-well cell culture plates with the density of 5000 cells per well and cultured in DMEM-F12. After incubation for 18 hours, the cells were washed with phosphate-buffered solution (PBS); 200 µl of the hydrogel extractive solution was then added into the culture well as culture medium. Crystal violet (C3886, Sigma, USA) was dissolved in 10% (v/v) ethanol (E7148, Sigma, USA). On day 1 and day 2, the cells were washed with PBS, and 50 µl of 0.2% (w/v) crystal solution was added into each culture well for 10 minutes. The crystal violet dye was carefully washed in running water, and then 100 µl of 33% (v/v) acetic acid was added in the dry well. The absorbance was measured using an enzyme-linked immunosorbent assay (ELISA, Sunrise remote, TECAN, USA) reader at the wavelength of 570 nm.
2.5 Hemolysis analysis 9
Hemolytic activity of the latanoprost-loaded C/G/GP hydrogel was tested by direct contact methods. Whole rabbit blood was used to evaluate the hemolysis of the specimens. Heparinized rabbit blood (1 ml) was added to 7 ml of PBS containing 1.4 g of latanoprost-loaded C/G/GP in triplicate. The positive and negative controls were H2O and PBS, respectively. The tubes were gently mixed and incubated at 37°C for 3 hours. After incubation, the suspension was centrifuged at 1000 rpm for 15 min, and the absorbance of the supernatant of each tube was measured by the ELISA reader at the wavelength of 545 nm. The percentage of hemolysis was calculated according to the following equation:
Hemolysis(%) =
ODsample − ODnegative × 100 OD positive − ODnegative
2.6 Subconjunctival delivery of latanoprost-loaded C/G/GP hydrogel
The animal experiment was approved by the Ethics Committee for Animal Research of the Taipei Veterans General Hospital. Ten male New Zealand albino rabbits (body weight approximately 2 kg) were used and maintained in accordance with the guidelines for the care and use of laboratory animals. All procedures were performed under general anesthesia with 10 mg/kg of tiletamine and zolazepam (Zoletil 50, Virbac, France) and topical anesthesia (0.5% of proparacaine hydrochloride). Ocular hypertension was induced by triamcinolone acetonide (TA) 10
[23-26]. Each rabbit was treated by an intravitreal injection of 0.1 ml of TA (4% of triamcinolone suspended Inj., Tai-yu Company, Taiwan) in the right eye at days 0 and 7. IOP measurement was performed with an icare® TONOLAB tonometer for laboratory research (TonoLab, Tiolat, Helsinki, Finland) at days 0 (pre-injection), 1, 3, 7, 10, 14, 17 and 21. After day 7, the IOP of the right eye was significantly elevated compared with the left eye (control group). At day 21, 100 µl of latanoprost-loaded C/G/GP hydrogel was injected into the subconjunctival space of the lower lid of the right eye using a 30-gauge needle. The IOP was then recorded at days 25, 29, 32, 39, 46, 53 and 60.
2.7 In-vivo release study
The concentration of latanoprost in aqueous humor was analyzed by an LC-MS/MS system (as described in section 2.3). Three male New Zealand albino rabbits (body weight approximately 2 kg) were used and maintained in accordance with the guidelines for the care and use of laboratory animals. The rabbits were anesthetized with 10 mg/kg of tiletamine and zolazepam (Zoletil 50, Virbac, France) and topical anesthesia (0.5% of proparacaine hydrochloride). One hundred microliters of latanoprost-loaded hydrogel was injected into the subconjunctival space of the lower lid of the right eye using a 30-gauge needle. Briefly, 100 µl of aqueous humor 11
from each eye was aspirated using a 30-gauge needle on days 3, 5, 7, 17 and 27. The samples were added to 300 µl of acetonitrile and then stored at 4 °C until analysis.
2.8 Histological analysis
The rabbits were euthanized at the end of the study period (day 60). The eyes were fixed in 10% formalin (HT501128, Sigma, USA) for 24 hours. The samples were dehydrated in a graded ethanol series and then embedded in paraffi n. The samples were cut into 5-µm sections and then stained with and Hematoxylin and Eosin (H&E, Muto Pure Chemicals, Japan).
2.9 Statistical analysis
Statistical analysis was calculated using one-way analysis of variance (ANOVA). The results are expressed as the mean ± standard deviation (SD) and considered significant at p＜0.05.
12
3 Results 3.1 Rheological measurement
Gelation temperature and time of the latanoprost-loaded C/G/GP solution were determined at the point when G’ is equal to G’’. As shown in Fig. 1 (a), the gelation temperature of the latanoprost-loaded C/G/GP solution was 32.14 ± 0.82 °C. The gelation time of the latanoprost-loaded C/G/GP solution was 51.15 ± 3.63 seconds at 37 °C (Fig. 1 (b)). The latanoprost-loaded C/G/GP solution was maintained in liquid form for at least 15 minutes at 25 °C (Fig. 1 (c)).
3.2 In-vitro drug release
The release of latanoprost from the C/G/GP hydrogel was calculated from the linear standard curve of latanoprost and expressed as cumulative drug release (%). Fig. 2 shows the release profile of latanoprost from the hydrogel. The percentage of cumulative release at 1, 7, 14 and 28 days was 1.34 ± 0.33, 26.80 ± 5.40, 45.03 ± 2.55 and 67.72 ± 4.25%, respectively.
3.3 Cytotoxicity of the latanoprost-loaded C/G/GP hydrogel on HCEC
Cell viability was determined by the crystal violet assay. As shown in Fig. 3 (b), the OD values of the control group at 24 and 48 hours were 1.192 ± 0.042 and 1.635 ± 13
0.018, respectively. The OD values of the latanoprost-loaded C/G/GP hydrogel group at 24 and 48 hours were 1.149 ± 0.054 and 1.524 ± 0.099, respectively. There was no significant difference in the OD values between the control and the latanoprost-loaded C/G/GP hydrogel group at 24 and 48 hours (n=4, p>0.05). The results showed no cytotoxic effects of latanoprost-loaded C/G/GP hydrogel on HCEC.
3.4 Hemolysis of latanoprost-loaded C/G/GP hydrogel
An in-vitro red blood cell hemolysis assay was used for the determination of the ocular irritation potential of latanoprost-loaded C/G/GP hydrogel. As shown in Table 1, the results show that there was no significant difference in hemolysis rate between the latanoprost-loaded C/G/GP hydrogel and the negative control group (Table 1).
3.5 IOP-lowering effects of subconjunctival injection of latanoprost-loaded C/G/GP hydrogel
IOP was periodically measured in both eyes of the rabbits during a 60-day study period. As shown in Fig. 4 (a), the mean IOP was significantly higher in the eyes that received an intravitreal injection of TA compared with that in the control eyes. The IOP elevation was first noted 3 days after injection and remained elevated until 60 days after the first TA injection. A series of IOP measurements after the injection of 14
latanoprost-loaded C/G/GP hydrogel were plotted for efficacy analysis (Fig. 4 (b)). Latanoprost-loaded C/G/GP hydrogel was injected into the subconjunctival space of the lower lid of the right eye at day 21. The mean IOP reduction was 2.4 mmHg (9.2%) at day 25. There was no significant difference in the mean IOP between the latanoprost-loaded C/G/GP group and the control group after day 29.
3.6 In-vivo release study
The drug level in the aqueous humor was evaluated using LC-MS/MS. After the administration of latanoprost-loaded C/G/GP hydrogel, the concentration of latanoprost in the aqueous humor at 3, 5, 7, 17 and 27 days was 2.77 ± 0.50, 2.64 ± 1.02, 4.30 ± 1.30, 4.04 ± 1.06 and 6.32 ± 2.70 ng/ml, respectively (Fig. 5).
3.7 Histological
examination
of
the
subconjunctival
delivery
of
latanoprost-loaded C/G/GP hydrogel
The histological analysis of the control and the latanoprost-loaded C/G/GP hydrogels are shown in Fig. 6 (a) and (b), respectively. After subconjunctival delivery of the latanoprost-loaded C/G/GP hydrogel, the histological analysis did not show any signs of inflammation at day 60 (Fig. 6 (b)).
15
4 Discussion
In this study, thermosensitive C/G/GP hydrogel was used as a sustained release carrier of latanoprost for controlling ocular hypertension, which is usually related to glaucoma. The gelation mechanism of thermosensitive C/G/GP hydrogels has been reported in our previous studies [20, 21]. Hydrophobic interactions might be the main driving force for the formation of hydrogels at 37°C. In this study, the results of rheological analyses indicated that the gelation temperature of the latanoprost-loaded C/G/GP hydrogel was 32.14°C (Fig. 1 (a)). The latanoprost-loaded C/G/GP solution could turn into a gel within 1 minute at 37°C and remain liquid for 15 minutes at 25°C (Fig. 1 (b) (c)). The rheological characterization of the latanoprost-loaded C/G/GP hydrogel was similar to the C/G/GP hydrogel [20]. The results suggested that 500 µg/ml of latanoprost may not affect the gelation properties of C/G/GP hydrogel.
Natarajan et al. [14, 15] used egg-phosphatidylcholine liposomes as a sustained delivery system for latanoprost. Their results showed that a slow and sustained release of 60% latanoprost was achieved at day 14 [14]. In our study, the C/G/GP hydrogel released approximately 70% of latanoprost at day 28 without the initial burst effect (Fig. 2). Our results suggest that the C/G/GP hydrogel provides a more prolonged-release of latanoprost. The dose of latanoprost released per day from the hydrogel was approximately 1.2 µg, lower than that of topical latanoprost eye drops 16
(1.5 µg/drop/day). It has been reported that only 1% to 7% of topically administered eye drops actually reach the anterior segment [27]. The subconjunctival delivery of latanoprost-loaded C/G/GP hydrogel might provide long-term sustained release and might maintain a constant drug concentration. It has been reported that the release of hydrophobic compounds from C/GP hydrogel can be sustained for one month [19]. In our study, the C/G/GP hydrogel showed the sustained release of latanoprost in-vitro for at least 28 days (Fig. 2). The release behavior of hydrophobic compounds from the C/G/GP hydrogel was similar to the C/GP hydrogel. However, the gelation time of the C/GP hydrogel was longer than 10 minutes at 37°C, as shown in our previous study [20]. Therefore, the gelation properties of the C/G/GP hydrogel may be more suitable for sustained release of latanoprost in clinical practice. Chitosan and gelatin are natural polymers that have good biocompatibility and biodegradation [28-31]. In the past few years, chitosan and gelatin have been widely used for various biomedical applications, including drug delivery and tissue engineering. It has been reported that the deacetylation degree of chitosan is strongly associated with biocompatibility. The higher deacetylated chitosan hydrogel had a longer residence time and resulted in no detectable inflammation [16, 32]. In the present study, the deacetylation degree of chitosan was higher than 95%. The results of the cytotoxicity assay showed that there were no harmful effects of 17
latanoprost-loaded C/G/GP hydrogel to HCEC (Fig. 3). The results of the hemolysis test indicated that the latanoprost-loaded C/G/GP hydrogel has no hemolytic action (Table 1). It has been demonstrated that the chitosan-based injectable hydrogel was safe and had no hemolytic effect [33]. The C/G/GP hydrogel also may have hemocompatibility and may be suitable for further drug delivery application. The elevation of IOP may lead to glaucoma, and the reduction of IOP is currently the only recognized effective method to decrease the risk of blindness resulting from glaucoma [34]. In this study, we established a rabbit model of ocular hypertension by intravitreal injection of TA, resulting in elevated IOP (~50% increase compared with the control eye) lasting for at least 60 days. After the subconjunctival injection of the latanoprost-loaded C/G/GP hydrogel, IOP was decreased effectively within 8 days and then remained at a normal level for the next 31 days (Fig. 4 (b)). The results of the in-vivo release study showed a steady drug concentration in the aqueous humor without noticeable burst release (Fig. 5). The results suggested that the effective concentration of latanoprost to treat ocular hypertension should be between 2.77 (day 3) and 4.30 (day 7) ng/ml because IOP was decreased effectively within 8 days (Fig. 4 (b)). The sustained release of latanoprost from the C/G/GP hydrogel might decrease IOP to the normal level within 8 days (Fig. 4 (b)). After the subconjunctival delivery of latanoprost-loaded C/G/GP hydrogel, the 18
histological analysis did not show any signs of inflammation at the end of the study period (Fig. 6). The results suggested that latanoprost-loaded C/G/GP hydrogel may have potential applications in glaucoma therapy, especially for those who are unable to apply eye drops by themselves due to physical limitations, vision impairment, or poor medication adherence.
19
5 Conclusion
In this study, we developed a thermosensitive latanoprost-loaded C/G/GP hydrogel which could gel within 1 minute at 37°C. The results of the release study showed a sustained release of latanoprost from C/G/GP hydrogel in-vitro and in-vivo. The newly developed hydrogel showed a good in-vitro and in-vivo biocompatibility. We demonstrated that 500 µg/ml of latanoprost-loaded C/G/GP hydrogel could significantly decrease the ocular hypertension induced by TA. These study results suggest that the subconjunctival delivery of latanoprost-loaded C/G/GP hydrogel could be applied as a sustained-release system for glaucoma therapy in the future.
6 Disclosures
The authors declare that there are no conflicts of interest.
20
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glaucoma. J Glaucoma 2010; 19:488-92. 14. Natarajan JV, Ang M, Darwitan A, Chattopadhyay S, Wong TT, Venkatraman SS. Nanomedicine for glaucoma: liposomes provide sustained release of latanoprost in the eye. Int J Nanomedicine 2012; 7:123-31. 15. Natarajan JV, Chattopadhyay S, Ang M, Darwitan A, Foo S, Venkatraman SS, et al. Sustained release of an anti-glaucoma drug: demonstration of efficacy of a liposomal formulation in the rabbit eye. PLoS One. 2011; 6:e24513. 16. Chenite A, Chaput C, Wang D, Combes C, Buschmann MD, Hoemann CD, et al. Novel injectable neutral solutions of chitosan form biodegradable gels in situ. Biomaterials 2000; 21:2155-61. 17. Roughley P, Hoemann C, DesRosiers E, Mwale F, Antoniou J, Alini M. The potential of chitosan-based gels containing intervertebral disc cells for nucleus pulposus supplementation. Biomaterials 2006; 27:388-96. 18. Berger J, Reist M, Chenite A, Felt-Baeyens O, Mayer JM, Gurny R. Pseudo-thermosetting chitosan hydrogels for biomedical application. Int J Pharm 2005; 288:197-206. 19. Ruel-Gariépy E, Shive M, Bichara A, Berrada M, Le Garrec D, Chenite A, et al. A thermosensitive chitosan-based hydrogel for the local delivery of paclitaxel. Eur J Pharm Biopharm. 2004; 57:53-63. 23
20. Cheng YH, Tang SH, Su WY, Chen YC, Yang KC, Lin FH, et al. thermosensitive chitosan-gelatin-glycerol phosphate hydrogels as a cell carrier for nucleus pulposus regeneration: an in-vitro study. Tissue Eng Part A 2010; 16:695-703. 21. Cheng YH, Yang SH, Lin FH. Thermosensitive chitosan-gelatin-glycerol phosphate hydrogel as a controlled release system of ferulic acid for nucleus pulposus regeneration. Biomaterials 2011; 32:6953-61. 22. Draize JH, Woodward G, Calvery HO. Methods for the study of irritation and toxicity of substances applied especially to the skin and mucous membranes. J Pharmacol. Exp. Ther 1944; 82:377-90. 23. Song Z, Gong Y, Liu H, Ren Q, Sun X. Glycyrrhizin could reduce ocular hypertension induced by triamcinolone acetonide in rabbits. Mol Vis. 2011; 17:2056-64. 24. Zarei-Ghanavati S, Malaekeh-Nikouei B, Pourmazar R, Seyedi S. Preparation, characterization, and in vivo evaluation of triamcinolone acetonide microspheres after intravitreal administration. J Ocul Pharmacol Ther. 2012; 28:502-6. 25. Kersey JP, Broadway DC. Corticosteroid-induced glaucoma: a review of the literature. Eye 2006; 20:407-16. 26. Smithen LM, Ober MD, Maranan L, Spaide RF. Intravitreal triamcinolone acetonide and intraocular pressure. Am J Ophthalmol. 2004; 138:740-3. 24
27. Ghate D, Edelhauser HF. Barriers to glaucoma drug delivery. J Glaucoma. 2008; 17:147-56. 28. Muzzarelli R.A.A., Greco, F., Busilacchi A., Sollazzo, V., Gigante, A. Chitosan, hyaluronan and chondroitin sulfate in tissue engineering for cartilage regeneration: a review. Carbohydrate Polymers 2012; 89:723-39. 29. Muzzarelli R.A.A. Chitins and chitosans for the repair of wounded skin, nerve, cartilage and bone. Carbohydrate Polymers 2009; 76:167-82. 30. Huang Y, Onyeri S, Siewe M, Moshfeghian A, Madihally SV. In vitro characterization of chitosan-gelatin scaffolds for tissue engineering. Biomaterials 2005; 26:7616-27. 31. Vandervoort J, Ludwig A. Preparation and evaluation of drug-loaded gelatin nanoparticles for topical ophthalmic use. Eur J Pharm Biopharm 2004; 57:251-61. 32. Molinaro G, Leroux JC, Damas J, Adam A. Biocompatibility of thermosensitive chitosan-based hydrogels: an in vivo experimental approach to injectable biomaterials. Biomaterials 2002; 23:2717-22. 33. Zhang YP, Zhang WF, Chen XG. Biocompatibility and characteristics of injectable
chitosan-based
thermosensitive
hydrogel
for
drug
delivery.
Carbohydrate Polymers 2011; 83:1643-51 34. Kwon YH, Fingert JH, Kuehn MH, Alward WL. Primary open-angle glaucoma. N 25
Engl J Med. 2009; 360:1113-24.
26
Fig. 1 Temperature and time dependence of storage modulus (G’) and loss modulus (G”) of latanoprost-loaded C/G/GP solution was gelled at pH 7.4. (a) The gelation temperature is 32.14 ± 0.82°C. The gelation time is (b) 51.15 ± 3.63 seconds at 37°C and (c) over 600 seconds at 25°C (n=3).
Fig. 2 In-vitro release study (a) Mass spectrum at a positive ionization mode of latanoprost, (b) Cumulative percent release of latanoprost from C/G/GP hydrogels in PBS at 37°C (n=5)
Fig. 3 Cytotoxicity of the latanoprost-loaded hydrogels to HCEC, (a) cell morphology at 48 hours (scale bar, 100 µm) and (b) crystal violet assay at 24 and 48 hours (n = 4, p＞0.05).
Fig. 4 IOP lowering effects of latanoprost-loaded C/G/GP hydrogel. Ocular hypertension was induced by triamcinolone acetonide (TA) at day 0 and 7. Single subconjunctival injection of latanoprost-loaded C/G/GP hydrogel was performed at day 21. The normal eyes (without treatment) were served as a control. The IOP was (a) increased after the intravitreal injection of TA and (b) decreased in the post-treatment of latanoprost-loaded C/G/GP hydrogel (n=5, * p < 0.05). 27
Fig. 5 Aqueous humor levels of latanoprost in rabbits after administration of latanoprost-loaded C/G/GP hydrogel at 3, 5, 7, 17 and 27 days (n=3).
Fig. 6 Histology revealed no abnormal scarring or damage in subconjunctiva after subconjunctival administration of latanoprost-loaded C/G/GP hydrogel (scale bar, 200 µm). * represents latanoprost-loaded C/G/GP hydrogel area.
Table 1 Hemolysis rate of latanoprost-loaded C/G/GP hydrogel
28
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Groups Positive control Latanoprost-loaded C/G/GP hydrogel Negative control
29
Hemolysis (%) 100 ± 1.1 0 ± 0.1 0 ± 0.1
Graphical Abstract
S1742-7061(14)00243-8 http://dx.doi.org/10.1016/j.actbio.2014.05.031 ACTBIO 3253
To appear in:
Acta Biomaterialia
Received Date: Revised Date: Accepted Date:
1 February 2014 14 May 2014 27 May 2014
Please cite this article as: Cheng, Y-H., Hung, K-H., Tsai, T-H., Lee, C-J., Ku, R-Y., Chiu, A.W-h., Chiou, S-H., Liu, C.J-l., Sustained Delivery of Latanoprost by Thermosensitive Chitosan-Gelatin-based Hydrogel for Controlling Ocular Hypertension, Acta Biomaterialia (2014), doi: http://dx.doi.org/10.1016/j.actbio.2014.05.031
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Sustained Delivery of Latanoprost by Thermosensitive Chitosan-Gelatin-based Hydrogel for Controlling Ocular Hypertension Yung-Hsin Chenga,b Kuo-Hsuan Hungc,d,e, Tung-Hu Tsaib,f, Chia-Jung Leeb,f, Ruy-Yu Kud, Allen Wen-hsiang Chiu d, Shih-Hwa Chioua,f,*, Catherine Jui-lin Liud,g,* a
Department and Institute of Pharmacology, National Yang-Ming University, Taipei, Taiwan b c
Department of Education and Research, Taipei City Hospital, Taipei, Taiwan Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan d
e
f g
National Yang-Ming University School of Medicine, Taipei ,Taiwan Division of Ophthalmology, National Yang-Ming University Hospital, I-Lan,
Taiwan Institute of Traditional Medicine, National Yang-Ming University, Taipei, Taiwan Department of Ophthalmology, Taipei Veterans General Hospital, Taipei, Taiwan
YUNG-HSIN CHENG: Ph.D. Assistant Professor Institute of Pharmacology, School of Medicine, National Yang-Ming University, No.155, Sec.2, Linong Street, Taipei, 112, Taiwan (R.O.C.) Adjunct Assistant Research Fellow Department of Education and Research, Taipei City Hospital, No.145, Zhengzhou Rd., Datong Dist., Taipei, 103, Taiwan (R.O.C.) Tel: 886-2-28712121 ext. 8431 Fax: 886-2-28720959 E-mail: [email protected]; [email protected]
KUO-HSUAN HUNG: M.D. Institute of Clinical Medicine, National Yang-Ming University, No.155, Sec.2, Linong Street, Taipei, 112, Taiwan (R.O.C.) Division of Ophthalmology, National Yang-Ming University Hospital, No.152, Xinmin Rd., Yilan City, 260, Taiwan (R.O.C.) National Yang-Ming University School of Medicine, No.155, Sec.2, Linong Street, Taipei, 112, Taiwan (R.O.C.) Tel: 886- 3-905-1688 Fax: 886-2-28720959 E-mail: [email protected]
TUNG-HU TSAI: Ph.D. Professor Institute of Traditional Medicine, School of Medicine, National Yang-Ming University, No.155, Sec.2, Linong Street, Taipei, 112, Taiwan (R.O.C.) Director Department of Education and Research, Taipei City Hospital, No.145, Zhengzhou Rd., Datong Dist., Taipei, 103, Taiwan (R.O.C.) Tel: 886-2-28267115 Fax: 886-2-28225044 E-mail: [email protected]
CHIA-JUNG LEE: Ph.D. Assistant Professor Institute of Traditional Medicine, School of Medicine, National Yang-Ming University, No.155, Sec.2, Linong Street, Taipei, 112, Taiwan (R.O.C.) Adjunct Assistant Research Fellow Department of Education and Research, Taipei City Hospital, No.145, Zhengzhou Rd., Datong Dist., Taipei, 103, Taiwan (R.O.C.) Tel: 886-2-28267115 Fax: 886-2-28225044 E-mail: [email protected]
RUY-YU KU: MS Institute of Pharmacology, School of Medicine, National Yang-Ming University, No.155, Sec.2, Linong Street, Taipei, 112, Taiwan (R.O.C.) Tel: 886-2-28712121 ext. 8453 Fax: 886-2-28720959 E-mail: [email protected]
1
ALLEN WEN-HSIANG CHIU: M.D., Ph.D. Dean National Yang-Ming University School of Medicine, No.155, Sec.2, Linong Street, Taipei, 112, Taiwan (R.O.C.) Professor National Yang-Ming University School of Medicine, No.155, Sec.2, Linong Street, Taipei, 112, Taiwan (R.O.C.) Tel: 886-2-28211699 Fax: 886-2-28202190 E-mail: [email protected]
SHIH-HWA CHIOU*: M.D., Ph.D. (Corresponding author) Professor Institute of Pharmacology, School of Medicine, National Yang-Ming University, No.155, Sec.2, Linong Street, Taipei, 112, Taiwan (R.O.C.) Tel: 886-2-28211699 Fax: 886-2-28202190 E-mail: [email protected]
CATHERINE JUI-LIN LIU*: M.D., Ph.D. (Corresponding author) Professor National Yang-Ming University School of Medicine, No.155, Sec.2, Linong Street, Taipei, 112, Taiwan (R.O.C.) Director Glaucoma Service, Department of Ophthalmology, Taipei Veterans General Hospital, No.201, Sec. 2, Shipai Rd., Beitou District, Taipei, 112, Taiwan (R.O.C.) Tel: 886-2-82757325 Fax: 886-2-28761351 E-mail: [email protected]
2
Abstract Glaucoma is an irreversible ocular disease that may lead to progressive visual field loss and eventually to blindness with inadequately controlled intraocular pressure (IOP). Latanoprost is one of the most potent ocular hypotensive compounds, the current first-line therapy in glaucoma. However, the daily instillation required for efficacy and undesirable side effects are major causes of treatment adherence failure and persistence in glaucoma therapy. In the present study, we developed an injectable thermosensitive chitosan/gelatin/glycerol phosphate (C/G/GP) hydrogel as a sustained-release
system
of
latanoprost
for
glaucoma
treatment.
The
latanoprost-loaded C/G/GP hydrogel can gel within 1 minute at 37°C. The results show a sustained release of latanoprost from C/G/GP hydrogel in-vitro and in-vivo. The latanoprost-loaded C/G/GP hydrogel showed a good in-vitro and in-vivo biocompatibility. A rabbit model of glaucoma was established by intravitreal injection of
triamcinolone
acetonide.
After
a
single
subconjunctival
injection
of
latanoprost-loaded C/G/GP hydrogel, IOP was significantly decreased within 8 days and then remained at a normal level. The results of the study suggest that latanoprost-loaded C/G/GP hydrogel may have a potential application in glaucoma therapy.
3
1. Introduction Glaucoma is an irreversible optic neuropathy that may lead to progressive visual field loss and blindness if intraocular pressure (IOP) is inadequately controlled. There are an estimated 67 million people worldwide with glaucoma, which is the second leading cause of irreversible blindness [1-2]. To date, IOP is still the only amendable therapeutic strategy target, regardless of diagnosed glaucoma type; thus, IOP-lowering medications play a key role in long-term treatment [3]. Various anti-glaucoma agents, including beta-blockers, prostaglandin analogs, a carbonic anhydrases inhibitor, an α-2 agonist and several fixed combination therapies, have been developed in different formulations for greater potency, longer release and less systemic or local side effects. Latanoprost belongs to the prostaglandins family and is one of the most potent ocular hypotensive compounds that reduces IOP by increasing uveoscleral outflow [4]. However, daily instillations of latanoprost eye drops are required to maintain drug efficacy, which is accompanied by undesirable local side effects, such as conjunctival hyperemia. Moreover, latanoprost eye drops contain an antimicrobial preservative that may cause ocular discomfort and a temporary burning sensation. These side effects and for those who are unable to apply eye drops by themselves due to physical limitations or vision impairment are the major causes of failure of adherence and persistence in glaucoma therapy [5-8]. 4
Several novel drug delivery systems of ocular hypertension agents are under investigation with the hope of reduced side effects and improved patient adherence to anti-glaucoma medications [9-12]. Compared with topically administered eye drops, injectable drug delivery systems, which have attracted considerable attention in recent years, can provide the sustained release of anti-glaucoma medication [13-15]. Thermosensitive hydrogel formation by simple sol-gel transition and without the use of organic crosslinking agents is attracting increased interest for a wide range of biomedical and pharmaceutical applications [16-18]. Chenite et al. [16] developed thermosensitive chitosan/β-glycerophosphate (C/GP) hydrogel, which has been used widely in the drug delivery of both hydrophobic and hydrophilic compounds [18, 19]. In
a
previous
study,
we
developed
a
thermosensitive
injectable
chitosan/gelatin/β-glycerophosphate (C/G/GP) system that is liquid at room temperature but gels at body temperature. The C/G/GP hydrogel showed better gel strength and optimal gelation time than those of C/GP hydrogel [20]. The C/G/GP hydrogel showed promising gelation properties and great biocompatibility that may be more suitable for clinical applications [20, 21]. In the present study, we aimed to evaluate the feasibility of using the thermosensitive C/G/GP hydrogel as a sustained release system of latanoprost to control ocular hypertension. The gelation temperature and time of latanoprost-loaded 5
C/G/GP hydrogel was evaluated using a rheometer. The release of latanoprost from C/G/GP hydrogel was analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). The biocompatibility of latanoprost-loaded C/G/GP hydrogel was analyzed by cell viability, Draize eye irritancy test and histology analysis. A rabbit model of glaucoma was established by intravitreal injection of triamcinolone acetonide (TA). After ocular hypertension had been successfully established, latanoprost-loaded C/G/GP hydrogel was injected subconjunctivally through a 30-gauge needle. The possible therapeutic effects of latanoprost-loaded C/G/GP hydrogel were evaluated by IOP measurement.
6
2. Materials and methods 2.1 Preparation of latanoprost-loaded thermosensitive chitosan/gelatin/β-glycerol phosphate (C/G/GP) hydrogel A combination of 2.5% chitosan (degree of deacetylation > 95%, viscosity ≒ 581 mPa·s, Xing Cheng Biochemical Factory Nantong, China) with 1% gelatin (G2500, Sigma, USA) was dissolved in 0.1 M acetic acid (242853, Sigma, USA) and sterilized by autoclave. A 44.4% (w/v) glycerol 2-phosphate disodium salt hydrate (GP, G5422, Sigma, USA) solution was sterilized by filtration (0.22 µm; Millex-GV, Millipore, USA). The GP solution was added dropwise into the C/G solution under stirring, and the pH value was adjusted to 7.4. The C/G/GP solution was stored at 4 °C and utilized as a controlled release system of latanoprost. Latanoprost (L1167, Sigma, USA) was added into the C/G/GP solution under stirring and cooling in an ice-water bath for 30 minutes. Then, 500 µg/ml of latanoprost-containing C/G/GP hydrogel was prepared under a laminar flow hood and stored at 4 °C until further use.
2.2 Rheological characterization of latanoprost-loaded C/G/GP hydrogel Rheological measurements were performed using a TA Instruments HR-1 rheometer equipped with a Peltier plate (Aluminum, 20-mm plate) in oscillatory mode (TA Instruments, New Castle, DE). The elastic modulus (G’) and viscous modulus (G’’) were measured at a gap of 1 mm and a fixed frequency of 1.0 Hz. In the gelation 7
temperature analysis, the samples were measured with a temperature range from 15 °C to 45 °C at a rate of 1 °C per minute. All samples were prepared and tested in triplicate.
2.3 In-vitro drug release Latanoprost-loaded C/G/GP solution (500 µg/ml) was added to the transwell (100 µl/well) mounted on 24-well plates, and 1.5 ml of PBS was added into each well and then incubated at 37 °C. The 1.5 ml of PBS was collected on days 1, 7, 14 and 28, and 1.5 ml of fresh PBS was replenished after each collection. The amount of latanoprost was analyzed by a LC-MS/MS system equipped with an electrospray ionization source. The LC-MS/MS consisted of a Sciex API 3000 tandem mass spectrometer and an Agilent 1100 series LC system. The separation of latanoprost was achieved using a Phenomenex Luna C18 column (particle size 5 µm, 50 mm×4.6 mm i.d.). The mobile phase consisted of water containing 0.1% formic acid (mobile phase A) and acetonitrile with 0.1% formic acid (mobile phase B) (A:B = 7:3, v/v) and delivered at 0.3 ml per minute. The mass spectrometer was operated in positive mode using multiple-reaction monitoring. The precursor to produce an ion transition of m/z 374.2→195.2 was monitored.
2.4 Cytotoxicity evaluation 8
The cytotoxicity of C/G/GP hydrogel on human corneal epithelial cells (HCEC) was evaluated by crystal violet assay. Hydrogel (0.1 g) was immersed in 1 ml of Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham (DMEM-F12, D8900, Sigma, USA) containing 5% fetal bovine serum (FBS, AXB30114, Hyclone, USA), 10 ng/ml epidermal growth factor (AF-100-15, PeproTech, USA) and 1% penicillin-streptomycin (15140-122, Gibco, USA) in a 48-well plate at 37°C, 5% carbon dioxide and 95% relative humidity for 72 hours to prepare the extraction medium for the cytotoxicity test. HCECs were seeded in the 96-well cell culture plates with the density of 5000 cells per well and cultured in DMEM-F12. After incubation for 18 hours, the cells were washed with phosphate-buffered solution (PBS); 200 µl of the hydrogel extractive solution was then added into the culture well as culture medium. Crystal violet (C3886, Sigma, USA) was dissolved in 10% (v/v) ethanol (E7148, Sigma, USA). On day 1 and day 2, the cells were washed with PBS, and 50 µl of 0.2% (w/v) crystal solution was added into each culture well for 10 minutes. The crystal violet dye was carefully washed in running water, and then 100 µl of 33% (v/v) acetic acid was added in the dry well. The absorbance was measured using an enzyme-linked immunosorbent assay (ELISA, Sunrise remote, TECAN, USA) reader at the wavelength of 570 nm.
2.5 Hemolysis analysis 9
Hemolytic activity of the latanoprost-loaded C/G/GP hydrogel was tested by direct contact methods. Whole rabbit blood was used to evaluate the hemolysis of the specimens. Heparinized rabbit blood (1 ml) was added to 7 ml of PBS containing 1.4 g of latanoprost-loaded C/G/GP in triplicate. The positive and negative controls were H2O and PBS, respectively. The tubes were gently mixed and incubated at 37°C for 3 hours. After incubation, the suspension was centrifuged at 1000 rpm for 15 min, and the absorbance of the supernatant of each tube was measured by the ELISA reader at the wavelength of 545 nm. The percentage of hemolysis was calculated according to the following equation:
Hemolysis(%) =
ODsample − ODnegative × 100 OD positive − ODnegative
2.6 Subconjunctival delivery of latanoprost-loaded C/G/GP hydrogel
The animal experiment was approved by the Ethics Committee for Animal Research of the Taipei Veterans General Hospital. Ten male New Zealand albino rabbits (body weight approximately 2 kg) were used and maintained in accordance with the guidelines for the care and use of laboratory animals. All procedures were performed under general anesthesia with 10 mg/kg of tiletamine and zolazepam (Zoletil 50, Virbac, France) and topical anesthesia (0.5% of proparacaine hydrochloride). Ocular hypertension was induced by triamcinolone acetonide (TA) 10
[23-26]. Each rabbit was treated by an intravitreal injection of 0.1 ml of TA (4% of triamcinolone suspended Inj., Tai-yu Company, Taiwan) in the right eye at days 0 and 7. IOP measurement was performed with an icare® TONOLAB tonometer for laboratory research (TonoLab, Tiolat, Helsinki, Finland) at days 0 (pre-injection), 1, 3, 7, 10, 14, 17 and 21. After day 7, the IOP of the right eye was significantly elevated compared with the left eye (control group). At day 21, 100 µl of latanoprost-loaded C/G/GP hydrogel was injected into the subconjunctival space of the lower lid of the right eye using a 30-gauge needle. The IOP was then recorded at days 25, 29, 32, 39, 46, 53 and 60.
2.7 In-vivo release study
The concentration of latanoprost in aqueous humor was analyzed by an LC-MS/MS system (as described in section 2.3). Three male New Zealand albino rabbits (body weight approximately 2 kg) were used and maintained in accordance with the guidelines for the care and use of laboratory animals. The rabbits were anesthetized with 10 mg/kg of tiletamine and zolazepam (Zoletil 50, Virbac, France) and topical anesthesia (0.5% of proparacaine hydrochloride). One hundred microliters of latanoprost-loaded hydrogel was injected into the subconjunctival space of the lower lid of the right eye using a 30-gauge needle. Briefly, 100 µl of aqueous humor 11
from each eye was aspirated using a 30-gauge needle on days 3, 5, 7, 17 and 27. The samples were added to 300 µl of acetonitrile and then stored at 4 °C until analysis.
2.8 Histological analysis
The rabbits were euthanized at the end of the study period (day 60). The eyes were fixed in 10% formalin (HT501128, Sigma, USA) for 24 hours. The samples were dehydrated in a graded ethanol series and then embedded in paraffi n. The samples were cut into 5-µm sections and then stained with and Hematoxylin and Eosin (H&E, Muto Pure Chemicals, Japan).
2.9 Statistical analysis
Statistical analysis was calculated using one-way analysis of variance (ANOVA). The results are expressed as the mean ± standard deviation (SD) and considered significant at p＜0.05.
12
3 Results 3.1 Rheological measurement
Gelation temperature and time of the latanoprost-loaded C/G/GP solution were determined at the point when G’ is equal to G’’. As shown in Fig. 1 (a), the gelation temperature of the latanoprost-loaded C/G/GP solution was 32.14 ± 0.82 °C. The gelation time of the latanoprost-loaded C/G/GP solution was 51.15 ± 3.63 seconds at 37 °C (Fig. 1 (b)). The latanoprost-loaded C/G/GP solution was maintained in liquid form for at least 15 minutes at 25 °C (Fig. 1 (c)).
3.2 In-vitro drug release
The release of latanoprost from the C/G/GP hydrogel was calculated from the linear standard curve of latanoprost and expressed as cumulative drug release (%). Fig. 2 shows the release profile of latanoprost from the hydrogel. The percentage of cumulative release at 1, 7, 14 and 28 days was 1.34 ± 0.33, 26.80 ± 5.40, 45.03 ± 2.55 and 67.72 ± 4.25%, respectively.
3.3 Cytotoxicity of the latanoprost-loaded C/G/GP hydrogel on HCEC
Cell viability was determined by the crystal violet assay. As shown in Fig. 3 (b), the OD values of the control group at 24 and 48 hours were 1.192 ± 0.042 and 1.635 ± 13
0.018, respectively. The OD values of the latanoprost-loaded C/G/GP hydrogel group at 24 and 48 hours were 1.149 ± 0.054 and 1.524 ± 0.099, respectively. There was no significant difference in the OD values between the control and the latanoprost-loaded C/G/GP hydrogel group at 24 and 48 hours (n=4, p>0.05). The results showed no cytotoxic effects of latanoprost-loaded C/G/GP hydrogel on HCEC.
3.4 Hemolysis of latanoprost-loaded C/G/GP hydrogel
An in-vitro red blood cell hemolysis assay was used for the determination of the ocular irritation potential of latanoprost-loaded C/G/GP hydrogel. As shown in Table 1, the results show that there was no significant difference in hemolysis rate between the latanoprost-loaded C/G/GP hydrogel and the negative control group (Table 1).
3.5 IOP-lowering effects of subconjunctival injection of latanoprost-loaded C/G/GP hydrogel
IOP was periodically measured in both eyes of the rabbits during a 60-day study period. As shown in Fig. 4 (a), the mean IOP was significantly higher in the eyes that received an intravitreal injection of TA compared with that in the control eyes. The IOP elevation was first noted 3 days after injection and remained elevated until 60 days after the first TA injection. A series of IOP measurements after the injection of 14
latanoprost-loaded C/G/GP hydrogel were plotted for efficacy analysis (Fig. 4 (b)). Latanoprost-loaded C/G/GP hydrogel was injected into the subconjunctival space of the lower lid of the right eye at day 21. The mean IOP reduction was 2.4 mmHg (9.2%) at day 25. There was no significant difference in the mean IOP between the latanoprost-loaded C/G/GP group and the control group after day 29.
3.6 In-vivo release study
The drug level in the aqueous humor was evaluated using LC-MS/MS. After the administration of latanoprost-loaded C/G/GP hydrogel, the concentration of latanoprost in the aqueous humor at 3, 5, 7, 17 and 27 days was 2.77 ± 0.50, 2.64 ± 1.02, 4.30 ± 1.30, 4.04 ± 1.06 and 6.32 ± 2.70 ng/ml, respectively (Fig. 5).
3.7 Histological
examination
of
the
subconjunctival
delivery
of
latanoprost-loaded C/G/GP hydrogel
The histological analysis of the control and the latanoprost-loaded C/G/GP hydrogels are shown in Fig. 6 (a) and (b), respectively. After subconjunctival delivery of the latanoprost-loaded C/G/GP hydrogel, the histological analysis did not show any signs of inflammation at day 60 (Fig. 6 (b)).
15
4 Discussion
In this study, thermosensitive C/G/GP hydrogel was used as a sustained release carrier of latanoprost for controlling ocular hypertension, which is usually related to glaucoma. The gelation mechanism of thermosensitive C/G/GP hydrogels has been reported in our previous studies [20, 21]. Hydrophobic interactions might be the main driving force for the formation of hydrogels at 37°C. In this study, the results of rheological analyses indicated that the gelation temperature of the latanoprost-loaded C/G/GP hydrogel was 32.14°C (Fig. 1 (a)). The latanoprost-loaded C/G/GP solution could turn into a gel within 1 minute at 37°C and remain liquid for 15 minutes at 25°C (Fig. 1 (b) (c)). The rheological characterization of the latanoprost-loaded C/G/GP hydrogel was similar to the C/G/GP hydrogel [20]. The results suggested that 500 µg/ml of latanoprost may not affect the gelation properties of C/G/GP hydrogel.
Natarajan et al. [14, 15] used egg-phosphatidylcholine liposomes as a sustained delivery system for latanoprost. Their results showed that a slow and sustained release of 60% latanoprost was achieved at day 14 [14]. In our study, the C/G/GP hydrogel released approximately 70% of latanoprost at day 28 without the initial burst effect (Fig. 2). Our results suggest that the C/G/GP hydrogel provides a more prolonged-release of latanoprost. The dose of latanoprost released per day from the hydrogel was approximately 1.2 µg, lower than that of topical latanoprost eye drops 16
(1.5 µg/drop/day). It has been reported that only 1% to 7% of topically administered eye drops actually reach the anterior segment [27]. The subconjunctival delivery of latanoprost-loaded C/G/GP hydrogel might provide long-term sustained release and might maintain a constant drug concentration. It has been reported that the release of hydrophobic compounds from C/GP hydrogel can be sustained for one month [19]. In our study, the C/G/GP hydrogel showed the sustained release of latanoprost in-vitro for at least 28 days (Fig. 2). The release behavior of hydrophobic compounds from the C/G/GP hydrogel was similar to the C/GP hydrogel. However, the gelation time of the C/GP hydrogel was longer than 10 minutes at 37°C, as shown in our previous study [20]. Therefore, the gelation properties of the C/G/GP hydrogel may be more suitable for sustained release of latanoprost in clinical practice. Chitosan and gelatin are natural polymers that have good biocompatibility and biodegradation [28-31]. In the past few years, chitosan and gelatin have been widely used for various biomedical applications, including drug delivery and tissue engineering. It has been reported that the deacetylation degree of chitosan is strongly associated with biocompatibility. The higher deacetylated chitosan hydrogel had a longer residence time and resulted in no detectable inflammation [16, 32]. In the present study, the deacetylation degree of chitosan was higher than 95%. The results of the cytotoxicity assay showed that there were no harmful effects of 17
latanoprost-loaded C/G/GP hydrogel to HCEC (Fig. 3). The results of the hemolysis test indicated that the latanoprost-loaded C/G/GP hydrogel has no hemolytic action (Table 1). It has been demonstrated that the chitosan-based injectable hydrogel was safe and had no hemolytic effect [33]. The C/G/GP hydrogel also may have hemocompatibility and may be suitable for further drug delivery application. The elevation of IOP may lead to glaucoma, and the reduction of IOP is currently the only recognized effective method to decrease the risk of blindness resulting from glaucoma [34]. In this study, we established a rabbit model of ocular hypertension by intravitreal injection of TA, resulting in elevated IOP (~50% increase compared with the control eye) lasting for at least 60 days. After the subconjunctival injection of the latanoprost-loaded C/G/GP hydrogel, IOP was decreased effectively within 8 days and then remained at a normal level for the next 31 days (Fig. 4 (b)). The results of the in-vivo release study showed a steady drug concentration in the aqueous humor without noticeable burst release (Fig. 5). The results suggested that the effective concentration of latanoprost to treat ocular hypertension should be between 2.77 (day 3) and 4.30 (day 7) ng/ml because IOP was decreased effectively within 8 days (Fig. 4 (b)). The sustained release of latanoprost from the C/G/GP hydrogel might decrease IOP to the normal level within 8 days (Fig. 4 (b)). After the subconjunctival delivery of latanoprost-loaded C/G/GP hydrogel, the 18
histological analysis did not show any signs of inflammation at the end of the study period (Fig. 6). The results suggested that latanoprost-loaded C/G/GP hydrogel may have potential applications in glaucoma therapy, especially for those who are unable to apply eye drops by themselves due to physical limitations, vision impairment, or poor medication adherence.
19
5 Conclusion
In this study, we developed a thermosensitive latanoprost-loaded C/G/GP hydrogel which could gel within 1 minute at 37°C. The results of the release study showed a sustained release of latanoprost from C/G/GP hydrogel in-vitro and in-vivo. The newly developed hydrogel showed a good in-vitro and in-vivo biocompatibility. We demonstrated that 500 µg/ml of latanoprost-loaded C/G/GP hydrogel could significantly decrease the ocular hypertension induced by TA. These study results suggest that the subconjunctival delivery of latanoprost-loaded C/G/GP hydrogel could be applied as a sustained-release system for glaucoma therapy in the future.
6 Disclosures
The authors declare that there are no conflicts of interest.
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Fig. 1 Temperature and time dependence of storage modulus (G’) and loss modulus (G”) of latanoprost-loaded C/G/GP solution was gelled at pH 7.4. (a) The gelation temperature is 32.14 ± 0.82°C. The gelation time is (b) 51.15 ± 3.63 seconds at 37°C and (c) over 600 seconds at 25°C (n=3).
Fig. 2 In-vitro release study (a) Mass spectrum at a positive ionization mode of latanoprost, (b) Cumulative percent release of latanoprost from C/G/GP hydrogels in PBS at 37°C (n=5)
Fig. 3 Cytotoxicity of the latanoprost-loaded hydrogels to HCEC, (a) cell morphology at 48 hours (scale bar, 100 µm) and (b) crystal violet assay at 24 and 48 hours (n = 4, p＞0.05).
Fig. 4 IOP lowering effects of latanoprost-loaded C/G/GP hydrogel. Ocular hypertension was induced by triamcinolone acetonide (TA) at day 0 and 7. Single subconjunctival injection of latanoprost-loaded C/G/GP hydrogel was performed at day 21. The normal eyes (without treatment) were served as a control. The IOP was (a) increased after the intravitreal injection of TA and (b) decreased in the post-treatment of latanoprost-loaded C/G/GP hydrogel (n=5, * p < 0.05). 27
Fig. 5 Aqueous humor levels of latanoprost in rabbits after administration of latanoprost-loaded C/G/GP hydrogel at 3, 5, 7, 17 and 27 days (n=3).
Fig. 6 Histology revealed no abnormal scarring or damage in subconjunctiva after subconjunctival administration of latanoprost-loaded C/G/GP hydrogel (scale bar, 200 µm). * represents latanoprost-loaded C/G/GP hydrogel area.
Table 1 Hemolysis rate of latanoprost-loaded C/G/GP hydrogel
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Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Groups Positive control Latanoprost-loaded C/G/GP hydrogel Negative control
29
Hemolysis (%) 100 ± 1.1 0 ± 0.1 0 ± 0.1
Graphical Abstract
