Generating nanoparticles containing a new 4-nitrobenzaldehyde thiosemicarbazone compound with antileishmanial activity

Materials Science and Engineering C 69 (2016) 1159–1166

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Generating nanoparticles containing a new 4-nitrobenzaldehyde thiosemicarbazone compound with antileishmanial activity Elizandra Aparecida Britta a, Cleuza Conceição da Silva b, Adley Forti Rubira b, Celso Vataru Nakamura a,⁎, Redouane Borsali c,⁎⁎ a b c

Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Estadual de Maringá, Brazil Departamento de Química, Universidade Estadual de Maringá, Brazil Centro de Pesquisas em Macromoléculas Vegetais, CERMAV, Grenoble, France

a r t i c l e

i n f o

Article history: Received 11 May 2016 Received in revised form 22 July 2016 Accepted 7 August 2016 Available online 8 August 2016 Keywords: Block copolymers Nanoparticles 4-Nitrobenzaldehyde thiosemicarbazone Antileishmanial activity Leishmania amazonensis

a b s t r a c t Thiosemicarbazones are an important class of compounds that have been extensively studied in recent years, mainly because of their broad profile of pharmacological activity. A new 4-nitrobenzaldehyde thiosemicarbazone compound (BZTS) that was derived from S-limonene has been demonstrated to have significant antiprotozoan activity. However, the hydrophobic characteristic of BZTS limits its administration and results in low oral bioavailability. In the present study, we proposed the synthesis of nanoparticle-based block copolymers that can encapsulate BZTS, with morphological evaluation of the nanoparticle suspensions being performed by transmission and cryo-transmission electronic microscopy. The mean particle sizes of the nanoparticle suspensions were determined by static light and dynamic light scattering (SLS/DLS), and the hydrodynamic radius (Rh) was determined using the Stokes-Einstein equation. The zeta potential (ζ) and polydispersity index (PDI) were also determined. The entrapment encapsulation efficiency of the BZTS nanoparticles was measured by ultraviolet spectrophotometry. In vitro activity of BZTS nanoparticle suspensions against intracellular amastigotes of Leishmania amazonensis and cytotoxic activity were also evaluated. The results showed the production of spherical nanoparticles with varied sizes depending on the hydrophobic portion of the amphiphilic diblock copolymers used. Significant concentration-dependent inhibitory activity against intracellular amastigotes was observed, and low cytotoxic activity was demonstrated against macrophages. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Thiosemicarbazones are substances with scientific importance and a wide pharmacological profile, constituting a class of compounds whose properties have been extensively studied. They exhibit various biological properties, such as antitumor, antibacterial, antiviral, and antiparasitic activity [1–4]. Recently, a series of novel thiosemicarbazones that were derived from the natural monoterpene R-limonene was synthesized, and antitumor activity was evaluated in vitro against 10 human cancer cell lines, including glioma (U251), melanoma (UACC-62), breast cancer (MCF7), ovarian cancer with a phenotype of multidrug resistance (NCIADR/RES), non-small-cell lung cancer (NCI-H460), kidney cancer

⁎ Correspondence to: C. V. Nakamura, Programa de Pós-graduação em Ciências Farmacêuticas, Universidade Estadual de Maringá, PR, Avenida Colombo, 5790, Jd. Universitário, Maringá, Paraná, Brazil. ⁎⁎ Corresponding author. E-mail addresses: [email protected] (C.V. Nakamura), [email protected] (R. Borsali).

http://dx.doi.org/10.1016/j.msec.2016.08.021 0928-4931/© 2016 Elsevier B.V. All rights reserved.

(746-0), prostate cancer (PC-3), ovarian cancer (OVCAR-03), colon cancer (HT-29), and leukemia (K-562). The majority of the synthetized and tested compounds presented considerable inhibitory effects on the growth of a wide range of cancer cell lines. Prostate cells (PC-3) were especially sensitive to almost all of the tested thiosemicarbazones [5]. Several studies have reported the activity of thiosemicarbazones against such protozoa as Plasmodium falciparum, Trichomonas vaginalis, Trypanosoma cruzi, and Entamoeba histolytica, among others [6–10]. Du et al. [11], Fujii et al. [12], and Siles et al. [13] reported potent inhibitory actions of thiosemicarbazone derivatives against cysteine protease (referred to as cruzain or cruzipain) and enzymes that are expressed in all stages of the life cycle of T. cruzi. These enzymes are essential for replication of the intracellular parasite. Additionally, Magalhães Moreira et al. [14] synthetized new aryl thiosemicarbazones and evaluated their anti-T. cruzi activity. These compounds significantly inhibited epimastigotes proliferation, exerted toxic effects against trypomastigotes, and induced T. cruzi cell death through an apoptotic process. Moreover, the activity of thiosemicarbazones against different Leishmania species has been reported in previous literature. Schröder et al. [15] found that semicarbazone and thiosemicarbazone demonstrated

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activity against Leishmania mexicana by inhibiting cysteine protease. Britta et al. [16] reported activity of a benzaldehyde thiosemicarbazone derivative of limonene complexed with copper against Leishmania amazonensis. A recent study by our group investigated the 4-nitrobenzaldehyde thiosemicarbazone compound BZTS, which is derived from S-limonene and found that it exhibited significant antileishmania and antitrypanosoma activity, demonstrating that it may be a potential agent against L. amazonensis and T. cruzi [17,18]. However, the hydrophobic characteristic of BZTS affects its administration, resulting in low oral bioavailability. In recent years, efforts to employ nanoparticle technologies have been growing exponentially for diverse applications in medicine. Nanobiotechnology offers multiple solutions for the prevention, diagnosis and treatment of infectious diseases. The development of nanoparticles in this area has been beneficial due to: the possibility of helping to overcome problems associated with poor solubility in water, the protection of therapeutic molecules, modification their blood circulation and tissue distribution, and the ability to focus their action on specific targets [19,20]. The use of nanoparticles for drug delivery systems to direct antileishmanial agents to the cells of the reticulo-endothelial system has been regarded as an effective strategy for disease treatment [21]. Kumari et al. [22] have developed PLGA–PEG encapsulated miltefosine nanoparticles to target the macrophage of tissues infected with L. donovani. Nanoparticles were observed in a size range of 10 to 20 nm with an increased localization in macrophages predominantly infested with the parasite. Polymeric materials, specifically block copolymers, are a promising approach for overcoming such problems because of their properties of self-association that give rise to particles with different morphological forms in suspension. Nanoparticles generally consist of biodegradable polymers or lipid material with a size smaller than 10–1000 nm, which can be prepared using various materials, such as proteins, polysaccharides and synthetic polymers [23–25]. The aim of the present study was to generate nanoparticle-based block copolymers that can encapsulate BZTS to increase its solubility and reduce cytotoxicity. The in vitro activity of BZTS nanoparticle suspensions against intracellular amastigotes of Leishmania amazonensis was also investigated. 2. Methods 2.1. Materials Diblock copolymers poly(ethylene oxide-b-ε-caprolactone) (PEO[5000]-PCL[5000] and PEO[5000]-PCL[10,000]) and poly(ethylene oxide-b-lactide) (PEO[550]-PLA[12,900]) with various molecular weights were purchased from Polymer Source (Dorval, Canada). An ultrapure deionization water system was used, with a resistivity of 18.2 MΩ.cm. All of the chemical reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used without further purification. 2.2. Preparation of nanoparticles Different methods were used to prepare nanoparticles for each block copolymer. Block copolymer PEO(5000)-PCL(5000) was dissolved in acetone (20 mg/mL), to which ultrapure Mili-Q water (aqueous phase) was added (5.08 mL/h) in the organic phase, initially prepared under magnetic stirring (750 rotations per minute [rpm]) at a 2:1 ultrapure water:acetone ratio. Block copolymer PEO(5000)-PCL(10,000) was dissolved in acetone (20 mg/mL), and this organic phase was poured into an aqueous phase under the same conditions described above. Block copolymer PEO(550)-PLA(12,900) was dissolved in acetone (2 mg/mL), and this organic phase was poured into an aqueous phase under magnetic stirring (1000 rpm) at a 1:1 ultrapure

water:acetone ratio. The volume was adjusted to 15 mL with ultrapure Mili-Q water. The acetone was then eliminated by evaporation under reduced pressure for each of the three methods. 2.3. Preparation of nanoparticle suspensions loaded with BZTS The nanoparticle suspensions that were loaded with BZTS were prepared as described above. Briefly, 40 mg of the PEO(5000)-PCL(10,000) copolymer and 1.6 mg BZTS were dissolved in 2 mL of acetone and stirred until the suspension dissolved completely. This organic phase was poured into 4 mL of ultrapure Mili-Q water under magnetic stirring. Finally, the acetone was eliminated by evaporation under reduced pressure. 2.4. Morphological analysis Morphological evaluation of the nanoparticle suspensions was performed by transmission electronic microscopy (JEOL) and cryo-transmission electronic microscopy (FEI, Hillsboro, OR) that operated at 80 kV. The nanoparticle suspensions were diluted in ultrapure Mili-Q water and deposited on carbon/formvar or carbon copper grids, followed by negative staining with 2% uranyl acetate solution and freezing in liquid nitrogen, respectively. 2.5. Particle size The mean particle sizes of the nanoparticle suspensions were determined by static light scattering and dynamic light scattering (SLS/DLS) using an ALV 5000 device (ALV-Langen, Germany) equipped with a red helium-neon laser at a wavelength of 632.8 nm. Sampling was performed over 300 s, and scattered light was measured at different angles, ranging from 40° to 140°, at 25 °C. The distributions of relaxation times (A[t]) were determined using CONTIN analysis applied to autocorrelation functions (C[q,t]). The hydrodynamic radius (Rh) was determined using the Stokes-Einstein equation, Rh = KBT/6πɳD, where KB is the Boltzmann constant (in J/K), T is the temperature (in K), D is the diffusion coefficient, and ɳ is the viscosity of the medium (i.e., ultrapure water; ɳ = 0.89 cP at 25 °C). 2.6. Zeta potential and polydispersity index determination The zeta potential (ζ) and polydispersity index (PDI) were determined using a Zetasizer Nano Series instrument (Malvern Instruments, Worcestershire, UK). The values for ζ were calculated as mean electrophoretic mobility values using Smoluchowski's equation. Each measure was repeated at least three times and the results are expressed as the mean ± standard error. 2.7. Drug entrapment efficiency determination Total BZTS content in the nanoparticles was determined by ultraviolet-visible spectrum spectroscopy at 365 nm. A defined amount of the BZTS-loaded nanoparticle suspension was first dissolved in acetone. The amount of encapsulated drug was then indirectly measured after centrifuging the BZTS-loaded nanoparticle suspension for 30 min at 10,000 rpm using a membrane concentrator (Amicon Ultra 0.5 ml, MWCO 10 K, Millipore, USA). Free BZTS was determined using the filtrate. The amount of BZTS that was loaded into the nanoparticles was calculated by subtracting the amount of free BZTS in the filtrate from the total amount of BZTS. The encapsulation efficiency is expressed as the mean and standard deviation of three evaluations independent. 2.8. In vitro cytotoxic effects of the nanoparticle suspensions AJ774A1 macrophage monolayer was suspended to yield 5 × 105 cells/mL in RPMI 1640 medium supplemented with 10% fetal

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bovine serum (FBS) and added to each well in 96-well microtiter plates. The plates were incubated in a 5% CO2-air mixture at 37 °C to obtain confluent cell growth. The macrophage monolayer was treated with different concentrations of the nanoparticle suspension for 72 h. After treatment, the medium was removed, the cell monolayer was washed with phosphate-buffered saline (PBS), and 50 μL of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide formazan; 2 mg/mL) was added. The microplate was then incubated for 4 h in a 5% CO2-air mixture at 37 °C. After incubation, 150 μL of dimethylsulfoxide was added, and the microplate was homogenized. Absorbance was read in a microplate reader (BIO-TEK Power Wave XS) at 570 nm. The percentage of viable cells was calculated relative to controls (i.e., macrophages cultured in medium without drug). The 50% cytotoxic concentration (CC50) was determined by logarithm regression analysis of the data. The results of cytotoxic activity are expressed as the mean and standard deviation from three independent experiments. 2.9. Activity of BZTS-loaded nanoparticle suspensions against intracellular amastigotes Peritoneal macrophages were collected from BALB/c mice by washing with cold PBS supplemented with 3% FBS. The animal protocol was approved by the Ethical Committee of the State University of Maringá (approval no. 074/2011). Sterile glass coverslips were placed in the wells of a 24-well microplate, and 5 × 105 cells/mL were added to each well in RPMI 1640 medium supplemented with 10% FBS. The microplate was incubated for 2 h at 37 °C in a 5% CO2-air mixture to adhere macrophages. The macrophage monolayer was infected with promastigote forms at a 7:1 parasite:macrophage ratio. After 4-h incubation at 34 °C in a 5% CO2-air mixture, the microplate was washed with RPMI 1640 medium to remove non-interiorized parasites. Afterward, the infected macrophages were treated with nanoparticle suspensions that were loaded with BZTS at concentrations of 200, 120, 80, and 40 μg/mL, followed by incubation for 72 h. The percentage of infected macrophages was evaluated after Giemsa staining by microscopically counting the number of amastigotes per macrophage. The results are expressed as the mean ± standard error from three independent experiments. The data were analyzed using one-way analysis of variance (ANOVA), and significant intergroup differences were analyzed using Tukey's test. All of the statistical analyses were performed at the p b 0.05 level of significance. 3. Results and discussion 3.1. Self-assembly of diblock copolymers The Rh, PDI, and ζ for nanoparticles are parameters that indicate the stability of the nanoparticle suspension. The PDI reflects the size distribution of the nanoparticles. Typically, smaller values indicate monodisperse nanoparticle suspensions. The ζ reflects the surface charge of the nanoparticles, and this parameter can be influenced by the composition of the particles, dispersing medium, pH, and ionic strength in solution [26]. Table 1 shows the values of these parameters for the nanoparticle suspensions that were obtained from amphiphilic diblock copolymers (poly[ethylene oxide]-modified poly[ε-caprolactone] [PEO-PCL] and

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PEO-PLA). The particle size of the PEO(5000)-PCL(5000) nanoparticle suspension had a diameter of approximately 36 nm, with a PDI of 0.3 ± 0.0 and ζ of −6.0 ± 0.7 mV. The particle size of the PEO-PLA nanoparticle suspension had a diameter of 110 nm, with a PDI of 0.1 ± 0.0 and ζ of − 29.2 ± 0.4 mV. The particle size of the PEO(5000)PCL(10,000) nanoparticle suspension had a diameter of 76 nm, with a PDI of 0.1 ± 0.0 and ζ of − 11.1 ± 3.8 mV. The results showed that the nanoparticle sizes varied according to the copolymer that was used and were found to depend on the hydrophobic portion mass of the amphiphilic diblock copolymers. All of the nanoparticle suspensions presented negative ζ values, but the PEO-PLA nanoparticle suspensions showed the most negative values. Assays were performed to encapsulate the BZTS with the three copolymers described above. However, BZTS encapsulation was only achieved using the diblock copolymer PEO(5000)-PCL(10,000), which had an Rh of 39.0 nm, PDI of 0.2 ± 0.0, and ζ of − 14.9 ± 2.5 mV (Table 1). Similar results were reported by Ma et al. [27] who investigated an amphiphilic block copolymer of PEO-PCL micelles with different molecular weights of PCL as the carrier for solubilization and controlled the delivery of curcumin, in which PEO-PCL polymers with longer PCL blocks more efficiently encapsulated curcumin. All the nanoparticle suspensions that were synthesized were self-assembled as described previously, and the resulting auto-organizations were probed using DLS. Figs. 1 and 2 show well-defined peaks in the relaxation-time distribution of the particles. Fig. 1 shows a typical TEM image and DLS analysis of the nanoparticle suspensions. Amphiphilic diblock copolymers formed spherical particles with a nanometric size. The mean diameter of the nanoparticle suspensions was confirmed by TEM and DLS analyses. Cryo-TEM images and the DLS analysis of empty PEO(5000)PCL(10,000) nanoparticle suspensions and BZTS-loaded nanoparticle suspensions are shown in Fig. 2. The nanoparticle suspensions had a spherical shape with a diameter b 100 nm. Furthermore, the cryo-TEM images revealed no size differences between the BZTS-loaded nanoparticle suspensions and empty nanoparticle suspensions. Fig. 3 shows typical angular variations (plotted as a function of q2) of the relaxation frequency, Γ = 1/τ, for BZTS-loaded nanoparticle suspensions. These variations were clearly attributable to Brownian diffusive motions of the particles. The Rh was calculated based on relaxation frequencies using the Stokes-Einstein equation. The Cryo-TEM image in Fig. 2D confirms the formation of mainly nanosized spherical structures with mean diameters of 39.0 nm. The drug entrapment efficiency (EE%) of BZTS in the nanoparticle suspensions was 99.6 ± 0.0%, indicating that BZTS was efficiently encapsulated in the nanoparticles. Biodegradable nanoparticles can be used as drug delivery systems to increase the bioavailability, solubility, and permeability of many potent drugs that are otherwise difficult to deliver orally. Nanoparticulate drug delivery systems are promising for the exploitation of many biological drugs that have poor aqueous solubility, poor permeability, and low bioavailability. Nanoparticles can minimize some of the unique problems associated with these drugs by preserving the drugs' stability and structure [24]. Diblock and triblock copolymers have generated much interest for use in biomedical applications. The amphiphilic characteristic of this copolymer enables the formation of core shell-type nanoparticles or polymeric micelles in water through a self-assembly process, with the

Table 1 Physical properties of amphiphilic diblock copolymers used. Hydrodymanic radius, zeta potential and polydispersity index of the nanoparticles suspension. Diblock copolymer

Mn × 103 PEO

Mn × 103 PCL/PLA

Hydrodynamic radius (nm)

Zeta potential (mV)a

Polydispersity indexa

PEO-PCL PEO-PLA PEO-PCL PEO-PCL + BZTS

5.0 0.55 5.0 5.0

5.0 12.9 10.0 10.0

18.0 55.0 38.0 39.0

−6.0 ± 0.7 −29.2 ± 0.4 −11.1 ± 3.8 −14.9 ± 2.5

0.3 ± 0.0 0.1 ± 0.0 0.1 ± 0.0 0.2 ± 0.0

a

Mean ± SD measured in triplicate. Mn: number average molecular weight.

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Fig. 1. Correlation function and decay time distribution obtained at 90° scattering angle for PEO(5000)-PCL(5000) nanoparticles (A) and PEO(550)-PLA(12,900) (B), and transmission electron micrographs PEO(5000)-PCL(5000) nanoparticles (C) and PEO(550)-PLA(12,900) (D).

hydrophobic segment serving as a core reservoir for drugs with poor solubility. Qi et al. [28] showed that a PEO-b-PCL diblock copolymer can be generated with nanoscale vesicles with various bilayer thicknesses, leading to vesicle degradation and the release of the encapsulated drug according to intended in vivo applications. Furthermore, many studies are being designed that employ block copolymers for the synthesis of nanoparticles as drug delivery systems to improve solubility, increase bioavailability, and consequently improve biological and pharmacological activity. Shenoy and Amiji [29] reported that tamoxifenloaded PEO-PCL nanoparticles exhibited a significant increase in accumulation of the drug within tumors with time and an extended presence in systemic circulation compared with controls. Additionally, Danhier et al. [30] showed that paclitaxel-loaded PEGylated PLGAbased nanoparticles caused greater TLT tumor growth inhibition in vivo compared with Taxol®. 3.2. In vitro cytotoxic effects of BZTS-loaded nanoparticle suspensions and activity against intracellular amastigotes of L. amazonensis The entry of sufficiently high therapeutic concentrations of antimicrobial agents into infected cells and intracellular niches where pathogens reside is necessary to achieve effective treatment against intracellular infections. However, most antimicrobial agents present poor cellular penetration, limited intracellular retention, unsatisfactory subcellular distribution, and low intracellular activity [31]. The main challenge for intracellular chemotherapy is to design and develop a carrier system for antimicrobial agents that can be efficiently endocytosed by phagocytic cells and are able to release the drug once inside the cell. Delivery systems that utilize liposomes, micro/nanoparticles, lipids, conjugates, and biological carriers may contribute to the increased therapeutic efficacy of antimicrobial agents by providing sustained drug

release, minimizing toxicity associated with encapsulated drugs, and increasing overall drug efficacy [32,33]. Leishmaniasis is an endemic disease in many countries that causes many deaths if not treated properly. It is caused by protozoa of the genus Leishmania and is transmitted through insect vectors. Leishmania are obligate intracellular parasites that cause infection only in the phagolysosomes of host macrophages. Many drugs are available that have anti-Leishmania activity, but their use is limited because of toxicity and drug resistance. The drugs of choice include pentavalent antimonials, amphotericin B, and pentamidines, but these are toxic to the liver, skin, and heart [34,35]. Because of this parasite's localization, the use of drug delivery systems may be very helpful. Nanoparticles that are generated from amphiphilic block copolymers have been extensively investigated because of their self-assembly behavior [36]. The present study evaluated the effects of BZTS-loaded nanoparticle suspensions on intracellular amastigotes, the clinically relevant form of the parasite. We observed significant concentration-dependent inhibitory activity against the parasites after 72-h incubation (Fig. 4). The survival indices were 370.4 ± 94.8 for the control and 123.5 ± 12.5, 83.2 ± 18.6, 65.0 ± 14.6, and 50.0 ± 29.6 for the 40, 80, 120 and 200 μg/mL concentrations, respectively. These results correspond to survival percentages of 30.3 ± 4.2%, 24.2 ± 11.3%, 18.7 ± 7.9%, and 13.8 ± 9.3%, respectively, compared with the control. Fig. 5 shows several bright-field optical microscopy images of infected peritoneal macrophages, revealing a decrease in the number of parasites and the presence of several empty parasitophorous vacuoles in infected macrophages that were treated with BZTS-loaded nanoparticle suspensions. We also evaluated the cytotoxic activity of the BZTS-loaded nanoparticle suspensions against J774A1 macrophages. Low cytotoxic activity was observed, with inhibition percentages of 27.5 ± 5.7%, 18.7 ± 7.1%, 17.8 ± 4.2%,

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Fig. 2. Correlation function and decay time distribution obtained at 90° scattering angle for PEO(5000)-PCL(10,000) nanoparticles (A) and BZTS-loaded PEO(5000)-PCL(10,000) nanoparticles (B), and transmission electron cryomicrographs PEO(5000)-PCL(10,000) nanoparticles (C) and BZTS-loaded PEO(5000)-PCL(10,000) nanoparticles (D).

and 14.6 ± 0.4% for the 100, 50, 25, and 12.5 μg/mL concentrations, respectively. According to previous literature, the main mechanism by which nanoparticles are captured by phagocytic cells follows several steps: stable adsorption into the cell membrane, vesicle internalization through an energy-dependent mechanism, the fusion of endocytic vesicles with lysosomes and degradation of the liposomes by lysosomal enzymes, leading to the release of the encapsulated drug. In vivo, the polymers are degraded enzymatically, non-enzymatically, or both enzymatically and non-enzymatically, yielding non-toxic products that are readily eliminated by the organism through usual metabolic routes [31–33]. The results of this study indicate that the nanoparticles were

Fig. 3. Inverse decay time vs. modulus of the scattering vector q2 obtained for sample BZTS-loaded nanoparticle suspension.

phagocytosed by macrophages, and BZTS was released intracellularly and exerted activity against intracellular amastigotes (Fig. 6). Want et al. [21] generated artemisinin-loaded polylactic co-glycolic acid (ALPLGA) nanoparticles and evaluated their effects on murine peritoneal macrophages that were infected with L. donovani. The ALPLGA nanoparticles significantly inhibited the growth of intracellular amastigotes compared with free artemisinin, whereas empty nanoparticles did not exhibit any antileishmanial activity. Additionally, the activity of formulations of amphotericin B (AmB) associated with poly (ε-caprolactone) nanospheres and coated with various amounts of a non-ionic surfactant

Fig. 4. Effect of BZTS-loaded nanoparticle suspension on the interaction between Leishmania amazonensis and mouse peritoneal macrophages. The survival percentages were 13.8 ± 9.3%, 18.7 ± 7.9%, 24.2 ± 11.3%, and 30.3 ± 4.2% for 200, 120, 80 and 40 μg/mL, respectively. The data are expressed as the means from three independent experiments. *p b 0.05, compared with the control group.

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Fig. 5. Light microscopy of intracellular amastigote forms of L. amazonensis treated with BZTS-loaded nanoparticles for 72 h. A–B Control parasite with many amastigotes inside parasitophorous vacuoles. C–D treatment with 40 μg/mL, E–F treatment with 80 μg/mL, G–H treatment with 120 μg/mL, and I–J treatment with 200 μg/mL, in which a significant reduction in the number of parasites and the presence of several empty parasitophorous vacuoles was observed. Arrow indicates intracellular amastigotes, and asterisk indicates parasitophorous vacuole. Bars = 20 μm.

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Fig. 6. Mechanisms of phagocytosis of nanoparticles by macrophage. The nanoparticles were phagocyted by macrophage (1), BZTS was released intracellulary (2) and exerted activity against intracellular amastigotes (3).

poloxamer 188 was evaluated against AmB-susceptible (WT) and AmBresistant (AmB-r) strains of L. donovani amastigotes in thioglycolateelicited peritoneal macrophages [37]. AmB-nanospheres were more active than free AmB only against wildtype amastigotes, suggesting that the association of AmB with nanospheres may improve the ability of the drug to interact with ergosterol. Activity was not influenced by the concentration of poloxamer 188, which was used to stabilize the nanospheres. Liposomes and polymeric particles have been developed for use with the antileishmanial drugs amphotericin B, miltefosine, and pentamidine [38,39]. In conclusion, the present study generated spherical nanoparticles with different sizes using different block copolymers based on nanoprecipitation methods. It was found that these nanoparticles had satisfactory particle size distributions and PDI and ζ values. BZTS was successfully encapsulated, and the rate of association of the BZTS-loaded nanoparticles positively affected water solubility. The BZTS nanoparticle suspensions presented satisfactory activity against intracellular amastigote forms of L. amazonensis and did not exert cytotoxic effects. Further studies are required to elucidate the pathway of internalization of BZTS-loaded nanoparticle suspensions into macrophages. Moreover, future studies should evaluate the in vivo activity of BZTS-loaded nanoparticle suspensions using models of cutaneous leishmaniasis. Acknowledgements This study was supported through grants from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Financiadora de Estudos e Projetos (FINEP), Programa de Núcleos de Excelência (PRONEX/Fundação Araucária), Complexo de Centrais de Apoio a Pesquisa (COMCAP), Programa de Pós-graduação em Ciências Farmacêuticas da Universidade Estadual de Maringá, and Centro de Pesquisas em Macromoléculas Vegetais (CERMAV). EAB had a Doctorate fellowship from CAPES. References [1] H. Beraldo, Semicarbazones and thiosemicarbazone: their wide pharmacological profile and clinical aplications, Quim Nova 27 (2004) 461–471. [2] R.P. Tenório, A.J.S. Góes, J.G. De Lima, A.R. De Faria, A.J. Alves, T.M. De Aquino, Tiossemicarbazonas: Métodos de obtenção, aplicações sintéticas e importância biológica, Quim Nova 28 (2005) 1030–1037. [3] A.C. Andriolli, D.D.S. Santos, S.C.G. Teixeira, L.R. Teixeira, H. Beraldo, R.L. Ziolli, Avaliação do potencial citotóxico de 2-piridiniformamida tiossemicarbazonas e de seus complexos de Fe (III) utilizando Artemia salina, Rev. Saúde Ambiente. 8 (2009) 19–23. [4] G. Pelosi, F. Bisceglie, F. Bignami, P. Ronzi, P. Schiavone, M.C. Re, C. Casoli, E. Pilotti, Antiretroviral activity of thiosemicarbazone metal complexes, J. Med. Chem. 53 (2010) 8765–8769.

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[5] F. Vandresen, H. Falzirolli, S.A. Almeida Batista, A.P.B. Da Silva-Giardini, D.N. De Oliveira, R.R. Catharino, A.L.T.G. Ruiz, J.E. De Carvalho, M.A. Foglio, C.C. Da Silva, Novel R-(+)-limonene-based thiosemicarbazones and their antitumor activity against human tumor cell lines, Eur. J. Med. Chem. 79 (2014) 110–116. [6] N. Bharti, K. Husain, M.T. Gonzalez Garza, D.E. Cruz-Vega, J. Castro-Garza, B.D. MataCardenas, F. Naqvi, A. Azam, Synthesis and in vitro antiprotozoal activity of 5nitrothiophene-2-carboxaldehyde thiosemicarbazone derivatives, Bioorg. Med. Chem. Lett. 12 (2002) 3475–3478. [7] N. Bharti-Shalendra, F. Naqvi, A. Azam, Synthesis, spectral studies and screening for amoebicidal activity of new palladium (II) complexes derived from thiophene-2carboxaldehyde thiosemicabazones, Bioorg. Med. Chem. Lett. 13 (2003) 689–692. [8] D.C. Greenbaum, Z. Mackay, E. Hansell, P. Doyle, J. Gut, C.R. Caffrey, J. Lehrman, P.J. Rosenthal, J.H. McKerrow, K. Chibale, Synthesis and structure - activity relationships of parasiticidal thios emicarbazone cysteine protease inhibitors against plasmodium falciparum, J. Med. Chem. 47 (2004) 3212–3219. [9] G. Aguirre, L. Boiani, H. Cerecetto, M. Fernández, M. González, A. Denicola, L. Otero, D. Gambino, C. Rigol, C. Olea-Azar, M. Faundez, In vitro activity and mechanism of action against the protozoan parasite Trypanosoma cruzi of 5-nitrofuryl containing thiosemicarbazones, Bioorg. Med. Chem. 12 (2004) 4885–4893. [10] A. Walcourt, M. Loyevsky, D.B. Lovejoy, V.R. Gordeuk, D.R. Richardson, Novel aroylhydrazone and thiosemicarbazone iron chelators with anti-malarial activity against chloroquine-resistant and -sensitive parasites, Int. J. Biochem. Cell Biol. 36 (2004) 401–407. [11] X. Du, C. Guo, E. Hansell, P.S. Doyle, C.R. Caffrey, T.P. Holler, J.H. McKerrow, F.E. Cohen, Synthesis and structure-activity relationship study of potent trypanocidal thio semicarbazone inhibitors of the trypanosomal cysteine protease cruzain, J. Med. Chem. 45 (2002) 2695–2707. [12] N. Fujii, J.P. Mallari, E.J. Hansell, Z. MacKey, P. Doyle, Y.M. Zhou, J. Gut, P.J. Rosenthal, J.H. McKerrow, R.K. Guy, Discovery of potent thiosemicarbazone inhibitors of rhodesain and cruzain, Bioorg. Med. Chem. Lett. 15 (2005) 121–123. [13] R. Siles, S. Chen, M. Zhou, K.G. Pinney, M.L. Trawick, Design, synthesis, and biochemical evaluation of novel cruzain inhibitors with potential application in the treatment of Chagas' disease, Bioorg. Med. Chem. Lett. 16 (2006) 4405–4409. [14] D.R. Magalhaes Moreira, A.D. de Oliveira, P.A. Teixeira de Moraes Gomes, C.A. de Simone, F.S. Villela, R.S. Ferreira, A.C. da Silva, T.A. dos Santos, M.C. Brelaz de Castro, V.R. Pereira, A.C. Leite, Conformational restriction of aryl thiosemicarbazones produces potent and selective anti-Trypanosoma cruzi compounds which induce apoptotic parasite death, Eur. J. Med. Chem. 75 (2014) 467–478. [15] J. Schröder, S. Noack, R.J. Marhöfer, J.C. Mottram, G.H. Coombs, P.M. Selzer, Identification of semicarbazones, thiosemicarbazones and triazine nitriles as inhibitors of Leishmania mexicana cysteine protease CPB, PLoS One 8 (2013) 1–12. [16] E.A. Britta, A.P. Silva, T. Ueda-Nakamura, B.P. Dias-Filho, C.C. Silva, R.L. Sernaglia, C.V. Nakamura, Benzaldehyde thiosemicarbazone derived from limonene complexed with copper induced mitochondrial dysfunction in Leishmania amazonensis, PLoS One 7 (2012) e41440. [17] E.A. Britta, D.B. Scariot, H. Falzirolli, T. Ueda-Nakamura, C.C. Silva, B.P.D. Filho, R. Borsali, C.V. Nakamura, Cell death and ultrastructural alterations in Leishmania amazonensis caused by new compound 4-nitrobenzaldehyde thiosemicarbazone derived from S-limonene, BMC Microbiol. 14 (2014) 236. [18] E.A. Britta, D.B. Scariot, T. Ueda-Nakamura, F.B.P. Dias, C.V. Nakamura, H. Falzirolli, S.C.C. da, R. Borsali, 4-Nitrobenzaldehyde thiosemicarbazone: a new compound derived from S-(−)-limonene that induces mitochondrial alterations in epimastigotes and trypomastigotes of Trypanosoma cruzi, Parasitology 142 (2015) 978–988. [19] N. Bertrand, J. Wu, X. Xu, N. Kamaly, O.C. Farokhzad, Cancer nanotechnology: the impact of passive and active targeting in the era of modern cancer biology, Adv. Drug Deliv. Rev. 66 (2014) 2–25. [20] E. Torres-Sangiao, A.M. Holban, M.C. Gestal, Advanced nanobiomaterials: vaccines, diagnosis and treatment of infectious diseases, Molecules 21 (2016) 1–22. [21] M.Y. Want, M. Islamuddin, G. Chouhan, A.K. Dasgupta, A.P. Chattopadhyay, F. Afrin, A new approach for the delivery of artemisinin: formulation, characterization, and ex-vivo antileishmanial studies, J. Colloid Interface Sci. 432 (2014) 258–269. [22] R. Kumari, G.C. Sahoo, K. Pandey, V.N.R. Das, R.K. Topno, M.Y. Ansari, S. Rana, P. Das, Development of PLGA–PEG encapsulated miltefosine based drug delivery system against visceral leishmaniasis, Mater. Sci. Eng. C 59 (2016) 748–753. [23] M. Hans, A. Lowman, Biodegradable nanoparticles for drug delivery and targeting, Curr. Opin. Solid State Mater. Sci. 6 (2002) 319–327. [24] A. Kumari, S.K. Yadav, S.C. Yadav, Biodegradable polymeric nanoparticles based drug delivery systems, Colloids Surf. B Biointerfaces 75 (2010) 1–18. [25] T.T.H. Pham, P.M. Loiseau, G. Barratt, Strategies for the design of orally bioavailable antileishmanial treatments, Int. J. Pharm. 454 (2013) 539–552. [26] V. Mohanraj, Y. Chen, M. Chen, Nanoparticles – a review, Trop. J. Pharm. Res. 5 (2006) 561–573. [27] Z. Ma, A. Haddadi, O. Molavi, A. Lavasanifar, R. Lai, J. Samuel, Micelles of poly(ethylene oxide)-b-poly(Ɛ-caprolactone) as vehicles for the solubilization, stabilization, and controlled delivery of curcumin, J. Biomed. Mater. Res., Part A 86 (2008) 300–310. [28] W. Qi, P.P. Ghoroghchian, G. Li, D.a. Hammer, M.J. Therien, Aqueous self-assembly of poly(ethylene oxide)-block-poly(ε-caprolactone) (PEO-b-PCL) copolymers: disparate diblock copolymer compositions give rise to nano- and meso-scale bilayered vesicles, Nanoscale 5 (2013) 10908–10915. [29] D.B. Shenoy, M.M. Amiji, Poly(ethylene oxide)-modified poly(Ɛ-caprolactone) nanoparticles for targeted delivery of tamoxifen in breast cancer, Int. J. Pharm. 293 (2005) 261–270. [30] F. Danhier, N. Lecouturier, B. Vroman, C. Jérôme, J. Marchand-Brynaert, O. Feron, V. Préat, Paclitaxel-loaded PEGylated PLGA-based nanoparticles: in vitro and in vivo evaluation, J. Control. Release 133 (2009) 11–17.

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E.A. Britta et al. / Materials Science and Engineering C 69 (2016) 1159–1166

[31] S. Xie, Y. Tao, Y. Pan, W. Qu, G. Cheng, L. Huang, D. Chen, X. Wang, Z. Liu, Z. Yuan, Biodegradable nanoparticles for intracellular delivery of antimicrobial agents, J. Control. Release 187 (2014) 101–117. [32] E. Imbuluzqueta, C. Gamazo, J. Ariza, M.J. Blanco-Prieto, Drug delivery systems for potential treatment of intracellular bacterial infections, Front. Biosci. 15 (2010) 397–417. [33] E. Briones, C. Isabel Colino, J.M. Lanao, Delivery systems to increase the selectivity of antibiotics in phagocytic cells, J. Control. Release 125 (2008) 210–227. [34] P. Mitropoulos, P. Konidas, M. Durkin-Konidas, New world cutaneous leishmaniasis: updated review of current and future diagnosis and treatment, J. Am. Acad. Dermatol. 63 (2010) 309–322. [35] D. Chaara, N. Haouas, J.P. Dedet, H. Babba, F. Pratlong, Leishmaniases in Maghreb: an endemic neglected disease, Acta Trop. 132 (2014) 80–93.

[36] M.S.E. Millán, D.M. Oliveira, O. Bruna-Romero, Technological perspectives in leishmaniasis, in: J.L. Costa (Ed.), Leishmanias E a Leishmaniose Tegumentar Nas Américas 2011, pp. 210–237 (Salvador, Bahia). [37] M.S. Espuelas, P. Legrand, P.M. Loiseau, C. Bories, G. Barratt, J.M. Irache, In vitro antileishmanial activity of amphotericin B loaded in poly(epsilon-caprolactone) nanospheres, J. Drug Target. 10 (2002) 593–599. [38] G. Banerjee, G. Nandi, S.B. Mahato, a. Pakrashi, M.K. Basu, Drug delivery system: targeting of pentamidines to specific sites using sugar grafted liposomes, J. Antimicrob. Chemother. 38 (1996) 145–150. [39] R. Durand, M. Paul, D. Rivollet, R. Houin, A. Astier, M. Deniau, Activity of pentamidine-loaded methacrylate nanoparticles against Leishmania infantum in a mouse model, Int. J. Parasitol. 27 (1997) 1361–1367.