International Journal of Pharmaceutics 489 (2015) 218–225

Contents lists available at ScienceDirect

International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

Hyaluronic acid based micelle for articular delivery of triamcinolone, preparation, in vitro and in vivo evaluation Ebrahim Saadat a , Naeeme Shakor a , Mehdi Gholami b , Farid A. Dorkoosh a,c, * a b c

Department of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran, Iran Medical Biomaterial Research Center, Tehran University of Medical Sciences, Tehran, Iran

A R T I C L E I N F O

A B S T R A C T

Article history: Received 7 February 2015 Received in revised form 30 April 2015 Accepted 3 May 2015 Available online 5 May 2015

A novel triamcinolone loaded polymeric micelle was synthesized based on hyaluronic acid and phospholipid for articular delivery. The newly developed micelle was characterized for physicochemical properties including size, zeta potential, differential scanning calorimetry (DSC) analysis and also morphology by means of transmission electron microscopy. The biocompatibility of micelle was explored by histopathological experiment in rat model. Also biological fate of micelle was investigated in rat by means of real time in vivo imaging system. Triamcinolone loaded micelle was in the size range of 186 nm with negative zeta potential charge. Micelles were spherical in shape with core shell like structure. Triamcinolone was released from micelle during 76 h with almost low burst effect. DSC analysis showed the conversion of crystalline triamcinolone from its crystalline state. Histopathological analysis showed no evidence of tissue damage or phagocytic accumulation in knee joint of rat. The real time in vivo imaging analysis suggested at least three days retention time of micellar system in knee joint post injection. ã 2015 Elsevier B.V. All rights reserved.

Keywords: Polymeric micelle Articular delivery Hyaluronic acid Triamcinolone In vivo real time imaging

1. Introduction Joint related disorders including arthritis rheumatism (RA) and osteoarthritis (OA) are among big challenges in recent years. This complication afflicted over 40 million people only in US and it is predicted to increase to 60 million till 2020 (Brooks, 2002). Surgery and pharmacotherapy are the most two indicated remedies for these kind of disorders the first of which is more invasive and indicated for end-stage patients only. The pharmacotherapy of articular disorders is based on the administration of non- steroidal anti-inflammatory (NSAIDs) drugs and also corticosteroids (Bradley et al., 1991; Minas and Nehrer, 1997). As most of these drugs are administered orally, various kinds of side effects like gastrointestinal complication, cataracts and ulcer may occur during therapy (Duru et al., 2007; Da Silva et al., 2006). One approach to bypass these side effects is the intra-articular (IA) injection of drugs to joints, which shows a relatively high concentration of the drug in the active site and eliminates gastrointestinal upset; but the major problems with this method is high drug clearance from joints

* Corresponding author at: Department of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran. Tel.: +98 21 88009440; fax: +98 21 88009440. E-mail address: [email protected] (F.A. Dorkoosh). http://dx.doi.org/10.1016/j.ijpharm.2015.05.001 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.

which needs frequent injections and can potentially lead to infection and joint disability and post injection flare (Gerwin et al., 2006). Moreover, due to the crystalline nature of some drugs and especially corticosteroids, inflammatory events may happen upon IA injection; a condition known as crystalline-induced arthritis (Ellman and Becker, 2006). Local articular delivery is a concept developed for articular complications treatment that has significant clinical outcomes including reduced joint inflammation, reduced side effects and increased patient compliance (Dingle et al., 1978). Till now, various kinds of delivery systems have been adapted for local delivery to joints. These systems include microspheres (Brown et al., 1998), nanospheres, liposomes (Lopez-Garcia et al., 1993) and hydrogels (Inoue et al., 2006) and have several advantages. Prolonged drug retention time in active site, reduced drug clearance from joints and also increased patient compliance through the reduction of administration frequency are among the advantages of these delivery systems (Raynauld et al., 2003; Horisawa et al., 2002a). Although these systems may improve drug efficacy, each of them has some limitations. Microspheres with the average size of 50–100 mm can be trapped by the articular immune system and triggered a granulation reaction. Moreover, microsphere drug delivery represented other implications like syringe clogging during administration and painful injection (Kempe and Mäder,

E. Saadat et al. / International Journal of Pharmaceutics 489 (2015) 218–225

2012). Nishide et al. (1999) reported an increase in the immune system responses following IA injection of microspheres from 5 to 40 mm. Beside microparticle related immune responses the shape of particles has been reported to be an important issue in increasing immunological responses. Ratcliffe et al. (1984) reported synovial membrane and subsynovial lining inflammation after IA injection of irregularly shaped PLA and poly (butyl cyanoacrilate) microparticles. Liposome drug delivery is an ideal platform for articular drug delivery but less stability and drug leakage are two important defects of this system (Gregoriadis and Davis, 1979). Hydrogel is another delivery system which is used in IA delivery. Due to high viscosity of this kind of delivery system a specific syringe with defined needle size is needed. Moreover, administration of this system needs slow and careful injection. Polymeric micelle is a nano-sized particle formed spontaneously in an aqueous media. This system is composed of at least two different block polymers with hydrophobic and hydrophilic characteristics. When such a co-polymer disperses in water, the hydrophilic part faces with water while the hydrophobic segment moves far from it in order to reach maximum stability. The hydrophobic and hydrophilic segments of these particles form the core and shell part of micelle, respectively. Due to hydrophobic characteristics of micelle core, it could be considered as a cargo part which can dissolve hydrophobic drugs. The hydrophilic part of micelle interacts with the surrounding water and makes micelle more stable (Nakanishi et al., 2001). Polymeric micelle has been used as drug delivery system due to its stability and solubilizing characteristics (Jiang et al., 2012). Moreover, micelles have the ability to target different tissues due to various targeting moieties which can be decorated on the surface of this system. Hyaluronic acid (HA) is a naturally occurring polymer in human tissues like cell matrix, connective tissues and also synovial fluid in different joints (Palumbo et al., 2006). HA is composed of repeating disaccharide units including D-glucuronic acid and N-acetyl glucosamine linked by b (1 ! 4) and b (1 ! 3) glucosidic bands (Brown and Jones, 2005). HA is a biocompatible and biodegradable polymer which has numerous applications in medicine and drug delivery. HA has been used as a scaffold and also different types of drug delivery systems including nanoparticles (Choi et al., 2010), polymeric micelles (Lee et al., 2009) and hydrogels (Luo et al., 2000). HA also has an important biological role in cell proliferation, differentiation and also angiogenesis by binding to the cell specific receptors like CD44 and receptor for HA-mediated motility (RHAMM) which are increasingly over-expressed on the surface of tumor cells. HA also has various functional groups like hydroxyl and carboxylic acid, which makes it an attractive polymer for further modification. Phospholipids (PEs) are a group of lipids which play an important role in cell structure and also exist in different tissues like the nervous system and lungs. Due to biocompatibility and biodegradability, they have been used excessively in different drug delivery systems such as liposomes, solid lipid nanoparticles (SLN) (Westesen and Siekmann, 1997) and also cochleates (Papahadjopoulos et al., 1975). PEs which are composed of one or two fatty acids esterifies with glycerol, are synthesized by most cells. Regarding the biocompatibility and also the desirable hydrophobic characteristics of these substances, they could be considered as a hydrophobic micelle cores. Wang et al. (2010) reported attractive micellar characteristics of dextran–PE conjugates for drug delivery. Also Gill et al. (2011) reported sustained release kinetic of the drug from PEG–PE micelle due to impressive hydrophobic core part of this micelle. Triamcinolone suppresses inflammation by increasing production of anti-inflammatory mediators like serum leuko-proteas inhibitors which leads to limited production of cyclooxygenases 1 and 2 (Mangal et al., 2014). Triamcinolone has limited solubility in

219

water around 0.018 mg/ml (Miro et al., 2012). In this study a novel polymeric micelle based on HA and 1,2-distearoylphosphatidylethanolamine (DSPE) was synthesized for sustained delivery of triamcinolone (Mw = 476, log P = 3.2), a highly used corticosteroid in OA and RO. Physicochemical properties of the drug loaded micelle and also the in vitro release profile of triamcinolone was investigated. Moreover, understanding the biological fate of micelles, IA injection of the labeled micelle into the knee joint of rat model was performed and in vivo real time imaging analysis was explored accordingly. 2. Materials and methods 2.1. Materials Hyaluronic acid (molecular weight of 10 kDa) was purchased from Freda Biochem Co., Ltd. (China). 1,2-Distearoylphosphatidylethanolamine (DSPE) were purchased from lipoid, (Germany). Triamcinolone was obtained from Jaber pharmaceutical company. N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and pyrene were bought from Sigma– Aldrich Co. (USA). Regenerated cellulose dialysis bag (molecular weight cut off 3500 Da) was purchased from Spectrum Laboratories Inc. (Canada). Cy 7.5 amine was bought from Lumiprobe Corporation (USA). All other reagents and materials are from analytical grade and used as received. 2.2. Methods 2.2.1. Synthesis of HA–DSPE co-polymer HA–DSPE co-polymer was synthesized as described previously (Saadat et al., 2014). 100 mg of HA was dissolved in dionized water with 2 molar excess of NHS and EDC and mixed for 2 h at room temperature in order to activate carboxylic acid groups. DSPE (5 M excess of HA) was dissolved in tert-butanol and deionized water (10%; v/v) in the presence of 0.1 mol triethylamine and mixed at 55  C. HA solution was added dropwise to DSPE solution and stirred at 60  C for 6 h followed by stirring at room temperature for additional 24 h. The resultant mixture was dialyzed against ethanol/water solution (70:30, 50:50, 30:70; v/v) for 24 h to remove impurities followed by lyophilization. 2.2.2. Preparation of triamcinolone loaded micelle For preparation of loaded micelle, dialysis method was selected. Briefly, 2 mg of triamcinolone was dissolved in methanol and added dropwise to 10 ml of HA–DSPE dispersion (1 mg/ml). The mixture was stirred for 12 h at room temperature. To remove the unloaded drug from the micellar system the whole mixture was poured in dialysis tube (Molecular cut off 3500 Da) and dialyzed against water/methanol (50:50; v/v) for 24 h. For further purification, the resultant dispersion was centrifuged (5000 rpm) for 15 min in order to remove any unloaded drugs and other impurities. The obtained polymeric micelle dispersion was lyophilized and kept in refrigerator for further experiments. 2.2.3. Characterization of triamcinolone loaded micelle The hydrodynamic size diameter and also zeta potential of triamcinolone loaded micelle were obtained by dynamic light scattering method (Zetasizer ZS, Malvern, UK). 1 ml of triamcinolone loaded micelle was diluted in deionized water and the micelle size determined at the wave length of 633 nm at 25  C with detection angle of 90 . For the evaluation of the loaded amount of drug in polymeric micelle, 500 ml of micelle dispersion was mixed with an equal amount of methanol and vortex vigorously. The whole mixture was centrifuged and 20 ml of supernatant was taken and analyzed by HPLC. The mobile phase was methanol with the

220

E. Saadat et al. / International Journal of Pharmaceutics 489 (2015) 218–225

flow rate of 1 ml/min through C18 column and the detection wave length of 254 nm at room temperature. The encapsulation efficiency (EE%) and drug loading (DL%) were calculated according to the following equations; EE% ¼

DL% ¼

Triamcinolone amount in micelle  100 Triamcinolone amount in feed

Triamcinolone amount in micelle Triamcinolone amount in micelle þ amountof HA  DSPE conjugate

100

2.2.4. Morphology determination of triamcinolone loaded micelle In order to explore the micelle shape, the transmission electron microscopy (TEM, CM 30, Philips, Eindhoven, the Netherlands) technique was applied. A small drop (30 ml) of triamcinolone loaded micelle was placed on a carbon-coated copper grid and fixed by gluteraldehyde 2% and negatively stained with uranuyl acetate 1% (w/v). The sample was explored with TEM equipment at 120 kV. 2.2.5. Differential scanning calorimetry (DSC) To investigate the physical state changes of triamcinolone in micelle core, differential scanning calorimetry analysis was performed using Mettler-Toledo (Greifensee; Switzerland). Analysis was performed for triamcinolone, HA–DSPE co-polymer,

physical mixture of triamcinolone and HA–PE co-polymer and also the triamcinolone loaded micelle freeze dried powder. The amount of triamcinolone in all samples was about 1 mg. All samples were analyzed in temperatures ranging from 20 to 300  C with heating rate of 10  C/min. 2.2.6. In vitro release evaluation The release profile of triamcinolone from micelle formulation was performed by dialysis method. 30 mg of the triamcinolone loaded micelle lyophilized powder was dispersed with 10 ml of water for injection and poured into the dialysis tube (molecular weight cut off 3500 Da). The dialysis tube was fully sealed and immersed into 200 ml of phosphate buffer saline (pH 7.4) and placed on a shaker bath (50 rpm) at 37  C. At predetermined time intervals 1 ml sample was removed and analyzed by HPLC using the method described above. To maintain sink condition after each sample removal 1 ml of fresh release medium was added. 2.2.7. Biocompatibility of HA–DSPE micelle In order to investigate the biocompatibility of the newly developed micelle, an in vivo experiment was performed. This part of the study was performed in accordance with approved guidelines by the ethics committee of Tehran University of Medical Sciences. 12 male Wistar rats were randomly divided into 2 groups of six. All rats were kept in lab temperature and humidity, allowed freely to access to food and water. The first group was examined with intra-articular injection of HA–DSPE co-polymer with concentration of 1 mg/ml (50 ml) and the second group was

Fig. 1. (a) Schematic presentation of HA–DSPE micellization in an aqueous media and drug loading and (b) endocytosis of HA–DSPE micelles by synovial phagocytes.

E. Saadat et al. / International Journal of Pharmaceutics 489 (2015) 218–225

221

Table 1 Physicochemical characteristic of triamcinolone loaded micelle. Data represented as mean  SD (n = 3). Formulation

Size (nm)

PdI

Zeta potential (mV)

EE%

DL%

HA–DSPE

186.4  7.3

0.19  0.02

34.3  2.6

52.41  0.37

7.91  0.06

treated with the same volume of sterile water for injection to the knee joint. Five days of post injection time, rats were sacrificed and knee joints were isolated for histopathological investigations. The isolated joints were fixed by formalin (10%) and embedded in paraffin wax. Thin sections were prepared by microtome and stained with eosin and hematoxylin (3%). Each section was explored for any sign of cyst formation, inflammation or microphage proliferation. 2.2.8. Intra-articular (IA) injection of HA–DSPE micelle For better understanding of micellar system retention time in joint cavity, intra-articular injection to the knee joint of rat was performed and monitored by means of real time in vivo imaging system (Kodak FX Pro). Six male Wistar rats were kept under laboratory standard condition. Firstly, the HA–DSPE micelle was labeled with cy 7.5 amine NIR fluorescent dye. The animals were anesthetized using xylazin 5% and ketamin 1% by intraperitoneal injection. 25 ml of HA–DSPE labeled micelle (10%; w/v) was injected into the right knee joint of each rat. In predetermined time intervals each anesthetized animal was put in the chamber of imaging system and pictures were captured. Fluorescence images were captured at 20 cm field of view and exposure time of 20 s and f stop of 2. Grayscale images with X-ray lamp were also obtained at 20 cm field of view and exposure time of 20 s and f stop of 2 for better understanding of micelle in knee joint. 2.2.9. Determination of triamcinolone levels in rat plasma Triamcinolone plasma concentrations were explored after intra articular injection of micellar formulation and also free drug. Twelve male Wistar rats with the average weight of 310 g were randomly divided in two groups of six and kept in standard laboratory condition with free access to food and water. 1 mg/kg of triamcinolone suspension and triamcinolone loaded micelle was injected to the knee joint of animals after being anesthetized with xylazin (5%) and ketamin (1%). At the predetermined time intervals (1, 6, 12, 18, 24, 48 h) post injection animals were tied up in chambers and 500 ml of blood samples was withdrawn from the tail vein of each animal. 200 ml aliquot of plasma samples was collected in centrifuge tube adding 1 ml of ethyl acetate. After well shaking, samples were centrifuged for 10 min at 4500 rpm and organic phase was collected and evaporated under nitrogen gas. The residue was dissolved in methanol and quantify with above mentioned HPLC method with some modifications.

(Kwon and Okano, 1996). Emulsification method needs various surfactants and emulsifiers which are mostly toxic, also sonication method needs more energy, which increases micellar dispersion temperature and may cause destructive reactions. Dialysis method is a simple and safe method for preparation of micellar dispersion as it does not need any emulsifiers and energy. Triamcinolone micellar dispersion was prepared by dialysis method with relatively high encapsulation efficiency (EE%). Table 1 shows the characterization of triamcinolone loaded micelle. As shown in Table 1 the EE% and DL% were calculated to be 52.41  0.37 and 7.91  0.06, respectively. The mean hydrodynamic diameter of micelles was found to be 186.4  7.3 nm with negative zeta potential of 34.3  2.6 mV. The negative zeta potential of micelle is due to the presence of carboxylic acid (COO) moieties in HA structure which is present on the surface of the micellar particles. The particle morphology of micelle was investigated by the transmission electron microscopy (TEM). As it can be observed in Fig. 2, the micelle particles have core-shell structures and smooth surfaces. The shape of the delivery system is an important factor for articular delivery. It has been reported that particles with irregular shape lead to increase tissue inflammation in comparison with round shaped particles. Liggins et al. (2004) reported that irregular milled chitosan particles induced joint inflammation despite of biocompatibility of this polymer. The size of the micelle was reported to be less than 150 nm by TEM which differs from previously mentioned DLS method. The difference between these two reports comes from different methods of analysis. As in TEM, the actual size of particle was investigated; in DLS method, the hydrodynamic diameter was evaluated. The DLS method estimated particle size indirectly via the Estock–Einestin equation by the diffusion of particle in an aqueous media. HA is a hydrophilic polymer which adsorbs water and swells to different values. In a previous study, it was reported that HA was swollen up to several folded, which makes particles much bigger than their actual size (Eenschooten et al., 2012). Apart from size evaluation method, the size of particles plays an important role in IA injection. Particles with bigger size may cause serious inflammatory reactions. It has been reported that PLGA microspheres cause inflammation reaction following the IA injection (Horisawa et al., 2002a). In another study two different sizes (265 nm and 26 mm) of PLGA particles were investigated. It

3. Results and discussion 3.1. Triamcinolone micelle preparation and characterization Like other amphipathic co-polymers, when HA–DSPE was surrounded by an aqueous media, the hydrophobic DSPE molecules escaped water and made a core; while hydrophilic HA remained on the surface of the particle. From the thermodynamic point of view, the tendency of DSPE molecules to aggregation is a driving force to form a core-shell structure in an aqueous media. The lipid core part of this particle is a cargo for the entrapment of hydrophobic drugs such as triamcinolone. Fig. 1a shows the schematic micellization of HA–DSPE co-polymer and drug entrapment. There are several methods for preparation of polymeric micelles including dialysis method, emulsification and sonication

Fig. 2. TEM image of triamcinolone loaded HA–DSPE micelle. Sample was prepared in PBS (pH 7.4) prior to deposition onto the carbon coated grid.

222

E. Saadat et al. / International Journal of Pharmaceutics 489 (2015) 218–225

was concluded that the PLGA nanoparticles were highly phagocyted and transported to the synovial membrane but the microparticles neither phagocyted nor transported, while triggered a granulation reaction by multinuclear giant cells (Horisawa et al., 2002b).

is one of the advantages of HA–DSPE micellar system. There are several studies have been reported the inflammatory condition which leads to crystal-induced arthritis after injection of steroids which are all have crystalline nature (Ellman and Becker, 2006; Solomon, 1973).

3.2. Differential scanning calorimetry (DSC) study

3.3. In vitro release of triamcinolone from micelle

Differential scanning calorimetry study is used to investigate the physical state of the molecules in different drug delivery systems (Siepmann and Peppas, 2001). Fig. 3 shows the DSC analysis of from top to bottom, triamcinolone, physical mixture of triamcinolone and HA–DSPE, freeze dried powder of triamcinolone loaded micelles and HA–DSPE co-polymer. As shown in this figure triamcinolone has sharp endothermic peak at 284  C which is attributed to its crystalline nature (Horisawa et al., 2002b). HA– DSPE has a melting endothermic peak around 60  C and also an exothermic peak at 310  C which are most probably related to the phase transition of DSPE molecules and HA–DSPE degradation, respectively (Luo et al., 2000). In the thermogram of physical mixture and also freeze dried triamcinolone loaded micelle powder, the sharp endothermic peak of triamcinolone was abolished which is attributed to the conversion of the crystalline molecules to the amorphous state. In the previous reports it has also been mentioned that the crystalline molecules of triamcinolone was converted to the amorphous state (Araujo et al., 2010). Conversion of the triamcinolone molecules to the amorphous state

Fig. 4 shows in vitro cumulative release of triamcinolone from HA–DSPE micelle. As seen in this graph, triamcinolone had small burst release at first 4 h of experiment which was followed by sustain release in next 72 h. The burst release of triamcinolone most probably is related to the drug molecules which were presented on the surface of the micelle and not incorporated in cargo part. Zeng et al. (2012) reported the burst release of paclitaxel from PEG–DSPE micelle and concluded that this burst effect was related to the unincorporated drug on the outer layer of the particles. The sustained release of triamcinolone from micelle is the result of hydrophobic interaction between drug molecules and lipid core part. On the other hand lipid core part of micelle acts as a reservoir of drug and released drug molecules slowly. Gill et al. (2011) reported the sustain release of paclitaxel from PEG–DSPE micelle and concluded that hydrophobic interaction of drug with lipid core cause that drug molecules entrapped between acyl chain of phospholoipids and released slowly. This impressive characteristic of HA–DSPE micelle is important to get the benefits of both sustain drug delivery along with solubilization property. 3.4. In vivo biocompatibility study One of the most important features of drug delivery systems is biocompatibility of these carriers. In order to evaluate the biocompatibility of the HA–DSPE micelle a histopathological analysis was performed. Fig. 5a and b shows the histological slides of the rat knees which received sterile water for injection and HA–DSPE micellar solution, respectively. As seen in this figure there is no sign of cyst formation, inflammation or accumulation of macrophages in groups which received HA–DSPE in comparison with groups which administered sterile water. Moreover, chondrocytes remained their structure and there were no any deformity in whole tissue. This experiment shows that HA–DSPE micelle is biocompatible with cartilage tissue and do not cause any undesired reaction.

Fig. 3. DSC thermograms of (a) triamcinolone, (b) physical mixture of triamcinolone and HA–PE, (c) freeze dried triamcinolone leaded HA–DSPE micelle and (d) HA–DSPE co-polymer.

Fig. 4. In vitro release of triamcinolone from HA–DSPE micelle in BPS (pH 7.4, temperature = 37  C). The error bars in the graph show standard deviation (n = 3).

E. Saadat et al. / International Journal of Pharmaceutics 489 (2015) 218–225

223

Fig. 5. Histological slide of knee joint which was examined by (a) water for injection and (b) HA–DSPE co-polymer with the concentration of 1 mg/ml.

3.5. In vivo real time imaging analysis In order to investigate the biological fate of micellar system in synovial space, the in vivo test was performed. Fig. 1b shows the anatomical structure of knee joint and also phagocytosis of micellar system. As seen in Fig. 6 the labeled micelle was remained in knee joint at least three days post injection and then disappeared at day six of the study. This profile suggests that micellar system remain in the knee cavity in spite of synovial clearance. Many researchers have explored the biological fate of nanoparticles in terms of absorption and retention time in joints.

For example Shaw et al. firstly reported long retention of liposome formulation in IA injection (Shaw et al., 1976). Bonanomi et al. (1987) suggested encapsulation of dexamethasone palmitate increased the retention time in comparison to triamcinolone acetonide. The increasing in retention time of drugs in different carriers comes from at least two different phenomena. The first scenario is up taking of drug carriers by phagocytic system and transporting the carrier to the underlying synovial membrane. Ratcliff et al. (1984) showed that phagocytosis of lactic acid oligomers by synovial macrophages improved the retention time of drug in the joint. The second reason for long retention time of

Fig. 6. In vivo real time imaging of Cy 7.5 labeled HA–DSPE micelle in rat knee joint. The analysis followed six days post injection.

224

E. Saadat et al. / International Journal of Pharmaceutics 489 (2015) 218–225

such systems in joint cavity is active uptake of drug carriers by cell surface receptors. One of the most important types of these surface receptors is CD44 which is responsible for uptake of hyaluronic acid. It has been reported that there are specific cells in the synovium and synovial fluid that actively uptake hyaluronic acid (Entwistle et al., 1996; Knudson et al., 2002; Homma et al., 2009). It has been reported that the IA injection half life of corticosteroids (like triamcinolone) and some non steroidal anti inflammatory (NSAIDs) such as naproxen and ketoprofen is about 1 to 2 h but in this study the retention time of labeled micelle was enhanced to at least three days (Knudson et al., 2002). This improvement in retention time of HA–PE micelle comes from to the active uptake of synovium cells which has specific receptors for HA. As a result of this phenomenon nanoparticles were up took from synovial fluid and preserved from synovial clearance. In the previous study it has been reported that methotrexate conjugated with HA had a higher retention time in joint (Homma et al., 2010). They concluded that the active uptake of HA by specific synovium cells resulted in superior effects compared to drug alone. 3.6. Plasma level of triamcinolone Fig. 7 shows the plasma concentrations of triamcinolone after IA injection of micellar and suspension formulations in rat. This plasma concentration pattern demonstrates high clearance of triamcinolone suspension from synovial fluid into the systemic circulation. This rapid clearance is a result of quick equilibrium between synovial fluid and plasma. The clearance of the triamcinolone loaded micelle was less than triamcinolone suspension which is due to the less exposure of the drug with the synovial fluid. Moreover, due to the uptake of the phagocytic and also CD44 presenting cells in synovial fluid, less micellar nanoparticles were available for clearance. In one previously report it was concluded that Brucin microparticles were washed out slower than Brucin solution from synovial fluid (Chen et al., 2012). Also in another study the plasma level of flubiprofen solution and its microsphere system were explored after IA injection (Kawadkar and Chauhan, 2012). The authors concluded that due to the high clearance of flubiprofen solution the plasma level of this drug was higher than that of the microsphere system. In addition to the lower clearance of the micellar system, the peak plasma concentration of micellar triamcinolone was found to be less than

Fig. 7. Plasma concentration of triamcinolone suspension and triamcinolone loaded micelle in rat after IA injection to the knee joint. Data are presented as mean  SD, (n = 6).

25% of the triamcinolone suspension which shows higher accumulation of drug in the joint tissue (Fig. 7). The lower plasma concentration of micellar system led to the minimum systemic side effects in comparison with free form of this corticosteroid. 4. Conclusion A novel polymeric micelle was synthesized based on hyaluronic acid and phospholipid. The newly developed micelle was used for delivery of triamcinolone to the knee join of rat by IA injection. Triamcinolone loaded micelle showed spherical core–shell particles with the size range of around 180 nm. The calorimetry analysis showed the conversion of triamcinolone molecules from its crystalline state in the core part of HA–DSPE micelle. The in vitro release analysis showed sustained release of drug for 76 h. In vivo biocompatibility investigation of HA–PE micelle showed no evidence of knee tissue disturbance or accumulation of phagocytic cells or cyst formation. In vivo real time imaging analysis showed longer retention time of micellar system in knee joint in comparison with free drug. Triamcinolone plasma concentration showed lower levels of drug for HA–DSPE formulation in comparison with suspension formulation. Conflict of interest The authors report no conflict of interest. Acknowledgment The authors would like to acknowledge Tehran University of Medical Sciences for the financial support of this study. References Araujo, J., Gonzalez-Mira, E., Egea, M., Garcia, M., Souto, E., 2010. Optimization and physicochemical characterization of a triamcinolone acetonide-loaded NLC for ocular antiangiogenic applications. Int. J. Pharm. 393, 168–176. Bonanomi, M., Velvart, M., Stimpel, M., Roos, K., Fehr, K., Weder, H., 1987. Studies of pharmacokinetics and therapeutic effects of glucocorticoids entrapped in liposomes after intraarticular application in healthy rabbits and in rabbits with antigen-induced arthritis. Rheumatol. Int. 7, 203–212. Bradley, J.D., Brandt, K.D., Katz, B.P., Kalasinski, L.A., Ryan, S.I., 1991. Comparison of an antiinflammatory dose of ibuprofen an analgesic dose of ibuprofen, and acetaminophen in the treatment of patients with osteoarthritis of the knee. N. Engl. J. Med. 325, 87–91. Brooks, P.M., 2002. Impact of osteoarthritis on individuals and society: how much disability? Social consequences and health economic implications. Curr. Opin. Rheumatol. 14, 573–577. Brown, M., Jones, S.A., 2005. Hyaluronic acid: a unique topical vehicle for the localized delivery of drugs to the skin. J. Eur. Acad. Dermatol. Venereol. 19, 308– 318. Brown, K.E., Leong, K., Huang, C.H., Dalal, R., Green, G.D., Haimes, H.B., Jimenez, P.A., Bathon, J., 1998. Gelatin/chondroitin 6-sulfate microspheres for the delivery of therapeutic proteins to the joint. Arthritis Rheum. 41, 2185–2195. Chen, Z., Liu, W., Liu, D., Xiao, Y., Chen, H., Chen, J., Li, W., Cai, H., Li, W., Cai, B., Pan, J., 2012. Development of brucine-loaded microsphere/thermally responsive hydrogel combination system for intra-articular administration. J. Control. Release 162, 628–635. Choi, K.Y., Chung, H., Min, K.H., Yoon, H.Y., Kim, K., Park, J.H., Kwon, I.C., Jeong, S.Y., 2010. Self-assembled hyaluronic acid nanoparticles for active tumor targeting. Biomaterials 31, 106–114. Da Silva, J.A., Jacobs, J.W., Kirwan, J.R., Boers, M., Saag, K.G., Inês, L.B., de koning, E.J.P., Buttgereit, F., Capell, H., Rau, R., Bijlsma, J.W.J., 2006. Safety of low dose glucocorticoid treatment in rheumatoid arthritis: published evidence and prospective trial data. Ann. Rheum. Dis. 65, 285–293. Dingle, J., Gordon, J., Hazleman, B., Knight, C., Thomas, D.P., Phillips, N., Shaw, I.H., Fildes, F.J.T., Oliver, J.E., Joney, G., Turney, E.H., Lowe, J.S., 1978. Novel treatment for joint inflammation. Nature 271, 372–373. Duru, N., Vander Goes, M.C., Jacobs, J.W.G., Andrews, T., Boers, M., Buttgereit, F., Caegers, N., Cutolo, M., Halliday, S., Dusilva, J.A.P., Kirwan, J.R., Ray, D., Rovensky, J., Severijns, G., Westhovens, R., Bijlsma, J.W.J., 2007. EULAR evidence-based recommendations on the management of systemic glucocorticoid therapy in rheumatic diseases. Ann. Rheum. Dis. 66, 1560–1567. Eenschooten, C., Vaccaro, A., Delie, F., Guillaumie, F., Tømmeraas, K., Kontogeorgis, G.M., Schwach-Abdellaovi, K., Borkovec, M., Gurny, R., 2012. Novel self-

E. Saadat et al. / International Journal of Pharmaceutics 489 (2015) 218–225 associative and multiphasic nanostructured soft carriers based on amphiphilic hyaluronic acid derivatives. Carbohydr. Polym. 87, 444–451. Ellman, M.H., Becker, M.A., 2006. Crystal-induced arthropathies: recent investigative advances. Curr. Rheum. Dis. 18, 249–255. Entwistle, J., Hall, C.L., Turley, E.A., 1996. HA receptors: regulators of signalling to the cytoskeleton. J. Cell. Biochem. 61, 569–577. Gerwin, N., Hops, C., Lucke, A., 2006. Intraarticular drug delivery in osteoarthritis. Adv. Drug Deliv. Rev. 58, 226–242. Gill, K.K., Nazzal, S., Kaddoumi, A., 2011. Paclitaxel loaded PEG(5000)–DSPE micelles as pulmonary delivery platform: formulation characterization, tissue distribution, plasma pharmacokinetics, and toxicological evaluation. Eur. J. Pharm. Biopharm. 79, 276–284. Gregoriadis, G., Davis, C., 1979. Stability of liposomes invivo and invitro is promoted by their cholesterol content and the presence of blood cells. Biochem. Biophys. Res. Commun. 89, 1287–1293. Homma, A., Sato, H., Okamachi, A., Emura, T., Ishizawa, T., Kato, T., Matsuura, T., Sato, S., Tamura, T., Higuchi, Y., Watanabe, T., Kitamura, H., Asanuma, K., Yamazaki, T., Ikemi, M., Kitagawa, H., Morikawa, T., Ikeya, H., Maeda, K., Takahashi, K., Nohmi, K., Izutani, N., Kanda, M., Suzuki, R., 2009. Novel hyaluronic acid-methotrexate conjugates for osteoarthritis treatment. Bioorg. Med. Chem. 17, 4647–4656. Homma, A., Sato, H., Tamura, T., Okamachi, A., Emura, T., Ishizawa, T., Kato, T., Matsuura, T., Sato, S., Higuchi, Y., Watanabe, T., Kitamura, H., Asanuma, K., Yamazaki, T., Ikemi, M., Kitagawa, H., Morikawa, T., Ikeya, H., Maeda, K., Takahashi, K., Nohmi, K., Izutani, N., Kanda, M., Suzuki, R., 2010. Synthesis and optimization of hyaluronic acid–methotrexate conjugates to maximize benefit in the treatment of osteoarthritis. Bioorg. Med. Chem. 18, 1062–1075. Horisawa, E., Hirota, T., Kawazoe, S., Yamada, J., Yamamoto, H., Takeuchi, H., Kawashima, Y., 2002a. Prolonged anti-inflammatory action of DL-lactide/ glycolide copolymer nanospheres containing betamethasone sodium phosphate for an intra-articular delivery system in antigen-induced arthritic rabbit. Pharm. Res. 19, 403–410. Horisawa, E., Kubota, K., Tuboi, I., Sato, K., Yamamoto, H., Takeuchi, H., Kawashima, Y., 2002b. Size-dependency of DL-lactide/glycolide copolymer particulates for intra-articular delivery system on phagocytosis in rat synovium. Pharm. Res. 19, 132–139. Inoue, A., Takahashi, K.A., Arai, Y., Tonomura, H., Sakao, K., Saito, M., Fujioka, M., Fujiwara, H., Tabata, Y., Kubo, T., 2006. The therapeutic effects of basic fibroblast growth factor contained in gelatin hydrogel microspheres on experimental osteoarthritis in the rabbit knee. Arthritis Rheum. 54, 264–270. Jiang, X., Li, L., Liu, J., Hennink, W.E., Zhuo, R., 2012. Facile fabrication of thermoresponsive and reduction-sensitive polymeric micelles for anticancer drug delivery. Macromol. Biosci. 12, 703–711. Kawadkar, J., Chauhan, M.K., 2012. Intra-articular delivery of genipin cross-linked chitosan microspheres of flurbiprofen: preparation, characterization, in vitro and in vivo studies. Eur. J. Pharm. Biopharm. 81, 563–572. Kempe, S., Mäder, K., 2012. In situ forming implants—an attractive formulation principle for parenteral depot formulations. J. Control. Release 161, 668–679. Knudson, W., Chow, G., Knudson, C.B., 2002. CD44-mediated uptake and degradation of hyaluronan. Matrix Biol. 21, 15–23. Kwon, G.S., Okano, T., 1996. Polymeric micelles as new drug carriers. Adv. Drug Deliv. Rev. 21, 107–116. Lee, H., Ahn, C.H., Park, T.G., 2009. Poly[lactic-co-(glycolic acid)]-grafted hyaluronic acid copolymer micelle nanoparticles for target-specific delivery of doxorubicin. Macromol. Biosci. 9, 336–342. Liggins, R., Cruz, T., Min, W., Liang, L., Hunter, W., Burt, H., 2004. Intra-articular treatment of arthritis with microsphere formulations of paclitaxel:

225

biocompatibility and efficacy determinations in rabbits. Inflamm. Res. 53, 363– 372. Lopez-Garcia, F., Vazquez-Autonim, I.M., Latoore, R., Moreno, F., Villalain, J., GomezFernandez, J.C., 1993. Intra-articular therapy of experimental arthritis with a derivative of triamcinolone acetonide incorporated in liposomes. J. Pharm. Pharmacol. 45, 576–578. Luo, Y., Kirker, K.R., Prestwich, G.D., 2000. Cross-linked hyaluronic acid hydrogel films: new biomaterials for drug delivery. J. Control. Release 69, 169–184. Mangal, D., Uboh, C.E., Soma, L.R., Liu, Y., 2014. Inhibitory effect of triamcinolone acetonide on synthesis of inflammatory mediators in the equine. Eur. J. Pharmacol. 736, 1–9. Minas, T., Nehrer, S., 1997. Current concept in the treatment of articular cartilage defects. Orthopedics 20, 525–538. Miro, A., Ungaro, F., Balzano, F., Masi, S., Musto, P., Manna, P., Barretta, G., Quaglia, F., 2012. Triamcinolone solubilization by (2-hydroxypropyl)-b-cyclodextrin: a spectroscopic and computational approach. Carbohydr. Polym. 90, 1288–1298. Nakanishi, T., Fukushima, S., Okamoto, K., Suzuki, M., Matsumura, Y., Yokoyama, M., Okano, T., Sakurai, Y., Kataoka, K., 2001. Development of the polymer micelle carrier system for doxorubicin. J. Control. Release 74, 295–302. Nishide, M., Kamei, S., Takakura, Y., Tamai, S., Tabata, Y., Ikada, Y., 1999. Fate of biodegradable DL-lactic acid oligomer microspheres in the articulus. J. Bioact. Compat. Polym. 14, 385–398. Palumbo, F.S., Pitarresi, G., Mandracchia, D., Tripodo, G., Giammona, G., 2006. New graft copolymers of hyaluronic acid and polylactic acid: synthesis and characterization. Carbohydr. Polym. 66, 379–385. Papahadjopoulos, D., Vail, W., Jacobson, K., Poste, G., 1975. Cochleate lipid cylinders: formation by fusion of unilamellar lipid vesicles. Biochim. Biophys. Acta 394, 483–491. Ratcliffe, J., Hunneyball, I., Smith, A., Wilson, C., Davis, S., 1984. Preparation and evaluation of biodegradable polymeric systems for the intra-articular delivery of drugs. J. Pharm. Pharmacol. 36, 431–436. Raynauld, J.P., Buckland-Wright, C., Ward, R., Choquette, D., Haraoui, B., MartelPelletier, J., Uthman, I., Khy, V., Tremblay, J.L., Bertrand, C., Pelletier, J.P., 2003. Safety and efficacy of long-term intraarticular steroid injections in osteoarthritis of the knee: a randomized, double-blind, placebo-controlled trial. Arthritis Rheum. 48, 370–377. Saadat, E., Amini, M., Dinarvand, R., Dorkoosh, F.A., 2014. Polymeric micelles based on hyaluronic acid and phospholipids: design, characterization, and cytotoxicity. J. Appl. Polym. Sci. 131, 40944. Shaw, I., Knight, C., Dingle, J., 1976. Liposomal retention of a modified antiinflammatory steroid. Biochem. J. 158, 473. Siepmann, J., Peppas, N., 2001. Modeling of drug release from delivery systems based on hydroxypropyl methylcellulose (HPMC). Adv. Drug Deliv. Rev. 48, 139– 157. Solomon, L., 1973. Drug-induced arthropathy and necrosis of the femoral head. J. Bone Joint Surg. Br. 55, 246–261. Wang, T., Xu, Q., Wu, Y., Zeng, A., Li, M., Gao, H., 2010. Preparation of dextran-1,2dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) copolymer and its micellar characteristics. Carbohydr. Polym. 80, 303–307. Westesen, K., Siekmann, B., 1997. Investigation of the gel formation of phospholipidstabilized solid lipid nanoparticles. Int. J. Pharm. 151, 35–45. Zeng, N., Hu, Q., Liu, Z., Gao, X., Hu, R., Song, Q., Gu, G., Xia, H., Yao, L., Pang, Z., Jiang, X., Chen, J., Fang, L., 2012. Preparation and characterization of paclitaxel-loaded DSPE–PEG–liquid crystalline nanoparticles (LCNPs) for improved bioavailability. Int. J. Pharm. 424, 58–66.