Magdalena Stevanović

DNA damage and alterations in expression of DNA damage responsive genes induced by TiO2 nanoparticles in human hepatoma HepG2 cells

Abstract We investigated the genotoxic responses to two types of TiO2 nanoparticles (<25 nm anatase: TiO2-An, and <100 nm rutile: TiO2-Ru) in human hepatoma HepG2 cells. Under the applied exposure conditions the particles were agglomerated or aggregated with the size of agglomerates and aggregates in the micrometer range, and were not cytotoxic. TiO2-An, but not TiO2-Ru, caused a persistent increase in DNA strand breaks (comet assay) and oxidized purines (Fpg-comet). TiO2-An was a stronger inducer of intracellular reactive oxygen species (ROS) than TiO2-Ru. Both types of TiO2 nanoparticles transiently upregulated mRNA expression of p53 and its downstream regulated DNA damage responsive genes (mdm2, gadd45α, p21), providing additional evidence that TiO2 nanoparticles are genotoxic. The observed differences in responses of HepG2 cells to exposure to anatase and rutile TiO2 nanoparticles support the evidence that the toxic potential of TiO2 nanoparticles varies not only with particle size but also with crystalline structure.

Poly(lactide-co-glycolide)-based Micro and Nanoparticles for the Controlled Drug Delivery of Vitamins

Controlled drug delivery systems and polymeric carriers have undergone significant development in recent years. Polymers like polylactides (PLA), polyglycolides (PGA), poly(lactide-co-glycolides) (PLGA), are approved by the World Health Organization (WHO) and Food and Drug Administration (FDA) as materials that can be used in medicine and pharmacy. Owing to their biodegradable nature, polymer materials, such as copolymer poly(DL-lactide-co-glycolide), are widely used in various medical applications; controlled release of delivering drugs, carriers in the tissue engineering, fixation of bone fractures, chirurgical strings, etc. Polymeric particles are used for the controlled delivery of several types of medicaments, including anticancer agents, antihypertensive agents, immunomodulatory drugs, hormones, vitamins and macromolecules, such as nucleic acid, proteins, peptides, antibodies, etc. Preparation of poly(lactide-co-glycolide) submicron spheres poses serious challenges. The present review attempts to address some important issues related to micro/nanoparticle-based delivery systems comprising poly(lactide-co-glycolide), with a special reference to PLGA for the controlled delivery of vitamins. A range of topics is discussed, including formulation aspects of micro- and nanoparticles, the effects of particle size and size distribution, most commonly used incorporation techniques, surface modification with stabilizers, surface functionalization, and factors affecting degradation and drug release rate. A post-print version of the article: Uskokovic, Dragan, and Magdalena Stevanovic. 2009. “Poly(lactide-Co-Glycolide)-Based Micro and Nanoparticles for the Controlled Drug Delivery of Vitamins.” Current Nanoscience 5 (1) (February 1): 1–14. doi:10.2174/157341309787314566.

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

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 5h 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.

Effect of poly-α, γ, L-glutamic acid as a capping agent on morphology and oxidative stress-dependent toxicity of silver nanoparticles

Highly stable dispersions of nanosized silver particles were synthesized using a straightforward, cost-effective, and ecofriendly method. Nontoxic glucose was utilized as a reducing agent and poly-α, γ, L-glutamic acid (PGA), a naturally occurring anionic polymer, was used as a capping agent to protect the silver nanoparticles from agglomeration and render them biocompatible. Use of ammonia during synthesis was avoided. Our study clearly demonstrates how the concentration of the capping agent plays a major role in determining the dimensions, morphology, and stability, as well as toxicity of a silver colloidal solution. Hence, proper optimization is necessary to develop silver colloids of narrow size distribution. The samples were characterized by Fourier transform infrared spectroscopy, ultraviolet-visible spectroscopy, field-emission scanning electron microscopy, transmission electron microscopy, and zeta potential measurement. MTT assay results indicated good biocompatibility of the PGA-capped silver nanoparticles. Formation of intracellular reactive oxygen species was measured spectrophotometrically using 2,7-dichlorofluorescein diacetate as a fluorescent probe, and it was shown that the PGA-capped silver nanoparticles did not induce intracellular formation of reactive oxygen species.

Composite PLGA/AgNpPGA/AscH Nanospheres with Combined Osteoinductive, Antioxidative, and Antimicrobial Activities

The global rise in the resistance of pathogens to conventional antibiotics has created an intensive search for alternative materials with antimicrobial properties. This study is performed with an intention to investigate the combined effects of poly(l-glutamic acid)-capped silver nanoparticles (AgNpPGA) and ascorbic acid (AscH) encapsulated within freeze-dried poly(lactide-co-glycolide) (PLGA) nanospheres to obtain a nanomaterial with simultaneous osteoinductive, antioxidative, and prolonged antimicrobial properties. The influence of PLGA/AgNpPGA/AscH particles on (i) viability and superoxide production of human umbilical vein endothelial cells in vitro, (ii) morphology and expression of osteogenic markers in osteoblastic MC3T3-E1 cells in vitro, and (iii) antimicrobial activity against a Gram-positive bacterium, methicillin-resistant Staphylococcus aureus, and a Gram-negative bacterium, Escherichia coli, was investigated. PLGA/AgNpPGA/AscH nanoparticles showed a superior and extended antibacterial activity against both types of bacteria. The nanoparticles appeared to be capable of delivering ascorbate to the cells, which was evidenced by the significant decrease in the level of superoxides in human umbilical vein endothelial cells and which could have a therapeutic potential in preventing oxidative stress. PLGA/AgNpPGA/AscH nanoparticles had a positive effect on MC3T3-E1 osteoblastic cells in vitro, promoting: (i) an intimate contact with the cells and preservation of their healthy morphologies; (ii) unreduced cell viability; and (iii) multiple-fold upregulation of two osteogenic markers: osteocalcin and type I procollagen. It is concluded that PLGA/AgNpPGA/AscH nanospheres present a promising new material for the treatment of infections and use in wound dressings and other prophylactic applications.

45S5Bioglass®-based scaffolds coated with selenium nanoparticles or with poly(lactide-co-glycolide)/selenium particles: Processing, evaluation and antibacterial activity

In the bone tissue engineering field, there is a growing interest in the application of bioactive glass scaffolds (45S5Bioglass(®)) due to their bone bonding ability, osteoconductivity and osteoinductivity. However, such scaffolds still lack some of the required functionalities to enable the successful formation of new bone, e.g. effective antibacterial properties. A large number of studies suggest that selenium (Se) has significant role in antioxidant protection, enhanced immune surveillance and modulation of cell proliferation. Selenium nanoparticles (SeNp) have also been reported to possess antibacterial as well as antiviral activities. In this investigation, uniform, stable, amorphous SeNp have been synthesized and additionally immobilized within spherical PLGA particles (PLGA/SeNp). These particles were used to coat bioactive glass-based scaffolds synthesized by the foam replica method. Samples were characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS) and transmission electron microscopy (TEM). SeNp, 45S5Bioglass(®)/SeNp and 45S5Bioglass(®)/PLGA/SeNp showed a considerable antibacterial activity against Gram positive bacteria, Staphylococcus aureus and Staphylococcus epidermidis, one of the main causative agents of orthopedic infections. The functionalized Se-coated bioactive glass scaffolds represent a new family of bioactive, antibacterial scaffolds for bone tissue engineering applications.

An innovative, quick and convenient labeling method for the investigation of pharmacological behavior and the metabolism of poly(DL-lactide-co-glycolide) nanospheres

Nanoparticles of poly(DL-lactide-co-glycolide) (PLGA) in the size range 90-150 nm were produced using the physicochemical method with solvent/non-solvent systems. The encapsulation of the ascorbic acid in the polymer matrix was performed by homogenization of the water and organic phases. In vitro degradation and release tests of PLGA nanoparticles with and without encapsulated ascorbic acid were studied for more than 60 days in PBS and it has been determined that PLGA completely degrades within this period, fully releasing all encapsulated ascorbic acid. The cytotoxicity of PLGA and PLGA/ascorbic acid 85/15% nanoparticles was examined with human hepatoma cell lines (HepG2 ECACC), in vitro. The obtained results indicate that neither PLGA nanospheres nor PLGA/ascorbic acid 85/15% nanoparticles significantly affected the viability of the HepG2 cells. The investigation of the distribution and pharmacokinetics of PLGA is crucial for the effective prediction of host responses to PLGA in particular applications. Thus we present a method of labeling PLGA nanospheres and PLGA/ascorbic acid 85/15 wt% nanoparticles by (99m)Tc which binds outside, leaving the cage intact. This enables a quick and convenient investigation of the pharmacological behavior and metabolism of PLGA. The biodistribution of (99m)Tc-labeled PLGA particles with and without encapsulated ascorbic acid after different periods of time of their installation into rats was examined. PLGA nanospheres with encapsulated ascorbic acid exhibit prolonged blood circulation accompanied by time-dependent reduction in the lungs, liver and spleen, and addition in the kidney, stomach and intestine. The samples were characterized by x-ray diffraction, scanning electron microscopy, stereological analysis, transmission electron microscopy, ultraviolet spectroscopy and instant thin layer chromatography.

The solvothermal synthesis of magnetic iron oxide nanocrystals and the preparation of hybrid poly(L-lactide)-polyethyleneimine magnetic particles.

We report a simple and green procedure for the preparation of magnetic iron oxide nanocrystals via solvothermal synthesis. The nanocrystal synthesis was carried out under mild conditions in the water-ethanol-oleic acid solvent system with the use of the oleate anion as a surface modifier of nanocrystals and glucose as a reducing agent. Specific conditions for homogenous precipitation achieved in such a reaction system lead to the formation of uniform high-quality nanocrystals down to 5 nm in diameter. The obtained hydrophobic nanocrystals can easily be converted to hydrophilic magnetic nanoparticles by being immobilized in a poly(L-lactide)-polyethyleneimine polymeric matrix. These hybrid nano-constructs may find various biomedical applications, such as magnetic separation, gene transfection and/or magnetic resonance imaging.

Preparation and characterization of poly(D,L-lactide-co-glycolide) nanoparticles containing ascorbic acid.

This paper is covering new, simplistic method of obtaining the system for controlled delivery of the ascorbic acid. Copolymer poly (D,L-lactide-co-glycolide) (DLPLG) nanoparticles are produced using physical method with solvent/nonsolvent systems where obtained solutions were centrifuged. The encapsulation of the ascorbic acid in the polymer matrix is performed by homogenization of water and organic phases. Particles of the DLPLG with the different content of ascorbic acid have different morphological characteristics, that is, variable degree of uniformity, agglomeration, sizes, and spherical shaping. Mean sizes of nanoparticles, which contain DLPLG/ascorbic acid in the ratio 85/150%, were between 130 to 200 nm depending on which stereological parameters are considered (maximal diameters Dmax, feret X, or feret Y). By introducing up to 15% of ascorbic acid, the spherical shape, size, and uniformity of DLPLG particles are preserved. The samples were characterized by infrared spectroscopy, scanning electron microscopy, stereological analysis, and ultraviolet spectroscopy.

Synthesis of poly(ɛ-caprolactone) nanospheres in the presence of the protective agent poly(glutamic acid) and their cytotoxicity, genotoxicity and ability to induce oxidative stress in HepG2 cells.

Nanospheres of poly(ɛ-caprolactone) (PCL) with sizes smaller than 200 nm were produced by combining the freeze drying method and the physicochemical solvent/non-solvent approach. The influence of various types of cryoprotectants (poly(glutamic acid) (PGA) or sacharose) and their concentrations on the outcome of freeze-dried poly(ɛ-caprolactone) particles was evaluated. The physiochemical properties, structural and morphological characteristics of thereby obtained PCL particles were determined by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). The cytotoxicity of the samples was examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT assay). The formation of intracellular reactive oxygen species was measured spectrophotometrically using a fluorescent probe (DCFH-DA assay). In addition, the genotoxic response of PCL particles obtained using PGA as a cryoprotectant was investigated by the Comet assay. This paper focuses on the role of PGA in the synthesis of PCL particles and demonstrates that PGA plays a dual role in the synthesis, i.e. it acts as a stabilizer but also as a cryoprotective agent. The sufficient and optimal concentration of PGA for producing uniform, spherical but also biocompatible PCL nanoparticles is established to be 0.05%.