Curcumin-loaded into PLGA nanoparticles
Priscilla P. Luz, Lizandra G. Magalhães, Ana Carolina Pereira, Wilson R. Cunha, Vanderlei Rodrigues & Marcio L. Andrade e Silva Parasitology Research Founded as Zeitschrift für Parasitenkunde ISSN 0932-0113 Volume 110 Number 2 Parasitol Res (2012) 110:593-598 DOI 10.1007/s00436-011-2527-9
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Author's personal copy Parasitol Res (2012) 110:593–598 DOI 10.1007/s00436-011-2527-9
ORIGINAL PAPER
Curcumin-loaded into PLGA nanoparticles Preparation and in vitro schistosomicidal activity Priscilla P. Luz & Lizandra G. Magalhães & Ana Carolina Pereira & Wilson R. Cunha & Vanderlei Rodrigues & Marcio L. Andrade e Silva
Received: 8 June 2011 / Accepted: 22 June 2011 / Published online: 8 July 2011 # Springer-Verlag 2011
Abstract The incorporation of the curcumin into poly(lacticco-glycolic)acid (PLGA) nanospheres by the nanoprecipitation technique, the characterization of the nanoparticles and the schistosomicidal activity of the curcumin-loaded into PLGA nanospheres were reported. The incorporation process occurred with high efficiency and the images of fieldemission scanning electron microscopy (FESEM) revealed the production of spherically shaped particles. According to the dynamic light scattering measurements, the particles are nanometric and monodisperse. The curcumin-loaded PLGA nanoparticles (50 and 100 μM) caused the death of all worms and a separation between 50% and 100% of Schistosoma mansoni couples at concentrations from 30 μM. Moreover, the curcumin-loaded PLGA nanoparticles also decreased the motor activity and caused partial alterations in the tegument of adult worms. This study marks the first time that schistosomicidal activity has been reported for curcumin-loaded PLGA nanoparticles.
Introduction Human schistosomiasis is a chronic debilitating disease in tropical and subtropical countries caused by parasites of the P. P. Luz : L. G. Magalhães : A. C. Pereira : W. R. Cunha : M. L. Andrade e Silva (*) Grupo de Pesquisas em Produtos Naturais, Núcleo de Pesquisas em Ciências Exatas e Tecnológicas da Universidade de Franca, Franca 14404-600 SP, Brazil e-mail:
[email protected] V. Rodrigues Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto 14049-900 SP, Brazil
genus Schistosoma (Gryseels et al. 2006). It is estimated that 200 million people in tropical and subtropical regions are infected by Schistosoma worms, causing more than 280,000 deaths per year and putting more than 600 million people at risk (van der Werf et al. 2003). Praziquantel and oxamniquine are the drugs available for the treatment of schistosomiasis (De Araújo et al. 2007). While praziquantel is active against all Schistosoma species and is the most widely used, the oxamniquine acts only on Schistosoma mansoni (Gryseels et al. 2006; Pica-Mattoccia et al. 2009). However, the existence of resistant strains for both drugs has been reported, leading to failure in schistosomiasis treatment and highlighting the importance of developing new and more effective drugs for this disease (Cioli et al. 1993, 1995; Pica-Mattoccia et al. 1993). In this context, the past years have been marked by increasing search for antiparasitic drugs from natural sources, especially from plants, which are the major source of biologically active compounds for the development of new treatments (Bastos et al. 1999; Magalhães et al. 2009; Silva et al. 2009). One of these compounds is curcumin [1,7-bis(4hydroxy-3methoxyphenyl)-1,6-heptadiene-3,5-dione] (Fig. 1), which is a naturally yellow pigment obtained from the rhizomes of Curcuma longa. In the field of medicine, the biological activities of curcumin have been explored, such as anti-inflammatory, antioxidant, antiviral, anti-infectious and antitumoral activities (Han et al. 2002; Maheshwari et al. 2006; Toda et al. 1985; Ruby et al. 1995; Song et al. 2009). Curcumin also possesses in vitro antiprotozoal activity, notably against Leishmaniasis, Giardiasis, and Trypanosomiasis (Nose et al. 1998; Pérez-Arriaga et al. 2006). In addition, in vitro and in vivo effects of the oil extract of C. longa and the curcumin against S. mansoni have been reported (Allam 2009; El-Ansary et al. 2007; ElBanhawey et al. 2007; Magalhães et al. 2009).
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Characterization of the nanoparticles Particle size analysis and morphology
Fig. 1 Chemical structure of curcumin
The studies concerning the production of new drugs that are more effective and that provoke fewer side effects are undoubtedly important and have been the focus of several researchers and industries. However, problems shown by the actual drugs, such as toxicity and poor solubility in water, may be overcome through their incorporation into drug delivery systems (DDS). The DDS should deliver a biologically active molecule at a desired rate for a desired duration and at a desired target, so as to maintain the drug level in the body at optimum therapeutic concentrations with minimum fluctuation (Kumar et al. 2009; Lakshmi and Cato 2006). There are several types of medicine applying the DSS technology, and some are already approved by the Food and Drug Administration (FDA). DDS applied for cancer treatment, for example, has been attracting much attention, but fewer studies are available on the literature relating to the incorporation of antiprotozoal drugs into DDS. These studies involve the use of DDS to treat Leishmaniasis, Malaria and Trypanosomiasis diseases, but fewer efforts are made for Schistosomiasis (Das et al. 2011; Nayaka et al. 2010; Romero and Morilla 2010). The aim of this work was to evaluate the in vitro schistosomicidal activity of curcumin incorporated into into poly(lactic-co-glycolic)acid (PLGA) nanoparticles.
Materials and methods Chemicals Curcumin, PLGA (copolymer ratio of lactide to glycolide of 50:50) and Pluronic F-68 were purchased from Sigma (St. Louis, MO). The acetonitrile analytical grade was obtained from EMD Chemicals (Gibbstown, NJ). Incorporation of curcumin into PLGA nanoparticles The curcumin-loaded PLGA nanoparticles were prepared by the nanoprecipitation method described by Fessi et al. (1989). In general, PLGA and curcumin (drug/polymer weight ratio of 1:10) were dissolved in acetonitrile and dropped into the stirred surfactant aqueous phase at room temperature by using a syringe. The suspension was stirred for 30 min and the evaporation under reduced pressure was used to remove the organic solvent and to concentrate the suspension.
The mean diameter and the polydispersity index (PDI) of nanoparticles in the suspension were evaluated by dynamic light scattering (DLS) using a Malvern Instruments 3000 HSa Zetasizer. The morphology of dried nanoparticles was examined using a Phillipis XL30 field-emission scanning electron microscope. The nanoparticles were coated with gold prior to examination by field-emission scanning electron microscopy (FESEM). Encapsulation efficiency This experiment was performed in triplicate, and then 1.0 ml of ethanol was added to each dried sample of the curcumin-loaded PLGA nanospheres. Ethanol was removed and the amount of the non-encapsulated curcumin was evaluated by absorption electronic spectroscopy at 422 nm using an HP 8453 Diode Array UV Spectrophotometer and a standard curve in ethanol. Later, 1.0 ml of acetonitrile was added to each nanoparticle sample and then centrifuged. The curcumin content inside the nanoparticles was also determined by absorption electronic spectroscopy at 422 nm and a standard curve in acetonitrile. In vitro schistosomicidal activity The adult S. mansoni worms (LE strain) were recovered under aseptic conditions by perfusion of the hepatic portal system of infected Balb/c mice after 8 weeks of infection with 200 cercariae. The worms were washed in Roswell Park Memorial Institute (RPMI) 1640 medium (Invitrogen), maintained at pH 7.5 with HEPES 20 mM, and supplemented with penicillin (100 UI ml−1), streptomycin (100 μg ml−1), and 10% bovine fetal serum (Gibco). After washing, pairs of adult worms were transferred to a 24-well culture plates containing 2 ml of the same medium and incubated at 37°C in a humid atmosphere containing 5% CO2 prior to use. At 24 h after incubation, curcumin-loaded PLGA nanoparticles were added to the medium containing the S. mansoni worms to give final concentrations of 20, 30, 40, 50, and 100 μM. The effects of curcumin-loaded PLGA nanoparticles on S. mansoni adult worms were monitored every 24 h during 120 h to evaluate their general condition: motor activity, alterations in the tegument, mortality rate and changes in pairing (Xiao et al. 2007). The worms were considered dead when no movement was observed for at least 2 min of examination (Manneck et al. 2010). Quadruplicate were carried out for each concentration used and was performed two experiments independents. RPMI 1640 medium and RPMI 1640 medium with empty
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PLGA nanoparticles (employed in the same amount used in the highest concentration of curcumin-loaded PLGA nanoparticles) were used as negative control groups. Statistical analysis Results are expressed as mean ± SEM. Data were statistically analyzed by one-way analysis of variance, followed by Dunnett’s comparison test.
Results and discussion Curcumin is a naturally occurring compound and the major constituent in the rhizome of C. longa (Zingiberaceae), commonly named turmeric. The curcumin is a yellow pigment responsible for the color of turmeric and exhibit several biological activity (Allam 2009; Magalhães et al. 2009). Recently, it has been reported that curcumin is active against S. mansoni in in vivo and in vitro studies (Allam 2009; Magalhães et al. 2009). However, no study with curcumin-loaded in polymeric nanoparticles against Schistosoma species has been performed. In addition, just one investigation involves the study of the employment of the DDS technology in the treatment of Schistosomiasis. In this study, De Araújo et al. (2007) reported the in vitro schistosomicidal activity of the nanoemulsion containing 2(butylamino)-1-phenyl-1-ethanethiosulfuric acid (BphEA) and compared its activity with that of free BphEA. In this study, we evaluated the biological activity of the curcumin incorporated into PLGA nanospheres against S. mansoni. The curcumin was successfully incorporated (94%) into PLGA nanoparticles by the nanopreciptation method. The nanoprecipitation or solvent displacement is a technique used to prepare nanoparticles instantly and in a single step, and requires the employment of miscible solvents, such as water and acetonitrile. At the beginning of the nanoprecipitation
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process, both the solvent and the polymer chains that compose the organic phase diffuse into the dispersing medium (in our case, the aqueous phase). The interaction between the solvent and the water causes a desolvation of the polymer chains carrying its aggregation. Then the polymer precipitates, entrapping the drug (e.g., curcumin) and forming the nanoparticles (Bilati et al. 2005). The morphology of the obtained particles was confirmed by FESEM and DLS. The FESEM image (Fig. 2a) shows that the produced particles are spherical and agglomerated, which was certainly caused by the centrifugation process. To acquire FESEM images, the suspension was primarily centrifuged at 10,000 rpm for several minutes, in order to be able to decant the particles. The nanosize was responsible for the long centrifugation time. The formation of spherical particles in the nanosize range was confirmed by DLS, and curcumin was incorporated into PLGA spheres which exhibit an average diameter of 108 nm (Fig. 2b). In addition, these spheres are monodisperse since the calculated PDI was 0.014. The schistosomicidal activity of the curcumin-loaded PLGA nanoparticles was also evaluated. As shown in Fig. 3, the curcumin-loaded PLGA nanoparticles caused the death of 100% of parasites at 50 and 100 μM at 120 and 24 h, respectively. The lethal effect was also observed at concentrations higher than of 40 μM at 24 h. In the negative controls (RPMI medium + PLGA empty nanoparticles or RPMI medium), no dead worms were observed. The results showed that both male and female parasites are susceptible to curcumin-loaded PLGA nanoparticles. In addition, Magalhães et al. (2009) showed that free curcumin caused 100% mortality of parasites at a concentration of 50 μM during 24 h of incubation, and no difference was observed between males and females. In contrast, it has been described that male worms of S. mansoni are often more susceptible than female worms in praziquantel resistance studies
Fig. 2 FEG-SEM image of the curcumin-loaded PLGA nanoparticles (a) and diameter size distribution of curcumin-loaded PLGA nanospheres (b)
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Fig. 3 Effect of different concentrations of curcumin-loaded PLGA nanoparticles on the mortality of adult S. mansoni worms. The adult worms were treated with the curcumin-loaded PLGA nanoparticles during 5 days, and the mortality was evaluated every 24 h by inverted microscopy. RPMI 1640 medium and RPMI 1640 medium with empty
PLGA nanoparticles (employed in the same amount used in the highest concentration of curcumin-loaded PLGA nanoparticles) were used as negative control groups. Data are expressed as the means ± SD of two independent experiments. *P
