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% CaCl2 and 0.5% (w/v) alginate and chitosan cross-linked with 20% CaCl2 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 (R2 = 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.