Parasitol Res (2014) 113:555–564 DOI 10.1007/s00436-013-3715-6
ORIGINAL PAPER
Pyrimethamine-loaded lipid-core nanocapsules to improve drug efficacy for the treatment of toxoplasmosis Kenia Pissinate & Érica dos Santos Martins-Duarte & Scheila Rezende Schaffazick & Catiúscia Padilha de Oliveira & Rossiane Cláudia Vommaro & Sílvia Stanisçuaski Guterres & Adriana Raffin Pohlmann & Wanderley de Souza
Received: 19 August 2013 / Accepted: 4 November 2013 / Published online: 29 November 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract We propose an innovative product based on the nanoencapsulation of pyrimethamine (PYR), aiming an improvement of drug efficacy for the treatment of toxoplasmosis. The in vitro cytotoxicity effect of encapsulated PYR and PYR-colloidal suspension was concomitantly evaluated against LLC-MK2 lineage and mouse peritoneal macrophage showing that the cells had similar tolerance for both PYR encapsulated or in the aqueous suspension. CF1 mice acutely infected with tachyzoites of Toxoplasma gondii RH strain treated with different doses (5.0–10 mg/kg/day) of PYRnanocapsules had survival rate higher than the animals treated with the same doses of non-encapsulated PYR. Thus,
Kenia Pissinate and Érica dos Santos Martins-Duarte contributed equally to this work K. Pissinate : É. dos Santos Martins-Duarte : S. R. Schaffazick : R. C. Vommaro : W. de Souza Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil É. dos Santos Martins-Duarte : R. C. Vommaro : W. de Souza Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil S. R. Schaffazick : C. P. de Oliveira : S. S. Guterres : A. R. Pohlmann Programa de Pós-Graduação em Ciências Farmacêuticas, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil K. Pissinate (*) : A. R. Pohlmann Departamento de Química Orgânica, Instituto de Química, Universidade Federal do Rio Grande do Sul, Caixa Postal 15003, Av. Bento Gonçalves, 9500, Porto Alegre 91501-970, Brazil e-mail: [email protected] W. de Souza Instituto Nacional de Metrologia e Qualidade Industrial—Inmetro, Rio de Janeiro, Brazil
encapsulation of PYR improved the efficacy of this drug against an acute model of toxoplasmosis in mice and can be considered an alternative for reducing the dose of PYR, which, in turn, could also reduce the side effects associated to the treatment.
Introduction Toxoplasma gondii is a world-spread protozoan and a significant pathogen associated with many medical areas, such as: pediatrics, ophthalmology, and infectology, regarding immunocompromised patients, principally the ones with AIDS. Cerebral toxoplasmosis is the most significant and common clinical presentation in immunocompromised patients and one of the main causes of morbidity and mortality in these patients (Montoya and Liesenfeld 2004). Pyrimethamine (PYR) is the most significant drug for the treatment of toxoplasmosis and its combination with sulfadiazine is the first-choice treatment of the disease. The mechanism of action of PYR consists in the inhibition of the enzyme dihydrofolate reductase (DHFR), which is essential for purine synthesis in parasites and humans (Anderson 2005). When patients do not tolerate the sulfa treatment, PYR combined with clindamycin or azithromycin is administrated as a second-line therapy. However, PYR administration is frequently associated with many side effects such as anorexia, vomiting, abdominal pain, diarrhea, megablastic anemia, leucopenia, and thrombocytopenia that can compromise the treatment continuation (Weniger 1979). To minimize the side effects, the concurrent administration of folinic acid is also used (Nissapatorn and Sawangjaroen 2011). New formulations for old drugs have been used as a strategy to improve the treatment of diverse infectious diseases. A new formulation combining enhancement of efficacy and reduced side effects is desirable.
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Nanocarriers have received considerable attention as potential drug delivery vehicles because they take the ultradisperse drugs to the specific intracellular targets, which are surrounded by complex physiological barriers (Prieto et al. 2006). Biodegradable polymeric nanoparticles are drug carrier systems which have distribution diameters of less than 1 μm and are widely researched for controlled release of drugs and for enhancing drug bioavailability (Pohlmann et al. 2013; Müller et al. 2007). Those nanoparticles are usually divided in lipid nanoparticles and polymeric nanoparticles. The lipid nanoparticles involve liposomes (Fig. 1a), solid lipid nanoparticles (Fig. 1b), and nanostructured lipid carriers (Fig. 1c), while the polymeric nanoparticles include nanocapsules (Fig. 1d) and nanospheres (Fig. 1e). Recently, we developed a hybrid biodegradable nanoparticle, called lipid-core nanocapsules. The lipid-core nanocapsules are vesicular structures constituted by a lipid-core composed of medium chain triacylglyceride and sorbitan monostearate, surrounded by a polymeric wall in general composed of poly(ε-caprolactone) and stabilized by polysorbate 80 at the particle/water interface (Fig. 1f) (Couvreur et al. 2002; Mora-Huertas et al. 2010). Lipid-core nanocapsule aqueous formulations are formed by the mechanism of self-assembly, based on the infinite dilution of the aggregation state of raw materials in the organic phase (Jornada et al. 2012). Hydrophobic drug incorporation to the internal oily cavity has been reported in various studies as a promising drug targeting strategy (Bernardi et al. 2009). The different types of drug distribution in lipid-core nanocapsules can be determined according to the log D value (Oliveira et al. 2012). Taking into account that the lipid-core nanocapsules are promising carriers to macrophage uptake (Poletto et al. 2012), this work reports the development of PYR-loaded lipid-core
Fig. 1 Type of biodegradable nanoparticle. Lipid nanoparticles involve liposomes (a), solid lipid nanoparticles (b), nanostructured lipid carriers (c); while the polymeric nanoparticles include nanospheres (d), polymeric nanocapsules (e) and hybrid biodegradable nanoparticle, called lipid-core nanocapsules (f)
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nanocapsules and its in vivo effect against mice acutely infected with RH strain of T. gondii.
Materials and methods Materials Pyrimethamine, sorbitan monostearate (Span® 60) and polysorbate 80 (Tween® 80) were obtained from Sigma-Aldrich (Strasbourg, France). Poly(ε-caprolactone) (CAPA® 6500, Mn 50 kDa, Perstorp UK Ltd) was kindly gifted by Perstorp Química do Brasil. Caprylic/capric triglyceride was acquired from Brasquim (Porto Alegre, Brazil). All solvents used were of analytical or pharmaceutical grade. Acetonitrile and methanol were of chromatographic grade. All solutions were prepared using ultrapure MILLI-Q® water. Preparation of pyrimethamine-loaded lipid-core nanocapsules Lipid-core nanocapsules were prepared by the self-assembling method previously reported (Jornada et al. 2012). The organic phase consisting of 0.10 g poly(ε-caprolactone) (PCL), 0.038 g sorbitan monostearate (MS), 0.160 mL oil capric/ caprylic triglyceride (CCT) and 27 mL acetone was maintained under stirring at 40 °C. In another vessel, the aqueous phase containing 0.077 g of polysorbate 80 (PS 80) in 53 mL of water. The organic phase was poured slowly into the aqueous phase under moderate agitation at room temperature. After 10 min, the organic solvent was eliminated and the final volume adjusted to 10 mL under reduced pressure. PYRloaded lipid-core nanocapsules (PYR-LNC) were prepared with 0.5 mg mL−1 of drug. Control formulation (LNC) was
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prepared omitting the drug. Aqueous suspension containing PYR and the hydrophilic surfactant (SU-PYR) was also prepared for comparison (Table 1).
determined after dispersing the sample in 10 mM NaCl aqueous solution, using the aforementioned Zetasizer® Nano ZS. pH measurements
Analytical method Pyrimethamine quantification in the samples was evaluated by HPLC (UV–Vis detector, Perkin Elmer) using C18 reverse phase column (Nova-Pak 3.9×300 mm, Waters) adapting a previously reported method (Boca et al. 2005). The mobile phase used consisted of acetonitrile/water (50:50, v/v) containing 0.1 % phosphoric acid (apparent pH 3.0 adjusted with 1 M NaOH). Flow-rate was 1.2 mL min−1; the volume injection was 50 μL and detection at 270 nm. The stock solution was carried out in acetonitrile at a concentration of 500 μg mL−1. The total concentration of PYR in each formulation was determined by HPLC after dissolution in 0.1 mL of the sample in 9.9 mL of acetonitrile. The concentration of free drug in the system (nonassociated) was analyzed by an ultrafiltration/centrifugation technique (Microcon® Centrifugal Filter Units NMWL, 10 (kDa) Millipore®, 5,000 rpm/5 min) (Poletto et al. 2008). The ultrafiltrate obtained from 0.3 mL of the suspensions was determined by HPLC. The encapsulation efficiency (EE) was determined in percentage by the equation: [(total drug−free drug)/total drug]×100. Lipid-core nanocapsule size distribution analysis Mean size distribution was determined by laser diffractometry in Mastersizer® 2000 equipment (Malvern Instruments) using distilled water for the dispersion of the samples (Keck and Muller 2008). Mean hydrodynamic diameter and zeta potential of lipid-core nanocapsule The mean particle size (z-average) and polydispersity index (PDI) values of the formulations were determined by photon correlation spectroscopy (PCS) using a Zetasizer® Nano ZS (Malvern Instruments) after diluting the samples with water (Milli-Q®). The zeta potential values of nanocapsule were Table 1 Composition of the formulations Formulation
PCL (g)
SM (g)
CCT (mL)
PYR (g)
PS 80 (g)
LNC PYR-LNC SU-PYR
0.1 0.1 –
0.038 0.038 –
0.16 0.16 –
– 0.005 0.005
0.077 0.077 0.077
Quantities of material per 10 mL of final formulation (after the evaporation step): PCL poly(ε-caprolactone); SM sorbitan monostearate; CCT capric/caprylic triglyceride; PYR pyrimethamine, PS 80 polysorbate 80
The pH values were determined at 25 °C directly in suspensions using a potentiometer (Denver) previously calibrated with buffer solution pH 4.0 and 7.0. The measurements were made in triplicate. Analysis by multiple light scattering The stability of the formulations was evaluated by optical characterization based on the multiple light scattering in Turbiscan™ Lab equipment (Formulaction, France) (Bordes et al. 2002). The principle measurement is based on the variation of the volume fraction or particle diameter resulting in a variation of the transmission signals (T) and backscatter (backscattering, BS). Samples were added in a transparent borosilicate glass cuvette (25 mm×55 mm) and acclimatized at 25 °C. The samples were scanned from the base to the top by an infrared pulse (880 nm) every 40 μm of the cuvette. A complete scan of the sample was made every 5 min over a period of 1 h. Field-emission scanning electron microscopy (FESEM) PYR-LNC was diluted in ultrapure water in a proportion of 1:3 (v/v). Diluted suspension was placed in coverslips coated with poly-L -lysine for 20 min and then washed with water. Coverslips were allowed to dry at room temperature and coated with a 10-nm thick gold coat and observed in FESEM at 5.0 kV at a working distance of 5 mm. Cell toxicity experiments The effect of LNC, PYR-LNC, and SU-PYR on the epithelial LLC-MK2 cells (kidney, Rhesus monkey, Macaca mulata— ATCC CCL7, Rockville, MD/USA) or mouse peritoneal macrophages was evaluated by the MTS assay. For LLC-MK2 assay 2×105 cells were placed in a 96-well tissue plate and for macrophage assay peritoneal macrophages from CF1 mice were harvested by washing them with RPMI medium (Gibco) and placed in 96-well tissue plate. Cells were treated for 72 h with different concentrations of LNC, PYR-LNC (1– 20 μM), SU-PYR (1–20 μM), and PYR diluted in DMSO (stock solution 5 mM; 1–10 μM) added to RPMI medium and 2 % FBS. In order to exclude any toxicity against host cells due to DMSO, for the experiments with PYR in DMSO cells treated with concentrations of 0.1 and 0.2 % were used as control. At the end of incubation time, cells were washed with PBS, each well was filled with 100 μL of 10 mM glucose in PBS and 20 μL of MTS/PMS (20:1) reagent (Promega,
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Madison WI, USA) was added. This methodology is based in the reduction of the MTS salt ((3-(4,5-dimethylthiazol-2-yl)5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H -tetrazolium) by mitochondrial dehydrogenases to the soluble formazan dye. The absorbance was read at 490 nm after 3 h of incubation and cytotoxicity was calculated as the percentage of viable cells versus untreated cells (control, 100 %).
chronicle infection). Mice presenting symptoms of disease in a period of 30 days after inoculation of the macerate were investigated for the presence of i.p. tachyzoites (positive bioassay). Mice without signs of disease or morbidity were considered free of disease (negative bioassay).
Parasite and mice
Non-infected female CF1 mice were treated i.p. with the same doses used above for ten consecutive days. Mice were weighed at the first day of treatment (day 1) up to the first day after the end of the treatment (day 11). At this time, mice were euthanized and the blood obtained by cardiac puncture. The serum levels of urea, glutamic oxalacetic transaminase (SGOT) and alkaline phosphatase (ALP) were determined using kits of diagnostic (Labtest Diagnostica S.A., Minas Gerais, Brazil). Two animals were used for each group of treatment.
Tachyzoites of the RH strain were used for the in vitro and in vivo tests and were obtained from the peritoneal cavities of CF1 mice 2 days after infection. Five-week-old female CF1 mice weighing 18–22 g at the beginning of the experiment were used for the in vivo experiments. Drinking water and food were given ad libitum. The experimental protocols for animal use in this study were approved by the Institutional Ethics Committee for Animal Use (approval ID: CEUA/CCS/UFRJ/IBCCF 99 and 100).
In vivo toxicity study
In vivo experiments
Results
For the in vivo study, mice were infected intraperitoneally (i.p.) with tachyzoites of the RH strain of T. gondii. Treatment was initiated 24 h post infection and groups of three or four mice were housed per cage and arbitrarily assigned to administration with LNC, PYR-LNC, SU-PYR, or left untreated as the control group. The chosen dosages of PYR were based in quantities of the drug that lead to low expansion in the mice survival compared to untreated animals, thus making possible to evaluate the improvement of LNC-PYR. The treatment lasted for 10 days and the drugs were administrated i.p. once a day. The mice and the mortality rates were monitored for a period of 60 days, at the end the surviving mice were euthanized and cyst brain investigated as described below. The survival curves and the median survival (day at which fractional survival equals 50 %) were calculated using the product limit method of Kaplan and Meier, and the comparison of the survival curves was carried out using the log-rank (Mantel– Cox) test in GraphPad Prism 5.0 (GraphPad Software Inc.). P ≤0.05 was considered statistically significant.
Physiochemical characterization of nanocapsules
Cyst brain evaluation and bioassay Mice brains were homogenized with 1 mL of sterile saline by serial passages through different needles (18, 20, 22, and 25G). For cyst number evaluation, three drops of 10 μL of brain suspensions were placed on slides and microscopically counted. The number of cysts per brain was calculated by multiplying the mean of three samples (mean of cysts×1, 000 μL/10 μL). Afterwards, the mouse brain homogenate was i.p. inoculated in a new mouse to verify the viability of the cysts (when visualized) or to confirm mice cure (lack of
PYR-LNC and drug-unloaded LNC consisting of 0.1 % PCL, 1.6 % caprylic/capric triglyceride, 0.38 % sorbitan monostearate and 0.77 % polysorbate 80 were prepared using the self-assembling method previously reported (Jordana et al. 2012). Macroscopically, LNC and PYR-LNC formulations had homogeneous and milky aspect. High-resolution scanning electron microscopy analysis of PYR-LNC showed spherically shaped particles with regular size (Fig. 2a). The particle size distributions obtained for different batches of LNC and PYR-LNC showed monomodal profiles with particle sizes below 1 μm (Fig. 2b, c). The average diameters (D 4, 3) determined by laser diffractometry of PYR-LNC and LNC were 273±17.5 nm and 150±5 nm, respectively (Table 2). According to the dynamic light scattering analysis, both formulations also presented monomodal profiles (data not shown). The z -average diameters were close to 200 nm (Table 2), being in agreement with those previously reported for similar formulations (Venturini et al. 2011). The polydispersity index of the LNC and PYR-LNC were below 0.11 demonstrating narrow size distributions. The pH value was 5.42±0.55 for LNC and 6.86±0.16 for PYR-LNC. Zeta potentials of the LNC and PYR-LNC formulations were −7.1± 0.9 mV and −10.9±0.1 mV, respectively (Table 3). The quantification method for PYR was linear with a correlation coefficient (r) of 0.995 for the concentration range studied (1–11 μg mL−1). Peak retention time of PYR was observed in the range from 1.6 to 1.8 min. The linear regression equation to determine the x (PRY-concentration) factor is: y =15,368.45+105,269.20x (n =6). The analytical method
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Fig. 2 High-resolution field-emission scanning electron microscopy image showing particles of PYR-LNC formulation (a). Particle size distribution profiles of LNC (b ) and PYR-LNC (c ) obtained by laser
diffactometry (n =3). Relative backscattering obtained by Turbiscan Lab® after 1 h of analysis of the formulations LNC (d) e PYR-LNC (e)
was selective and specific since the other components did not interfere in the analysis. The method showed reproducibility having a relative standard deviation lower than 4 %. The concentration of PYR in the PYR-LNC formulation was 424.6±19.5 μg mL−1 with an encapsulation efficiency of 92.5±3.8 % (Table 3). The physical stability of the formulations was analyzed by multiple light scattering using a Turbiscan Lab®. After 1 h of
analysis, the variation of the backscattering at the top and the bottom of the cuvettes was lower than 5 %. The biggest particles are responsible for the variations in backscattering at the top in the LNC formulation (Fig. 2d) and at the bottom in the PYR-LNC showing those minimal tendencies of migration (Fig. 2e). At the middle of the cuvettes, no change was observed for both the formulations indicating that the particles did not agglomerate.
Table 2 Physicochemical characterization of drug-unloaded formulation (LNC) and pyrimethamine-loaded nanocapsules (PYR-LNC) (0.5 mg mL−1) Formulation
D[4,3]a (nm)
d(0,5)n a (nm)
Spana
z-average diameterb (nm)
PDIb
LNC PYR-LNC
150 (± 5) 273 (± 17)
66(±0.003) 78 (±0.009)
1.63 (±0.10) 1.34 (±0.14)
179 (±10) 184 (±17)
0.06(±0.01) 0.10 (±0.02)
a
D[4,3]=Volume weighted mean diameter; d(0,5)n =particle diameter determined at 50th percentile of the undersize particle distribution curve obtained by number of particles, and span (polydispersity) obtained by laser diffractometry
b Mean hydrodynamic diameter obtained by dynamic light scattering using the method of cumulants; PDI polydispersity index (variance) determined by dynamic light scattering
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Table 3 Physicochemical characterization of drug-unloaded formulation (LNC) and pyrimethamine-loaded nanocapsules (PYR-LNC) (0.5 mg mL−1), and drug-aqueous suspension (SU-PYR) Formulation
ζ (mV)
pH
Drug content (μg mL−1)
E.E (%)
LNC PYR-LNC SU-PYR
−7.1 (± 0.9) −10.9 (± 0.1) nd
5.42(± 0.55) 6.86(± 0.16) 6.87(± 0.53)
nd 424.6(±19.5) nd
– 92.5(±3.8) –
ζ zeta potential by analysis by light scattering; EE efficiency of encapsulation, nd not determined
Cytotoxicity against cells In order to evaluate the cytotoxicity of LNC, PYR-LNC or SU-PYR, LLC-MK2 cells and mouse peritoneal macrophages were incubated with the formulations at concentrations of 1– 20 μM (0.25-5.0 μg mL−1) for 72 h (Table 4). The viability evaluated by MTS assay showed that none of the preparations affected peritoneal macrophages viability. LNC did not affect LLC-MK2, but PYR-LNC, SU-PYR and PYR dissolved in DMSO inhibited LLC-MK2 proliferation with TC50 values of 6.0, 7.7, and 6.0 μM, respectively. Statistical analysis (Mann– Whitney test) showed that the effect of PYR-LNC against LLC-MK2 did not have significant difference compared to Su-PYR (P =0.82) and PYR in DMSO (P =0.82). Effect in a mouse model of acute toxoplasmosis Infected mice were i.p. administrated with LNC, PYR-LNC, and SU-PYR for 10 days (Fig. 3). Untreated mice and mice administrated with LNC died after 8 days of treatment. The treatment with PYR-LNC showed better results than SUPYR, mice treated with 5 mg/kg/day of PYR-LNC had survival rate similar to the treatment with 7.5 mg/kg/day of SUPYR resulting in a median survivals of 14 and 12 days,
Table 4 Toxicity of preparations against LLC-MK2 cells and mouse peritoneal macrophages in vitro Formulation
LLC-MK2 TC50a
LNC PYR-LNC Su-PYR PYR DMSO
>20 μM e.q.vb (5.0 μg. mL−1) 6.0±1.2 μM (1.5 μg. mL−1) 7.7±2.8 μM (1.9 μg. mL−1) 6.1±1.4 μM (1.5 μg. mL−1 )
Mouse peritoneal macrophage TC50a >20 μM e.q.v (5.0 μ g. mL−1) >20 μM e.q.v (5.0 μ g. mL−1) >20 μM e.q.v (5.0 μ g. mL−1) –
a TC50 is the concentration necessary to inhibit 50 % of proliferation after 72 h of treatment b e.q.v means equivalent volume of PYR-LNC. The difference between groups for LLC-MK2 assays was not statistically significant
respectively. Log-rank test analysis showed that the difference of survival between the groups treated with 5 mg/kg/day PYR-LNC and 5 mg/kg/day SU-PYR was statistically significant (P
ORIGINAL PAPER
Pyrimethamine-loaded lipid-core nanocapsules to improve drug efficacy for the treatment of toxoplasmosis Kenia Pissinate & Érica dos Santos Martins-Duarte & Scheila Rezende Schaffazick & Catiúscia Padilha de Oliveira & Rossiane Cláudia Vommaro & Sílvia Stanisçuaski Guterres & Adriana Raffin Pohlmann & Wanderley de Souza
Received: 19 August 2013 / Accepted: 4 November 2013 / Published online: 29 November 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract We propose an innovative product based on the nanoencapsulation of pyrimethamine (PYR), aiming an improvement of drug efficacy for the treatment of toxoplasmosis. The in vitro cytotoxicity effect of encapsulated PYR and PYR-colloidal suspension was concomitantly evaluated against LLC-MK2 lineage and mouse peritoneal macrophage showing that the cells had similar tolerance for both PYR encapsulated or in the aqueous suspension. CF1 mice acutely infected with tachyzoites of Toxoplasma gondii RH strain treated with different doses (5.0–10 mg/kg/day) of PYRnanocapsules had survival rate higher than the animals treated with the same doses of non-encapsulated PYR. Thus,
Kenia Pissinate and Érica dos Santos Martins-Duarte contributed equally to this work K. Pissinate : É. dos Santos Martins-Duarte : S. R. Schaffazick : R. C. Vommaro : W. de Souza Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil É. dos Santos Martins-Duarte : R. C. Vommaro : W. de Souza Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil S. R. Schaffazick : C. P. de Oliveira : S. S. Guterres : A. R. Pohlmann Programa de Pós-Graduação em Ciências Farmacêuticas, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil K. Pissinate (*) : A. R. Pohlmann Departamento de Química Orgânica, Instituto de Química, Universidade Federal do Rio Grande do Sul, Caixa Postal 15003, Av. Bento Gonçalves, 9500, Porto Alegre 91501-970, Brazil e-mail: [email protected] W. de Souza Instituto Nacional de Metrologia e Qualidade Industrial—Inmetro, Rio de Janeiro, Brazil
encapsulation of PYR improved the efficacy of this drug against an acute model of toxoplasmosis in mice and can be considered an alternative for reducing the dose of PYR, which, in turn, could also reduce the side effects associated to the treatment.
Introduction Toxoplasma gondii is a world-spread protozoan and a significant pathogen associated with many medical areas, such as: pediatrics, ophthalmology, and infectology, regarding immunocompromised patients, principally the ones with AIDS. Cerebral toxoplasmosis is the most significant and common clinical presentation in immunocompromised patients and one of the main causes of morbidity and mortality in these patients (Montoya and Liesenfeld 2004). Pyrimethamine (PYR) is the most significant drug for the treatment of toxoplasmosis and its combination with sulfadiazine is the first-choice treatment of the disease. The mechanism of action of PYR consists in the inhibition of the enzyme dihydrofolate reductase (DHFR), which is essential for purine synthesis in parasites and humans (Anderson 2005). When patients do not tolerate the sulfa treatment, PYR combined with clindamycin or azithromycin is administrated as a second-line therapy. However, PYR administration is frequently associated with many side effects such as anorexia, vomiting, abdominal pain, diarrhea, megablastic anemia, leucopenia, and thrombocytopenia that can compromise the treatment continuation (Weniger 1979). To minimize the side effects, the concurrent administration of folinic acid is also used (Nissapatorn and Sawangjaroen 2011). New formulations for old drugs have been used as a strategy to improve the treatment of diverse infectious diseases. A new formulation combining enhancement of efficacy and reduced side effects is desirable.
556
Nanocarriers have received considerable attention as potential drug delivery vehicles because they take the ultradisperse drugs to the specific intracellular targets, which are surrounded by complex physiological barriers (Prieto et al. 2006). Biodegradable polymeric nanoparticles are drug carrier systems which have distribution diameters of less than 1 μm and are widely researched for controlled release of drugs and for enhancing drug bioavailability (Pohlmann et al. 2013; Müller et al. 2007). Those nanoparticles are usually divided in lipid nanoparticles and polymeric nanoparticles. The lipid nanoparticles involve liposomes (Fig. 1a), solid lipid nanoparticles (Fig. 1b), and nanostructured lipid carriers (Fig. 1c), while the polymeric nanoparticles include nanocapsules (Fig. 1d) and nanospheres (Fig. 1e). Recently, we developed a hybrid biodegradable nanoparticle, called lipid-core nanocapsules. The lipid-core nanocapsules are vesicular structures constituted by a lipid-core composed of medium chain triacylglyceride and sorbitan monostearate, surrounded by a polymeric wall in general composed of poly(ε-caprolactone) and stabilized by polysorbate 80 at the particle/water interface (Fig. 1f) (Couvreur et al. 2002; Mora-Huertas et al. 2010). Lipid-core nanocapsule aqueous formulations are formed by the mechanism of self-assembly, based on the infinite dilution of the aggregation state of raw materials in the organic phase (Jornada et al. 2012). Hydrophobic drug incorporation to the internal oily cavity has been reported in various studies as a promising drug targeting strategy (Bernardi et al. 2009). The different types of drug distribution in lipid-core nanocapsules can be determined according to the log D value (Oliveira et al. 2012). Taking into account that the lipid-core nanocapsules are promising carriers to macrophage uptake (Poletto et al. 2012), this work reports the development of PYR-loaded lipid-core
Fig. 1 Type of biodegradable nanoparticle. Lipid nanoparticles involve liposomes (a), solid lipid nanoparticles (b), nanostructured lipid carriers (c); while the polymeric nanoparticles include nanospheres (d), polymeric nanocapsules (e) and hybrid biodegradable nanoparticle, called lipid-core nanocapsules (f)
Parasitol Res (2014) 113:555–564
nanocapsules and its in vivo effect against mice acutely infected with RH strain of T. gondii.
Materials and methods Materials Pyrimethamine, sorbitan monostearate (Span® 60) and polysorbate 80 (Tween® 80) were obtained from Sigma-Aldrich (Strasbourg, France). Poly(ε-caprolactone) (CAPA® 6500, Mn 50 kDa, Perstorp UK Ltd) was kindly gifted by Perstorp Química do Brasil. Caprylic/capric triglyceride was acquired from Brasquim (Porto Alegre, Brazil). All solvents used were of analytical or pharmaceutical grade. Acetonitrile and methanol were of chromatographic grade. All solutions were prepared using ultrapure MILLI-Q® water. Preparation of pyrimethamine-loaded lipid-core nanocapsules Lipid-core nanocapsules were prepared by the self-assembling method previously reported (Jornada et al. 2012). The organic phase consisting of 0.10 g poly(ε-caprolactone) (PCL), 0.038 g sorbitan monostearate (MS), 0.160 mL oil capric/ caprylic triglyceride (CCT) and 27 mL acetone was maintained under stirring at 40 °C. In another vessel, the aqueous phase containing 0.077 g of polysorbate 80 (PS 80) in 53 mL of water. The organic phase was poured slowly into the aqueous phase under moderate agitation at room temperature. After 10 min, the organic solvent was eliminated and the final volume adjusted to 10 mL under reduced pressure. PYRloaded lipid-core nanocapsules (PYR-LNC) were prepared with 0.5 mg mL−1 of drug. Control formulation (LNC) was
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prepared omitting the drug. Aqueous suspension containing PYR and the hydrophilic surfactant (SU-PYR) was also prepared for comparison (Table 1).
determined after dispersing the sample in 10 mM NaCl aqueous solution, using the aforementioned Zetasizer® Nano ZS. pH measurements
Analytical method Pyrimethamine quantification in the samples was evaluated by HPLC (UV–Vis detector, Perkin Elmer) using C18 reverse phase column (Nova-Pak 3.9×300 mm, Waters) adapting a previously reported method (Boca et al. 2005). The mobile phase used consisted of acetonitrile/water (50:50, v/v) containing 0.1 % phosphoric acid (apparent pH 3.0 adjusted with 1 M NaOH). Flow-rate was 1.2 mL min−1; the volume injection was 50 μL and detection at 270 nm. The stock solution was carried out in acetonitrile at a concentration of 500 μg mL−1. The total concentration of PYR in each formulation was determined by HPLC after dissolution in 0.1 mL of the sample in 9.9 mL of acetonitrile. The concentration of free drug in the system (nonassociated) was analyzed by an ultrafiltration/centrifugation technique (Microcon® Centrifugal Filter Units NMWL, 10 (kDa) Millipore®, 5,000 rpm/5 min) (Poletto et al. 2008). The ultrafiltrate obtained from 0.3 mL of the suspensions was determined by HPLC. The encapsulation efficiency (EE) was determined in percentage by the equation: [(total drug−free drug)/total drug]×100. Lipid-core nanocapsule size distribution analysis Mean size distribution was determined by laser diffractometry in Mastersizer® 2000 equipment (Malvern Instruments) using distilled water for the dispersion of the samples (Keck and Muller 2008). Mean hydrodynamic diameter and zeta potential of lipid-core nanocapsule The mean particle size (z-average) and polydispersity index (PDI) values of the formulations were determined by photon correlation spectroscopy (PCS) using a Zetasizer® Nano ZS (Malvern Instruments) after diluting the samples with water (Milli-Q®). The zeta potential values of nanocapsule were Table 1 Composition of the formulations Formulation
PCL (g)
SM (g)
CCT (mL)
PYR (g)
PS 80 (g)
LNC PYR-LNC SU-PYR
0.1 0.1 –
0.038 0.038 –
0.16 0.16 –
– 0.005 0.005
0.077 0.077 0.077
Quantities of material per 10 mL of final formulation (after the evaporation step): PCL poly(ε-caprolactone); SM sorbitan monostearate; CCT capric/caprylic triglyceride; PYR pyrimethamine, PS 80 polysorbate 80
The pH values were determined at 25 °C directly in suspensions using a potentiometer (Denver) previously calibrated with buffer solution pH 4.0 and 7.0. The measurements were made in triplicate. Analysis by multiple light scattering The stability of the formulations was evaluated by optical characterization based on the multiple light scattering in Turbiscan™ Lab equipment (Formulaction, France) (Bordes et al. 2002). The principle measurement is based on the variation of the volume fraction or particle diameter resulting in a variation of the transmission signals (T) and backscatter (backscattering, BS). Samples were added in a transparent borosilicate glass cuvette (25 mm×55 mm) and acclimatized at 25 °C. The samples were scanned from the base to the top by an infrared pulse (880 nm) every 40 μm of the cuvette. A complete scan of the sample was made every 5 min over a period of 1 h. Field-emission scanning electron microscopy (FESEM) PYR-LNC was diluted in ultrapure water in a proportion of 1:3 (v/v). Diluted suspension was placed in coverslips coated with poly-L -lysine for 20 min and then washed with water. Coverslips were allowed to dry at room temperature and coated with a 10-nm thick gold coat and observed in FESEM at 5.0 kV at a working distance of 5 mm. Cell toxicity experiments The effect of LNC, PYR-LNC, and SU-PYR on the epithelial LLC-MK2 cells (kidney, Rhesus monkey, Macaca mulata— ATCC CCL7, Rockville, MD/USA) or mouse peritoneal macrophages was evaluated by the MTS assay. For LLC-MK2 assay 2×105 cells were placed in a 96-well tissue plate and for macrophage assay peritoneal macrophages from CF1 mice were harvested by washing them with RPMI medium (Gibco) and placed in 96-well tissue plate. Cells were treated for 72 h with different concentrations of LNC, PYR-LNC (1– 20 μM), SU-PYR (1–20 μM), and PYR diluted in DMSO (stock solution 5 mM; 1–10 μM) added to RPMI medium and 2 % FBS. In order to exclude any toxicity against host cells due to DMSO, for the experiments with PYR in DMSO cells treated with concentrations of 0.1 and 0.2 % were used as control. At the end of incubation time, cells were washed with PBS, each well was filled with 100 μL of 10 mM glucose in PBS and 20 μL of MTS/PMS (20:1) reagent (Promega,
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Madison WI, USA) was added. This methodology is based in the reduction of the MTS salt ((3-(4,5-dimethylthiazol-2-yl)5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H -tetrazolium) by mitochondrial dehydrogenases to the soluble formazan dye. The absorbance was read at 490 nm after 3 h of incubation and cytotoxicity was calculated as the percentage of viable cells versus untreated cells (control, 100 %).
chronicle infection). Mice presenting symptoms of disease in a period of 30 days after inoculation of the macerate were investigated for the presence of i.p. tachyzoites (positive bioassay). Mice without signs of disease or morbidity were considered free of disease (negative bioassay).
Parasite and mice
Non-infected female CF1 mice were treated i.p. with the same doses used above for ten consecutive days. Mice were weighed at the first day of treatment (day 1) up to the first day after the end of the treatment (day 11). At this time, mice were euthanized and the blood obtained by cardiac puncture. The serum levels of urea, glutamic oxalacetic transaminase (SGOT) and alkaline phosphatase (ALP) were determined using kits of diagnostic (Labtest Diagnostica S.A., Minas Gerais, Brazil). Two animals were used for each group of treatment.
Tachyzoites of the RH strain were used for the in vitro and in vivo tests and were obtained from the peritoneal cavities of CF1 mice 2 days after infection. Five-week-old female CF1 mice weighing 18–22 g at the beginning of the experiment were used for the in vivo experiments. Drinking water and food were given ad libitum. The experimental protocols for animal use in this study were approved by the Institutional Ethics Committee for Animal Use (approval ID: CEUA/CCS/UFRJ/IBCCF 99 and 100).
In vivo toxicity study
In vivo experiments
Results
For the in vivo study, mice were infected intraperitoneally (i.p.) with tachyzoites of the RH strain of T. gondii. Treatment was initiated 24 h post infection and groups of three or four mice were housed per cage and arbitrarily assigned to administration with LNC, PYR-LNC, SU-PYR, or left untreated as the control group. The chosen dosages of PYR were based in quantities of the drug that lead to low expansion in the mice survival compared to untreated animals, thus making possible to evaluate the improvement of LNC-PYR. The treatment lasted for 10 days and the drugs were administrated i.p. once a day. The mice and the mortality rates were monitored for a period of 60 days, at the end the surviving mice were euthanized and cyst brain investigated as described below. The survival curves and the median survival (day at which fractional survival equals 50 %) were calculated using the product limit method of Kaplan and Meier, and the comparison of the survival curves was carried out using the log-rank (Mantel– Cox) test in GraphPad Prism 5.0 (GraphPad Software Inc.). P ≤0.05 was considered statistically significant.
Physiochemical characterization of nanocapsules
Cyst brain evaluation and bioassay Mice brains were homogenized with 1 mL of sterile saline by serial passages through different needles (18, 20, 22, and 25G). For cyst number evaluation, three drops of 10 μL of brain suspensions were placed on slides and microscopically counted. The number of cysts per brain was calculated by multiplying the mean of three samples (mean of cysts×1, 000 μL/10 μL). Afterwards, the mouse brain homogenate was i.p. inoculated in a new mouse to verify the viability of the cysts (when visualized) or to confirm mice cure (lack of
PYR-LNC and drug-unloaded LNC consisting of 0.1 % PCL, 1.6 % caprylic/capric triglyceride, 0.38 % sorbitan monostearate and 0.77 % polysorbate 80 were prepared using the self-assembling method previously reported (Jordana et al. 2012). Macroscopically, LNC and PYR-LNC formulations had homogeneous and milky aspect. High-resolution scanning electron microscopy analysis of PYR-LNC showed spherically shaped particles with regular size (Fig. 2a). The particle size distributions obtained for different batches of LNC and PYR-LNC showed monomodal profiles with particle sizes below 1 μm (Fig. 2b, c). The average diameters (D 4, 3) determined by laser diffractometry of PYR-LNC and LNC were 273±17.5 nm and 150±5 nm, respectively (Table 2). According to the dynamic light scattering analysis, both formulations also presented monomodal profiles (data not shown). The z -average diameters were close to 200 nm (Table 2), being in agreement with those previously reported for similar formulations (Venturini et al. 2011). The polydispersity index of the LNC and PYR-LNC were below 0.11 demonstrating narrow size distributions. The pH value was 5.42±0.55 for LNC and 6.86±0.16 for PYR-LNC. Zeta potentials of the LNC and PYR-LNC formulations were −7.1± 0.9 mV and −10.9±0.1 mV, respectively (Table 3). The quantification method for PYR was linear with a correlation coefficient (r) of 0.995 for the concentration range studied (1–11 μg mL−1). Peak retention time of PYR was observed in the range from 1.6 to 1.8 min. The linear regression equation to determine the x (PRY-concentration) factor is: y =15,368.45+105,269.20x (n =6). The analytical method
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Fig. 2 High-resolution field-emission scanning electron microscopy image showing particles of PYR-LNC formulation (a). Particle size distribution profiles of LNC (b ) and PYR-LNC (c ) obtained by laser
diffactometry (n =3). Relative backscattering obtained by Turbiscan Lab® after 1 h of analysis of the formulations LNC (d) e PYR-LNC (e)
was selective and specific since the other components did not interfere in the analysis. The method showed reproducibility having a relative standard deviation lower than 4 %. The concentration of PYR in the PYR-LNC formulation was 424.6±19.5 μg mL−1 with an encapsulation efficiency of 92.5±3.8 % (Table 3). The physical stability of the formulations was analyzed by multiple light scattering using a Turbiscan Lab®. After 1 h of
analysis, the variation of the backscattering at the top and the bottom of the cuvettes was lower than 5 %. The biggest particles are responsible for the variations in backscattering at the top in the LNC formulation (Fig. 2d) and at the bottom in the PYR-LNC showing those minimal tendencies of migration (Fig. 2e). At the middle of the cuvettes, no change was observed for both the formulations indicating that the particles did not agglomerate.
Table 2 Physicochemical characterization of drug-unloaded formulation (LNC) and pyrimethamine-loaded nanocapsules (PYR-LNC) (0.5 mg mL−1) Formulation
D[4,3]a (nm)
d(0,5)n a (nm)
Spana
z-average diameterb (nm)
PDIb
LNC PYR-LNC
150 (± 5) 273 (± 17)
66(±0.003) 78 (±0.009)
1.63 (±0.10) 1.34 (±0.14)
179 (±10) 184 (±17)
0.06(±0.01) 0.10 (±0.02)
a
D[4,3]=Volume weighted mean diameter; d(0,5)n =particle diameter determined at 50th percentile of the undersize particle distribution curve obtained by number of particles, and span (polydispersity) obtained by laser diffractometry
b Mean hydrodynamic diameter obtained by dynamic light scattering using the method of cumulants; PDI polydispersity index (variance) determined by dynamic light scattering
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Table 3 Physicochemical characterization of drug-unloaded formulation (LNC) and pyrimethamine-loaded nanocapsules (PYR-LNC) (0.5 mg mL−1), and drug-aqueous suspension (SU-PYR) Formulation
ζ (mV)
pH
Drug content (μg mL−1)
E.E (%)
LNC PYR-LNC SU-PYR
−7.1 (± 0.9) −10.9 (± 0.1) nd
5.42(± 0.55) 6.86(± 0.16) 6.87(± 0.53)
nd 424.6(±19.5) nd
– 92.5(±3.8) –
ζ zeta potential by analysis by light scattering; EE efficiency of encapsulation, nd not determined
Cytotoxicity against cells In order to evaluate the cytotoxicity of LNC, PYR-LNC or SU-PYR, LLC-MK2 cells and mouse peritoneal macrophages were incubated with the formulations at concentrations of 1– 20 μM (0.25-5.0 μg mL−1) for 72 h (Table 4). The viability evaluated by MTS assay showed that none of the preparations affected peritoneal macrophages viability. LNC did not affect LLC-MK2, but PYR-LNC, SU-PYR and PYR dissolved in DMSO inhibited LLC-MK2 proliferation with TC50 values of 6.0, 7.7, and 6.0 μM, respectively. Statistical analysis (Mann– Whitney test) showed that the effect of PYR-LNC against LLC-MK2 did not have significant difference compared to Su-PYR (P =0.82) and PYR in DMSO (P =0.82). Effect in a mouse model of acute toxoplasmosis Infected mice were i.p. administrated with LNC, PYR-LNC, and SU-PYR for 10 days (Fig. 3). Untreated mice and mice administrated with LNC died after 8 days of treatment. The treatment with PYR-LNC showed better results than SUPYR, mice treated with 5 mg/kg/day of PYR-LNC had survival rate similar to the treatment with 7.5 mg/kg/day of SUPYR resulting in a median survivals of 14 and 12 days,
Table 4 Toxicity of preparations against LLC-MK2 cells and mouse peritoneal macrophages in vitro Formulation
LLC-MK2 TC50a
LNC PYR-LNC Su-PYR PYR DMSO
>20 μM e.q.vb (5.0 μg. mL−1) 6.0±1.2 μM (1.5 μg. mL−1) 7.7±2.8 μM (1.9 μg. mL−1) 6.1±1.4 μM (1.5 μg. mL−1 )
Mouse peritoneal macrophage TC50a >20 μM e.q.v (5.0 μ g. mL−1) >20 μM e.q.v (5.0 μ g. mL−1) >20 μM e.q.v (5.0 μ g. mL−1) –
a TC50 is the concentration necessary to inhibit 50 % of proliferation after 72 h of treatment b e.q.v means equivalent volume of PYR-LNC. The difference between groups for LLC-MK2 assays was not statistically significant
respectively. Log-rank test analysis showed that the difference of survival between the groups treated with 5 mg/kg/day PYR-LNC and 5 mg/kg/day SU-PYR was statistically significant (P
