Michał Arabski

Effect of surface modification of silica nanoparticles on toxicity and cellular uptake by human peripheral blood lymphocytes in vitro

Abstract Silica nanoparticles have an interesting potential in drug delivery, gene therapy and molecular imaging due to the possibility of tailoring their surface reactivity that can be obtained by surface modification. Despite these potential benefits, there is concern that exposure of humans to certain types of silica nanomaterials may lead to significant adverse health effects. The motivation of this study was to determine the kinetics of cellular binding/uptake of the vinyl- and the aminopropyl/vinyl-modified silica nanoparticles into peripheral blood lymphocytes in vitro, to explore their genotoxic and cytotoxic properties and to compare the biological properties of modified silica nanoparticles with those of the unmodified ones. Size of nanoparticles determined by SEM varied from 10 to 50 nm. The average hydrodynamic diameter and zeta potential also varied from 176.7 nm (+18.16 mV) [aminopropyl/vinyl-modified] and 235.4 nm (−9.49 mV) [vinyl-modified] to 266.3 (−13.32 mV) [unmodified]. Surface-modified silica particles were internalized by lymphocytes with varying efficiency and expressed no cytotoxic nor genotoxic effects, as determined by various methods (cell viability, apoptosis/necrosis, oxidative DNA damage, chromosome aberrations). However, they affected the proliferation of the lymphocytes as indicated by a decrease in mitotic index value and cell cycle progression. In contrast, unmodified silica nanoparticles exhibited cytotoxic and genotoxic properties at high doses as well as interfered with cell cycle.

Laser interferometric and cultivation methods for measurement of colistin/ampicilin and saponin interactions with smooth and rough of Proteus mirabilis lipopolysaccharides and cells

Laser interferometry is commonly used in permeability studies of soluble substances. In this study a modification that allowed testing partially insoluble mixtures is presented. The modification relies on the measurement of diffusion from 1% agarose gel. As a model for this study, two Proteus mirabilis strains were used that differ in polysaccharide content: smooth P. mirabilis S1959 strain and its Re-type mutant, strain R45. By laser interferometry and precipitation it is shown that R45 lipopolysaccharide is more effective in binding colistin. It has been shown with the laser interferometric method that saponins, which are detergent-like substances of plant origin, partially enhance the interaction of colistin with the S and Re types of P. mirabilis. These results were confirmed with whole cell Proteus studies. The saponin partially inhibited the growth of the S and Re P. mirabilis strains at doses of 31-500 microg/ml. A sub-inhibitory dose--15 microg/ml of saponins alone do not reduced the numbers of P. mirabilis S1959 and R45 cells. However, the presence of colistin or amipicillin and 15 microg/ml of saponins reduced the amount of P. mirabilis S1959 and R45 cells. The saponins enhanced sensitivities of S and R P. mirabilis cells towards colistin and amipicillin. One may proposed that saponins binds to lipid A part of LPS may resulted on an increase in bacterial cell wall outer-membrane permeabilities and by that facilitated antibiotics penetration into the bacterial cells. In conclusion, the laser interferometric method is a useful tool for studies of lipopolysaccharide-antibiotic interactions even if the tested substances are not fully soluble in water.

Effects of Saponins against Clinical E. coli Strains and Eukaryotic Cell Line

Saponins are detergent-like substances showing antibacterial as well as anticancer potential. In this study, the effects of saponins from Quillaja saponaria were analyzed against prokaryotic and eukaryotic cells. Multidrug-resistant clinical E. coli strains were isolated from human urine. As eukaryotic cells, the CHO-K1 cell lines were applied. Antibacterial effect of ampicillin, streptomycin, and ciprofloxacin in the presence of saponins was measured by cultivation methods. Properties of saponins against CHO-K1 cells were measured by the MTT test, hemolysis assay and flow cytometry. Saponin from Quillaja saponaria has a cytotoxic effect at concentrations higher than 25 μg/mL and in the range of 12–50 μg/mL significantly increases the level of early apoptotic cells. Saponin at dose of 12 μg/mL enhances the six E. coli strains growth. We postulate that saponins increase the influx of nutrients from the medium into E. coli cells. Saponins do not have synergetic effects on antibacterial action of tested antibiotics. In contrary, in the presence of saponins and antibiotics, more CFU/mL E. coli cells were observed. This effect was similar to saponins action alone towards E. coli cells. In conclusion, saponins was cytotoxic against CHO-K1 cells, whereas against E. coli cells this effect was not observed.

A proposed integrated approach for the preclinical evaluation of phage therapy in Pseudomonas infections.

Bacteriophage therapy is currently resurging as a potential complement/alternative to antibiotic treatment. However, preclinical evaluation lacks streamlined approaches. We here focus on preclinical approaches which have been implemented to assess bacteriophage efficacy against Pseudomonas biofilms and infections. Laser interferometry and profilometry were applied to measure biofilm matrix permeability and surface geometry changes, respectively. These biophysical approaches were combined with an advanced Airway Surface Liquid infection model, which mimics in vitro the normal and CF lung environments, and an in vivo Galleria larvae model. These assays have been implemented to analyze KTN4 (279,593 bp dsDNA genome), a type-IV pili dependent, giant phage resembling phiKZ. Upon contact, KTN4 immediately disrupts the P. aeruginosa PAO1 biofilm and reduces pyocyanin and siderophore production. The gentamicin exclusion assay on NuLi-1 and CuFi-1 cell lines revealed the decrease of extracellular bacterial load between 4 and 7 logs and successfully prevents wild-type Pseudomonas internalization into CF epithelial cells. These properties and the significant rescue of Galleria larvae indicate that giant KTN4 phage is a suitable candidate for in vivo phage therapy evaluation for lung infection applications.

DNA damage in human colonic mucosa cells evoked by nickel and protective action of quercetin - involvement of free radicals?

Nickel is a toxic and carcinogenic environmental and occupational pollutant and quercetin is a dietary flavonoid that is reported to modulate effects of many mutagens and carcinogens. We investigated the ability of nickel chloride to induce DNA damage in human colonic mucosa cells in the presence of quercetin, using the alkaline comet assay. Nickel chloride (5–250 μmol/L) evoked dose-dependent DNA damage and quercetin at 50 μmol/L decreased the extent of this damage. The cells exposed to nickel chloride progressively removed their DNA damage and the presence of 50 μmol/L quercetin in the repair-incubation medium did not affect the repair kinetics. Cells exposed to nickel and treated with endonuclease III, an enzyme recognizing oxidized bases, displayed a greater extent of DNA damage than those not treated with the enzyme. Quercetin did not exert a significant effect on the production of oxidized bases by nickel. Pretreatment of the cells with a nitrone spin trap, N-tert-butyl-α-phenylnitrone, decreased the extent of DNA damage evoked by nickel. Quercetin caused a further decrease in the extent of the damage in the presence of the trap. The results obtained suggest that reactive oxygen species, including free radicals, might be involved in the formation of DNA lesions induced by nickel chloride in colonic mucosa cells and that quercetin may exert protective effects in these cells.

Correction: Characterization of the Newly Isolated Lytic Bacteriophages KTN6 and KT28 and Their Efficacy against Pseudomonas aeruginosa Biofilm

We here describe two novel lytic phages, KT28 and KTN6, infecting Pseudomonas aeruginosa, isolated from a sewage sample from an irrigated field near Wroclaw, in Poland. Both viruses show characteristic features of Pbunalikevirus genus within the Myoviridae family with respect to shape and size of head/tail, as well as LPS host receptor recognition. Genome analysis confirmed the similarity to other PB1-related phages, ranging between 48 and 96%. Pseudomonas phage KT28 has a genome size of 66,381 bp and KTN6 of 65,994 bp. The latent period, burst size, stability and host range was determined for both viruses under standard laboratory conditions. Biofilm eradication efficacy was tested on peg-lid plate assay and PET membrane surface. Significant reduction of colony forming units was observed (70-90%) in 24 h to 72 h old Pseudomonas aeruginosa PAO1 biofilm cultures for both phages. Furthermore, a pyocyanin and pyoverdin reduction tests reveal that tested phages lowers the amount of both secreted dyes in 48-72 h old biofilms. Diffusion and goniometry experiments revealed the increase of diffusion rate through the biofilm matrix after phage application. These characteristics indicate these phages could be used to prevent Pseudomonas aeruginosa infections and biofilm formation. It was also shown, that PB1-related phage treatment of biofilm caused the emergence of stable phage-resistant mutants growing as small colony variants.

The presence of anti-LPS antibodies and human serum activity against Proteus mirabilis S/R forms in correlation with TLR4 (Thr399Ile) gene polymorphism in rheumatoid arthritis.

OBJECTIVES Proteus mirabilis strains are human pathogens responsible for urinary tract infections, which may also be involved in rheumatoid arthritis (RA). DESIGN AND METHODS We determined whether the binding site of anti-LPS antibodies on the O-polysaccharide part of P. mirabilis LPS correlates with the level of TLR4 (Thr399Ile) gene polymorphism in the sera of RA patients. We investigated the deposition of C3d and C5b complement components on the P. mirabilis LPS. The ELISA method used in this study was optimized with LAL test and laser interferometry. RESULTS Depending on LPS P. mirabilis used in these studies, the amount of antibodies in RA patients sera varied. We did not observe a correlation between anti-LPS antibodies binding and the level of TLR4 (Thr399Ile) gene polymorphism. We found that the lower complement components deposition by O49 in contrast to O9 LPS correlates with its reduced sensitivities to human complement-mediated killing. CONCLUSION The immunological response against P. mirabilis LPS might play a role in rheumatoid arthritis.

Morphological changes in Proteus mirabilis O18 biofilm under the influence of a urease inhibitor and a homoserine lactone derivative

Proteus mirabilis is a pathogenic gram-negative bacterium that frequently causes kidney infections, typically established by ascending colonization of the urinary tract. The present study is focused on ureolytic activity and urease inhibition in biofilms generated by P. mirabilis O18 cells. Confocal microscopy revealed morphological alterations in biofilms treated with urea and a urease inhibitor (acetohydroxamic acid, AHA), as some swarmer cells were found to protrude from the biofilm. The presence of a quorum-sensing molecule (N-butanoyl homoserine lactone, BHL) increased biofilm thickness and its ureolytic activity. Laser interferometric determination of diffusion showed that urea easily diffuses through P. mirabilis biofilm, while AHA is blocked. This may suggest that the use of urease inhibitors in CAUTIs may by less effective than in other urease-associated infections. Spectroscopic studies revealed differences between biofilm and planktonic cells indicating that polysaccharides and nucleic acids are involved in extracellular matrix and biofilm formation.

Laser interferometry analysis of ciprofloxacin and ampicillin diffusion from liposomal solutions to water phase

The paper presents experimental investigations of diffusion of antibiotics (ciprofloxacin or ampicillin) into the water phase from mixtures of neutral or negatively charged liposomes, and antibiotic–liposome interactions. Using the laser interferometry technique, the amounts and fluxes of released antibiotics, concentration field evolution, and the velocity of the concentration boundary layer’s “growth” were determined. To avoid the limitations of membranes, a measurement system without the artificial boundary of phases with a free water–solution interface has been proposed. It was found that the diffusion of anionic and neutral liposomes into the water phase was insignificant and mainly the diffusion of antibiotics was measured. Differences in the diffusion kinetics of ciprofloxacin and ampicillin from liposomal solutions to the water phase were observed. Ampicillin diffused more efficiently than ciprofloxacin regardless of the liposomal solution type. Moreover, the amount of ampicillin and ciprofloxacin released from the anionic liposomal phase was higher than that from the neutral one. Our results confirm that ciprofloxacin at neutral pH shows little tendency to bind neutral liposomes. Additionally, it was also observed that ciprofloxacin disrupts negatively charged liposomes as a final effect of antibiotic–lipid interactions.

Human complement activation by smooth and rough Proteus mirabilis lipopolysaccharides.

IntroductionProteus mirabilis bacilli play an important role in human urinary tract infections, bacteremia, and rheumatoid arthritis. The authors previously studied human complement C3 conversion by smooth-form P. mirabilis O10, O23, O30, and O43 lipopolysaccharides (LPSs) and showed that smooth Proteus LPSs fragmented C3 in a dose- and time-dependent manner. In the present study, one smooth P. mirabilis S1959 and its two polysaccharide-truncated LPSs isolated from an R mutant strain were used to study the C3 conversion.Materials and MethodsThe conversion of C3 to C3c by smooth and rough P. mirabilis LPSs was studied by capture ELISA and crossed immunoelectrophoresis. Proteins isolated from the outer membrane were analyzed by discontinuous sodium dodecyl sulfate gel electrophoresis.ResultsThe smooth P. mirabilis S1959 (O3) strain was resistant to the bactericidal activity of human serum, in contrast to the Ra and Re mutant strains. The presence of an exposed core oligosaccharide in R110 LPS was not sufficient to protect the strain from serum-dependent killing. In addition to LPS structure, the outer-membrane proteins may also play roles in protecting the smooth P. mirabilis S1959 (O3) strain from the bactericidal action of serum. It was shown that the Ra P. mirabilis R110 and the Re P. mirabilis R45 mutants possess very different OMP compositions from that of the P. mirabilis S 1959 strain.ConclusionRegardless of the complement resistance of the P. mirabilis strains, the S1959, R110, and R45 LPSs fragmented C3 and induced C3c neo-antigen exposure. The use of complement-deficient human serum allows the conclusion that the Re-type P. mirabilis R45 LPS fragmented C3 by the antibody-independent classical pathway.