DNA encapsulation within membrane-coated alginate beads

Alginate beads produced by both external and internal gelation techniques were Studied so as to determine the optimal bead matrix structure within which DNA can be immobilized for in vim application. Alginates were characterized for guluronic/marmuronic acid (G/M) content and average molecular weight. Non-homogeneous calcium, alginate and DNA distributions were found within gels made by the external gelation method because of the external calcium source used. In contrast, the internal gelation method produces more homogeneous gels. The encapsulation yield of DNA was over 97 and 80% for beads formed using external and internal gelation methods, respectively. DNA molecular weight cut-off was determined to be 394 kDa for both external and internal gelled beads.; DNA alginate beads produced using both external and internal calcium sources were coated with chitosan, poly-L-lysine or co-guanidine membranes. For chitosan and poly-L-lysine, membrane thickness increased with decreasing molecular weight and increasing degree of deacetylation (chitosan). Co-guanidine membranes were shown to form intact ionically complexed membranes on alginate beads, serving as an alternative to the commonly used polymers; poly-L-lysine and chitosan. The co-guanidine membranes thickness increased with increasing concentration and coating time. The beads were assayed with DNAse nuclease to determine optimal membrane matrix combinations offering the highest level of DNA protection from nucleic acid hydrolysis, simulating gastrointestinal exposure. An extractive electrophoresis method was also developed to extract and assay intracapsular DNA by measuring its molecular size range.; Almost total hydrolysis of DS-DNA was observed in alginate beads and chitosan coated beads following nuclease exposure. The casting of membranes reduced the permeability of alginate beads, shown by the enhanced retention of DNA residuals (i.e. double- and single-stranded DNA, polynucleatides, bases) after DNAse exposure. The highest level of DNA protection was obtained with high molecular weight (197.1 kDa) poly-L-lysine and high concentration (5 mg/ml) co-guanidine membranes coated on beads formulated using an external calcium source, where over 80 and 90% of the DS-DNA remained after 40 min of DNAse exposure. Guanidine membranes were capable of fully excluding DNAse nuclease with a molecular weight of 31 kDa. Lyophilization and rehydration of DNA beads also reduced permeability to nucleases, resulted in DNA recoveries following nuclease exposure of 60% for chitosan coated, 90% for poly-L-lysine coated, and 95% for alginate beads.; Soluble chitosan and poly-L-lysine are readily hydrolyzed using lysozyme or chitmanase for chitosan, and trypsin, chymotrypsin or proteinase K for poly-L-lysine. In contrast, chitosan and poly-L-lysine membranes were almost inert to the respective hydrolytic enzymes, with less than 2% of the membrane weight being hydrolyzed. It appeared that either membrane material would be stable for in vivo application, and in particular in the protection of DNA during gastrointestinal transit; Intracapsular DNA was accessible to the carcinogen ethidium bromide, which showed a 4 fold increase in uptake in uncoated beads and 2 fold uptake in co-guanidine coated beads compared to beads lacking DNA. Low molecular weight cut-off co-guanidine membranes appear to provide free diffusional access to low molecular weight carcinogens or mutagens.