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

We have previously demonstrated that transferrin-polycation conjugates are efficient carrier molecules for the introduction of genes into eucariotic cells. We describe here a more specific method for conjugation of transferrin with DNA-binding compounds involving attachment at the transferrin carbohydrate moiety. We used the polycation poly(L-lysine) or the DNA intercalator, ethidium homodimer as DNAbinding domains. Successful transferrin-receptor-mediatedd elivery and expression of the Photinus pyralis luciferase gene in K562 cells has been shown with these new transferrin conjugates. The activity of the transferrin-ethidium homodimer (TfEtD) conjugates is low relative to transferrin-polylysine conjugates; probably because of incomplete condensation of the DNA. However, DNA delivery with TfEtD is drastically improved when ternary complexes of the DNA with TfEtD and the DNA condensing agent polylysine are prepared. The gene delivery with the carbohydrate-linked transferrin-polylysine conjugates is equal or superior to described conjugates containing disulfide linkage. The new ligation method facilitates the synthesis of large quantities (>lo0 mg) of conjugates. INTRODUCTION Transferrin-polycation conjugates are efficient carriers for the uptake of DNA into eucariotic cells (I). This gene transfer technique, termed tramferrinfection, is based on receptor-mediated endocytosis of DNA complexed with polycation-transferrin conjugates (2,3). Our initial conjugate synthesis (1) involved the modification of one to two amino groups on the transferrin molecule with the bifunctional reagent succinimidyl34 2-pyridy1dithio)propionate (SPDP), followed by ligation to similarly modified polycations (polylysine or protamine) through the formation of disulfide bonds. Because there are more than 50 lysines on the large (about 80 kDa) transferrin protein, the actual site (or sites) of ligation to the polycation is unknown with this method. In this paper we describe the synthesis of new transferrin conjugates that are ligated with DNA-binding compounds in a specific manner through modification of the transferrin carbohydrate moiety. The conjugates thus obtained are free of any groups derived from chemical linking agents, since the connecting atoms are already present within the starting compounds. The carbohydrate group acts as anatural spacer that puts a 32-atom distance between the transferrin and the DNA binding moiety. This spacer effect may be important for appropriate presentation of the ligand to its receptor. As a DNA-binding compound, the polycation polylysine was used, similar to the use described in ref 1 or to the asialo-orosomucoid conjugates prepared by Wu and Wu (4). We have also prepared a novel type of transferrin conjugate that contains the DNA intercalator ethidium homodimer (5) as the DNAbinding group and demonstrate successful receptormediated gene delivery with these conjugates. EXPERIMENTAL PROCEDURES Human 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-piperazineethanesulfonic acid

Niosomes and polymeric chitosan based vesicles bearing transferrin and glucose ligands for drug targeting

PURPOSE: To prepare polymeric vesicles and niosomes bearing glucose or transferrin ligands for drug targeting. METHODS: A glucose-palmitoyl glycol chitosan (PGC) conjugate was synthesised and glucose-PGC polymeric vesicles prepared by sonication of glucose-PGC/cholesterol. N-palmitoylglucosamine (NPG) was synthesised and NPG niosomes also prepared by sonication of NPG/ sorbitan monostearate/ cholesterol/ cholesteryl poly-24-oxyethylene ether. These 2 glucose vesicles were incubated with colloidal concanavalin A gold (Con-A gold), washed and visualised by transmission electron microscopy (TEM). Transferrin was also conjugated to the surface of PGC vesicles and the uptake of these vesicles investigated in the A431 cell line (over expressing the transferrin receptor) by fluorescent activated cell sorter analysis. RESULTS: TEM imaging confirmed the presence of glucose units on the surface of PGC polymeric vesicles and NPG niosomes. Transferrin was coupled to PGC vesicles at a level of 0.60+/-0.18 g of transferrin per g polymer. The proportion of FITC-dextran positive A431 cells was 42% (FITC-dextran solution), 74% (plain vesicles) and 90% (transferrin vesicles). CONCLUSIONS: Glucose and transferrin bearing chitosan based vesicles and glucose niosomes have been prepared. Glucose bearing vesicles bind Con-A to their surface. Chitosan based vesicles are taken up by A431 cells and transferrin enhances this uptake

Delivery of drugs, proteins and genes into cells using transferrin as a ligand for receptor-mediated endocytosis

Transferrin, an iron-transporting serum glycoprotein, is efficiently taken up into cells by the process of receptor-mediated endocytosis. Transferrin receptors are found on the surface of most proliferating cells, in elevated numbers on erythroblasts and on many kinds of tumors. The efficient cellular mechanism for uptake of transferrin has been subverted for the delivery of low-molecular-weight drugs, protein toxins, and liposomes by linkage of these agents to transferrin or to anti-transferrin receptor antibodies. Linkage may be via chemical conjugation procedures or by the generation of chimeric fusion proteins. Transferrin conjugated to DNA-binding compounds (e.g. polycations or intercalating agents) has been successfully used for the import of DNA molecules into cells. High-level gene expression is obtained only if endosome-disruptive agents such as influenza hemagglutinin peptides or adenovirus particles are included which release the DNA complex from intracellular vesicles into the cytoplasm

Transferrin-polycation conjugates as carriers for DNA uptake into cells.

We have developed a high-efficiency nucleic acid delivery system that uses receptor-mediated endocytosis to carry DNA macromolecules into cells. We accomplished this by conjugating the iron-transport protein transferrin to polycations that bind nucleic acids. Human transferrin, as well as the chicken homologue conalbumin, has been covalently linked to the small DNA-binding protein protamine or to polylysines of various sizes through a disulfide linkage. These modified transferrin molecules maintain their ability to bind their cognate receptor and to mediate efficient iron transport into the cell. The transferrin-polycation molecules form electrophoretically stable complexes with double-stranded DNA, single-stranded DNA, and modified RNA molecules independent of nucleic acid size (from short oligonucleotides to DNA of 21 kilobase pairs). When complexes of transferrin-polycation and a bacterial plasmid DNA containing the gene for Photinus pyralis luciferase are supplied to eukaryotic cells, high-level expression of the luciferase gene occurs, demonstrating transferrin receptor-mediated endocytosis and expression of the imported DNA. We refer to this delivery system as "transferrinfection.

N-glycans of human amniotic fluid transferrin stimulate progesterone production in human first trimester trophoblast cells in vitro

Aims: During pregnancy, the placenta produces a variety of steroid hormones and proteins. Several of these substances have been shown to exert immunomodulatory effects. Progesterone is thought to mediate some of these effects by regulating uterine responsiveness. The aim of this study was to clarify the effect of amniotic fluid transferrin and its N-glycans on the release of progesterone by first trimester trophoblast cells in vitro. Methods: Cytotrophoblast cells were prepared from human first trimester placentae by trypsin-DNAse dispersion of villous tissue followed by a percoll gradient centrifugation and depletion of CD45 positive cells by magnetic cell sorting. Trophoblasts were incubated with varying concentrations (50-300 mug/ml) of transferrin from human amniotic fluid and serum as well as with N-glycans obtained from amniotic fluid transferrin. Culture supernatants were assayed for progesterone by enzyme-immunometric methods. Results: The release of progesterone increased in amniotic fluid transferrin- and N-glycan-treated trophoblast cell cultures compared to untreated trophoblast cells. There was no stimulating effect of serum transferrin on the progesterone production of trophoblast cells. Conclusions: The results suggest that amnion-transferrin and especially its N-glycans modulate the endocrine function of trophoblasts in culture by up regulating progesterone secretion

Transferrin-polycation-DNA complexes. The effect of polycations on the structure of the complex and DNA delivery to cells.

We have previously described a gene delivery system based upon the receptor-mediated endocytosis of DNA complexed with transferrin-polycation conjugates. This delivery system has been found to be very effective for both the internalization and the expression of genetic material in cells that have many transferrin receptors. Upon scrutinization of the parameters involved in this method, which we have termed transferrinfection, we note two important features of the process: the polycation in polycation-transferrin conjugates, as expected, serves to attach the transferrin moiety to the DNA and, in addition, the polycation functions to condense the DNA into a doughnut structure. Electron microscopic analysis of a range of poorly active to highly active transferrinfection samples reveals a strong correlation between DNA condensation and cellular DNA uptake. Furthermore, we demonstrate that the transfection activity of the DNA complex can be increased by addition of free polycation as long as a sufficient quantity of polycation-transferrin conjugates remains in the complex to ensure its binding to the cellular receptor

Delivery of the vitamin E compound tocotrienol to cancer cells

Tocotrienol, a member of the vitamin E family of compounds, is currently receiving increased attention because of its highly promising anti-cancer effects. However, its potential in cancer therapy is limited by its poor bioavailability and its inability to specifically reach tumors at therapeutic concentrations after intravenous administration. In order to remediate to these problems, various delivery strategies have been proposed, such as the inclusion of tocotrienol in γ-cyclodextrins, prodrugs and emulsions, entrapment in lipid nanoparticles and vesicles. Among these approaches, we demonstrated that the entrapment of tocotrienol within vesicles bearing transferrin, whose receptors are overexpressed on numerous cancer cells, significantly improved the uptake by cancer cells overexpressing transferrin receptors. Consequently, the intravenous administration of tocotrienol entrapped in transferrin-bearing vesicles led to tumor regression and even complete tumor suppression in some cases in a murine tumor model, as well as improvement of animal survival. Transferrin-bearing vesicles are therefore highly promising for the delivery of tocotrienol to cancer cells in vitro and in vivo and should be further investigated to optimize the anti-cancer therapeutic effect of tocotrienol

Human amniotic fluid glycoproteins expressing sialyl Lewis carbohydrate antigens stimulate progesterone production in human trophoblasts in vitro

Background: Progesterone is thought to mediate immune modulator effects by regulating uterine responsiveness. The aim of the study was to clarify the effect of transferrin and glycodelin A (former name PP14) as sialyl Lewis X-expressing glycoproteins on the release of progesterone by trophoblast cells in vitro. Methods: Cytotrophoblast cells were prepared from human term placentas by standard dispersion of villous tissue followed by a Percoll gradient centrifugation step. Trophoblasts were incubated with varying concentrations (50-300 mug/ml) of human amniotic fluid- and serum-transferrin as well as with glycodelin A. Culture supernatants were assayed for progesterone, human chorionic gonadotropin (hCG) and cortisol by enzyme immunometric methods. Results: The release of progesterone is increased in amniotic fluid transferrin- and glycodelin A-treated trophoblast cell cultures compared to untreated trophoblast cells. There is no relation between transferrin and the hCG or cortisol production of trophoblast cells. Conclusion: The results suggest that sialyl Lewis carbohydrate antigen-expressing amniotic fluid glycoproteins modulate the endocrine function of trophoblasts in culture by upregulating progesterone production. Copyright (C) 2004 S. Karger AG, Basel

Role of transferrin, transferrin receptors, and iron in macrophage listericidal activity.

It is not yet known what properties distinguish macrophages which can kill facultative intracellular bacteria, such as Listeria monocytogenes, from those which cannot. Listeria is an organism which requires iron for growth, yet macrophage listericidal mechanisms are also likely to be iron dependent. We show here that resident peritoneal macrophages and thioglycollate-elicited macrophages cannot kill listeria, but proteose peptone-elicited and FCS-elicited macrophages can. All these cell populations phagocytose listeria. Transferrin receptor expression is low on resident cells, intermediate on peptone- and FCS-elicited cells, and high on thioglycollate-elicited cells. Transferrin transports iron into cells via the transferrin receptor: thus, iron content of resident cells is low, of peptone- and FCS-elicited cells is intermediate, and of thioglycollate-elicited cells is high. Moreover, antibody to transferrin, which prevents it binding its receptor, inhibits listericidal macrophages from killing this bacterium. Finally, nonlistericidal cells with high transferrin receptor expression and high intracellular iron become listericidal if they are incubated with apotransferrin, an iron-free ligand which prevents iron uptake by cells. These data suggest that macrophages must have enough available intracellular iron to support listericidal mechanisms, but too much iron favors growth of the bacterium, which no longer can be killed by the macrophage