ToxSci Advance Access published June 5, 2015

Nanoparticles made from xyloglucan-block-polycaprolactone copolymers: safety assessment for drug delivery Letícia Mazzarino*, Gecioni Loch-Neckel*, Lorena dos Santos Bubniak†, Fabiana Ourique§, Issei Otsuka¶, Sami Halila¶, Rozangela Curi Pedrosa§, Maria Cláudia Santos-Silva†, Elenara Lemos-Senna*, Edvani Curti Muniz#, and Redouane Borsali¶

Departamento de Ciências Farmacêuticas, †Departamento de Análises Clínicas,

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Departamento de Bioquímica, Universidade Federal de Santa Catarina (UFSC), Campus Universitário Trindade, 88040-900, Florianópolis, SC, Brazil

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Departamento de Química, Universidade Estadual de Maringá (UEM), 87020-970, Maringá, PR, Brazil ¶

Centre de Recherches sur les Macromolécules Végétales (CERMAV, UPR-CNRS 5301), Université Grenoble Alpes, BP 53, F 38041 Grenoble Cedex 9, France

Corresponding author: Phone: +55 48 3721 5067; Fax: +55 48 3721 9350; E-mail: [email protected] (L. Mazzarino)

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*

Abstract Xyloglucan-block-polycaprolactone (XGO-PCL) copolymer nanoparticles have been proposed as nanocarriers for drug delivery. However, the possible harmful effects of exposure to nanoparticles still remain a concern. Therefore, the aim of this study is to evaluate the potential toxicity of XGO-PCL nanoparticles using in vitro and in vivo assays. Cytotoxicity and genotoxicity studies were conducted on MRC-5 human fetal lung fibroblast

and no DNA damage were observed at the different concentrations tested. Erythrocyte toxicity was assessed by the incubation of nanoparticles with human blood. XGO-PCL nanoparticles induced a haemolytic ratio of less than 1%, indicating good blood compatibility. Finally, the subacute toxicity of XGO-PCL nanoparticles (10 mg kg-1 day-1) was evaluated in BALB/c mice when administered orally or intraperitoneally for 14 days. Results of the in vivo toxicity study showed no clinical signs of toxicity, mortality, weight loss, or haematological and biochemical alterations after treatment with nanoparticles. Also, microscopic analysis of the major organs revealed no histopathological abnormalities, corroborating the previous results. Thus, it can be concluded that XGO-PCL nanoparticles induced no effect indicative of toxicity, indicating their potential use as drug delivery systems.

Keywords: nanoparticle; nanotoxicology; cytotoxicity; genotoxicity; haemolysis; in vivo toxicity.

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cells upon exposure to XGO-PCL nanoparticles. No significant reduction in the cell viability

1. INTRODUCTION

Nanoparticles have demonstrated extraordinary potential for biomedical purposes, including diagnosis, imaging, and drug delivery. Due to their unique physicochemical properties, small size and large surface area, nanoparticles are able to overcome some limitations found in conventional therapeutic and diagnostic agents (Cheng et al., 2015).

at the molecular level, and improve the sensitivity and specificity of magnetic resonance imaging. As drug delivery systems, nanoparticles acts as carriers for therapeutic molecules, permitting increased stability of the drug, controlled release and site-specific targeting, reduced side effects, and improved the solubility of hydrophobic molecules (Zhang et al., 2008). Besides, nanoparticles can be administered through different routes (oral, buccal, nasal, ocular, transdermal, pulmonary, and parenteral). Currently, several types of nanoparticles have been used in pre-clinical studies and it is expected that they will be increasingly used in commercial products in the coming years (Aillon et al., 2009; Nel et al., 2006). Despite remarkable advances in pharmaceutical and medical fields, the toxicity and health risks of exposure to nanomaterials are attracting considerable and increasing concern worldwide (Agarwal et al., 2013). Due to the complexity of nanomaterials, current studies have shown different point of views on their use and safety (Aillon et al., 2009). It has become clear that the toxicity of nanoparticles depends on their physicochemical properties such as chemical composition, particle size, size distribution, shape, surface charge, and solubility (Li et al., 2012;

Oberdorster, 2010). Therefore, a detailed assessment of

nanoparticles through toxicological studies is crucial to assure their biological safety.

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Nanoparticle-based imaging agents allow the detection of abnormalities and disease markers

The majority of nanotoxicity studies have focused on the health effects of inorganic nanoparticles, such as silver and gold nanoparticles (Hong et al., 2014; Lasagna-Reeves et al., 2010), carbon nanotubes (Kido et al., 2014), and fullerenes (Kolosnjaj et al., 2007). However, there are still few reports on toxicological research of polymeric nanoparticles. Some studies have demonstrated the lack of toxicity of polymeric nanoparticles proposed as drug delivery systems. The incubation of human colorectal carcinoma (Caco-2) and human epithelial carcinoma (HeLa) cells with poly(lactide-co-glycolide) (PLGA) nanoparticles over

cell viability greater than 80%. Additionally, no pathological lesion suggestive of toxicity was observed in the tissues of BALB/c mice treated orally with PLGA nanoparticles for 7 days (Semete et al., 2010). Recently, the in vivo toxicity evaluation of poly(ε-caprolactone) (PCL) lipid-core nanocapsules after intradermal administration has also been reported. Nanocapsule formulations showed a narrow size distribution with a mean particle size of about 245 nm, zeta potential of -7.5 mV, and spherical shape. Wistar rats treated with PCL nanocapsules (1.8, 3.6, and 5.4 x 1012 nanoparticles/Kg) showed no clinical signs of toxicity (no mortality, no haematological and histopathological alterations, and no genotoxicity) after 28 consecutive days of treatment (Bulcao et al., 2014). Nanoparticles formed by amphiphilic diblock copolymers have been considered effective vehicles for the solubilisation of hydrophobic drugs due to their incorporation within the hydrophobic cores of polymeric nanoparticles. There are many examples of block copolymers that form nanoparticles in aqueous media; however, only a small number of these copolymers are suitable as drug delivery systems due to the requirement for biocompatibility of the copolymer (Burt et al., 1999). Recently, we have described the elaboration of nanoparticles based on the new xyloglucan-block-polycaprolactone (XGO-PCL) diblock copolymer (Mazzarino et al., 2014). Due to the biocompatibility and biodegradability of the

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a size range of 200-350 nm at different concentrations (1, 10, and 100 µg mL-1) resulted in a

XGO-PCL copolymer, the nanoparticles might have potential application in drug delivery, especially as hydrophobic drug carriers. In previous studies, XGO-PCL nanoparticles exhibited a high ability to associate curcumin, a hydrophobic natural drug, increasing its apparent water solubility by 300 times. Moreover, preliminary cytotoxicity studies showed that the incubation of XGO-PCL nanoparticles with L929 mouse fibroblast and B16F10 melanoma cells maintained the cellular viability higher than 80% after 72 hours. However, until now, further toxicological studies on XGO-PCL nanoparticles in order to assure the

In this context, the aim of this study is to assess the toxicological properties of XGOPCL nanoparticles using both in vitro and in vivo studies. In vivo toxicity was investigated by the daily oral and parenteral administration of nanoparticle suspensions in BALB/c mice for 14 days. Clinical observation, weight monitoring, haematological/biochemical tests, and histopathological examination were carried out to evaluate the toxicity of XGO-PCL nanoparticles on animals. Also, cytotoxicity and genotoxicity studies on MRC-5 human fetal lung fibroblast cells, and erythrocyte toxicity using human blood were conducted.

2. MATERIALS AND METHODS

2.1. Materials

Dulbecco’s Modified Eagle’s medium (DMEM), 3-(4,5-dimethiazol-zyl)-2-5diphenyltetrazolium bromide (MTT), low melting point agarose, and normal melting point agarose were purchased from Sigma-Aldrich (St. Louis, MO, USA). Water was purified by a Milli-Q water purification system. The synthesis of diblock copolymer containing

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safety use of these systems have not been performed.

xyloglucooligosaccharide (XGO, Mw = 1,360) and polycaprolactone (PCL, Mw = 10,320) was reported elsewhere (Mazzarino et al., 2014).

2.2. Nanoparticles Preparation

XGO-PCL copolymer nanoparticles (Cp = 1.0 mg mL-1) were prepared using the

PCL copolymer was dissolved in 2 mL of THF in a closed vial. Then, the formation of nanoparticles was induced by addition of the copolymer solution in 10 mL of Milli-Q water. Finally, the organic solvent was removed by evaporation under reduced pressure and the colloidal suspension concentrated to 5 mL.

2.3. Nanoparticle Characterisation

2.3.1. Particle Size and Zeta Potential The mean particle size and zeta potential were determined by dynamic light scattering (DLS) and laser-Doppler anemometry, respectively, using a Zetasizer Nano Series (Malvern Instruments, Worcestershire, UK). The measurements were made at 25°C after appropriate dilution of the XGO-PCL nanoparticle suspensions in Milli-Q water. Size analyses were performed at a fixed scattering angle of 173°. For measurements of zeta potential (ζ), samples were placed in an electrophoretic cell, where a potential of ± 150 mV was established. The ζ potential values were calculated from mean electrophoretic mobility

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cosolvent method, as reported previously by Mazzarino et al. (2014). Briefly, 5 mg of XGO-

values using Smoluchowski’s equation: ζ = 4π(µη/ε), where µ is the electrophoretic mobility of particles, η is the viscosity of the medium, and ε is the dielectric constant of the medium.

2.3.2. Morphology The morphology of nanoparticles was examined using a CM200 Philips transmission electron microscope (FEI Company, Hillsboro, USA). A drop of nanoparticle suspension

negatively stained with 2% (w/v) uranyl acetate. The grids were subsequently dried overnight at room temperature and then observed in the electron microscope.

2.4. In vitro Toxicity Studies

2.4.1. Cell Preparation In vitro toxicity studies were conducted on MRC-5 human fetal lung fibroblast cells (Rio de Janeiro, Brazil). MRC-5 cells were cultured in DMEM pH 7.4 supplemented with 10% fetal bovine serum, 100 U mL-1 penicillin, 100 µg mL-1 streptomycin and 10 mM HEPES in a 5% CO2 humidified atmosphere at 37°C. For haemolysis studies, erythrocytes were obtained from a healthy female blood donor. Blood samples were collected into test tubes containing 3.2% sodium citrate and erythrocytes were immediately separated by centrifugation (1,500 rpm, 10 min). After, cells were washed three times with saline solution (0.9% NaCl) and diluted with saline to obtain a stock dispersion with a fixed concentration of haemoglobin (50% haematocrit).

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previously diluted in Milli-Q® water was deposited on a carbon-coated copper grid and

2.4.2. Cytotoxicity The viability of nanoparticle-treated cells was assessed using the MTT assay (van de Loosdrecht et al., 1991). XGO-PCL nanoparticle suspensions at different concentrations (5, 10, 20, 50, and 100 µg mL-1) were added to MRC-5 cells (5.0 x 104 cells well-1) and incubated at 37 °C for 24 hours. The concentration of nanoparticles used in the cytotoxicity evaluation was determined in a previous study (Mazzarino et al., 2014). After incubation, the

Cells were then centrifuged and the formazan precipitated dissolved with 100 µL of 0.04 N isopropyl alcohol/HCl solution. The absorbance was determined at 540 nm using a microplate reader. The control group was only plated with cell medium and MTT reagent. Experiments were performed in triplicate.

2.4.3. Apoptosis Analysis Apoptotic death was verified as described by Geng et al. (2003). MRC-5 cells (5 x 105 cells well-1) were incubated with XGO-PCL nanoparticles at 150 µg mL-1 for 24 hours. Subsequently, the coverslips covering the bottom of the plate were removed, washed with PBS and treated with 40 µL of acridine orange (10 µg mL-1) and ethidium bromide (5 µg mL1

) solution. Then, cells were examined under a fluorescence microscope (Olympus BX-FLA)

and representative fields were photographed using a digital camera (Olympus BX40, Japan). Viable cells exhibited green fluorescence (acridine orange staining), whereas apoptotic cells exhibited an orange-red nuclear fluorescence (ethidium bromide staining).

2.4.4. Genotoxicity

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cells were resuspended in fresh medium and treated with MTT solution at 37°C for 3 hours.

DNA damage by XGO-PCL nanoparticles was evaluated using the alkaline comet assay (Singh et al., 1988). MRC-5 cells (5 x 104 cells well-1) were incubated with different concentrations of nanoparticles (100, 250, and 500 µg mL-1) for 24 h. Treated cells were washed with ice-cold phosphate buffered saline (PBS), trypsinised, and resuspended in complete medium. Cells were then centrifuged and mixed with 100 µL of low melting point agarose and placed on the slides, previously coated with a thin layer of 1% normal melting point agarose. Agarose was allowed to solidify at 4ºC for 5 min. In order to remove cellular

cold lysis solution (2.5 M NaCl, 10 mM Tris, 100 mM EDTA, 1% Triton X-100 and 10% DMSO, pH 10) for least 2 h at 4ºC. Afterwards the slides were placed on a horizontal electrophoresis chamber and covered with fresh buffer (300 mM NaOH, 1 mM EDTA, pH 13.0) to allow DNA unwinding and expression of alkali-labile sites. Electrophoresis was performed at 25 V and 300 mA for 20 minutes on ice. All of the above steps were performed in the dark to prevent additional DNA damage. The slides were immersed in neutralisation buffer (0.4 M Tris, pH 7.5), washed with water, and stained with ethidium bromide (0.5 mg mL-1). DNA damage was evaluated according to tail length by fluorescence microscopy (Olympus BX41, Japan, excitation and emission filters at 510 nm and 590 nm, respectively). Images of 100 cells per slide were individually analysed and scored. Each nuclei received an arbitrary value range from 0 to 4 (0, undamaged; 4 maximally damaged) (Ross et al., 1995), and damage index ranged from 0 (completely undamaged: 100 cells x 0) to 400 (maximum damage: 100 cells x 4) (Burlinson et al., 2007). Milli-Q water was used as a negative control and hydrogen peroxide (H2O2 , 50 µM) as a positive control (Benhusein et al., 2010).

2.4.5. Erythrocyte Haemolysis

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proteins and membranes, leaving the DNA as “nucleoids”, the slides were incubated in ice-

The erythrocyte toxicity assay was conducted as described by Wang et al. (2009) with modifications. In brief, 50 µL of the erythrocyte stock dispersion was added to 950 µL of saline containing different concentrations of XGO-PCL nanoparticles (10, 25, 50, and 100 µg mL-1) and incubated in an Eppendorf Thermomixer (Hamburg, Germany) at a mixing frequency of 450 rpm and a temperature of 37°C for 1 h. Intact erythrocytes were removed by centrifugation at 10,000 rpm for 5 min. Then, the absorbance of the resulting supernatant was

lysis) and distilled water as a positive control (100% lysis). The haemolysis rate was calculated according to the following equation: Hemolysis rate % =

D − D x 100 D − D

Where Dt, Dnc, and Dpc are the absorbance of the tested sample, negative control, and positive control, respectively (Zhang et al., 2007). The experiments were performed in triplicate.

2.5. In vivo Toxicity Studies

2.5.1. Animals and Treatment Ten- to twelve-week-old female BALB/c mice were housed in a room with controlled temperature (23 ± 2°C) and humidity (60 ± 10%) under a 12-h light/dark cycle with access to food and water ad libitum. Animals were allowed to acclimatise to this facility for at least one week before being used in the experiment. The experimental procedures were previously approved by the Ethics Committee from Universidade Federal de Santa Catarina (CEUA/UFSC, protocol number PP00902).

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measured at 540 nm. Saline solution (0.9% NaCl) was employed as a negative control (0%

A total of 32 mice were randomly divided into 4 groups of eight mice each. Mice were administered daily, orally or intraperitoneally, with XGO-PCL nanoparticle suspension (10 mg kg-1 day-1) or saline solution (control, 0.9% NaCl) for 14 days. Animals were observed daily according to clinical signs of toxicity (e.g., tremors, ataxia, mortality) and total body weights verified on every 2 days. After 14 days of experiment, all animals were euthanized for the collection of blood and organs.

Blood samples (six mice per group) were taken from the heart by cardiac puncture for biochemical and haematological analysis. The haematological parameters such as erythrocytes, haemoglobin, haematocrit, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), red cell distribution width (RDW), platelets, leukocytes, neutrophils, lymphocytes, and monocytes were measured. For biochemical analysis, whole blood samples were centrifuged to separate plasma within 4 hours of collection. The evaluation of hepatic function was performed by measuring the biomarkers aspartate aminotransferase (AST) and alanine aminotransferase (ALT), while renal function was assessed by determination of urea and creatinine.

2.5.3. Histopathological Analysis After collecting blood samples, vital organs like the heart, lungs, liver, spleen, and kidneys were removed carefully and fixed in 10% formalin in PBS solution pH 7.4. The tissues were embedded in paraffin blocks, sectioned (5 µm thickness), placed onto glass slides, and stained with haematoxylin and eosin (H/E). The histological examination was performed by a pathologist, and images were taken using a light microscope (400 x).

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2.5.2. Biochemical and Haematological Analysis

2.6. Statistical Analysis

Statistical comparisons were carried out by analysis of variance (ANOVA) followed by Bonferroni’s post-hoc test, using the Graph-Pad Prism software (GraphPad Software Inc., San Diego, CA, USA). P-values less than 0.05 (P < 0.05) were considered significant. Data are presented as mean ± standard error of the mean (SEM), except for results of the

3. RESULTS AND DISCUSSION

3.1. Nanoparticle Characterisation

The increased interest in the use of nanotechnology has resulted in a large diversity of nanoparticles with different compositions and physicochemical properties. These properties determine not only the utility of nanoparticles for biomedical applications, but also their potential toxicity (Luyts et al., 2013). Studies have reported that the physicochemical characteristics of nanoparticles, such as chemical composition, solubility, particle size, charge, and surface area, have crucial influences on the possible toxic effect (Hsiao and Huang, 2011; Luyts et al., 2013; Powers et al., 2006). Despite the progress of research in the nanotoxicology field, correlations between the toxicity of nanoparticles and their physicochemical properties have not reached consensus (Hsiao and Huang, 2011). On the other hand, it is clear that the chemical composition of nanoparticles is one of the most important factors affecting their toxicity. In the development

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physicochemical characterisation, which are expressed as mean ± standard deviation (SD).

process, the choice of biodegradable and biocompatible materials constitutes one important strategy to prevent their accumulation in the body and to minimize the possibility of toxic effects (Garnett and Kallinteri, 2006). Thus, in this study, self-assembled nanoparticles based on the biocompatible and biodegradable copolymer xyloglucan-polycaprolactone, were prepared as previously described by Mazzarino et al. (2014). The diblock copolymer XGOPCL was synthesised by “click” reaction with propargyl-XGO and PCL-N3. The development of nanoparticles from XGO-PCL copolymer is proposed as a strategy for

irritation and damage. Samples were characterised in terms of particle size, polydispersity, morphology, and surface charge. According to DLS measurements (Figure 1A), the average diameter and polydispersity index of XGO-PCL nanoparticles were 104.35 ± 0.35 nm and 0.158 ± 0.009, respectively, indicating an almost monodisperse size distribution. The particle size as well as the shape influences the toxicity of the material by affecting the site of deposition, clearance, and biological responses (Powers et al., 2006). Smaller nanoparticles possess higher surface area, and increased surface reactivity, and, consequently, have a greater ability to react with the biological components and produce toxic effects when compared to larger particles (Aillon et al., 2009; Luyts et al., 2013). TEM examination (Figure 1B) showed that the nanoparticles have a well-defined spherical shape with a narrow size distribution, showing good agreement with the results obtained by DLS studies. The shape of nanomaterials may affect their cellular uptake and distribution in the body, however the relationship between particle shape and uptake mechanism is not completely understood (Luyts et al., 2013). The surface charge of nanoparticles is one of the most important physicochemical characteristics of nanoparticles, since this determines the dispersion of particles in suspension (stability) and influences the adsorption of biomolecules on the particle surface (Powers et

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obtaining colloidal systems without the use of surfactants, which may induce potential

al., 2006). The values of zeta potential (a function of the surface charge) for XGO-PCL nanoparticles were close to neutral (0.112 ± 0.377 mV), which is probably related to the external non-ionic xyloglucan blocks. The role of the surface chemistry and surface charge of nanoparticles on their potential in vitro toxicity has been investigated by Mura et al. (2011). In this study, poly(lactide-co-glycolide) nanoparticles were modified by different stabilising agents, i.e. chitosan, poloxamer, and poly(vinyl alcohol), in order to obtain positively and negatively charged as well as neutral nanoparticles, respectively. However, the internalisation

inflammatory response were observed, regardless of the surface charge of the nanoparticles.

3.2. In vitro Toxicity Studies

In vitro studies including the use of cell cultures have been widely employed to assess acute and chronic toxicity of new therapeutic drugs or materials. Cellular systems are mainly used for screening purposes and to provide more detailed information about the toxicological profile of experimental compounds, avoiding the unnecessary exposure of animals to highly cytotoxic materials, for example (Castell and Gómez-Lechón, 1997; Eisenbrand et al., 2002). The cell viability assay is a crucial step in toxicology studies and estimates the cellular response to new materials, such as cell death, survival rate, and metabolic activity (AshaRani et al., 2009). Cytotoxic studies of XGO-PCL nanoparticles were carried out on MRC-5 human fibroblast cells. MRC-5 cells are often employed as normal human cell lines to evaluate the in vitro toxicity, since fibroblasts are easily maintained in culture and exhibit a finite life span (Castell and Gómez-Lechón, 1997). The cytotoxicity results obtained for XGO-PCL

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of nanoparticles by human bronchial Calu-3 cells, a very low cytotoxicity and no

nanoparticles at different concentrations (5, 10, 20, 50, and 100 µg mL-1) after 24 hours of incubation are shown in Figure 2. The incubation of MRC-5 cells with nanoparticles at the tested concentrations did not cause a significant reduction in the number of viable cells when compared with the control group. In agreement with these results, previous studies reported that unloaded XGO-PCL nanoparticles showed no cytotoxic effect on L929 mouse fibroblast and B16F10 mouse melanoma cells after 24 h incubation. A slight reduction in the number of viable cells was only observed after 72 h of incubation (Mazzarino et al., 2014).

investigated using acridine orange/ethidium bromide staining (Figure 3). Acridine orange stains both live and dead cells with a green colour. Live cells appear uniformly green, while early apoptotic cells are stained green with bright green dots in the nuclei, as a consequence of chromatin condensation and nuclear fragmentation. On the other hand, ethidium bromide stains only those cells that have lost their membrane integrity (late apoptotic cells) with an orange/red colour (Kasibhatla et al., 2006). Both cells treated with vehicle only (control group) and treated with XGO-PCL nanoparticles for 24 h showed a green colour with uniform intensity and nuclei integrity, which indicate that nanoparticles were not able to induce significant cell death, and confirm the results of cell viability. Studies have reported that some nanoparticles are able to enter cells and reach their nucleus, which may cause potential risks to human health. In this sense, assessment of the ability of nanoparticles to cause DNA damage has been considered particularly important in nanotoxicological research (Elsaesser and Howard, 2012). Genotoxicity studies of XGO-PCL nanoparticles were performed using a comet assay to detect possible DNA damage in MRC-5 human fibroblast cells (Figure 4A). The comet assay is frequently used in both in vitro and in vivo genotoxicity investigations on nanomaterials, since it is very sensitive and can provide

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The ability of nanoparticles to induce apoptosis on MRC-5 fibroblast cells was

valuable information about potential hazards of nanoparticles (Karlsson, 2010; Landsiedel et al., 2009). No DNA damage was observed after the treatment of MRC-5 cells with XGO-PCL nanoparticles at concentrations of 100, 250, and 500 µg mL-1 (Figure 4A). The DNA damage index after 24 hours of treatment with samples was equal to zero, indicating that all cells remained completely undamaged when exposed to nanoparticles. Hydrogen peroxide, a

diffusion through cellular membranes, hydrogen peroxide can easily reach the nucleus and causes multiple lesions in DNA (e.g., base damage, sugar damage, strand breaks, abasic sites, and DNA-protein cross-links) by generating highly reactive hydroxyl radicals (Benhusein et al., 2010; Dizdaroglu, 1992). The different levels of DNA damage observed on MCR-5 cells treated with hydrogen peroxide are illustrated in Figure 4B. The evaluation of haemocompatibility of nanoparticles is considered critical in the design of nanoparticle for in vivo applications. The various assays that can be used to evaluate haemocompatibility include erythrocyte composure. Erythrocytes act as perfect osmometers, where disturbances in osmotic and physical changes in blood can cause their lysis. Erythrocyte destruction causes the release of haemoglobin and its excretion in the urine leads to haemoglobinuria, which is a diagnosis for “blood poisoning”; here, erythrocyte lysis becomes a good indicator of toxicity of any foreign material to the blood cells. In addition, nanoparticles can be responsible for bridging erythrocytes, causing haemagglutination. This effect has serious consequences in humans, leading to the obstruction of blood cells, imbalance in osmolarity of blood, and unavailability of erythrocytes to carry out normal functions (Evani and Ramasubramanian, 2011). In this study, the haemocompatibility of the nanocarriers was evaluated in terms of haemolysis using human blood. The haemolytic properties of XGO-PCL nanoparticles were investigated at different concentrations (10, 25,

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typical DNA damage-inducing agent, was used as a positive control. Because of the free

50, and 100 µg mL-1). Milli-Q water and saline solution were used as positive and negative controls, respectively. As shown in Figure 5, the incubation of blood with nanoparticles for 1 hour resulted in a haemolytic ratio of less than 1% for all of the tested concentrations. According to ISO/TR 7406, samples with haemolytic rates less than 5% (the critical safe haemolytic ratio for biomaterials) are considered non-haemolytic, which suggests that XGOPCL nanoparticles possess good blood compatibility.

Recently, nanotoxicity studies have reported the health risks after pulmonary (Bakand et al., 2012; Seiffert et al., 2015), oral (Hadrup and Lam, 2014; Yun et al., 2015), and dermal (Crosera et al., 2009; Sadrieh et al., 2010; Smijs and Bouwstra, 2010) exposures to ultrafine particles. These studies have mainly focused on the local toxicity, such as lung toxicity after nanoparticle inhalation. However, the growing interest in the use of nanomaterials like nanoparticles as therapeutic and imaging tools renders the systemic toxicity a critical point to be considered in the toxicity evaluation (Aillon et al., 2009). A preliminary acute toxicity study (data not shown) conducted on BALB/c mice revealed that, at the highest tested dose of XGO-PCL nanoparticles, no signs of toxicity (death and behavioural changes) were observed after oral and intraperitoneal administration, indicating that the maximal tolerance dose of nanoparticles was higher than 10 mg kg-1 b.w. Therefore, the repeat-dose toxicity test is proposed in this study in order to evaluate the safety of the daily administration of XGO-PCL nanoparticles over a period of 14 days. The subacute toxicity study was performed following the oral and intraperitoneal administration of nanoparticles at 10 mg kg-1 day-1 dose in BALB/c mice.

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3.3. In vivo Toxicity Studies

3.3.1. General Conditions and Body Weight Subacute oral and intraperitoneal administration of XGO-PCL nanoparticles in mice showed no evidence of toxicity of nanoparticles as assessed by daily behavioural observations, weight control, haematological, biochemical, and histopathological analysis. All animals survived to an exposure time of 14 days and no clinical signs of toxicity were

urine were apparently normal. The daily observation and handling of the animals committed to subacute and subchronic studies is considered an important aspect, since subtle differences between groups such as appearance, social behaviour, irritability, and lack of interest in food and water may indicate signs of toxicity (Ecobichon, 1997). Following the administration of XGO-PCL nanoparticles (10 mg kg-1 day-1) injected intraperitoneally or orally, no significant loss of body weight was observed during the period of monitoring when compared to the respective control group (Figure 6). A recent study led by the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) established a body weight loss limit of 10% for rat and dog in short-term toxicity studies of up to 1 week in duration, while body weight loss limits of 15% and 20% have been acceptable for longer time period. In vivo toxicity studies have reported that upper body weight loss is usually associated with additional clinical symptoms and may be considered an early indicator of adverse effects (Chapman et al., 2013). Therefore, these findings suggest that the nanoparticles at the dose tested are essentially non-toxic and safe for oral and intraperitoneal administration.

3.3.2. Haematology and Biochemistry

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detected. No behavioural abnormalities, vomiting or oedema were observed and faeces and

Haematological and biochemical parameters are considered good indicators of the physiological conditions, and are often used to assess the risk of toxicity in animals. Haematological and biochemical profiles of mice are shown in Tables 1 and 2, respectively. The treatment with nanoparticles (oral and intraperitoneal) caused no significant differences in haematology values in comparison with the respective control groups, except for the leukocyte count (P < 0.05). Animals treated with nanoparticles had a significant reduction in the number of leukocytes (oral administration: control group 4.1x103 mm-3 vs. nanoparticle

nanoparticle group 3.3x103 mm-3); however, these values are in agreement with the reference range for female BALB/c mice (1.0-5.5x103 mm-3) (Araújo, 2012). All haematological and biochemical values are in agreement with the reference intervals of previous studies (Charles River International Laboratories, 2012; Araújo, 2012; Spinelli et al., 2014). Furthermore, no significant biochemical changes on AST, ALT, urea, and creatinine were observed between control and nanoparticle-treated groups. AST and ALT constitutes the most common biomarkers of hepatocellular injury. Significant alterations in the levels of these enzymes are associated with hepatocyte necrosis, liver inflammation, and toxic injury (Johnston, 1999). On the other hand, urea and creatinine are useful indicators in the evaluation of nephrotoxicity. Changes in urea and creatinine concentrations may estimate alterations in the glomerular filtration process, glomerular and tubular damage (Diezi and Biollaz, 1979). Then, the subacute administration of nanoparticles induced no alteration in hepatocytes, kidneys, and normal metabolism of the animals. Normal haematological and biochemical values observed confirm that the XGO-PCL nanoparticles affect neither the hematopoietic system nor hepatic and renal functions when administered orally or intraperitoneally at 10 mg kg-1 day-1 dose for 14 days.

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group 3.3x103 mm-3; intraperitoneal administration: control group 4.8x103 mm-3 vs.

3.3.3. Histopathology Since the dosage of 10 mg kg-1 day-1 of XGO-PCL nanoparticles caused no mortality or toxic signs on BALB/c mice, the histopathological changes of XGO-PCL nanoparticles on major organs were observed after 14 days of treatment. Figures 7 and 8 show the micrographs of histological sections of heart, lungs, liver, spleen, and kidneys of mice administered with nanoparticles or saline solution (control group) by oral gavage and

observed by microscopic analysis as a result of the administration of nanoparticles, corroborating the haematological and biochemical findings. As shown in Figures 7 and 8 (A, B), the micrograph of the cardiac muscle presents cardiac myocytes, clear and arranged in longitudinal orientation, with no histopathological lesions, haemorrhage, necrosis or inflammatory exudate. No morphological and structural changes were observed in lung tissue of mice treated with nanoparticles when compared with control group [Figures 7 and 8 (C, D)]. No lesions in lung such as pulmonary oedema, haemorrhage, emphysema, congestion, and leukocyte infiltration around the bronchial branches were detected. Tissue sections of liver from nanoparticle-treated mice showed no indication of hepatotoxicity lesions, similar to their respective control groups [Figures 7 and 8 (E, F)]. Micrographs illustrated normal hepatocytes, with eosinophilic cytoplasm and a round welldefined nucleus. Also, no significant hepatocellular degeneration and cell infiltration were observed. In the spleen, no morphological changes were observed between nanoparticletreated mice and their respective control groups [Figures 7 and 8 (G, H)]. Finally, no lesion was visualised in mice kidney as seen in Figures 7 and 8 (I, J). Renal glomerulus showed normal structure, and no haemorrhage, degeneration or necrosis was observed.

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intraperitoneal injection, respectively. No significant histopathological abnormalities were

XGO-PCL nanoparticles were found to be non-toxic in both in vitro and in vivo assessments performed in this study. The high cell viability on MRC-5 human fibroblast as well as on L929 mouse fibroblast and B16F10 mouse melanoma cell lines (Mazzarino et al., 2014) were the first indications of the lack of toxicity. The inability to induce apoptotic cell death confirmed the excellent biocompatibility of the nanoparticles. The biocompatibility of nanoparticles was probably influenced not only by the safety of the bulk material (XGO-PCL copolymer), but also by their physicochemical characteristics like monodisperse size

to genetic mutations and erythrocyte toxicity was detected, since nanoparticles showed no DNA damage on MRC-5 cells and good compatibility with human blood, respectively. Finally, no clinical and physiological signs of toxicity were observed on mice treated orally and intraperitoneally with XGO-PCL nanoparticles at a dose of 10 mg kg-1 day-1 for 14 days, evidencing their safe administration at tested conditions.

4. CONCLUSIONS

Biodegradable nanoparticles based on XGO-PCL diblock copolymer are proposed as novel nanocarriers for drug delivery. XGO-PCL nanoparticles were shown to be biocompatible and non-toxic in a range of in vitro and in vivo evaluations. In vitro studies showed that nanoparticles did not produce haemolytic activity on human erythrocytes, or cytotoxic and genotoxic effects on human fibroblast cells, indicating their cellular compatibility. Additionally, in vivo studies on the evaluation of subacute (14 days) oral and intraperitoneal toxicity in mouse showed no evidence of toxicity of XGO-PCL nanoparticles at a dose of 10 mg kg-1 day-1, as assessed by haematological, biochemical and

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distribution, spherical shape, and neutral surface charge. No potential hazard that could lead

histopathological analysis. In conclusion, this study suggests that XGO-PCL nanoparticles may be considered a safe drug carrier for biomedical applications. FUNDING INFORMATION

This work was supported by CNRS, CAPES [CAPES/PNPD 3041/2010], CNPq [grant numbers 400702/2012-6, 308337/2013-1, and 150890/2013-3], Institut Carnot Polynat,

REFERENCES

Charles River Laboratories International (2012). BALB/C mouse hematology. Available at: http://www.criver.com. Accessed March 25 2014. Agarwal, M., Murugan, M. S., Sharma, A., Rai, R., Kamboj, A., Sharma, H., and Roy, S. K. (2013). Nanoparticles and its toxic effects: a review. IJCMAS 2(10), 76-82. Aillon, K. L., Xie, Y., El-Gendy, N., Berkland, C. J., and Forrest, M. L. (2009). Effects of nanomaterial physicochemical properties on in vivo toxicity. Adv Drug Deliv Rev 61(6), 45766. Araújo, F. T. M. (2012). Estabelecimento de valores de referência para parâmetros hematológicos e bioquímicos e avaliação do perfil imunológico de linhagens de camundongos produzidas nos biotérios do Centro de Pesquisas René Rachou/FIOCRUZ Minas e do Centro de Criação de Animais de Laboratório / FIOCRUZ. Master, Fundação Oswaldo Cruz.

22

Downloaded from http://toxsci.oxfordjournals.org/ at Mount Allison University on July 27, 2015

and Labex Arcane ANR-11-LABX-0003-01.

AshaRani, P. V., Low Kah Mun, G., Hande, M. P., and Valiyaveettil, S. (2009). Cytotoxicity and genotoxicity of silver nanoparticles in human cells. ACS Nano 3(2), 279-290. Bakand, S., Hayes, A., and Dechsakulthorn, F. (2012). Nanoparticles: a review of particle toxicology following inhalation exposure. Inhal Toxicol 24(2), 125-35. Benhusein, G. M., Mutch, E., Aburawi, S., and Williams, F. M. (2010). Genotoxic effect induced by hydrogen peroxide in human hepatoma cells using comet assay. Libyan J Med 5.

Sauer, E., Cassini, C., Cerski, C. T., Zielinsky, P., Salvador, M., Pohlmann, A. R., Guterres, S. S., and Garcia, S. C. (2014). In vivo toxicological evaluation of polymeric nanocapsules after intradermal administration. Eur J Pharm Biopharm 86(2), 167-77. Burlinson, B., Tice, R. R., Speit, G., Agurell, E., Brendler-Schwaab, S. Y., Collins, A. R., Escobar, P., Honma, M., Kumaravel, T. S., Nakajima, M., Sasaki, Y. F., Thybaud, V., Uno, Y., Vasquez, M., and Hartmann, A. (2007). Fourth International Workgroup on Genotoxicity testing: results of the in vivo Comet assay workgroup. Mutat Res 627(1), 31-5. Burt, H. M., Zhang, X. C., Toleikis, P., Embree, L., and Hunter, W. L. (1999). Development of copolymers of poly(D,L-lactide) and methoxypolyethylene glycol as micellar carriers of paclitaxel. Colloids Surf B Biointerfaces 16(1-4), 161-171. Castell, J. V., and Gómez-Lechón, M. J. (1997). In vitro methods in pharmaceutical research. First ed. Academic Press, Valencia, Spain. Chapman, K., Sewell, F., Allais, L., Delongeas, J. L., Donald, E., Festag, M., Kervyn, S., Ockert, D., Nogues, V., Palmer, H., Popovic, M., Roosen, W., Schoenmakers, A., Somers, K., Stark, C., Stei, P., and Robinson, S. (2013). A global pharmaceutical company initiative: an evidence-based approach to define the upper limit of body weight loss in short term toxicity studies. Regul Toxicol Pharmacol 67(1), 27-38.

23

Downloaded from http://toxsci.oxfordjournals.org/ at Mount Allison University on July 27, 2015

Bulcao, R. P., de Freitas, F. A., Dallegrave, E., Venturini, C. G., Baierle, M., Durgante, J.,

Cheng, C. J., Tietjen, G. T., Saucier-Sawyer, J. K., and Saltzman, W. M. (2015). A holistic approach to targeting disease with polymeric nanoparticles. Nat Rev Drug Discov doi: 10.1038/nrd4503. Crosera, M., Bovenzi, M., Maina, G., Adami, G., Zanette, C., Florio, C., and Filon Larese, F. (2009). Nanoparticle dermal absorption and toxicity: a review of the literature. Int Arch Occup Environ Health 82(9), 1043-1055.

Pharmacol Ther B 5(1-3), 135-45. Dizdaroglu, M. (1992). Oxidative damage to DNA in mammalian chromatin. Mutat Res 275(3-6), 331-342. Ecobichon, D. J. (1997). The basis of the toxicity testing. 2 ed. CRC Press LLC, Florida. Eisenbrand, G., Pool-Zobel, B., Baker, V., Balls, M., Blaauboer, B. J., Boobis, A., Carere, A., Kevekordes, S., Lhuguenot, J. C., Pieters, R., and Kleiner, J. (2002). Methods of in vitro toxicology. Food Chem Toxicol 40(2-3), 193-236. Elsaesser, A., and Howard, C. V. (2012). Toxicology of nanoparticles. Adv Drug Deliv Rev 64(2), 129-137. Evani, S. J., and Ramasubramanian, A. K. (2011). Hemocompatibility of nanoparticles. In Nanobiomaterials Handbook (B. Sitharaman, Ed.) Eds.) doi: 10.1201/b10970-32 , pp. 1-17. CRC Press. Garnett, M. C., and Kallinteri, P. (2006). Nanomedicines and nanotoxicology: some physiological principles. Occup Med 56(5), 307-311.

24

Downloaded from http://toxsci.oxfordjournals.org/ at Mount Allison University on July 27, 2015

Diezi, J., and Biollaz, J. (1979). Renal function tests in experimental toxicity studies.

Geng, C. X., Zeng, Z. C., and Wang, J. Y. (2003). Docetaxel inhibits SMMC-7721 human hepatocellular carcinoma cells growth and induces apoptosis. World J Gastroenterol 9(4), 696-700. Hadrup, N., and Lam, H. R. (2014). Oral toxicity of silver ions, silver nanoparticles and colloidal silver-a review. Regul Toxicol Pharmacol 68(1), 1-7. Hong, J. S., Kim, S., Lee, S. H., Jo, E., Lee, B., Yoon, J., Eom, I. C., Kim, H. M., Kim, P.,

repeated-dose toxicity study of silver nanoparticles with the reproduction/developmental toxicity screening test. Nanotoxicology 8(4), 349-62. Hsiao, I. L., and Huang, Y. J. (2011). Effects of various physicochemical characteristics on the toxicities of ZnO and TiO nanoparticles toward human lung epithelial cells. Sci. Total Environ. 409(7), 1219-1228. Johnston, D. E. (1999). Special considerations in interpreting liver function tests. Am Fam Physician 59(8), 2223-30. Karlsson, H. L. (2010). The comet assay in nanotoxicology research. Anal Bioanal Chem 398(2), 651-666. Kasibhatla, S., Amarante-Mendes, G. P., Finucane, D., Brunner, T., Bossy-Wetzel, E., and Green, D. R. (2006). Acridine Orange/Ethidium Bromide (AO/EB) Staining to Detect Apoptosis. CSH Protoc. doi: 10.1101/pdb.prot4493(3). Kido, T., Tsunoda, M., Kasai, T., Sasaki, T., Umeda, Y., Senoh, H., Yanagisawa, H., Asakura, M., Aizawa, Y., and Fukushima, S. (2014). The increases in relative mRNA expressions of inflammatory cytokines and chemokines in splenic macrophages from rats exposed to multi-walled carbon nanotubes by whole-body inhalation for 13 weeks. Inhal Toxicol 26(12), 750-758.

25

Downloaded from http://toxsci.oxfordjournals.org/ at Mount Allison University on July 27, 2015

Choi, K., Lee, M. Y., Seo, Y. R., Kim, Y., Lee, Y., Choi, J., and Park, K. (2014). Combined

Kolosnjaj, J., Szwarc, H., and Moussa, F. (2007). Toxicity studies of fullerenes and derivatives. Adv Exp Med Biol 620, 168-80. Landsiedel, R., Kapp, M. D., Schulz, M., Wiench, K., and Oesch, F. (2009). Genotoxicity investigations on nanomaterials: methods, preparation and characterization of test material, potential artifacts and limitations--many questions, some answers. Mutat Res 681(2-3), 241258.

Ramanujam, V. M., Urayama, A., Vergara, L., Kogan, M. J., and Soto, C. (2010). Bioaccumulation and toxicity of gold nanoparticles after repeated administration in mice. Biochem Biophys Res Commun 393(4), 649-55. Li, X., Wang, L., Fan, Y., Feng, Q., and Cui, F.-z. (2012). Biocompatibility and toxicity of nanoparticles and nanotubes. J. Nanomaterials 2012, 6-6. Luyts, K., Napierska, D., Nemery, B., and Hoet, P. H. M. (2013). How physico-chemical characteristics of nanoparticles cause their toxicity: complex and unresolved interrelations. Env Sci Process Impact 15(1), 23-38. Mazzarino, L., Otsuka, I., Halila, S., Bubniak, L. D., Mazzucco, S., Santos-Silva, M. C., Lemos-Senna, E., and Borsali, R. (2014). Xyloglucan-block-poly(-caprolactone) copolymer nanoparticles coated with chitosan as biocompatible mucoadhesive drug delivery system. Macromol Biosci 14(5), 709-719. Mura, S., Hillaireau, H., Nicolas, J., Le Droumaguet, B., Gueutin, C., Zanna, S., Tsapis, N., and Fattal, E. (2011). Influence of surface charge on the potential toxicity of PLGA nanoparticles towards Calu-3 cells. Int J Nanomedicine 6, 2591-605. Nel, A., Xia, T., Madler, L., and Li, N. (2006). Toxic potential of materials at the nanolevel. Science 311(5761), 622-627.

26

Downloaded from http://toxsci.oxfordjournals.org/ at Mount Allison University on July 27, 2015

Lasagna-Reeves, C., Gonzalez-Romero, D., Barria, M. A., Olmedo, I., Clos, A., Sadagopa

Oberdorster, G. (2010). Safety assessment for nanotechnology and nanomedicine: concepts of nanotoxicology. J Intern Med 267(1), 89-105. Powers, K. W., Brown, S. C., Krishna, V. B., Wasdo, S. C., Moudgil, B. M., and Roberts, S. M. (2006). Research strategies for safety evaluation of nanomaterials. Part VI. Characterization of nanoscale particles for toxicological evaluation. Toxicol Sci 90(2), 296303.

electrophoresis assay (comet assay): technical aspects and applications. Mutat Res 337(1), 57-60. Sadrieh, N., Wokovich, A. M., Gopee, N. V., Zheng, J., Haines, D., Parmiter, D., Siitonen, P. H., Cozart, C. R., Patri, A. K., McNeil, S. E., Howard, P. C., Doub, W. H., and Buhse, L. F. (2010). Lack of significant dermal penetration of titanium dioxide from sunscreen formulations containing nano- and submicron-size TiO(2) particles. Toxicol Sci 115(1), 156166. Seiffert, J., Hussain, F., Wiegman, C., Li, F., Bey, L., Baker, W., Porter, A., Ryan, M. P., Chang, Y., Gow, A., Zhang, J., Zhu, J., Tetley, T. D., and Chung, K. F. (2015). Pulmonary toxicity of instilled silver nanoparticles: influence of size, coating and rat strain. PLoS ONE 10(3), e0119726. Semete, B., Booysen, L., Lemmer, Y., Kalombo, L., Katata, L., Verschoor, J., and Swai, H. S. (2010). In vivo evaluation of the biodistribution and safety of PLGA nanoparticles as drug delivery systems. Nanomedicine 6(5), 662-671. Singh, N. P., McCoy, M. T., Tice, R. R., and Schneider, E. L. (1988). A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175(1), 184-191.

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Downloaded from http://toxsci.oxfordjournals.org/ at Mount Allison University on July 27, 2015

Ross, G. M., McMillan, T. J., Wilcox, P., and Collins, A. R. (1995). The single cell microgel

Smijs, T. G., and Bouwstra, J. A. (2010). Focus on skin as a possible port of entry for solid nanoparticles and the toxicological impact. J Biomed Nanotechnol 6(5), 469-84. Spinelli, M. O., Motta, M. C., Cruz, R. J., and Godoy, C. M. S. C. (2014). Reference intervals for hematological parameters of animals bred and kept at the vivarium of the Faculty of Medicine of the State University of São Paulo. Acta Sci Health Sci 36(1), 1-4. van de Loosdrecht, A. A., Nennie, E., Ossenkoppele, G. J., Beelen, R. H. J., and

monocytes and macrophages in a modified colorimetric MTT assay: A methodological study. J Immunol Methods 141(1), 15-22. Wang, J. J., Liu, K. S., Sung, K. C., Tsai, C. Y., and Fang, J. Y. (2009). Lipid nanoparticles with different oil/fatty ester ratios as carriers of buprenorphine and its prodrugs for injection. Eur J Pharm Sci 38(2), 138-146. Yun, J.-W., Kim, S.-H., You, J.-R., Kim, W. H., Jang, J.-J., Min, S.-K., Kim, H. C., Chung, D. H., Jeong, J., Kang, B.-C., and Che, J.-H. (2015). Comparative toxicity of silicon dioxide, silver and iron oxide nanoparticles after repeated oral administration to rats. J Appl Toxicol 35(6), 681-693. Zhang, J., Chen, X. G., Li, Y. Y., and Liu, C. S. (2007). Self-assembled nanoparticles based on hydrophobically modified chitosan as carriers for doxorubicin. Nanomedicine 3(4), 258265. Zhang, L., Gu, F. X., Chan, J. M., Wang, A. Z., Langer, R. S., and Farokhzad, O. C. (2008). Nanoparticles in medicine: therapeutic applications and developments. Clin Pharmacol Ther 83(5), 761-769.

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Langenhuijsen, M. M. A. C. (1991). Cell mediated cytotoxicity against U 937 cells by human

FIGURE CAPTIONS

Figure 1. Characterisation of XGO-PCL nanoparticles. (A) Size distribution of XGO-PCL nanoparticles obtained at a scattering angle of 173°. (B) TEM image of XGO-PCL nanoparticles (scale bar, 100 nm).

on MRC-5 human fibroblast cells after 24 hours of incubation. Optical density of control (vehicle-treated cells) was taken as 100% cell viability. The results are the mean ± SEM of three independent experiments.

Figure 3. Detection of apoptosis on MRC-5 human fibroblast cells using the acridine orange/ethidium bromide staining assay (400x magnification). Cells were incubated with XGO-PCL nanoparticles at a concentration of 150 µg mL-1 for 24 hours. Viable cells exhibit green fluorescence (acridine orange staining), whereas apoptotic cells exhibit orange-red nuclear fluorescence (ethidium bromide staining). The group treated with vehicle only was taken as the control group.

Figure 4. Effect of XGO-PCL nanoparticles on DNA damage in MRC-5 human fibroblast cells assessed by the comet assay. (A) Cells after 24 hours incubation with different concentrations of nanoparticles (NP, 100, 250, and 500 µg mL-1). (B) Visual scoring of DNA damage in MRC-5 cells treated with hydrogen peroxide, from 0 (undamaged cells) to 4 (maximally damaged cells) according to comet appearance.

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Figure 2. Cytotoxic effect of XGO-PCL nanoparticles (NP, 5, 10, 20, 50, and 100 µg mL-1)

Figure 5. Haemolysis assay on XGO-PCL nanoparticles. (A) Relative rate of haemolysis in human erythrocytes following 1 hour incubation with different concentrations of XGO-PCL nanoparticle suspension at 37°C. Data are presented as mean ± SEM (n = 3). (B) Image of samples after centrifugation at 10,000 rpm for 5 minutes: negative control (NC, saline), positive control (PC, water), and nanoparticle suspension (NP, 10, 25, 50, and 100 µg mL-1).

positive control tube.

Figure 6. Changes in body weight of BALB/c mice treated orally (A) and intraperitoneally (B) with control (open circles) and XGO-PCL nanoparticles (filled circles) for 14 days. Nanoparticles were administered at a dose of 10 mg kg-1 day-1. Data are presented as mean ± SEM (n = 8).

Figure 7. Light micrographs of the organs of cardiac muscle, lung, liver, spleen, and kidney of mice from the control group (A, C, E, G, I) and nanoparticle-treated group (oral administration, B, D, F, H, J), respectively. Histological sections of organs were examined microscopically after H/E staining with magnification x 400.

Figure 8. Light micrographs of the organs of cardiac muscle, lung, liver, spleen, and kidney of mice from the control group (A, C, E, G, I) and nanoparticle-treated group (intraperitoneal administration, B, D, F, H, J), respectively. Histological sections of organs were examined microscopically after H/E staining with magnification x 400.

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The presence of large amounts of haemoglobin in the supernatant is only observed in the

TABLES WITH CAPTIONS

Table 1. Haematological parameters in BALB/c mice after oral and intraperitoneal administration of XGO-PCL nanoparticles (10 mg kg-1 day-1) for 14 days. Intraperitoneal administration

Control

NP XGO-PCL

Control

NP XGO-PCL

Erythrocytes (106 mm-3) 9.2 ± 0.2

9.9 ± 1.0

10.3 ± 1.2

9.1 ± 0.2

Haemoglobin (g dL-1)

13.5 ± 0.5

14.7 ± 1.9

15.0 ± 2.2

12.9 ± 0.5

Haematocrit (%)

43.5 ± 1.2

47.8 ± 5.3

50.2 ± 5.5

45.6 ±1.4

MCV (fL)

47.2 ± 0.3

48.0 ± 0.5

48.0 ± 0.7

50.3 ± 0.9

MCH (pg)

14.6 ± 0.1

14.6 ± 0.3

14.1 ± 0.7

14.1 ± 0.2

MCHC (g dL-1)

30.9 ± 0.2

30.5 ± 0.5

29.5 ± 1.2

28.2 ± 0.8

RDW (%)

13.9 ± 0.1

13.7 ± 0.2

13.9 ± 0.1

14.0 ± 0.4

Platelets (103 mm-3)

1122.2 ± 125.4 930.8 ± 62.0

1380.0 ± 61.4

1219.8 ± 87.6

Leukocytes (103 mm-3)

4.1 ± 0.4

3.3 ± 0.2*

4.8 ± 0.3

3.3 ± 0.1*

Neutrophils (%)

26.5 ± 3.7

21.5 ± 1.4

20.5 ± 1.6

22.6 ± 2.7

Lymphocytes (%)

69.6 ± 4.3

74.0 ± 1.5

76.0 ± 1.7

73.4 ± 3.2

Monocytes (%)

4.3 ± 0.7

4.5 ± 0.3

3.5 ± 0.3

4.0 ± 0.5

Data are presented as mean ± SEM (n = 6). MCV: mean corpuscular volume; MCH: mean corpuscular haemoglobin; MCHC: mean corpuscular haemoglobin concentration; RDW: red cell distribution width. *P < 0.05 compared with their respective control groups.

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Oral administration

Table 2. Biochemical parameters in BALB/c mice after oral and intraperitoneal administration of XGO-PCL nanoparticles (10 mg kg-1 day-1) for 14 days. Intraperitoneal administration

Control

NP XGO-PCL

Control

NP XGO-PCL

41.8 ± 1.1

41.7 ± 1.2

48.5 ± 2.5

44.7 ± 1.9

Creatinine (mg dL-1) 0.4 ± 0.03

0.4 ± 0.02

0.4 ± 0.03

0.4 ± 0.04

ALT (U L-1)

37.5 ± 6.2

46.1 ± 9.4

32.4 ± 2.9

29.3 ± 2.0

AST (U L-1)

71.7 ± 11.2

51.0 ± 8.0

56.0 ± 8.3

58.4 ± 9.9

Urea (mg dL-1)

Data are presented as mean ± SEM (n = 6). ALT: alanine aminotransferase; AST: aspartate aminotransferase.

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Oral administration

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Figure 1. Characterisation of XGO-PCL nanoparticles. (A) Size distribution of XGO-PCL nanoparticles obtained at a scattering angle of 173°. (B) TEM image of XGO-PCL nanoparticles (scale bar, 100 nm). 229x81mm (300 x 300 DPI)

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Figure 2. Cytotoxic effect of XGO-PCL nanoparticles (NP, 5, 10, 20, 50, and 100 µg mL-1) on MRC-5 human fibroblast cells after 24 hours of incubation. Optical density of control (vehicle-treated cells) was taken as 100% cell viability. The results are the mean ± SEM of three independent experiments. 100x88mm (300 x 300 DPI)

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Figure 3. Detection of apoptosis on MRC-5 human fibroblast cells using the acridine orange/ethidium bromide staining assay (400x magnification). Cells were incubated with XGO-PCL nanoparticles at a concentration of 150 µg mL-1 for 24 hours. Viable cells exhibit green fluorescence (acridine orange staining), whereas apoptotic cells exhibit orange-red nuclear fluorescence (ethidium bromide staining). The group treated with vehicle only was taken as the control group. 112x90mm (300 x 300 DPI)

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Figure 4. Effect of XGO-PCL nanoparticles on DNA damage in MRC-5 human fibroblast cells assessed by the comet assay. (A) Cells after 24 hours incubation with different concentrations of nanoparticles (NP, 100, 250, and 500 µg mL-1). (B) Visual scoring of DNA damage in MRC-5 cells treated with hydrogen peroxide, from 0 (undamaged cells) to 4 (maximally damaged cells) according to comet appearance. 218x121mm (300 x 300 DPI)

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Figure 5. Haemolysis assay on XGO-PCL nanoparticles. (A) Relative rate of haemolysis in human erythrocytes following 1 hour incubation with different concentrations of XGO-PCL nanoparticle suspension at 37°C. Data are presented as mean ± SEM (n = 3). (B) Image of samples after centrifugation at 10,000 rpm for 5 minutes: negative control (NC, saline), positive control (PC, water), and nanoparticle suspension (NP, 10, 25, 50, and 100 µg mL-1). The presence of large amounts of haemoglobin in the supernatant is only observed in the positive control tube. 106x113mm (300 x 300 DPI)

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Figure 6. Changes in body weight of BALB/c mice treated orally (A) and intraperitoneally (B) with control (open circles) and XGO-PCL nanoparticles (filled circles) for 14 days. Nanoparticles were administered at a dose of 10 mg kg-1 day-1. Data are presented as mean ± SEM (n = 8). 171x86mm (300 x 300 DPI)

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Figure 7. Light micrographs of the organs of cardiac muscle, lung, liver, spleen, and kidney of mice from the control group (A, C, E, G, I) and nanoparticle-treated group (oral administration, B, D, F, H, J), respectively. Histological sections of organs were examined microscopically after H/E staining with magnification x 400. 120x230mm (300 x 300 DPI)

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Figure 8. Light micrographs of the organs of cardiac muscle, lung, liver, spleen, and kidney of mice from the control group (A, C, E, G, I) and nanoparticle-treated group (intraperitoneal administration, B, D, F, H, J), respectively. Histological sections of organs were examined microscopically after H/E staining with magnification x 400. 122x230mm (300 x 300 DPI)

Nanoparticles Made From Xyloglucan-Block-Polycaprolactone Copolymers: Safety Assessment for Drug Delivery.

Xyloglucan-block-polycaprolactone (XGO-PCL) copolymer nanoparticles have been proposed as nanocarriers for drug delivery. However, the possible harmfu...
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