DNA-binding transferrin conjugates as functional gene-delivery agents: synthesis by linkage of polylysine or ethidium homodimer to the transferrin carbohydrate moiety

摘要

We have previously demonstrated that transferrin-polycation conjugates are efficient carrier moleculesfor the introduction of genes into eucariotic cells. We describe here a more specific method for conjugationof transferrin with DNA-binding compounds involving attachment at the transferrin carbohydratemoiety. We used the polycation poly(L-lysine) or the DNA intercalator, ethidium homodimer as DNAbindingdomains. Successful transferrin-receptor-mediatedd elivery and expression of the Photinuspyralis luciferase gene in K562 cells has been shown with these new transferrin conjugates. The activityof the transferrin-ethidium homodimer (TfEtD) conjugates is low relative to transferrin-polylysineconjugates; probably because of incomplete condensation of the DNA. However, DNA delivery withTfEtD is drastically improved when ternary complexes of the DNA with TfEtD and the DNA condensingagent polylysine are prepared. The gene delivery with the carbohydrate-linked transferrin-polylysineconjugates is equal or superior to described conjugates containing disulfide linkage. The new ligationmethod facilitates the synthesis of large quantities (>lo0 mg) of conjugates.INTRODUCTIONTransferrin-polycation conjugates are efficient carriersfor the uptake of DNA into eucariotic cells (I). This genetransfer technique, termed tramferrinfection, is basedon receptor-mediated endocytosis of DNA complexed withpolycation-transferrin conjugates (2,3). Our initial conjugatesynthesis (1) involved the modification of one totwo amino groups on the transferrin molecule with thebifunctional reagent succinimidyl34 2-pyridy1dithio)propionate(SPDP), followed by ligation to similarly modifiedpolycations (polylysine or protamine) through the formationof disulfide bonds. Because there are more than50 lysines on the large (about 80 kDa) transferrin protein,the actual site (or sites) of ligation to the polycation isunknown with this method.In this paper we describe the synthesis of new transferrinconjugates that are ligated with DNA-bindingcompounds in a specific manner through modification ofthe transferrin carbohydrate moiety. The conjugates thusobtained are free of any groups derived from chemicallinking agents, since the connecting atoms are alreadypresent within the starting compounds. The carbohydrategroup acts as anatural spacer that puts a 32-atom distancebetween the transferrin and the DNA binding moiety. Thisspacer effect may be important for appropriate presentationof the ligand to its receptor. As a DNA-bindingcompound, the polycation polylysine was used, similar tothe use described in ref 1 or to the asialo-orosomucoidconjugates prepared by Wu and Wu (4). We have alsoprepared a novel type of transferrin conjugate that containsthe DNA intercalator ethidium homodimer (5) as the DNAbindinggroup and demonstrate successful receptormediatedgene delivery with these conjugates.EXPERIMENTAL PROCEDURESHuman transferrin (iron-free), conalbumin (iron-free),and poly(L-lysine) were obtained from Sigma. Liquid chro-Abbreviations used: FITC, fluorescein ieothiocyenate; TfEtD,traneferrin-ethidium homodimer conjugate; TfpL, traneferrinpolytL-lysine) conjugate; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonicacid.