Transfersomes (TFs) are highly deformable hydrophilic lipid vesicles that are able to penetrate the skin barrier spontaneously because of their characteristics. Transfersomes are able to transport noninvasively low- and high-molecular-weight molecules into the body. We describe the formulation and several biologic characteristics of interleukin-2 (IL-2)- and interferon-alpha (IFNalpha )-containing TFs. TFs contain natural phosphatidylcholine and sodium cholate. Recombinant human IL-2 and human hybrid IFNalpha were added to TFs and incubated for 24 hours at 4 degrees C. Immunotransfersomes were isolated from free IL-2 and IFNalpha by filtration (Centrisart, Sartorius). The biologic activity of immunotransfersomes was measured by a cytotoxic lymphoid line assay for IL-2 and by an A549-encephalomyocarditis virus assay for IFN; concentrations of proteins were determined by the enzyme-linked immunosorbent assay (ELISA). It was possible to incorporate a large amount of IL-2 and IFN in TFs (75-80%), and the incorporated IL-2, and IFN were biologically active. The increased lipid/protein ratio (90.9/1.0) led to a growing probability of association. We were thus able to show that IL-2 and IFN are trapped by transfersomes in a biologically active form and in sufficient concentrations for immunotherapy. In upcoming experiments these IL-2- and IFN-containing TFs will be used for a transdermal approach in the murine RENCA cell line model.