TiO2 nanoparticles have generally low toxicity in the in vitro systems although some toxicity is expected to originate in the TiO2-associated photo-generated radical production, which can however be ...modulated by the radical trapping ability of the serum proteins. To explore the role of serum proteins in the phototoxicity of the TiO2 nanoparticles we measure viability of the exposed cells depending on the nanoparticle and serum protein concentrations.
Fluorescence and spin trapping EPR spectroscopy reveal that the ratio between the nanoparticle and protein concentrations determines the amount of the nanoparticles' surface which is not covered by the serum proteins and is proportional to the amount of photo-induced radicals. Phototoxicity thus becomes substantial only at the protein concentration being too low to completely coat the nanotubes' surface.
These results imply that TiO2 nanoparticles should be applied with ligands such as proteins when phototoxic effects are not desired - for example in cosmetics industry. On the other hand, the nanoparticles should be used in serum free medium or any other ligand free medium, when phototoxic effects are desired - as for efficient photodynamic cancer therapy.
High economic burden is associated with foodborne illnesses. Different disinfection methods are therefore employed in food processing industry; such as use of ultraviolet light or usage of surfaces ...with copper-containing alloys. However, all the disinfection methods currently in use have some shortcomings. In this work we show that copper doped TiO2 nanotubes deposited on existing surfaces and illuminated with ceiling mounted fluorescent lights can retard the growth of Listeria Innocua by 80% in seven hours of exposure to the fluorescent lights at different places in a food processing plant or in the laboratory conditions with daily reinocuation and washing. The disinfection properties of the surfaces seem to depend mainly on the temperature difference of the surface and the dew point, where for the maximum effectiveness the difference should be about 3 degrees celsius. The TiO2 nanotubes have a potential to be employed for an economical and continuous disinfection of surfaces.
Bacterial infections acquired in healthcare facilities including hospitals, the so called healthcare acquired or nosocomial infections, are still of great concern worldwide and represent a ...significant economical burden. One of the major causes of morbidity is infection with Methicillin Resistant Staphylococcus aureus (MRSA), which has been reported to survive on surfaces for several months. Bactericidal activity of copper-TiO2 thin films, which release copper ions and are deposited on glass surfaces and heated to high temperatures, is well known even when illuminated with very weak UVA light of about 10 μW/cm2. Lately, there is an increased intrerest for one-dimensional TiO2 nanomaterials, due to their unique properties, low cost, and high thermal and photochemical stability. Here we show that copper doped TiO2 nanotubes produce about five times more ·OH radicals as compared to undoped TiO2 nanotubes and that effective surface disinfection, determined by a modified ISO 22196:2011 test, can be achieved even at low intensity UVA light of 30 μW/cm2. The nanotubes can be deposited on a preformed surface at room temperature, resulting in a stable deposition resistant to multiple washings. Up to 103 microorganisms per cm2 can be inactivated in 24 hours, including resistant strains such as Methicillin-resistant Staphylococcus aureus (MRSA) and Extended-spectrum beta-lactamase Escherichia coli (E. coli ESBL). This disinfection method could provide a valuable alternative to the current surface disinfection methods.
Magnesium oxide (MgO) is recognised as exhibiting a contact‐based antibacterial activity. However, a comprehensive study of the impact of atomic‐scale surface features on MgO's antibacterial activity ...is lacking. In this study, the nature and abundance of the native surface defects on different MgO powders are thoroughly investigated. Their impacts on the hydrolysis kinetics, antibacterial activity against Escherichia coli (ATCC 47076), Staphylococcus epidermidis and Pseudomonas aeruginosa and the reactive oxygen species (ROS) generation potential are determined and explained. It is shown that a reduction in the abundance of low‐coordinated oxygen atoms on the surface of the MgO improves its resistance to both hydrolysis and antibacterial activity. The ROS generation potential, determined in‐situ using a fluorescence microplate assay and electron paramagnetic resonance spectroscopy, is not an inherent property of the studied MgO, rather it is a side product of hydrolysis (only for the most highly defected MgO particles) and/or a consequence of the MgO/bacteria interaction. The evaluation of the mutual correlations of the hydrolysis, the antibacterial activity and the ROS generation, with their origin in the surface defects' peculiarities, led to the conclusion that the acid/base reaction between the MgO surface and the bacterial wall contributes considerably to the MgO's antibacterial activity.
The manipulation of native defects at the surface of MgO is an effective tool to regulate surface hydrolysis and antibacterial activity. The processing of an oxygen‐deficient MgO surface leads to optimal bactericidal activity due to slower hydrolysis, while reactive oxygen species are generated only as a side‐product of chemical MgO/bacteria interactions.
Designing efficient 'vectors', to deliver therapeutics across endothelial barriers, in a controlled manner, remains one of the key goals of drug development. Recently, transcytosis of liposome ...encapsulated fluorescence marker calcein across a tight cell barrier was studied. The most efficient liposomes were found to be liposomes containing sufficient amount of alkyl phospholipid (APL) perifosine. APLs have similar structure as lysophosphatidyl choline (LPC), since APLs were synthesized as metabolically stable analogues of LPC, which increases endothelial permeability directly by inducing endothelial cell contraction, resulting in formation of gaps between endothelial cells. Since one of the unique properties of lysolipid, containing liposomal formulations is dynamic equilibrium of lysolipids, which are distributed among liposomes, micelles, and free form, such liposomes represent a reservoir of free lysolipids. On the other hand lysolipid containing liposomes also represent a reservoir of an encapsulated hydrophilic drug.
We hypothesize that free lysolipids, with highest concentration in vicinity of drug carrying liposomes, compromise endothelial integrity, primarily where concentrations of liposomes is the highest, in a similar manner as LPC, by formation of gaps between endothelial cells. Liposome encapsulated drug, which leaks from liposomes, due to liposome destabilization, caused by lysolipid depletion, can therefore be efficiently transported across the locally compromised endothelial barrier.
This hypothesis could be verified: by measuring binding of perifosine and other lysolipids to albumin and to lysophospholipid receptor (LPL-R) group; formation of stress fibers and subsequent cell contraction; activation of RhoA, and endothelial barrier dysfunction; by a synthesis of other LPC analogues with high critical micellar concentration and measuring their effect on transendothelial permeability in presence and absence of albumin.
We propose that lysolipid containing liposomal formulations might be used as nonspecific transendothelial transport vector, since leakage of liposome encapsulated active drug occurs simultaneously with the release of the lysolipids. The concentration of the active drug is therefore expected to be the highest at the site of compromised endothelial barrier. By appropriate choice of the lysolipids an endothelial barrier would stay open only for a short time. Use of such liposomes would potentially maximize the delivery of the drug while limiting the passage of toxic substances and pathogens across the endothelial barrier. Combining lysolipid containing liposomes with superparamagnetic iron oxide nanoparticles or a targeting ligand might be required to efficiently localize drug delivery to a disease affected tissue and to avoid endothelial disruption over the entire body.
Clinical studies have demonstrated a correlation between elevated levels of FIX and the risk of coronary heart disease, while reduced plasma FIX causes hemophilia B. FIXa interacts with FVIIIa in the ...presence of Ca2+ and phosphatidylserine (PS)-containing membranes to form a factor X-activating complex (Xase) that is key to propagation of the initiated blood coagulation process in human. We test the hypothesis that PS in these membranes up-regulates the catalytic activity of this essential enzyme. We used a soluble form of phosphatidylserine, 1, 2-dicaproyl-sn-glycero-3-phospho-L-serine (C6PS), as a tool to do so. C6PS and PS in membranes are reported to regulate the homologous FXa nearly identically. FIXa binds a molecule of C6PS at each of with two sites with such different affinities (∼100-fold) that these appear to be independent. A high affinity C6PS binding site (Kd∼1.4 µM) regulates structure, whereas a low-affinity binding site (Kd∼140 µM) regulates activity. Equilibrium dialysis experiments were analyzed globally with four other data sets (proteolytic and amidolytic activities, intrinsic fluorescence, ellipticity) to unequivocally demonstrate stoichiometries of one for both sites. Michaelis-Menten parameters for FIXa proteolytic activity were the same in the presence of C6PS or PS/PC membranes. We conclude that the PS molecule and not a membrane surface is the key regulator of both factors Xa and IXa. Despite some minor differences in the details of regulation of factors Xa and IXa, the similarities we found suggest that lipid regulation of these two proteases may be similar, a hypothesis that we continue to test.
•Perifosine (OPP) increases membrane fluidity of liposomes.•OPP promotes release of neutral liposome encapsulated molecules at physiological temperature.•OPP promotes release of charged molecules ...only at low concentration (10mol%).•Content release is about 10× faster for neutral than for charged molecules.•OPP simultaneously promotes transendothelial transfer of liposome encapsulated content and its release from liposomes.
Perifosine (OPP) containing liposomal formulation was previously found to deliver almost half of liposome encapsulated content through a tight cellular barrier in vitro. In order to understand the role of different liposome components, especially perifosine, in transendothelial transport the physical characteristics of liposome membranes composed of phosphatidylcholine, and cholesterol, as a main lipid constituents, and variable amount of helper lipids: dioleoyl phosphatidylethanolamine (DOPE), and alkylphospholipid perifosine.
For this purpose, electron paramagnetic resonance (EPR) with computer aided EPR spectra simulation and fluorescence polarization spectroscopy were used to investigate how different membrane components influence membrane characteristics and the release of liposome entrapped substances. Beside methylester of palmitic acid with nitroxide group at different position on acyl chain usually used for such studies, the spin labeled and fluorescent labeled analog of perifosine were introduced.
OPP increases membrane fluidity of liposomes as well as the release of liposome encapsulated content. The release of neutral molecules increases with OPP concentration, while the release of charged molecules is about an order of magnitude slower. Optimal OPP concentration, for release of charged molecules, is about 15mol%.
These results are one step further toward the conclusion that the lysolipid-containing liposomes could be promising trans endothelial delivery system, since lysolipids, such as OPP, open tight cellular barriers, as was published before, and in the same time induce the release of liposome encapsulated content at physiological temperature, as shown here. Since many drug delivery systems are being developed, which mainly exploit the transcellular route of delivery through barrier-forming cells, we hope that the uniqueness of lysolipid-containing liposomes, exploiting the paracellular route, and thus avoiding efflux transporters, will foster further research in formulating other lysolipid-containing liposomes as drug delivery systems.
Nanomaterial (NM) characteristics may affect the pulmonary toxicity and inflammatory response, including specific surface area, size, shape, crystal phase or other surface characteristics. Grouping ...of TiO2 in hazard assessment might be challenging because of variation in physicochemical properties. We exposed C57BL/6 J mice to a single dose of four anatase TiO2 NMs with various sizes and shapes by intratracheal instillation and assessed the pulmonary toxicity 1, 3, 28, 90 or 180 days post-exposure. The quartz DQ12 was included as benchmark particle. Pulmonary responses were evaluated by histopathology, electron microscopy, bronchoalveolar lavage (BAL) fluid cell composition and acute phase response. Genotoxicity was evaluated by DNA strand break levels in BAL cells, lung and liver in the comet assay. Multiple regression analyses were applied to identify specific TiO2 NMs properties important for the pulmonary inflammation and acute phase response. The TiO2 NMs induced similar inflammatory responses when surface area was used as dose metrics, although inflammatory and acute phase response was greatest and more persistent for the TiO2 tube. Similar histopathological changes were observed for the TiO2 tube and DQ12 including pulmonary alveolar proteinosis indicating profound effects related to the tube shape. Comparison with previously published data on rutile TiO2 NMs indicated that rutile TiO2 NMs were more inflammogenic in terms of neutrophil influx than anatase TiO2 NMs when normalized to total deposited surface area. Overall, the results suggest that specific surface area, crystal phase and shape of TiO2 NMs are important predictors for the observed pulmonary effects of TiO2 NMs.
•Inflammation and acute phase response was highest and more persistent for TiO2 tube.•TiO2 tube and DQ12 caused pulmonary alveolar proteinosis.•Rutile TiO2 NMs were more inflammogenic in terms of neutrophil influx than anatase.•BET surface area strongly correlated with neutrophil influx for both crystal phases.•BET surface area, crystal phase and shape of TiO2 NMs are predictors for the effects.
Cell detachment procedures can cause severe damage to cells. Many studies require cells to be detached before measurements; therefore, research on cells that have been grown attached to the bottom of ...the culture dish and later detached represents a special problem with respect to the experimental results when the properties of cell membranes undergo small changes such as in spectroscopic studies of membrane permeability. We characterized the influence of three different detachment procedures: cell scraping by rubber policeman, trypsinization and a citrate buffer treatment on V‐79 cells in the plateau phase of growth (arrested in G1). We have measured cell viability by a dye‐exclusion test; nitroxide reduction kinetics and membrane fluidity by EPR (electron paramagnetic resonance) method using the lipophilic spin‐probe MeFASL(10,3) (5‐doxylpalmitoyl‐methylester), which partitions mainly in cell membranes and the hydrophilic spin‐probe TEMPONE (4‐oxo‐2,2,6,6‐tetramethylpiperidine‐1‐oxyl). The resulting cell damage due to the detachment process was observed with SEM (scanning electron microscopy). We found out that cell viability was 91% for trypsin treatment, 85% for citrate treatment and 70% for cell scraping. Though the plasma membrane was mechanically damaged by scraping, the membrane domain structure was not significantly altered compared with other detachment methods. On the other hand, the spin‐probe reduction rate, which depends both on the transport across plasma membrane as well as on metabolic properties of cells, was the highest for trypsin method, suggesting that metabolic rate was the least influenced. Only the reduction rate of trypsin‐treated cells stayed unchanged after 4 h of stirring in suspension. These results suggest that, compared with scraping cells or using citrate buffer, the most suitable detachment method for V‐79 cells is detachment by trypsin and keeping cells in the stirred cell suspension until measurement. This method provides the highest cell viability, less visible damage on SEM micrographs and leaves the metabolic rate of cells unchanged.
PDF
