Chitosan nanoparticles were prepared by ionic cross-linking with tripolyphosphate (TPP). The major effect on encapsulation and release of protein in chitosan-TPP nanoparticles was investigated in order to control the loading and release efficiency. A set of the same molecular weight (MW) proteins with different pI and a set of the same pI proteins with different MW were studied. The influence of protein concentration, pH of solution, and the activity of released protein were examined. It was found that the encapsulation efficiency (EE) of a set of the different MW protein decreased with increasing of MW of protein and protein concentration. The protein with having pI higher than pH of solution was attracted to the positively charged chitosan, resulting in increasing of EE. The release of protein from the nanoparticles showed that the protein release decreased with increasing of chitosan concentration, high MW protein, low pH, and less swelling of the particle. The released protein in chitosan-TPP matrix was still active in the buffer solution.
The back-extraction of proteins encapsulated in AOT reverse micelles was performed by adding a counterionic surfactant, either TOMAC or DTAB. This novel backward transfer method gave higher backward extraction yields compared to the conventional method with high salt and high pH of the aqueous stripping solution. The protein activity was maintained in the resulting aqueous phase, which in this case had a near neutral pH and low salt concentration. A sharp decrease of the water content was observed in the organic phase corresponding to protein back-extraction using TOMAC. The backward transfer mechanism was postulated to be caused by electrostatic interaction between oppositely charged surfactant molecules, which lead to the collapse of the reverse micelles. The back-extraction process with TOMAC was found to be very fast; more than 100 times faster than back-extraction with the conventional method, and as much as 3 times faster than forward extraction. The formation of 1:1 complexes of AOT and TOMAC in the solvent phase was observed, and these hydrophobic complexes could be efficiently removed from the solvent using adsorption onto Montmorillonite in order for the organic solvent to be reused. A second cationic surfactant, DTAB, confirmed the general applicability of counterionic surfactants for the backward transfer of proteins.
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