In order to incorporate hydrophilic macromolecular drugs into lipid-based formulations (LBF), HIP has shown great potential. In this study, different HIP methods were compared with each other. Hydrophobic complexes were formed between bovine serum albumin (BSA) and either dodecyl sulfate, cetyl trimethylammonium or 1,2-dipalmitoyl-sn-glycero-3-phosphate applying the organic solvent-free method, Bligh-Dyer method and biphasic metathesis reaction either with ethyl acetate or chloroform as organic phase. Complex formation efficiency was determined. Hydrophobicity of the obtained complexes was characterized by their apparent partition coefficient between 1-butanol and water. The highest complex formation efficiency was achieved with the Bligh-Dyer method, followed by the organic solvent-free method and the biphasic metathesis reaction. When applying the organic solvent-free method, complex formation efficiency was hampered at higher surfactant concentrations due to the formation of micelles. Furthermore, this method could only be applied for water-soluble compounds. On the contrary, the Bligh-Dyer method was robust towards high surfactant concentrations. Moreover, it enables the use of water-insoluble compounds. The rank order Bligh-Dyer method > organic solvent-free method > biphasic metathesis reaction was confirmed by the log D. According to these results, the Bligh-Dyer method appears advantageous for HIP. However, the organic-solvent free method is an adequate alternative for water-soluble compounds.
