Light-induced antibacterial activity of electrospun chitosan-based material containing photosensitizer.

Increasing antimicrobial resistance requires the development of novel materials and approaches for treatment of various infections. Utilization of photodynamic therapy represents an advanced alternative to antibiotics and metal-based agents. Here, we report the fabrication of electrospun material that possesses benefits of both topical antimicrobial and photodynamic therapies. This material combines chitosan, as a biocompatible polymer, and a second generation photosensitizer. The incorporation of photosensitizer doesn't affect the material morphology and its nearly uniform distribution in fibers structure was observed by confocal Raman microscopy. Owing to photosensitizer the prepared material exhibits the light-induced and spatially limited antimicrobial activity that was demonstrated against Staphylococcus aureus, an important etiological infectious agent. Such material can be potentially used in antibacterial therapy of chronic wounds, infections of diabetic ulcers, and burns, as well as rapidly spreading and intractable soft-tissue infections caused by resistant bacteria.

Photosensitizer-loaded electrospun chitosan-based scaffolds for photodynamic therapy and tissue engineering.

Novel chitosan-based nanofibrous composite materials containing different amounts of the photosensitizer Photosens were obtained by electrospinning and were characterized by scanning electron microscopy and by confocal laser scanning microscopy. The release of Photosens from the materials was investigated in water and in phosphate-buffered saline. A noncancerous (MC3T3-E1 murine osteoblasts) and a cancerous [T-47D (mammary gland)] cell line were cultivated on Photosens-containing scaffolds, and cell growth and metabolic activity were examined by confocal laser scanning microscopy and by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-nyltetrazolium bromide assay, respectively. The viability of both cell lines on Photosens-containing fibers decreased in a spatial manner upon laser irradiation of an appropriate wavelength and power density. Interestingly, the noncancerous MC3T3-E1 cells grown on Photosens -containing scaffolds were less affected by the irradiation. We conclude that the Photosens-containing electrospun chitosan nanofibers described here are of potential interest for biomedical applications, particularly topical photodynamic therapy and tissue engineering.

In vitro inhibition of fungal activity by macrophage-mediated sequestration and release of encapsulated amphotericin B nanosupension in red blood cells.

The efficacy of antifungal treatment has been diminished by the biodistribution limitations of amphotericin B (AmB) due to its pharmacological profile, as well as the severe side effects it causes. A cellular drug-delivery system, which incorporates human erythrocytes (RBCs) loaded with an AmB nanosuspension (AmB-NS), is developed in order to improve antifungal treatment. AmB-NS encapsulation in RBCs is achieved by using hypotonic hemolysis, leading to intracellular AmB amounts of 3.81 +/- 0.47 pg RBC(-1) and an entrapment efficacy of 15-18%. Upon phagocytosis of AmB-NS-RBCs, leukocytes show a slow AmB release over ten days, and no alteration in cell viability. This results in an immediate, permanent inhibition of intra- and extracellular fungal activity. AmB-NS-RBC-leukocyte-mediated delivery of AmB is efficient in amounts 1000 times lower than the toxic dose. This drug-delivery method is effective for the transport of water-insoluble substances, such as AmB, and this warrants consideration for further testing.

Magnetite-loaded carrier erythrocytes as contrast agents for magnetic resonance imaging.

Superparamagnetic iron oxide nanoparticles (SPIONs) are commonly used in magnetic resonance imaging (MRI), but their fast phagocytosis makes them less than ideal for this application. To circumvent the lymphocyte-macrophage system, we encapsulated SPIONs into red blood cells (RBCs). For loading, the RBC's membrane was opened by swelling under hypoosmotic conditions and subsequently resealed. In this work, we demonstrate that SPIONs can be loaded into RBCs in a concentration sufficient to obtain strong contrast enhancement in MRI.

Composite lipid polyelectrolyte capsules templated on red blood cells: fabrication and structural characterisation.

Charged phospholipids and mixtures of charged phospholipids with zwitterionic lipids were adsorbed onto polyelectrolyte capsules templated on erythrocytes. The assembly was proved by means of electrophoretic mobility measurements, confocal laser scanning microscopy and flow cytometry. Freeze-fracture electron microscopy proved that the phospholipids assemble as bilayers or multilayers. Single particle light scattering showed that bilayers composed of anionic lipids can be intercalated between subsequent polyelectrolyte inter-layers in a regular manner. Neutral lipids can form multilayers. A pronounced decrease in capsule permeability for small polar dyes upon lipid adsorption was followed by confocal laser scanning microscopy.

Freeze-fracture electron microscopy of lipid membranes on colloidal polyelectrolyte multilayer coated supports.

Lipid membranes were assembled on polyelectrolyte (PE)-coated colloidal particles. The assembly was studied by means of confocal microscopy, flow cytometry, scanning force microscopy, and freeze-fracture electron microscopy. A homogeneous lipid coverage was established within the limits of optical resolution. Flow cytometry showed that the lipid coverage was uniform. Freeze-fracture electron microscopy revealed that the lipid was adsorbed as a bilayer, which closely followed the surface profile of the polyelectrolyte support. Additional adsorption of polyelectrolyte layers on top of the lipid bilayer introduced inhomogeneities as evident from jumps in the fracture plane. Characteristic lipid multilayers have not been seen with freeze-fracture electron microscopy.

Permeation of macromolecules into polyelectrolyte microcapsules.

Polyelectrolyte microcapsules (PEMCs) have been prepared by coating red blood cells with the polyelectrolytes poly(styrenesulfonate), poly(allylamine hydrochloride), and dextran sulfate applying the layer-by-layer technique with subsequent dissolution of the core. The capsule permeability for human serum albumin (HSA) was studied as a function of the ionic strength and pH by means of confocal microscopy. PEMCs produced with dextran sulfate and poly(allylamine hydrochloride) show a significant increase in permeability for HSA at salt concentrations over 1 mM. For PEMCs prepared with poly(styrenesulfonate) and poly(allylamine hydrochloride) the limiting salt concentration is 5 mM. No pH dependence for permeation was observed. A correlation between the permeation and adsorption of HSA on the PEMC walls was investigated. Finally, a mechanism for the permeability, combining electrostatic interactions, and the presence of pores in the polymer layers is presented confirmed by the considerable increase of permeation of charged molecules in the presence of salt and the permeation of neutral molecules regardless of the ionic strength.

Electrophoretic and aggregation behavior of bovine, horse and human red blood cells in plasma and in polymer solutions.

The electrophoretic mobility of native and glutaraldehyde-fixed bovine, human, and horse red blood cells (RBC) was investigated as a function of ionic strength (5-150 mM) and concentration of 464 kDa dextran (2 and 3 g/dl); RBC aggregation in autologous plasma and in dextran solutions was also measured. In agreement with previous observations, human and horse RBC form stable rouleaux whereas bovine RBC do not aggregate in either plasma or in dextran 464 kDa solutions. Electrophoretic measurements showed a species-dependent adsorption and depletion of dextran that can be theoretically evaluated. Adsorption of polymer is not a prerequisite for RBC aggregation (bovine RBC show the highest amount of adsorbed dextran yet do not aggregate). Aggregate formation thus occurs as long as the Gibbs free energy difference, given by the osmotic pressure difference between the bulk phase and the polymer-depleted region between two RBC, is larger than the steric and electrostatic repulsive energy contributed by the macromolecules present on the RBC surface. With increasing bulk-phase polymer concentration the depletion layer thickness decreases and the amount of adsorbed macromolecules increases, thereby resulting in an increase of the repulsive component of the interaction energy and decreased aggregation. We thus view electrophoretic measurements of RBC in various media as an important tool for understanding polymer behavior near the red cell surface and hence the mechanisms involved in RBC aggregation.

Biological cells as templates for hollow microcapsules.

Microcapsules in the micrometer size range with walls of nanometer thickness are of both scientific and technological interest, since they can be employed as micro- and nano-containers. Liposomes represent one example, yet their general use is hampered due to limited stability and a low permeability for polar molecules. Microcapsules formed from polyelectrolytes offer some improvement, since they are permeable to small polar molecules and resistant to chemical and physical influences. Both types of closed films are, however, limited by their spherical shape which precludes producing capsules with anisotropic properties. Biological cells possess a wide variety of shapes and sizes, and, thus, using them as templates would allow the production of capsules with a wide range of morphologies. In the present study, human red blood cells (RBC) as well as Escherichia coli bacteria were used; these cells were fixed by glutardialdehyde prior to layer-by-layer (LbL) adsorption of polyelectrolytes. The growth of the layers was verified by electrophoresis and flow cytometry, with morphology investigated by atomic force and electron microscopy; the dissolution process of the biological template was followed by confocal laser scanning microscopy. The resulting microcapsules are exact copies of the biological template, exhibit elastic properties, and have permeabilities which can be controlled by experimental parameters; this method for microcapsule fabrication, thus, offers an important new approach for this area of biotechnology.

Release of WBC-derived IL-1 receptor antagonist into supernatants of RBCs: influence of storage time and filtration.

BACKGROUND: Transfusion-associated immunodepression may be related to the transfer of immunoinhibitory cytokines with blood components. STUDY DESIGN AND METHODS: After evidence of increasing concentrations of IL-1 receptor antagonist (IL-1RA) but not of IL-10 was obtained in supernatants of stored RBC units that were WBC-reduced by centrifugation (C-RBCs) in a pilot study, IL-1RA concentrations were determined weekly in supernatants of C-RBCs and in units that underwent prestorage WBC reduction by in-line filtration (F-RBCs) over a 49-day storage. For assessing total IL-1RA content, complete cell lysis by repeated freezing and thawing was done. The results were related to the changes in WBC count during storage. The dependency of IL-1RA content on preparation procedures was assessed. RESULTS: The prestorage IL-1RA concentration in C-RBCs (859 +/- 218 pg/mL) was significantly higher than in F-RBC (75 +/- 13 pg/mL). Whereas no changes were seen in F-RBCs during storage, IL-1RA levels in C-RBC supernatants drastically increased to levels about 50 times those in normal plasma (16,327 +/- 2,686 pg/mL on Day 49). Follow-up analysis revealed stringent correlation between IL-1RA release into supernatants and the current loss of WBCs (r = 0.79, n = 42; p<0.001). The total IL-1RA content did not change during storage and was directly dependent on prestorage WBC count. Preparation procedures altered the IL-1RA content only by WBC reduction. CONCLUSION: The immunosuppressive cytokine IL-1RA is transmitted by RBCs in relation to WBC content and storage time.

Electrophoretic mobility of human red blood cells coated with poly(ethylene glycol).

Poly(ethylene glycol), abbreviated as PEG, was covalently attached to the surface of human red blood cells (RBC) and the effects of such coating on the regions near the cell's glycocalyx were explored by means of cell electrophoresis. RBC electrophoretic mobilities were measured, in polymer-free buffers of various ionic strengths, as functions of PEG molecular mass (3.35, 18.5, 35.0, 35.9 kDa), geometry, (linear or 8-arm branched) and polymer/RBC ratio during attachment. The results indicate marked decreases of the mobility (up to 85%) which were affected by polymer molecular mass and geometry. Since PEG is neutral and its covalent attachment only removes positively-charged amino groups on the cell membrane, such decreases of mobility likely reflect structural changes near and within the RBC glycocalyx. Experimental results were analyzed using an extended "hairy sphere" model to consider friction and thickness of the polymer layer. Calculated polymer layer thickness increased with molecular mass for linear PEGs and was less extended for a branched PEG of similar molecular mass. Friction within the polymer layer increased with polymer/RBC ratio and for the linear PEGs was inversely related to molecular mass; friction was greatest for the branched PEG. Our results are consistent with the effects of attached PEGs on RBC aggregation and surface antigenic site masking, and suggest the usefulness of electrophoretic mobility techniques for studies of bound neutral polymers.

Plastic behaviour of polyelectrolyte microcapsules derived from colloid templates.

The deformability and osmotic properties of hollow microcapsules were studied by means of the micropipette video microscopic technique. The microcapsules were prepared by consecutive multiple adsorption of the polyanion, poly(styrene sulphonate), and the polycation, poly(allylamine hydrochloride), onto melamine formaldehyde resin latex of 5 microm diameter, which was decomposed after completing the coating by transferring to hydrochloric acid of pH 1.1. The polyelectrolyte microcapsules reacted to micropipette suction with plastic deformation. If lipids are added to the polyelectrolyte layers, the capsules cannot be visibly deformed by micropipette suction up to 10(4) N/m2. However, plastic shrinking was observed if the stress was generated by the osmotic pressure of a sucrose solution of 10(6) N/m2.

Low frequency electrorotation of fixed red blood cells.

Electrorotation of fixed red blood cells has been investigated in the frequency range between 16 Hz and 30 MHz. The rotation was studied as a function of electrolyte conductivity and surface charge density. Between 16 Hz and 1 kHz, fixed red blood cells undergo cofield rotation. The maximum of cofield rotation occurs between 30 and 70 Hz. The position of the maximum depends weakly on the bulk electrolyte conductivity and surface charge density. Below 3.5 mS/m, the cofield rotation peak is broadened and shifted to higher frequencies accompanied by a decrease of the rotation speed. Surface charge reduction leads to a decrease of the rotation speed in the low frequency range. These observations are consistent with the recently developed electroosmotic theory of low frequency electrorotation.

Electrophoresis of human red blood cells and platelets. Evidence for depletion of dextran.

A theoretical description of polymer depletion layers near smooth and hairy surfaces is developed and used for interpretation of experiments. Electrophoretic mobility measurements of human red blood cells and platelets in aqueous electrolyte solutions were performed in the presence of dextran (MW = 70 kD) to study the interaction of dextran with the cell surface. The electrophoretic mobility in the presence of dextran was considerably larger than expected from the viscosity. This behavior was interpreted as evidence for the existence of a polymer depletion layer. Depending on ionic strength, depletion layer thicknesses ranging from 2.9 to 4.4 nm were found. The dextran concentration at the outer border of the glycocalyx was only 10% of the bulk value. One cannot exclude the possibility that this small amount reflected adsorption. In the case of platelets, the degree of the mobility reduction depended on ionic strength. Depletion of dextran from the platelet surface apparently became smaller with decreasing ionic strength. This indicated a more complex pattern of interaction of dextran with the platelet surface than with the RBC surface. Both adsorption and polymer penetration into the glycocalyx were discussed quantitatively. It was concluded that ionic strength-dependent penetration of dextran into the hairy layer is more likely than ionic strength dependent adsorption changes. As in the case of red cells, some adsorption of dextran might be present.

Electrophoretic fingerprinting and multiparameter analysis of cells and particles.

A recently developed electrophoretic instrumentation based on a real-time image processing system has been applied to electrophoretic fingerprinting and multiparameter analysis of cells and other particles. The comparison between theoretical and experimental electrophoretic fingerprints, completed by the analysis of differences between measured fingerprints, offers a new methodology for better understanding and controlling of the processes at solid/liquid interfaces. Moreover, the multi-parameter analysis including electrophoretic mobility, size, density and shape of cells can complete the diagnosis and prognosis of diseases.

[Mechanisms of metabolic changes after in-line filtration of erythrocytes]. / Mechanismen der metabolischen Veränderungen nach einer In-Line-Filtration von roten Blutzellen.

BACKGROUND: The reasons for the improved RBC storage after in-line filtration are not fully understood. MATERIAL AND METHODS: Red blood cell concentrates were in-line filtered and stored for 6 weeks at 4 degrees C in SAGM. Parameter 2,3-DPG, p50, density, cell electrophoresis, pH were measured. RESULTS/CONCLUSION: The obtained results may explain the metabolic findings: Cell retention (during filtration older, rigide RBC are retained in the filter- a relative increase of younger RBC); changes in RBC membrane permeability (a changed electrophoretic mobility is attributed to bound RBC membrane proteins to the filter material); blood cell depletion (a high depletion efficiency prevents toxic influences of degenerating/disintegrating WBC and PLT); release of substances from filter and blood bag material (during passage of anticoagulated blood some compounds may be released in ppm concentrations - the density of in-line filtered RBC is higher than unfiltered RBC after filtration procedure and hypothermic storage).

[Serial infrared and ultraviolet whole body irradiation and placebo and ultraviolet irradiation of autologous venous blood in peripheral arterial occlusive disease. 1. Treadmill ergometry, metabolic, rheologic and hemodynamic parameters]. / Serielle Infrarot- und UV-Ganzkörperbestrahlung sowie Schein- und UV-Bestrahlung venösen Eigenblutes bei peripherer arterieller Verschlusskrankheit. 1. Laufbandergometrie, metabolische, rheologische und hämodynamische Parameter.

In 21 patients suffering from obstructive peripheral arterial disease stage II according to Fontaine, therapeutic efficacy of serial whole body irradiations (infrared or ultraviolet radiation) and pretended or real ultraviolet light blood irradiations was evaluated. Before, during and after treatment the following parameters were monitored: walking distance, oxygen partial pressure (quasi-arterial/venous), flow properties of blood (appearant blood viscosity, hemodynamics (peak flow, ultrasonics). There were no significant changes following both modalities of whole body irradiations either by sunshine-like ultraviolet light nor by infrared radiation, nor by pretended blood irradiation. In the same patients mean walking distance was prolonged threefold after ultraviolet irradiation of the patient's own venous blood and subsequent retransfusion. Simultaneously, oxygen utilisation was improved (enlarged arterial/venous difference), lactate concentration was decreased and apparent blood viscosity was diminished, whereas blood flow remained unchanged or only slightly improved. In this way the circulus vitiosus of obstructive peripheral arterial disease can be overcome. As a consequence of blood irradiation walking distance enlarges, providing better chances for physical training, which helps to extend walking distance furthermore.