Multifunctional PLGA particles containing poly(l-glutamic acid)-capped silver nanoparticles and ascorbic acid with simultaneous antioxidative and prolonged antimicrobial activity

Abstract

A water-soluble antioxidant (ascorbic acid, vitamin C) was encapsulated together with poly(l-glutamic acid)-capped silver nanoparticles (AgNpPGA) within a poly(lactide-co-glycolide) (PLGA) polymeric matrix and their synergistic effects were studied. The PLGA/AgNpPGA/ascorbic acid particles synthesized by a physicochemical method with solvent/non-solvent systems are spherical, have a mean diameter of 775 nm and a narrow size distribution with a polydispersity index of 0.158. The encapsulation efficiency of AgNpPGA/ascorbic acid within PLGA was determined to be >90%. The entire amount of encapsulated ascorbic acid was released in 68 days, and the entire amount of AgNpPGAs was released in 87 days of degradation. The influence of PLGA/AgNpPGA/ascorbic acid on cell viability, generation of reactive oxygen species (ROS) in HepG2 cells, as well as antimicrobial activity against seven different pathogens was investigated. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay indicated good biocompatibility of these PLGA/AgNpPGA/ascorbic acid particles. We measured the kinetics of ROS formation in HepG2 cells by a DCFH-DA assay, and found that PLGA/AgNpPGA/ascorbic acid caused a significant decrease in DCF fluorescence intensity, which was 2-fold lower than that in control cells after a 5 h exposure. This indicates that the PLGA/AgNpPGA/ascorbic acid microspheres either act as scavengers of intracellular ROS and/or reduce their formation. Also, the results of antimicrobial activity of PLGA/AgNpPGA/ascorbic acid obtained by the broth microdilution method showed superior and extended activity of these particles. The samples were characterized using Fourier transform infrared spectroscopy, field-emission scanning electron microscopy, transmission electron microscopy, zeta potential and particle size analysis. This paper presents a new approach to the treatment of infection that at the same time offers a very pronounced antioxidant effect.

Introduction

Among the most promising nanomaterials with antibacterial and antiviral properties are metallic nanoparticles (silver, gold, platinum, etc.), which exhibit increased chemical activity due to their large surface-to-volume ratio and crystallographic surface structure [1], [2], [3], [4]. However, several recent studies indicate that, at a cellular level, metal nanoparticles interact with biological molecules within mammalian cells and can interfere with the antioxidant defense mechanism leading to the generation of reactive oxygen species (ROS). Such species, in excess, can cause damage to biological components through oxidation of lipids, proteins and DNA. Oxidative stress may play a role in the induction or enhancement of inflammation through upregulation of redox-sensitive transcription factors (e.g. NF-κB), activator protein-1 and kinases involved in inflammation [5], [6], [7]. Although some studies suggest that the toxicity of silver nanoparticles (AgNps) is mainly due to oxidative stress and independent of silver ions [8], other authors report that both silver ions and AgNps contribute to the toxicity [9], [10]. Recently, Beer et al. investigated to what level the silver ions present in the AgNp suspensions contribute to the toxicity of AgNps and how much toxicity is related to the AgNps themselves [11]. They concluded that free silver ions in AgNp preparations play a significant role in the toxicity of AgNp suspensions. Unrelated to silver ions, many studies also support the concept according to which AgNp toxicity is associated with the nanoparticles’ bare metallic surface, while particles protected by an organic layer are much more biocompatible, and hence less toxic [12]. It is also now known that the potential interaction with tissues and cells and the potential toxicity greatly depend on the actual composition of the particle formulation. There are many existing methods for delivering metal nanoparticles to human body. Polymer/metal nanoparticle composites have the ability to produce a synergistic combination of excellent properties that cannot be obtained from individual components. Until now, various polymers, such as carboxy methyl cellulose, polyurethane, sodium alginate, poly(acrylamide), chitosan, poly(e-caprolactone), poly(styrene), poly(methylmethacrylate), montmorillonite, polyvinyl alcohol, starch, etc., have been employed to prepare composites with AgNps [13]. These have provided materials in the form of films, scaffolds, fibers or grafts. The production of poly(l-lactide) and PLGA nanofibers containing AgNps using an electrospinning method [14], as well as of PLGA/silver composite grafts by extraction methods [15], have also been described in literature.

Our study investigates the possibility of the simultaneous encapsulation of poly(l-glutamic acid) (PGA)-capped silver nanoparticles (AgNpPGAs) together with an antioxidant, vitamin C (ascorbic acid), within poly(lactide-co-glycolide) (PLGA) spheres in order to obtain a system possessing simultaneously antioxidative and prolonged antimicrobial activity. PLGA is currently the most frequently used biodegradable and biocompatible matrix former for controlled drug delivery [16], [17], [18], [19]. Several products are available on the market [4], e.g. PLGA-based microparticles loaded with leuprolide (Lupron Depot) or triptorelin (Trelstar) [20]. Common methods to produce PLGA micro- and nanospheres include emulsification/solvent-evaporation, emulsification/solvent-extraction and phase separation [13], [16]. However, from a technical point of view, it is difficult to load smaller molecules into PLGA nanoparticles with high loading and encapsulation efficiency via conventional preparation methods. Conventional techniques for encapsulating active substances into polymeric nanoparticles, such as the double emulsion–solvent diffusion method, frequently suffer from low encapsulation efficiency because the drug rapidly partitions to an external aqueous phase [21]. A common strategy to increase the encapsulation efficiency is to complex the drug with ionic excipients, such as dioleyltrimethylammoniumpropane, polyethyleneimine or polyamines, in order to enhance the affinity between the drug and the particle matrix, thereby increasing the loading percentage and encapsulation efficiency of drug [20], [21]. However, such excipients are often associated with toxicity and may retard drug release. In this study, AgNpPGAs/ascorbic acid-loaded PLGA particles were prepared by a physicochemical method with solvent/non-solvent systems, in which PGA was employed as a capping agent for AgNps in an aqueous medium. PGA is a water-soluble, anionic, biodegradable and edible biopolymer produced by Bacillus subtilis. It has wide-ranging potential applications in foods, pharmaceuticals, healthcare, water treatment and other fields. PGA was chosen as the capping agent to make AgNps more biocompatibile, to prevent them from agglomerating in the medium, as well as to enhance their affinity with the PLGA polymer matrix. Compared to other methods, one of the major advantages of this type of processing of AgNps clearly arises from the use of PGA as a capping agent. The use of PGA acid as a capping agent has already been reported by another research group [22], but they used ammonia in the synthesis, which was avoided in our method. The use of ammonia, which is a very corrosive and hazardous chemical, was avoided in a number of other methods, in which, however, other chemicals were used as reducing, and/or capping agents (sodium borohydride, dimethyl formamide, cetyltrimethylammonium bromide, etc.) [23]; there is a general concern that these chemicals may pose environmental and biological risks.

AgNpPGAs, together with ascorbic acid, were additionally encapsulated within spherical PLGA particles (PLGA/AgNpPGAs/ascorbic acid) to ensure their release over an extended period of time. The antioxidant effects of ascorbic acid have been demonstrated in many experiments in vitro. In this particular case, we used ascorbic acid to promote the antioxidative effect, reduce free silver ions in AgNp preparations, and improve the effectiveness and safety of AgNps during administration. Recently, Posgai et al. have reported that two mechanisms through which ascorbic acid may reverse the toxicity of nanosilver are directly through the reduction of ROS and/or through the chelation of silver ions released by AgNps [24]. As far as we are aware, the present study is the first report of a strategy to produce a silver-based antimicrobial with prolonged activity and, at the same time, antioxidant effect based on PLGA spherical particles containing stable PGA-capped AgNps and ascorbic acid. In this study, we have evaluated (i) the in vitro degradation process and release of AgNpPGAs/ascorbic acid from the PLGA polymer matrix; (ii) the cytotoxic potential towards HepG2 human hepatoma cells; (iii) the antimicrobial activity against several different gram-positive, gram-negative bacteria and yeast; and (iv) the antioxidative properties of PLGA/AgNpPGAs/ascorbic acid particles. The method described herein provides a generally high-yield, low-cost route to the preparation of PLGA/AgNpPGAs/ascorbic acid particles which, from a materials and device development perspective, represent a promising pharmaceutical material, especially in orthopedic surgery or as an ocular drug for retinal therapies.