http://informahealthcare.com/phd ISSN: 1083-7450 (print), 1097-9867 (electronic) Pharm Dev Technol, Early Online: 1–7 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/10837450.2015.1026607
RESEARCH ARTICLE
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Thermosensitive in situ nanogel as ophthalmic delivery system of curcumin: development, characterization, in vitro permeation and in vivo pharmacokinetic studies Rui Liu1*, Lu Sun1*, Shiming Fang2, Shuting Wang1, Jingjing Chen1, Xuefeng Xiao1, and Changxiao Liu3 1
School of Traditional Chinese Materia Medica and 2Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin, P.R. China, and 3Center for Drug Evaluation Research, Tianjin Institute of Pharmaceutical Research, Tianjin, P.R. China
Abstract
Keywords
Context: The nanogel combining cationic nanostructured lipid carriers (CNLC) and thermosensitive gelling agent could enhance preocular retention and ocular permeation capacity of curcumin (CUR). Objective: The purpose of the study was to develop and characterize a thermosensitive ophthalmic in situ nanogel of CUR-CNLC (CUR-CNLC-GEL) and evaluate in vitro and in vivo properties of the formulations. Materials and methods: The physicochemical properties, in vitro release and corneal permeation, were evaluated. Ocular irritation and preocular retention capacity were also conducted. Finally, pharmacokinetic study in the aqueous humor was investigated by microdialysis technique. Results: The solution–gel transition temperature of the optimized formulation diluted by simulated tear fluid was 34 ± 1.0 C. The CUR-CNLC-GEL displayed zero-order release kinetics. The apparent permeability coefficient (Papp) and the area under the curve (AUC0!1) of CUR-CNLC-GEL were 1.56-fold and 9.24-fold, respectively, than those of curcumin solution (CUR-SOL, p50.01). The maximal concentration (Cmax) was significantly improved (p50.01). The prolonged mean residence time (p50.01) indicated that CUR-CNLC-GEL is a controlled release formulation. Discussion and conclusion: Those results demonstrated that CUR-CNLC-GEL could become a potential formulation for increasing the bioavailability of CUR in the aqueous humor by enhancing corneal permeation and retention capacity.
Cationic nanostructured lipid carriers, microdialysis, ocular drug delivery, pharmacokinetics, thermosensitive in situ nanogel
Introduction The unique characteristics of ocular tissues and the anatomical and physicochemical barriers of ocular globe make the permeation of ocular drugs a difficult task1. Only less than 5% of the administrated drugs for traditional formulations can penetrate the cornea2. There are several reasons for the low therapeutic efficacy of traditional ophthalmic dosage forms, such as blinking reflex, tear dilution and nasolacrimal duct drainage and so on.3 Hence, traditional pharmaceuticals are not appropriate for administration due to their low bioavailability. In order to overcome these shortcomings and increase ocular drug bioavailability, several strategies including microparticles, colloidal carriers [e.g. micelles, drug nanosuspensions, nanoemulsions, liposomes, nanostructured lipid carriers (NLC) and polymeric nanoparticles], and in situ nanogel have been developed and investigated1,2,4–8. Among these new delivery systems, NLC
*These co-first authors contributed equally to this work. Address for correspondence: Xuefeng Xiao, School of Traditional Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine, 312 Anshanwest Road, Nankai District, Tianjin 300193, P.R. China. Tel: +86 02259596221. E-mail: [email protected]
History Received 30 December 2014 Revised 17 February 2015 Accepted 25 February 2015 Published online 29 May 2015
composed of a solid lipid matrix with a certain content of spatially incompatible liquid lipid appear to be the most promising candidate for ocular drug delivery9. NLC can improve drug bioavailability either by facilitating transcorneal penetration or by prolonging the precorneal residence time10. Cationic NLC (CNLC) has been recently investigated for targeting ocular mucosa, namely the posterior segment of the eye. This is a smart strategy that combines the positive surface charge of the particles and the negative surface charge of ocular mucosa by means of an electrostatic attraction11. Chitosan is a suitable cationic material for mucosal delivery due to its biodegradability and biocompatibility12. Previous studies have presented chitosan as a well-tolerated material for ophthalmic administration13. Amphipathic octadecyl-quaternized carboxymethyl chitosan (QACMC), a kind of commonly used chitosan, applied in this study could improve the water solubility of lipid soluble drugs, increase the stability of nanoparticles and prolong the circulation time of the CNLC14. Nowadays, a kind of droppable gel called ‘‘in situ nanogel’’ consisting of certain polymers which can undergo solution–gel phase transition has been developed. It makes a major progress in ophthalmic gel technology by increasing bioavailability. In particular, thermosensitive in situ nanogel used as ophthalmic product vehicle possesses liquid characteristic at low temperature and becomes gel when it contacts with eyes. Poloxamer 407 and
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Poloxamer 188 are the most common polymer types possessing thermosensitive in situ nanogel characteristics. They are triblock copolymer showing amphiphilic behavior due to the hydrophilic ethylene oxide domains and hydrophobic propylene oxide domains15. Curcumin (CUR) is a natural phenolic compound isolated from the rhizomes of Curcuma. Evidence suggests various beneficial pharmacological effects of CUR, including antioxidant, anti-inflammatory, anti-carcinogenic, anti-bacterial and anti-coagulantion16,17. CUR has also been proved effective for ophthalmic use in various ocular fundus pathologies. It inhibits proliferation of human lens epithelial cells and protects retinal cells, retinal ganglion cells and corneal epithelial cells18,19. However, owing to main drawbacks such as low solubility, instability, poor bioavailability, the clinical application of CUR is limited. In this study, a novel formulation of CUR for ophthalmic application was formulated by using a combination of CNLC and Poloxamer as thermosensitive gelling agent. The developed thermosensitive in situ nanogel system combines the advantage of CNLC as a drug carrier with the virtue of in situ gelling delivery systems to address the problem of low ocular bioavailability caused by poor aqueous solubility of the drug and rapid drug loss caused by ocular protective mechanisms. The objective of this study was to develop and evaluate the formulation of thermosensitive CNLC in situ nanogel of CUR (CUR-CNLC-GEL), characterize physicochemical properties, in vitro release, in vivo ophthalmic absorption and evaluate the potential for being non-irritant.
Pharm Dev Technol, Early Online: 1–7
407, 0.2 g Poloxamer 188 and 15 mL of distilled water was used as a gel base solution. Six microliters of CUR-CNLC was added to the base solution at 4 C with continuous stirring to form homogeneous mixture. Finally, the mixture was incubated at 35 C for gelation. Characterization of CUR-CNLC-GEL Determination of visual appearance, clarity and pH Appearance and clarity were determined visually against white background at room temperature (20 C). The pH of the formulations was determined using pH meter (PB-10, Sartorius Scientific Instruments Co., Ltd., Beijing, China). Determination of flow ability and the gelation temperature The phase behavior and gelation temperature of the examined formulations were determined by tube inversion method21,22. Briefly, test tubes were filled with 1 g in situ nanogel sample and incubated at 5 ± 1 C (storage temperature), 25 ± 1 C (average room temperature in China) and 35 ± 1 C (precorneal temperature), respectively23. The tubes were shaken at 50 rpm. Samples flowing at 5 ± 1 and 25 ± 1 C but not at 35 ± 1 C within 30 s were accepted as optimal thermosensitive in situ nanogel in this study. Samples were investigated under two different conditions. One condition was with dilution by simulated tear fluid (STF) at a ratio of 40:7 (CUR-CNLC-GEL:STF, v/v), the other condition was without dilution by STF15,23. Each measurement was repeated three times. Viscosity test
Materials and methods Materials CUR was purchased from Zelang Medical Technology Co., Ltd. (98%, Jiangsu, China). Glycerin monostearate (GMS) and Tween80 were supplied by Tianjin Guangfu Chemical Research Institute (Tianjin, China). Gelucire 44/14 was gifted by Gattefosse S.A. (Saint-Priest, France). MiglyolÕ 812 and Myri 52 were generously provided by Fengli Jingqiu Commerce and Trade Co., Ltd. (Beijing, China). Poloxamer 188 and Poloxamer 407 were gifted by BASF Chemical Company (Ludwigshafen, Germany). QACMC was supplied by Nantong Lushen Biological Engineering Co., Ltd. (Jiangsu, China). All other reagents were of analytical grade. Preparation of CUR-CNLC-GEL CUR-CNLC was prepared by film-ultrasonic technique. Briefly, ethanol was added to the lipid phase containing 2 mg CUR, 25.45 mg GMS and 14.55 mg miglyolÕ 812 appropriately. Then the lipid phase was heated up to 80 C to melt the lipids. After that a rotary evaporator (RE-52AA, Ya Rong Biochemical Instrument Co., Ltd., Shanghai, China) was used to remove ethanol in the lipid phase at 80 C. Meanwhile, 50.10 mg Myri 52, 21.21 mg Gelucire 44/14 and 10.00 mg QACMC were added to 50 mL of distilled water heated to 80 C. The solution was stirred by intelligent magnetic heating stirrer (SZCL-4B, Yu Hua Instrument Co., Ltd., Henan, China) to obtain a homogeneous lipid solution. A small amount of ethanol was added to the hot lipid phase, and then it was dispersed in the aqueous phase to form a pre-emulsion. After stirring at 80 C for 0.5 h, the pre-emulsion was treated by an ultrasonic cleaner (KH 2200B, He Chuang Ultrasonic Co., Ltd., Shanghai, China) at the same temperature for 0.5 h. The resulting hot O/W nano-emulsion was cooled at 0 C and then restirred to form the CUR-CNLC. Next, the CUR-CNLC-GEL was prepared by cold method20. A mixture of 4.8 g Poloxamer
Viscosity measurement was performed using a rheometer (DV-III ULTRA, Brookfield Engineering Laboratories Inc., Middleborough, MA) at different temperatures. The method consulted the paper of Sun et al.24. The rotor model was SC4-18 and SC4-64. Revolving speed was 60 rpm. The study was performed in triplicate. Morphological study Optimized CUR-CNLC-GEL formulation was examined morphologically by transmission electron microscope (TEM, HT7700, Hitachi, Tokyo, Japan). After diluting the sample 20-fold with purified water, a drop of the suspension was immediately spread on a copper grid, and the excess liquid was removed with filter paper. Then the sample was allowed to dry at ambient atmosphere. In vitro release study In vitro release behavior of CUR from CUR-CNLC-GEL was investigated at 35 C in PBS solution with 5% Tween-80 (v/v). The Tween-80 was added into the release medium according to the experimental requirements. The volume of Tween-80 was adjusted considering its ability of increasing the solubility of CUR and sink conditions. Briefly, 1 mL of CUR-CNLC-GEL was prepared in a 10 mL glass bottle followed by the addition of 2.4 mL of PBS solution with 5% Tween-80 as release medium. Thereafter, the samples were incubated at water bath at 35 C with the rotary speed of 100 rpm. At predetermined time points, all release medium was withdrawn for detection of drug concentration by high performance liquid chromatography (HPLC, e2695, Waters Ltd., Milford, CT). Another freshly prepared solution was added into glass bottle for continuous study. HPLC analysis was carried out on a reversed phase C18 column (4.6 mm 200 mm, 5 mm, Diamonsil C18) and the column temperature was set at 35 C. The mobile phase was composed of methanol and 0.05%
Nanogel as ophthalmic delivery system of curcumin
DOI: 10.3109/10837450.2015.1026607
formic acid (80/20, v/v). The flow rate was 1.0 mLmin1 and the eluent was detected by a UV 2998 detector at 425 nm. Corneal permeation evaluation and hydration level
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In vitro corneal permeation profiles of curcumin solution (CURSOL) and CUR-CNLC-GEL formulations were evaluated using excised corneas of rabbits. The method consulted the paper of Liu et al.25. The solution in receiver was fresh glutathione bicarbonate Ringer’s solution (GBR) with 3% Tween-80 (v/v). At the end of the corneal permeation experiment, each cornea was weighed to calculate the corneal hydration level. The study was performed in triplicate. In vivo eye irritation test
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where Cd, Cp and Cm are the concentrations of the drug in the dialysate, perfusate and aqueous humor, respectively. R is the value of the slope for the plot of Cd Cp versus Cp. Two-hundred microliters of CUR-SOL (0.5 mg mL1) and CUR-CNLC-GEL (0.5 mg mL1) were instilled into the eyes of two groups of rabbits. The dialysate was collected every 20 min at the first 2 h and every 30 min thereafter. The study was performed in triplicate and all samples were analyzed by HPLC. Statistical analysis The results were presented as mean (X) ± standard deviations (SD). The data of in vivo study were statistically evaluated by two one-sided t test. Statistical analysis of the other results was assessed by one-way analysis of variance (ANOVA) using SPSS software (17.0 version, SPSS Inc., Chicago, IL) and the statistical significance was p50.05.
Eye irritation test was performed according to Draize technique on six New Zealand white rabbits26. New Zealand white rabbits weighing 2.5–3.0 kg were used in the study. The animals housed in a light-controlled room at 25 ± 2 C and 50 ± 5% relative humidity were fed with a standard pellet diet and water. Procedures involving animal care and management were reviewed and approved by the Animal Ethical Committee at Tianjin University of Traditional Chinese Medicine. CUR-CNLC-GEL was instilled into the left eyes and PBS (pH 7.4) was instilled into the right eyes, 0.1 mL every 4 h, four times a day for a period of 21 days. The degree of eye irritation was evaluated by scoring lesions of conjunctiva, cornea and iris, at specific intervals.
The developed CUR-CNLC-GEL was light yellow and clear. The system was flowable liquid at lower temperature about 5 C, while it turned into the unflowable gel at physiological temperature about 35 C. This characteristic indicated that it was suitable for ocular application. Moreover, as a drug delivery system for ocular application, CUR-CNLC-GEL was ideally transparent. The pH of formulations was 6.34 ± 0.03.
Preocular retention time study
Determination of flow ability and gelation temperature
In vivo preocular retention capability of CUR-CNLC-GEL formulation was investigated as described by Liu et al.25. The fluorescent CUR-SOL and CUR-CNLC-GEL formulations were prepared by adding a certain amount of coumarin 6. The fluorescent CUR-CNLC-GEL formulation (0.1 mL) was instilled into the left rabbit eye and CUR-SOL into the right eye as a control. Preocular retention time was observed at appropriate time intervals by examining with a slit lamp (YZ2, Kangjie Medical Instrument Company, Suzhou, China). The time course of fluorescence on the corneal surface and in the conjunctival sac was evaluated by the same operator.
The flow ability and solution–gel transition temperature results for different samples are shown in Table 1. The 24% Poloxamer 407 formulation diluted by STF is supposed to exhibit solution characteristic at 25 C and change into gel at the body temperature. The STF diluting method is used to simulate the dilution after formulation administered topically. Solution–gel transition temperatures of formulations containing different content of Poloxamer 407 were compared. The result suggests that formulations containing more Poloxamer 407 would exhibit a lower solution–gel transition temperature. Poloxamer 407 consisting of higher ratio of hydrophobic poly(propylene oxide) units/hydrophilic poly(ethylene oxide) units per mole compared to Poloxamer 188 could decrease the hydrophobic interaction between each Poloxamer micelle. Therefore, the solution–gel transition of the system was lower28. However, the solution–gel transition temperature of the formulation should be higher than the room temperature to avoid refrigeration. Thus, the formulation of 24% Poloxamer 407 (V/V) was chosen for further study.
Pharmacokinetic study Pharmacokinetic study in the aqueous humor was carried out by microdialysis technique. Three rabbits were locally anesthetized around the ocular region with 20 mg kg1 of lidocaine hydrochloride administered intravenously. A microdialysis probe (MD 2000, Bioanalytical Systems, Inc, West Lafayette, IN) was implanted into the anterior chamber of the eye by a 20 G needle, thereafter the needle was removed. The probe was adjusted, and the microdialysis membrane totally resided in the anterior chamber. Thereafter, it was perfused with GBR containing 0.06% Tween-80 at a rate of 3 mL min1 by a microdialysis pump (CMA106, CMA Microdialysis Co., Ltd, Stockholm, Sweden). The corneal wound was treated with 0.3% (w/v) ofloxacin ophthalmic drops and allowed to stabilize for 1 day before topical drug administration. Following a 1 h equilibration period with perfusion of GBR containing 0.06% Tween-80 (v/v), standard drug solution of different concentrations was perfused and the dialysate was collected for 15 min after 30 min of perfusion. In vivo recovery (R) is calculated by the following equation27: R% ¼
Cd Cp 100, Cm Cp
ð1Þ
Results and discussion Determination of visual appearance, clarity and pH
Viscosity test The viscosity of CUR-CNLC-GEL was estimated by a rheological instrument to predict retention behavior and physical integrity in vivo. As presented in Figure 1, when the temperature reached 34 C, a transition occurred which suggested that the system was more viscous. The increase in viscosity is expected to trigger prolonged retention of formulation on the corneal surface. Morphological study The TEM image in Figure 2 revealed that the optimized CURCNLC-GEL formulation was dispersed as individual nanostructured lipid carrier and a network of Poloxamer. The particle size of the optimized CUR-CNLC formulation was 158.1 nm and polydispersity index was 0.29. CUR-CNLC were spherical in shape and homogeneously in distribution.
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Table 1. Evaluation of transparency and flow ability of the CUR-CNLC-GEL (X ± SD, n ¼ 3).
Formulation
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20% 22% 23% 24% 22% 23% 24%
Poloxamer Poloxamer Poloxamer Poloxamer Poloxamer Poloxamer Poloxamer
407 407 407 407 407-STF 407-STF 407-STF
Transparency at temperaturea ( C)
Flow ability at temperatureb ( C)
5±1
25 ± 1
35 ± 1
5±1
25 ± 1
35 ± 1
Tsolution–gel
+++ +++ +++ +++ +++ +++ +++
+++ +++ +++ +++ +++ +++ +++
+++ +++ +++ +++ +++ +++ +++
+++ +++ +++ +++ +++ +++ +++
+++ +++ ++ – +++ +++ +++
++ – – – +++ +++ –
48 ± 1.0 35 ± 0.6 29 ± 0.6 24 ± 1.2 81 ± 0.6 71 ± 0.6 34 ± 1.0
Tsol-gel ¼ solution–gel transition temperature. a Transparency: +++, transparent; ++, slightly translucent; +, translucent; , turbid. b Flow ability: +++, very good; ++, good; +, average; , not flow.
Figure 1. Temperature-induced viscosity change of the optimal CUR-CNLC-GEL formulation.
Figure 3. In vitro release profile of CUR from the optimal CUR-CNLCGEL formulation (n ¼ 3). N is the cumulative released percentage of CUR from in situ nanogel, and the experiment was conducted at 25 C.
Figure 2. TEM micrograph of the optimal CUR-CNLC-GEL formulation.
In vitro release study The percentage of CUR released from in situ nanogel was plotted as a function with time. As depicted in Figure 3, 94.91% of CUR was released from CUR-CNLC-GEL within 4 h. Release profiles
evaluated using different models (zero order, first order, and Higuchi square-root, Hixson–Crowell models, Ritger–Peppas model and Weibull) are shown in Table 2. Drug release fitted to zero-order kinetics. Linearity in the zero-order equation plot (R2 ¼ 0.9961) suggested that the release of drug from formulation was only time dependent29. Polymeric controlled release delivery systems can be classified as matrix and reservoir systems. In a matrix system, the drug is distributed throughout the polymeric matrix. In a reservoir system, however, the drug is present in the core, surrounded by a polymeric film that is rate-limiting.
Nanogel as ophthalmic delivery system of curcumin
DOI: 10.3109/10837450.2015.1026607
Table 2. Parameters of the mathematical models of regression for CURCNLC-GEL. Equation
R2
Y ¼ 0.2511t 0.03635 ln(1 Y) ¼ 0.8053t + 0.7378 Y ¼ 0.7076t1/2 0.4934 (1 Y)1/3 ¼ 0.1721t + 1.118 ln Y ¼ 1.085lnt 1.532 ln ln[1/(1 Y)] ¼ 1.617lnt 1.418
0.9961 0.8902 0.9798 0.9552 0.9958 0.9652
Model Zero order First order Higuchi Hixcon–Crowell Ritger–Peppas Weibull
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Y, the percentage of CUR released from CUR-CNLC-GEL; t, time point; R, regression coefficient. Table 3. Corneal permeability coefficients (X±SD, n ¼ 3). Parameters 7
1
Papp 10 (cm s ) Jss 106 (mg s1 cm2) HL (%)
CUR-SOL
CUR-CNLC-GEL
3.778 ± 0.2940 1.889 ± 0.1470 78.25 ± 1.919
5.894 ± 0.2601** 2.330 ± 0.1028* 79.70 ± 2.692
Papp, the apparent permeability coefficient; Jss, the steady-state flux; HL, hydration level. *p50.05 versus CUR-SOL; **p50.01 versus CUR-SOL.
Reservoir systems typically allow zero-order or constant drug release30. Poloxamer hydrogel as reservoir systems was formed mainly by the physical aggregation of micelles, which easily collapse when placed in PBS solution within a very short period of time. However, with the collapse of Poloxamer hydrogel, the encapsulated CUR-CNLC is released, which might be able to serve as the controlled release system for CUR2. Therefore, the release of CUR from CUR-CNLC-GEL appears to be controlled by gel dissolution and nanoparticles decomposition. In vitro corneal permeation evaluation Table 3 compared the corneal permeability coefficients of CURSOL and CUR-CNLC-GEL formulation. CUR-CNLC-GEL formulation showed significantly higher Papp value (1.56-fold) than that of CUR-SOL (p50.01). The results revealed significantly higher corneal permeation of CUR from the CUR-CNLC-GEL formulation compared to the conventional formulation based on aqueous solution. The normal corneal hydration level is reported to be 75–80%31. Thus, corneal hydration level in the present study was within limits indicating integrity of corneal epithelium and endothelium. During earlier studies, drugs formulated as nanoparticulate carriers have been reported to provide a higher corneal permeation which was attributed to the endocytic uptake32. Consequently, enhanced permeation of CUR from CUR-CNLC-GEL across excised rabbit cornea might be attributed to the endocytic uptake after the collapse of Poloxamer hydrogel. In vivo ocular irritation test Ocular irritation level of CUR-CNLC-GEL was 2 ± 0 (score53) which indicated that CUR-CNLC-GEL is non-irritant. Although there was a slight hyperemia in conjunctival, excellent ocular tolerance was noted. Preocular retention time study As shown in Table 4, CUR-SOL formed a weak and short-lived fluorescent film and disappeared rapidly from corneal surface. However, the CUR-CNLC-GEL formulation formed a rather stable precorneal film and retained longer on the corneal surface (p50.01) and in the conjunctival sac compared with those of
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Table 4. Evaluation of the preocular retention time (X ± SD, n ¼ 3). Formulation
Corneal surface (h)
Conjunctival sac (h)
CUR-SOL CUR-CNLC-GEL
0.2778 ± 0.09623 1.333** ± 0.1667
0.5000 ± 0.1667 4.778** ± 0.1925
**p50.01 versus CUR-SOL.
CUR-SOL (p50.01). The prolonged drug retention is due to not only the high viscosity of the in situ nanogel but also the following reasons: first, nanoparticles possess bioadhesive property due to the extremely small particle size and increased surface area33; second, the cationic character and the viscosity of QACMC are able to prolong the retention of drug and improve its bioavailability in ocular administration34. Therefore, these factors ensure an intimate contact with the epithelial mucosal surface of the eye, preventing tear wash out and consequently prolong drug retention time. Pharmacokinetics study Before pharmacokinetic study, in vivo probe recovery was determined to ensure that the implanted probes were in good function. The in vivo recovery was 50.75 ± 3.91% (n ¼ 3). The concentration–time profiles following topical administration of CUR-SOL and CUR-CNLC-GEL are illustrated in Figure 4. The corresponding pharmacokinetic parameters are summarized in Table 5. As shown in Figure 4, the drug was immediately absorbed into the aqueous humor and reached maximal concentration of CUR (Cmax) at 30 min after ocular application of CURSOL. After that, concentration of drug remarkably decreased in the following hours. However, after an instillation of CUR-CNLCGEL, concentrations of drug were much higher than those of CUR-SOL. CUR could exert antioxidant and antiglycating effects through inhibition of lipid peroxidation, advanced glycated end products and protein aggregation at very low concentrations35. The common experimental diet level of CUR in diabetes mellitus is usually below 0.01%36. The topical application dosage of CUR in our research was 0.5 mg mL1 (0.05%), which is much higher than the common level mentioned above. Compared with CUR-SOL, area under the concentration–time curve (AUC) value was enhanced 9.24-fold for CUR-CNLC-GEL (p50.01), and the Cmax value of CUR-CNLC-GEL was improved 3.38-fold (p50.01). There was statistical difference in mean residence time (MRT) compared with those of CUR-SOL (p50.01). There was no statistical difference in either elimination rate constant (ke) or Tmax value of CUR-CNLC-GEL compared with those of CUR-SOL. The higher ocular bioavailability of the CUR-CNLC-GEL could be related to its biological adhesive ability to prolong the residence time on the corneal epithelial layer33. Previous study37 has demonstrated that micelles/nanoparticles formulation could greatly facilitate the drug across the corneal barrier, yet resulting in the enhancement of bioavailability. However, the major shortcoming for micelles/nanoparticles formulation is their quick clearance from ocular tissue. Poloxamer hydrogel has received great attentions due to its noncytotoxic and prolonged residence time on the corneal surface. So, it is able to compensate for the shortcomings of NLC for ophthalmic delivery38.
Conclusions In this article, CUR-CNLC-GEL was developed and physicochemical properties were evaluated including appearance, clarity, pH, flow ability, gelation temperature, viscosity and morphology.
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Figure 4. CUR concentration–time profiles following a 200 mL topical administration at dose of 0.5 mg mL1 in aqueous humor of conscious rabbits (X ± SD, n ¼ 3).
Table 5. Pharmacokinetic parameters of CUR in the aqueous humor after topical administration in the conscious rabbits (X ± SD, n ¼ 3). Formulation CUR-SOL CUR-CNLC-GEL
ke 103
Tmax (min)
Cmax (ng mL1)
AUC (ng mL1 min)
MRT (min)
3.465 ± 2.026 4.755 ± 0.346
30.00 ± 0.000 23.33 ± 11.55
11.56 ± 0.9576 39.13 ± 4.549**
601.4 ± 17.67 5559 ± 453.1**
43.74 ± 0.604 111.8 ± 5.249**
ke, elimination rate constant; Tmax, time to maximal concentration; Cmax, maximal concentration of CUR; AUC, area under the curve; MRT, mean residence time. **p50.01 versus CUR-SOL.
The developed CUR-CNLC-GEL provided controlled CUR release and the prolonged precorneal residence time. In vivo eye irritation test suggested that the developed CUR-CNLC-GEL was non-irritating and it might be suitable for various ocular applications. In vivo pharmacokinetic study indicated that the developed CUR-CNLC-GEL could significantly increase the bioavailability of CUR and maintain drug concentration in aqueous humor after administration compared with that of the CUR-SOL. The CUR-CNLC-GEL could increase the bioavailability of CUR by improving corneal permeation and retention capacity in the aqueous humor. Therefore, our results suggested that the combination of CNLC and thermosensitive in situ gel has a great potential to be an ophthalmic delivery system of CUR.
Acknowledgements We are thankful to Gattefosse´ SAS (Saint-Priest, France), BASF (Ludwigshafen, German) and Beijing Fengli Jingqiu Commerce and Trade Co., Ltd. (Beijing, China) for samples support of this work.
Declaration of interest This project was supported by National Natural Science Foundation of China (No. 81303142), Specialized Research Fund for the Doctoral Program of Higher Education of China (No. 20121210120001) and Tianjin Research Program of Application Foundation and Advanced Technology (No. 14CQNJC13100). The authors report no declarations of interest.
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DOI: 10.3109/10837450.2015.1026607
13. Tiyaboonchai W. Chitosan nanoparticles: a promising system for drug delivery. (Naresuan Univ J). 2013;11: 51–66. 14. Kono H, Kusumoto R. Preparation, structural characterization, and flocculation ability of amphoteric cellulose. React Funct Polym 2014;82:111–119. 15. Asasutjarit R, Thanasanchokpibull S, Fuongfuchat A, Veeranondha S. Optimization and evaluation of thermoresponsive diclofenac sodium ophthalmic in situ gels. Int J Pharm 2011;411:128–135. 16. Dev S, Prabhakaran P, Filgueira L, et al. Microfluidic fabrication of cationic curcumin nanoparticles as an anti-cancer agent. Nanoscale 2012;4:2575–2579. 17. Onoue S, Takahashi H, Kawabata Y, et al. Formulation design and photochemical studies on nanocrystal solid dispersion of curcumin with improved oral bioavailability. J Pharm Sci 2010;99:1871–1881. 18. Burugula B, Ganesh BS, Chintala SK. Curcumin attenuates staurosporine-mediated death of retinal ganglion cells. Invest Ophthalmol Vis Sci 2011;52:4263–4273. 19. Hu Y-h, Huang X-r, Qi M-x, Hou B-y. Curcumin inhibits proliferation of human lens epithelial cells: a proteomic analysis. J Zhejiang Univ Sci B 2012;13:402–407. 20. Heilmann S, Ku¨chler S, Wischke C, et al. A thermosensitive morphine-containing hydrogel for the treatment of large-scale skin wounds. Int J Pharm 2013;444:96–102. 21. Garripelli VK, Kim J-K, Son S, et al. Matrix metalloproteinasesensitive thermogelling polymer for bioresponsive local drug delivery. Acta Biomater 2011;7:1984–1992. 22. Sugihara S, Iwata K, Miura S, et al. Synthesis of dual thermoresponsive ABA triblock copolymers by both living cationic vinyl polymerization and RAFT polymerization using a dicarboxylic RAFT agent. Polymer 2013;54:1043–1052. 23. Lihong W, Xin C, Yongxue G, et al. Thermoresponsive ophthalmic poloxamer/tween/carbopol in situ gels of a poorly water-soluble drug fluconazole: preparation and in vitro–in vivo evaluation. Drug Dev Indus Pharm 2013;40:1402–1410. 24. Sun S-z, Wang Y-j, Zhang D-q, et al. Rheological characteristics of Carbopol 981. Tianjin J Tradit Chin Med 2012;2:025. 25. Liu R, Liu Z, Zhang C, Zhang B. Nanostructured lipid carriers as novel ophthalmic delivery system for mangiferin: improving in vivo ocular bioavailability. J Pharm Sci 2012;101:3833–3844.
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26. Deshmukh PK, Gattani SG. In vitro and in vivo consideration of novel environmentally responsive ophthalmic drug delivery system. Pharm Dev Technol 2013;18:950–956. 27. Aggarwal D, Pal D, Mitra AK, Kaur IP. Study of the extent of ocular absorption of acetazolamide from a developed niosomal formulation, by microdialysis sampling of aqueous humor. Int J Pharm 2007; 338:21–26. 28. Joao Garcia M, Ruivo J, Oliveira R, Figueiras A. Preparation of supramolecular hydrogels containing poloxamers and methylb-cyclodextrin. Lett Drug Des Discov 2014;11:922–929. 29. Aerry S, De A, Kumar A, et al. Synthesis and characterization of thermoresponsive copolymers for drug delivery. J Biomed Mater Res A 2013;101:2015–2026. 30. Thakur A, Kompella UB. Drug delivery systems for diseases of the back of the eye. Treat Ocular Drug Deliv 2013;1:114–139. 31. Kalam A, Sultana Y, Ali A, et al. Part II: enhancement of transcorneal delivery of gatifloxacin by solid lipid nanoparticles in comparison to commercial aqueous eye drops. J Biomed Mater Res A 2013;101:1828–1836. 32. Sharma R, Ahuja M, Kaur H. Thiolated pectin nanoparticles: preparation, characterization and ex vivo corneal permeation study. Carbohydr Polym 2012;87:1606–1610. 33. Kompella UB, Amrite AC, Pacha Ravi R, Durazo SA. Nanomedicines for back of the eye drug delivery, gene delivery, and imaging. Progr Retinal Eye Res 2013;36:172–198. 34. Jøraholmen MW, Vanic´ Zˇ, Tho I, Sˇkalko-Basnet N. Chitosan-coated liposomes for topical vaginal therapy: assuring localized drug effect. Int J Pharm 2014;472:94–101. 35. Pescosolido N, Giannotti R, Plateroti AM, et al. Curcumin: therapeutical potential in ophthalmology. Planta Med 2014;80: 249–254. 36. Libby P, Plutzky J. Inflammation in diabetes mellitus: role of peroxisome proliferator-activated receptor-a and peroxisome proliferator-activated receptor-g agonists. Am J Cardiol 2007;99: 27–40. 37. Li X, Zhang Z, Li J, et al. Diclofenac/biodegradable polymer micelles for ocular applications. Nanoscale 2012;4:4667–4673. 38. Casolaro M, Casolaro I, Lamponi S. Stimuli-responsive hydrogels for controlled pilocarpine ocular delivery. Eur J Pharm Biopharm 2012;80:553–561.
RESEARCH ARTICLE
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Thermosensitive in situ nanogel as ophthalmic delivery system of curcumin: development, characterization, in vitro permeation and in vivo pharmacokinetic studies Rui Liu1*, Lu Sun1*, Shiming Fang2, Shuting Wang1, Jingjing Chen1, Xuefeng Xiao1, and Changxiao Liu3 1
School of Traditional Chinese Materia Medica and 2Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin, P.R. China, and 3Center for Drug Evaluation Research, Tianjin Institute of Pharmaceutical Research, Tianjin, P.R. China
Abstract
Keywords
Context: The nanogel combining cationic nanostructured lipid carriers (CNLC) and thermosensitive gelling agent could enhance preocular retention and ocular permeation capacity of curcumin (CUR). Objective: The purpose of the study was to develop and characterize a thermosensitive ophthalmic in situ nanogel of CUR-CNLC (CUR-CNLC-GEL) and evaluate in vitro and in vivo properties of the formulations. Materials and methods: The physicochemical properties, in vitro release and corneal permeation, were evaluated. Ocular irritation and preocular retention capacity were also conducted. Finally, pharmacokinetic study in the aqueous humor was investigated by microdialysis technique. Results: The solution–gel transition temperature of the optimized formulation diluted by simulated tear fluid was 34 ± 1.0 C. The CUR-CNLC-GEL displayed zero-order release kinetics. The apparent permeability coefficient (Papp) and the area under the curve (AUC0!1) of CUR-CNLC-GEL were 1.56-fold and 9.24-fold, respectively, than those of curcumin solution (CUR-SOL, p50.01). The maximal concentration (Cmax) was significantly improved (p50.01). The prolonged mean residence time (p50.01) indicated that CUR-CNLC-GEL is a controlled release formulation. Discussion and conclusion: Those results demonstrated that CUR-CNLC-GEL could become a potential formulation for increasing the bioavailability of CUR in the aqueous humor by enhancing corneal permeation and retention capacity.
Cationic nanostructured lipid carriers, microdialysis, ocular drug delivery, pharmacokinetics, thermosensitive in situ nanogel
Introduction The unique characteristics of ocular tissues and the anatomical and physicochemical barriers of ocular globe make the permeation of ocular drugs a difficult task1. Only less than 5% of the administrated drugs for traditional formulations can penetrate the cornea2. There are several reasons for the low therapeutic efficacy of traditional ophthalmic dosage forms, such as blinking reflex, tear dilution and nasolacrimal duct drainage and so on.3 Hence, traditional pharmaceuticals are not appropriate for administration due to their low bioavailability. In order to overcome these shortcomings and increase ocular drug bioavailability, several strategies including microparticles, colloidal carriers [e.g. micelles, drug nanosuspensions, nanoemulsions, liposomes, nanostructured lipid carriers (NLC) and polymeric nanoparticles], and in situ nanogel have been developed and investigated1,2,4–8. Among these new delivery systems, NLC
*These co-first authors contributed equally to this work. Address for correspondence: Xuefeng Xiao, School of Traditional Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine, 312 Anshanwest Road, Nankai District, Tianjin 300193, P.R. China. Tel: +86 02259596221. E-mail: [email protected]
History Received 30 December 2014 Revised 17 February 2015 Accepted 25 February 2015 Published online 29 May 2015
composed of a solid lipid matrix with a certain content of spatially incompatible liquid lipid appear to be the most promising candidate for ocular drug delivery9. NLC can improve drug bioavailability either by facilitating transcorneal penetration or by prolonging the precorneal residence time10. Cationic NLC (CNLC) has been recently investigated for targeting ocular mucosa, namely the posterior segment of the eye. This is a smart strategy that combines the positive surface charge of the particles and the negative surface charge of ocular mucosa by means of an electrostatic attraction11. Chitosan is a suitable cationic material for mucosal delivery due to its biodegradability and biocompatibility12. Previous studies have presented chitosan as a well-tolerated material for ophthalmic administration13. Amphipathic octadecyl-quaternized carboxymethyl chitosan (QACMC), a kind of commonly used chitosan, applied in this study could improve the water solubility of lipid soluble drugs, increase the stability of nanoparticles and prolong the circulation time of the CNLC14. Nowadays, a kind of droppable gel called ‘‘in situ nanogel’’ consisting of certain polymers which can undergo solution–gel phase transition has been developed. It makes a major progress in ophthalmic gel technology by increasing bioavailability. In particular, thermosensitive in situ nanogel used as ophthalmic product vehicle possesses liquid characteristic at low temperature and becomes gel when it contacts with eyes. Poloxamer 407 and
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Poloxamer 188 are the most common polymer types possessing thermosensitive in situ nanogel characteristics. They are triblock copolymer showing amphiphilic behavior due to the hydrophilic ethylene oxide domains and hydrophobic propylene oxide domains15. Curcumin (CUR) is a natural phenolic compound isolated from the rhizomes of Curcuma. Evidence suggests various beneficial pharmacological effects of CUR, including antioxidant, anti-inflammatory, anti-carcinogenic, anti-bacterial and anti-coagulantion16,17. CUR has also been proved effective for ophthalmic use in various ocular fundus pathologies. It inhibits proliferation of human lens epithelial cells and protects retinal cells, retinal ganglion cells and corneal epithelial cells18,19. However, owing to main drawbacks such as low solubility, instability, poor bioavailability, the clinical application of CUR is limited. In this study, a novel formulation of CUR for ophthalmic application was formulated by using a combination of CNLC and Poloxamer as thermosensitive gelling agent. The developed thermosensitive in situ nanogel system combines the advantage of CNLC as a drug carrier with the virtue of in situ gelling delivery systems to address the problem of low ocular bioavailability caused by poor aqueous solubility of the drug and rapid drug loss caused by ocular protective mechanisms. The objective of this study was to develop and evaluate the formulation of thermosensitive CNLC in situ nanogel of CUR (CUR-CNLC-GEL), characterize physicochemical properties, in vitro release, in vivo ophthalmic absorption and evaluate the potential for being non-irritant.
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407, 0.2 g Poloxamer 188 and 15 mL of distilled water was used as a gel base solution. Six microliters of CUR-CNLC was added to the base solution at 4 C with continuous stirring to form homogeneous mixture. Finally, the mixture was incubated at 35 C for gelation. Characterization of CUR-CNLC-GEL Determination of visual appearance, clarity and pH Appearance and clarity were determined visually against white background at room temperature (20 C). The pH of the formulations was determined using pH meter (PB-10, Sartorius Scientific Instruments Co., Ltd., Beijing, China). Determination of flow ability and the gelation temperature The phase behavior and gelation temperature of the examined formulations were determined by tube inversion method21,22. Briefly, test tubes were filled with 1 g in situ nanogel sample and incubated at 5 ± 1 C (storage temperature), 25 ± 1 C (average room temperature in China) and 35 ± 1 C (precorneal temperature), respectively23. The tubes were shaken at 50 rpm. Samples flowing at 5 ± 1 and 25 ± 1 C but not at 35 ± 1 C within 30 s were accepted as optimal thermosensitive in situ nanogel in this study. Samples were investigated under two different conditions. One condition was with dilution by simulated tear fluid (STF) at a ratio of 40:7 (CUR-CNLC-GEL:STF, v/v), the other condition was without dilution by STF15,23. Each measurement was repeated three times. Viscosity test
Materials and methods Materials CUR was purchased from Zelang Medical Technology Co., Ltd. (98%, Jiangsu, China). Glycerin monostearate (GMS) and Tween80 were supplied by Tianjin Guangfu Chemical Research Institute (Tianjin, China). Gelucire 44/14 was gifted by Gattefosse S.A. (Saint-Priest, France). MiglyolÕ 812 and Myri 52 were generously provided by Fengli Jingqiu Commerce and Trade Co., Ltd. (Beijing, China). Poloxamer 188 and Poloxamer 407 were gifted by BASF Chemical Company (Ludwigshafen, Germany). QACMC was supplied by Nantong Lushen Biological Engineering Co., Ltd. (Jiangsu, China). All other reagents were of analytical grade. Preparation of CUR-CNLC-GEL CUR-CNLC was prepared by film-ultrasonic technique. Briefly, ethanol was added to the lipid phase containing 2 mg CUR, 25.45 mg GMS and 14.55 mg miglyolÕ 812 appropriately. Then the lipid phase was heated up to 80 C to melt the lipids. After that a rotary evaporator (RE-52AA, Ya Rong Biochemical Instrument Co., Ltd., Shanghai, China) was used to remove ethanol in the lipid phase at 80 C. Meanwhile, 50.10 mg Myri 52, 21.21 mg Gelucire 44/14 and 10.00 mg QACMC were added to 50 mL of distilled water heated to 80 C. The solution was stirred by intelligent magnetic heating stirrer (SZCL-4B, Yu Hua Instrument Co., Ltd., Henan, China) to obtain a homogeneous lipid solution. A small amount of ethanol was added to the hot lipid phase, and then it was dispersed in the aqueous phase to form a pre-emulsion. After stirring at 80 C for 0.5 h, the pre-emulsion was treated by an ultrasonic cleaner (KH 2200B, He Chuang Ultrasonic Co., Ltd., Shanghai, China) at the same temperature for 0.5 h. The resulting hot O/W nano-emulsion was cooled at 0 C and then restirred to form the CUR-CNLC. Next, the CUR-CNLC-GEL was prepared by cold method20. A mixture of 4.8 g Poloxamer
Viscosity measurement was performed using a rheometer (DV-III ULTRA, Brookfield Engineering Laboratories Inc., Middleborough, MA) at different temperatures. The method consulted the paper of Sun et al.24. The rotor model was SC4-18 and SC4-64. Revolving speed was 60 rpm. The study was performed in triplicate. Morphological study Optimized CUR-CNLC-GEL formulation was examined morphologically by transmission electron microscope (TEM, HT7700, Hitachi, Tokyo, Japan). After diluting the sample 20-fold with purified water, a drop of the suspension was immediately spread on a copper grid, and the excess liquid was removed with filter paper. Then the sample was allowed to dry at ambient atmosphere. In vitro release study In vitro release behavior of CUR from CUR-CNLC-GEL was investigated at 35 C in PBS solution with 5% Tween-80 (v/v). The Tween-80 was added into the release medium according to the experimental requirements. The volume of Tween-80 was adjusted considering its ability of increasing the solubility of CUR and sink conditions. Briefly, 1 mL of CUR-CNLC-GEL was prepared in a 10 mL glass bottle followed by the addition of 2.4 mL of PBS solution with 5% Tween-80 as release medium. Thereafter, the samples were incubated at water bath at 35 C with the rotary speed of 100 rpm. At predetermined time points, all release medium was withdrawn for detection of drug concentration by high performance liquid chromatography (HPLC, e2695, Waters Ltd., Milford, CT). Another freshly prepared solution was added into glass bottle for continuous study. HPLC analysis was carried out on a reversed phase C18 column (4.6 mm 200 mm, 5 mm, Diamonsil C18) and the column temperature was set at 35 C. The mobile phase was composed of methanol and 0.05%
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formic acid (80/20, v/v). The flow rate was 1.0 mLmin1 and the eluent was detected by a UV 2998 detector at 425 nm. Corneal permeation evaluation and hydration level
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In vitro corneal permeation profiles of curcumin solution (CURSOL) and CUR-CNLC-GEL formulations were evaluated using excised corneas of rabbits. The method consulted the paper of Liu et al.25. The solution in receiver was fresh glutathione bicarbonate Ringer’s solution (GBR) with 3% Tween-80 (v/v). At the end of the corneal permeation experiment, each cornea was weighed to calculate the corneal hydration level. The study was performed in triplicate. In vivo eye irritation test
3
where Cd, Cp and Cm are the concentrations of the drug in the dialysate, perfusate and aqueous humor, respectively. R is the value of the slope for the plot of Cd Cp versus Cp. Two-hundred microliters of CUR-SOL (0.5 mg mL1) and CUR-CNLC-GEL (0.5 mg mL1) were instilled into the eyes of two groups of rabbits. The dialysate was collected every 20 min at the first 2 h and every 30 min thereafter. The study was performed in triplicate and all samples were analyzed by HPLC. Statistical analysis The results were presented as mean (X) ± standard deviations (SD). The data of in vivo study were statistically evaluated by two one-sided t test. Statistical analysis of the other results was assessed by one-way analysis of variance (ANOVA) using SPSS software (17.0 version, SPSS Inc., Chicago, IL) and the statistical significance was p50.05.
Eye irritation test was performed according to Draize technique on six New Zealand white rabbits26. New Zealand white rabbits weighing 2.5–3.0 kg were used in the study. The animals housed in a light-controlled room at 25 ± 2 C and 50 ± 5% relative humidity were fed with a standard pellet diet and water. Procedures involving animal care and management were reviewed and approved by the Animal Ethical Committee at Tianjin University of Traditional Chinese Medicine. CUR-CNLC-GEL was instilled into the left eyes and PBS (pH 7.4) was instilled into the right eyes, 0.1 mL every 4 h, four times a day for a period of 21 days. The degree of eye irritation was evaluated by scoring lesions of conjunctiva, cornea and iris, at specific intervals.
The developed CUR-CNLC-GEL was light yellow and clear. The system was flowable liquid at lower temperature about 5 C, while it turned into the unflowable gel at physiological temperature about 35 C. This characteristic indicated that it was suitable for ocular application. Moreover, as a drug delivery system for ocular application, CUR-CNLC-GEL was ideally transparent. The pH of formulations was 6.34 ± 0.03.
Preocular retention time study
Determination of flow ability and gelation temperature
In vivo preocular retention capability of CUR-CNLC-GEL formulation was investigated as described by Liu et al.25. The fluorescent CUR-SOL and CUR-CNLC-GEL formulations were prepared by adding a certain amount of coumarin 6. The fluorescent CUR-CNLC-GEL formulation (0.1 mL) was instilled into the left rabbit eye and CUR-SOL into the right eye as a control. Preocular retention time was observed at appropriate time intervals by examining with a slit lamp (YZ2, Kangjie Medical Instrument Company, Suzhou, China). The time course of fluorescence on the corneal surface and in the conjunctival sac was evaluated by the same operator.
The flow ability and solution–gel transition temperature results for different samples are shown in Table 1. The 24% Poloxamer 407 formulation diluted by STF is supposed to exhibit solution characteristic at 25 C and change into gel at the body temperature. The STF diluting method is used to simulate the dilution after formulation administered topically. Solution–gel transition temperatures of formulations containing different content of Poloxamer 407 were compared. The result suggests that formulations containing more Poloxamer 407 would exhibit a lower solution–gel transition temperature. Poloxamer 407 consisting of higher ratio of hydrophobic poly(propylene oxide) units/hydrophilic poly(ethylene oxide) units per mole compared to Poloxamer 188 could decrease the hydrophobic interaction between each Poloxamer micelle. Therefore, the solution–gel transition of the system was lower28. However, the solution–gel transition temperature of the formulation should be higher than the room temperature to avoid refrigeration. Thus, the formulation of 24% Poloxamer 407 (V/V) was chosen for further study.
Pharmacokinetic study Pharmacokinetic study in the aqueous humor was carried out by microdialysis technique. Three rabbits were locally anesthetized around the ocular region with 20 mg kg1 of lidocaine hydrochloride administered intravenously. A microdialysis probe (MD 2000, Bioanalytical Systems, Inc, West Lafayette, IN) was implanted into the anterior chamber of the eye by a 20 G needle, thereafter the needle was removed. The probe was adjusted, and the microdialysis membrane totally resided in the anterior chamber. Thereafter, it was perfused with GBR containing 0.06% Tween-80 at a rate of 3 mL min1 by a microdialysis pump (CMA106, CMA Microdialysis Co., Ltd, Stockholm, Sweden). The corneal wound was treated with 0.3% (w/v) ofloxacin ophthalmic drops and allowed to stabilize for 1 day before topical drug administration. Following a 1 h equilibration period with perfusion of GBR containing 0.06% Tween-80 (v/v), standard drug solution of different concentrations was perfused and the dialysate was collected for 15 min after 30 min of perfusion. In vivo recovery (R) is calculated by the following equation27: R% ¼
Cd Cp 100, Cm Cp
ð1Þ
Results and discussion Determination of visual appearance, clarity and pH
Viscosity test The viscosity of CUR-CNLC-GEL was estimated by a rheological instrument to predict retention behavior and physical integrity in vivo. As presented in Figure 1, when the temperature reached 34 C, a transition occurred which suggested that the system was more viscous. The increase in viscosity is expected to trigger prolonged retention of formulation on the corneal surface. Morphological study The TEM image in Figure 2 revealed that the optimized CURCNLC-GEL formulation was dispersed as individual nanostructured lipid carrier and a network of Poloxamer. The particle size of the optimized CUR-CNLC formulation was 158.1 nm and polydispersity index was 0.29. CUR-CNLC were spherical in shape and homogeneously in distribution.
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Table 1. Evaluation of transparency and flow ability of the CUR-CNLC-GEL (X ± SD, n ¼ 3).
Formulation
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20% 22% 23% 24% 22% 23% 24%
Poloxamer Poloxamer Poloxamer Poloxamer Poloxamer Poloxamer Poloxamer
407 407 407 407 407-STF 407-STF 407-STF
Transparency at temperaturea ( C)
Flow ability at temperatureb ( C)
5±1
25 ± 1
35 ± 1
5±1
25 ± 1
35 ± 1
Tsolution–gel
+++ +++ +++ +++ +++ +++ +++
+++ +++ +++ +++ +++ +++ +++
+++ +++ +++ +++ +++ +++ +++
+++ +++ +++ +++ +++ +++ +++
+++ +++ ++ – +++ +++ +++
++ – – – +++ +++ –
48 ± 1.0 35 ± 0.6 29 ± 0.6 24 ± 1.2 81 ± 0.6 71 ± 0.6 34 ± 1.0
Tsol-gel ¼ solution–gel transition temperature. a Transparency: +++, transparent; ++, slightly translucent; +, translucent; , turbid. b Flow ability: +++, very good; ++, good; +, average; , not flow.
Figure 1. Temperature-induced viscosity change of the optimal CUR-CNLC-GEL formulation.
Figure 3. In vitro release profile of CUR from the optimal CUR-CNLCGEL formulation (n ¼ 3). N is the cumulative released percentage of CUR from in situ nanogel, and the experiment was conducted at 25 C.
Figure 2. TEM micrograph of the optimal CUR-CNLC-GEL formulation.
In vitro release study The percentage of CUR released from in situ nanogel was plotted as a function with time. As depicted in Figure 3, 94.91% of CUR was released from CUR-CNLC-GEL within 4 h. Release profiles
evaluated using different models (zero order, first order, and Higuchi square-root, Hixson–Crowell models, Ritger–Peppas model and Weibull) are shown in Table 2. Drug release fitted to zero-order kinetics. Linearity in the zero-order equation plot (R2 ¼ 0.9961) suggested that the release of drug from formulation was only time dependent29. Polymeric controlled release delivery systems can be classified as matrix and reservoir systems. In a matrix system, the drug is distributed throughout the polymeric matrix. In a reservoir system, however, the drug is present in the core, surrounded by a polymeric film that is rate-limiting.
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DOI: 10.3109/10837450.2015.1026607
Table 2. Parameters of the mathematical models of regression for CURCNLC-GEL. Equation
R2
Y ¼ 0.2511t 0.03635 ln(1 Y) ¼ 0.8053t + 0.7378 Y ¼ 0.7076t1/2 0.4934 (1 Y)1/3 ¼ 0.1721t + 1.118 ln Y ¼ 1.085lnt 1.532 ln ln[1/(1 Y)] ¼ 1.617lnt 1.418
0.9961 0.8902 0.9798 0.9552 0.9958 0.9652
Model Zero order First order Higuchi Hixcon–Crowell Ritger–Peppas Weibull
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Y, the percentage of CUR released from CUR-CNLC-GEL; t, time point; R, regression coefficient. Table 3. Corneal permeability coefficients (X±SD, n ¼ 3). Parameters 7
1
Papp 10 (cm s ) Jss 106 (mg s1 cm2) HL (%)
CUR-SOL
CUR-CNLC-GEL
3.778 ± 0.2940 1.889 ± 0.1470 78.25 ± 1.919
5.894 ± 0.2601** 2.330 ± 0.1028* 79.70 ± 2.692
Papp, the apparent permeability coefficient; Jss, the steady-state flux; HL, hydration level. *p50.05 versus CUR-SOL; **p50.01 versus CUR-SOL.
Reservoir systems typically allow zero-order or constant drug release30. Poloxamer hydrogel as reservoir systems was formed mainly by the physical aggregation of micelles, which easily collapse when placed in PBS solution within a very short period of time. However, with the collapse of Poloxamer hydrogel, the encapsulated CUR-CNLC is released, which might be able to serve as the controlled release system for CUR2. Therefore, the release of CUR from CUR-CNLC-GEL appears to be controlled by gel dissolution and nanoparticles decomposition. In vitro corneal permeation evaluation Table 3 compared the corneal permeability coefficients of CURSOL and CUR-CNLC-GEL formulation. CUR-CNLC-GEL formulation showed significantly higher Papp value (1.56-fold) than that of CUR-SOL (p50.01). The results revealed significantly higher corneal permeation of CUR from the CUR-CNLC-GEL formulation compared to the conventional formulation based on aqueous solution. The normal corneal hydration level is reported to be 75–80%31. Thus, corneal hydration level in the present study was within limits indicating integrity of corneal epithelium and endothelium. During earlier studies, drugs formulated as nanoparticulate carriers have been reported to provide a higher corneal permeation which was attributed to the endocytic uptake32. Consequently, enhanced permeation of CUR from CUR-CNLC-GEL across excised rabbit cornea might be attributed to the endocytic uptake after the collapse of Poloxamer hydrogel. In vivo ocular irritation test Ocular irritation level of CUR-CNLC-GEL was 2 ± 0 (score53) which indicated that CUR-CNLC-GEL is non-irritant. Although there was a slight hyperemia in conjunctival, excellent ocular tolerance was noted. Preocular retention time study As shown in Table 4, CUR-SOL formed a weak and short-lived fluorescent film and disappeared rapidly from corneal surface. However, the CUR-CNLC-GEL formulation formed a rather stable precorneal film and retained longer on the corneal surface (p50.01) and in the conjunctival sac compared with those of
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Table 4. Evaluation of the preocular retention time (X ± SD, n ¼ 3). Formulation
Corneal surface (h)
Conjunctival sac (h)
CUR-SOL CUR-CNLC-GEL
0.2778 ± 0.09623 1.333** ± 0.1667
0.5000 ± 0.1667 4.778** ± 0.1925
**p50.01 versus CUR-SOL.
CUR-SOL (p50.01). The prolonged drug retention is due to not only the high viscosity of the in situ nanogel but also the following reasons: first, nanoparticles possess bioadhesive property due to the extremely small particle size and increased surface area33; second, the cationic character and the viscosity of QACMC are able to prolong the retention of drug and improve its bioavailability in ocular administration34. Therefore, these factors ensure an intimate contact with the epithelial mucosal surface of the eye, preventing tear wash out and consequently prolong drug retention time. Pharmacokinetics study Before pharmacokinetic study, in vivo probe recovery was determined to ensure that the implanted probes were in good function. The in vivo recovery was 50.75 ± 3.91% (n ¼ 3). The concentration–time profiles following topical administration of CUR-SOL and CUR-CNLC-GEL are illustrated in Figure 4. The corresponding pharmacokinetic parameters are summarized in Table 5. As shown in Figure 4, the drug was immediately absorbed into the aqueous humor and reached maximal concentration of CUR (Cmax) at 30 min after ocular application of CURSOL. After that, concentration of drug remarkably decreased in the following hours. However, after an instillation of CUR-CNLCGEL, concentrations of drug were much higher than those of CUR-SOL. CUR could exert antioxidant and antiglycating effects through inhibition of lipid peroxidation, advanced glycated end products and protein aggregation at very low concentrations35. The common experimental diet level of CUR in diabetes mellitus is usually below 0.01%36. The topical application dosage of CUR in our research was 0.5 mg mL1 (0.05%), which is much higher than the common level mentioned above. Compared with CUR-SOL, area under the concentration–time curve (AUC) value was enhanced 9.24-fold for CUR-CNLC-GEL (p50.01), and the Cmax value of CUR-CNLC-GEL was improved 3.38-fold (p50.01). There was statistical difference in mean residence time (MRT) compared with those of CUR-SOL (p50.01). There was no statistical difference in either elimination rate constant (ke) or Tmax value of CUR-CNLC-GEL compared with those of CUR-SOL. The higher ocular bioavailability of the CUR-CNLC-GEL could be related to its biological adhesive ability to prolong the residence time on the corneal epithelial layer33. Previous study37 has demonstrated that micelles/nanoparticles formulation could greatly facilitate the drug across the corneal barrier, yet resulting in the enhancement of bioavailability. However, the major shortcoming for micelles/nanoparticles formulation is their quick clearance from ocular tissue. Poloxamer hydrogel has received great attentions due to its noncytotoxic and prolonged residence time on the corneal surface. So, it is able to compensate for the shortcomings of NLC for ophthalmic delivery38.
Conclusions In this article, CUR-CNLC-GEL was developed and physicochemical properties were evaluated including appearance, clarity, pH, flow ability, gelation temperature, viscosity and morphology.
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Figure 4. CUR concentration–time profiles following a 200 mL topical administration at dose of 0.5 mg mL1 in aqueous humor of conscious rabbits (X ± SD, n ¼ 3).
Table 5. Pharmacokinetic parameters of CUR in the aqueous humor after topical administration in the conscious rabbits (X ± SD, n ¼ 3). Formulation CUR-SOL CUR-CNLC-GEL
ke 103
Tmax (min)
Cmax (ng mL1)
AUC (ng mL1 min)
MRT (min)
3.465 ± 2.026 4.755 ± 0.346
30.00 ± 0.000 23.33 ± 11.55
11.56 ± 0.9576 39.13 ± 4.549**
601.4 ± 17.67 5559 ± 453.1**
43.74 ± 0.604 111.8 ± 5.249**
ke, elimination rate constant; Tmax, time to maximal concentration; Cmax, maximal concentration of CUR; AUC, area under the curve; MRT, mean residence time. **p50.01 versus CUR-SOL.
The developed CUR-CNLC-GEL provided controlled CUR release and the prolonged precorneal residence time. In vivo eye irritation test suggested that the developed CUR-CNLC-GEL was non-irritating and it might be suitable for various ocular applications. In vivo pharmacokinetic study indicated that the developed CUR-CNLC-GEL could significantly increase the bioavailability of CUR and maintain drug concentration in aqueous humor after administration compared with that of the CUR-SOL. The CUR-CNLC-GEL could increase the bioavailability of CUR by improving corneal permeation and retention capacity in the aqueous humor. Therefore, our results suggested that the combination of CNLC and thermosensitive in situ gel has a great potential to be an ophthalmic delivery system of CUR.
Acknowledgements We are thankful to Gattefosse´ SAS (Saint-Priest, France), BASF (Ludwigshafen, German) and Beijing Fengli Jingqiu Commerce and Trade Co., Ltd. (Beijing, China) for samples support of this work.
Declaration of interest This project was supported by National Natural Science Foundation of China (No. 81303142), Specialized Research Fund for the Doctoral Program of Higher Education of China (No. 20121210120001) and Tianjin Research Program of Application Foundation and Advanced Technology (No. 14CQNJC13100). The authors report no declarations of interest.
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