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Manfred Ogris

Publications and source records attributed to Manfred Ogris.

28 records · Page 2Linked to original sources
PubMed2003

Nanoparticles bearing polyethyleneglycol-coupled transferrin as gene carriers: preparation and in vitro evaluation.

The aims of this work were to determine the stability of pDNA against various conditions during microencapsulation, prepare transferrin (TF)-conjugated PEGylated polycyanoacrylate nanoparticles (TF-PEG-nanoparticles), and assess its physicochemical characteristics and in vitro targeting cells association. The open circular forms of pDNA obviously increased when pDNA was emulsified into organic solution under sonification. When pDNA solution (pH 7.0) contained 1, 3 or 5% (w/v) PVA, after sonification, average 48.2, 59.4 and 62.1% of double-supercoiled DNA (dsDNA) were preserved, respectively. When medium of pDNA was 0.9% NaCl (pH 7.0), 0.1M NaHCO(3) (pH 8.0) or phosphate buffer (pH 8.0), average 53.1, 69.3 and 56.9% of dsDNA remained after sonification, respectively. Poly(aminopoly(ethylene glycol)cyanoacrylate-co-hexadecyl cyanoacrylate) (poly(H(2)NPEGCA-co-HDCA)) showed a slight influence on pDNA in 0.1M NaHCO(3) (pH 8.0) when its concentration increased from 0.5 to 4% (w/v). TF-PEG-nanoparticles loading pDNA were spherical in shape with size under 200nm and entrapment efficiency 35-50%. 0.1M NaHCO(3) with 3% PVA (w/v) could largely reduce the damage of pDNA during microencapsulation. TF-PEG-nanoparticles bore 1-3% of the total PEG chains conjugated to TF molecules, and exhibited the burst effect with over 30% drug release within 1 day. After the first phase, pDNA release profiles displayed a sustained release. The amount of cumulated pDNA release over 7 days was: 86.3, 81.5 and 74.4% for 1, 2 and 4% polymer nanoparticles, respectively. The degree of target K562 cell binding of TF-PEG-nanoparticles was greater than that of non-targeted PEG-nanoparticles at 4 degrees C. The presence of free TF decreased significantly the degree of cell binding of TF-PEG-nanoparticles, which revealed that the binding of TF-PEG-nanoparticles to K562 cells was indeed receptor specific. These results suggested that TF-PEG-nanoparticles were useful for delivery of pDNA to target cells.

Buffers↗
PubMed2003

Failure to generate atheroprotective apolipoprotein AI phenotypes using synthetic RNA/DNA oligonucleotides (chimeraplasts).

BACKGROUND: Elevated plasma high-density lipoprotein (HDL), and its major constituent apolipoprotein AI (apoAI), are cardioprotective. Paradoxically, two natural variants of apoAI, termed apoAI(Milano) and apoAI(Paris), are associated with low HDL, but nevertheless provide remarkable protection against heart disease for heterozygous carriers and may even lead to longevity. Both variants arise from point mutations and have Arg(173) and Arg(151) to Cys substitutions, respectively, which allow disulphide-linked dimers to form. Potentially, synthetic RNA/DNA oligonucleotides (chimeraplasts) can permanently correct single point mutations in genomic DNA. Here, we use a variation of such targeted gene repair technology, 'gain-of-function chimeraplasty', and attempt to enhance the biological activity of apoAI by altering a single genomic base to generate the atheroprotective phenotypes, apoAI(Milano) and apoAI(Paris). METHODS: We targeted two cultured cell lines that secrete human apoAI, hepatoblastoma HepG2 cells and recombinant CHO-AI cells, using standard 68-mer chimeraplasts with polyethyleneimine (PEI) as carrier and then systematically varied several experimental conditions. As a positive control we targeted the dysfunctional APOE2 gene, which we have previously converted to wild-type APOE3. RESULTS: Conversion of wild-type apoAI to apoAI(Milano) proved refractory, with limited correction in CHO-AI cells only. However, a successful conversion to apoAI(Paris) was achieved, as demonstrated by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis and direct genomic sequencing. Unexpectedly, attempts with a new batch of 68-mer chimeraplast to enhance conversion, by using different delivery vehicles, including chemically modified PEI, failed to show a base change; nor could conversion be detected with an 80-mer or a 52-76-mer series. In contrast, when a co-culture of CHO-E2 and CHO-AI cells was co-targeted, a clear conversion of apoE2 to apoE3 was seen, whereas no apoAI(Paris) could be detected. When the individual chimeraplasts were analysed by denaturing electrophoresis only the active apoE2-to-E3 chimeraplast gave a sharp band. CONCLUSIONS: Our findings suggest that different batches of chimeraplasts have variable characteristics and that their quality may be a key factor for efficient targeting and/or base conversion. We conclude that, although an evolving technology with enormous potential, chimeraplast-directed gene repair remains problematical.

Animals↗
PubMed2002

Targeting tumors with non-viral gene delivery systems.

Targeting therapeutic genes to tumors is an attractive concept in curing malignant diseases. Systemic gene delivery systems are needed for therapeutic applications in which the target cells are not directly accessible, and which can only be reached via the systemic route. Recent developments in the field of non-viral gene delivery have shown that, based on (poly)cationic carrier molecules, DNA can be efficiently targeted to tumors via the bloodstream. Tailor-made synthetic vectors can be used to achieve predominant gene expression in tumor tissue. Therapeutic concepts based, for example, on suicide genes or cytokines, showed encouraging results in preclinical and also in first clinical evaluations.

Animals↗
PubMed2002

Importance of lateral and steric stabilization of polyelectrolyte gene delivery vectors for extended systemic circulation.

Gene therapy for systemic diseases requires intravenous administration, but existing vectors are not suitable for systemic delivery, often showing rapid elimination from the bloodstream that restricts potential transfection sites to "first-pass" organs. To develop long-circulating vectors, here we have compared polyplexes containing DNA and poly-L-lysine (PLL) or polyethylenimine (PEI), surface-modified with either monovalent polyethylene glycol (PEG) or multivalent copolymers of N-(2-hydroxypropyl)methacrylamide (PHPMA), correlating their biophysical properties with their distribution following intravenous injection. A key difference between the two types of coating is the introduction of lateral stabilization by surface attachment of multivalent PHPMA, in addition to the steric stabilization provided by both types of polymers. The alpha-half-life for bloodstream clearance of polycation/DNA polyplexes (typically <5 minutes in mice) could be extended using multivalent PHPMA coating to >90 minutes. We found that the dose administered, as well as the amount and molecular weight of the coating PHPMA, had important effects on circulation properties. Multivalent PHPMA coating allows, for the first time, considerably extended circulation time using polyplex systems-a prerequisite for systemic gene delivery.

Acrylamides↗
PubMed2002

Tumor-targeted gene transfer with DNA polyplexes.

Systemic gene delivery systems are needed for therapeutic applications; in some situations, target cells might be spread throughout the organism, as in the case of cancer metastases, which can be reached only via the systemic route. Within the class of nonviral vectors, polymer-based transfection particles named DNA polyplexes and lipid-based systems named DNA lipoplexes are being developed for this purpose. For systemic circulation, masking the surface charge of DNA complexes has to be accomplished to avoid interactions with plasma components, erythrocytes, and the reticuloendothelial system. Among other vector formulations, polyplexes based on polyethylenimine (PEI), shielded with polyethylene glycol (PEG), and linked to the receptor binding ligands transferrin (Tf) or epidermal growth factor (EGF) have been developed. Complexes were found to mediate efficient gene transfer into tumor cell lines in a receptor-dependent and cell-cycle-dependent manner. Systemic administration of surface-shielded Tf-PEI polyplexes into the tail vein of mice resulted in preferential gene delivery into distantly growing subcutaneous tumors. In contrast, application of positively charged PEI polyplexes directed gene transfer primarily to the lung.

Animals↗
PubMed2002

Development of long-circulating polyelectrolyte complexes for systemic delivery of genes.

Extended systemic circulation of vectors for gene delivery has long been a goal for cancer gene therapy. In recent years, several groups have evaluated the feasibility of gaining extended circulation of polyplex vectors by endowing them with steric stabilisation, resulting from the presence of a surface layer of hydrophilic polymer. Although this approach has been in itself mainly unsuccessful, remarkable progress has been gained when steric stabilisation has been combined with "lateral stabilisation", using a range of agents to crosslink the surface of the complexes. Lateral stabilisation has been achieved using low molecular weight crosslinking agents, and also using multivalent polymers designed for cooperative surface attachment to the polyplex. In this way plasma clearance kinetics can be substantially extended, with a half lives for clearance in excess of 90 min. In order to enable transgene expression within target cells it can be necessary for the vectors to undergo triggered activation following arrival at the target site. The most promising way to achieve this is by reductive activation of the vector within the cytoplasm or nucleus of the target cell. State of the art in this rapidly progressing field is reviewed.

Animals↗
PubMedDate not supplied

Novel shielded transferrin-polyethylene glycol-polyethylenimine/DNA complexes for systemic tumor-targeted gene transfer.

Tumor-targeting DNA complexes which can readily be generated by the mixing of stable components and freeze-thawed would be very advantageous for their subsequent application as medical products. Complexes were generated by the mixing of plasmid DNA, linear polyethylenimine (PEI22, 22 kDa) as the main DNA condensing agent, PEG-PEI (poly(ethylene glycol)-conjugated PEI) for surface shielding, and Tf-PEG-PEI (transferrin-PEG-PEI) to provide a ligand for receptor-mediated cell uptake. Within the shielding conjugates, PEG chains of varying size (5, 20, or 40 kDa) were conjugated with either linear PEI22 (22 kDa) or branched PEI25 (25 kDa). The three polymer components were mixed together at various ratios with DNA; particle size, surface charge, in vitro transfection activity, and systemic gene delivery to tumors was investigated. In general, increasing the proportion of shielding conjugate in the complex reduced surface charge, particle size, and in vitro transfection efficiency in transferrin receptor-rich K562 cells. The particle size or surface charge of the complexes containing the PEG-PEI conjugate did not significantly change after freeze-thawing, while complexes without the shielding conjugate aggregated. Complexes containing PEG-PEI conjugate efficiently transfected K562 cells after freeze-thawing. Furthermore the systemic application of freeze-thawed complexes exhibited in vivo tumor targeted expression. For complexes containing the luciferase reporter gene the highest expression was found in tumor tissue of mice. An optimum formulation for in vivo application, PEI22/Tf-PEG-PEI/PEI22-PEG5, containing plasmid DNA encoding for the tumor necrosis factor (TNF-alpha), inhibited tumor growth in three different murine tumor models. These new DNA complexes offer simplicity and convenience, with tumor targeting activity in vivo after freeze-thawing.

Animals↗
PubMedDate not supplied

Temperature dependent gene expression induced by PNIPAM-based copolymers: potential of hyperthermia in gene transfer.

The objective of this work was to obtain gene delivery vectors with high efficiency induced by application of local hyperthermia. As a building construct for the polyplex particles, block copolymers were used, in which one block represents poly(ethyleneimine) (PEI) and another block a statistical copolymer of poly(N-isopropylacryamide) (PNIPAM) and different hydrophilic monomers (acrylamide or vinylpyrrolidinone). The block copolymers were synthesizized by radical polymerization of the corresponding monomers directly onto PEI. The complexation of DNA with these copolymers led to small, charge neutral particles, which aggregated upon increasing the temperature from 37 degrees C to 42 degrees C. This aggregation was found to be responsible for the enhanced transfection efficiency of these formulations under hyperthermic conditions. Gene expression in cells treated by hyperthermia was found to be nearly 2 orders of magnitude higher in comparison to cells transfected at physiological temperature. The mechanism by which hyperthermia influences the gene transfection efficiency is proposed.

Acrylic Resins↗
PubMedDate not supplied

Gene carriers based on hexanediol diacrylate linked oligoethylenimine: effect of chemical structure of polymer on biological properties.

Two biodegradable polycations based on hexanediol diacrylate linked oligoethylenimine (OEI) were synthesized by applying different reaction temperatures, 20 degrees C (LT-OEI-HD) and 60 degrees C (HT-OEI-HD). Their structural properties were analyzed by NMR, FTIR, and SEC/MALLS (size exclusion chromatography coupled with multiangle laser light scattering detection). Reaction temperature strongly influenced molecular weight and ester/amide ratio and thus resulted in polycations with different biological activities and degradation profiles. LT-OEI-HD was an ester-based polycation of 8.7 kDa which degraded rapidly at pH 7 and pH 9 respectively. HT-OEI-HD had a molecular weight of 26.6 kDa, was mainly based on amides, and degraded more slowly than LT-OEI-HD. Both polymers mediated gene transfer as efficiently as linear polyethylenimine of 22 kDa in two cell lines while being less toxic at their optimal conjugate/plasmid (C/P) ratios. LT-OEI-HD needed higher C/P ratios for gene delivery; however, it was significantly less toxic than HT-OEI-HD.

Absorbable Implants↗