New approach to measuring oxygen diffusion and consumption in encapsulated living cells, based on electron spin resonance microscopy.

Cell microencapsulation within biocompatible polymers is an established technology for immobilizing living cells that secrete therapeutic products.  These can be transplanted into a desired site in the body for the controlled and continuous delivery of the therapeutic molecules.  One of the most important properties of the material that makes up the microcapsule is its oxygen penetrability, which is critical for the cells' survival.  Oxygen reaches the cells inside the microcapsules via a diffusion process.  The diffusion coefficient for the microcapsules' gel material is commonly measured using bulk techniques, where the gel in a chamber is first flushed with nitrogen and the subsequent rate of oxygen diffusion back into it is measured by an oxygen electrode placed in the chamber.  This technique does not address possible heterogeneities between microcapsules, and also cannot reveal O2 heterogeneity inside the microcapsule resulting from the living cells' activity.  Here we develop and demonstrate a proof of principle for a new approach to measuring and imaging the partial pressure of oxygen (pO2) inside a single microcapsule by means of high-resolution and high-sensitivity electron spin resonance (ESR).  The proposed methodology makes use of biocompatible paramagnetic microparticulates intercalated inside the microcapsule during its preparation.  The new ESR approach was used to measure the O2 diffusion properties of two types of gel materials (alginate and extracellular matrix - ECM), as well as to map a 3D image of the oxygen inside single microcapsules with living cells. STATEMENT OF SIGNIFICANCE: The technology of cell microencapsulation offers major advantages in the sustained delivery of therapeutic agents used for the treatment of various diseases ranging from diabetes to cancer. Despite the great advances made in this field, it still faces substantial challenges, preventing it from reaching the clinical practice. One of the primary challenges in developing cell microencapsulation systems is providing the cells with adequate supply of oxygen in the long term. Nevertheless, there is still no methodology good enough for measuring O2 distribution inside the microcapsule with sufficient accuracy and spatial resolution without affecting the microcapsule and/or the cells' activity in it. In the present work, we introduce a novel magnetic resonance technique to address O2 availability within cell-entrapping microcapsules. For the first time O2 distribution can be accurately measured and imaged within a single microcapsule. This new technique may be an efficient tool in the development of more optimal microencapsulation systems in the future, thus bringing this promising field closer to clinical application.

Investigation of the influence of different physico-chemical parameters upon the susceptibility of planktonic and adherent Escherichia coli cells to beta-lactams and quinolones.

The purpose of this study was to evaluate the influence of different physico-chemical parameters on Escherichia coli susceptibility to ceftriaxone (CRO), cefotaxime (CTX), imipenem (IMP), and nalidixic acid (as marker for resistance by impermeability). The influence of chemical composition of culture medium was evaluated by the comparative assessment of inhibition growth diameters on different solid media: Mueller Hinton Medium (MH), Plate Count Agar Medium (PCA), MacConkey Medium (MC) and Eosin Methylen Blue Medium (EMB). In order to evaluate the differences in antibiotic susceptibility between the biofilm embedded and planktonic cells, an original, simple experimental model was used, by including the bacterial cells in an agar layer, mimicking the biofilm matrix. Our results demonstrated that the inhibition diameter zone was much larger on PCA, EMB and MC than on MH, considered as general standard medium for the antibiosusceptibility testings (CLSI). When bacterial cells were included in the agar matrix, the growth inhibition diameters obtained for different beta-lactams proved to be different of planktonic cells, i.e.: for CTX, a narrow inhibition diameter was obtained, demonstrating the low efficiency of this antibiotic in the treatment of biofilm associated infections, whereas the CRO proved the same efficiency against planktonic as well as to agar embedded bacteria. The different susceptibility results obtained for the cells embedded in the agar matrix by an adapted disk diffusion method are pleading for the necessity to assess new adapted standard methods and specific parameters in the purpose to determine the antibiotic resistance of bacterial cells isolated from biofilm associated infections.

Bile acid sequestrants based on cationic dextran hydrogel microspheres. 2. Influence of the length of alkyl substituents at the amino groups of the sorbents on the sorption of bile salts.

Cationic dextran hydrogel microspheres with pendant quaternary ammonium groups having alkyl substituents (C(2)-C(12)) at quaternary nitrogen were synthesized. The in vitro sorption of sodium salts of four bile acids (glycocholic, cholic, taurocholic, and deoxycholic acids) with these hydrogels was studied as a function of substituent alkyl chain length and bile acid hydrophobicity. Sorption experiments were performed in phosphate buffer solutions (pH 7.4) containing one bile salt (individual sorption) or mixtures of several bile salts (competitive sorption). Parameters for individual sorption were calculated taking into consideration the stoichiometric and cooperative binding of bile salts to oppositely charged polymer hydrogels. The results show that the increase in the length of the alkyl chain of the substituent leads to an increase in both ionization constant K(0) and overall stability constant of binding K, but decreases the cooperativity parameter u. The competitive sorption studies indicate that the hydrogels display a good affinity for both dihydroxylic and trihydroxylic bile salts. The molar ratio of maximum amounts bound for the two types of bile acid is 2 to 1, which is much lower than those reported for other cationic polymers recommended as bile acid sequestrants. The binding constants for the sorption of bile salts by some dextran hydrogels are 20-30 times higher than those obtained for cholestyramine under similar sorption conditions.

Sodium cholate sorption on cationic dextran hydrogel microspheres. 1. Influence of the chemical structure of functional groups.

New hydrogel microspheres based on crosslinked dextran, containing pendant quaternary ammonium groups with different chemical structures have been synthesized and tested as possible bile acid sorbents The in vitro sodium cholate sorption by these hydrogels has been followed in the absence or in the presence of competing anions The sorption results have indicated a strong influence of the chemical structure of functional groups on both the affinity and selectivity towards cholate ions. The best sorption performances were obtained with hydrogels having in the structure of functional groups an alkyl substituent with the length higher than C(8).

Aminated polysaccharides as bile acid sorbents: in vitro study.

Dextran, pullulan, and microcrystalline cellulose were cross-linked with 1-chloro-2,3-epoxypropane and reacted with N-(1-chloroethyl)-N,N-diethylamine or N-glycidyl-N,N,N-trialkylammonium chloride in order to obtain sorbents containing tertiary amino and/or quaternary ammonium groups. In vitro equilibrium sorption of cholic acid on these sorbents was studied in comparison with Cholestyramine, and in vitro dissociation of ionic complexes of cholic acid-sorbents was determined under dynamic conditions. The sorption capacity and the affinity of these sorbents for cholic acid were investigated in relation to the nature of the polymeric support, the swelling porosity of sorbent, the basicity of amino groups, and the nature of the substituents at the nitrogen atom. The maximum sorption capacity increases with the increase in amino group content, their basicity, and the length of alkyl substituents at the nitrogen atom. The affinity for cholic acid of all polysaccharide-based sorbents is higher than that of Cholestyramine. Dextran-based sorbents display the highest sorption affinity. It was found that there exists an optimum swelling porosity for the polysaccharide sorbents to attain the highest affinity for cholic acid. The dissociation rate of ionic complexes depends also on the nature of the polysaccharide and the swelling porosity and its lower for sorbents with higher sorption affinity.