International Journal of Pharmaceutics 463 (2014) 31–37
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical Nanotechnology
In vitro evaluation of permeation, toxicity and effect of praziquantel-loaded solid lipid nanoparticles against Schistosoma mansoni as a strategy to improve efficacy of the schistosomiasis treatment Ana Luiza Ribeiro de Souza a,b , Tatiana Andreani b,c , Rosimeire Nunes de Oliveira d , Charlene Priscila Kiill a , Fernanda Kolenyak dos Santos a , Silmara Marques Allegretti d , Marco Vinícius Chaud e , Eliana B. Souto f,g,∗ , Amélia M. Silva b,c , Maria Palmira Daflon Gremião a,∗∗ a
School of Pharmaceutical Sciences, UNESP – University Estadual Paulista, Rodovia Araraquara-Jau Km 1, Araraquara, SP 14801-902, Brazil Department of Biology and Environment, University of Trás-os-Montes e Alto Douro (UTAD), P.O. Box 1013, 5001-801 Vila Real, Portugal c Centre for Research and Technology of Agro-Environmental and Biological Sciences (CITAB/UTAD), Vila Real, Portugal d Institute of Biology, Department of Animal Biology, Laboratory of Helminthology, State University of Campinas, Rua Monteiro Lobato 255, CEP. 13083-862, Campinas, SP, Brazil e Department of Pharmacy, College of Pharmaceutical, Sorocaba University – UNISO, 18023-000 Sorocaba, SP, Brazil f Faculty of Health Sciences, Department of Pharmaceutical Technology, Fernando Pessoa University, UFP, Rua Carlos da Maia 296, Office S.1, P-4200-150 Porto, Portugal g Institute of Biotechnology and Bioengineering, Centre of Genomics and Biotechnology, University of Trás-os-Montes e Alto Douro (IBB-CGB/UTAD), Vila Real, Portugal b
a r t i c l e
i n f o
Article history: Received 10 November 2013 Received in revised form 10 December 2013 Accepted 14 December 2013 Available online 24 December 2013 Keywords: Praziquantel Solid lipid nanoparticles Schistosomiasis Schistosoma mansoni Cytotoxicity HepG2 cells
a b s t r a c t Solid lipid nanoparticles (SLN) are a promising drug delivery system for oral administration of poorlywater soluble drugs because of their capacity to increase the solubility of drug molecules when loaded in their lipid matrices, with the resulting improvement of the drug bioavailability. In the present work, we have developed praziquantel (PZQ)-loaded SLN and explored the biological applications of this system for intestinal permeation of PZQ. The effect in vitro on Schistosoma mansoni culture and the cytotoxicity in HepG2 line cell were also evaluated. The results showed a significant decrease in the intestinal absorption of PZQ loaded in SLN compared to free PZQ, suggesting that the SLN matrix could act as reservoir system. In culture of S. mansoni, we observed that PZQ-loaded SLN were more effective than free PZQ, leading the death of the parasites in less time. The result was proportional to doses of PZQ (25 and 50 g mL−1 ) and lipid concentration. Regarding cytotoxicity, the encapsulation of PZQ into SLN decreased the toxicity in HepG2 cells in comparison to the free PZQ. From the obtained results, PZQ-loaded SLN could be a new drug delivery system for the schistosomiasis treatment especially in marginalized communities, improving the therapeutic efficacy and reducing the toxic effects of PZQ. © 2014 Published by Elsevier B.V.
1. Introduction Schistosomiasis is a parasitic disease caused by blood flukes of the genus Schistosoma, being a serious public health problem
∗ Corresponding author at: Faculty of Health Sciences of Fernando Pessoa University, Rua Carlos da Maia 296, Office S.1, P-4200-150 Porto, Portugal. Tel.: +351 22 507 4630x3056; fax: +351 22 550 4637. ∗∗ Corresponding author at: School of Pharmaceutical Sciences of Paulista State University, Rodovia Araraquara-Jau Km 1, CEP 14801-902 Araraquara, SP, Brazil. Tel.: +55 16 3301 6975; fax: +55 16 3301 6960. E-mail addresses: [email protected], [email protected] (E.B. Souto), [email protected] (M.P.D. Gremião). 0378-5173/$ – see front matter © 2014 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.ijpharm.2013.12.022
in tropical areas without potable water and poor sanitary conditions. It is estimated that at least 230 million of people need treatment against schistosomiasis infection per year (World Health Organization, 2013). Praziquantel (PZQ) (Fig. 1) is currently being used as the main drug for the schistosomiasis treatment and to preventive chemotherapy (Cioli, 1998; Hotez et al., 2007). Although PZQ is effective against all species of Schistosoma, this drug is a class II compound, and thus, presents poor solubility in water and, consequently, low absorption through the gastrointestinal tract (GIT) (Lindenberg et al., 2004). In addition, PZQ chemotherapy has low effectiveness on schistosomula, resulting in failures of mass treatment in the high endemicity areas (Doenhoff et al., 2008).
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Technologies, Alfagene, Portugal). The water used throughout the experiments was purified obtained from a Milli® Q Plus, Millipore (Germany). All other reagents and solvents were of analytical grade. 2.2. Preparation of PZQ-SLN
Fig. 1. Chemical structure of praziquantel.
Moreover, large doses are required in order to achieve adequate concentrations due to its high liposolubility and its significant firstpass metabolism after oral administration which convert PZQ in a less potent compound (Mourão et al., 2005; Xiao et al., 1985). Several reports have described many technological alternatives to improve the oral absorption of PZQ (Almeida et al., 2012; Mainardes et al., 2006; Mourão et al., 2005; Souza et al., 2011; Yang et al., 2009). Among them, studies using solid lipid nanoparticles (SLN) for lipophilic drug delivery have gained great attention in the biopharmaceutical field (Muller et al., 2006; Silva et al., 2012, 2007). SLN have many advantages, comparing with the traditional systems, including drug protection, high physical stability, as well as controlled and target drug delivery (Müller et al., 2000). Since SLN matrix are composed of physiological components and/or excipients of accepted status (e.g., GRAS status for oral and topical administration), these systems can reduce the risk of acute and chronic toxicity (Souto and Müller, 2007). The use of hydrogenatedcastor oil in SLN improved the bioavailability of PZQ, as well as increased the residence time of the drug after oral administration (Xie et al., 2010). Since lipids may promote the oral absorption of poorly water soluble drugs, a technological approach based on SLN formulations for improving oral delivery of PZQ offers a new perspective for schistosomiasis treatment. Our previous work demonstrated that SLN were a suitable carrier for loading PZQ with high drug encapsulation efficacy and long-term stability (Souza et al., 2011). In the present study, PZQ-loaded SLN (PZQ-SLN) were produced via o/w microemulsion technique and applied to S. mansoni culture to evaluate the schistosomal activity. The in vitro intestinal permeation of PZQ loaded into SLN was also assessed. In addition, cytotoxicity studies were performed to estimate the possible toxicity of SLN on HepG2 cell line.
PZQ-SLN were prepared by oil-in-water microemulsion as described previously (Souza et al., 2011). Briefly, an amount of PZQ was added to 5% (w/v) of SA and melted at about 90 ◦ C. Then, 1% (w/v) of Poloxamer 188 was dissolved in purified water and heated up to approximately the same temperature. The hot lipid phase was poured into the hot water-phase under high shear homogenization at 8000 rpm (Ultra-Turrax® , T 25, IKA), for 10 min. The temperature was maintained at 90 ◦ C during the homogenization step. The obtained coarse emulsion was immediately dispersed in 10 mL of cooled distilled water (in ice bath) followed by homogenization at 3400 rpm for 1 min. Subsequently, the obtained dispersions were allowed to recrystallize under refrigerated temperature to obtain PZQ-SLN. For the production of empty SLN, the drug was replaced by solid lipid, following the same process as described above. 2.3. Intestinal permeation assay
2. Experimental
All the animal studies were done after approval of the protocol by the Institutional Animal Ethics Care Committee of São Carlos Federal University (protocol number 003/2011). The everted gut sac model was carried out in order to evaluate the effect of lipid formulation in the efficiency of PZQ transport through the intestinal membrane. The samples tested were PZQ dispersed in TC-199 buffer solution (PZQ-F) and PZQ-SLN. The TC-199 buffer is a solution used for tissue culture composed of NaCl, KCl, CaCl2 , Na2 PO4 and glucose. Rats were anaesthetized by intra-peritoneal sodium pentobarbital and a segment of around 6 cm in length of the small intestine was removed, washed several times with oxygenated TC199 buffer without glucose at 10 ◦ C and carefully inverted. The intestine was filled with TC-199 buffer and the ends were clamped using braided silk sutures. The gut sacs were placed into 20 mL of TCC-199 buffer containing samples of 250 g mL−1 of PZQ or PZQ-SLN (equivalent quantity of PZQ), for incubation. After 60 min, the sacs were removed, washed with TC-199 buffer followed by the filtration of content present in the interior of everted rat small intestine segments through cellulose acetate membrane (0.22 m). All the content of PZQ was determined by HPLC method, as previously described (Cinto et al., 2009). Briefly, all the content of PZQ was determined by a ProStar HPLC equipment from Varian. The instrument is equipped with a binary pump solvent delivery system and a ProStar 330 UV–vis photodiode array detector. The analysis was performed employing an analytical column RP-18 (Varian, Chromsep) (250 mm × 4.6 mm i.d., 5 m particle size), under isocratic conditions with acetonitrile and water (45:55, v/v) at 25 ◦ C, and flow rate was 1.0 mL min−1 . Detection of the PZQ was carried out at 215 nm. The injection volume was set to 100 L using a sample loop (Cinto et al., 2009).
2.1. Materials
2.4. In vitro effect of SLN and PZQ against Schistosoma mansoni
PZQ was obtained from Henrifarma® (Brazil). Stearic acid (SA) was obtained from Acofarma (Portugal). Poloxamer 188 (Lutrol® F68) was purchased from BASF ChemTrade GmbH (Germany). Pentobarbital sodium was purchased from Abbott (Brazil). Dulbecco’s modified Eagle’s medium (DMEM), antibiotics (penicillin/streptomycin), l-glutamine, 0.05% trypsin–EDTA and fetal bovine serum (FBS) were from Gibco® (Life Technologies, Invitrogen, Alfagene, Portugal). RPMI-1640 cell culture medium was obtained from Invitrogen (USA). AlamarBlue® and HepG2 (ATCC n◦ HB-8065) cell line were obtained from Invitrogen (Life
S. mansoni (BH strain from Belo Horizonte, MG, Brazil) life cycle is maintained Biomphalaria glabrata as intermediate at the Department of Animal Biology, IB, UNICAMP. As definitive host Swiss/SPF female mice, weighing ∼20–22 g and 4 weeks of age, were previously infected by means of exposure to suspension containing 70 cercariae, using the tail immersion technique as described by Olivier and Stirewalt (1952). For the in vitro studies, adult worms recovered by perfusion of the hepatic portal system and mesenteric veins after 60 days of infection of definitive host were washed in RPMI-1640
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(Nutricell® ) medium supplemented with 0.05 g L−1 of streptomycin, 10,000 UI mL−1 of penicillin, 0.3 g L−1 of l-glutamine, 2.0 g L−1 of d-glucose, 2.0 g L−1 of NaHCO3 and 5.958 g L−1 of HEPES buffer. After washing, a worm’s couple was incubated in a 24-well culture plate (TTP) containing the same medium RPMI-1640. The samples SLN, PZQ-SLN or PZQ dispersed in PBS solution in two different concentrations (25 g mL−1 and 50 g mL−1 of PZQ and equivalent bulk of SLN) were applied directly about the worm couple of S. mansoni and the plates were incubated in CO2 greenhouse at 5% and 37 ◦ C (de Oliveira et al., 2012; Xiao et al., 2007). The negative control group was incubated in RPMI-1640 medium. All tested samples were carried out in five replicates for each concentration, to evaluate the effect of samples in oviposition, morphological changes, and susceptibility of parasites at regular intervals of 2, 4, 6, 24, 48 and 72 h by using an inverted Leica DM-500 microscope (de Oliveira et al., 2012). 2.5. Effect of PZQ and PZQ-SLN in HepG2 cell culture 2.5.1. HepG2 cell culture The human hepatoma cell line HepG2 (ATCC n◦ HB-8065) was used as cell model to evaluate the cytotoxicity in this study. The experiments were conducted at the Cell Biology and Bioenergetic Laboratory of the Center for Research and Technology of AgroEnvironmental and Biological Sciences, Vila Real, Portugal. HepG2 cells were maintained in culture with Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 2 mM l-glutamine, antibiotics (200 UI mL−1 penicillin and 200 g mL−1 streptomycin) and 10% (v/v) of FBS, under standard culture conditions (5% CO2 in 95% air, with controlled humidity, at 37 ◦ C). The HepG2 cells were subcultured every 7 days by trypsinization with 0.05% trypsin–EDTA solution, to avoid a decrease in mitotic index and even cell death (Phelan, 2007) and media was changed in between. 2.5.2. Cell treatment HepG2 cells were seeded in 96-well microplates at a cell density of 5 × 104 cells/mL (100 L per well) and incubated at 37 ◦ C under 5% CO2 in fresh cell culture medium. After 24 h of incubation, the medium was replaced with FBS-free culture medium supplemented with the test compounds (PZQ dissolved in ethanol and PZQ-SLN). Three concentrations of PZQ were tested (30, 60 and 120 g mL−1 ). Ethanol toxicity at final concentrations was also studied (data not showed).
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Table 1 Mean particle size and polydispersity index of free-SLN, and PZQ-loaded SLN. Samples
Size (nm)
PdI
SLN PZQ-SLN
396.8 ± 2.8 505.6 ± 27.2
0.156 ± 0.036 0.264 ± 0.044
oxidized (εox ) and reduced (εred ) forms. A1 and A2 represent absorbance of test wells at 570 nm and 620 nm, respectively. A 2 and A 1 represent absorbance of negative control wells at 620 and 570 nm, respectively. After 24 h of cell incubation with the test compounds, 10 L/well of AlamarBlue® solution was added to each well (10%, v/v) and the plate was incubated for an additional 2 h (37 ◦ C, 5% CO2 , in the incubator). The AB reduction was followed by a change in color which was monitored by reading the absorbance at 570 nm and 620 nm, using a multi-well plate reader (Multiskan EX, Labsystem). The experiments were performed in triplicates (n = 3). 2.6. Statistical analysis The statistical analysis to evaluate the differences in the treatments among the different groups was carried out by a one-way analysis of variance (ANOVA) test. A p-value < 0.05 was considered statically significant. 3. Results and discussion 3.1. Evaluation of intestinal permeation PZQ plays an important role in parasite control on schistosomiasis treatment. However, due to low solubility in water and poor bioavailability, the traditional therapy does not ensure the total efficacy against all stages of Schistosoma development. PZQ loaded in SLN were produced and the particle size characterization is given in Table 1. In the present study, the intestinal transport of PZQ loaded in SLN was evaluated by the everted rat gut sac method. This technique is used to identify the important parameters in the transport of the drug from the pharmaceutical matrix and thus make a rational decision about the system of interest (Barthe et al., 1998). Fig. 2 shows the PZQ concentration measured in the inner side of the everted gut sac from the two samples. It can be observed that
2.5.3. AlamarBlue® assay AlamarBlue® assay was used to estimate the viability and proliferation rate of HepG2 cells, by monitoring the reding power of the cell, by the conversion of resazurin (blue) into resorufin (pink/red). Viable cells continuously convert resazurin to resorufin, and thus producing a quantitative measure of cell viability (or cytotoxicity) Since that AlamarBlue® is stable and non-toxic to the cells, this test has been considerable superior to the classical cell proliferation assessments, such as those performed by (4,5-dimethylthiazol-2yl)2,5-diphenyl-tetrazolium bromide (MTT) (Fields and Lancaster, 1993). Cell viability correlates with the magnitude of dye reduction and may be expressed as percentage of AlamarBlue® reduction (% AB reduction). Owing to overlap in the absorbance spectrum of oxidized and reduced form of AlamarBlue® , the % AB reduction was calculated according to Eq. (1) derived by Willard et al. (1965), and recommended by the manufacturer’s (Larson et al., 1997): %AB reduction =
(εox 2 )(A1 ) − (εox 1 )(A2 ) × 100 (εred 1 )(A 2 ) − (εred 2 )(A 1 )
(1)
In the formula, ε2 and ε1 are constants representing the molar extinction coefficient of AB at 620 and 570 nm, respectively, in the
Fig. 2. Intestinal permeation of PZQ from different samples by everted intestinal sac method. Data are means ± SD. Symbol indicate statistically significant difference in comparison with PZQ-F (*p < 0.05).
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the cumulative permeation profile of free PZQ transported across the duodenal segment (22.57 ± 3.49%) was significantly higher than those from the PZQ loaded in SLN (11.97 ± 5.85%). The decrease in the intestinal permeation by PZQ loaded in SLN may be attributed to a reduced availability of drug in mucosal bulk, at time of 60 min, due to its incorporation into SLN, suggesting that the SLN matrix acts as reservoir system, leading to less drug diffusion, and thus, PZQ could be more effective against the parasites located in mesenteric veins of intestine. The same mechanism was proposed by Mainardes et al. (2006) when they observed that the intestinal permeation of PZQ loaded in PLGA nanoparticles was 4-fold smaller than the intestinal permeation of free PZQ. Fig. 3. Adult worms of S. mansoni incubated with RPMI 1640 (control). Image with 100× magnification.
3.2. Comparison of PZQ vs PZQ-SLN efficacy against S. mansoni Adult worms of S. mansoni were placed into RPMI-1640 medium and exposed to PZQ (25 and 50 g mL−1 , in PBS) or equivalent amounts of SLN or PZQ-SLN. The results obtained in the S. mansoni after their incubation with SLN, PZQ-SLN and PZQ are summarized in Table 2.
Fig. 3 shows a couple of worms incubated with RPMI 1640 medium (control group). From the results, the presence of the female in the ginecophoric channel of the male was observed, indicating the couple’s non-separation. Further, no evident alterations in the worms’ tegument were seen.
Fig. 4. Adult worms of S. mansoni exposed to: (a) PZQ in PBS (25 g mL−1 ); (b) PZQ in PBS (50 g mL−1 ); (c) SLN (Eq. 25 g mL−1 ); (d) SLN (Eq. 50 g mL−1 ); (e) PZQ-SLN (25 g mL−1 of PZQ); (f) PZQ-SLN (50 g mL−1 of PZQ). Arrows indicate nanoparticles in worm tegument (magnification of 100×).
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Table 2 In vitro effect of SLN, PZQ-SLN and PZQ on adult worms of S. mansoni. Evaluation was performed at several time points along an incubation period of 72 h. Samples
Period of incubation (h)
Couples separated worms (%)
Control (RPMI-1640)
2 4 6 24 48 72
0 0 0 0 0 20
PZQ (25 g mL−1 )
2 4 6 24 48 72
SLN (Eq. 25 g mL−1 )
Dead worms (%)
Oviposition
Tegumentary changes in the worms
0 0 0 0 0 0
+ + + + + +
I I I I I I
0 0 0 20 20 20
0 0 0 20 20 60
− − − − − −
S S S S S S
2 4 6 24 48 72
20 20 40 60 60 60
20 40 100 100 100 100
− − − − − −
S S S S S S
PZQ-SLN (25 g mL−1 )
2 4 6 24 48 72
0 0 20 100 100 100
40 40 100 100 100 100
− − − − − −
S S S S S S
PZQ (50 g mL−1 )
2 4 6 24 48 72
0 0 0 40 40 40
0 0 0 40 40 70
− − − − − −
S S S S S S
SLN (Eq. 50 g mL−1 )
2 4 6 24 48 72
20 0 0 0 0 0
100 100 100 100 100 100
− − − − − −
S S S S S S
PZQ-SLN (50 g mL−1 )
2 4 6 24 48 72
60 60 60 60 60 60
100 100 100 100 100 100
− − − − − −
S S S S S S
I: insignificant; S: significant; +: oviposition; − no eggs.
When compared to the control group, the effect of free PZQ in culture of S. mansoni was significant, since 60% and 70% of the worms died in a period of 72 h at the drug concentration of 25 g mL−1 and 50 g mL−1 , respectively (Table 2). Moreover, the PZQ caused changes in the tegument and contraction of worms (Fig. 4a and b). In the case of incubation with empty SLN (Fig. 4c and 4d a), couples separation, contraction of parasite, tegumentar and also internal organs damage were observed and the sample with higher lipid caused the death of the parasites in less time (2 h vs 6 h). These results indicate that SLN are able to induce the death of S. mansoni parasites and it could be due to the presence of nanoparticles in worm tegument. Similar results were reported by Mourão et al. (2005) when S. mansoni strain LE was incubated with phosphatidylcholine liposomes. The worms exposed to PZQ-SLN (Fig. 4e and f) revealed aspects similar to those incubated with empty SLN, suggesting a synergic effect of SLN and the drug. According to Brindley et al. (1989), the morphological and tegumentar changes induced by PZQ are related to increase of antigen exposure in the surface of parasites. However, earlier studies by Caffrey (2007) and Doenhoff et al. (2008)
described that PZQ promotes a rapid influx of extracelullar calcium, followed by contraction, paralysis and destruction of the tegument. It is believed that this ion influx is due to the activation of adenosine receptors – known calcium channel modulators – existing in the parasite. Regarding oviposition, earlier studies by Xiao et al. (1981) reports that the PZQ induces changes in the ovaries and alters the vitelline glands of adult females of S. japonicum. In this study, the results obtained with all samples revealed absence of oviposition at all concentrations evaluated (Table 2). According to Katz (2008) and Wilson et al. (2007) the absence of oviposition in in vitro studies is important, since the pathology of schistosomiasis is associated with the presence of parasite eggs in the tissues of the host. 3.3. Comparison of PZQ vs PZQ-SLN efficacy in HepG2 cell culture Another important parameter to be evaluated in the development of drug delivery systems is the toxicity, since there is a growing debate about the potential benefits and side effects in living cells resulting from the use of nanoparticles. Therefore, it is necessary to understand the risk associated with the human and
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the lipid matrix. The encapsulation of PZQ into SLN can lead to an improvement of oral administration with minimal cytotoxicity events. Therefore, the PZQ-loaded SLN could be a promising system for the schistosomiasis control especially in marginalized communities, improving the therapeutic efficacy and reducing the toxic effects of PZQ. Acknowledgements Financial support provided by CAPES, CNPq and FAPESP, and by FCT (Fundac¸ão para a Ciência e Tecnologia) for Tatiana Andreani under the reference SFRH/BD/60640/2009 are acknowledged. The authors also acknowledge FCT and the European Union Funds (FEDER/COMPETE – Operational Competitiveness Programme) under the project references PTDC/SAUFAR/113100/2009, FCOMP-01-0124-FEDER-022696, and PEstC/AGR/UI4033/2011. References
Fig. 5. Cell viability (% of control) of HepG2 cells treated with (a) PZQ; (b) PZQ30, loaded SLN. Results are expressed as mean ± standard deviation (n = 3). 60, and 120 g mL−1 of PZQ.
environment exposure to nanoparticles, as well as the molecular mechanisms of toxicity, since these particles have the ability to reach the blood through the skin, lung and GIT, and deposit in various organs causing several biological adverse reactions (Souto et al., 2009). Although PZQ is generally described as non-toxic, some side effects are known, such as nausea, vomiting, headache and hepatomegaly (Montero and Ostrosky, 1997). Moreover, Omar et al. (2005) reported that the PZQ (1500 mg kg−1 , weekly for 6 weeks) induced hepatotoxicicity, genotoxicicity and carcinogenic effects in albino rats. In this study, the cytotoxicity of tested compounds was carried out applying the human hepatoma cell line HepG2. Cells were incubated with three concentrations of PZQ pre-dissolved in ethanol and then diluted in FBS-free culture medium and PZQ-SLN. After 24 h of incubation, all concentrations of free PZQ show low cytotoxicity on HepG2 cells (cell viability between 68% and 79% of the control). However, after 48 h of incubation, it was clearly observed that free PZQ caused cytotoxicity in the HepG2 cells as observed by the reduction on the cell viability and/or proliferation rate with values between 20% and 30% of the control and these values remained constant over the time (Fig. 5a). Comparing the data from Fig. 5a with those from Fig. 5b, the incorporation of PZQ into SLN demonstrated lower toxicity to HepG2 cells in comparison to PZQ solution at an equivalent concentration. PZQ-SLN decreased the cell viability and cell proliferation in dose-dependent manner which is more evident after 48 h of exposure. No cytotoxicity effect on cell proliferation and viability was observed at low concentration (30 g mL−1 ). These results are in agreement with other studies (Nassimi et al., 2010; Silva et al., 2011), demonstrating high biocompatibility of SLN-based formulations. This parameter is essential in the development of suitable systems designed for nanomedical applications. 4. Conclusions The results obtained in the present work demonstrated that the SLN provided a controlled release of PZQ, higher schistosomicidal activity on the S. mansoni culture in comparison to PZQ suspension due to the interaction between the parasite tegument and
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A.L.R.d. Souza et al. / International Journal of Pharmaceutics 463 (2014) 31–37 Phelan, M.C., 2007. Basic Techniques in Mammalian Cell Tissue Culture. John Wiley & Sons, Inc. Silva, A.C., González-Mira, E., García, M.L., Egea, M.A., Fonseca, J., Silva, R., Santos, D., Souto, E.B., Ferreira, D., 2011. Preparation, characterization and biocompatibility studies on risperidone-loaded solid lipid nanoparticles (SLN): high pressure homogenization versus ultrasound. Colloids Surf. B: Biointerfaces 86, 158–165. Silva, A.C., Lopes, C.M., Fonseca, J., Soares, M.E., Santos, D., Souto, E.B., Ferreira, D., 2012. Risperidone release from solid lipid nanoparticles (SLN): validated HPLC method and modelling kinetic profile. Curr. Pharm. Anal. 8, 307–316. Silva, A.C., Santos, D., Ferreira, D.C., Souto, E.B., 2007. Oral delivery of drugs by means of solid lipid nanoparticles. Minerva Biotechnol. 19, 1–5. Souto, E.B., Martins-Lopes, P., Lopes, C.M., Gaivão, I., Silva, A.M., Guedes-Pinto, H., 2009. A note on regulatory concerns and toxicity assessment in lipid-based delivery systems (LDS). J. Biomed. Nanotechnol. 5, 317–322. Souto, E.B., Müller, R.H., 2007. Lipid nanoparticles (SLN and NLC) for drug delivery. In: Domb, J., Tabata, Y., Ravi Kumar, M.N.V., Farber, S. (Eds.), Nanoparticles for Pharmaceutical Applications. American Scientific Publishers, Los Angeles, CA, pp. 103–122. Souza, A.L.R., Andreani, T., Nunes, F.M., Cassimiro, D.L., Almeida, A.E., Ribeiro, C.A., Sarmento, V.H.V., Gremião, M.P.D., Silva, A.M., Souto, E.B., 2011. Loading of raziquantel in the crystal lattice of solid lipid nanoparticles: studies by DSC and SAXS. J. Therm. Anal. Calorim. 108, 353–360.
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Willard, H.H., Merritt, L.L., Dean, J.A., 1965. Ultraviolet and visible absorption methods. Instrumental Methods of Analysis, Norstrand, D.V./Princeton, NJ. Wilson, M.S., Mentink-Kane, M.M., Pesce, J.T., Ramalingam, T.R., Thompson, R., Wynn, T.A., 2007. Immunopathology of schistosomiasis. Immunol. Cell Biol. 85, 148–154. World Health Organization, W., 2013. Schistosomiasis. Xiao, S.H., Catto, B.A., Webster, L.T., 1985. Effects of praziquantel on different developmental stages of Schistosoma mansoni in vitro and in vivo. J. Infect. Dis. 151, 1130–1137. Xiao, S.H., Keiser, J., Chollet, J., Utzinger, J., Dong, Y.X., Endriss, Y., Vennerstrom, J.L., Tanner, M., 2007. In vitro and in vivo activities of synthetic trioxolanes against major human schistosome species. Antimicrob. Agents Chemother. 51, 1440–1445. Xiao, S.H., Yu, Y.G., Wang, C.Y., Jiao, P.Y., Yuan, X.J., Liang, Y., 1981. The uptake and distribution of [3 H]-pyquiton in Schistosoma japonicum. Chung Kuo Yao Li Hsuch Pao 16, 488–493. Xie, S., Pan, B., Wang, M., Zhu, L., Wang, F., Dong, Z., Wang, X., Zhou, W., 2010. Formulation, characterization and pharmacokinetics of praziquantel-loaded hydrogenated castor oil solid lipid nanoparticles. Nanomedicine 5, 693–701. Yang, L., Geng, Y., Li, H., Zhang, Y., You, J., Chang, Y., 2009. Enhancement the oral bioavailability of praziquantel by incorporation into solid lipid nanoparticles. Pharmazie 64, 86–89.
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical Nanotechnology
In vitro evaluation of permeation, toxicity and effect of praziquantel-loaded solid lipid nanoparticles against Schistosoma mansoni as a strategy to improve efficacy of the schistosomiasis treatment Ana Luiza Ribeiro de Souza a,b , Tatiana Andreani b,c , Rosimeire Nunes de Oliveira d , Charlene Priscila Kiill a , Fernanda Kolenyak dos Santos a , Silmara Marques Allegretti d , Marco Vinícius Chaud e , Eliana B. Souto f,g,∗ , Amélia M. Silva b,c , Maria Palmira Daflon Gremião a,∗∗ a
School of Pharmaceutical Sciences, UNESP – University Estadual Paulista, Rodovia Araraquara-Jau Km 1, Araraquara, SP 14801-902, Brazil Department of Biology and Environment, University of Trás-os-Montes e Alto Douro (UTAD), P.O. Box 1013, 5001-801 Vila Real, Portugal c Centre for Research and Technology of Agro-Environmental and Biological Sciences (CITAB/UTAD), Vila Real, Portugal d Institute of Biology, Department of Animal Biology, Laboratory of Helminthology, State University of Campinas, Rua Monteiro Lobato 255, CEP. 13083-862, Campinas, SP, Brazil e Department of Pharmacy, College of Pharmaceutical, Sorocaba University – UNISO, 18023-000 Sorocaba, SP, Brazil f Faculty of Health Sciences, Department of Pharmaceutical Technology, Fernando Pessoa University, UFP, Rua Carlos da Maia 296, Office S.1, P-4200-150 Porto, Portugal g Institute of Biotechnology and Bioengineering, Centre of Genomics and Biotechnology, University of Trás-os-Montes e Alto Douro (IBB-CGB/UTAD), Vila Real, Portugal b
a r t i c l e
i n f o
Article history: Received 10 November 2013 Received in revised form 10 December 2013 Accepted 14 December 2013 Available online 24 December 2013 Keywords: Praziquantel Solid lipid nanoparticles Schistosomiasis Schistosoma mansoni Cytotoxicity HepG2 cells
a b s t r a c t Solid lipid nanoparticles (SLN) are a promising drug delivery system for oral administration of poorlywater soluble drugs because of their capacity to increase the solubility of drug molecules when loaded in their lipid matrices, with the resulting improvement of the drug bioavailability. In the present work, we have developed praziquantel (PZQ)-loaded SLN and explored the biological applications of this system for intestinal permeation of PZQ. The effect in vitro on Schistosoma mansoni culture and the cytotoxicity in HepG2 line cell were also evaluated. The results showed a significant decrease in the intestinal absorption of PZQ loaded in SLN compared to free PZQ, suggesting that the SLN matrix could act as reservoir system. In culture of S. mansoni, we observed that PZQ-loaded SLN were more effective than free PZQ, leading the death of the parasites in less time. The result was proportional to doses of PZQ (25 and 50 g mL−1 ) and lipid concentration. Regarding cytotoxicity, the encapsulation of PZQ into SLN decreased the toxicity in HepG2 cells in comparison to the free PZQ. From the obtained results, PZQ-loaded SLN could be a new drug delivery system for the schistosomiasis treatment especially in marginalized communities, improving the therapeutic efficacy and reducing the toxic effects of PZQ. © 2014 Published by Elsevier B.V.
1. Introduction Schistosomiasis is a parasitic disease caused by blood flukes of the genus Schistosoma, being a serious public health problem
∗ Corresponding author at: Faculty of Health Sciences of Fernando Pessoa University, Rua Carlos da Maia 296, Office S.1, P-4200-150 Porto, Portugal. Tel.: +351 22 507 4630x3056; fax: +351 22 550 4637. ∗∗ Corresponding author at: School of Pharmaceutical Sciences of Paulista State University, Rodovia Araraquara-Jau Km 1, CEP 14801-902 Araraquara, SP, Brazil. Tel.: +55 16 3301 6975; fax: +55 16 3301 6960. E-mail addresses: [email protected], [email protected] (E.B. Souto), [email protected] (M.P.D. Gremião). 0378-5173/$ – see front matter © 2014 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.ijpharm.2013.12.022
in tropical areas without potable water and poor sanitary conditions. It is estimated that at least 230 million of people need treatment against schistosomiasis infection per year (World Health Organization, 2013). Praziquantel (PZQ) (Fig. 1) is currently being used as the main drug for the schistosomiasis treatment and to preventive chemotherapy (Cioli, 1998; Hotez et al., 2007). Although PZQ is effective against all species of Schistosoma, this drug is a class II compound, and thus, presents poor solubility in water and, consequently, low absorption through the gastrointestinal tract (GIT) (Lindenberg et al., 2004). In addition, PZQ chemotherapy has low effectiveness on schistosomula, resulting in failures of mass treatment in the high endemicity areas (Doenhoff et al., 2008).
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Technologies, Alfagene, Portugal). The water used throughout the experiments was purified obtained from a Milli® Q Plus, Millipore (Germany). All other reagents and solvents were of analytical grade. 2.2. Preparation of PZQ-SLN
Fig. 1. Chemical structure of praziquantel.
Moreover, large doses are required in order to achieve adequate concentrations due to its high liposolubility and its significant firstpass metabolism after oral administration which convert PZQ in a less potent compound (Mourão et al., 2005; Xiao et al., 1985). Several reports have described many technological alternatives to improve the oral absorption of PZQ (Almeida et al., 2012; Mainardes et al., 2006; Mourão et al., 2005; Souza et al., 2011; Yang et al., 2009). Among them, studies using solid lipid nanoparticles (SLN) for lipophilic drug delivery have gained great attention in the biopharmaceutical field (Muller et al., 2006; Silva et al., 2012, 2007). SLN have many advantages, comparing with the traditional systems, including drug protection, high physical stability, as well as controlled and target drug delivery (Müller et al., 2000). Since SLN matrix are composed of physiological components and/or excipients of accepted status (e.g., GRAS status for oral and topical administration), these systems can reduce the risk of acute and chronic toxicity (Souto and Müller, 2007). The use of hydrogenatedcastor oil in SLN improved the bioavailability of PZQ, as well as increased the residence time of the drug after oral administration (Xie et al., 2010). Since lipids may promote the oral absorption of poorly water soluble drugs, a technological approach based on SLN formulations for improving oral delivery of PZQ offers a new perspective for schistosomiasis treatment. Our previous work demonstrated that SLN were a suitable carrier for loading PZQ with high drug encapsulation efficacy and long-term stability (Souza et al., 2011). In the present study, PZQ-loaded SLN (PZQ-SLN) were produced via o/w microemulsion technique and applied to S. mansoni culture to evaluate the schistosomal activity. The in vitro intestinal permeation of PZQ loaded into SLN was also assessed. In addition, cytotoxicity studies were performed to estimate the possible toxicity of SLN on HepG2 cell line.
PZQ-SLN were prepared by oil-in-water microemulsion as described previously (Souza et al., 2011). Briefly, an amount of PZQ was added to 5% (w/v) of SA and melted at about 90 ◦ C. Then, 1% (w/v) of Poloxamer 188 was dissolved in purified water and heated up to approximately the same temperature. The hot lipid phase was poured into the hot water-phase under high shear homogenization at 8000 rpm (Ultra-Turrax® , T 25, IKA), for 10 min. The temperature was maintained at 90 ◦ C during the homogenization step. The obtained coarse emulsion was immediately dispersed in 10 mL of cooled distilled water (in ice bath) followed by homogenization at 3400 rpm for 1 min. Subsequently, the obtained dispersions were allowed to recrystallize under refrigerated temperature to obtain PZQ-SLN. For the production of empty SLN, the drug was replaced by solid lipid, following the same process as described above. 2.3. Intestinal permeation assay
2. Experimental
All the animal studies were done after approval of the protocol by the Institutional Animal Ethics Care Committee of São Carlos Federal University (protocol number 003/2011). The everted gut sac model was carried out in order to evaluate the effect of lipid formulation in the efficiency of PZQ transport through the intestinal membrane. The samples tested were PZQ dispersed in TC-199 buffer solution (PZQ-F) and PZQ-SLN. The TC-199 buffer is a solution used for tissue culture composed of NaCl, KCl, CaCl2 , Na2 PO4 and glucose. Rats were anaesthetized by intra-peritoneal sodium pentobarbital and a segment of around 6 cm in length of the small intestine was removed, washed several times with oxygenated TC199 buffer without glucose at 10 ◦ C and carefully inverted. The intestine was filled with TC-199 buffer and the ends were clamped using braided silk sutures. The gut sacs were placed into 20 mL of TCC-199 buffer containing samples of 250 g mL−1 of PZQ or PZQ-SLN (equivalent quantity of PZQ), for incubation. After 60 min, the sacs were removed, washed with TC-199 buffer followed by the filtration of content present in the interior of everted rat small intestine segments through cellulose acetate membrane (0.22 m). All the content of PZQ was determined by HPLC method, as previously described (Cinto et al., 2009). Briefly, all the content of PZQ was determined by a ProStar HPLC equipment from Varian. The instrument is equipped with a binary pump solvent delivery system and a ProStar 330 UV–vis photodiode array detector. The analysis was performed employing an analytical column RP-18 (Varian, Chromsep) (250 mm × 4.6 mm i.d., 5 m particle size), under isocratic conditions with acetonitrile and water (45:55, v/v) at 25 ◦ C, and flow rate was 1.0 mL min−1 . Detection of the PZQ was carried out at 215 nm. The injection volume was set to 100 L using a sample loop (Cinto et al., 2009).
2.1. Materials
2.4. In vitro effect of SLN and PZQ against Schistosoma mansoni
PZQ was obtained from Henrifarma® (Brazil). Stearic acid (SA) was obtained from Acofarma (Portugal). Poloxamer 188 (Lutrol® F68) was purchased from BASF ChemTrade GmbH (Germany). Pentobarbital sodium was purchased from Abbott (Brazil). Dulbecco’s modified Eagle’s medium (DMEM), antibiotics (penicillin/streptomycin), l-glutamine, 0.05% trypsin–EDTA and fetal bovine serum (FBS) were from Gibco® (Life Technologies, Invitrogen, Alfagene, Portugal). RPMI-1640 cell culture medium was obtained from Invitrogen (USA). AlamarBlue® and HepG2 (ATCC n◦ HB-8065) cell line were obtained from Invitrogen (Life
S. mansoni (BH strain from Belo Horizonte, MG, Brazil) life cycle is maintained Biomphalaria glabrata as intermediate at the Department of Animal Biology, IB, UNICAMP. As definitive host Swiss/SPF female mice, weighing ∼20–22 g and 4 weeks of age, were previously infected by means of exposure to suspension containing 70 cercariae, using the tail immersion technique as described by Olivier and Stirewalt (1952). For the in vitro studies, adult worms recovered by perfusion of the hepatic portal system and mesenteric veins after 60 days of infection of definitive host were washed in RPMI-1640
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(Nutricell® ) medium supplemented with 0.05 g L−1 of streptomycin, 10,000 UI mL−1 of penicillin, 0.3 g L−1 of l-glutamine, 2.0 g L−1 of d-glucose, 2.0 g L−1 of NaHCO3 and 5.958 g L−1 of HEPES buffer. After washing, a worm’s couple was incubated in a 24-well culture plate (TTP) containing the same medium RPMI-1640. The samples SLN, PZQ-SLN or PZQ dispersed in PBS solution in two different concentrations (25 g mL−1 and 50 g mL−1 of PZQ and equivalent bulk of SLN) were applied directly about the worm couple of S. mansoni and the plates were incubated in CO2 greenhouse at 5% and 37 ◦ C (de Oliveira et al., 2012; Xiao et al., 2007). The negative control group was incubated in RPMI-1640 medium. All tested samples were carried out in five replicates for each concentration, to evaluate the effect of samples in oviposition, morphological changes, and susceptibility of parasites at regular intervals of 2, 4, 6, 24, 48 and 72 h by using an inverted Leica DM-500 microscope (de Oliveira et al., 2012). 2.5. Effect of PZQ and PZQ-SLN in HepG2 cell culture 2.5.1. HepG2 cell culture The human hepatoma cell line HepG2 (ATCC n◦ HB-8065) was used as cell model to evaluate the cytotoxicity in this study. The experiments were conducted at the Cell Biology and Bioenergetic Laboratory of the Center for Research and Technology of AgroEnvironmental and Biological Sciences, Vila Real, Portugal. HepG2 cells were maintained in culture with Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 2 mM l-glutamine, antibiotics (200 UI mL−1 penicillin and 200 g mL−1 streptomycin) and 10% (v/v) of FBS, under standard culture conditions (5% CO2 in 95% air, with controlled humidity, at 37 ◦ C). The HepG2 cells were subcultured every 7 days by trypsinization with 0.05% trypsin–EDTA solution, to avoid a decrease in mitotic index and even cell death (Phelan, 2007) and media was changed in between. 2.5.2. Cell treatment HepG2 cells were seeded in 96-well microplates at a cell density of 5 × 104 cells/mL (100 L per well) and incubated at 37 ◦ C under 5% CO2 in fresh cell culture medium. After 24 h of incubation, the medium was replaced with FBS-free culture medium supplemented with the test compounds (PZQ dissolved in ethanol and PZQ-SLN). Three concentrations of PZQ were tested (30, 60 and 120 g mL−1 ). Ethanol toxicity at final concentrations was also studied (data not showed).
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Table 1 Mean particle size and polydispersity index of free-SLN, and PZQ-loaded SLN. Samples
Size (nm)
PdI
SLN PZQ-SLN
396.8 ± 2.8 505.6 ± 27.2
0.156 ± 0.036 0.264 ± 0.044
oxidized (εox ) and reduced (εred ) forms. A1 and A2 represent absorbance of test wells at 570 nm and 620 nm, respectively. A 2 and A 1 represent absorbance of negative control wells at 620 and 570 nm, respectively. After 24 h of cell incubation with the test compounds, 10 L/well of AlamarBlue® solution was added to each well (10%, v/v) and the plate was incubated for an additional 2 h (37 ◦ C, 5% CO2 , in the incubator). The AB reduction was followed by a change in color which was monitored by reading the absorbance at 570 nm and 620 nm, using a multi-well plate reader (Multiskan EX, Labsystem). The experiments were performed in triplicates (n = 3). 2.6. Statistical analysis The statistical analysis to evaluate the differences in the treatments among the different groups was carried out by a one-way analysis of variance (ANOVA) test. A p-value < 0.05 was considered statically significant. 3. Results and discussion 3.1. Evaluation of intestinal permeation PZQ plays an important role in parasite control on schistosomiasis treatment. However, due to low solubility in water and poor bioavailability, the traditional therapy does not ensure the total efficacy against all stages of Schistosoma development. PZQ loaded in SLN were produced and the particle size characterization is given in Table 1. In the present study, the intestinal transport of PZQ loaded in SLN was evaluated by the everted rat gut sac method. This technique is used to identify the important parameters in the transport of the drug from the pharmaceutical matrix and thus make a rational decision about the system of interest (Barthe et al., 1998). Fig. 2 shows the PZQ concentration measured in the inner side of the everted gut sac from the two samples. It can be observed that
2.5.3. AlamarBlue® assay AlamarBlue® assay was used to estimate the viability and proliferation rate of HepG2 cells, by monitoring the reding power of the cell, by the conversion of resazurin (blue) into resorufin (pink/red). Viable cells continuously convert resazurin to resorufin, and thus producing a quantitative measure of cell viability (or cytotoxicity) Since that AlamarBlue® is stable and non-toxic to the cells, this test has been considerable superior to the classical cell proliferation assessments, such as those performed by (4,5-dimethylthiazol-2yl)2,5-diphenyl-tetrazolium bromide (MTT) (Fields and Lancaster, 1993). Cell viability correlates with the magnitude of dye reduction and may be expressed as percentage of AlamarBlue® reduction (% AB reduction). Owing to overlap in the absorbance spectrum of oxidized and reduced form of AlamarBlue® , the % AB reduction was calculated according to Eq. (1) derived by Willard et al. (1965), and recommended by the manufacturer’s (Larson et al., 1997): %AB reduction =
(εox 2 )(A1 ) − (εox 1 )(A2 ) × 100 (εred 1 )(A 2 ) − (εred 2 )(A 1 )
(1)
In the formula, ε2 and ε1 are constants representing the molar extinction coefficient of AB at 620 and 570 nm, respectively, in the
Fig. 2. Intestinal permeation of PZQ from different samples by everted intestinal sac method. Data are means ± SD. Symbol indicate statistically significant difference in comparison with PZQ-F (*p < 0.05).
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the cumulative permeation profile of free PZQ transported across the duodenal segment (22.57 ± 3.49%) was significantly higher than those from the PZQ loaded in SLN (11.97 ± 5.85%). The decrease in the intestinal permeation by PZQ loaded in SLN may be attributed to a reduced availability of drug in mucosal bulk, at time of 60 min, due to its incorporation into SLN, suggesting that the SLN matrix acts as reservoir system, leading to less drug diffusion, and thus, PZQ could be more effective against the parasites located in mesenteric veins of intestine. The same mechanism was proposed by Mainardes et al. (2006) when they observed that the intestinal permeation of PZQ loaded in PLGA nanoparticles was 4-fold smaller than the intestinal permeation of free PZQ. Fig. 3. Adult worms of S. mansoni incubated with RPMI 1640 (control). Image with 100× magnification.
3.2. Comparison of PZQ vs PZQ-SLN efficacy against S. mansoni Adult worms of S. mansoni were placed into RPMI-1640 medium and exposed to PZQ (25 and 50 g mL−1 , in PBS) or equivalent amounts of SLN or PZQ-SLN. The results obtained in the S. mansoni after their incubation with SLN, PZQ-SLN and PZQ are summarized in Table 2.
Fig. 3 shows a couple of worms incubated with RPMI 1640 medium (control group). From the results, the presence of the female in the ginecophoric channel of the male was observed, indicating the couple’s non-separation. Further, no evident alterations in the worms’ tegument were seen.
Fig. 4. Adult worms of S. mansoni exposed to: (a) PZQ in PBS (25 g mL−1 ); (b) PZQ in PBS (50 g mL−1 ); (c) SLN (Eq. 25 g mL−1 ); (d) SLN (Eq. 50 g mL−1 ); (e) PZQ-SLN (25 g mL−1 of PZQ); (f) PZQ-SLN (50 g mL−1 of PZQ). Arrows indicate nanoparticles in worm tegument (magnification of 100×).
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Table 2 In vitro effect of SLN, PZQ-SLN and PZQ on adult worms of S. mansoni. Evaluation was performed at several time points along an incubation period of 72 h. Samples
Period of incubation (h)
Couples separated worms (%)
Control (RPMI-1640)
2 4 6 24 48 72
0 0 0 0 0 20
PZQ (25 g mL−1 )
2 4 6 24 48 72
SLN (Eq. 25 g mL−1 )
Dead worms (%)
Oviposition
Tegumentary changes in the worms
0 0 0 0 0 0
+ + + + + +
I I I I I I
0 0 0 20 20 20
0 0 0 20 20 60
− − − − − −
S S S S S S
2 4 6 24 48 72
20 20 40 60 60 60
20 40 100 100 100 100
− − − − − −
S S S S S S
PZQ-SLN (25 g mL−1 )
2 4 6 24 48 72
0 0 20 100 100 100
40 40 100 100 100 100
− − − − − −
S S S S S S
PZQ (50 g mL−1 )
2 4 6 24 48 72
0 0 0 40 40 40
0 0 0 40 40 70
− − − − − −
S S S S S S
SLN (Eq. 50 g mL−1 )
2 4 6 24 48 72
20 0 0 0 0 0
100 100 100 100 100 100
− − − − − −
S S S S S S
PZQ-SLN (50 g mL−1 )
2 4 6 24 48 72
60 60 60 60 60 60
100 100 100 100 100 100
− − − − − −
S S S S S S
I: insignificant; S: significant; +: oviposition; − no eggs.
When compared to the control group, the effect of free PZQ in culture of S. mansoni was significant, since 60% and 70% of the worms died in a period of 72 h at the drug concentration of 25 g mL−1 and 50 g mL−1 , respectively (Table 2). Moreover, the PZQ caused changes in the tegument and contraction of worms (Fig. 4a and b). In the case of incubation with empty SLN (Fig. 4c and 4d a), couples separation, contraction of parasite, tegumentar and also internal organs damage were observed and the sample with higher lipid caused the death of the parasites in less time (2 h vs 6 h). These results indicate that SLN are able to induce the death of S. mansoni parasites and it could be due to the presence of nanoparticles in worm tegument. Similar results were reported by Mourão et al. (2005) when S. mansoni strain LE was incubated with phosphatidylcholine liposomes. The worms exposed to PZQ-SLN (Fig. 4e and f) revealed aspects similar to those incubated with empty SLN, suggesting a synergic effect of SLN and the drug. According to Brindley et al. (1989), the morphological and tegumentar changes induced by PZQ are related to increase of antigen exposure in the surface of parasites. However, earlier studies by Caffrey (2007) and Doenhoff et al. (2008)
described that PZQ promotes a rapid influx of extracelullar calcium, followed by contraction, paralysis and destruction of the tegument. It is believed that this ion influx is due to the activation of adenosine receptors – known calcium channel modulators – existing in the parasite. Regarding oviposition, earlier studies by Xiao et al. (1981) reports that the PZQ induces changes in the ovaries and alters the vitelline glands of adult females of S. japonicum. In this study, the results obtained with all samples revealed absence of oviposition at all concentrations evaluated (Table 2). According to Katz (2008) and Wilson et al. (2007) the absence of oviposition in in vitro studies is important, since the pathology of schistosomiasis is associated with the presence of parasite eggs in the tissues of the host. 3.3. Comparison of PZQ vs PZQ-SLN efficacy in HepG2 cell culture Another important parameter to be evaluated in the development of drug delivery systems is the toxicity, since there is a growing debate about the potential benefits and side effects in living cells resulting from the use of nanoparticles. Therefore, it is necessary to understand the risk associated with the human and
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the lipid matrix. The encapsulation of PZQ into SLN can lead to an improvement of oral administration with minimal cytotoxicity events. Therefore, the PZQ-loaded SLN could be a promising system for the schistosomiasis control especially in marginalized communities, improving the therapeutic efficacy and reducing the toxic effects of PZQ. Acknowledgements Financial support provided by CAPES, CNPq and FAPESP, and by FCT (Fundac¸ão para a Ciência e Tecnologia) for Tatiana Andreani under the reference SFRH/BD/60640/2009 are acknowledged. The authors also acknowledge FCT and the European Union Funds (FEDER/COMPETE – Operational Competitiveness Programme) under the project references PTDC/SAUFAR/113100/2009, FCOMP-01-0124-FEDER-022696, and PEstC/AGR/UI4033/2011. References
Fig. 5. Cell viability (% of control) of HepG2 cells treated with (a) PZQ; (b) PZQ30, loaded SLN. Results are expressed as mean ± standard deviation (n = 3). 60, and 120 g mL−1 of PZQ.
environment exposure to nanoparticles, as well as the molecular mechanisms of toxicity, since these particles have the ability to reach the blood through the skin, lung and GIT, and deposit in various organs causing several biological adverse reactions (Souto et al., 2009). Although PZQ is generally described as non-toxic, some side effects are known, such as nausea, vomiting, headache and hepatomegaly (Montero and Ostrosky, 1997). Moreover, Omar et al. (2005) reported that the PZQ (1500 mg kg−1 , weekly for 6 weeks) induced hepatotoxicicity, genotoxicicity and carcinogenic effects in albino rats. In this study, the cytotoxicity of tested compounds was carried out applying the human hepatoma cell line HepG2. Cells were incubated with three concentrations of PZQ pre-dissolved in ethanol and then diluted in FBS-free culture medium and PZQ-SLN. After 24 h of incubation, all concentrations of free PZQ show low cytotoxicity on HepG2 cells (cell viability between 68% and 79% of the control). However, after 48 h of incubation, it was clearly observed that free PZQ caused cytotoxicity in the HepG2 cells as observed by the reduction on the cell viability and/or proliferation rate with values between 20% and 30% of the control and these values remained constant over the time (Fig. 5a). Comparing the data from Fig. 5a with those from Fig. 5b, the incorporation of PZQ into SLN demonstrated lower toxicity to HepG2 cells in comparison to PZQ solution at an equivalent concentration. PZQ-SLN decreased the cell viability and cell proliferation in dose-dependent manner which is more evident after 48 h of exposure. No cytotoxicity effect on cell proliferation and viability was observed at low concentration (30 g mL−1 ). These results are in agreement with other studies (Nassimi et al., 2010; Silva et al., 2011), demonstrating high biocompatibility of SLN-based formulations. This parameter is essential in the development of suitable systems designed for nanomedical applications. 4. Conclusions The results obtained in the present work demonstrated that the SLN provided a controlled release of PZQ, higher schistosomicidal activity on the S. mansoni culture in comparison to PZQ suspension due to the interaction between the parasite tegument and
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