Cross-Linked Alginate Film Pore Size Determination Using Atomic Force Microscopy and Validation Using Diffusivity Determinations

Abstract

The deficit of organ donors has fueled the need for advances in tissue engineering and regenerative medicine. Microencapsulation in alginate immuno-isolation membranes has been used to treat many disabling metabolic disorders, namely, phenylketonuria, kidney failure and diabetes mellitus. Systematic nutrient flux determinations are hindered by the lack of experimental data on alginate-based membrane topography and the pore size thus preventing the full therapeutic potential of the bio-membranes to be reached. In this study, samples of cross-linked alginate membranes were subjected to the following analytical characterization: 1) pore size characterization using atomic force microscopy operated in contact mode to detect and measure pore size; 2) differential scanning calorimetry to confirm biopolymer cross-linking; and 3) diffusivity measurements using spectrophotometry and fluorescence microscopy to confirm the presence of through pores and to calculate reflection coefficients. The pore sizes for the pre-clinical standard formulation of 1.5% (w/v) medium viscosity alginate cross-linked with 1.5% CaCl 2 and 0.5% (w/v) alginate and chitosan cross-linked with 20% CaCl 2 are 5.2 nm ± 0.9 nm and 7.0 nm ± 3.1 nm, respectively. An increase in the glass transition temperatures as a function of cross-linker concentration was observed. Diffusivity values obtained from the inward diffusivity of creatinine into macrocapsules (d = 1000 µm ± 75 µm) and the outward diffusivity of FITC dextrans from macrocapsules (d = 1000 µm ± 75 µm) and microcapsules (d = 40 µm ± 5 µm) were shown to correlate strongly (R 2 = 0.9835) with the ratio of solute to pore sizes, confirming the presence of through pores. Reflection coefficients approaching and exceeding unity correlate with the lack of permeability of the membranes to MW markers that are 70 kDa and greater.

Quantifying osmotic membrane fouling to enable comparisons across diverse processes

“…For pores with diameters on the order of nanometers (true of alginate gels [17], and likely true of most foulant cakes because particles over 0.45 µm in diameter generally do not accumulate on the membrane [18], and pores are unlikely to be larger than the particle size), flow through the porous media is laminar. The friction factor can be estimated by the correlation for laminar flow through round tubes,…”

“…Klein et al [37] who found pore diameters of approximately 6.8-16.6 nm for three alginates through inverse steric exclusion chromatography. Imaging methods tend to identify larger pore sizes (likely due to the drying necessary), but three diffusion-based methods reviewed in [17] find pore sizes that are also in the 10 nm range. Shoichet et al [38] find that a higher degree of cross-linking leads to smaller pores.…”

“…With just six equations (4, 6, and [16][17][18][19], it is insufficient to specify just the flux or structural parameter. As with FO, a known relationship between foulant pore diameter and local conditions can add one equation.…”

Production of Biodiesel Using Immobilized Lipase and the Characterization of Different Co-Immobilizing Agents and Immobilization Methods

“…In order to better exhibit the difference between three immobilized methods (catalysts), the structure of free lipase and three immobilized lipase were examined by AFM [23] and SEM. The images of free lipase scanned by AFM were shown in Figure 6(A1-A3).…”

“…Lipase and substrate can fully contact each other through the pores to obtain a high catalytically activity which was proved by the data shown in Figure 5. In order to better exhibit the difference between three immobilized methods (catalysts), the structure of free lipase and three immobilized lipase were examined by AFM [23] and SEM. The images of free lipase scanned by AFM were shown in Figure 6(A1-A3).…”