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

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

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. ..

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

Biomaterials in RNAi therapeutics: quo vadis?

The fifteen years of utilizing RNAi present a surprise story, ranging from the unexpected discovery and publication of RNA interference in 1998, rewarded by the nobel prize in 2006, the introduction of synthetic short siRNAs for the specific gene silencing in mammalian cells in 2001, or the discovery of more than 1600 human microRNAs naturally regulating about one third of our genes. Therapeutic applications started amazingly fast and resulted in the first recent successes in therapy. Synthetic siRNAs are under evaluation for knocking down disease-associated target mRNAs, microRNA mimics for turning on or antagonists (antagomirs) for turning off microRNA activity. Modified oligonucleotides comprise a special class of therapeutics with a new chemical profile; the precise synthetic molecules are much smaller than protein or gene vector drugs, but they are larger than conventional drugs and thus cannot passively diffuse into their target cells. The main current strategies for solving the delivery problem are discussed. We now face the interesting question of alternative future directions: should oligonucleotide molecules be chemically further minimized into small drug-like chemical entities? Or should multiple RNAi molecules be wrapped up into larger virus-like nanoparticles for delivery? Biomaterials in therapeutic RNA interference, quo vadis

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

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