Chitosan-based matrix as a carrier for bacteriophages.

Wound healing is a dynamic and complex process where infection prevention is essential. Chitosan, thanks to its bactericidal activity against gram-positive and gram-negative bacteria, as well as anti-inflammatory and hemostatic properties, is an excellent candidate to design dressings for difficult-to-heal wound treatment. The great advantage of this biopolymer is its capacity to be chemically modified, which allows for the production of various functional forms, depending on the needs and subsequent use. Moreover, chitosan can be an excellent polymer matrix for bacteriophage (phage) packing as a novel alternative/supportive antibacterial therapy approach. This study is focused on the preparation and characteristics of chitosan-based material in the form of a film with the addition of Pseudomonas lytic phages (KTN4, KT28, and LUZ19), which would exhibit antibacterial activity as a potential dressing that accelerates the wound healing. We investigated the method of producing a polymer based on microcrystalline chitosan (MKCh) to serve as the matrix for phage deposition. We described some important parameters such as average molar mass, swelling capacity, surface morphology, phage release profile, and antibacterial activity tested in the Pseudomonas aeruginosa bacterial model. The chitosan polysaccharide turned out to interact with phage particles immobilizing them within a material matrix. Nevertheless, with the high hydrophilicity and swelling features of the prepared material, the external solution of bacterial culture was absorbed and phages went in direct contact with bacteria causing their lysis in the polymer matrix. KEY POINTS: • A novel chitosan-based matrix with the addition of active phages was prepared • Phage interactions with the chitosan matrix were determined as electrostatic • Phages in the matrix work through direct contact with the bacterial cells.

The impact of agarose immobilization on the activity of lytic Pseudomonas aeruginosa phages combined with chemicals.

The implementation of non-traditional antibacterials is currently one of the most intensively explored areas of modern medical and biological sciences. One of the most promising alternative strategies to combat bacterial infections is the application of lytic phages combined with established and new antibacterials. The presented study investigates the potential of agarose-based biocomposites containing lytic Pseudomonas phages (KT28, KTN4, and LUZ19), cupric ions (Cu2+), strawberry furanone (HDMF), and gentamicin (GE) as antibacterials and anti-virulent compounds for novel wound dressings. Phages (KT28, KTN4, LUZ19, and triple-phage cocktail) alone and in combination with a triple-chemical mixture (Cu + GE + HDMF) when applied as the liquid formulation caused a significant bacterial count reduction and biofilm production inhibition of clinical P. aeruginosa strains. The immobilization in the agarose scaffold significantly impaired the bioavailability and diffusion of phage particles, depending on virion morphology and targeted receptor specificity. The antibacterial potential of chemicals was also reduced by the agarose scaffold. Moreover, the Cu + GE + HDMF mixture impaired the lytic activity of phages depending on viral particles' susceptibility to cupric ion toxicity. Therefore, three administration types were tested and the optimal turned out to be the one separating antibacterials both physically and temporally. Taken together, the additive effect of phages combined with chemicals makes biocomposite a good solution for designing new wound dressings. Nevertheless, the phage utilization should involve an application of aqueous cocktails directly onto the wound, followed by chemicals immobilized in hydrogel dressings which allow for taking advantage of the antibacterial and anti-virulent effects of all components. KEY POINTS: • The immobilization in the agarose impairs the bioavailability of phage particles and the Cu + GE + HDMF mixture. • The cupric ions are toxic to phages and are sequestrated on phage particles and agarose matrix. • The elaborated TIME-SHIFT administration effectively separates antibacterials both physically and temporally.

The Antibacterial Effect of PEGylated Carbosilane Dendrimers on P. aeruginosa Alone and in Combination with Phage-Derived Endolysin.

The search for new microbicide compounds is of an urgent need, especially against difficult-to-eradicate biofilm-forming bacteria. One attractive option is the application of cationic multivalent dendrimers as antibacterials and also as carriers of active molecules. These compounds require an adequate hydrophilic/hydrophobic structural balance to maximize the effect. Herein, we evaluated the antimicrobial activity of cationic carbosilane (CBS) dendrimers unmodified or modified with polyethylene glycol (PEG) units, against planktonic and biofilm-forming P. aeruginosa culture. Our study revealed that the presence of PEG destabilized the hydrophilic/hydrophobic balance but reduced the antibacterial activity measured by microbiological cultivation methods, laser interferometry and fluorescence microscopy. On the other hand, the activity can be improved by the combination of the CBS dendrimers with endolysin, a bacteriophage-encoded peptidoglycan hydrolase. This enzyme applied in the absence of the cationic CBS dendrimers is ineffective against Gram-negative bacteria because of the protective outer membrane shield. However, the endolysin-CBS dendrimer mixture enables the penetration through the membrane and then deterioration of the peptidoglycan layer, providing a synergic antimicrobial effect.

Spectroscopic and Small-angle X-ray scattering analysis of binding between Copper(II) -1-allylimidazole complex, a potential anti-tumor agent, and bovine serum albumin.

Interactions between transport proteins and compounds with therapeutic potential are pharmacologically important. In this study, using fluorescence, circular dichroism (CD), and small-angle X-ray Scattering (SAXS), we investigated the interaction between bovine serum albumin (BSA) and a copper(II)-1-allylimidazole complex with potential anti-cancer properties. The results revealed dynamic fluorescence quenching of the model carrier protein BSA by the copper(II) complex. The enthalpy change (ΔH), free energy (ΔG), and entropy change (ΔS) were calculated to be 108 kJ/mol, -16.47 kJ/mol, and 419 J/mol K, respectively, according to the Van't Hoff equation. The reaction was an endothermic and spontaneous process, and hydrophobic interactions played a major role in binding. The results indicate a much lower affinity (Kb âˆ¼ 102-103) for the metal complex compared with similar compounds (Kb âˆ¼ 103-105). CD showed that the studied copper(II) complex does not change the secondary structure of the protein, while SAXS showed that the this compound may attach to the protein surface and stimulate interactions between proteins. The results suggest that the copper(II) complex with 1-allylimidazole binds weakly to BSA, leading to aggregation of albumin in solution, thereby altering its pharmacokinetic properties. The findings are pertinent to drug design.

Coralyne Radiosensitizes A549 Cells by Upregulation of CDKN1A Expression to Attenuate Radiation Induced G2/M Block of the Cell Cycle.

Coralyne is a synthetic analog of berberine related to protoberberine-isoquinoline alkaloids. Isoquinoline derivatives and analogs are renowned as potent radiosensitizers with potential medical application. In the present study, we investigated the effect of coralyne on the cell death, cytoskeletal changes and cell cycle progression of irradiated A549 cells. A clonogenic assay revealed that coralyne pretreatment decreased the viability of A549 cells in a time- and dose-dependent manner. Moreover, exposure to coralyne and ionizing radiation (IR) markedly altered the filamentous actin cytoskeletal architecture and integrin-ß binding sites of A549 cells. Treatment with 1-25 µM coralyne in combination with 2 Gy of IR significantly reduced the percentage of cells in G2/M phase compared with 2 Gy IR alone. These results indicate that coralyne is a potent radiosensitizing agent that may find an application in medicine.

New Approach to Antifungal Activity of Fluconazole Incorporated into the Porous 6-Anhydro-α-l-Galacto-ß-d-Galactan Structures Modified with Nanohydroxyapatite for Chronic-Wound Treatments-In Vitro Evaluation.

New fluconazole-loaded, 6-Anhydro-α-l-Galacto-ß-d-Galactan hydrogels incorporated with nanohydroxyapatite were prepared and their physicochemical features (XRD, X-ray Diffraction; SEM-EDS, Scanning Electron Microscopy-Energy Dispersive X-ray Spectroscopy; ATR-FTIR, Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy), fluconazole release profiles and enzymatic degradation were determined. Antifungal activity of pure fluconazole was tested using Candida species (C. albicans, C. tropicalis, C. glabarata), Cryptococcus species (C. neoformans, C. gatti) and Rhodotorula species (R. mucilaginosa, R. rubra) reference strains and clinical isolates. Standard microdilution method was applied, and fluconazole concentrations of 2-250 µg/mL were tested. Moreover, biofilm production ability of tested isolates was tested on the polystyrene surface at 28 and 37 ± 0.5 °C and measured after crystal violet staining. Strains with the highest biofilm production ability were chosen for further analysis. Confocal microscopy photographs were taken after live/dead staining of fungal suspensions incubated with tested hydrogels (with and without fluconazole). Performed analyses confirmed that polymeric hydrogels are excellent drug carriers and, when fluconazole-loaded, they may be applied as the prevention of chronic wounds fungal infection.

Experimental and Theoretical Analysis of Metal Complex Diffusion through Cell Monolayer.

The study of drugs diffusion through different biological membranes constitutes an essential step in the development of new pharmaceuticals. In this study, the method based on the monolayer cell culture of CHO-K1 cells has been developed in order to emulate the epithelial cells barrier in permeability studies by laser interferometry. Laser interferometry was employed for the experimental analysis of nickel(II) and cobalt(II) complexes with 1-allylimidazole or their chlorides' diffusion through eukaryotic cell monolayers. The amount (mol) of nickel(II) and cobalt(II) chlorides transported through the monolayer was greater than that of metals complexed with 1-allylimidazole by 4.34-fold and 1.45-fold, respectively, after 60 min. Thus, laser interferometry can be used for the quantitative analysis of the transport of compounds through eukaryotic cell monolayers, and the resulting parameters can be used to formulate a mathematical description of this process.

Poly(propylene imine) dendrimers can bind to PEGylated albumin at PEG and albumin surface: Biophysical examination of a PEGylated platform to transport cationic dendritic nanoparticles.

Cationic dendrimers are considered one of the best drug transporters in the body. However, in order to improve their biocompatibility, modification of them is required to reduce toxicity. In this way, many dendrimers may lose their original properties, for example, anticancer. To improve biocompatibility of dendrimers, it is possible to complex them with albumin, as is done very often in drug delivery. However, the interaction of dendrimers with albumin can lead to protein structure disruption or no complexation at all. Therefore, the investigation of the interaction between cationic poly-(propylene imine) dendrimers and polyethylene glycol (PEG)-albumin by fluorescence, circular dichroism, small angle X-ray scattering (SAXS), and transmission electron microscopy was carried out. Results show that cationic dendrimers bind to PEGylated albumin at PEG and albumin surfaces. The obtained results for 5k-PEG indicate a preferential binding of the dendrimers to PEG. For 20k-PEG binding of dendrimers to PEG and protein could induce a collapse of the PEG chain onto the protein surface. This opens up new possibilities to the use of PEGylated albumin as a platform to carry dendrimers without changing the albumin structure and improve the pharmacokinetic properties of dendrimers without further modification.

Antitumor Activity of Pt(II), Ru(III) and Cu(II) Complexes.

Metal complexes are currently potential therapeutic compounds. The acquisition of resistance by cancer cells or the effective elimination of cancer-affected cells necessitates a constant search for chemical compounds with specific biological activities. One alternative option is the transition metal complexes having potential as antitumor agents. Here, we present the current knowledge about the application of transition metal complexes bearing nickel(II), cobalt(II), copper(II), ruthenium(III), and ruthenium(IV). The cytotoxic properties of the above complexes causing apoptosis, autophagy, DNA damage, and cell cycle inhibition are described in this review.

The correlation of crystalline and elemental composition of urinary stones with a history of bacterial infections: TXRF, XRPD and PCR-DGGE studies.

The aim of this study was to analyze the correlation between past bacterial infections and the type and chemical composition of urinary stones experienced by human patients. Bacteria have been recognized to contribute to urinary stones; however, the role of uropathogens in the development of specific stones has not been extensively investigated. The detection of past bacterial infection (eleven different bacterial species) in urinary stones from 83 patients was made on a DNA level using polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) and correlated with the chemical composition of urinary stones measured using X-ray powder diffraction (XPRD) technique and their elemental composition by total reflection X-ray fluorescence (TXRF). In this study, two scenarios of urinary stones formation mediated by Proteus sp. or Escherichia coli are presented. The first one is associated with Proteus spp. which dominated in 84% of infectious urinary stones and is strongly correlated with struvite and calcium phosphate, in whose matrix additionally strontium, phosphorus, potassium, nickel and zinc are detected. The formation of these stones is closely correlated with urease activity. The second scenario for urinary stone mineralization is associated with E. coli identified in weddellite stones, in which matrix iron was detected. In conclusion, the statistical correlations of bacterial infections with crystalline and elemental composition showed that in mixed bacterial infections, one scenario dominated and excluded the second one.

The influence of cationic dendrimers on antibacterial activity of phage endolysin against P. aeruginosa cells.

Nowadays, the researchers make a big effort to find new alternatives to overcome bacterial drug resistance. One option is the application of bacteriophage endolysins enable to degrade peptidoglycan (PG) what in consequence leads to bacterial cell lysis. In this study we examine phage KP27 endolysin mixed with poly(propyleneimine) dendrimers to evaluate an antimicrobial effect against Pseudomonas aeruginosa. Polycationic compounds destabilize bacterial outer membrane (OM) helping endolysins to gain access to PG. We found out that not only bacterial lipopolysaccharide (LPS) is the main hindrance for highly charged cationic dendrimers to disrupt OM and make endolysin reaching the target but also the dendrimer surface modification. The reduction of a positive charge and concentration in maltose poly(propyleneimine) dendrimers significantly increased an antibacterial effect of endolysin. The application of recombinant lysins against Gram-negative bacteria is one of the future therapy options, thus OM permeabilizers such as cationic dendrimers may be of high interest to be combined with PG-degrading enzymes.

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 interferometric analysis of glucose and sucrose diffusion in agarose gel.

The paper presents the investigation results of glucose and sucrose diffusion in agarose gel studied with laser interferometry method and the results of fluorescence analysis of the macroscopic gel structure. The diffusion kinetics of these substances released from aqueous solutions of a molar concentration of 0.05 M into the agarose solutions of concentrations of 0.5% and 3% in two gravitational configurations of measuring system was analysed. In the first configuration the solute diffused according, whereas in the second one - opposite to the gravitational force. The diffusion was analysed in the time period between 120 and 2400 s with a time interval of Δt = 120 s. We observed that the convective instabilities were damped well by the agarose gel, which gives the possibility of the interferometric studies of the diffusive transport for other substances in different gravitational configurations of the system. The time characteristics of glucose and sucrose fluxes in both configurations of the system and the gravitational polarisation coefficient values were obtained. The substantial differences in fluxes of glucose and sucrose diffused according and opposite to the gravitational force were observed. Additionally, we observed the differences between the diffusive fluxes of these substances in both configurations in dependence on the gel solution concentration (which is associated with gel porosity dependent on its concentration) and the kind of diffused substance.

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.

Dendritic systems for bacterial outer membrane disruption as a method of overcoming bacterial multidrug resistance.

The alarming rise of multi-drug resistant microorganisms has increased the need for new approaches through the development of innovative agents that are capable of attaching to the outer layers of bacteria and causing permanent damage by penetrating the bacterial outer membrane. The permeability (disruption) of the outer membrane of Gram-negative bacteria is now considered to be one of the main ways to overcome multidrug resistance in bacteria. Natural and synthetic permeabilizers such as AMPs and dendritic systems seem promising. However, due to their advantages in terms of biocompatibility, antimicrobial capacity, and wide possibilities for modification and synthesis, highly branched polymers and dendritic systems have gained much more interest in recent years. Various forms of arrangement, and structure of the skeleton, give dendritic systems versatile applications, especially the possibility of attaching other ligands to their surface. This review will focus on the mechanisms used by different types of dendritic polymers, and their complexes with macromolecules to enhance their antimicrobial effect, and to permeabilize the bacterial outer membrane. In addition, future challenges and potential prospects are illustrated in the hope of accelerating the advancement of nanomedicine in the fight against resistant pathogens.

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.

Subdiffusion equation with Caputo fractional derivative with respect to another function in modeling diffusion in a complex system consisting of a matrix and channels.

Anomalous diffusion of an antibiotic (colistin) in a system consisting of packed gel (alginate) beads immersed in water is studied experimentally and theoretically. The experimental studies are performed using the interferometric method of measuring concentration profiles of a diffusing substance. We use the g-subdiffusion equation with the fractional Caputo time derivative with respect to another function g to describe the process. The function g and relevant parameters define anomalous diffusion. We show that experimentally measured time evolution of the amount of antibiotic released from the system determines the function g. The process can be interpreted as subdiffusion in which the subdiffusion parameter (exponent) α decreases over time. The g-subdiffusion equation, which is more general than the "ordinary" fractional subdiffusion equation, can be widely used in various fields of science to model diffusion in a system in which parameters, and even a type of diffusion, evolve over time. We postulate that diffusion in a system composed of channels and a matrix can be described by the g-subdiffusion equation, just like diffusion in a system of packed gel beads placed in water.

The effect of surface modification of dendronized gold nanoparticles on activation and release of pyroptosis-inducing pro-inflammatory cytokines in presence of bacterial lipopolysaccharide in monocytes.

Biomedical applications of gold nanoparticles (AuNPs) may be limited by their toxicological effects. Although surface-modified AuNPs can induce apoptosis, less is known about whether they can induce other types of cell death. Pyroptosis, an inflammatory type of programmed cell death, can be induced in immune cells, especially macrophages, by bacterial endotoxins. Therefore, in this study, dendronized AuNPs were combined with bacterial lipopolysaccharides (LPSs) as the main stimulators of pro-inflammatory responses to test the induction of pyroptosis in THP-1 myeloid cell line. These AuNPs induced caspase-1 activity (3-4 times more compared to control) and enhanced the release of interleukin (IL)-18 and IL-1ß without inducing gasdermin D cleavage and related pore formation. The production of pro-inflammatory cytokines occurred mainly visible during LPS treatment, although their secretion was observed only after administration of dendronized AuNPs (release of IL-1ß to supernatant up to 80 pg/mL). These findings suggest that dendronized AuNPs can induce pyroptosis-like inflammatory mechanisms and that these mechanisms are enhanced in the presence of bacterial LPS. The intensity of this effect was dependent on AuNP surface modification. These results shed new light on the cytotoxicity of metal NPs, including immune responses, indicating that surface modifications play crucial roles in their nanotoxicological effects.

[Analysis of antibiotic diffusion from agarose gel by spectrophotometry and laser interferometry methods]. / Analiza dyfuzji antybiotyków z zelu agarozowego metodami spektrofotometrii i interferometrii laserowej.

The aim of this study was to analysis of antibiotics (ampicilin, streptomycin, ciprofloxacin or colistin) release from agarose gel by spectrophotmetry and laser interferometry methods. The interferometric system consisted of a Mach-Zehnder interferometer with a He-Ne laser, TV-CCD camera, computerised data acquisition system and a gel system. The gel system under study consists of two cuvettes. We filled the lower cuvette with an aqueous 1% agarose solution with the antibiotics at initial concentration of antibiotics in the range of 0.12-2 mg/ml for spectrophotmetry analysis or 0.05-0.5 mg/ml for laser interferometry methods, while in the upper cuvette there was pure water. The diffusion was analysed from 120 to 2400 s with a time interval of deltat = 120 s by both methods. We observed that 0.25-1 mg/ml and 0,05 mg/ml are minimal initial concentrations detected by spectrophotometric and laser interferometry methods, respectively. Additionally, we observed differences in kinetic of antibiotic diffusion from gel measured by both methods. In conclusion, the laser interferometric method is a useful tool for studies of antibiotic release from agarose gel, especially for substances are not fully soluble in water, for example: colistin.

Emerging Phage Resistance in Pseudomonas aeruginosa PAO1 Is Accompanied by an Enhanced Heterogeneity and Reduced Virulence.

Bacterial surface structures of a proteinic nature and glycoconjugates contribute to biofilm formation and provide shields to host defense mechanisms (e.g., the complement system and phagocytosis). A loss or alteration of these molecules, leading to phage resistance, could result in fewer virulent bacteria. In this study, we evaluate the biology and phenotype changes in Pseudomonas aeruginosa PAO1 phage-resistant clones, which emerge in phage-treated biofilms. We characterize these clones for phage-typing patterns, antibiotic resistance, biofilm formation, pathogenicity, and interactions with the innate immune system. Another important question that we address is whether phage-resistant mutants are also generated incidentally, despite the phage treatment-selective pressure, as the natural adaptation of the living biofilm population. It is found that the application of different phages targeting a particular receptor selects similar phage resistance patterns. Nevertheless, this results in a dramatic increase in the population heterogeneity, giving over a dozen phage-typing patterns, compared to one of the untreated PAO1 sessile forms. We also confirm the hypothesis that "phage-resistant bacteria are more susceptible to antibiotics and host-clearance mechanisms by the immune system". These findings support phage application in therapy, although the overall statement that phage treatment selects the less virulent bacterial population should be further verified using a bigger collection of clinical strains.