Abstract:
Partitioning of proteins in aqueous two-phase systems (ATPS) has been shown to provide a powerful method for preparing protein-loaded hydrogel microparticles from an all aqueous process. There are many factors known which affect partitioning of therapeutic proteins in ATPS. The value of the partition coefficient relies on the physico-chemical properties of protein drug and other molecules present and their interactions with those of the chosen ATPS, e.g. the polarity of the “incompatible” polymers. In this work, ATPS consisting of hydroxyethyl starch (HES), polyethylene glycol and water were used. We investigated the partitioning of lysozyme as a model protein and sucrose, glycine and trehalose as cryo-protectants in both phases of the ATPS and in particular their mutual interaction.
It was shown that with increasing concentrations of cryo-protectants in the ATPS both the density ratio (density of the PEG phase divided by density of the HES phase) and the drying residue ration (drying residue of the PEG phase divided by drying residue of the HES phase) increased. However, the partition coefficient kcryo-protectant (concentration of the cryo-protectant in the PEG phase devided by the concentration of the cryo-protectant in the HES phase), determined by measuring the concentration of radio-labelled cryo-protectants in both phases, remains constant over the investigated concentration range. Moreover it could be shown that sucrose and trehalose accumulate slightly in the PEG-phase. Partition does neither depend on concentration nor is it affected by the addition of lysozyme. Glycine distributes almost homogenously in both phases, which is also independent from its concentration and added lysozyme.
The partition coefficient of lysozyme as a model protein was determined using fluorimetry and ELISA for quantitative measurements of the protein. Furthermore, the influence of sucrose on its partition behaviour was determined. Without sucrose lysozyme accumulated approx. 60-fold in the HES phase. Accumulation in the HES phase was reduced upon addition of sucrose leading to lysozyme concentrations in the HES phase which were 30- to 40-fold higher than in the PEG phase.
On the basis of this ATPS microparticles were prepared by means of a radical polymerisation. For this purpose a modified ATPS was used, consisting of hydroxyethyl starch-hydroxyethyl methacrylate (HES-HEMA), PEG and water. Microparticles were prepared in the presence of 0, 5, 10, or 15 % sucrose, respectively. The resulting microparticles were characterized with respect to morphology, particle size, and encapsulation efficiency of lysozyme and FITC-dextran which were used as model drugs. Moreover, release rate and release kinetic from the hydrogel microparticles were studied in dependence from the degree of substitution of HES-HEMA and the sucrose content.
The obtained microparticles showed a narrow particle size distribution with an average particle size of 10-15 µm. The release rate of the model drugs was strongly affected by the degree of substitution and the release medium, but not by the sucrose content. In contrast, the release kinetic was affected only by the release medium, but not by the degree of substitution and the sucrose content.