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Polymer 53 (2012) 2818e2828
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Poly(lactide-co-glycolide)/silver nanoparticles: Synthesis, characterization, antimicrobial activity, cytotoxicity assessment and ROS-inducing potential b Magdalena M. Stevanovi c a, *, Sre co D. Skapin , Ines Bra cko b, Marina Milenkovi c c, Jana Petkovi c d, ca Metka Filipi c d, Dragan P. Uskokovi a
Centre for Fine Particles Processing and Nanotechnologies, Institute of Technical Sciences of SASA, Knez Mihailova 35/IV, 11000 Belgrade, Serbia Advanced Materials Department, Jozef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia Department of Microbiology and Immunology, Faculty of Pharmacy, University of Belgrade, 11000 Belgrade, Serbia d Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Vecna pot 111, 1000 Ljubljana, Slovenia b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 February 2012 Received in revised form 24 April 2012 Accepted 28 April 2012 Available online 11 May 2012
Silver nanoparticles (AgNps) were prepared by modified chemical reduction with poly (a, g, L-glutamic acid) (PGA) as capping agent. These Ag/PGA nanoparticles (AgNpPGAs) were highly stable over long periods of time without signs of precipitation. In addition to obtaining stable AgNpPGAs, a further aim was to examine their encapsulation in the poly(L-lactide-co-glycolide) (PLGA) polymer matrix. The current interest of polymer-AgNps in biomedical applications is because a versatile system must have antimicrobial activity upon target contact, without the release of toxic biocides. The synthesis of these PLGA/AgNpPGAs used physicochemical methods with solvent/non-solvent systems. Degradation of these PLGA/AgNpPGAs and the release rate of their AgNPs were studied in physiological solution over three months. The antimicrobial activity of the samples was investigated towards six laboratory control strains from the American Type Culture Collection (ATCC) and one clinical isolate methicillin-resistant Staphylococcus aureus strain by the broth microdilution method and the results showed superior and extended activity of PLGA/AgNpPGAs. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay indicated good biocompatibility of these PLGA/AgNpPGAs. The formation of intracellular reactive oxygen species was measured spectrophotometrically using a fluorescent probe, which showed that these PLGA/ AgNpPGAs are not inducers of such species. The samples were characterized by UVeVIS spectrometry, Xray diffraction, zeta potential measurements, field-emission scanning electron microscopy, and transmission electron microscopy. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Silver nanoparticles PLGA Nanocomposites
1. Introduction Poly (L-lactide-co-glycolide) (PLGA) is a biocompatible and biodegradable polymer widely used in various medical applications, such as controlled release of delivering drugs, scaffolds in tissue engineering, fixing of bone fractures, and chirurgical strings [1,2]. PLGA particles are used for controlled delivery of several types of medicaments, including anticancer agents, antihypertensive agents, immunomodulatory drugs, hormones, vitamins and antibiotics [3,4]. Antibiotic resistance is a widespread problem, which the U.S. Centers for Disease Control and Prevention has called “one of the world’s most pressing public health problems”. The available data
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suggest that resistance has reached unacceptable levels in the pathogens that are most common in developing countries, and that these trends are further increasing. Resistance appears to have emerged and spread rapidly in many areas, with important consequences for patients and public health [5e7]. Predominance of multi-antibiotic resistant organisms has renewed interest in the use of antiseptic silver as an effective, but less toxic, antimicrobial with decreased potential for bacterial resistance [8]. The mechanism of action of silver nanoparticles (AgNps) as antimicrobial agents is due to their binding to proteins and interference with bacterial and viral processes [9,10]. The literature describes different methods for obtaining AgNps, including chemical reduction, solid-state synthesis, sonochemical synthesis, in-situ radical polymerization, and spray pyrolysis [11]. Through the optimization of experimental conditions, it is possible to synthesize nanoparticles of different sizes and morphologies. Such optimization relates to concentrations of reactants, temperature, pH, reducing agents, different surfactants and reaction
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media, and these can significantly affect the stability of the resulting particles [12]. PLGA is used for predictable controlled delivery of antibiotics [2,3,13], although in addition, PLGA with AgNps and with antibiotics has the potential for enhanced efficacy against bacteria and viruses. A first goal of the present study was the synthesis of new, stable, biocompatibile PLGA/AgNps and PLGA/Ag microparticles for improved effects under controlled drug delivery. The study exploited the prior experience of our laboratory with obtaining PLGA spheres and with encapsulation of active substances within the PLGA polymer matrix [2]. In the present study, the synthesis of AgNps is reported according to a modified chemical method, which represents an easy, simple and convenient way for the preparation of AgNps in the nanometer range. However, it has been reported that bare silver nanoparticles can be toxic [14]. This supports the concept that this toxicity is associated to the presence of the bare metallic nanoparticle surface, while particles protected by an organic layer are much more biocompatible, and thereby less toxic. PGA is an anionic polyelectrolyte that is a water-soluble, safe, and edible biomaterial that is naturally synthesized by Bacillus subtilis, in which the aamino and g-carboxy groups of glutamic acid are polymerized by gamide linkages [15]. These properties of PGA make it a promising biopolymer for use as a thickener, an osteoporosis-preventing factor, a stabilizer, and a moisturizer in cosmetics, and also promising for various biomedical product applications [15]. In the present study, PGA was used as the organic layer (a capping agent) for AgNps obtained using saccharose as a reducing agent. PGA was chosen as the capping agent to make the AgNps more biocompatibile and to protect them from agglomerating in the medium. PGA-capped AgNps (AgNpPGAs) were additionally encapsulated within PLGA particles (PLGA/AgNpPGAs) to ensure their release over an extended period of time, and therefore their extended antimicrobial effects. One of the basic requirements for the controlled and balanced release of the medicament in the body is ideal spherical shape of the PLGA particles and narrow distribution of their sizes. The size and shape of the particles play key role in their adhesion and interaction with the cell [2]. Polymer degradation also has a key role in medicament release from sustained-release polyester systems. Therefore, to investigate the mechanisms governing this release, it appears essential to analyze the in-vitro degradation behavior of such complexes. The degradation of these PLGA/AgNpPGAs and the release rate of the silver particles were studied over three months in physiological solution. The antimicrobial activities of bare and PGA-capped AgNps, both immediately after preparation and after two months, and the AgNp release from these PLGA polymer matrices after different degradation times were evaluated against the Gram-positive bacteria methicillin-resistant Staphylococcus aureus (MRSA; ATCC 43300), a clinical isolate of MRSA (hospital strain), and Fnterococcus faecalis (ATCC 29212), the Gram-negative bacteria Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 13889), and Pseudomonas aeruginosa (ATCC 27853), and the yeast Candida albicans (ATCC 10231). MRSA is also called multidrug-resistant Staphylococcus aureus and it is a bacteria responsible for several difficult-to-treat infections in humans. MRSA is especially troublesome in hospitals, where patients with open wounds, invasive devices, and weakened immune systems are at greater risk of infection than the general public. As has been shown in toxicokinetic studies, the liver is the major target organ of the systemic toxicity of AgNps (reviewed in Christensen et al. [16]). Therefore, in the present study, we used a test system with HepG2 human hepatoma cells to evaluate the in-vitro cytotoxic potential of the AgNps, the PGA-capped AgNps (AgNpPGAs), and the AgNpPGAs encapsulated within the PLGA polymer
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matrix (PLGA/AgNpPGAs). To reveal possible cytotoxicity activities of AgNps, AgNpPGAs and PLGA/AgNpPGAs towards these HepG2 cells, we used the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. As recent studies have indicated that the higher toxic potential of nanomaterials in comparison to their larger counterparts appears to be due to oxidative stress as a consequence of increased production of reactive oxygen species (ROS) [17,18], it was also important to determine the ROS-inducing potential of our samples. Therefore, we also determined whether these AgNps, AgNpPGAs and PLGA/AgNpPGAs result in increased production of ROS. To date, the literature has described the synthesis of composites of AgNps with polymers such as cellulose [19], polyurethane [20], poly(acrylamide) [21], chitosan [22], poly(e-caprolactone) [23], poly(styrene) [24], poly(methylmethacrylate) [25], montmorillonite [26], and polyvinyl alcohol [27]. These have provided materials in the forms of films, scaffolds, fibers or grafts. The literature has also describes the obtaining of poly(L-lactide) and PLGA nanofibers containing AgNps using an electrospinning method [28], as well as the obtaining of PLGA/silver composite grafts by extraction methods [8]. Our study thus reports on obtaining PLGA microcomposite and nanocomposite spheres with stable, biocompatible AgNps finely dispersed in a polymer matrix, which represent an important system in the field of medicine, nanomedicine, pharmacy and controlled drug delivery, and in particular to overcome resistance to antibiotics. 2. Experimental section 2.1. Materials The reagents used were of analytical grade: AgNO3 (Mr, 169.88; Centrohem, Serbia), NaOH (Mr, 40.00; Kemika, Croatia) and NH4OH (Mr, 35.046; Zorka Pharma, Serbia). Saccharose was purchased from VWR BDH Prolabo, Belgium. PGA was obtained from Guilin Peptide Technology Limited, China (Mr, 20,000e40,000 g/mol). PLGA was purchased from Bio Invigor Corporation, Taiwan (lactide-to-glycolide ratio, 50:50; Mr, 40,000e50,000 g/mol). Polyvinyl pyrrolidone (povidone, PVP) was obtained from Merck Chemicals Ltd (k-25, Merck, Germany). The following agents and chemicals used for the determination of cytotoxicity and the formation of ROS were obtained from Sigma Aldrich (St. Louis, USA): Eagle’s Minimal Essential Medium, penicillin/streptomycin, L-glutamine, phosphate-buffered saline, trypsin, fetal bovine serum, non-essential amino-acid solution (100), MTT, dimethyl sulfoxide, tert-butyl hydroperoxide and 2,7-dichlorofluorescein diacetate (DCFH-DA). 2.2. Synthesis of bare AgNps and AgNpPGAs The AgNps were prepared by chemical reduction, as described in the literature [29] with major modifications. AgNO3 was used as the precursor, saccharose as the reducing agent, and PGA as the capping agent. Ten milliliters of a 0.001 g/ml aqueous solution of AgNO3 was mixed on a magnetic stirrer (300 rpm) and 60 C, and then 10 ml 0.01 g/ml NaOH was added. The AgNO3 turned a dark color due to Ag2O precipitate. The addition of 10 ml 0.005 g/ml NH4OH then turned this solution clear. An aqueous solution of saccharose (10 ml, 1%w/w) was heated to boiling over 35e45 min, and then added drop-wise into the mixture. The formation of the AgNps promoted another change in color as these small AgNps are yellow-green. The addition of 0.5 ml 0.1wt% PGA then stabilized these AgNps, prevented their agglomeration, and made them more biocompatible, due to a layer of absorbed glutamic acid anions on
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their surface. This reaction mixture was stirred for over 3 h before the AgNpPGAs were collected. 2.3. Incorporation of AgNpPGAs within the PLGA polymer matrix The synthesis of the PLGA/AgNpPGA nanocomposites was performed according to our previously described protocol [30]; i.e. using a physicochemical method with solvent/non-solvent systems, where the solutions obtained were centrifuged. The synthesis was carried out by homogenization of the colloidal silver (AgNpPGAs) and the polymeric solution (PLGA) in acetone, followed by precipitation with ethanol and stabilization with PVP. PLGA commercial granules (220 mg) were dissolved in 20 ml acetone over approximately 2 h at room temperature. Then, 3 ml of the aqueous solution containing AgNpPGA was added to this PLGA solution, with continuous homogenization at 200 rpm over 30 min. This was followed by precipitation by addition of 25 ml ethanol, with the solution obtained was poured very slowly into 40 ml aqueous 0.05% (w/w) PVP solution, with continuous stirring at 1200 rpm. This final solution was centrifuged at 5000 rpm for 120 min, at 10 C (Universal 320R, Hettich, Germany). 2.4. Characterization of the AgNps UV measurements were performed on a GBC Cintra UVeVIS spectrophotometer in the frequency interval of 200e600 nm to estimate the formation and stability of the AgNps and AgNpPGAs, and the formation of the PLGA/AgNpPGAs. For identification of the phase composition (phase analysis) of the samples, X-ray diffraction was used, with a Philips PW 1050 diffractometer with Cu-Ka1,2 radiation (Ni filter). The measurements were carried out in the 2q range of 10 e90 , with a scanning step width of 0.05 , and 2 s per step. To define the microstructures of these AgNps, AgNpPGAs and PLGA/AgNpPGAs, field-emission scanning electron microscopy (FESEM) was carried out, on a SUPRA 35 VP Carl Zeiss instrument. The samples were prepared by re-dispersion in ethanol in an ultrasonic bath, and filtering of the dispersions using polycarbonate membranes. The carbon coating was used to prevent their charging. Transmission electron microscopy (TEM) using a JEOL JEM-2100 instrument provided further morphological characterization of the AgNpPGAs encapsulated within the PLGA polymeric matrix, based on the exploration of the individual nanostructures. The structural characteristics of the AgNps were determined by selected-area electron diffraction (SAED) and high-resolution TEM (HRTEM). Samples for TEM analysis were prepared by dispersing the PLGA/ AgNpPGA powders in distilled water, using an ultrasonic bath. The suspensions were then dripped onto lacey carbon film supported by a 300-mesh copper grid. The zeta potential was measured using a Zetasizer (Nano ZS, Model ZEN3600) with a 5 nm to 10 mm particle size range for zeta potential determination (Malvern Instruments, Malvern, UK), following the principle of electrophoretic mobility under an electric field. The zeta potential is the function of dispersion/suspension pH that determines the particle stability in the dispersion. 2.5. In-vitro release PLGA particles without and with AgNpPGAs were kept over three months (90 days) in a physiological solution at 37 1 C (VIMS electric, SCG) in the absence of light. Naþ and Cl ions dominate in extracellular fluids, so the composition of this physiological solution was 154 mM Naþ, 154 mM Cl, with its osmolarity thus corresponding to the osmolarity of extracellular fluids. The supernatant was sampled (and replaced with fresh solution)
approximately every fifth day over these 90 days, and the AgNps released from the PLGA/AgNpPGA spheres were quantified using UV spectroscopy in the frequency interval of 300 nme600 nm. The supernatant was also collected for examination of its antimicrobial activity. The morphological characteristics of the PLGA/AgNpPGAs during their degradation were examined using FESEM. All of the measurements were conducted in triplicate, and the mean values are reported. 2.6. Antimicrobial activities and determination of minimal inhibitory concentrations The antimicrobial activities of the compounds tested were evaluated against Gram-positive (two MRSA isolates and F. faecalis) and gram-negative (E. coli, K. pneumoniae and P. aeruginosa) bacteria, and against a yeast (C. albicans). The broth microdilution method was used to determine the minimal inhibitory concentrations (MICs) of the compounds tested, according to the Clinical and Laboratory Standards Institute (CLSI, 2005) [31]. These tests were performed in MüllereHinton broth for the bacterial strains, and in Sabouraud dextrose broth for C. albicans. Overnight broth cultures were prepared for each strain, and the final concentration in each well was adjusted to 2 106 CFU/ml for the bacteria, and 2 105 CFU/ml for the yeast. Compounds were dissolved in 1% dimethylsulfoxide and serial doubling dilutions (over the range 1000e12.5 ml/ml) were prepared, in MüllereHinton broth for bacteria and Sabouraud broth for C. albicans, in a 96-well microtiter plate. In the tests, 0.05% triphenyl tetrazolium chloride (Aldrich Chemical Company Inc., USA) was also added to the culture medium as a growth indicator. As a positive control of growth, wells containing only the microorganisms in the broth were used. Microbial growth was determined after 24 h of incubation at 37 C for the bacteria, and after 48 h at 26 C for C. albicans. The MICs are defined as the lowest concentration of a compound at which the microorganism does not demonstrate visible growth. All of the MIC determinations were performed in duplicate, and two positive growth controls were included. 2.7. Determination of cytotoxicity of the AgNps, AgNpPGAs and PLGA/AgNpPGAs: the MTT assay HepG2 cells were obtained from the European Collection of Cell Cultures (ECACC). The cells were grown in Eagle’s Minimal Essential Medium containing 10% fetal bovine serum, 1% non-essential amino-acids solution, 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin, at 37 C in humidified atmosphere and under 5% CO2. The cytotoxicities of the AgNps, AgNpPGAs and PLGA/AgNpPGAs were determined with MTT, according to Mosmann [32], with minor modifications [2]. This assay measures the conversion of MTT to the insoluble formazan by dehydrogenase enzymes of intact mitochondria of living cells. The HepG2 cells were seeded into 96-well microplates (Nunc, Naperville IL, USA) at a density of 40,000 cells/ml, and incubated for 20 h at 37 C to attach. The medium was then replaced by fresh complete medium without and with 0.01%, 0.1% and 1% (v/v) AgNps, AgNpPGAs and PLGA/AgNpPGAs, and incubated for a further 24 h. A negative control (non-treated cells) was always included. MTT (final concentration, 0.5 mg/ml) was then added, with an incubated for an additional 3 h, after which the medium with MTT was removed and the formazan crystals formed were dissolved in dimethyl sulfoxide. The optical density at 570 nm (OD570) was measured (reference filter, 690 nm) using a microplate-reading spectrofluorimeter (Tecan GENios, Austria). Cell
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survival (viability) was determined by comparing the OD570 of the wells containing the treated cells with those of the non-treated cells. Three independent experiments were performed, each with five replicates per concentration. The data are expressed as the means of these three independent experiments. The students’ t-test was used for the statistical significance between the treated and control cells; P < 0.05 was considered significant. 2.8. Determination of intracellular reactive oxygen species formation: the DCFH-DA assay The formation of intracellular ROS was measured spectrophotometrically using the fluorescent probe DCFH-DA, as described by Osseni et al., [33], with minor modifications [34]. DCFH-DA readily diffuses through the cell membrane and is hydrolyzed by intracellular esterases to the non-fluorescent 20 ,70 -dichlorofluorescin. In the presence of ROS, this is rapidly oxidized to the highly fluorescent 20 ,70 -dichlorofluorescein (DCF). The DCF fluorescence intensity is proportional to the amount of ROS formed intracellularly, with H2O2 as the principle ROS responsible for oxidation of DCFH-DA to DCF [35]. The HepG2 cells were seeded at a density of 75,000 cells/ml into 96-well, black, tissue-culture-treated microtiter plates (Nunc, Naperville IL, USA), as five replicates. After 20 h of incubation at 37 C in 5% CO2, the cells were incubated with 20 mM DCFH-DA. After 30 min, the DCFH-DA was removed and the cells were left untreated or were treated with 0.01%, 0.1%, and 1% (v/v) AgNps, AgNpPGAs and PLGA/AgNpPGAs in phosphate-buffered saline. Negative controls (non-treated cells) and positive controls (0.5 mM tert-butyl hydroperoxide treated) were always included. For kinetic analyses, the dishes were maintained at 37 C, and the fluorescence intensities were determined every 30 min over a 5 h incubation, using a microplate-reading spectrofluorimeter (Tecan, Genios) at 485 nm excitation wavelength and 530 nm emission wavelength. Statistically significant differences between the treated groups and controls were determined by one-way analysis of variance (KruskaleWallis) with Dunnett’s post-test. P < 0.05 was considered to be statistically significant. Two independent experiments were performed, each with five replicates. 3. Results and discussion 3.1. Ultravioletevisible spectroscopy The successful synthesis of the AgNps was first indicated by the specific colors of the colloidal solution. After 30 min of the reaction, the colorless solution was colored into pale yellow. From the changes in solution color and spectra measurements we can conclude that the reaction was complete within 45 min, under magnetic stirring. This was shown by the absence of any further color change after this time, with the yellow-green color of the final product persisting even after an extended periods of stirring. UVeVIS absorption spectra have been shown to be relatively sensitive to the formation of silver colloids, as AgNps have an intense absorption band due to surface plasmon excitation [36]. The spectra of noble-metal particles and clusters show pronounced resonance lines that are caused by the collective excitation of their conduction electrons. These excitations are known as particle plasmons, Mie plasmons, or surface plasmons [36]. Mie theory (classical electromagnetic theory of spherical particles) has a crucial role in describing the optical properties of metal nanoparticles, and especially of AgNps [37]. The spectral position of the particle plasmon depends on the dielectric properties of the cluster metal, the size and shape of the particles and clusters, the influence
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of the surrounding medium, and their chemical interactions with their environment. From the literature, there is the well-known phenomenon of polaritonic red-shift with increasing cluster size for sizes greater than approximately 10 nm, due to electromagnetic delay [36]. In addition, resonances of higher multipolar orders appear as well as the usual dipolar resonance when the cluster size is increased, such that the optical field becomes non-uniform across the cluster. In clusters of non-spherical shape, the single dipolar resonance of the spherical case splits into two or more nondegenerate plasmon modes that differ in their oscillation directions [36,37]. This UVeVIS absorption confirmed the formation of AgNps prepared in the solution by a modified chemical reduction (AgNO3 reduced by the saccharose disaccharide) (Fig. 1). In the present study, the UV/VIS spectra showed only one dominant, broad absorption band, at around 420 nm, which represents the typical signature of dipolar plasmon resonance of spherical AgNps [36e38]. This thus confirmed the formation of AgNps after 30 min of reaction. UV spectroscopy was used to show the formation of the bare AgNps, AgNpPGAs and PLGA/AgNpPGAs (Fig. 1). The absorption spectra of solutions containing AgNpPGAs and PLGA/AgNpPGAs recorded immediately after preparation and after two months confirmed the very good stability of these samples (Fig. 1B,C). Knowing the molar absorptivity of the solution containing the AgNpPGAs at a particular wavelength, and measuring the absorbance of the solution containing PLGA/AgNpPGAs at that wavelength, we calculated the concentration of these encapsulated AgNpPGAs within the PLGA polymer matrix. The concentration of the AgNpPGAs was 2.33 106 mol/l. The UV/VIS absorption spectra of the AgNpPGAs that were synthesized using PGA as the stabilizer show a narrow surface plasmon absorption band at a wavelength of 418 nm (Fig. 1A). In the case of the bare AgNps obtained from the reaction without PGA addition over the same time (45 min), the absorption band is at 435 nm, which means that the AgNpPGAs have a smaller size. The blue shift of the plasmon resonance from 435 nm to 418 nm is due to the change in the local refractive index of the nanoparticles that results from the interaction between PGA and the metal surface [36,38]. The formation rate of AgNpPGAs was also investigated using UVeVIS spectroscopy. The absorption spectra of the water solutions of AgNpPGAs were recorded at various reaction times (from 30 min to 90 min of the reaction) (Fig. 2). This showed that the reaction time was an important factor in the particle formation. The maximum absorption of the surface plasmon resonance band changed to 429 nm from 30 min to 44 min, and to 418 nm at 45 min. This absorption maximum displacement to smaller wavelengths (blue shift) after 45 min of reaction indicates the change to a smaller particle size. These data thus indicate the process of the forming of these particles: larger particles were dominant at the beginning of the reaction, and then the smaller particles became prevalent. 3.2. X-ray diffraction To determine the phase composition of the PLGA/AgNpPGAs, X-ray diffraction was applied. PLGA did not show any crystalline peaks under our experimental conditions because it is amorphous. The characteristic crystalline peaks of silver appeared after its encapsulation in PLGA particles. This indicates that the AgNpPGAs are integrated within the PLGA polymer matrix. The X-ray diffraction confirmed the silver phase, with characteristic reflections (111) (200), (220) and (311) of face-centered cubic (fcc) Ag (JCPDS No.40783) [27,39].
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Fig. 2. Representative UVeVIS spectra of the AgNpPGAs at different times during the reaction: (1) 30 min; (2) 32 min; (3) 33 min; (4) 35 min; (5) 36 min; (6) 38 min; (7) 40 min; (8) 44 min; (9) 45 min; and (10) 90min.
samples were obtained at the macroscopic, as well as microscopic, level. From the micrograph of the bare AgNps (Fig. 3A), these nanoparticles are spherical, with a smooth surface and a high level of uniformity, although also a high level of agglomeration. These bare AgNps were thus joined together, and formed clusters, fibers and rope-like structures (Fig. 3A). The main reason for this is the lack of the stabilizer. In contrast to these bare AgNps, from the FESEM images of the AgNpPGAs, it can be seen that they were very uniform, with diameters on the nanometer scale and with much less agglomeration (Fig. 3B). These AgNpPGAs have a spherical shape and no cracks or pores on their surface. The size distribution of the AgNpPGAs was also unimodal, with diameters of about 5 nme40 nm. Fig. 4 shows the FESEM of the microstructure of the PLGA/ AgNpPGAs. The morphological characteristics of these nanoparticles are extremely important for controlled drug delivery and tissue engineering, and particularly influence their adhesion and interactions with cells (e.g. intracellular uptake). Along with other factors, the dynamics of the release (rate and concentration) of encapsulated medicaments greatly depend on the morphology of such nanoparticles [2,30]. These PLGA/AgNpPGAs have diameters around and below 1 mm, and as can be seen, they have a uniform spherical morphology and are not agglomerated (Fig. 4).
3.4. Transmission electron microscopy
Fig. 1. Representative UVeVIS spectra. (A) Bare AgNps and AgNpPGAs recorded immediately after preparation (as indicated). (B) AgNpPGAs recorded after two months. (C) PLGA without and with encapsulated AgNpPGA (PLGA/AgNpPGAs) recorded immediately and after two months of the preparation (as indicated).
3.3. Morphology studies The morphological characteristics of the AgNps, AgNpPGAs and PLGA/AgNpPGAs were observed by FESEM. By using PGA as a stabilizer, the different morphological characteristics of the
Fig. 5a shows a bright-field TEM image of PLGA spherically shaped particles with encapsulated PGA-capped silver nanoparticles. Larger, 15e30 nm sized AgNpPGA nanoparticles, within the PLGA polymeric matrix, are randomly distributed clusters of few Ag nanoparticles (Fig. 5a) while individual spherical PGAcapped silver nanoparticles with average diameter of 4.5 nm are homogeneously encapsulated within the PLGA polymeric matrix (Fig. 5b). The typical selected-area electron diffraction pattern of AgNpPGAs within the PLGA matrix in Fig. 5c shows the coexistence of AgNps with cubic and hexagonal crystal structures. This formation of cubic and hexagonal AgNps is in agreement with previously reported X-ray diffraction analyses [27]. Since in our case, due to the small amount of characterized sample, the existence of AgNps with a hexagonal crystal structure was not seen by
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Fig. 3. Representative FESEM images of AgNps. (A) Obtained without PGA. (B) Obtained with PGA (AgNpPGAs). Different fields and magnifications are shown.
X-ray diffraction analysis, the preferential formation of cubic AgNps within the polymeric matrix is assumed. The high-resolution TEM images of Fig. 5d,e shows the growth characteristics of these AgNps within the spheres of the PGA
capping agent when they are encapsulated in the PLGA polymeric structure as PLGA/AgNpPGAs. When individual AgNps are capped by PGA to form AgNpPGAs, their aggregation is prevented and single crystalline AgNpPGAs with an average diameter of 4.5 nm are
Fig. 4. Representative FESEM images of PLGA/AgNpPGAs spherical particles, with different fields and magnifications shown.
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Fig. 5. (a) Bright-field TEM image of PLGA particles with encapsulated individual 15e30 nm sized AgNpPGA nanoparticles (clusters), (b) Bright-field TEM image of clusters of AgNpPGA nanoparticles randomly distributed within PLGA matrix and AgNpPGA nanoparticles with average size of 4.5 nm homogeneously distributed within PLGA matrix and (c) SAED pattern of PLGA/AgNpPGA particles HRTEM image of (d) 5 nm sized single crystalline Ag nanoparticles within PLGA matrix and (e) Larger (w30 nm sized) polycrystalline Ag nanoparticle on the edge of PLGA polymer matrix.
obtained, as shown in Fig. 5d. The larger, 15e30-nm diameter AgNpPGAs within the PLGA polymeric matrix appear to be composed of differently oriented smaller units (Fig. 5e). The polycrystalline structure of these AgNps suggests that the smaller initially formed AgNps of different orientations in close proximity to each other are confined within the PGA layer, due to their high surface energy, and they tend to assemble into larger clusters by a random-aggregation mechanism.
(Table 1). The sizes of these particles from dispersion of the same samples determined using a Malvern particle analyzer were greater than those obtained by FESEM and TEM for the particles in the same samples. The explanation of this phenomenon lies in the hydrodynamic effects, the layers of ions around the particles, and the aggregates of the particles [41]. The polydispersity index (PDI), which is a dimensionless measure that indicates the width of the size distribution, has values between 0 and 1 here (with 0 being for monodispersed particles) (Table 1).
3.5. Zeta potential measurements The zeta potential gives the overall charge that a particle acquires in a specific medium. The magnitude of the zeta potential provides an indication of the potential stability of colloidal systems [40]. The stabilizer used here, as PGA for the synthesis of AgNpPGAs or as PVP for the incorporation of AgNpPGAs within the PLGA polymer matrix, creates negatively charged particles, which induces a specific zeta potential. The stabilizer, as PGA or PVP, reduces the agglomeration because the particles of the same charge are not attracted to each other. If the particles have low zeta potentials, then there is little or no force to prevent the particles coming together, which leads to dispersion instability. A dividing line between stable and unstable aqueous dispersions is generally taken to be at either þ30 mV or 30 mV [40]. Particles with zeta potentials more positive than þ30 mV or more negative than 30 mV are normally considered to be stable. The zeta potentials of our samples of AgNpPGAs and PLGA/AgNpPGAs were greater than 30 mV, which indicates high stability of our samples
3.6. In-vitro release An in-vitro “drug-release” study was performed to investigate the release of the AgNps from the PLGA/AgNpPGAs (Fig. 6). This followed a continuous and a multiphase pattern over 90 days (Fig. 6A). The first phase represents a “burst” effect, which is caused by the release of the drug that was adsorbed to the outer particle surface. Here, initially, 7% of the AgNpPGAs was released over the Table 1 Characteristics of the AgNpPGAs and PLGA/AgNpPGAs in the dispersions. Preparation
Particle size (nm)
Polydispersity index
Zeta potential (mV)
AgNpPGAs PLGA/AgNpPGAs
39 5 811 5
0.385 0.127
54.9 1.5 32.5 0.9
Data are mean standard deviation (n ¼ 5). The zeta potentials are in the pH range 4.30e4.37.
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Fig. 6. (A) Cumulative in-vitro release of AgNps from PLGA/AgNpPGAs over time in physiological solution as a degradation medium (pH 7.4; 37 1 C). Data are means SD (n ¼ 3). Inset: UV spectra for PLGA/AgNpPGAs after different degradation times. (B) Representative FESEM micrograph of dry degraded PLGA/AgNpPGAs, after 40 days of degradation.
first day of degradation. The second phase is characterized by relatively slow release due to the diffusion of the AgNpPGAs out of the matrix (from the first to the 20th day). The third phase is a phase of increased AgNpPGAs release that is caused by (extensive) polymer degradation, which results in increased permeability of the AgNpPGAs from the polymer matrix. More than 96% of the AgNpPGAs were released by the end of 90 days. FESEM was used to assess the changes in the morphology of the samples during this degradation, with a representative FESEM micrograph of the dry degraded PLGA/AgNpPGAs shown in Fig. 6B. The particles are in very close contact during the degradation process, which leads to higher agglomeration, with the simultaneous release of the previously encapsulated AgNpPGAs. 3.7. Antimicrobial activity The antibacterial and fungicidal activities are an important characteristic of any material that is intended for biomedical applications. The mechanisms of the antimicrobial activity of AgNps have been the subject of intense investigations. Ag ions and Ag-based compounds are highly toxic to microorganisms, and they show strong biocidal effects on at least 12 species of bacteria [10]. In the present study, we also determined the MICs for AgNps, AgNpPGAs and PLGA/AgNpPGAs against seven different pathogens (Table 2) and compared these MICs values with the MICs for commercial antibiotic drugs ampicillin, amikacin and nystatin (Table 3). All as-synthesized samples exhibited much better antimicrobial activity then these antibiotics. The PLGA/AgNpPGAs achieved bacterial reductions at levels superior to the AgNps and AgNpPGAs (Table 2, Fig. 7). These growth inhibition effects were also concentration-dependent.
Analyzing antimicrobial activity of many polymers has been reported by many authors [42e49]. We hypothesized that several factors (components) might act synergically with the AgNps for the killing of these pathogens. The PVP used for the stabilization of the PLGA/AgNpPGAs, the PGA used as the capping agent for the AgNpPGAs as well as the PLGA in which AgNpPGAs are encapsulated also might have antimicrobial activities [42e45]. The high molecular weight nature of polymers makes them systemically non-absorbed, thus providing a number of advantages including long-term safety profiles over traditional small molecule drug products. In addition, multiple functional groups in the polymers incorporate polyvalent binding interactions that can result in pharmaceutical properties not found in small molecule drugs [46,47]. Most bacterial infections are initiated by adhesion of microorganisms to the mucosal surfaces of the host [46]. Cluster effects from polyvalent ligands would lead to amplification of weak non-covalent bonding interactions between the bacterial surface receptors and the polymer. The cell membrane of Gram-positive and Gram-negative bacteria has an overall charge because of the presence of teichoic acids and lipopolysaccharide, respectively. PVP and PGA are different types of polyelectrolites and the potent bactericidal activity of these polymers could be explained by strong interaction between these polymers and the charged cell membrane of bacteria [46e49]. Aggregation and precipitation of bacteria by polyvalent ligands is potentially another favorable feature. Finally, polyvalent ligands utilizing multi-point attachments could enhance lysis of the bacterial cell membrane/wall [46e49]. It was evident from these results (Table 2) that PLGA micro- and nanospheres are a carrier system with adjuvant properties for the
Table 2 Minimal inhibitory concentrations of the AgNps, AgNpPGAs and PLGA/AgNpPGAs using the broth microdilution assay, according to time from preparation and during degradation. Microorganism
MIC (mg/ml) AgNps
MRSA (ATCC 43300) MRSA (clinical isolate) E. faecalis (ATCC 29212) E. coli (ATCC 25922) K. pneumoniae (ATCC 13889) P. aeruginosa (ATCC27853) C. albicans (ATCC 10231)
AgNpPGAs
PLGA/AgNpPGAs
Post-preparation
Two months
Post-preparation
Two months
One day
30 days
50 days
85 days
0.24 0.24 0.39 0.24 0.48 0.60 0.12
>1.20 >1.20 1.20 0.36 0.36 0.36 0.24
0.13 0.13 0.38 0.20 0.16 0.20 0.13
0.13 0.13 0.20 0.20 0.20 0.25 0.13
0.0055 0.004 0.017 0.007 0.008 0.008 0.007
0.005 0.003 0.009 0.007 0.007 0.009 0.005
0.005 0.005 0.020 0.011 0.011 0.013 0.005
0.006 0.006 0.018 0.006 0.009 0.009 0.006
MRSA e methicillin-resistant S. aureus, ATCC e American type culture collection.
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3.8. In-vitro cytotoxicity and induction of intracellular ROS formation by AgNps, AgNpPGAs and PLGA/AgNpPGAs
Table 3 Minimal inhibitory concentrations of standard antibiotics. Microorganism
S. aureus (ATCC 25923) S. epidermidis (ATCC 12228) M. luteus (ATCC 9341) M. flavus (ATCC 10240) E. faecalis (ATCC 29212) B. subtilis (ATCC 6633) E. coli (ATCC 25922) K. pneumoniae (ATCC 13883) P. aeruginosa (ATCC 27853) C. albicans (ATCC 10259)
MIC (mg/ml) Ampicillin
Amikacin
Nystatin
0.5 0.2 2 3 0.5 n.t. 2 4 3 n.t.
2 n.t. n.t. n.t. n.t. n.t. 5 n.t. 0.5 n.t.
n.t. n.t. n.t. n.t. n.t. n.t. n.t. n.t. n.t. 3
ATCC e American type culture collection. n.t. e not tested.
delivery of PGA-capped silver nanoparticles. Considering that the AgNp release from the PLGA/AgNpPGAs was prolonged for up to 85 days, antimicrobial activities can be expected, and were seen, for prolonged periods of time (Table 2).
Cell viability assays represent a vital step in toxicology investigations, to explain the cell response to a toxicant. They give information on the effects of the material tested on cell survival, metabolic activity and death [50]. The cytotoxicity of these AgNps, AgNpPGAs and PLGA/AgNpPGAs were determined by measurement of mitochondrial activity with the MTT assay after treatment of HepG2 cells without or with 0.01%, 0.1%, and 1% (v/v) AgNps, AgNpPGAs and PLGA/AgNpPGAs for 24 h. Only the bare AgNps at the highest applied concentration (1%), reduced the viability of these HepG2 cells (>90%) (Fig. 8A). This cytotoxicity of these AgNps is in line with several previous studies [50e53]. It is considered that nanoparticles, including AgNps, induce toxicity predominately through oxidative stress, by the generation of ROS, which have specific effects in cells, including oxidative damage to DNA, protein and lipid [16, 53-59].
Fig. 7. Representative microplate wells for antimicrobial actions. The darker color indicates a more viable microbial. Top row: AgNps immediately after preparation (left) and after two months (right). Second row: AgNpPGAs immediately after preparation (left) and after two months (right). Third row: PLGA/AgNpPGAs after one day (left) and after 30 days (right). Bottom row: PLGA/AgNpPGAs after 50days (left) and 85days (right) of degradation. 1, MRSA (ATCC 43300); 2, MRSA (clinical isolate); 3, E. faecalis (ATCC 25922); 4, E. coli (ATCC 25922); 5, K. pneumoniae (ATCC 27853); 6, P. aeruginosa (ATCC 27853); and 7, C. albicans (ATCC 10231). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 8. Quantification of in-vitro cytotoxicity and induction of intracellular ROS formation. (A) Effects of AgNps, AgNpPGAs and PLGA/AgNpPGAs on cell viability, as indicated. Data are means S.D. of three independent experiments, each carried out as five replicates. * Significant difference over relevant control (students’ t-test, P < 0.05). (B) Effects of AgNps, AgNpPGAs and PLGA/AgNpPGAs on intracellular ROS formation, as indicated. Data are means SD of two independent experiments. *, Significant difference over relevant control (ANOVA, KruskaleWallis, P < 0.05).
To determine the influence of these AgNps, AgNpPGAs and PLGA/AgNpPGAs on intracellular ROS formation, we used the DCFH-DA assay to measured ROS formation in the HepG2 cells. The bare AgNps, the capped AgNpPGAs and the PLGA/AgNpPGAs did not induce any increases in DCF fluorescence intensity (Fig. 8B). This indicates that the observed cytotoxicity of the AgNps at the highest applied dose (1%) was not caused by oxidative stress. Several previous studies have shown that silver ions (Agþ) are more cytotoxic than AgNps [60e62], and thus it is possible that the cytotoxicity of these AgNps was due to the release of Agþ. Interestingly, there were some significant decreases in DCF fluorescence compared to the controls, for the lowest dose of the AgNps (0.01%), for the AgNpPGAs at 0.1% and 1%, and for the PLGA/ AgNpPGAs at 0.1% (Fig. 8B). This can be explained through a recent study by Arora et al. [51], who showed that in-vitro in rat fibroblasts and liver cells, lower doses of AgNps can provide protective effects against oxidative stress characterized by decreases in lipid peroxidation and increases in the antioxidant protective enzymes glutathione transferase and superoxide dismutase. These data show that the toxic potential of these bare AgNps can be reduced by coating them with organic layers through capping with PGA (AgNpPGAs), and additionally by their encapsulation within a PLGA polymer matrix (PLGA/AgNpPGAs), thus increasing their biocompatibility. These findings are very encouraging for future applications of these AgNps. 4. Conclusions Highly stable, uniform, spherical PGA-capped AgNps (AgNpPGAs) in the size range of 5 nme40 nm were successful synthesized and then encapsulated within a PLGA polymer matrix (PLGA/ AgNpPGAs). These PLGA/AgNpPGAs are spherical, uniform and do not agglomerate. Our study thus shows that the toxic potential of bare AgNps can be reduced by the coating of these nanoparticles with organic layers, with capping with PGA (AgNpPGAs), and additionally by their encapsulation within a PLGA polymer matrix (PLGA/AgNpPGAs). The MTT assay data indicate good biocompatibility of these AgNpPGAs and PLGA/AgNpPGAs. Thus these AgNpPGAs and PLGA/AgNpPGAs are biocompatible, and we show that they do not induce increased production of ROS in HepG2 cells in-vitro. The in-vitro release of the AgNps from these PLGA/AgNpPGAs follows a continuous and multiphase pattern over 90 days. These PLGA/AgNpPGAs can achieve bacterial inhibition at levels superior to the AgNps and AgNpPGAs as well as three different commercial antibiotics. Considering that AgNps
release from these PLGA/AgNpPGAs is prolonged for over 85 days, this antimicrobial activity can be expected to last for prolonged periods of time. These data are very encouraging for future applications of AgNps. The PLGA/AgNpPGAs thus provide a system that is a new and simple solution in the battle against antibiotic resistance, which is a huge and widespread problem. Acknowledgements This study was supported by the Ministry of Science and Technological Development of the Republic of Serbia, under Grant No. III45004: Molecular designing of nanoparticles with controlled morphological and physicochemical characteristics and functional materials based on them. The authors would like to thank to Igor Savanovi c for technical assistance, Miodrag Mitri c and Ljiljana Kandi c for the X-ray measurements, and Mirjana Markovi c for the zeta potential measurements. References [1] Acharya S, Sahoo SK. Advanced Drug Delivery Reviews 2011;63(3):170e83. [2] Stevanovi c M, Maksin T, Petkovi c J, Filipi c M, Uskokovi c D. Nanotechnology 2009;20:335102 (12pp). [3] Hong Y, Fujimoto K, Hashizume R, Guan J, Stankus JJ, Tobita K, et al. Biomacromolecules 2008;9(4):1200e7. [4] Stevanovi c M, Uskokovi c D. Current Nanoscience 2009;5(1):1e14. [5] Okeke IN, Laxminarayan R, Bhutta ZA, Duse AG, Jenkins P, O’Brien TF, et al. Lancet Infectious Diseases 2005;5:481e93. [6] Shehab N, Patel PR, Srinivasan A, Budnitz DS. Clinical Infectious Diseases 2008; 47:735e43. [7] Kumarasamy KK, Toleman MA, Walsh TR, Bagaria J, Butt F, Balakrishnan R, et al. Lancet Infectious Diseases 2010;10:597e602. [8] Zheng Z, Yin W, Zara JN, Li W, Kwak J, Mamidi R, et al. Biomaterials 2010;31: 9293e300. [9] Xing ZC, Chae WP, Baek JY, Choi M-J, Jung Y, Kang I-K. Biomacromolecules 2010;11(5):1248e53. [10] Kim J, Kuk E, Yu K, Kim J, Park S, Lee H, et al. Nanomedicine: Nanotechnology, Biology and Medicine 2007;3:95e101. [11] Stevanovi c M, Savanovi c I, Uskokovi c V, Skapin SD, Bra cko I, Jovanovi c U, et al. Colloid and Polymer Science 2012;290:221e31. [12] Stevanovi c M, Kova cevi c B, Petkovi c J, Filipi c M, Uskokovi c D. International Journal of Nanomedicine 2011;6:2837e47. [13] Pillai R, Somayaji SN, Rabinovich M, Hudson MC, Gonsalves KE. Biomedical Matrials 2008;3:034114 (7pp). [14] Schrand AM, Rahman MF, Hussain SM, Schlager JJ, Smith DA, Syed AF. WIREs Nanomedicine and Nanobiotechnology 2010;2:544e68. [15] Takashi T, Kimura J, Takeuchi Y, Uyama H, Park C, Sung MH. Journal of Microbiology and Biotechnology 2010;20(10):1436e9. [16] Christensen FM, Johnston HJ, Stone V, Aitken RJ, Hankin S, Peters S, et al. Nanotoxicology 2010;4(3):284e95. [17] Kim S, Choi JE, Choi J, Chung KH, Park K, Yi J, et al. Toxicology In Vitro 2009; 23(6):1076e84. [18] Schins RP, Knaapen AM. Inhalation Toxicology 2007;19(1):189e98.
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