Colloids and Surfaces B: Biointerfaces 114 (2014) 111–120
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Fabrication of a composite system combining solid lipid nanoparticles and thermosensitive hydrogel for challenging ophthalmic drug delivery Jifu Hao a,∗,1 , Xiaodan Wang a,1 , Yanping Bi a , Yufang Teng a , Jianzhu Wang a , Fei Li a , Qiankui Li a , Jimei Zhang a , Fengguang Guo a , Jiyong Liu b,∗ a b
College of Pharmacy, Taishan Medical University, Taian 271016, PR China Department of Pharmacy, Changhai Hospital, Second Military Medical University, Shanghai 200433, PR China
a r t i c l e
i n f o
Article history: Received 16 June 2013 Received in revised form 25 September 2013 Accepted 29 September 2013 Available online 11 October 2013 Keywords: Resina Draconis Central composite design Solid lipid nanoparticle Thermosensitive hydrogels Ocular drug delivery
a b s t r a c t The purpose of this study was to explore a composite thermosensitive in situ gelling formulation using the distribution of solid lipid nanoparticles (SLNs) among poloxamer-based hydrogels as a potential carrier for novel ocular drug delivery. SLNs containing the model drug Resina Draconis were prepared using a melt-emulsion ultrasonication method. A central composite design (CCD) was adopted to screen the thermosensitive hydrogel (THG) formulation. After aqueous SLNs were dispersed into the THG matrices, the physicochemical properties of the SLNs were characterized before and after their incorporation into hydrogels. The in vitro corneal penetration experiment, ocular irritant test and transcorneal mechanism across the cornea have been previously described to predict the feasibility for the proposed ophthalmic application. Finally, the optimal THGs consisted of 27.8% (w/v) poloxamer 407 and 3.55% (w/v) poloxamer 188. The particle size of the SLNs remained within the colloidal range. In vitro corneal penetration studies revealed a nearly steady sustained drug release. The hen’s egg test-chorioallantoic membrane (HETCAM) test indicated that all of the tested polymer systems were non-irritant. Coumarin-6 labeled SLNs formulated into THGs displayed a more homogeneous fluorescence with a deeper penetration intensity into the cornea at various times. Taken together, these results suggest that the SLN-based THG system can be used as a potential vehicle for ocular application. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Topical ophthalmic application of drugs is generally regarded as the most popular and well-accepted approach for the treatment of various eye diseases [1]. However, the efficient protective mechanisms of the eye render it difficult to achieve a desired drug concentration at the target site. The unique physiological constraints of the eye, such as the blinking reflex, lachrymal secretion and nasolacrimal drainage, contribute to this organ’s exquisitely imperviousness to foreign substances. Moreover, the anatomy and safeguard barrier of the cornea compromise the rapid absorption of drugs [2]. Currently, eye drops are a common type of topical ocular medication in clinical practice, where a drop of an ophthalmic solution, irrespective of the instilled volume, often rapidly eliminates irritants after administration, despite the small amount
∗ Corresponding authors. Tel.: +86 538 6229751; fax: +86 21 3116 2308. E-mail addresses: [email protected] (J. Hao), [email protected] (J. Liu). 1 These two authors contributed equally to this study. 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.09.059
that actually reaches the intraocular tissue [3]. Thus, this method cannot provide and maintain an adequate concentration of drug in the precorneal area [4]. Consequently, various routine ophthalmic vehicles, such as viscous solutions, ointments, gels, or polymeric inserts, have been devised to enhance ocular bioavailability and the duration of drug action. In consideration of these preparations, these limitations of conventional dosage forms cannot address the problem of low bioavailability presented in actual ophthalmic administration. Thus, an ideal dosage form is needed for ophthalmic drug delivery that not only increases the drug’s corneal penetration capability but also prolongs the retention time of the vehicle on the ocular surface [5]. The emergence of colloidal delivery systems, which offers a valuable route for enhancing the potency of corneal penetration, are preferable methods toward a settlement of the former strategy [6], such as liposomes, cubosomes, solid lipid nanoparticles (SLNs), nanostructured lipid carriers (NLCs) and microemulsions. These nanocarriers help to overcome anatomical barriers and delivers the drug to the desired site, minimizing systemic exposure and severe adverse effects [7].
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J. Hao et al. / Colloids and Surfaces B: Biointerfaces 114 (2014) 111–120
Among these various nanocarriers for ocular administration, the introduction of SLNs loaded with an active ingredient to enhance the penetration capability across the cornea has gained increasing interest in the field of ocular drug delivery within the past few years, due to the obvious advantages of SLNs, such as its ability to encapsulate and protect the lipophilic drug, prevent tear wash out, enhance ocular tolerance, improve penetration efficiency and increase corneal uptake [8,9]. Nevertheless, SLNs are still aqueous dispersal systems, which display low viscosity as a result of the difficult retention capability on the ocular surface. To overcome this issue, SLNs can be incorporated into traditional semi-solid systems (e.g., hydrogels) to increase the consistency of the final formulations and to improve the long-term stability of the incorporated nanoparticles [10,11]. Another alternative method to prolong the precorneal residence time may be achieved using delivery systems based on in situ gel forming solutions, which consist of phase transition systems when instilled in a liquid form that shifts to the gel or solid phase. According to the different factors that cause sol-to-gel phase transitions on the eye surface, the phase transition is triggered by the pH of tears, the temperature at the eye surface or the electrolytes present in the tear film [1]. Several in situ gelling systems have been developed to prolong the precorneal residence time of a drug and to improve ocular bioavailability [12]. Poloxamers are nonionic triblock copolymers consisting of a central hydrophobic chain of polyoxypropylene oxide (PPO) flanked by two hydrophilic chains of polyoxyethylene oxide (PEO) and are known to exhibit the phenomenon of reverse thermal gelation under a specific concentration and temperature. Because of its unique thermoreversible gelation properties, poloxamer analogs have become one of the most extensively investigated temperature-responsive materials [13]. Resina Draconis (also called “Dragon’s blood” in China), a deep red resin, has been used in traditional medicine since ancient times in many cultures. Pharmacological studies have shown that it demonstrates beneficial effects on the treatment of blood stasis syndrome, trauma, tumors, inflammation, gynecopathy, allergic dermatitis, among others. Taken together, these results indicated that Resina Draconis has enormous potential for further study [14]. However, the poor solubility of Resina Draconis limits its therapeutic efficacy and clinical application. It is generally accepted that SLNs have been proposed as a significant drug carrier system, thus loading Resina Draconis into the SLNs carrier may be regarded as a potential approach for ocular administration to improve its bioavailability. The aim of the present study was to fabricate a composite hybrid thermosensitive in situ gelling formulation using the distribution SLNs among poloxamer-based hydrogels as a potential carrier for novel ocular drug delivery. This composite SLNs-based thermosensitive hydrogel (THG) system combines the advantage of SLNs 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 nasolachrymal drainage. SLNs containing the drug Resina Draconis were prepared using the melt-emulsion ultrasonication and low temperature solidification method. After prepared aqueous SLNs were dispersed into the THG matrices, their physicochemical properties were characterized before and after their incorporation into the hydrogels. A central composite design (CCD) was performed for the optimization and development of THG formulation containing poloxamer 407 in combination with poloxamer 188. The in vitro corneal penetration experiment, ocular irritant test and transcorneal mechanism across the cornea using confocal laser scanning microscopy of the same optimal formulations have also been described to predict their feasibility for the proposed ophthalmic application.
2. Materials and methods 2.1. Materials and animals Resina Draconis was purchased from Hunan Dongtian Pharmaceutical Co. Ltd. (China). GMS was provided by Shanghai Chemical Reagent Co., Ltd. (China). Poloxamer 188 and 407 were purchased from Beijing Fengli Jingqiu Commerce and Trade Co., Ltd. (China). Coumarin-6 (C6 ) was obtained from Sigma–Aldrich (USA). Acetonitrile was of high performance liquid chromatography (HPLC) grade. All other reagents and solvents were of analytical reagent grade. New Zealand albino rabbits free of any ocular damage were obtained from the Taishan Medical University Animal Center (Taian, China). All animal studies were handled according to the Principles of Laboratory Animal Care, and the protocols were approved by the Taishan Medical University Animal Ethical Committee. 2.2. Preparation of Resina Draconis-loaded SLNs The Resina Draconis-loaded SLNs were prepared using the melt-emulsion ultrasonication and low temperature-solidification method as previously described with some modification [15]. Briefly, the lipid matrix was melted at 5–10 ◦ C above its melting point. The Resina Draconis was dissolved in the melt lipid phase. The hot lipid phase was dispersed into a hot water-surfactant solution at the same temperature, then the pre-emulsion was formed under constant mechanical agitation (DC-40, Hangzhou Electrical Engineering Instruments, China) at 1000 rpm for 15 min at 70 ◦ C. The original warm emulsion was further treated for 5 min (work 2 s and stand 3 s) using a Lab ultrasonic cell pulverizer (JY92-II, Ningbo Scientz Biotechnology Co., Ltd. China) at 600 W to form a nanoemulsion. The emulsion was rapidly cooled by immersing the beaker into ice-cold water (0 ◦ C). Agitation continued until the nanoemulsion yielded a uniform dispersion of nanoparticles. The drug concentration in the supernatant was analyzed using the HPLC method. The chromatography system consisted of a Shimadzu LC-10AT solvent delivery pump (Kyoto, Japan) equipped with a 20 L loop and a UV visible detector. The eluate was monitored at 280 nm. The mobile phase was acetonitrile and water (33:67, v/v) with a flow speed of 1.2 mL min−1 at room temperature. The entrapment efficiency (EE) of the Resina Draconis incorporated in the SLNs was determined after centrifugation (CS120GXL, Hitachi, Japan) at 50,000 rpm for 15 min. EE was performed after 48 h of storage at 4 ◦ C to evaluate the drug leakage following storage as a colloidal dispersion. To perform confocal laser scanning microscopy (CLSM), C6 -labeled SLNs were also prepared following the procedure previously described. 2.3. Preparation of SLNs-based thermosensitive hydrogels THGs consisting of Poloxamer 407 and Poloxamer 188 were prepared using the cold process described in previously published studies [16]. For preparation of this composite hybrid system, a specific amount of polymers (P407 and P188) were dispersed in a cool nanosuspension and stored in the refrigerator until the polymer completely dissolved to form a clear solution. Other excipients, such as glycerin and benzalkonium chloride, were added as an isotonicity agent and preservative, respectively. The concentration of the isotonicity adjustment agent that rendered the formulations isotonic with eye fluid was calculated using the freezing point depression method on a STY-2 osmometer (Tianjin, China). The resulting formulation was kept at 4 ◦ C for further study.
J. Hao et al. / Colloids and Surfaces B: Biointerfaces 114 (2014) 111–120
2.4. Measurement of the sol–gel transition temperature The phase transition temperature of this composite hybrid system was measured using the test tube inverting method [17]. A 2-mL sample was introduced into a screw capped test tube and examined for gelation, which was said to have occurred when the meniscus could no longer tilting through 90◦ . The hydrogel samples were heated from 4 ◦ C to 60 ◦ C at a rate of 1 ◦ C/min. Each sample was measured at least in triplicate. To simulate the in vivo phase transition process, the gelation temperatures were also measured after the poloxamer formulations were diluted by artificial tear fluid (ATF) in a ratio of 40:7. 2.5. Central composite factorial design It has been reported that the poloxamer polymer is present in the gel form only between two critical transition temperatures, which vary with the polymer composition and concentration [18]. The poloxamer analogs may be interdependent to form the optimum in situ gel and the effect of multiple variables should be studied simultaneously. Thus, the establishment of a rational mathematical experimental design for the effective optimization of poloxamer in situ gel formulation is essential, which enables for simultaneous and multidimensional testing of the many causal factors that determine the target quality and to preclude the performance of a large number of unnecessarily independent runs. Thus, a two-factor, five-level CCD was constructed to explore the optimum levels of this in situ gel formulation. Two selected independent variables (the feasible concentrations of P407 (X1 ) and P188 (X2 ) for tailoring the thermosensitive gel) were studied as factors. The gelation temperatures were significant variables, and should be taken into account in the process of formula optimization. Due to the dilution effect from the tear fluid, it should be considered during the design of ophthalmic formulations, which in theory, the smaller the difference in the gelation temperatures before and after ATF dilution, the stronger the ability of the thermosensitive gel to endure the dilution by ATF [19]. Thus, the gelation temperatures before and after ATF dilution (Y1 , Y2 , respectively) and the difference in value of the gelation temperatures before and after ATF dilution (Y3 ) were selected as important objective parameters. The coded and actual values of the variables are provided in Table 1. When it came to the prediction of the best suitable formulation, the fitness of the model among the linear, two-factor interaction model and quadratic model was assessed due to the analysis of variance p-value and focus on the model maximizing multiple correlation coefficient r2 , predicted r2 and adjusted r2 as quality indicators in the model summary statistic list. A p-value of less than 0.05 was considered statistically significant. Optimization was performed using an objective function to obtain the optimal points concerning the predetermined constraints in which the gelation temperature before and after the ATF dilution and their different values were answered for the demand of ophthalmic THGs. The selected optimal formulation was prepared for further evaluation of this hybrid system. 2.6. Physicochemical characterization 2.6.1. Morphology and particle size The particle size and polydispersion index (PI) of the SLNs and hydrogel were measured using a Zetasizer (3000SH, Malvern Instruments Ltd., UK). In addition, the particle size measurement to assess the stability of the resulting SLN dispersions after 24 h of storage at 4 ◦ C was performed according to the cold method process for hydrogel preparation. In addition, the SLN stability after they were embedded in the hydrogel formulations in terms of particle size after 30 days of storage at room temperature was also
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evaluated. The results were reported as the mean ± standard deviation (SD) of three replicates (n = 3). The surface morphology of the SLNs before and after its incorporation into the hydrogel was studied using scanning electron microscopy (SEM). A drop of the sample was deposited on to a silicon wafer, dried, and sputtered for 20 s with gold, and finally, analyzed using SEM (Inspect F50, FEI, USA). 2.6.2. Thermal analysis using differential scanning calorimetry The thermal behavior of these composite hydrogels was determined using differential scanning calorimetry (DSC Q10, TA, USA) at a heating rate of 10 ◦ C/min. This was performed under a dry nitrogen atmosphere and Al2 O3 was used as a reference. The DSC measurements were performed on the following samples: (A) lyophilized blank hydrogel without cryoprotectant; (B) Resina Draconis; (C) lyophilized Resina Draconis-loaded SLNs without cryoprotectant; (D) lyophilized SLN-based hydrogel hybrid system; and (E) lyophilized blank SLNs. 2.7. In vitro corneal penetration experiments The transcorneal penetration behavior of the Resina Draconis from the hybrid hydrogel complex was performed on isolated rabbit corneas with a conventional vertical Franz cell diffusion apparatus (RYJ-2B, Huanghai medical and testing instruments, China). The receptor compartment of the apparatus was filled with glutathione bicarbonate ringer (GBR) buffer that had been prewarmed to a temperature of 37 ◦ C. The Resina Draconis-SLNs and hybrid hydrogel were placed in the donor compartment, respectively. At predetermined time intervals of up to 6 h, 0.2 mL sample was withdrawn from the receiving compartment and was immediately replaced with an equal volume of preheated GBR buffer. The amount of drug that permeated across the cornea was assayed using HPLC. The penetration parameter was calculated by plotting the amounts of drug permeating through the cornea per unit area (g/cm2 ) versus time (min). The steady-state flux (J) values across the cornea were evaluated by determining the slope of the linear portion of the permeation graphs using the relationship given below: J (g/(cm2 s)) = dQ/A dt; where Q indicates the cumulative amount of drug permeated at time t, and A is the exposed corneal surface area. The corneal permeability coefficient (Kp, cm/s) was determined using the expression given below: Kp (cm/s) = J/C0 ; where C0 is the initial concentration of drug in the donor compartment. All experiments were performed in triplicate. At the end of the penetration study, each cornea was carefully removed from the sclera ring and weighed (Ww ). It was then dessicated overnight at 70 ◦ C and reweighed (Wd ). The corneal hydration level (HL %), defined as [1 − (Wd /Ww )] × 100, was calculated [20,21]. 2.8. Ocular tolerance test (HET-CAM test) To evaluate the ocular tolerance of the developed formulation, a modified HET-CAM test was performed as previously reported [22]. Briefly, freshly fertilized hen’s eggs were incubated at 37 ± 0.5 ◦ C for nine days, and then each egg was tested with a cold lamp to ensure viable and optimal illumination of the chorioallantoic membrane by detailed observation of the embryo development. On day 10, the chorioallantoic membrane (CAM) was carefully dissected (without injuring any underlying blood vessels) and exposed by removing a portion of the eggshell and inner membrane above the air cell. Subsequently, the surface of the CAM was exposed to 300 L of the test substance, 0.1 M sodium hydroxide (NaOH) solution and 70% isopropyl alcohol served as the positive controls, and a saline solution was used as a negative control. The irritant effects on blood vessels such as hyperemia, hemorrhage or coagulation were examined using a stereo microscope (SZX16, Olympus, Japan) before exposure and at different time points post-application for 5 min. Scoring of
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Table 1 Independent variables and their levels of experimental design. Independent variables
X1 : concentration of P407 (%, w/v) X2 : concentration of P188 (%, w/v)
Levels −1.414
−1
0
1
1.414
21 0
22.17 2.2
25 7.5
27.83 12.8
29 15
Dependent variables
Constraints
Y1 : gelation temperature before ATF dilution (◦ C) Y2 : gelation temperature after ATF dilution (◦ C) Y3 : difference in gelation temperature before and after ATF dilution (◦ C)
In range: In range:
25 ◦ C ≤ Y1 ≤ 35 ◦ C 30 ◦ C ≤ Y1 ≤ 35 ◦ C Minimize
each test substance or formulation was assigned. The mean score value of the product was designated as nonirritant (irritation score 0–0.9), slight irritant (score 1–4.9), moderate irritant (score 5–8.9), and strong irritant (score 9–21) using a classification system previously described by Kalweit et al. [23]. In addition, images were obtained before application and at 30 s, 2 min, and 5 min after exposure. The mean detection time in seconds after exposure for the appearance of different degrees of abnormal irritant effect was determined for each solution based on 4 eggs.
3.2. Analysis of the response surfaces
2.9. Transcorneal mechanism across the cornea
Y1 = 35.24 − 2.91X1 + 5.75X2 − 1.04X1 X2 − 0.85X12 − 3.88X22
An investigation on the transport mechanism across the cornea of SLNs-based THGs was performed in rabbits in vivo. New Zealand albino rabbits (male, weighing 2.5–3.0 kg) free of any ocular damage were selected and utilized to perform this experiment. The transport process across the corneal epithelium was located by C6 , a fluorescent marker. The corneal C6 disposition was investigated by examining the samples of rabbit cornea according to the CLSM. All experiments were approved and monitored by the Institutional Animal Care and Use Committee of Taishan Medical University. Fifty microliters of the formulation were administered into the lower fornix of the conjunctival sac. The contralateral eye was used as the control and received no treatment. The animals were euthanized and then sacrificed at predetermined time points after administration of the hydrogel formulation, and the freshly harvested cornea tissue was then rinsed with saline, flash frozen in liquid nitrogen, cut into 10-m thick sections and mounted onto slides and a coverslipped. Transcorneal characteristics were observed and recorded using the Leica Confocal apparatus (MP5, Leica, Germany) for image acquisition. An Ar excitation laser (excitation 488 nm) and a HeNe excitation laser (excitation 543 nm) were selected for C6 .
The model F-value of 91.54 indicated that this model was significant (p < 0.0001). The obtained results in this design suggested that independent factors affecting Y1 included the concentration of P407 (X1 ), P188 (X2 ) and the quadratic term of P188 (X22 ), with a p-value of
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Fabrication of a composite system combining solid lipid nanoparticles and thermosensitive hydrogel for challenging ophthalmic drug delivery Jifu Hao a,∗,1 , Xiaodan Wang a,1 , Yanping Bi a , Yufang Teng a , Jianzhu Wang a , Fei Li a , Qiankui Li a , Jimei Zhang a , Fengguang Guo a , Jiyong Liu b,∗ a b
College of Pharmacy, Taishan Medical University, Taian 271016, PR China Department of Pharmacy, Changhai Hospital, Second Military Medical University, Shanghai 200433, PR China
a r t i c l e
i n f o
Article history: Received 16 June 2013 Received in revised form 25 September 2013 Accepted 29 September 2013 Available online 11 October 2013 Keywords: Resina Draconis Central composite design Solid lipid nanoparticle Thermosensitive hydrogels Ocular drug delivery
a b s t r a c t The purpose of this study was to explore a composite thermosensitive in situ gelling formulation using the distribution of solid lipid nanoparticles (SLNs) among poloxamer-based hydrogels as a potential carrier for novel ocular drug delivery. SLNs containing the model drug Resina Draconis were prepared using a melt-emulsion ultrasonication method. A central composite design (CCD) was adopted to screen the thermosensitive hydrogel (THG) formulation. After aqueous SLNs were dispersed into the THG matrices, the physicochemical properties of the SLNs were characterized before and after their incorporation into hydrogels. The in vitro corneal penetration experiment, ocular irritant test and transcorneal mechanism across the cornea have been previously described to predict the feasibility for the proposed ophthalmic application. Finally, the optimal THGs consisted of 27.8% (w/v) poloxamer 407 and 3.55% (w/v) poloxamer 188. The particle size of the SLNs remained within the colloidal range. In vitro corneal penetration studies revealed a nearly steady sustained drug release. The hen’s egg test-chorioallantoic membrane (HETCAM) test indicated that all of the tested polymer systems were non-irritant. Coumarin-6 labeled SLNs formulated into THGs displayed a more homogeneous fluorescence with a deeper penetration intensity into the cornea at various times. Taken together, these results suggest that the SLN-based THG system can be used as a potential vehicle for ocular application. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Topical ophthalmic application of drugs is generally regarded as the most popular and well-accepted approach for the treatment of various eye diseases [1]. However, the efficient protective mechanisms of the eye render it difficult to achieve a desired drug concentration at the target site. The unique physiological constraints of the eye, such as the blinking reflex, lachrymal secretion and nasolacrimal drainage, contribute to this organ’s exquisitely imperviousness to foreign substances. Moreover, the anatomy and safeguard barrier of the cornea compromise the rapid absorption of drugs [2]. Currently, eye drops are a common type of topical ocular medication in clinical practice, where a drop of an ophthalmic solution, irrespective of the instilled volume, often rapidly eliminates irritants after administration, despite the small amount
∗ Corresponding authors. Tel.: +86 538 6229751; fax: +86 21 3116 2308. E-mail addresses: [email protected] (J. Hao), [email protected] (J. Liu). 1 These two authors contributed equally to this study. 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.09.059
that actually reaches the intraocular tissue [3]. Thus, this method cannot provide and maintain an adequate concentration of drug in the precorneal area [4]. Consequently, various routine ophthalmic vehicles, such as viscous solutions, ointments, gels, or polymeric inserts, have been devised to enhance ocular bioavailability and the duration of drug action. In consideration of these preparations, these limitations of conventional dosage forms cannot address the problem of low bioavailability presented in actual ophthalmic administration. Thus, an ideal dosage form is needed for ophthalmic drug delivery that not only increases the drug’s corneal penetration capability but also prolongs the retention time of the vehicle on the ocular surface [5]. The emergence of colloidal delivery systems, which offers a valuable route for enhancing the potency of corneal penetration, are preferable methods toward a settlement of the former strategy [6], such as liposomes, cubosomes, solid lipid nanoparticles (SLNs), nanostructured lipid carriers (NLCs) and microemulsions. These nanocarriers help to overcome anatomical barriers and delivers the drug to the desired site, minimizing systemic exposure and severe adverse effects [7].
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J. Hao et al. / Colloids and Surfaces B: Biointerfaces 114 (2014) 111–120
Among these various nanocarriers for ocular administration, the introduction of SLNs loaded with an active ingredient to enhance the penetration capability across the cornea has gained increasing interest in the field of ocular drug delivery within the past few years, due to the obvious advantages of SLNs, such as its ability to encapsulate and protect the lipophilic drug, prevent tear wash out, enhance ocular tolerance, improve penetration efficiency and increase corneal uptake [8,9]. Nevertheless, SLNs are still aqueous dispersal systems, which display low viscosity as a result of the difficult retention capability on the ocular surface. To overcome this issue, SLNs can be incorporated into traditional semi-solid systems (e.g., hydrogels) to increase the consistency of the final formulations and to improve the long-term stability of the incorporated nanoparticles [10,11]. Another alternative method to prolong the precorneal residence time may be achieved using delivery systems based on in situ gel forming solutions, which consist of phase transition systems when instilled in a liquid form that shifts to the gel or solid phase. According to the different factors that cause sol-to-gel phase transitions on the eye surface, the phase transition is triggered by the pH of tears, the temperature at the eye surface or the electrolytes present in the tear film [1]. Several in situ gelling systems have been developed to prolong the precorneal residence time of a drug and to improve ocular bioavailability [12]. Poloxamers are nonionic triblock copolymers consisting of a central hydrophobic chain of polyoxypropylene oxide (PPO) flanked by two hydrophilic chains of polyoxyethylene oxide (PEO) and are known to exhibit the phenomenon of reverse thermal gelation under a specific concentration and temperature. Because of its unique thermoreversible gelation properties, poloxamer analogs have become one of the most extensively investigated temperature-responsive materials [13]. Resina Draconis (also called “Dragon’s blood” in China), a deep red resin, has been used in traditional medicine since ancient times in many cultures. Pharmacological studies have shown that it demonstrates beneficial effects on the treatment of blood stasis syndrome, trauma, tumors, inflammation, gynecopathy, allergic dermatitis, among others. Taken together, these results indicated that Resina Draconis has enormous potential for further study [14]. However, the poor solubility of Resina Draconis limits its therapeutic efficacy and clinical application. It is generally accepted that SLNs have been proposed as a significant drug carrier system, thus loading Resina Draconis into the SLNs carrier may be regarded as a potential approach for ocular administration to improve its bioavailability. The aim of the present study was to fabricate a composite hybrid thermosensitive in situ gelling formulation using the distribution SLNs among poloxamer-based hydrogels as a potential carrier for novel ocular drug delivery. This composite SLNs-based thermosensitive hydrogel (THG) system combines the advantage of SLNs 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 nasolachrymal drainage. SLNs containing the drug Resina Draconis were prepared using the melt-emulsion ultrasonication and low temperature solidification method. After prepared aqueous SLNs were dispersed into the THG matrices, their physicochemical properties were characterized before and after their incorporation into the hydrogels. A central composite design (CCD) was performed for the optimization and development of THG formulation containing poloxamer 407 in combination with poloxamer 188. The in vitro corneal penetration experiment, ocular irritant test and transcorneal mechanism across the cornea using confocal laser scanning microscopy of the same optimal formulations have also been described to predict their feasibility for the proposed ophthalmic application.
2. Materials and methods 2.1. Materials and animals Resina Draconis was purchased from Hunan Dongtian Pharmaceutical Co. Ltd. (China). GMS was provided by Shanghai Chemical Reagent Co., Ltd. (China). Poloxamer 188 and 407 were purchased from Beijing Fengli Jingqiu Commerce and Trade Co., Ltd. (China). Coumarin-6 (C6 ) was obtained from Sigma–Aldrich (USA). Acetonitrile was of high performance liquid chromatography (HPLC) grade. All other reagents and solvents were of analytical reagent grade. New Zealand albino rabbits free of any ocular damage were obtained from the Taishan Medical University Animal Center (Taian, China). All animal studies were handled according to the Principles of Laboratory Animal Care, and the protocols were approved by the Taishan Medical University Animal Ethical Committee. 2.2. Preparation of Resina Draconis-loaded SLNs The Resina Draconis-loaded SLNs were prepared using the melt-emulsion ultrasonication and low temperature-solidification method as previously described with some modification [15]. Briefly, the lipid matrix was melted at 5–10 ◦ C above its melting point. The Resina Draconis was dissolved in the melt lipid phase. The hot lipid phase was dispersed into a hot water-surfactant solution at the same temperature, then the pre-emulsion was formed under constant mechanical agitation (DC-40, Hangzhou Electrical Engineering Instruments, China) at 1000 rpm for 15 min at 70 ◦ C. The original warm emulsion was further treated for 5 min (work 2 s and stand 3 s) using a Lab ultrasonic cell pulverizer (JY92-II, Ningbo Scientz Biotechnology Co., Ltd. China) at 600 W to form a nanoemulsion. The emulsion was rapidly cooled by immersing the beaker into ice-cold water (0 ◦ C). Agitation continued until the nanoemulsion yielded a uniform dispersion of nanoparticles. The drug concentration in the supernatant was analyzed using the HPLC method. The chromatography system consisted of a Shimadzu LC-10AT solvent delivery pump (Kyoto, Japan) equipped with a 20 L loop and a UV visible detector. The eluate was monitored at 280 nm. The mobile phase was acetonitrile and water (33:67, v/v) with a flow speed of 1.2 mL min−1 at room temperature. The entrapment efficiency (EE) of the Resina Draconis incorporated in the SLNs was determined after centrifugation (CS120GXL, Hitachi, Japan) at 50,000 rpm for 15 min. EE was performed after 48 h of storage at 4 ◦ C to evaluate the drug leakage following storage as a colloidal dispersion. To perform confocal laser scanning microscopy (CLSM), C6 -labeled SLNs were also prepared following the procedure previously described. 2.3. Preparation of SLNs-based thermosensitive hydrogels THGs consisting of Poloxamer 407 and Poloxamer 188 were prepared using the cold process described in previously published studies [16]. For preparation of this composite hybrid system, a specific amount of polymers (P407 and P188) were dispersed in a cool nanosuspension and stored in the refrigerator until the polymer completely dissolved to form a clear solution. Other excipients, such as glycerin and benzalkonium chloride, were added as an isotonicity agent and preservative, respectively. The concentration of the isotonicity adjustment agent that rendered the formulations isotonic with eye fluid was calculated using the freezing point depression method on a STY-2 osmometer (Tianjin, China). The resulting formulation was kept at 4 ◦ C for further study.
J. Hao et al. / Colloids and Surfaces B: Biointerfaces 114 (2014) 111–120
2.4. Measurement of the sol–gel transition temperature The phase transition temperature of this composite hybrid system was measured using the test tube inverting method [17]. A 2-mL sample was introduced into a screw capped test tube and examined for gelation, which was said to have occurred when the meniscus could no longer tilting through 90◦ . The hydrogel samples were heated from 4 ◦ C to 60 ◦ C at a rate of 1 ◦ C/min. Each sample was measured at least in triplicate. To simulate the in vivo phase transition process, the gelation temperatures were also measured after the poloxamer formulations were diluted by artificial tear fluid (ATF) in a ratio of 40:7. 2.5. Central composite factorial design It has been reported that the poloxamer polymer is present in the gel form only between two critical transition temperatures, which vary with the polymer composition and concentration [18]. The poloxamer analogs may be interdependent to form the optimum in situ gel and the effect of multiple variables should be studied simultaneously. Thus, the establishment of a rational mathematical experimental design for the effective optimization of poloxamer in situ gel formulation is essential, which enables for simultaneous and multidimensional testing of the many causal factors that determine the target quality and to preclude the performance of a large number of unnecessarily independent runs. Thus, a two-factor, five-level CCD was constructed to explore the optimum levels of this in situ gel formulation. Two selected independent variables (the feasible concentrations of P407 (X1 ) and P188 (X2 ) for tailoring the thermosensitive gel) were studied as factors. The gelation temperatures were significant variables, and should be taken into account in the process of formula optimization. Due to the dilution effect from the tear fluid, it should be considered during the design of ophthalmic formulations, which in theory, the smaller the difference in the gelation temperatures before and after ATF dilution, the stronger the ability of the thermosensitive gel to endure the dilution by ATF [19]. Thus, the gelation temperatures before and after ATF dilution (Y1 , Y2 , respectively) and the difference in value of the gelation temperatures before and after ATF dilution (Y3 ) were selected as important objective parameters. The coded and actual values of the variables are provided in Table 1. When it came to the prediction of the best suitable formulation, the fitness of the model among the linear, two-factor interaction model and quadratic model was assessed due to the analysis of variance p-value and focus on the model maximizing multiple correlation coefficient r2 , predicted r2 and adjusted r2 as quality indicators in the model summary statistic list. A p-value of less than 0.05 was considered statistically significant. Optimization was performed using an objective function to obtain the optimal points concerning the predetermined constraints in which the gelation temperature before and after the ATF dilution and their different values were answered for the demand of ophthalmic THGs. The selected optimal formulation was prepared for further evaluation of this hybrid system. 2.6. Physicochemical characterization 2.6.1. Morphology and particle size The particle size and polydispersion index (PI) of the SLNs and hydrogel were measured using a Zetasizer (3000SH, Malvern Instruments Ltd., UK). In addition, the particle size measurement to assess the stability of the resulting SLN dispersions after 24 h of storage at 4 ◦ C was performed according to the cold method process for hydrogel preparation. In addition, the SLN stability after they were embedded in the hydrogel formulations in terms of particle size after 30 days of storage at room temperature was also
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evaluated. The results were reported as the mean ± standard deviation (SD) of three replicates (n = 3). The surface morphology of the SLNs before and after its incorporation into the hydrogel was studied using scanning electron microscopy (SEM). A drop of the sample was deposited on to a silicon wafer, dried, and sputtered for 20 s with gold, and finally, analyzed using SEM (Inspect F50, FEI, USA). 2.6.2. Thermal analysis using differential scanning calorimetry The thermal behavior of these composite hydrogels was determined using differential scanning calorimetry (DSC Q10, TA, USA) at a heating rate of 10 ◦ C/min. This was performed under a dry nitrogen atmosphere and Al2 O3 was used as a reference. The DSC measurements were performed on the following samples: (A) lyophilized blank hydrogel without cryoprotectant; (B) Resina Draconis; (C) lyophilized Resina Draconis-loaded SLNs without cryoprotectant; (D) lyophilized SLN-based hydrogel hybrid system; and (E) lyophilized blank SLNs. 2.7. In vitro corneal penetration experiments The transcorneal penetration behavior of the Resina Draconis from the hybrid hydrogel complex was performed on isolated rabbit corneas with a conventional vertical Franz cell diffusion apparatus (RYJ-2B, Huanghai medical and testing instruments, China). The receptor compartment of the apparatus was filled with glutathione bicarbonate ringer (GBR) buffer that had been prewarmed to a temperature of 37 ◦ C. The Resina Draconis-SLNs and hybrid hydrogel were placed in the donor compartment, respectively. At predetermined time intervals of up to 6 h, 0.2 mL sample was withdrawn from the receiving compartment and was immediately replaced with an equal volume of preheated GBR buffer. The amount of drug that permeated across the cornea was assayed using HPLC. The penetration parameter was calculated by plotting the amounts of drug permeating through the cornea per unit area (g/cm2 ) versus time (min). The steady-state flux (J) values across the cornea were evaluated by determining the slope of the linear portion of the permeation graphs using the relationship given below: J (g/(cm2 s)) = dQ/A dt; where Q indicates the cumulative amount of drug permeated at time t, and A is the exposed corneal surface area. The corneal permeability coefficient (Kp, cm/s) was determined using the expression given below: Kp (cm/s) = J/C0 ; where C0 is the initial concentration of drug in the donor compartment. All experiments were performed in triplicate. At the end of the penetration study, each cornea was carefully removed from the sclera ring and weighed (Ww ). It was then dessicated overnight at 70 ◦ C and reweighed (Wd ). The corneal hydration level (HL %), defined as [1 − (Wd /Ww )] × 100, was calculated [20,21]. 2.8. Ocular tolerance test (HET-CAM test) To evaluate the ocular tolerance of the developed formulation, a modified HET-CAM test was performed as previously reported [22]. Briefly, freshly fertilized hen’s eggs were incubated at 37 ± 0.5 ◦ C for nine days, and then each egg was tested with a cold lamp to ensure viable and optimal illumination of the chorioallantoic membrane by detailed observation of the embryo development. On day 10, the chorioallantoic membrane (CAM) was carefully dissected (without injuring any underlying blood vessels) and exposed by removing a portion of the eggshell and inner membrane above the air cell. Subsequently, the surface of the CAM was exposed to 300 L of the test substance, 0.1 M sodium hydroxide (NaOH) solution and 70% isopropyl alcohol served as the positive controls, and a saline solution was used as a negative control. The irritant effects on blood vessels such as hyperemia, hemorrhage or coagulation were examined using a stereo microscope (SZX16, Olympus, Japan) before exposure and at different time points post-application for 5 min. Scoring of
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Table 1 Independent variables and their levels of experimental design. Independent variables
X1 : concentration of P407 (%, w/v) X2 : concentration of P188 (%, w/v)
Levels −1.414
−1
0
1
1.414
21 0
22.17 2.2
25 7.5
27.83 12.8
29 15
Dependent variables
Constraints
Y1 : gelation temperature before ATF dilution (◦ C) Y2 : gelation temperature after ATF dilution (◦ C) Y3 : difference in gelation temperature before and after ATF dilution (◦ C)
In range: In range:
25 ◦ C ≤ Y1 ≤ 35 ◦ C 30 ◦ C ≤ Y1 ≤ 35 ◦ C Minimize
each test substance or formulation was assigned. The mean score value of the product was designated as nonirritant (irritation score 0–0.9), slight irritant (score 1–4.9), moderate irritant (score 5–8.9), and strong irritant (score 9–21) using a classification system previously described by Kalweit et al. [23]. In addition, images were obtained before application and at 30 s, 2 min, and 5 min after exposure. The mean detection time in seconds after exposure for the appearance of different degrees of abnormal irritant effect was determined for each solution based on 4 eggs.
3.2. Analysis of the response surfaces
2.9. Transcorneal mechanism across the cornea
Y1 = 35.24 − 2.91X1 + 5.75X2 − 1.04X1 X2 − 0.85X12 − 3.88X22
An investigation on the transport mechanism across the cornea of SLNs-based THGs was performed in rabbits in vivo. New Zealand albino rabbits (male, weighing 2.5–3.0 kg) free of any ocular damage were selected and utilized to perform this experiment. The transport process across the corneal epithelium was located by C6 , a fluorescent marker. The corneal C6 disposition was investigated by examining the samples of rabbit cornea according to the CLSM. All experiments were approved and monitored by the Institutional Animal Care and Use Committee of Taishan Medical University. Fifty microliters of the formulation were administered into the lower fornix of the conjunctival sac. The contralateral eye was used as the control and received no treatment. The animals were euthanized and then sacrificed at predetermined time points after administration of the hydrogel formulation, and the freshly harvested cornea tissue was then rinsed with saline, flash frozen in liquid nitrogen, cut into 10-m thick sections and mounted onto slides and a coverslipped. Transcorneal characteristics were observed and recorded using the Leica Confocal apparatus (MP5, Leica, Germany) for image acquisition. An Ar excitation laser (excitation 488 nm) and a HeNe excitation laser (excitation 543 nm) were selected for C6 .
The model F-value of 91.54 indicated that this model was significant (p < 0.0001). The obtained results in this design suggested that independent factors affecting Y1 included the concentration of P407 (X1 ), P188 (X2 ) and the quadratic term of P188 (X22 ), with a p-value of
