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

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.

Nanodiamond as a vector for siRNA delivery to Ewing sarcoma cells

We investigated the ability of diamond nanoparticles (nanodiamonds, NDs) to deliver small interfering RNA (siRNA) in Ewing sarcoma cells, in the perspective of in vivo anti-cancer nucleic acid drug delivery. siRNA was adsorbed onto NDs previously coated with cationic polymer. Cell uptake of NDs has been demonstrated by taking advantage of NDs intrinsic fluorescence coming from embedded color center defects. Cell toxicity of these coated NDs was shown to be low. Consistent with the internalization efficacy, we have shown a specific inhibition of EWS/Fli-1 gene expression at the mRNA and protein level by the ND vectorized siRNA in a serum containing medium

Comparative study of osteogenic activity of multilayers made of synthetic and biogenic polyelectrolytes

Polyelectrolyte multilayer (PEM) coatings on biomaterials are applied to tailor adhesion, growth, and function of cells on biomedical implants. Here, biogenic and synthetic polyelectrolytes (PEL) are used for layer-by-layer assembly to study the osteogenic activity of PEM with human osteosarcoma MG-63 cells in a comparative manner. Formation of PEM is achieved with biogenic PEL fibrinogen (FBG) and poly-l-lysine (PLL) as well as biotinylated chondroitin sulfate (BCS) and avidin (AVI), while poly(allylamine hydrochloride) (PAH) and polystyrene sulfonate (PSS) represent a fully synthetic PEM used as a reference system here. Surface plasmon resonance measurements show highest layer mass for FBG/PLL and similar for PSS/PAH and BCS/AVI systems, while water contact angle and zeta potential measurements indicate larger differences for PSS/PAH and FBG/PLL but not for BCS/AVI multilayers. All PEM systems support cell adhesion and growth and promote osteogenic differentiation as well. However, FBG/PLL layers are superior regarding MG-63 cell adhesion during short-term culture, while the BCS/AVI system increases alkaline phosphatase activity in long-term culture. Particularly, a multilayer system based on affinity interaction like BCS/AVI may be useful for controlled presentation of biotinylated growth factors to promote growth and differentiation of cells for biomedical applications

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

Interaction of aluminium hydrolytic species with biomolecules

In this contribution the formation of bioinorganic assemblies between the basic globular protein lysozyme and aqueous aluminium species including Al 13 -mer, Al 30 -mer and colloidal aluminium hydroxide have been explored and comparison made to previous interaction studies performed with bovine serum albumin (BSA). Specific charge-stabilised bioinorganic assemblies involving aluminium species and lysozyme were observed to form in contrast to the gel like structures formed on interaction of BSA with aluminium species. As demonstrated by infrared spectroscopy (structural assignment, 2D correlation spectroscopy), interactions mostly involve acidic surface groups of the proteins (Asp, Glu), with strong complexation and deprotonation in the case of BSA interacting with Al 13 and Al 30 and through hydrogen bonding for lysozyme interacting with the same species and aluminium hydroxide particles interacting with both biomolecules

Gene transfer into hepatocytes using asialoglycoprotein receptor mediated endocytosis of DNA complexed with an artificial tetra-antennary galactose ligand

We have constructed an artificial ligand for the hepatocyte-specific asialoglycoprotein receptor for the purpose of generating a synthetic delivery system for DNA. This ligand has a tetra-antennary structure, containing four terminal galactose residues on a branched carrier peptide. The carbohydrate residues of this glycopeptide were introduced by reductive coupling of lactose to the alpha- and epsilon-amino groups of the two N-terminal lysines on the carrier peptide. The C-terminus of the peptide, containing a cysteine separated from the branched N-terminus by a 10 amino acid spacer sequence, was used for conjugation to 3-(2-pyridyldithio)propionate-modified polylysine via disulfide bond formation. Complexes containing plasmid DNA bound to these galactose-polylysine conjugates have been used for asialoglycoprotein receptor-mediated transfer of a luciferase gene into human (HepG2) and murine (BNL CL.2) hepatocyte cell lines. Gene transfer was strongly promoted when amphipathic peptides with pH-controlled membrane-disruption activity, derived from the N-terminal sequence of influenza virus hemagglutinin HA-2, were also present in these DNA complexes. Thus, we have essentially borrowed the small functional domains of two large proteins, asialoglycoprotein and hemagglutinin, and assembled them into a supramolecular complex to generate an efficient gene-transfer system

Effect of heating rate on gas emissions and properties of fired clay bricks and fired clay bricks incorporated with cigarette butts

In general, the firing process of clay bricks generates a range of gas emissions into the atmosphere. At high concentrations, these volatile emissions can be a serious source of environmental pollutions. The main purpose of this study was to evaluate the effect of different heating rates on gas emissions and properties during the firing of clay bricks and clay bricks incorporated with cigarette butts (CBs). In this investigation, four different heating rates were used: 0.7 °C min−1−1, 2 °C min−1, 5 °C min−1 and 10 °C min. The samples were fired in solid form from room temperature to 1050 °C. During the firing cycles, carbon monoxide, carbon dioxide, nitrogen oxides,hydrogen cyanide and chlorine emissions were measured at different heating rates. All bricks were also tested for their physical and mechanical properties including dry density, compressive strength, tensile strength, water absorption and initial rate of absorption. Results show that gas emissions were reduced significantly with higher heating rates (10 °C min) followed by 5 °C min−1−1 and 2 °C min for both types of brick samples. Higher heating rates also decrease the compressive strength and tensile strength value but demonstrate an insignificant effect on the water absorption properties respectively. In conclusion, a higher heating rate is preferable in terms of decreasing gas emissions and it is also able to produce adequate physical and mechanical properties especially for the CB brick

SINTESIS LEMPUNG TERPILAR CetylTrimethylAmmonium-Aluminium (CTMA-Al) DARI LEMPUNG ALAM SERTA PEMANFAATANNYA SEBAGAI ADSORBEN ZAT WARNA INDIGO KARMINA

Dilakukan sintesis lempung terpilar karena lempung mempunyai pori yang tidak tetap yaitu akan mengkerut jika dipanaskan (dehidrasi) dan akan mengembang jika terjadi hidrasi. Lempung terpilar disintesis dengan pertukaran kation CetylTriMethylAmmonium CTMA dilanjutkan dengan polikation Al pada lempung alam. Lempung terpilar CTMA-Al (CetylTriMethylAmmonium-Aluminium) yang disintesis digunakan untuk mengadsorpsi indigo karmina pada larutan air relatif terhadap lempung alam. Proses pilarisasi pada lempung dilakukan dengan interkalasi surfaktan CTMA dilanjutkan dengan interkalasi polikation Al pada lempung alam. Hasil sintesis lempung terpilar CTMA-Al dikarakterisasi dengan difraktometer sinar-X. Uji kemampuan lempung terpilar CTMA-Al digunakan sebagai adsorben terhadap larutan indigo karmina. Analisis larutan indigo karmina sebelum dan setelah adsorpsi dilakukan menggunakan spektrofotometer UV-Vis. Hasil difraksi sinar-X menunjukkan bahwa penambahan surfaktan CTMA-Br dan polikation Al mampu meningkatkan ukuran basal spacing lempung alam dari 15,46 Å menjadi 19,99 Å, tetapi kristalinitas lempung terpilar CTMA-Al yang disintesis menurun. Lempung terpilar CTMA-Al 0,5 gram mampu menyerap warna dengan baik sampai dengan 125 mL indigo karmina (8,58x10-5 M). Sedangkan pada lempung alam kemampuan adsorpsiya jauh lebih kecil daripada lempung terpilar CTMA-Al, yaitu dengan volume larutan indigo karmina yang sama (15 mL) lempung alam mempunyai kemampuan adsorpsi 37,07 %, dan lempung terpilar CTMA-Al mempunyai kemampuan adsorpsi 99,56 %