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Antitumor activity and systemic effects of PVM/MA-shelled selol nanocapsules in lung adenocarcinoma-bearing mice
This content has been downloaded from IOPscience. Please scroll down to see the full text. 2015 Nanotechnology 26 505101 (http://iopscience.iop.org/0957-4484/26/50/505101) View the table of contents for this issue, or go to the journal homepage for more
Download details: IP Address: 142.66.3.42 This content was downloaded on 19/04/2016 at 07:04
Please note that terms and conditions apply.
Nanotechnology Nanotechnology 26 (2015) 505101 (13pp)
doi:10.1088/0957-4484/26/50/505101
Antitumor activity and systemic effects of PVM/MA-shelled selol nanocapsules in lung adenocarcinoma-bearing mice Ludmilla Regina de Souza1, Luis Alexandre Muehlmann2, Lívia Carneiro Matos3, Rosana Simón-Vázquez4, Zulmira Guerreiro Marques Lacava3, Alfredo Maurício Batista De-Paula5, Ewa Mosiniewicz-Szablewska6, Piotr Suchocki7,8, Paulo César Morais9,10, África González-Fernández4, Sônia Nair Báo3 and Ricardo Bentes Azevedo3,11 1
Institute of Biological Sciences, Molecular Biology Programme, University of Brasília, Brasília/DF, 70910-900, Brazil 2 Faculty of Ceilandia, University of Brasília, Brasília/DF, 72220-900, Brazil 3 Institute of Biological Sciences, University of Brasília, Brasília/DF, 70910-900, Brazil 4 Immunology, Biomedical Research Center (CINBIO), University of Vigo, Instituto de Investigación Biomédica (IBI), Xerencia de Xestión Integrada de Vigo/Pontevedra/Ourense-SERGAS, E-36310 Vigo (Pontevedra), Spain 5 Institute of Odontology, State University of Montes Claros, Montes Claros/MG, 39401-001, Brazil 6 Institute of Physics, Polish Academy of Sciences, Warsaw, 02-668, Poland 7 Department of Bioanalysis and Drugs Analysis, Warsaw Medical University, Warsaw, 02-097, Poland 8 Department of Pharmaceutical Chemistry, National Medicines Institute, Warsaw, 00-725, Poland 9 Institute of Physics, University of Brasília, Brasília/DF, 70910-900, Brazil 10 School of Automation, Huazhong University of Science and Technology, Wuhan/Hubei, 430074, People’s Republic of China E-mail: [email protected], [email protected], [email protected], rosana. [email protected], [email protected], [email protected], [email protected], piotr.suchocki@wum. edu.pl, [email protected], [email protected], [email protected] and [email protected] Received 29 July 2015, revised 18 September 2015 Accepted for publication 12 October 2015 Published 17 November 2015 Abstract
Selol is a semi-synthetic compound containing selenite that is effective against cancerous cells and safer for clinical applications in comparison with other inorganic forms of selenite. Recently, we have developed a formulation of poly(methyl vinyl ether-co-maleic anhydride)shelled selol nanocapsules (SPN), which reduced the proliferative activity of lung adenocarcinoma cells and presented little deleterious effects on normal cells in in vitro studies. In this study, we report on the antitumor activity and systemic effects induced by this formulation in chemically induced lung adenocarcinoma-bearing mice. The in vivo antitumor activity of the SPN was verified by macroscopic quantification, immunohistochemistry and morphological analyses. Toxicity analyses were performed by evaluations of the kidney, liver, and spleen; analyses of hemogram and plasma levels of alanine aminotransferase, aspartate transaminase, urea, and creatinine; and DNA fragmentation and cell cycle activity of the bone marrow cells. Furthermore, we investigated the potential of the SPN formulation to cause hemolysis, activate the complement system, provoke an inflammatory response and change the conformation of the plasma proteins. Our results showed that the SPN reduced the area of the surface tumor nodules but not the total number of tumor nodules. The biochemical 11
Author to whom any correspondence should be addressed.
0957-4484/15/505101+13$33.00
1
© 2015 IOP Publishing Ltd Printed in the UK
Nanotechnology 26 (2015) 505101
L R de Souza et al
and hematological findings were suggestive of the low systemic toxicity of the SPN formulation. The surface properties of the selol nanocapsules point to characteristics that are consistent with the treatment of the tumors in vivo: low hemolytic activity, weak inflammatory reaction with no activation of the complement system, and mild or absent conformational changes of the plasma proteins. In conclusion, this report suggests that the SPN formulation investigated herein exhibits anti-tumoral effects against lung adenocarcinoma in vivo and is associated with low systemic toxicity and high biocompatibility. Keywords: selenium, selenite, lung cancer, poly(methyl vinyl ether-co-maleic anhydride), biocompatibility, toxicity, experimental model (Some figures may appear in colour only in the online journal) 1. Introduction
2. Experimental details
Selenium is an essential micronutrient found in the body, mainly incorporated into the active center of selenoproteins [1–3]. Selenoproteins are involved in oxidoreduction, redox signaling, and antioxidant defense [4]. On the other hand, some selenium compounds were shown to trigger the production of oxygen reactive species, inducing cell cycle arrest and apoptosis in cancer cells, and have thus been suggested as potential anticancer drugs [5, 6]. The most oxidative form of selenium is selenite [7]. However, inorganic selenite is poorly selective for cancer cells and thus presents severe systemic toxicity, limiting its clinical application. Selol is a mixture of selenitetriacylglycerides that has low cumulative toxicity [8, 9], whereas its anticancer activity has been associated with the production of oxygen reactive species, which eventually leads to irreversible cell damage, cell cycle arrest, and the sensitization of multidrug-resistant cells [10–13]. Lung adenocarcinoma stands out for its high incidence of metastatic disease and clinical, pathological and molecular heterogeneity, limiting the success of therapies and the survival of individuals [14–16]. In this context, selol appears to prevent the proliferative activity of the lung adenocarcinoma cells while presenting less effect on normal cells [13]. The cytostatic activity of different selenium compounds against lung adenocarcinoma cells was also observed by other studies [17–19]. With the advancement of therapeutic nanoparticle technology in the last decade, innovative approaches have been used to improve the therapeutic index of anticancer drugs [20, 21]. Aiming at improving selol pharmacokinetics, our research group recently developed a delivery system for selol, which consists of poly(methyl vinyl ether-co-maleic anhydride) (PVM/MA)-shelled selol nanocapsules [13]. While performing in vitro studies with lung adenocarcinoma A549 cells, we found that the nanocapsules showed anticancer activity comparable to the free drug and significantly reduced the toxicity to normal cells [13]. This study aimed to evaluate the effects of the selol nanocapsules against lung adenocarcinoma cells in vivo, the corresponding potential systemic toxicity, and the immunogenicity and interaction with plasma proteins.
2.1. Materials
Selol containing 5% selenium (w/w) was provided by Warsaw Medical University, Poland. PVM/MA (Gantrez AN 119) was kindly provided by the ISP Corp, Brazil. Acetone, ethanol and sodium chloride were purchased from J. T. Baker, Brazil, and used as received. Phosphate buffered saline (PBS) was purchased from Laborclin, Brazil. Urethane, bovine serum albumin (BSA), agar, yeast extract, citric acid, dry milk, Mayer’s hematoxylin, Zymosan A, 5-bromo-4-chloro3-indolyl-phosphate and lectin from Phaseolus vulgaris were purchased from Sigma, USA, and used as received. Biotinylated secondary antibody and avidin-biotin peroxidase kit was purchased from Diagnostic BioSystems, USA. Diaminobenzidine was purchased from Spring, USA, and used without further purification. RNase A was obtained from Promega, USA. Paraffin, hydrogen peroxide and paraformaldehyde were purchased from Vetec, Brazil, and used as received. Eosin and Harris hematoxylin were purchased from Lafan Química Fina, Brazil. Advia reagents were purchased from Siemens, Germany. RPMI 1640 medium, lipopolysaccharide from E. coli and propidium iodide were purchased from Invitrogen, USA. DL-dithiothreitol, Laemmli sample buffer and polyvinylidene difluoride membranes were purchased from Bio-Rad, USA. Tris and Triton X-100 were purchased from Merck, Brazil. Ficoll-Paque PLUS™ was purchased from GE Healthcare, USA. Tryptone was purchased from Fluka, USA. Fetal bovine serum, penicillin and streptomycin were purchased from Gibco, USA. FlowCytomix kit was purchased from eBioscience, USA. 2.2. Preparation and characterization of the selol nanocapsules
The preparation and characterization of the selol nanocapsules (SPN) were described in detail in the literature [13]. Briefly, selol and PVM/MA were dissolved in acetone at room temperature. The subsequent addition of ethanol and distilled water, in this sequence, under mild stirring, formed a yellowish, opaque dispersion of nanocapsules. The organic solvents were removed by distillation at 45 °C under reduced pressure (80 mbar). Next, the nanocapsules were centrifuged, the supernatant was removed, and the pellet was resuspended 2
Nanotechnology 26 (2015) 505101
L R de Souza et al
in PBS (pH 7.4). The characterization of the selol nanocapsules revealed spherical morphology, hydrodynamic diameter of 344±5 nm, polydispersion index of 0.061±0.005 and zeta potential of −29±2 [13].
2.7. Morphological analysis
Lung samples were fixed with 4% (w/v) paraformaldehyde, dehydrated with ethanol and embedded in paraffin. All five lung lobes were embedded at the same plane, and the entire lung was represented in nine semi-serial sections of 5 μm taken from each 50 μm of continuous tissue. Sections were stained with hematoxylin and eosin for morphological analysis, which was carried out by a trained pathologist without prior knowledge about the case and control groups. Lung tumors were classified as adenoma or adenocarcinoma based on criteria of Nikitin et al [25] and Stearman et al [26]. The adenomas were also characterized as solid, papillary and mixed histological subtypes according to descriptions of Nikitin et al [25] and Malknson [27].
2.3. Assessment of microbial contamination in the SPN formulation
To assess the sterility of the SPN formulation, 50 μL of the formulation was spread over Petri dishes containing LB agar medium (10 g l−1 tryptone, 5 g l−1 yeast extract, 10 g L−1 sodium chloride and 15 g l−1 agar, pH 7.0), incubated at 37 °C for 24 h and evaluated for bacterial growth [22]. After that time, non-specific bacterial growth was evaluated and our findings showed that no contamination was detected in the SPN formulation; therefore, it was used in subsequent studies.
2.8. Immunohistochemical reactions
Two 3 μm thick sections of lung tissue were made with paraffin-embedded samples. Antigens were retrieved in sodium citrate buffer (0.21 g l−1 citric acid, pH 6.0, 5 min at 100 °C). The blocking of endogenous peroxidases was performed with 3% (v/v) hydrogen peroxide for 30 min at room temperature. Nonspecific binding was blocked with 3% (w/v) dry milk for 30 min at room temperature. The primary antibody anti-ki67 (polyclonal, rabbit, ab15580, Abcam), a cell proliferation biomarker, was prepared in 3% (w/v) BSA at 1:100 dilution, incubated by 12 h at 4 °C in a humid environment and detected using a biotinylated secondary antibody and an avidin-biotin peroxidase complex. A diaminobenzidine chromogen was used as the substrate of reaction, and slides were counterstained with Mayer’s hematoxylin. Normal spleen sections were used as positive controls. The negative control was obtained by replacing the primary antibody by 3% (w/v) BSA. Only cells that presented a strong brown nuclear staining were considered positive. The evaluations of labeled and unlabeled tumor cells were performed under an optical microscope (Olympus®, Japan) with a 40× objective with the aid of an ocular lattice (0.092 mm2) superimposed on different nodules. Ten fields were evaluated per sample, and the tumor proliferation index (PI) was defined according to the percentage of positive cells relative to the total cells. Photomicrographs were performed using AnalySIS getIT software (Olympus®, Japan).
2.4. Lung tumor induction
Female A/J mice were provided by the Fundação Oswaldo Cruz (Fiocruz, Rio de Janeiro, Brazil) and maintained under appropriate conditions. Tumors were induced by intraperitoneal injections of urethane (1 g kg−1) in eight-week-old mice, once a week, during four weeks. Treatments started on the 16th week after the last urethane injection, as reported in the literature [23, 24]. Mice were weekly monitored regarding their weight loss, behavioral and clinical changes, and survival. 2.5. Treatments
Tumor-bearing animals were divided into three groups (seven animals each). The SPN formulation was administered 10 times every 48 h into the tail vein, at a selenium dose of 5 mg kg−1 or 10 mg/kg/application. Tumor-bearing animals that received saline solution (PBS) were used as the control group. A group of five mice without tumors was treated with PBS and used as healthy controls. Animals were weighed prior to each treatment and just before euthanasia. Euthanasia was performed on anesthetized animals by cervical dislocation. The blood, bone marrow, lung, kidney, liver and spleen were collected. Organs were subjected to macroscopic examination and weighed. Due to the wide variation in body weight between healthy and tumor-bearing animals, the organ weights were normalized to body weight.
2.9. Systemic toxicity assessment
Blood samples were collected prior to euthanasia for the assessment of hematological and biochemical parameters. A hemogram was performed by an automated hematology analyzer (pocH-100iV Diff, Sysmex, Brazil). Alanine aminotransferase (ALT) and aspartate transaminase (AST) activities, plus urea and creatinine concentrations, were measured from serum samples using Advia protocols and reagents and evaluated on the ADVIA 2400 (Siemens, Germany) analyzer [28]. DNA fragmentation and cell cycle progression were evaluated in bone marrow cells and used as indicators of hematopoietic toxicity. Briefly, femoral bone marrow cells
2.6. Quantification of the number and area of superficial lung tumors
The lung lobes of the mice were separated and photographed with a stereo microscope (Stemi 2000 Zeiss, Germany) connected to an Axio Cam HRM camera (Zeiss, Germany). The images were taken with the objective lens 2.5×, optovar lens 1.0×, and camera 0.63×. The number and area of superficial tumors were quantified with Zen lite software (Zeiss, Germany). Under the conditions described, each pixel corresponded to 4.2 μm. 3
Nanotechnology 26 (2015) 505101
L R de Souza et al
were collected, fixed and stored overnight with 70% ethanol at 4 °C. Next, cells were rinsed with PBS, incubated with 50 μg ml−1 RNase A for 30 min at 37 °C and stained with 50 μg ml−1 propidium iodide for 30 min at room temperature [13]. The cells were analyzed with a CyFlow® space cytometer (Partec, Germany) with 10 000 events counted per sample. The cell cycle was analyzed in the DNA content range of 2n–4n. Fragmented DNA was identified in sub-G1 (DNA content
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My IOPscience
Antitumor activity and systemic effects of PVM/MA-shelled selol nanocapsules in lung adenocarcinoma-bearing mice
This content has been downloaded from IOPscience. Please scroll down to see the full text. 2015 Nanotechnology 26 505101 (http://iopscience.iop.org/0957-4484/26/50/505101) View the table of contents for this issue, or go to the journal homepage for more
Download details: IP Address: 142.66.3.42 This content was downloaded on 19/04/2016 at 07:04
Please note that terms and conditions apply.
Nanotechnology Nanotechnology 26 (2015) 505101 (13pp)
doi:10.1088/0957-4484/26/50/505101
Antitumor activity and systemic effects of PVM/MA-shelled selol nanocapsules in lung adenocarcinoma-bearing mice Ludmilla Regina de Souza1, Luis Alexandre Muehlmann2, Lívia Carneiro Matos3, Rosana Simón-Vázquez4, Zulmira Guerreiro Marques Lacava3, Alfredo Maurício Batista De-Paula5, Ewa Mosiniewicz-Szablewska6, Piotr Suchocki7,8, Paulo César Morais9,10, África González-Fernández4, Sônia Nair Báo3 and Ricardo Bentes Azevedo3,11 1
Institute of Biological Sciences, Molecular Biology Programme, University of Brasília, Brasília/DF, 70910-900, Brazil 2 Faculty of Ceilandia, University of Brasília, Brasília/DF, 72220-900, Brazil 3 Institute of Biological Sciences, University of Brasília, Brasília/DF, 70910-900, Brazil 4 Immunology, Biomedical Research Center (CINBIO), University of Vigo, Instituto de Investigación Biomédica (IBI), Xerencia de Xestión Integrada de Vigo/Pontevedra/Ourense-SERGAS, E-36310 Vigo (Pontevedra), Spain 5 Institute of Odontology, State University of Montes Claros, Montes Claros/MG, 39401-001, Brazil 6 Institute of Physics, Polish Academy of Sciences, Warsaw, 02-668, Poland 7 Department of Bioanalysis and Drugs Analysis, Warsaw Medical University, Warsaw, 02-097, Poland 8 Department of Pharmaceutical Chemistry, National Medicines Institute, Warsaw, 00-725, Poland 9 Institute of Physics, University of Brasília, Brasília/DF, 70910-900, Brazil 10 School of Automation, Huazhong University of Science and Technology, Wuhan/Hubei, 430074, People’s Republic of China E-mail: [email protected], [email protected], [email protected], rosana. [email protected], [email protected], [email protected], [email protected], piotr.suchocki@wum. edu.pl, [email protected], [email protected], [email protected] and [email protected] Received 29 July 2015, revised 18 September 2015 Accepted for publication 12 October 2015 Published 17 November 2015 Abstract
Selol is a semi-synthetic compound containing selenite that is effective against cancerous cells and safer for clinical applications in comparison with other inorganic forms of selenite. Recently, we have developed a formulation of poly(methyl vinyl ether-co-maleic anhydride)shelled selol nanocapsules (SPN), which reduced the proliferative activity of lung adenocarcinoma cells and presented little deleterious effects on normal cells in in vitro studies. In this study, we report on the antitumor activity and systemic effects induced by this formulation in chemically induced lung adenocarcinoma-bearing mice. The in vivo antitumor activity of the SPN was verified by macroscopic quantification, immunohistochemistry and morphological analyses. Toxicity analyses were performed by evaluations of the kidney, liver, and spleen; analyses of hemogram and plasma levels of alanine aminotransferase, aspartate transaminase, urea, and creatinine; and DNA fragmentation and cell cycle activity of the bone marrow cells. Furthermore, we investigated the potential of the SPN formulation to cause hemolysis, activate the complement system, provoke an inflammatory response and change the conformation of the plasma proteins. Our results showed that the SPN reduced the area of the surface tumor nodules but not the total number of tumor nodules. The biochemical 11
Author to whom any correspondence should be addressed.
0957-4484/15/505101+13$33.00
1
© 2015 IOP Publishing Ltd Printed in the UK
Nanotechnology 26 (2015) 505101
L R de Souza et al
and hematological findings were suggestive of the low systemic toxicity of the SPN formulation. The surface properties of the selol nanocapsules point to characteristics that are consistent with the treatment of the tumors in vivo: low hemolytic activity, weak inflammatory reaction with no activation of the complement system, and mild or absent conformational changes of the plasma proteins. In conclusion, this report suggests that the SPN formulation investigated herein exhibits anti-tumoral effects against lung adenocarcinoma in vivo and is associated with low systemic toxicity and high biocompatibility. Keywords: selenium, selenite, lung cancer, poly(methyl vinyl ether-co-maleic anhydride), biocompatibility, toxicity, experimental model (Some figures may appear in colour only in the online journal) 1. Introduction
2. Experimental details
Selenium is an essential micronutrient found in the body, mainly incorporated into the active center of selenoproteins [1–3]. Selenoproteins are involved in oxidoreduction, redox signaling, and antioxidant defense [4]. On the other hand, some selenium compounds were shown to trigger the production of oxygen reactive species, inducing cell cycle arrest and apoptosis in cancer cells, and have thus been suggested as potential anticancer drugs [5, 6]. The most oxidative form of selenium is selenite [7]. However, inorganic selenite is poorly selective for cancer cells and thus presents severe systemic toxicity, limiting its clinical application. Selol is a mixture of selenitetriacylglycerides that has low cumulative toxicity [8, 9], whereas its anticancer activity has been associated with the production of oxygen reactive species, which eventually leads to irreversible cell damage, cell cycle arrest, and the sensitization of multidrug-resistant cells [10–13]. Lung adenocarcinoma stands out for its high incidence of metastatic disease and clinical, pathological and molecular heterogeneity, limiting the success of therapies and the survival of individuals [14–16]. In this context, selol appears to prevent the proliferative activity of the lung adenocarcinoma cells while presenting less effect on normal cells [13]. The cytostatic activity of different selenium compounds against lung adenocarcinoma cells was also observed by other studies [17–19]. With the advancement of therapeutic nanoparticle technology in the last decade, innovative approaches have been used to improve the therapeutic index of anticancer drugs [20, 21]. Aiming at improving selol pharmacokinetics, our research group recently developed a delivery system for selol, which consists of poly(methyl vinyl ether-co-maleic anhydride) (PVM/MA)-shelled selol nanocapsules [13]. While performing in vitro studies with lung adenocarcinoma A549 cells, we found that the nanocapsules showed anticancer activity comparable to the free drug and significantly reduced the toxicity to normal cells [13]. This study aimed to evaluate the effects of the selol nanocapsules against lung adenocarcinoma cells in vivo, the corresponding potential systemic toxicity, and the immunogenicity and interaction with plasma proteins.
2.1. Materials
Selol containing 5% selenium (w/w) was provided by Warsaw Medical University, Poland. PVM/MA (Gantrez AN 119) was kindly provided by the ISP Corp, Brazil. Acetone, ethanol and sodium chloride were purchased from J. T. Baker, Brazil, and used as received. Phosphate buffered saline (PBS) was purchased from Laborclin, Brazil. Urethane, bovine serum albumin (BSA), agar, yeast extract, citric acid, dry milk, Mayer’s hematoxylin, Zymosan A, 5-bromo-4-chloro3-indolyl-phosphate and lectin from Phaseolus vulgaris were purchased from Sigma, USA, and used as received. Biotinylated secondary antibody and avidin-biotin peroxidase kit was purchased from Diagnostic BioSystems, USA. Diaminobenzidine was purchased from Spring, USA, and used without further purification. RNase A was obtained from Promega, USA. Paraffin, hydrogen peroxide and paraformaldehyde were purchased from Vetec, Brazil, and used as received. Eosin and Harris hematoxylin were purchased from Lafan Química Fina, Brazil. Advia reagents were purchased from Siemens, Germany. RPMI 1640 medium, lipopolysaccharide from E. coli and propidium iodide were purchased from Invitrogen, USA. DL-dithiothreitol, Laemmli sample buffer and polyvinylidene difluoride membranes were purchased from Bio-Rad, USA. Tris and Triton X-100 were purchased from Merck, Brazil. Ficoll-Paque PLUS™ was purchased from GE Healthcare, USA. Tryptone was purchased from Fluka, USA. Fetal bovine serum, penicillin and streptomycin were purchased from Gibco, USA. FlowCytomix kit was purchased from eBioscience, USA. 2.2. Preparation and characterization of the selol nanocapsules
The preparation and characterization of the selol nanocapsules (SPN) were described in detail in the literature [13]. Briefly, selol and PVM/MA were dissolved in acetone at room temperature. The subsequent addition of ethanol and distilled water, in this sequence, under mild stirring, formed a yellowish, opaque dispersion of nanocapsules. The organic solvents were removed by distillation at 45 °C under reduced pressure (80 mbar). Next, the nanocapsules were centrifuged, the supernatant was removed, and the pellet was resuspended 2
Nanotechnology 26 (2015) 505101
L R de Souza et al
in PBS (pH 7.4). The characterization of the selol nanocapsules revealed spherical morphology, hydrodynamic diameter of 344±5 nm, polydispersion index of 0.061±0.005 and zeta potential of −29±2 [13].
2.7. Morphological analysis
Lung samples were fixed with 4% (w/v) paraformaldehyde, dehydrated with ethanol and embedded in paraffin. All five lung lobes were embedded at the same plane, and the entire lung was represented in nine semi-serial sections of 5 μm taken from each 50 μm of continuous tissue. Sections were stained with hematoxylin and eosin for morphological analysis, which was carried out by a trained pathologist without prior knowledge about the case and control groups. Lung tumors were classified as adenoma or adenocarcinoma based on criteria of Nikitin et al [25] and Stearman et al [26]. The adenomas were also characterized as solid, papillary and mixed histological subtypes according to descriptions of Nikitin et al [25] and Malknson [27].
2.3. Assessment of microbial contamination in the SPN formulation
To assess the sterility of the SPN formulation, 50 μL of the formulation was spread over Petri dishes containing LB agar medium (10 g l−1 tryptone, 5 g l−1 yeast extract, 10 g L−1 sodium chloride and 15 g l−1 agar, pH 7.0), incubated at 37 °C for 24 h and evaluated for bacterial growth [22]. After that time, non-specific bacterial growth was evaluated and our findings showed that no contamination was detected in the SPN formulation; therefore, it was used in subsequent studies.
2.8. Immunohistochemical reactions
Two 3 μm thick sections of lung tissue were made with paraffin-embedded samples. Antigens were retrieved in sodium citrate buffer (0.21 g l−1 citric acid, pH 6.0, 5 min at 100 °C). The blocking of endogenous peroxidases was performed with 3% (v/v) hydrogen peroxide for 30 min at room temperature. Nonspecific binding was blocked with 3% (w/v) dry milk for 30 min at room temperature. The primary antibody anti-ki67 (polyclonal, rabbit, ab15580, Abcam), a cell proliferation biomarker, was prepared in 3% (w/v) BSA at 1:100 dilution, incubated by 12 h at 4 °C in a humid environment and detected using a biotinylated secondary antibody and an avidin-biotin peroxidase complex. A diaminobenzidine chromogen was used as the substrate of reaction, and slides were counterstained with Mayer’s hematoxylin. Normal spleen sections were used as positive controls. The negative control was obtained by replacing the primary antibody by 3% (w/v) BSA. Only cells that presented a strong brown nuclear staining were considered positive. The evaluations of labeled and unlabeled tumor cells were performed under an optical microscope (Olympus®, Japan) with a 40× objective with the aid of an ocular lattice (0.092 mm2) superimposed on different nodules. Ten fields were evaluated per sample, and the tumor proliferation index (PI) was defined according to the percentage of positive cells relative to the total cells. Photomicrographs were performed using AnalySIS getIT software (Olympus®, Japan).
2.4. Lung tumor induction
Female A/J mice were provided by the Fundação Oswaldo Cruz (Fiocruz, Rio de Janeiro, Brazil) and maintained under appropriate conditions. Tumors were induced by intraperitoneal injections of urethane (1 g kg−1) in eight-week-old mice, once a week, during four weeks. Treatments started on the 16th week after the last urethane injection, as reported in the literature [23, 24]. Mice were weekly monitored regarding their weight loss, behavioral and clinical changes, and survival. 2.5. Treatments
Tumor-bearing animals were divided into three groups (seven animals each). The SPN formulation was administered 10 times every 48 h into the tail vein, at a selenium dose of 5 mg kg−1 or 10 mg/kg/application. Tumor-bearing animals that received saline solution (PBS) were used as the control group. A group of five mice without tumors was treated with PBS and used as healthy controls. Animals were weighed prior to each treatment and just before euthanasia. Euthanasia was performed on anesthetized animals by cervical dislocation. The blood, bone marrow, lung, kidney, liver and spleen were collected. Organs were subjected to macroscopic examination and weighed. Due to the wide variation in body weight between healthy and tumor-bearing animals, the organ weights were normalized to body weight.
2.9. Systemic toxicity assessment
Blood samples were collected prior to euthanasia for the assessment of hematological and biochemical parameters. A hemogram was performed by an automated hematology analyzer (pocH-100iV Diff, Sysmex, Brazil). Alanine aminotransferase (ALT) and aspartate transaminase (AST) activities, plus urea and creatinine concentrations, were measured from serum samples using Advia protocols and reagents and evaluated on the ADVIA 2400 (Siemens, Germany) analyzer [28]. DNA fragmentation and cell cycle progression were evaluated in bone marrow cells and used as indicators of hematopoietic toxicity. Briefly, femoral bone marrow cells
2.6. Quantification of the number and area of superficial lung tumors
The lung lobes of the mice were separated and photographed with a stereo microscope (Stemi 2000 Zeiss, Germany) connected to an Axio Cam HRM camera (Zeiss, Germany). The images were taken with the objective lens 2.5×, optovar lens 1.0×, and camera 0.63×. The number and area of superficial tumors were quantified with Zen lite software (Zeiss, Germany). Under the conditions described, each pixel corresponded to 4.2 μm. 3
Nanotechnology 26 (2015) 505101
L R de Souza et al
were collected, fixed and stored overnight with 70% ethanol at 4 °C. Next, cells were rinsed with PBS, incubated with 50 μg ml−1 RNase A for 30 min at 37 °C and stained with 50 μg ml−1 propidium iodide for 30 min at room temperature [13]. The cells were analyzed with a CyFlow® space cytometer (Partec, Germany) with 10 000 events counted per sample. The cell cycle was analyzed in the DNA content range of 2n–4n. Fragmented DNA was identified in sub-G1 (DNA content
