Cryoprotective Mechanism of DMSO Induced by the Inhibitory Effect on Eutectic NaCl Crystallization.

Cryopreservation is a critical procedure in autologous hematopoietic stem cell transplantation. Dimethyl sulfoxide (DMSO) is the cryoprotectant of choice. Optimization of the cryopreservation protocol in the past revealed a dramatic loss of cell viability associated with a reduction of the DMSO concentration below 2 vol % in the freezing medium. The cryoprotective mechanism of DMSO is usually ascribed to the ability to suppress ice formation and reduce the adverse effects of the freeze-concentrated solution. This work proposes an alternative hypothesis considering the detrimental impact of NaCl eutectic crystallization on cell viability. Thermoanalytical and microstructural analysis of the DMSO effect on eutectic phase transformation of cryoprotective mixtures revealed a correlation between the loss of cell viability and eutectic NaCl crystallization. DMSO inhibits the eutectic crystallization of NaCl and preserves cell viability. Thermodynamic description of the inhibitory action and possible mechanism of cryoinjury are provided.

In Vitro Interactions of Amphiphilic Phosphorous Dendrons with Liposomes and Exosomes-Implications for Blood Viscosity Changes.

Drug delivery by dendron-based nanoparticles is widely studied due to their ability to encapsulate or bind different ligands. For medical purposes, it is necessary (even if not sufficient) for these nanostructures to be compatible with blood. We studied the interaction of amphiphilic dendrons with blood samples from healthy volunteers using standard laboratory methods and rheological measurements. We did not observe clinically relevant abnormalities, but we found a concentration-dependent increase in whole blood viscosity, higher in males, presumably due to the formation of aggregates. To characterize the nature of the interactions among blood components and dendrons, we performed experiments on the liposomes and exosomes as models of biological membranes. Based on results obtained using diverse biophysical methods, we conclude that the interactions were of electrostatic nature. Overall, we have confirmed a concentration-dependent effect of dendrons on membrane systems, while the effect of generation was ambiguous. At higher dendron concentrations, the structure of membranes became disturbed, and membranes were prone to forming bigger aggregates, as visualized by SEM. This might have implications for blood flow disturbances when used in vivo. We propose to introduce blood viscosity measurements in early stages of investigation as they can help to optimize drug-like properties of potential drug carriers.

On crystallization of water confined in liposomes and cryoprotective action of DMSO.

In this work, the phase behavior of cryoprotective mixtures based on dimethyl sulfoxide (DMSO) mixed with a lipid bilayer consisting of dimyristoyl phosphatidylcholine (DMPC) was studied. This system represented a model of a biological cell and its membrane. The aim of the work was to clarify the origin of the cryoprotective action of low-concentrated mixtures (1-10 vol%) DMSO in water, representing mixtures used in cryopreservation in cell therapy. The combination of experimental techniques of differential scanning calorimetry (DSC) and positron annihilation lifetime spectroscopy (PALS) allowed a study of crystallization behavior of water confined in liposomes imitating the intracellular environment. The ability of liposomes to show the fundamental aspects of water phase behavior seen during freezing of biological cells was proved. The presence of an amorphous freeze-concentrated phase of DMSO in the frozen state was confirmed and its possible crystallization into the DMSO trihydrate and ice during thawing was demonstrated. Correlation between the critical temperature range for the loss of cell viability during slow thawing and the temperatures of freeze-concentrated phase crystallization was found. Based on this finding, possible mechanisms of DMSO cryoprotection are discussed with support brought by results for the studied model system. Quantification of the ice phase fraction in the frozen mixtures revealed that even low concentrations of DMSO can induce a considerable decrease in the amount of ice present.

Microporous carbon fibers as electroconductive immobilization matrixes: Effect of their structure on operational parameters of laccase-based amperometric biosensor.

In this study, we describe the fabrication of sensitive biosensor for the detection of phenolic substrates using laccase immobilized onto two types of microporous carbon fibers (CFs). The main characteristics of microporous CFs used for preparation of biosensors are given. Two CFs were characterized by different specific surface area, CFA (<1 m2·g-1) and CFB (1448 m2·g-1), but with comparable size of the micropores estimated by positron annihilation lifetime spectroscopy. The structural analysis was shown that CFA is formed by thin interwoven fibers forming a highly porous structure, as well as CFB - by granular formations with uneven edges that shape a cellulose membrane of lower porosity. The results of amperometric analysis revealed that the laccase-bound CFs possesses better electrochemical behavior for laccase than non-modified rod carbon electrodes (control). Using chronoamperometric analysis, the operational parameters of the CFs-modified bioelectrodes were compared to control bioelectrodes. The bioelectrodes based on CFs have demonstrated 2.4-2.7 folds enhanced maximal current at substrate saturation (Imax) values, 1.2-1.4 folds increased sensitivity and twice wide linearity compared with control bioelectrodes. The sensitivity of the developed CFs-based bioelectrodes was improved compared with the laccase-bound electrodes, described in literature. The developed biosensor was tested for catechol analysis in the real communal wastewater sample.

Microstructural free volume and dynamics of cryoprotective DMSO-water mixtures at low DMSO concentration.

This work investigates the free-volume properties of the dimethyl sulfoxide (DMSO)-water mixtures by positron annihilation lifetime spectroscopy over a wide temperature range of 20-320 K. The processes of melting and solidification of the water, DMSO and the DMSO-water mixtures at 1.8, 2.0 and 10% vol. DMSO respectively were studied. It was found that the recrystallization during heating of the water-DMSO cryoprotective mixtures above 160 K at low DMSO concentrations is affected by the amount of DMSO in the mixture. The amount of amorphous phase formed during cooling influences the hysteresis between cooling and heating cycles which could be crucial for cell survival. Experiments also show the time dependence of crystallization which indicates that rapid heating can suppress this secondary crystallization which is undesirable during the cell thawing process. Similar concentrations of DMSO (1.8% and 2% vol. DMSO in water) where a 2% vol. DMSO mixture secures cell survival but 1.8 vol% does not, showed differences in structural and dynamic properties that are key factors in cell survival. These results were supported by differential scanning calorimetry and low frequency dielectric spectroscopy measurements. The obtained data are in strong agreement with the observed cryoprotective efficacy of the DMSO-water mixtures on living cells.