Dragan Uskoković

Nanosized hydroxyapatite and other calcium phosphates: Chemistry of formation and application as drug and gene delivery agents

The first part of this review looks at the fundamental properties of hydroxyapatite (HAP), the basic mineral constituent of mammalian hard tissues, including the physicochemical features that govern its formation by precipitation. A special emphasis is placed on the analysis of qualities of different methods of synthesis and of the phase transformations intrinsic to the formation of HAP following precipitation from aqueous solutions. This serves as an introduction to the second part and the main subject of this review, which relates to the discourse regarding the prospects of fabrication of ultrafine, nanosized particles based on calcium phosphate carriers with various therapeutic and/or diagnostic agents coated on and/or encapsulated within the particles. It is said that the particles could be either surface-functionalized with amphiphiles, peptides, proteins, or nucleic acids or injected with therapeutic agents, magnetic ions, or fluorescent molecules. Depending on the additive, they could be subsequently used for a variety of applications, including the controlled delivery and release of therapeutic agents (extracellularly or intracellularly), magnetic resonance imaging and hyperthermia therapy, cell separation, blood detoxification, peptide or oligonucleotide chromatography and ultrasensitive detection of biomolecules, and in vivo and in vitro gene transfection. Calcium phosphate nanoparticles as carriers of therapeutic agents that would enable a controlled drug release to treat a given bone infection and at the same be resorbed in the body so as to regenerate hard tissue lost to disease are emphasized hereby as one of the potentially attractive smart materials for the modern medicine.

Synthesis and properties of hydroxyapatite/poly-L-lactide composite biomaterials.

Calcium hydroxyapatite (HAp) and poly-L-lactide (PLLA) were synthesized chemically. The obtained HAp was of high purity and, after special thermal treatment, of high crystallinity as well. Synthesis of PLLA was performed using L-lactide as a monomer and nontoxic initiator. In this way a polymer of large molar weight (about 400,000) was obtained. The HAp and PLLA obtained were used as constituents of the HAp/PLLA composite biomaterial, a potential material for implants. The composite was obtained by mixing completely dissolved PLLA with granules of HAp. The composite was compacted by cold and hot pressing at pressures of 49.0-490.5 MPa and temperatures of 20-184 degrees C. The material obtained at optimum process parameters had a density of 99.6% and compressive strength of 93.2 MPa.

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.

A study of HAp/PLLA composite as a substitute for bone powder, using FT-IR spectroscopy.

Chemically synthesized hydroxyapatite/poly-L-lactide (HAp/PLLA) composite biomaterial was studied in vivo. The biocomposite was implanted into Balb/c Singen mice and after 1 and 3 weeks removed from their organisms and analyzed by the FT-IR spectroscopy. After 1 week of testing in vivo the implanted sample gave a spectrum in which absorption bands arising from newly formed functional groups of amine and peptide can be seen. After 3 weeks, a spectrum with pronounced absorption bands at 3420 and 1650cm(-1) assigned to newly generated collagen, a component of the extracellular connective-tissue matrix, was registered. Also, decrease of the intensity absorption band at 1760cm(-1) originating from the C=O group of PLLA indicates bioresorption of the PLLA used. Analysis of the microstructure of the sample surface by scanning electron microscopy before and after implantation revealed bioresorption of the PLLA polymer phase and generation of collagen fibers at the sites of implanted bioresorptive PLLA. A mixture of autologous bone powder and HAp/PLLA biocomposite was also examined. After implantation, the same final products as in the case of HAp/PLLA composite biomaterial used alone were found.

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.

Influence of size scale and morphology on antibacterial properties of ZnO powders hydrothemally synthesized using different surface stabilizing agents

Metal oxide nanoparticles represent a new class of important materials that are increasingly being developed for use in research and health-related applications. Although the antibacterial activity and efficiency of bulk zinc oxide were investigated in vitro, the knowledge about the antibacterial activity of ZnO nanoparticles remains deficient. In this study, we have synthesized ZnO particles of different sizes and morphologies with the assistance of different types of surface stabilizing agents - polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA) and poly (α,γ, l-glutamic acid) (PGA) - through a low-temperature hydrothermal procedure. The characterization of the prepared powders was preformed using X-ray diffraction (XRD) method and field emission scanning electron microscopy (FE SEM), as well as Malverns Mastersizer instrument for particle size distribution. The specific surface area (SSA) of the ZnO powders was measured by standard Brunauer-Emmett-Teller (BET) technique. The antibacterial behavior of the synthesized ZnO particles was tested against gram-negative and gram-positive bacterial cultures, namely Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), respectively. We compared the results of the antibacterial properties of the synthesized ZnO samples with those of the commercial ZnO powder. According to the obtained results, the highest microbial cell reduction rate was recorded for the synthesized ZnO powder consisting of nanospherical particles. In all of the examined samples, ZnO particles demonstrated a significant bacteriostatic activity.

TiO2 films prepared by ultrasonic spray pyrolysis of nanosize precursor

Abstract A methodology for the preparation of the TiO 2 films, based on the process of ultrasonic spray pyrolysis and usage of TiO 2 nanoparticles, with the mean diameter of 45 A as a precursor, was developed. The X-ray diffraction (XRD) analysis of the TiO 2 films revealed the appearance of rutile phase at 620 °C. Morphology of the TiO 2 films deposited at different temperatures on titanium substrates was investigated by scanning electron microscopy (SEM). The SEM analysis revealed that morphology of the TiO 2 films can be adjusted by temperature. It seems that the process of ultrasonic spray pyrolysis provides an easy way to prepare either compact smooth films or films with a porous structure.

Preparation of fine spherical ZnO powders by an ultrasonic spray pyrolysis method

Abstract High-purity, fine agglomerate-free, spherical ZnO powders were synthesized by means of an ultrasonic spray pyrolysis method. The solution was sprayed using an aerosol generator which was operated at 2.5 MHz. The droplets generated were fed to the reaction furnace with a carrier gas (compressed air) with flow rates of 80 l/h. The corresponding residence time of the carrier gas in the furnace was estimated to be 37.5 s, the gas velocity was 0.02 m/s, and the flow was laminar. For the reaction temperature of 1173 K, the corresponding droplets/particles heating rate was approximately 50 K/s. The particle size and the perimeter form factor distribution indicated that the most particles are in the range from 0.5 to 1.1 (μ with the mean particle size of 0.81 μ, and the particle shape is predominantly spherical.

Synthetical bone-like and biological hydroxyapatites: a comparative study of crystal structure and morphology

Phase composition, crystal structure and morphology of biological hydroxyapatite (BHAp) extracted from human mandible bone, and carbonated hydroxyapatite (CHAp), synthesized by the chemical precipitation method, were studied by x-ray powder diffraction (XRD), Fourier transform infrared (FTIR) and Raman (R) spectroscopy techniques, combined with transmission electron microscopy (TEM). Structural and microstructural parameters were determined through Rietveld refinement of recorded XRD data, performed using the FullProf computing program, and TEM. Microstructural analysis shows anisotropic extension along the [00l] crystallographic direction (i.e. elongated crystallites shape) of both investigated samples. The average crystallite sizes of 10 and 8 nm were estimated for BHAp and CHAp, respectively. The FTIR and R spectroscopy studies show that carbonate ions substitute both phosphate and hydroxyl ions in the crystal structure of BHAp as well as in CHAp, indicating that both of them are mixed AB-type of CHAp. The thermal behaviour and carbonate content were analysed using thermogravimetric and differential thermal analysis. The carbonate content of about 1 wt.% and phase transition, at near 790 °C, from HAp to β-tricalcium phosphate were determined in both samples. The quality of synthesized CHAp powder, particularly, the particle size distribution and uniformity of morphology, was analysed by a particle size analyser based on laser diffraction and field emission scanning electron microscopy, respectively. These data were used to discuss similarity between natural and synthetic CHAp. Good correlation between the unit cell parameters, average crystallite size, morphology, carbonate content and crystallographic positions of carbonate ions in natural and synthetic HAp samples was found.

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.