Life Sciences xxx (2014) xxx–xxx

LFS-14139; No of Pages 8

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Meloxicam-loaded nanocapsules have antinociceptive andantiedematogenic effects in acute models of nociception

Benonio T. Villalba a, Francine R. Ianiski a, Ethel A. Wilhelm b,⁎, Renata S. Fernandes a,Marta P. Alves c, Cristiane Luchese a,b,⁎⁎a Centro de Ciências Tecnológicas, Centro Universitário Franciscano, CEP 97010-032 Santa Maria, RS, Brazilb Centro de Ciências Químicas, Farmacêuticas e de Alimentos, Universidade Federal de Pelotas, Pelotas CEP 96010-900, RS, Brazilc Centro de Ciências da Saúde, Universidade Federal de Santa Maria, CEP 97105-900 Santa Maria, RS, Brazil

⁎ Correspondence to: E. Wilhelm, Programa de PósBioprospecção, Centro de Ciências Químicas, FarmUniversidade Federal de Pelotas, Campus Capão do LeãBrazil. Tel.: +55 53 32757356.⁎⁎ Correspondence to: C. Luchese, Programa de PósBioprospecção, Centro de Ciências Químicas, FarmUniversidade Federal de Pelotas, Campus Capão do LeãBrazil. Tel.: +55 53 3275 7233.

E-mail addresses: ethelwilhelm@yahoo.com.br (E.A. Wcristiane_luchese@yahoo.com.br (C. Luchese).

http://dx.doi.org/10.1016/j.lfs.2014.09.0020024-3205/© 2014 Elsevier Inc. All rights reserved.

Please cite this article as: Villalba BT, et al, Menociception, Life Sci (2014), http://dx.doi.org

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 29 May 2014Accepted 5 September 2014Available online xxxx

Keywords:MeloxicamNanoparticlesAntinociceptiveAntiedematogenicPain

Aims: The development of new treatments for inflammation and pain continues to be of high interest, since long-acting effect is critical for patients. The present study investigated whether the polymeric nanocapsules, a drugdelivery system, have pharmacological effect on acute nociceptive and inflammatory models in mice.Main methods: Swiss mice (20–25 g) were previously pre-treated with meloxicam-loaded nanocapsules (M-NC)or free meloxicam (M-F) or suspension without drug (B-NC), at a dose of 5 mg/kg (per oral) at different times(0.5–120 h). Antinociceptive and antiedematogenic effects were evaluated by chemical (acetic acid-induced ab-dominal writhing, nociception and paw edema induced by formalin and glutamate, croton oil-induced earedema) and thermal (tail immersion and hot-plate) tests.Key findings:M-NC reduced the licking time- and paw edema-induced by glutamate and formalin, whileM-F didnot have an effect. In the acetic acid-induced abdominal writhing and croton oil-induced ear edema, analysis of

time-course revealed that M-NC showed a response more prolonged than M-F. In the hot-plate test, a thermaltest, the time-course analysis indicated a similar increase in the latency response to thermal stimuli of M-NCand M-F, while in the tail-immersion test M-F had an effect at 0.5 h and M-NC at 24 h.Significance: Polymeric nanoparticles had antinociceptive, anti-inflammatory and antiedematogenic effects in theformalin and glutamate tests, and prolonged the effect in acetic acid and croton oil tests, but not in thermal tests,supporting the idea that the inflammatory process in tissues facilitates the vectoring of polymeric nanoparticles.

© 2014 Elsevier Inc. All rights reserved.

Introduction

Pain is outlined by the International Association for the Study of Pain(IASP) as an unpleasant sensory or emotional experience associatedwith tissue damage, that can be related or not to inflammation (IASP,1994). In this context, life quality is negatively affected by pain, whichinterferes in multiple aspects of health and wellbeing, such as work ca-pacity, relationships and cognitive abilities (Rustoen et al., 2008). Sever-al diseases have pain as major symptom and it begins through noxiousthermal, mechanical, or chemical stimulus that excites the peripheral

-graduação em Bioquímica eacêuticas e de Alimentos,o, Pelotas CEP 96010-900, RS,

-graduação em Bioquímica eacêuticas e de Alimentos,o, Pelotas CEP 96010-900, RS,

ilhelm),

loxicam-loaded nanocapsules/10.1016/j.lfs.2014.09.002

terminals of neurons, called nociceptors (Ferreira et al., 2004). It hasbeen reported that inflammation and pain are related processes thathave common effectors and mediators. In inflammation there is the re-cruitment of leukocytes and plasma proteins to a site of affected tissueas an adaptive response (Medzhitov, 2008). Moreover, classical signsas heat, redness, swelling, pain and consequent function loss of the af-fected local/organ are present in exacerbated inflammation processes(Lawrence et al., 2002).

Although there is an arsenal of effective analgesics and anti-inflammatory, the clinical use is problematic due to the concern regard-ing safety, side-effects and bioavailability (Jage, 2005). In this context,new alternatives for the pain pre-treatment have been studied world-wide. Nowadays, nanotechnology is quickly progressing and this areahas become a major research field in this century (Arora et al., 2012).During the last decade, nanotechnology has demonstrated priceless ad-vantages in different areas such as pharmacy, medicine, computer sci-ence and engineering. In fact, the development of new nanocarriersystems to target the drug delivery is considered the possible future ofpharmaceutical therapy (Couvreur and Vauthier, 2006). Over the lastfew years, biodegradable and biocompatible nanoparticles have been

have antinociceptive and antiedematogenic effects in acutemodels of

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reported as the most promising for delivery of pharmacologically activedrugs (Adair et al., 2010; Zhang et al., 2011).

Nanocapsules are polymeric nanoparticles that promote drug con-trolled delivery in specific body sites (Rieux et al., 2006). Several advan-tages are associated with the incorporation of drugs into polymericnanocapsules, such as the reduction of side effects (Faraji and Wipf,2009; Guterres et al., 2001; Schaffazick et al., 2003) and the increaseof bioavailability (Couvreur and Vauthier, 2006).

\In this context, many efforts have been made to study the improve-ment of the pharmacological effects of these systems (Badran et al.,2014; Bender et al., 2012; Cheng et al., 2008). Accordingly, meloxicam-loaded polymeric nanocapsules were effective in protecting learningand memory impairment, neuronal loss and oxidative stress in a mousemodel of Alzheimer's disease induced by β-amiloid peptide (Ianiskiet al., 2012). Moreover, Khachane et al. (2011) demonstrated that thepolymeric nanoparticles containing meloxicam resulted in lesserulcerogenicity than free suspension.

Meloxicam is a non-steroidal anti-inflammatory drug (NSAID) thatpossesses analgesic action and reduces pain and inflammation (Oginoet al., 1997; Pairet et al., 1998). In order to produce its effects,meloxicaminhibits the cyclooxygenase-2 (COX-2) activity (Pairet et al., 1998),blocking the prostaglandin synthesis and arachidonic acid metabolism(Gupta et al., 2002; Lopez-Garcia and Laird, 1998; Vane et al., 1998).

In view of our interest in the pharmacology of nanocarrier systems,the present study aimed to investigate whether polymeric nanocapsules,a drug delivery system, have pharmacological effect on acute nociceptivemodels in mice, by analysis of time-course of antinociceptive, anti-inflammatory and antiedematogenic responses.

Materials and methods

Materials

Meloxicam and Carbopol® 940 were obtained from Henrifarma(São Paulo, Brazil). Sorbitanmonostearate, polysorbate 80 andpoly(ε-caprolactone) MW = 80,000 were purchased from Sigma-Aldrich (Strasbourg, France). Triglycerides of capric/caprylic acidand triethanolamine were obtained from Via Farma (São Paulo,Brazil), while methylparaben, propylparaben, sorbitol and propyl-ene glycol from Alpha Quimica (Porto Alegre, Brazil).

All drugs used in induction of nociception were dissolved in saline(0.9%). All other chemicals were acquired from standard commercialsuppliers.

Nanocapsule preparation

Suspensions of meloxicam-loaded nanocapsules (M-NC) were pre-pared by the method of interfacial deposition of preformed polymer(adapted from Fessi et al., 1989) at a concentration of 0.3 mg/ml(Ianiski et al., 2012). After preparation of suspensions, polymeric nano-particles were incorporated into semisolid formulations (Ianiski et al.,2012). The particle diameter was around 283 and 285 nm for M-NCand blank nanocapsules (B-NC), respectively. The zeta potential was ap-proximately −14.53 mV for M-NC and −16.21 mV for B-NC. The con-tent of meloxicam nanocoated was 99.97%.

Animals

Male adult Swiss mice (20–25 g) were obtained from local breedingcolony of Federal University of Santa Maria. The mice were maintainedat 22–25 °C with free access to water and food, under a 12:12 h light/dark cycle (with lights on at 7:00 a.m.). The animals were acclimatizedto the behavior room for at least 1 h before test and theywere used onlyonce in each test.

The experimentwas conducted according to the institutional and in-ternational guidelines for the care and use of animals. The Local

Please cite this article as: Villalba BT, et al, Meloxicam-loaded nanocapsulesnociception, Life Sci (2014), http://dx.doi.org/10.1016/j.lfs.2014.09.002

Committee for Care and Use of Laboratory Animals of the FranciscanUniversity Center (Santa Maria, Brazil) approved the research (codenumber: 001/2011). The animals were used according to ethical guide-lines for investigations of experimental pain in conscious animals(Zimmermann, 1983). All efforts were taken to minimize the numberof animals used and their suffering. The number of animals and intensi-ties of noxius stimuli used were necessarily at theminimum to demon-strate the consistent effects of the drug pre-treatment. At the end of theexperimental procedure the mice were killed by cervical dislocation.

Themicewere divided into three groups (7–8 animals per group) foreach pre-treatment time, as follows: group I received B-NC and servedas control (17 ml/kg; oral route), groups II and III received M-NC andfree meloxicam (M-F), respectively, at a dose of 5 mg/kg by the oralroute (Ianiski et al., 2012). Different groups of animals were used foreach nociceptive test. Each time of pre-treatment had a control groupthat received B-NC. However, all results of group I were condensed(n= 7–8) because they did not have significant difference between an-imals at different times of pre-treatment.

Acetic acid-induced abdominal writhing

Intraperitoneal (i.p.) injection of acetic acid (1.6%) was used to in-duce the abdominal writhing (Nogueira et al., 2003). The mice werepre-treated (0.5–72 h)with B-NC,M-NC orM-F before acetic acid injec-tion. After acetic acid injection, the abdominal writhing (full extensionof both hind paws) were counted cumulatively over a period of 20 min.

Formalin-induced nociception and paw edema

The formalin testwas carried out as described byHunskaar and Hole(1987). M-NC, M-F or B-NC was administered 0.5–72 h before formalininjection. An intraplantar (i.pl.) injection of formalin (2.5%/paw, 20 μl)(v/v) was administered into the dorsal right hind paw (ipsilateral) ofmice. After formalin injection, the time spent licking or biting theinjected paw was recorded during the periods of 0–5 min (earlyphase) and 15–30 min (late phase). The paw edema was measured bycomparing the difference between the weight of the formalin-injectedpaw and the weight of the contralateral paw (non-treated paw). Forthis purpose, the animals were killed by cervical dislocation 30 minafter formalin injection and both paws were cut at the ankle joint andweighed on an analytical balance.

Glutamate-induced nociception and paw edema

The procedurewas carried out according to the procedure describedpreviously by Beirith et al.(2002). The mice were pre-treated with M-NC,M-F or B-NC 0.5–72 h before i.pl. injection of glutamate (20 μmol/paw,20 μl) on the right hind paw. The amount of time spent licking or bitingthe injected paw was recorded during 15 min following glutamate injec-tion. The difference between the weight of the glutamate-injected pawand the weight of the saline 0.9%-injected paw (contralateral paw) wasused as a measure of paw edema. For this purpose, the mice were killedby cervical dislocation 15 min after treatment with glutamate and bothpaws were cut at the ankle joint and weighed on an analytical balance.

Ear edema induced by croton oil

The antiedematogenic effect of M-NC or M-F was assessed by in-duction of inflammation by topical application of 2.5% croton oil inacetone (10 μl/ear) in the right ear of each mouse, according toRomay et al. (1998). The mice were pre-treated with M-NC, M-F orB-NC at 0.5–48 h before test. Four hours after phlogistic agent, theanimals were killed by cervical dislocation. A segment of 8 mm ofeach ear was obtained to weighing on an analytical balance. Theswelling was determined by comparing the weight difference be-tween the injected ear and untreated ear.

have antinociceptive and antiedematogenic effects in acutemodels of

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Tail-immersion-induced nociception

The lower 3.5 cm portions of the tails were marked and the animalswere then pre-treated with M-NC, M-F or B-NC at 0.5–48 h before test.Immersion of the tail (the lower 3.5 cm) was placed into a water bathwith constant temperature (55 °C) until the tail withdrawal was ob-served (Janssen et al., 1963). In order to avoid damage to the tails ofthe animals, the time standing on the water was limited to 7 s. Tail-flick latency, Δt (s), was calculated according to the formula: Δt (s) =post-drug latency − pre-drug latency (Pinardi et al., 2003).

Hot-plate test

The change in thermal withdrawal latencies was recorded beforetreatment and at 0.5–48 h after pre-treatments with M-NC, M-F or B-NC. In this test, the mice were placed in a glass cylinder on a heatedmetal plate maintained at 55 ± 1 °C (Woolfre and MacDonald, 1944).In order to avoid damage to the paws of the animals, the time standingon the platewas limited to 30 s. Nociceptive response latency (licking orshaking one of the paws or jumping) was recorded as the reaction time.The change of latency, Δt (s), was calculated according to the formula:Δt (s) = post-drug latency − pre-drug latency.

Open field test

The animals were observed at 0.5–120 h after pre-treatment with M-NC or M-F or B-NC. The open-field test was made in a box of plywood(40 cm length × 40 cm width × 30 cm height) with the floor dividedinto 9 squares (3 rows of 3). Each mouse was placed at the center of thebox and observed for 4min to record the locomotor (number of segmentscrossed with the four paws) and exploratory activities (expressed by thenumber of time rearing on the hind limbs) (Walsh and Cummins, 1976).

Statistical analysis

Data are expressed as mean ± S.E.M. Statistical analysis was per-formed using a one-way ANOVA followed by the Newman–Keuls.Values of p b 0.05 were considered statistically significant.

Results

Acetic acid-induced abdominal writhing

The antinociceptive effect of M-NC started at 0.5 h and remainedsignificant up to 48 h after administration, while M-F inhibited aceticacid-induced abdominal constriction only at 0.5 h of pre-treatment(F8,58 = 7.078, p b 0.0001) (Fig. 1).

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Fig. 1. Time-course analysis of meloxicam-loaded nanocapsules (M-NC) and free meloxicam (MM-NC or M-F before test. Results are expressed as means ± S.E.M. of 7 to 8 animals. **p b 0.01

Please cite this article as: Villalba BT, et al, Meloxicam-loaded nanocapsulesnociception, Life Sci (2014), http://dx.doi.org/10.1016/j.lfs.2014.09.002

Formalin-induced nociception and paw edema

I.p. injection of 2.5% formalin evoked a characteristic biphasic lickingresponse. The duration of licking in control groups was 87± 5 s for theearly phase (0–5 min) and 247 ± 47 s for the late phase (15–30 min)(Fig. 2A and B, respectively). Pre-treatment of up to 96 h with M-NCproduced a marked reduction of the licking time in first and secondphases of the formalin test (Fig. 2A and B, respectively). While M-Fhad no effect in licking behavior at both phases of the formalin test(F12,91 = 10.73, p b 0.0001 for first phase; F12,84 = 6.477, p b 0.0001for second phase) (Fig. 2A and B, respectively).

Pre-treatment with M-NC markedly reduced the paw edema for-mation up to 120 h. M-F had no effect against the paw edema(F12,84 = 13.76; p b 0.0001) (Fig. 2C).

Glutamate-induced nociception and paw edema

Fig. 3A shows that pre-treatment of up to 72 h with M-NC producedamarked reduction of the licking time induced by glutamate, whileM-Fhad no significant effect against the duration of licking (F10,77 = 13.27;p b 0.0001).

Pre-treatment with M-NC, but not with M-F, reduced the by gluta-mate up to 72 h (F10,77 = 9.523, p b 0.0001) (Fig. 3B).

Ear edema induced by croton oil

The antiedematogenic effect of M-NC started at 0.5 h after the ad-ministration and remained significant up to 96 h. M-F inhibited earedema at 0.5 h after administration (F14,104 = 14.42, p b 0.0001)(Fig. 4).

Tail-immersion-induced nociception

Pre-treatment with M-NC significantly increased the change of la-tency at 24 h, while M-F enhanced the change of latency at 0.5 h(F8,54 = 5.527, p b 0.0001) (Fig. 5).

Hot-plate test

In the hot-plate test, pre-treatments with M-NC and M-F signifi-cantly increased the change of latency up to 24 h (F6,49 = 8.749,p b 0.0001) (Fig. 6).

Open field test

The spontaneous locomotor and exploratory activities measured inthe open-field test did not differ significantly between the groups(F12,78 = 1.065, p = 0.4006 for crossing, and F12,78 = 0.7363, p =0.7119 for rearing) (data not shown).

**

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t time (h)0.5 42 84 27

M-F

-F) effects in acetic acid-induced writhing. The animals were pre-treated (0.5–72 h) withand ***p b 0.001 compared to the control group (blank nanocapsules (B-NC)).

have antinociceptive and antiedematogenic effects in acutemodels of

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Fig. 2. Time-course analysis of meloxicam-loaded nanocapsules (M-NC) and free meloxicam (M-F) effects on first (A) and second (B) phases and paw edema (C) in formalin injection inmice. The animalswere pre-treated (0.5–120 h)withM-NC orM-F before test. Results are expressed asmeans± S.E.M. of 7 to 8 animals. *pb 0.05, **p b 0.01 and ***pb 0.001 as comparedto the control group (blank nanocapsules (B-NC)).

4 B.T. Villalba et al. / Life Sciences xxx (2014) xxx–xxx

Discussion

The findings of the present study indicated that the oral administra-tion of M-NC, at low dose, produced significant antinociceptive, anti-inflammatory and antiedematogenic responses in different acutemodels of nociception, when compared to M-F, without modifying thelocomotor activity of mice in the open field test. In this context, poly-meric nanocapsules demonstrated superior antinociceptive effect thanM-F. In the present study, the mice were pre-treated with M-NC or M-F at a dose of 5 mg/kg, which was based on previous studies from ourresearch group that demonstrated protection ofM-NC in neuronal dam-age induced by aβ peptide (Ianiski et al., 2012).

The potential actions of M-NC and M-F were evaluated in the aceticacid, formalin, glutamate and croton oil tests, which diverge in sensibil-ity and specificity (Le Bars et al., 2001). In the acetic acid test, writhingbehavior is associated to the activation of acid-sensing ion channelsand vanilloid receptors (Holzer, 2011), and this test is described to beresponsive to antinociceptive drugs. In this test, M-NC elicited

Please cite this article as: Villalba BT, et al, Meloxicam-loaded nanocapsulesnociception, Life Sci (2014), http://dx.doi.org/10.1016/j.lfs.2014.09.002

antinociceptive action more prolonged than M-F. Although acetic acid-inducedwrithing test has a good sensitivity, this method has poor spec-ificity (Hunskaar and Hole, 1987) and therefore, to confirm the results,we choose acute nociception testsmore specific, such as formalin, gluta-mate, and croton oil tests.

In fact, M-NC produced a marked reduction of the licking time- andpaw edema-induced by glutamate and formalin. Therefore, these resultsindicate that M-NC had antiedematogenic and antinociceptive threshold,whileM-F did not have effect in these tests. Response in the formalin testis described to be very predicative, involving two distinct phases: the firstphase corresponds to acute neurogenic pain with direct activation ofnociceptors, while the second phase corresponds to inflammatory noci-ceptive response with sensitization of nociceptors by different inflamma-tion mediators released in the first phase (Tjolsen et al., 1992). It isdifficult to explain why meloxicam, a NSAID, had antinociceptive effectin the first phase of formalin test. However, similar to the results obtainedby us, other studies showed the effect in the first phase of the formalintest (Santos et al., 1998; Gonzalez et al., 2011).

have antinociceptive and antiedematogenic effects in acutemodels of

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Fig. 3. Time-course analysis of meloxicam-loaded nanocapsules (M-NC) and free meloxicam (M-F) effects on licking- (A) and paw edema- (B) induced by glutamate inmice. The animalswere pre-treated (0.5–96 h) with M-NC or M-F before the test. Results are expressed as means ± S.E.M. of 7 to 8 animals. ***p b 0.001 as compared to the control group (blanknanocapsules (B-NC)).

5B.T. Villalba et al. / Life Sciences xxx (2014) xxx–xxx

Moreover, Malmberg and Yaksh (1995) demonstrated that i.pl. injec-tion of formalin produced a significant increase in spinal levels of gluta-mate, aspartate, taurine, glycine, citrulline, serine, asparagine, glutamineand PGE2 in thefirst phase. During the secondphase a significant increasein the release of citrulline, PGE2, glutamate and aspartate was observed.Therefore, the formalin test involves differentmediators, such as excitato-ry amino acids, PGE2, nitric oxide and tachykinins (Malmberg and Yaksh,1995; Vaz et al., 1996). Thus,meloxicam could elicit antinociceptive effectin the first phase of formalin test by the mechanisms described above.

Additionally, the antinociceptive effect of pre-treatments in theglutamate-induced licking behavior indicates, at least in part, an inter-action of meloxicam with the glutamatergic system. In addition, M-NCcaused antiedematogenic response more prolonged than M-F in reduc-ing the ear edema formation caused by croton oil in mice. In fact, the

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Fig. 4. Time-course analysis of meloxicam-loaded nanocapsules (M-NC) and free meloxicam (Mwith M-NC or M-F before test. Results are expressed as means ± S.E.M. of 7 to 8 animals. ***p

Please cite this article as: Villalba BT, et al, Meloxicam-loaded nanocapsulesnociception, Life Sci (2014), http://dx.doi.org/10.1016/j.lfs.2014.09.002

reduction of paw and ear edema after M-NC pre-treatment indicated aperipheral action, probably related to the arachidonic acid cascade (LeBars et al., 2001) and an antiedematogenic action due to an interactionwith the inflammatory system. Thus, polymeric nanocapsules modifiedantinociceptive and antiedematogenic effects of meloxicam in acutemodels of nociception.

Several studies have demonstrated antinociceptive, antiedematogenicand anti-inflammatory effects ofmeloxicamor otherNSAIDs onmodels ofnociception and inflammation (Choi et al., 2001; Cruz et al., 2008; DeMelo et al., 2005; Pinardi et al., 2003; Santos et al., 1998). Indeed, Santoset al. (1998) reported that treatment ofmicewithmeloxicam caused a re-duction of acetic acid-induced writhing and an inhibition of the lickingtime- and paw edema-induced by formalin. However, it is difficult tocompare the study of Santos et al. (1998) with our study. Santos et al.

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tment time (h)

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-F) effects on ear edema-induced by croton oil. The animals were pre-treated (0.5–120 h)b 0.001 as compared to the control group (blank nanocapsules (B-NC)).

have antinociceptive and antiedematogenic effects in acutemodels of

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Fig. 5. Time-course analysis ofmeloxicam-loaded nanocapsules (M-NC) and freemeloxicam (M-F) effects in the hot-plate test. The animalswere pre-treated (0.5–48h)withM-NC orM-Fbefore test. Results are expressed as means ± S.E.M. of 7 to 8 animals.*p b 0.05 and ***p b 0.001 as compared to the control group (blank nanocapsules (B-NC)).

6 B.T. Villalba et al. / Life Sciences xxx (2014) xxx–xxx

(1998) demonstrated the effect of meloxicam administered i.p. 30 minprior to the tests, while in this present study M-F was administered24 h prior to the tests by the oral route.

Our results suggest that polymeric nanocapsules promote asustained release of meloxicam in inflamed tissues, allowing drug tobe delivered to the cells in different magnitudes of time, providing asustained blockage of nociception and inflammation. However, morestudies are necessary to support this assumption, such as the absorp-tion, distribution, metabolism or excretion of M-NC. Indeed, studiesshowed an increase in retention and sustained release of drugs to thetissues when drugs were associated with polymeric nanoparticles(Rao and Geckeler, 2011; Sun et al., 2010; Tsai et al., 2011). Further-more, sustained-release forms of drugs have advantages when com-pared to immediate-release forms of the same drug, such as a lessfrequent dosage (Pietkiewicz et al., 2010). Normally, release of thedrug of pharmacological nanocarriers, as polymeric nanocapsules, is de-scribed in two phases. The first stage is the initial rupture of thenanocapsule, followed by a sustained release with the drug pharmaco-logical effect lasting longer (Lamprecht, 2002; Mora-Huertas et al.,2011; Musumeci et al., 2006).

In the present study M-NC was prepared with poly-ε-caprolactone,which is a biocompatible and biodegradable polymer (Filipovic et al.,2013; Wei et al., 2009). Furthermore, nanocapsules used in this studycontain polysorbate 80 that is an emulsifier, which can be used to retardopsonization (Bender et al., 2012). In fact, studies have demonstratedthat the carrier size, polymer type, emulsifier type and surface charac-teristics could induce steric stabilization of nanoparticles, increasing

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Fig. 6. Time-course analysis of meloxicam-loaded nanocapsules (M-NC) and freemeloxicam (Mor M-F before test. Results are expressed as means ± S.E.M. of 7 to 8 animals. ***p b 0.001 as c

Please cite this article as: Villalba BT, et al, Meloxicam-loaded nanocapsulesnociception, Life Sci (2014), http://dx.doi.org/10.1016/j.lfs.2014.09.002

blood circulation (Bernardi et al., 2009a,b; Frozza et al., 2010; Jageret al., 2007; Merisko-Liversidge and Liversidge, 2008). Therefore, wecan propose that nanocapsules promote a sustained release by slowlydeliver the meloxicam, prolonging the time and therapeutic activity ofdrug. In accordance with the results demonstrated here, other studieshave shown a superior efficacy of polymeric nanocapsules when com-pared to free drug in different models (Bernardi et al., 2009a,b, 2010;Ianiski et al., 2012; Khachane et al., 2011).

Furthermore, when drugs were given orally, the bioavailability maybe reduced due to poor absorption in the gastro-intestinal tract and theinstability in gastric and intestinal fluids (Kofi-Tsekpo, 1994). In thisway, antinociceptive and antiedematogenic actions of M-NC might bebased on a sustained release via the oral route, where consequentlymeloxicam is not freely available for absorption from the intestinallumen and elimination. In addition, it is reported that nanocarriers canbe translocated across the gut wall (Florence et al., 1995), retainingthe controlled drug release properties of nanocapsules, which may in-crease the bioavailability of the drug in the plasma (Modi et al., 2009).

Moreover, nanoencapsulated systemshave other advantages such asencapsulation efficiency due to optimized of drug solubility in the core,protection against degradation factors like pH and light and the reduc-tion of tissue irritation due to the polymeric shell and the simplicity ofthe polymerization process (Anton et al., 2008; Pinto et al., 2006).Thus, all these factors were important for the best effect of M-NC to-wards M-F. In addition, Khachane et al. (2011) demonstrated that thepolymeric nanoparticles containing meloxicam resulted in lesserulcerogenicity than free suspension.

****

B-NCM-NCM-F

eatment time (h)

0.5 24 4848

-F) effects in the tail-immersion test. The animals were pre-treated (0.5–48 h)withM-NCompared to the control group (blank nanocapsules (B-NC)).

have antinociceptive and antiedematogenic effects in acutemodels of

7B.T. Villalba et al. / Life Sciences xxx (2014) xxx–xxx

Furthermore, thermal tests (tail immersion and hot-plate) were car-ried out in this study with the aim of further demonstrating the efficacyof M-NC in non-inflammatory conditions. The results obtained with M-NC in thermal tests further support our hypothesis, since polymericnanocapsules did not prolong the antinociceptive action of meloxicamin the hot-plate test, andM-NC andM-F showed similar antinociceptiveeffect in time-course analysis. In the tail immersion test, M-NC was ef-fective only at 24 h of pre-treatment, while M-F at 0.5 h. These resultsindicate that M-NC and M-F blocked thermal nociception at differentlevels of central nervous system, since hot-plate mainly reflectssupraspinal response and tail-immersion reflects spinal response(Langerman et al., 1995). In fact, Pinardi et al. (2003) showed thatmeloxicamhad a similar antinociceptive activity tomorphine and cloni-dine in a somatic acute pain model (tail-flick), acting by spinal andsupraspinal muscarinic cholinergic receptors.

Lastly, evidence has been found to suggest that control of the motoractivity can interfere in the nociceptive response (Le Bars et al., 2001),therefore, the spontaneous locomotor and exploratory behavior wasassessed in the open-field test. No significant difference was found be-tween mice pre-treated with M-NC or M-F and control animals,discarding a possible interference of drug on themotor activity and con-sequently a false-positive response in the nociceptive models.

Conclusion

In conclusion, in the acute nocipeptive chemical tests, time-courseanalysis revealed that polymeric nanoparticles presented antinociceptiveand antiedematogenic responses in the formalin and glutamate tests, andprolonged the effect in the acetic acid and croton oil tests, but not in ther-mal tests, supporting the idea that the inflammatory process in tissues fa-cilitates the vectoring of polymeric nanoparticles. Thus, M-NC may be ofpotential interest in developing new drugs for pre-treatment of acutepain. Further studies are needed to confirm this hypothesis and to inves-tigate the effect of M-NC in chronic pain models.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgments

The financial support by FAPERGS (#1924-2551/13-1), is gratefullyacknowledged.

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