Nucleic Acid Carriers Based on Precise Polymer Conjugates

Polymer polydispersity, random conjugation of functional groups, and poorly understood structure–activity relationships have constantly hampered progress in the development of nucleic acid carriers. This review focuses on the synthetic concepts for the generation of precise polymers, site-specific conjugation strategies, and multifunctional conjugates for nucleic acid transport. Dendrimers, defined peptide carriers, sequence-defined polyamidoamines assembled by solid-phase supported synthesis, and precise lipopeptides or lipopolymers have been characterized for pDNA and siRNA delivery. Conjugation techniques such as click chemistries and peptide ligation are available for conjugating polymers with functional transport elements such as targeting or shielding domains and for direct covalent modification of therapeutic nucleic acids in a site-specific mode

Novel Fmoc-Polyamino Acids for Solid-Phase Synthesis of Defined Polyamidoamines

A versatile solid-phase approach to sequence-defined polyamidoamines was developed. Four different Fmoc-polyamino acid building blocks were synthesized by selective protection of symmetrical oligoethylenimine precursors followed by introduction of a carboxylic acid handle using cyclic anhydrides and subsequent Fmoc-protection. The novel Fmoc-polyamino acids were used to construct polyamidoamines demonstrating complete compatibility to standard Fmoc reaction conditions. The straightforward synthesis of the building blocks and the high efficiency of the solid-phase coupling reactions allow the versatile synthesis of defined polycations

Ein Trockenmauerring am Südosthang des großen Feldberges im Taunus

Am 26. August 1916 fielen mir südöstlich des Feldberggipfels, in etwa 650 m Meereshöhe, an der in dem Meßtischblattausschnitt auf der Beilage mit einem Pfeil bezeichneten Stelle, die Reste eines eigenartigen, bisher nirgends beschriebenen Bauwerks auf, dessen Grundriß aus der Skizze auf der Beilage ersichtlich ist. Das Terrain, auf dem die Anlage steht, ist eben, aber etwa im Verhältnis von 1:10 nach Südosten geneigt. Es handelt sich um einen teilweise zerstörten elliptischen Steinring von etwa 1,2 ha Flächeninhalt, der aus rohen, aber auffällig regelmäßig gebrochenen Quarzitplatten und Blöcken, ohne Mörtel und ohne erkennbare Verbandlagerung errichtet, sich in seinen besterhaltenen Teilen noch heute bis zu 50 cm über dem Erdboden erhebt. ..

Effective incorporation of 2'-O-methyl-oligoribonucleotides into liposomes and enhanced cell association through modification with thiocholesterol

Cholesterol was linked to 2'-O-methyl-oligoribonucleotides (2'-OMe-RNA) via a disulfide bond by reacting the 3'-(pyridyldithio)-modified 2'-OMe-RNA with thiocholesterol in dichloromethane-methanol solution. This ligation reaction was made possible by a novel strategy in which the highly charged oligonucleotide was rendered soluble in nonaqueous solvent through conversion to a lipophilic amidinium salt. The biodegradable lipophilic modification of 2'-OMe-RNA resulted in a large increase in incorporation of such oligonucleotides into liposomes prepared by reversephase evaporation. Furthermore, association of these modified oligonucleotides with cultured TIB 73 cells was 100-fold higher than that seen with unmodified 2'-OMe-RNA in serum-free medium and about 10 to 30-fold higher in the presence of 10% calf serum. During incubation with cells, release of the internalized oligonucleotide from the thiocholesteryl moiety can be demonstrated

miR-200c sensitizes breast cancer cells to doxorubicin treatment by decreasing TrkB and Bmi1 expression.

Acquired resistance to classical chemotherapeutics is a major obstacle in cancer treatment. Doxorubicin is frequently used in breast cancer therapy either as single-agent or in combination with other drugs like docetaxel and cyclophosphamide. All these chemotherapies have in common that they are administered sequentially and often result in chemoresistance. Here, we mimicked this pulse therapy of breast cancer patients in an in vitro cell culture model, where the epithelial breast cancer cell line BT474 was sequentially treated with doxorubicin for several treatment cycles. In consequence, we obtained chemoresistant cells displaying a mesenchymal-like phenotype with decreased levels of miR-200c. To investigate the involvement of miR-200c in resistance formation, we inhibited and overexpressed miR-200c in different cell lines. Thereby, the cells were rendered more resistant or susceptible to doxorubicin treatment. Moreover, the receptor tyrosine kinase TrkB and the transcriptional repressor Bmi1 were identified as miR-200c targets mediating the drug resistance. Hence, we provide a mechanism of acquired resistance to doxorubicin that is caused by the loss of miR-200c. Along with this, our study demonstrates the complex network of microRNA mediated chemoresistance highlighting the challenges in cancer therapy and the importance of novel microRNA-modulating anticancer agents

Lipopolysaccharide is a frequent contaminant of plasmid DNA preparations and can be toxic to primary cells in the presence of adenovirus

Endotoxin (lipopolysaccharide, LPS) is commonly found as a contaminant in plasmid DNA preparations. We demonstrate here that the quantities of LPS typically contaminating DNA preparations can generate a toxicity to primary cells (primary human skin fibroblasts, primary human melanoma cells) in the presence of entry-competent adenovirus particles. Toxicity can be observed with as little as 100 ng/ml free LPS or 100 pg/ml LPS when the LPS is assembled into polylysine/adenovirus complexes. Simple and effective methods of removing the contaminating LPS using either a polymyxin B resin or Triton X-114 extraction are described. Treatment of DNA samples to remove LPS eliminates the toxicity to primary cells

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

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.

Simple Modifications of Branched PEI Lead to Highly Efficient siRNA Carriers with Low Toxicity

Polymer carriers like PEI which proved their efficiency in DNA delivery were found to be far less effective for the applications with siRNA. In the current study, we generated a number of nontoxic derivates of branched PEI through modification of amines by ethyl acrylate, acetylation of primary amines, or introduction of negatively charged propionic acid or succinic acid groups to the polymer structure. The resulting products showed high efficiency in siRNA-mediated knockdown of target gene. In particular, succinylation of branched PEI resulted in up to 10-fold lower polymer toxicity in comparison to unmodified PEI. Formulations of siRNA with succinylated PEI were able to induce remarkable knockdown (80% relative to untreated cells) of target luciferase gene at the lowest tested siRNA concentration of 50 nM in Neuro2ALuc cells. The polyplex stability assay revealed that the efficiency of formulations which are stable in physiological saline is independent of the affinity of siRNA to the polymer chain. The improved properties of modified PEI as siRNA carrier are largely a consequence of the lower polymer toxicity. In order to achieve significant knockdown of target gene, the PEI-based polymer has to be applied at higher concentrations, required most probably for sufficient accumulation and proton sponge effects in endosomes. Unmodified PEI is highly toxic at such polymer concentrations. In contrast, the far less toxic modified analogues can be applied in concentrations required for the knockdown of target genes without side effects