Life Sciences 90 (2012) 944–949 Contents lists available at SciVerse ScienceDirect Life Sciences j ourna l homepage: www.e lsev ie r .com/ locate / l i fesc ie 15d-PGJ2-loaded in nanocapsules enhance the antinociceptive properties into rat temporomandibular hypernociception Juliana T. Clemente-Napimoga a, Juscelaine A. Moreira b, Renato Grillo c,d, Nathalie F.S. de Melo c,d, Leonardo F. Fraceto c,d, Marcelo H. Napimoga e,⁎ a Laboratory of Orofacial Pain, Department of Physiology, Piracicaba Dental School, State University of Campinas, Brazil b Laboratory of Biopathology and Molecular Biology, University of Uberaba, Brazil c Department of Biochemistry, State University of Campinas, Brazil d Department of Environmental Engineering, São Paulo State University, Brazil e Laboratory of Immunology and Molecular Biology, São Leopoldo Mandic Institute and Research Center, Campinas, Brazil ⁎ Corresponding author at: Laboratory of Immunolo Leopoldo Mandic Institute and Research Center, R. José São Paulo, 13045‐755, Brazil. Tel.: +55 19 3211 3659; E-mail addresses: marcelo.napimoga@gmail.com, na (M.H. Napimoga). 0024-3205/$ – see front matter © 2012 Elsevier Inc. All doi:10.1016/j.lfs.2012.04.035 a b s t r a c t a r t i c l e i n f o Article history: Received 28 January 2012 Accepted 20 April 2012 Keywords: 15d-PGJ2 PPAR-gamma Nociception Temporomandibular joint Inflammation Aims: To verify whether the nanoencapsulation of 15d-PGJ2 in poly(D,L-lactide-co-glycolide) (PLGA) nanocapsules (15d-PGJ2-NC) might potentialize its antinociceptive activity into rats’ temporomandibular joint (TMJ). Main methods: Transmission electron microscopy (TEM) and atomic force microscopy (AFM) were used to evaluate the morphology and suspension of the PLGA nanocapsules. Rats were pretreated (15 min) with an intra-TMJ injection of unloaded 15d-PGJ2 or 15d-PGJ2-NC at concentrations of 10, 100 or 1000 pg followed by an ipsilateral intra-TMJ injection of 1.5% formalin. The nociceptive behavioral response was observed during 45 min; animals were then sacrificed and the periarticular tissue was removed for IL- 1β measurements. Key finding: TEM and AFM analyses showed that 15d-PGJ2-NC is spherical without any aggregates or ad- hesion confirming that this formulation is a good drug carrier system for 15d-PGJ2. Pretreatment with 15d-PGJ2-NC (100 and 1000 pg/TMJ), but not unloaded 15d-PGJ2, was found to significantly decrease the release of IL-1β cytokine and the animals’ nociceptive behavioral response induced by intra-TMJ injec- tion of formalin. Significance: The compound 15d-PGJ2-NC might be used as a potential antinociceptive and anti-inflammatory agent to treat temporomandibular disorders in clinical practice. © 2012 Elsevier Inc. All rights reserved. Introduction Temporomandibular disorders (TMD) involve multifactorial eti- ology and might result in temporomandibular joint (TMJ) and/or masticatory muscle pain leading, in many cases, to chronic orofacial pain (Cairns, 2010). Since traditional strategies to control TMD-related pain are unsatisfactory, an alternative and efficient approach to treat such condition is of great interest to both pa- tients and clinicians (Cairns, 2010). Therefore, the development of new drugs and/or new formulation to treat chronic inflammato- ry diseases continues to be of considerable importance to re- searchers (Bernardi et al., 2009). gy and Molecular Biology, São Rocha Junqueira, 13 Campinas, fax: +55 19 3211 3712. pimogamh@yahoo.com rights reserved. Peroxisome proliferators-activated receptor-γ (PPARγ) is a ligand-activated transcription factor of the nuclear hormone recep- tor superfamily (Escher and Wahli, 2000). Synthetic PPAR-γ ago- nists of the thiazolidinedione class act as insulin sensitizers and have become important in the treatment of type 2 diabetes (Lehrke and Lazar, 2005). PPAR ligands represent a promising ther- apeutic strategy for other diseases such as arthritis, sepsis, peritoni- tis, and colitis (Chima et al., 2008; Cuzzocrea et al., 2003; Kaplan et al., 2005; Napimoga et al., 2008a, 2008b; Shan et al., 2004), espe- cially when it involves inflammatory pain (Pena-dos-Santos et al., 2009). Otherwise, PPAR-γ agonists are neuroprotective in animal models of acute central nervous system injury including focal ische- mia, spinal cord injury and surgical trauma (Hyong et al., 2008; McTigue et al., 2007; Park et al., 2007; Pereira et al., 2006; Sundararajan et al., 2005; Tureyen et al., 2007; Zhao et al., 2005, 2006). Considering the neuroprotective effect of PPAR-γ agonists, 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), which is a natural li- gand for PPAR-γ (Ricote et al., 1998; Schoonjans et al., 1997), was found to have a peripheral antinociceptive effect on the TMJ via http://dx.doi.org/10.1016/j.lfs.2012.04.035 mailto:marcelo.napimoga@gmail.com mailto:napimogamh@yahoo.com http://dx.doi.org/10.1016/j.lfs.2012.04.035 http://www.sciencedirect.com/science/journal/00243205 945J.T. Clemente-Napimoga et al. / Life Sciences 90 (2012) 944–949 PPAR-γ with the co-participation of κ/δ opiod receptors (Pena-dos- Santos et al., 2009). Nanomedicine has emerged as a new field of study and is considered one of the most promising pathways for the development of effective targeted therapies (Huynh et al., 2009). Polymeric nanoparticles are colloidal structures below 1 μm and have been used to encapsulate lipophilic drugs to target organs and/or tissues, to avoid drug degradation, to improve its efficacy, and to circumvent its toxicity (Adair et al., 2010; Couvreur et al., 2002). Nanoencapsulation of drugs was observed to greatly prolong their pharmacological activity and decrease their toxicity (Alves et al., 2011; Bernardi et al., 2009; Elron-Gross et al., 2009; Grillo et al., 2010). In particular, the systemic administration of 15d-PGJ2-NC, when compared to unloaded 15d-PGJ2, was found to improve the la- tency and the anti-inflammatory effect of the 15d-PGJ2 at a much smaller dose (Alves et al., 2011). Therefore, the present study aimed to analyze if the nanoencapsulation of 15d-PGJ2 might keep or en- hance the antinociceptive effects of peripheral injection on acute in- flammatory TMJ nociception in a rat model. Material and methods Preparation of the PLGA nanocapsules with 15d-PGJ2 The poly (D,L-lactic-co-glycolic acid, 50:50) (PLGA) nanocapsules were prepared by the nanoprecipitation method (Fessi et al., 1989), which involves mixing an organic phase into an aqueous phase. The organic phase consisted of PLGA polymer (100 mg), acetone (30 mL), 15d-PGJ2 (100 μg), sorbitan monostereate (40 mg) and cap- rylic/capric acid triglyceride (200 mg). The aqueous phase was com- posed of polysorbate 80 (60 mg) and deionized water (30 mL). After dissolution of the components of both phases, the organic phase was gradually added to the aqueous phase, and the suspension maintained under agitation for 10 min. The solvent (acetone) was re- moved by evaporation and the suspension was concentrated to a vol- ume of 10 mL under low pressure, using a rotary evaporator, in order to obtain a suspension of 15d-PGJ2 with a final concentration of 10 μg/ mL. After evaporation no traces of acetone were observed in the for- mulation (data not shown). A control formulation (without 15d- PGJ2) was also prepared, following the methodology described above. All parameters such as size and polydispersion measurements, Zeta potential measurements and efficiency of association of 15d- PGJ2 in the PLGA nanocapsules were employed as described previous- ly (Alves et al., 2011). Transmission electron microscopy (TEM) The morphology and structure of the PLGA nanocapsules with 15d-PGJ2 were examined in a JEOL 1200EX II microscope (Jeol ltda, Akishima, Japan) operating at 80 kV. In order to perform the TEM ob- servations, the 15d-PGJ2-NC was first diluted in water and after that the sample was diluted with a 2% uranyl acetate solution (w/v). One drop of the mixture was deposited on a standard copper grid covered by a carbon film and dried at ambient temperature before TEM analysis. Atomic force microscopy (AFM) The microscopy studies of suspension of PLGA nanocapsules with 15d-PGJ2 were performed with a Nanosurf Easy Scan 2 Basic atomic force microscope (BT02217, Nanosurf, Switzerland). The suspension was deposited onto a silicon surface and the immobilized sample was air-jet dried and analyzed using in contact mode. The analysis was made using a commercial Contr 10 cantilever. The diameters of PLGA nanoparticles were measured and a size distribution was per- formed using Nanosurf software. Animals This study was carried out with male Wistar rats (150–250 g) maintained in a temperature-controlled room (23°±1 °C) with a 12‐hour light–dark cycle. All experiments were conducted in accordance to the IASP guidelines on using laboratory animals for investigations of experimental pain in conscious animals. All animal experimental procedures and protocols were approved by the Committee on Animal Research of the University of Uberaba (# 047/2009). The animals suffering and number per group were kept at a minimum and each animal was used once. Testing procedure for TMJ pain Testing sessions took place during the light phase (between 9:00 AM and 5:00 PM) in a quiet room maintained at 23 °C±1 °C. Each animal was manipulated for 7 days to be habituated to the ex- perimental manipulation. After this period, the animal was placed in a test chamber (30×30×30 cm mirrored wood chamber with a glass at the front side) for 15 min habituation period to minimize stress. Animals were briefly anesthetized by inhalation of halothane to allow the TMJ injection, which was performed with a 30-gauge needle connected to a 50‐μL Hamilton syringe (Roveroni et al., 2001). Each animal regained consciousness approximately 30 s after discontinuing the anesthetic and was returned to the test chamber for counting nociceptive responses. The nociceptive re- sponse score was defined as the cumulative total number of seconds that the animal spent rubbing the orofacial region asym- metrically with the ipsilateral fore or hind paw plus the number of head flinches counted during the observation period as described previously. Since head flinches followed a uniform pattern of 1 s of duration, each flinch was expressed as 1 s. Results are expressed as the duration time of nociceptive behavior (Roveroni et al., 2001). All animals received a final volume of 30 μL into TMJ. All experi- ments were conducted in a double blind fashion in which the person who injected the solutions was different from the one who made the behavioral assessment. Experimental protocols Effect of 15d-PGJ2-NC on formalin-induced TMJ nociception Rats were pretreated (15 min) with an intra-TMJ injection of unloaded 15d-PGJ2 or 15d-PGJ2-loaded nanocapsules (15d-PGJ2-NC) in the concentrations of 10, 100 or 1000 pg (n=6; 15 μL/TMJ) followed by ipsilateral intra-TMJ injection of 1.5% formalin in a final volume of 30 μL. In order to test whether empty nanocapsules could affect formalin-induced TMJ nociception, a control group of rats were pretreated with empty nanocapsules (the amount of nanocapsules corresponding to the highest dose used, 1000 pg/TMJ, diluted in saline in a final volume of 15 μL) followed by injection of saline or 1.5% formalin into the TMJ. Behavioral nociception response was evaluated for a 45‐minute observation period. In order to confirm the peripheral 15d-PGJ2-mediated antinociception, the highest dose of 15d-PGJ2 was also injected in the contralateral TMJ that received injection of 1.5% formalin. Effect of 15d-PGJ2-NC on formalin-induced IL-1β cytokine release After the evaluation of the formalin-induced TMJ nociception, the animals were sacrificed and the periarticular tissues were removed and homogenized in cold RIPA buffer (20 mM Tris–HCl, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β- glycerophosphate, 1 mM Na3VO4, 1 μg/ml leupeptin, pH 7.5). The samples were centrifuged at 10,000 g for 10 min at 4 °C. The super- natant was removed and centrifuged again. IL-1β levels were detected by ELISA (Enzyme Linked Immunosorbent Assay) using Fig. 1. Transmission electron microscopy images of a spherical PLGA NC observed after negative fixation. 946 J.T. Clemente-Napimoga et al. / Life Sciences 90 (2012) 944–949 protocols supplied by the manufacturer (R&D Systems, Minneapolis, USA). After all standard procedures, the optical density (O.D.) was measured at 490 nm. Results are expressed as pg/mg of the cyto- kine, based on the standard curves. Statistical analysis To determine if there were significant differences (pb0.05) among treatment groups, the data was analyzed using the t-test or one-way ANOVA as appropriate. If there was a significant between-subjects main effect of treatment group following one-way ANOVA, post-hoc contrasts, using the Bonferroni test, were performed to determine the basis of the significant difference. Data are presented in figures as means±S.E.M. Fig. 2. Atomic force microscopy images of 15d-PGJ2-NC. A) bi-dimensional im Results Characterization of the 15d-PGJ2-NC The images of transmission electron microscopy (Fig. 1) showed that 15d-PGJ2-NC were spherical, contained no aggregates, and had a size distribution between 182 and 220 nm. This range is lower than that observed by photon correlation spectroscopy and, in this technique, the samples were dried. Another analysis of morphology of the PLGA 15d-PGJ2-NC was determined by AFM confirming that 15d-PGJ2-NC were spherical and without aggregates (Fig. 2). An in- teresting fact observed in this image was that the size distribution of 15d-PGJ2-NC had a size range between 250 and 600 nm and this in- crease in the diameter of the nanoparticles can be explained by the flattening or deformation of nanocapsules when the formulation was dripped on the surface of silicon. The 3D view of the AFM image showed that the height of the nanocapsules was only 26 nm, clearly showing that the technique of dripping or a possible interac- tion with the substrate structure resulted in the formation of the flat- tened structure of the nanocapsules. Effect of 15d-PGJ2-NC on formalin-induced TMJ nociception Compared with saline administration, the injection of formalin into the TMJ (1.5%) significantly increased the nociceptive behavior (Fig. 3A and B). On the other hand, pretreatment with 15d-PGJ2-NC (100 and 1000 pg/TMJ) strongly decreased the nociceptive behavior induced by intraarticular injection of formalin (Fig. 3A; Pb0.05). Interestingly, pretreatment with unloaded 15d-PGJ2 (10, 100 and 1000 pg/TMJ) did not reduce the nociceptive behavioral (Fig. 3B). The injection of 15d-PGJ2-NC at the highest concentration in the contralateral TMJ, did not decrease the formalin-induced age; B) three-dimensional image and C) size distribution of 15d-PGJ2-NC. image of Fig.�2 947J.T. Clemente-Napimoga et al. / Life Sciences 90 (2012) 944–949 TMJ nociception (data not shown) demonstrating that the anti- nociceptive effect is local. In order to test whether the empty nanocapsules induced any al- teration in the nociceptive behavior, it was injected empty nanocapsules followed by saline or formalin administration. In nei- ther case was there any significant change (P>0.05) in the nocicep- tive behavior (Fig. 3C). Effect of low doses of 15d-PGJ2-NC on IL-1β releases The possible interference of 15d-PGJ2-NC in the release of IL-1β into the periarticular tissue was investigated since this is an impor- tant nociceptive mediator. There was a dose-dependent decrease in the levels of IL-1β in the periarticular tissue of rats pretreated with Fig. 3. Effect of 15d-PGJ2-NC on formalin-induced TMJ nociception. (A) 15d- PGJ2-loaded nanocapsules (15d-PGJ2-NC) (1000, 100 but not 10 ng/TMJ) significantly reduced the magnitude of 1.5% formalin-induced nociceptive responses (pb0.05). (B) Pre-treatment with unloaded 15d-PGJ2 (1000, 100 and 10 ng/TMJ) did not reduce the magnitude of 1.5% formalin-induced nociceptive responses (p>0.05). (C) Pre- treatment with empty nanocapsules (NC) did not change the behavioral response of animal that received intra-TMJ injection of saline or 1.5% formalin (p>0.05). The sym- bol (#) indicates statistical significance compared to saline; the symbol (*) indicates statistical significance (pb0.05, ANOVA, Bonferroni test) compared to 1.5% formalin. Fig. 4. Effect of low doses of 15d-PGJ2-NC on IL-1β release. (A) Pre-treatment with 15d- PGJ2-NC (1000, 100 but not 10 ng/TMJ) significantly reduced the release of formalin- induced IL-1β cytokine (pb0.05). (B) Pre-treatment with unloaded 15d-PGJ2 (1000, 100 and 10 ng/TMJ) did not reduce the release of IL-1β (p>0.05). The symbol (#) in- dicates statistical significance (pb0.05, Bonferroni test) compared to saline; the symbol (*) indicates statistical significance (pb0.05, ANOVA, Bonferroni test) compared to 1.5% formalin. 15d-PGJ2-NC, in contrast to rats pretreated with saline and injected with formalin (Fig. 4A). On the other hand, there was no statistical re- duction of cytokine levels in the periarticular tissue of animals treated with the same concentration (10, 100 and 1000 pg/TMJ) of non- encapsulated 15d-PGJ2 (Fig. 4B). Discussion Developing new chemical and biological compounds intended for therapeutics is a great challenge for researchers’ worldwide (Huynh et al., 2009). The carrier system is considered a reliable approach to target the drug delivery site (Couvreur et al., 2002; Couvreur and Vauthier, 2006). Among the different nanocarrier systems, biodegrad- able nanoparticles have been reported as potential drug delivery ve- hicles over the last few years. A new versatile nanodelivery system for the targeted delivery of therapeutic compounds has shown poten- tial activity against several diseases (Adair et al., 2010). The biocompatibility of nanoparticles is one of the major con- cerns in biomedical applications. Lipid nanocapsules are potential vectors for the delivery of drugs into the inner ear after round win- dow membrane application without morphological or cochlear neu- ral functional changes (Zhang et al., 2011). PLGA nanocapsules with 15d-PGJ2 were used in the present study which is an FDA-approved polymer used for the preparation of nanoparticles. In particular, intra-articular injections of PLGA nanocapsules causes no alteration in articular tissue functions in the knee or TMJ of healthy rats or of those undergoing degenerative or inflammatory conditions such as arthritis and/or osteoarthritis, suggesting that PLGA-nanocapsules image of Fig.�3 image of Fig.�4 948 J.T. Clemente-Napimoga et al. / Life Sciences 90 (2012) 944–949 could be used as a safe drug delivery system to treat articular dis- eases, allowing a wide range of encapsulating molecules (Zille et al., 2010; Mountziaris et al., 2010). In addition, polymers containing PLGA, in previous animal experiments, were found to be biocom- patible and to have low immunogenicity and little toxicity (Alves et al., 2011; Ishihara et al., 2010; Shive and Anderson, 1997). In a previous study, 15d-PGJ2-containing PLGA-nanoparticles (hy- drodynamic diameter: 100 to 400 nm; polydispersity: b0.2; and zeta potential: −30 mV) were reported as an efficient carrier system (Alves et al., 2011). In the present study, morphological analysis of this carrier system using transmission electron microscopy (TEM) and atomic force microscopy (AFM) showed that 15d-PGJ2-NC is spherical without any aggregates or adhesions, which are important characteristics of colloidal stability in solution. Relieving TMJ pain is a challenge since temporomandibular dis- orders involve deep tissues, making it difficult to target the trigem- inal neural system (Cairns, 2010). The nanoparticles can accumulate in inflamed tissues due to greater microvascular permeability in those sites (Bernardi et al., 2009). In the present study, an intra- articular injection of 15d-PGJ2-NC (100 pg/TMJ), at a dose 1000 times lower than that (100 ng/TMJ) used for the unloaded 15d- PGJ2 (Pena-dos-Santos et al., 2009), enhanced its temporomandibu- lar peripheral antinociceptive effect. This might be due to its spherical morphology, containing no aggregates, as well as the ability of the nanoparticles to reach or release 15d-PGJ2 in the cells. The literature shows different mechanisms for the endocytosis of nanoparticles, such as pinocytosis, formation of caveolae and clathrin, and caveolae/clathrin-independent uptake (Dobrovolskaia and McNeil, 2007). Thus, the morphological properties of 15d- PGJ2-NC might allow active compounds to enter the cells via differ- ent mechanisms, initiating different interactions with organelles and macromolecules, resulting in different pharmacological activi- ties (Nel et al., 2009; Gratton et al., 2008). It is well known that 15d-PGJ2 is a natural ligand for PPAR-γ (Ricote et al., 1998; Schoonjans et al., 1997). PPAR-γ agonists repre- sent a promising therapeutic alternative for inflammatory diseases (Chima et al., 2008; Cuzzocrea et al., 2003; Kaplan et al., 2005; Napimoga et al., 2008a, 2008b; Pena-dos-Santos et al., 2009; Shan et al., 2004) and, in particular, they are extremely neuroprotective (Collino et al., 2006; Hyong et al., 2008; McTigue et al., 2007; Park et al., 2007; Pereira et al., 2006; Sundararajan et al., 2005; Tureyen et al., 2007; Zhao et al., 2005, 2006). PPAR-γ was observed to be more expressive in ischemic neurons in rats with transient cerebral ischemia, suggesting that neuronal injury might alter PPAR-γ signal- ing (Victor et al., 2006). Pharmacological activation of PPAR-γ in the brain and spinal cord rapidly inhibits the spinal transmission of nox- ious inflammatory signals and local edema. These results suggest that PPAR-γ plays an important role in pain modulation in the central ner- vous system (Morgenweck et al., 2010) as well as in peripheral tis- sues and in peripheral endings of somatic afferents (Napimoga et al., 2008a; Pena-dos-Santos et al., 2009). With the co-participation of κ/δ opioid receptors mediated by the activation of the intracellular L-Arginine-NO/cGMP/K(+)ATP, 15d-PGJ2 was found to activate PPAR-γ in the TMJ, inducing a pe- ripheral antinociceptive effect (Pena-dos-Santos et al., 2009). TMJ inflammatory conditions result in the release of several pro- inflammatory cytokines, especially tumor necrosis factor-α (TNF- α) and interleukins (Kopp, 2001), both of which contribute to joint remodeling and cartilage degradation (Vernal et al., 2008). These cytokines induce the release of a number of pro-nociceptive compounds, such as potassium chloride, leukotriene B4, prostaglan- din E2 (PGE2), bradykinin, serotonin, histamine, glutamate and adenosine triphosphate (ATP), all of which have been shown to ex- cite and induce spontaneous discharge in the TMJ (Flake and Gold, 2005; Kopp, 2001; Oliveira et al., 2005; Rodrigues et al., 2006). In- terestingly, in the present study, low doses of 15d-PGJ2-NC, but not unloaded 15d-PGJ2, were found to inhibit the release of the pro- inflammatory cytokine IL-1β. Since IL-1β is one of the major hypernociceptive-mediator (Verri et al., 2006), we may speculate that this reduced levels of IL-1β might enhance the antinociceptive activity of 15d-PGJ2-NC. Conclusion Nanoencapsulation improves drug efficacy and drug bioavailabil- ity by providing a more sustained drug release to the inflamed site resulting in facilitating a 15d-PGJ2 target trigeminal pathway that in turn enhances the peripheral antinociceptive effect of loading 15d- PGJ2. Taken together with the biodegradable, biocompatible, and low toxic properties of the nanoparticle, these results suggest a strong potential use of this compound as novel pharmacological agents for antinociceptive and anti-inflammatory therapy in clinical practice. Conflict of interest statement The manuscript in its submitted form has been read and approved by all authors before submission, and none of them has any potential financial conflict of interests re- lated to this manuscript. Acknowledgments This work was supported by the Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP, Brazil)—Grant #2010/15014-9 and Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq, Brazil)—Grant #303080/2010-8. The manuscript in its submit- ted form has been read and approved by all authors before submis- sion, and none of them has any potential financial conflict of interests related to this manuscript. References Adair JH, Parette MP, Altinoğlu EI, Kester M. Nanoparticulate alternatives for drug de- livery. ACS Nano 2010;4:4967–70. Alves C, de Melo N, Fraceto L, de Araújo D, Napimoga M. Effects of 15d-PGJ -loaded poly(D,L-lactide-co-glycolide) nanocapsules on inflammation. Br J Pharmacol 2011;162:623–32. Bernardi A, Zilberstein AC, Jäger E, Campos MM, Morrone FB, Calixto JB, et al. Effects of indomethacin-loaded nanocapsules in experimental models of inflammation in rats. Br J Pharmacol 2009;158:1104–11. Cairns BE. Pathophysiology of TMD pain—basic mechanisms and their implications for pharmacotherapy. J Oral Rehabil 2010;37:391–410. Chima RS, Hake PW, Piraino G, Mangeshkar P, Denenberg A, Zingarelli B. Ciglitazone ameliorates lung inflammation by modulating the inhibitor kappaB protein kinase/nuclear factor-kappaB pathway after hemorrhagic shock. Crit Care Med 2008;36:2849–57. Collino M, Aragno M, Mastrocola R, Gallicchio M, Rosa AC, Dianzani C, et al. Modulation of the oxidative stress and inflammatory response by PPAR-gamma agonists in the hippocampus of rats exposed to cerebral ischemia/reperfusion. Eur J Pharmacol 2006;530:70–80. Couvreur P, Vauthier C. Nanotechnology: intelligent design to treat complex disease. Pharm Res 2006;23:1417–50. Couvreur P, Barratt G, Fattal E, Legrand P, Vauthier C. Nanocapsule technology: a re- view. Crit Rev Ther Drug Carrier Syst 2002;19:99-134. Cuzzocrea S, Ianaro A, Wayman NS, Mazzon E, Pisano B, Dugo L, et al. The cyclopentenone prostaglandin 15-deoxy-delta (12,14)-PGJ2 attenuates the devel- opment of colon injury caused by dinitrobenzene sulphonic acid in the rat. Br J Pharmacol 2003;138:678–88. Dobrovolskaia MA, McNeil SE. Immunological properties of engineered nanomaterials. Nat Nanotechnol 2007;2:469–78. Elron-Gross I, Glucksam Y, Biton IE, Margalit R. A novel Diclofenac-carrier for local treatment of osteoarthritis applying live-animal MRI. J Control Release 2009;135: 65–70. Escher P, Wahli W. Peroxisome proliferator-activated receptors: insight into multiple cellular functions. Mutat Res 2000;448:121–38. Fessi H, Puiseiux F, Devissaguet JP, Ammoury N, Benita S. Nanocapsule formation by in- terfacial polymer deposition following solvent displacement. Int J Pharm 1989;55: R1–4. Flake NM, Gold MS. Inflammation alters sodium currents and excitability of temporo- mandibular joint afferents. Neurosci Lett 2005;384:294–9. 949J.T. Clemente-Napimoga et al. / Life Sciences 90 (2012) 944–949 Gratton SE, Ropp PA, Pohlhaus PD, Luft JC, Madden VJ, Napier ME, et al. The effect of particle design on cellular internalization pathways. Proc Natl Acad Sci U S A 2008;105:11613–8. Grillo R, de Melo NFS, de Araújo DR, de Paula E, Rosa AH, Fraceto LF. Polymeric alginate nanoparticles containing the local anesthetic bupivacaine. J Drug Target 2010;18: 688–99. Huynh NT, Passirani C, Saulnier P, Benoit JP. Lipid nanocapsules: a new platform for nanomedicine. Int J Phytoremediation 2009;379:201–9. Hyong A, Jadhav V, Lee S, Tong W, Rowe J, Zhang JH, et al. Rosiglitazone, a PPAR gamma agonist, attenuates inflammation after surgical brain injury in rodents. Brain Res 2008;1215:218–24. Ishihara T, Takahashi M, Higaki M, Mizushima Y, Mizushima T. Preparation and charac- terization of a nanoparticulate formulation composed of PEG-PLA and PLA as anti- inflammatory agents. Int J Pharm 2010;385:170–5. Kaplan JM, Cook JA, Hake PW, O'Connor M, Burroughs TJ, Zingarelli B. 15-Deoxy- delta(12,14)-prostaglandin J(2) (15D-PGJ(2)), a peroxisome proliferator activated receptor gamma ligand, reduces tissue leukosequestration and mortality in endo- toxic shock. Shock 2005;24:59–65. Kopp S. Neuroendocrine, immune, and local responses related to temporomandibular disorders. J Orofac Pain 2001;15:9-28. Lehrke M, Lazar MA. The many faces of PPARgamma. Cell 2005;123:993–9. McTigue DM, Tripathi R, Wei P, Lash AT. The PPAR gamma agonist pioglitazone im- proves anatomical and locomotor recovery after rodent spinal cord injury. Exp Neurol 2007;205:396–406. Morgenweck J, Abdel-Aleem OS, McNamara KC, Donahue RR, Badr MZ, Taylor BK. Activation of peroxisome proliferator-activated receptor gamma in brain in- hibits inflammatory pain, dorsal horn expression of Fos, and local edema. Neuropharmacology 2010;58:337–45. Mountziaris PM, Sing DC, Mikos AG, Kramer PR. Intra-articular microparticles for drug delivery to the TMJ. J Dent Res 2010;89:1039–44. Napimoga MH, Souza GR, Cunha TM, Ferrari LF, Clemente-Napimoga JT, Parada CA, et al. 15d-prostaglandin J2 inhibits inflammatory hypernociception: involvement of peripheral opioid receptor. J Pharmacol Exp Ther 2008a;324:313–21. Napimoga MH, Vieira SM, Dal-Secco D, Freitas A, Souto FO, Mestriner FL, et al. Peroxisome proliferator-activated receptor-gamma ligand, 15-deoxy-Delta12,14- prostaglandin J2, reduces neutrophil migration via a nitric oxide pathway. J Immunol 2008b;180:609–17. Nel AE, Madler L, Velegol D, Xia T, Hoek EMV, Somasundaran P, et al. Understanding biophysicochemical interactions at the nano-bio interface. Nat Mater 2009;8: 543–57. Oliveira MC, Parada CA, Veiga MC, Rodrigues LR, Barros SP, Tambeli CH. Evidence for the involvement of endogenous ATP and P2X receptors in TMJ pain. Eur J Pain 2005;9:87–93. Park SW, Yi JH, Miranpuri G, Satriotomo I, Bowen K, Resnick DK, et al. Thiazolidinedione class of peroxisome proliferator-activated receptor gamma ago- nists prevents neuronal damage, motor dysfunction, myelin loss, neuropathic pain, and inflammation after spinal cord injury in adult rats. J Pharmacol Exp Ther 2007;320:1002–12. Pena-dos-Santos DR, Severino FP, Pereira SA, Rodrigues DB, Cunha FQ, Vieira SM, et al. Activation of peripheral kappa/delta opioid receptors mediates 15- deoxy-(Delta12,14)-prostaglandin J2 induced-antinociception in rat temporoman- dibular joint. Neuroscience 2009;163:1211–9. Pereira MP, Hurtado O, Cardenas A, Bosca L, Castillo J, Davalos A, et al. Rosiglitazone and 15-deoxy-delta12, 14-prostaglandin J2 cause potent neuroprotection after experi- mental stroke through noncompletely overlapping mechanisms. J Cereb Blood Flow Metab 2006;26:218–29. Ricote M, Li AC, Willson TM, Kelly CJ, Glass CK. The peroxisome proliferator-activated receptor-γ is a negative regulator of macrophage activation. Nature 1998;391: 79–82. Rodrigues LR, Oliveira MC, Pelegrini-da-Silva A, de Arruda Veiga MC, Parada CA, Tambeli CH. Peripheral sympathetic component of the temporomandibular joint inflammatory pain in rats. J Pain 2006;7:929–36. Roveroni RC, Parada CA, Cecilia M, Veiga FA, Tambeli CH. Development of a behavioral model of TMJ pain in rats: the TMJ formalin test. Pain 2001;94:185–91. Schoonjans K, Martin G, Staels B, Auwerx J. Peroxisome proliferator activated receptors, orphans with ligands and functions. Curr Opin Lipidol 1997;8:159–66. Shan ZZ, Masuko-Hongo K, Dai SM, Nakamura H, Kato T, Nishioka K. A potential role of 15-deoxy-delta(12,14)- prostaglandin J2 for induction of human articular chon- drocyte apoptosis in arthritis. J Biol Chem 2004;279:37939–50. Shive MS, Anderson JM. Biodegradation and biocompatibility of PLA and PLGA micro- spheres. Adv Drug Deliv Rev 1997;28:5-24. Sundararajan S, Gamboa JL, Victor NA, Wanderi EW, Lust WD, Landreth GE. Peroxisome proliferator-activated receptor-gamma ligands reduce inflammation and infarction size in transient focal ischemia. Neuroscience 2005;130:685–96. Tureyen K, Kapadia R, Bowen K, Satriotomo I, Liang J, Feinstein D, et al. Peroxisome proliferators-activated receptor-γ agonists induce neuroprotection following tran- sient focal ischemia in normotensive, normoglycemic as well as hypertensive and type-2 diabetic rodents. J Neurochem 2007;101:41–6. Vernal R, Velasquez E, Gamonal J, Garcia-Sanz JA, Silva A, Sanz M. Expression of proinflammatory cytokines in osteoarthritis of the temporomandibular joint. Arch Oral Biol 2008;53:910–5. Verri Jr WA, Cunha TM, Parada CA, Poole S, Cunha FQ, Ferreira SH. Hypernociceptive role of cytokines and chemokines: targets for analgesic drug development? Pharmacol Ther 2006;112:116–38. Victor NA, Wanderi EW, Gamboa J, Zhao X, Aronowski J, Deininger K, et al. Altered PPARgamma expression and activation after transient focal ischemia in rats. Eur J Neurosci 2006;24:1653–63. Zhang Y, Zhang W, Löbler M, Schmitz KP, Saulnier P, Perrier T, et al. Inner ear biocom- patibility of lipid nanocapsules after round window membrane application. Int J Pharm 2011;404:211–9. Zhao Y, Patzer A, Gohlke P, Herdegen T, Culman J. The intracerebral application of the PPARgamma-ligand pioglitazone confers neuroprotection against focal ischaemia in the rat brain. Eur J Neurosci 2005;22:278–82. Zhao Y, Patzer A, Herdegen T, Gohlke P, Culman J. Activation of cerebral peroxisome proliferator-activated receptors gamma promotes neuroprotection by attenuation of neuronal cyclooxygenase-2 overexpression after focal cerebral ischemia in rats. FASEB J 2006;20:1162–75. Zille H, Paquet J, Henrionnet C, Scala-Bertola J, Leonard M, Six JL, et al. Evaluation of intra-articular delivery of yaluronic acid functionalized biopolymeric nanoparticles in healthy rat knees. Biomed Mater Eng 2010;20:235–42. 15d-PGJ2-loaded in nanocapsules enhance the antinociceptive properties into rat temporomandibular hypernociception Introduction Material and methods Preparation of the PLGA nanocapsules with 15d-PGJ2 Transmission electron microscopy (TEM) Atomic force microscopy (AFM) Animals Testing procedure for TMJ pain Experimental protocols Effect of 15d-PGJ2-NC on formalin-induced TMJ nociception Effect of 15d-PGJ2-NC on formalin-induced IL-1β cytokine release Statistical analysis Results Characterization of the 15d-PGJ2-NC Effect of 15d-PGJ2-NC on formalin-induced TMJ nociception Effect of low doses of 15d-PGJ2-NC on IL-1β releases Discussion Conclusion Conflict of interest statement Acknowledgments References