Preparation and Characterization of Liposomal-Lipophilic Tumor Necrosis Factor

The liposomal delivery of rccombinanl human tumor necrosis factor (rHuTNF) has been shown to be effective in reducing its toxic effects and in its targeting of organs rich in cells of the rcticuloendothelial system. However, native recombinant human TNT shows only poor affinity for liposomes, presumably due to its low hydrophobicity. In an attempt to increase the efficiency of association with liposomes, we have modified TNF to increase its hydrophobicity by selective substitution of its amino groups with fatty acid side chains. N-Hydroxysuccinimide esters of saturated fatty acids ranging from Cs to C18 were reacted with recombinant human TNF. Modification with esters of C8 to C14 acids occurred as determined by consumption of positively charged amino groups monitored by native polyacrylamide gel electrophoresis; however, esters of longer chain lengths (C16, C18) were much less capable of introducing these chains via amide linkages, and thus these adducts were not further characterized. Biological assays revealed that retention of activity was inversely dependent both on the number of chains introduced and on their length; activity was most conserved (>50%) in a TNF preparation modified with only -1-2.5 caprylic acid (CK)residues/trimer. This preparation was found to bind with high efficiency (-50%) to preformed dipalmitoylphosphatidylcholinc-small unilamcllar vesicles. The extent of binding closely paralleled both the number of chains introduced and their length; binding was even more efficient (80-90%) for TNF modified cither with -3.5 caprylic acid residues/trimer or with -1.5 residues of myristic acid (CM). However, the biological activity of these acylated TNFs was further reduced by this more extensive chemical modification (<50% activity for Cx and <10% for C,4). The biological activity of dipalmitoylphosphatidylcholine-small unilamellar vesicle-C8- TNF was found to be comparable to that of the nonliposomal C8-TNF. Thus, biologically active preparations of liposomal-lipophilic TNF can be prepared with high efficiency.