J Polym Environ (2011) 19:196–202 DOI 10.1007/s10924-010-0262-4
ORIGINAL PAPER
Evaluation of Cyto- and Genotoxicity of Poly(lactide-co-glycolide) Nanoparticles Renata de Lima • Anderson do Espirito Santo Pereira • Raquel Martins Porto • Leonardo Fernandes Fraceto
Published online: 4 November 2010 Ó Springer Science+Business Media, LLC 2010
Abstract This work reports on an analysis of the cytoand genotoxicity of poly(lactide-co-glycolide) polymer nanoparticles, in an attempt to evaluate their mutagenic effects. Fibroblast (3T3) and human lymphocyte cell cultures were exposed to solutions containing three different concentrations of nanoparticles (5.4, 54 and 540 lg/mL, polymer mass/volume of solution). The nanoparticles were characterized in terms of their hydrodynamic diameters, zeta potentials and polydispersity indices. The morphology of the particles was determined by atomic force microscopy. The PLGA nanospheres presented a size of 95 nm, a zeta potential of -20 mV and a spherical morphology. Cellular viability assays using fibroblast cells showed no significant alterations compared with the negative control. A cytogenetic analysis of human lymphocyte cells showed no significant changes in the mitotic index in relation to the control, indicating that in the concentration range tested, the particles used in the experimental models did not present cyto- or genotoxicity. For the tests conducted in this work we can conclude that biodegradable and biocompatible PLGA nanospheres are not toxic in the cell cultures tested (fibroblast and lymphocyte cells) and in the
R. de Lima A. do Espirito Santo Pereira R. M. Porto Department of Biotechnology, University of Sorocaba, Sorocaba, SP, Brazil L. F. Fraceto (&) Department of Environmental Engineering, Sa˜o Paulo State University Ju´lio de Mesquita Filho, Campus Sorocaba, Av. Treˆs de Marc¸o, 511, 18087-180 Sorocaba, SP, Brazil e-mail:
[email protected] L. F. Fraceto Department of Biochemistry, Institute of Biology, UNICAMP, Cidade Universita´ria Zeferino Vaz, s/n, Campinas, SP, Brazil
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range of concentrations employed. The results provide new information concerning the toxic effects of particles produced using PLGA. Keywords Polymeric nanoparticles Cytotoxicity Genotoxicity Environmental toxicity
Introduction Progress in nanotechnology in various fields of science and technology, including biology, chemistry, physics and engineering, has led to substantial benefits to society, the economy and the environment [1]. Many nanometric materials have been developed and commercialized in electronic, optical and textile products, in medical devices, cosmetics, fuel cells, biosensors, and bioremediation agents, amongst other uses [2–4]. However, evaluations of the impacts of these nanoproducts on the part of regulatory agencies are still limited [5], although many research centers around the world have already initiated programs to determine the effects of such materials on the environment and on living systems [1]. Many studies have addressed the toxicity of nanoparticulate inorganic materials including metallic nanoparticles [6], semiconductor quantum dots [7], and carbon nanotubes [8]. In mammals, it has been reported that particulate materials with dimensions of 0.1–10 lm are toxic to the respiratory system [9], while other studies have reported negative and positive effects of nanoparticles in some organisms [1, 10]. The nanometric materials produced today include polymer nanoparticles (NP). These are structures with sizes in the range \100 nm, which are used for a variety of purposes, and especially as delivery systems for bioactive
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compounds such as drugs, proteins, DNA, pesticides, nutrients and other substances [11–14]. Several polymers are used for the preparation of NP; however, aliphatic polyesters are the most attractive for injectable systems due to their biodegradability, availability, biocompatibility, absence of toxicity, and their ability to incorporate a wide variety of drugs [15]. The aliphatic polyesters most commonly employed are poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly(lactide-co-glycolide) (PLGA), poly-e-caprolactone (PCL), poly(hydroxyvalerate) (PHV) and poly(hydroxybutyrate) (PHB) [16]. Poly(lactic-co-glycolic acid), PLGA, a biodegradable aliphatic polyester polymer obtained from hydroxyl acids and approved for use by the Food and Drug Administration, is widely used in many pharmaceutical and medical areas. The hydrolysis of this polymer produces lactic and glycolic acids, which are metabolized to carbon dioxide and water in the tricarboxylic acid cycle [17, 18]. However, the cyto- and genotoxicities of nanoparticles prepared with PLGA (and its formulation components) have been little studied, even though this polymer is one of the most widely used in the preparation of polymer nanoparticles. Studies of the impacts of these nanostructures on living organisms and on the environment are therefore needed so that the safety of these nanosystems can be assessed before they become even more widely commercialized. The literature describes several assays that can be used to determine toxicity in polymeric nanostructured systems, including assays involving cell viability analyses of mammalian cells [19–25], such as tetrazolium salt reduction assays [26], MTT assays, and genotoxicity assays [27]. In other studies plants have been used in genotoxicity assays such as the Allium cepa chromosome aberration test [28–30]. The objective of the present work was to prepare, characterize and investigate the cyto- and genotoxicity of a formulation of poly(lactide-co-glycolide) (PLGA) nanospheres, using different assays. To this end, cytotoxicity assays were performed with mouse fibroblasts (3T3 cells) to assess cell viability when exposed to different concentrations of nanoparticle preparations. The genotoxicity of PLGA nanospheres was also investigated, based on genotoxicity assays using human lymphocyte cell cultures, since it was considered that these assays could provide improved information on the impact of the nanoparticles in the experimental models used here. Two different cell types were used in order to identify possible impacts on cells obtained from different organisms and therefore exhibiting different cellular metabolisms, as described previously for analysis of the toxicity of various nanoparticles on different cell types using different tests [19, 20, 22, 23].
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Experimental Reagents and Chemicals Poly(lactide-co-glycolide) (MW 45,000 g/mol, 50:50), polyvinyl alcohol (PVA), 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide (MTT), and Dulbecco’s Modified Eagle’s Medium (Sigma Chem. Co) were supplied by Nutricell (Brazil). All other chemicals were reagent grade. Methods Particle Preparation The preparation of the poly(lactide-co-glycolide) nanoparticles (PLGA-NP) was based on a solvent removal process [31]. Twenty milligram of PLGA was first dissolved in 25 mL of acetone. This organic phase was then mixed with 30 mL of water containing 100 mg of PVA, under moderate magnetic stirring. The organic solvent was evaporated under reduced pressure at 45 °C, and the final volume of the aqueous suspension adjusted to 10 mL to give a final concentration of 2 mg/mL of polymer in solution (stock solution). In the cyto- and genotoxicity tests, the stock solution was diluted with deionized water to give final concentrations of 540.0, 54.0 and 5.4 lg/mL. PLGA-NP Characterization Particle Size, Zeta Potential and pH The mean diameter of the nanoparticles in the dispersion was determined by photon correlation spectroscopy (PCS), using a laser lightscattering instrument (Zetasizer, Malvern Inc.) at a fixed angle of 90° and temperature of 25 °C. The zeta potential was determined using a Zeta potential analyzer (Zetasizer, Malvern Inc.) at the same temperature. All the preparations were diluted 1:20 with 1 mM KCl solution, and measured in triplicate. The pH of the aqueous suspension was measured with a pH meter (Tecnal), inserted directly into the mixture. Atomic Force Microscopy (AFM) The suspension of PLGA nanospheres was examined by atomic force microscopy, using 10 lL of sample deposited onto a mica surface. The immobilized sample was air-jet dried and analyzed using a Nanosurf Easy Scan 2 Basic atomic force microscope (BT02217, Nanosurf, Switzerland) in noncontact mode. The analysis was made using a commercial Contr 10 cantilever [32]. Transmission Electron Microscopy (TEM) TEM images were obtained using a transmission electron microscope operating at an acceleration voltage of 60 kV. Copper grids
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were pre-coated with a thin film of Formvar. PLGA nanosphere solution was applied to the grids, and after evaporation of the water (at room temperature) the samples were stained with uranyl acetate and then analyzed in the microscope. Fibroblast Culture and Cell Viability Assays Balb/c mouse fibroblasts (3T3 cells) were cultured in DMEM (Dulbecco’s Modified Eagle’s Medium) supplemented with 10% fetal bovine serum, 100 UI/mL penicillin and 100 lg/mL streptomycin sulfate (pH 7.2–7.4), in a humidified atmosphere at 37 °C and with 5% CO2. Cells were seeded (2 9 104 cells/well) in 48-well tissue culture plates and cultured for 48 h. The cells were then cultured for 24 h with the PLGA-NP at three different concentrations: 5.4, 54 and 540 lg/mL. Culture medium was used as a negative control. Cell viability was assessed by tetrazolium reduction assays (MTT test). 1 mg/mL MTT was incubated with treated 3T3 cells for 1 h at 37 °C. The number of viable cells was determined by measuring the amount of MTT converted to insoluble formazan dye by mitochondrial dehydrogenases. The resulting formazan crystals were dissolved in a 1 M HCl-isopropyl alcohol mixture (1:24 v/v) and shaken for 20 min at room temperature. The colorimetric change was measured with a scanning multiwall spectrophotometer (Labsystems). Cytogenetic Assays Peripheral blood samples were collected from 4 different individuals (samples 1–4), all non-smokers with regular eating habits. The volunteers participated after signing a Free Prior Informed Consent form (Research Ethics Committee, University of Sorocaba, Protocol no. 008/08). Each volunteer contributed 2.5 mL of peripheral blood, which was used in four different cultures, each of which employed 500 lL. One of the samples served as a negative control, and three were used for the treatments with different concentrations of PLGA-NP suspensions (concentrations of 5.4, 54 and 540 lg/mL at the beginning of the culture). All the cultures were incubated for 72 h in 5 mL of final solution, which consisted of 78% RPMI culture medium (Cultilab) supplemented with 20% fetal bovine serum and 2% phytohemagglutinin, at 37 °C under controlled humidity and with 5% CO2. Approximately 30 min before the end of the incubation period, 15 lL aliquots of colchinin were added to each culture, and at the end of the 72 h cell hypotony was performed with a solution of 0.075 M KCl at 37 °C for 1 h. The metaphases were fixed using a mixture of methanol and acetic acid (3:1, v/v). The slides were mounted and stained with Giemsa dye, after which they were analyzed by
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microscopy. The mitotic index was determined by counting the number of metaphases versus the total number of cells. Statistical Analysis The cyto- and genotoxicity assays were analyzed statistically by one-way analysis of variance (One-way ANOVA) with Tukey–Kramer as a post-hoc test. Statistical significance was defined as p \ 0.05.
Results and Discussion PLGA-NP Characterization The measures of size (hydrodynamic diameter), polydispersion and zeta potential of particles are parameters indicative of their stability in suspension. Polydispersion indicates the nanoparticle size distribution, and for colloidal suspensions a value less than 0.2 is normally considered to be a good indication of uniformity in the preparation. The measure of zeta potential reflects the nanoparticle surface charge, which may be influenced by the particle composition, the dispersion medium, pH and the ionic strength of the solution. The PLGA nanospheres produced in this work had a hydrodynamic size of 95 ± 6 nm, a polydispersion index of 0.102, a pH of 6.6, and a zeta potential of -20.5 mV. These characteristics are compatible with nanostructured colloidal systems [16]. The morphology of the nanoparticles was analyzed by atomic force microscopy and transmission electron microscopy (Fig. 1). The PLGA nanospheres were spherical in shape and the size values obtained were compatible with those determined by dynamic light scattering. Effect of PLGA-NP on 3T3 Cell Viability Many earlier studies have used the colorimetric MTT reduction assay to evaluate cell viability, and hence the cytotoxicity of nanoparticulate systems. Materials examined include chitosan [33], iron oxide [34], titanium dioxide [35], cobalt-chromium [25] and silica [36]. Papageorgiou et al. [25] demonstrated the effect of microand nanoparticles of cobalt-chromium alloy in in vitro assays with human fibroblasts. The nanoparticles induced cytotoxicity more rapidly than the microparticles, and the mechanism of cell damage appeared to be different when the cells were exposed to either nano- or microparticles. Semete et al. [23] performed in vitro (cytotoxicity in Caco-2 and Hela cells) and in vivo (histopathological analysis and distribution in Balb/C tissues) tests using PLGA nanoparticles with sizes between 200 and 350 nm. It
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Fig. 1 a 3D AFM images of PLGA-NS visualized in amplitude mode (left) and height mode (right); b transmission electron microphotograph of PLGA nanospheres (arrow indicates a representation of one particle)
was shown that nanoparticle concentrations between 1 and 100 mg/mL did not cause any damage to the cell types studied, and did not present any negative influences in the histopathological analyses. In the present work, we evaluated the effect of PLGA polymer nanoparticles on cultures of mouse fibroblast cells (3T3 cells), an approach different to that of Semete et al. [23]. The viability of the cells was measured as a function of the concentration of the PLGA nanospheres. The cells were treated with three different concentrations of nanoparticles (5.4, 54 and 540 lg/mL), to which they were exposed for a period of 24 h. This concentration range was chosen because it was the same as that used earlier in an investigation of the use of these nanoparticles as a pharmaceutical carrier system [31]. The highest concentration level used was five times greater than the maximum concentration employed by Semete et al. [23]. Figure 2 shows the cell viability results, indicating that increasing nanoparticle concentrations did not alter their cytotoxicity, since the results were the same as those obtained using the negative control (where the cells were not exposed to any external chemical agent). These data indicate that, at the concentrations tested and in the model used here, the nanoparticles did not affect cell viability.
Fig. 2 Cytotoxic effects of PLGA nanospheres, at concentrations of 5.4, 54 and 540 lg/mL, on Balb/c 3T3 cells incubated for 24 h at 37 °C and with 5% CO2, evaluated using the MTT reduction test. Data expressed as % cell viability (Mean ± SD, n = 8 experiments)
Cytogenetic Investigation of PLGA-NP Using Lymphocyte Cells Another test commonly employed to evaluate the toxicity of nanoparticles is the measurement of cytotoxicity using
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Fig. 3 Examples of metaphasis of human lymphocyte cells (2n = 46) obtained after treatment with PLGA nanospheres (5.4, 54 and 540 lg/mL), and of the negative control (example obtained from Sample 1)
human lymphocyte cell cultures [37]. In this work, we examined the effect on human lymphocyte cells of increasing the concentration of PLGA polymer nanoparticles, in four different samples. As in the fibroblast culture cytotoxicity assays, three different nanoparticle concentrations were tested (5.4, 54 and 540 lg/mL, polymer mass/volume of solution). Figure 3 illustrates the results obtained after exposure of the human lymphocyte cells to the polymer nanoparticles (Sample 1). There was no alteration in the chromosomes of the lymphocyte cells as a function of the different concentrations of sample tested, i.e. no typical alterations were observed, such as chromatid gaps, chromatid breaks, chromosome breaks or other effects characteristic of the exposure of these cells to a genotoxic chemical agent [38]. The mitotic indices obtained for the different concentrations were very similar to that of the negative control (Table 1; Fig. 4), and are in agreement with indices previously reported [39]. Moreover, no significant differences were found among the three concentrations tested (p [ 0.05). Therefore, this assay did not show any genetic alterations in the cells studied. This result, together with those obtained using the fibroblast cell viability assays (which also showed no alterations in the cells) is indicative of the suitability of PLGA nanostructures for use as carriers of chemical compounds. In their review article, Nel et al. [40] showed that various factors are important in the interactions between nanomaterials and biological systems. Especially significant are: (1) The characteristics of the nanoparticles, such as size, shape,
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surface area, surface charge, functional groups, ligands, hydrophobicity and hydrophilicity; (2) the nature of the medium in which the particles are suspended, such as the presence of water molecules, acids and bases, salts and multivalent ions, surfactants, polymers and polyelectrolytes; (3) the solid–liquid interface, considering surface hydration and dehydration, sorption of steric molecules and toxins, aggregation, dispersion and dissolution, and hydrophilic and hydrophobic interactions; (4) the nano–bio interface, considering membrane interactions, receptor–ligand binding and biomolecule interactions (involving lipids, proteins and DNA) that lead to structural and functional changes, mitochondrial and lysosomal damage, and other effects. The recent work of Semete et al. [23] involved in vitro and in vivo studies of the toxicity of PLGA nanospheres using different cell types (Caco-2 and Hela), as well as histopathological studies and measurement of the distribution of PLGA nanoparticles in different Balb/C mouse tissues. Semete et al. [23] used 200–350 nm nanospheres in a concentration range of 1.0–100 lg/mL. The low toxicity of the PLGA nanoparticles was suggested to be unrelated to the toxic effects reported for other particles [40], caused by the greater surface area of nanostructured systems. Semete et al. [23] suggested that the absence of toxicity was related to the chemical nature of PLGA, which consists of polyesters susceptible to hydrolysis and enzymatic degradation in organisms, which produces biologically compatible compounds that are readily metabolized and therefore do not cause toxic effects. In addition, degradation of PLGA is a slow process that does not interfere in cellular metabolism.
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Table 1 Mitotic index, mean mitotic index, and relative mitotic index obtained from the analyses of human lymphocyte cells after the different treatments (540, 54.0, 5.4 lg/mL) and of the negative control of the various samples analyzed (samples 1–4) Mitotic index (%)
Mean ± SD (%)
Relative mitotoic index
Sample 1
3.27
4.05±1.02
1.00
Sample 2
5.33
Sample 3
3.20
Sample 4
4.40 3.26±0.29
0.80
Control
to the types of cells studied. This demonstrates that even given a high particle surface area, concentrations of particles in the range 5.4–540 lg/mL were incapable of causing cellular toxicity, corroborating the explanations presented by [23]. Another important factor highlighted by Nel et al. [40] is that cationic particles (with positive zeta potential) are more cytotoxic than particles possessing more negative zeta potentials. The nanoparticles prepared in the present work showed zeta potential values around -20 mV, so that their surface reactivity was probably low.
5.4 lg/mL Sample 1
3.23
Sample 2
3.09
Sample 3
3.68
Sample 4 54 lg/mL
3.03
Sample 1
4.63
Sample 2
4.75
Sample 3
4.53
Sample 4
4.12
Conclusions
4.51±0.27
1.11
4.46±0.56
1.10
540 lg/mL Sample 1
4.36
Sample 2
4.44
Sample 3
3.83
Sample 4
5.20
This work involved the preparation, characterization and evaluation of the cyto- and genotoxicities of PLGA polymer nanoparticles. Physicochemical characterization indicated that the particles were spherical, and were characteristic of a nanoparticulate colloidal system. Cyto- and genotoxicity assays demonstrated that, in the concentration range tested, the PLGA nanospheres (95 nm diameter) caused no detectable effects either on cell (fibroblast) viability or on mitotic indices measured using human lymphocyte cell assays. It can therefore be concluded that neither the surface area of the particles nor the chemical reactivity of groups present on the surfaces of the PLGA nanoparticles were able to promote toxic effects (at the concentrations tested and using the experimental models adopted). However, it should be pointed out that the study reported here was restricted to a small number of assays and cell types, and that a more indepth investigation is ongoing and aims to obtain further details concerning the safety of this type of nanoparticle. This investigation is expected to generate further information on the possible toxicological risks involved in the use of these nanoparticles. Acknowledgments This research was supported by FAPESP, CNPq and FUNDUNESP. The authors are also indebted to the company Exsor for the AFM analyses.
Fig. 4 Mean values of the mitotic indices obtained from analyses of cells after different treatments (5.4, 54 and 540 lg/mL of PLGA nanospheres), and for the negative controls
References There have been various previous reports concerning the influence of the surface of nanostructured materials on their toxicity in biological systems [33, 41, 42]. These studies suggest that, in general, the toxicity of nanostructured systems increases as particle size diminishes. The results presented here show that the PLGA nanospheres prepared, which had a diameter of 95 nm (smaller than the particles used by Semete et al. [23], and therefore possessing both a greater surface area and an increased presence of functional groups), did not cause any damage
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