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Preparation and Characterization of Liposomal-Lipophilic Tumor Necrosis Factor¹

Department of Tumor Biology, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

The liposomal delivery of recombinant 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 reticuloendothelial system. However, native recombinant human TNF 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 C₈ 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 (C₈) residues/trimer. This preparation was found to bind with high efficiency (50%) to preformed dipalmitoylphosphatidylcholine-small unilamellar 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 either with 3.5 caprylic acid residues/trimer or with 1.5 residues of myristic acid (C₁₄). However, the biological activity of these acylated TNFs was further reduced by this more extensive chemical modification (<50% activity for C₈ and <10% for C₁₄). 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.

¹ Supported by American Cancer Society Grant IM-571.

² To whom requests for reprints should be addressed, at Box 108, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe, Houston, TX 77030.

Received 12/ 4/90. Accepted 4/19/91.

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