Biomedical subjects

David Oupický

Publications and source records attributed to David Oupický.

9 recordsLinked to original sources

PubMed2007

Synthesis of temperature-responsive heterobifunctional block copolymers of poly(ethylene glycol) and poly(N-isopropylacrylamide).

Heterobifunctional block copolymers of poly(ethylene glycol) (PEG) and poly(N-isopropylacrylamide) (PNIPAM) were synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization of NIPAM using a macromolecular trithiocarbonate PEG-based chain transfer agent. The polymerization showed all the expected features of living radical polymerization and allowed the synthesis of copolymers with different lengths of the PNIPAM block. The synthesized block copolymers contained a carboxylic acid group from L-lysine at the focal point and a trithiocarbonate group at the terminus of the PNIPAM block. The trithiocarbonate functionality was converted into a thiol group and used for conjugation of biotin to the end of the PNIPAM block. The copolymers exhibited temperature-dependent association behavior in aqueous solution with a phase transition of approximately 32 degrees C. The described heterobifunctional block copolymers show promise for surface modifications with the potential for stimulus-controlled surface presentation of ligands attached to the terminus of the PNIPAM block.

Acrylic Resins↗

PubMed2006

Disassembly of layer-by-layer films of plasmid DNA and reducible TAT polypeptide.

This paper reports the disassembly of layer-by-layer (LbL) films of plasmid DNA and a reducible cationic polypeptide. To utilize a reducing microenvironment of cellular plasma membrane as a potential trigger, LbL films are assembled to contain both DNA and the TAT-based polypeptide (PTAT) with reducible disulfide bonds in the backbone. The assembly and disassembly processes are monitored by goniometry, ellipsometry, and atomic force microscopy (AFM). The structure of the PTAT films is compared with that of non-reducible poly(L-lysine) (PLL) films. Both PTAT and PLL films exhibit exponential growth but with the contact angle alternating between characteristic values. Ellipsometry and AFM show a gradual and complete disassembly of the PTAT but not the PLL films in a 24h period in the reducing environment in vitro. This study suggests a potential of using reducible LbL films for controlled DNA delivery.

DNA↗

PubMed2006

Polyplex gene delivery modulated by redox potential gradients.

Polyplexes sensitive to redox potential gradients represent a promising class of vectors for delivery of nucleic acids. This review focuses on the recent advances in the development of these vectors. The biological rationale for the design of redox-sensitive polyplexes is discussed together with the basic synthetic approaches for introducing reducible disulfide bonds into the structure of the polyplexes. The biological properties of the redox-sensitive polyplexes of plasmid DNA, mRNA, antisense oligonucleotides and siRNA are reviewed with emphasis on in vitro cellular delivery, cytotoxicity and in vivo activity. Overall, redox-sensitive polyplexes represent a promising platform for further development as vectors for delivery of a wide variety of therapeutic nucleic acids.

Animals↗

PubMed2003

Vectors based on reducible polycations facilitate intracellular release of nucleic acids.

BACKGROUND: Inefficient intracellular delivery of nucleic acids limits the therapeutic usefulness of synthetic vectors such as poly(L-lysine) (PLL)/DNA polyplexes. This article reports on the characterisation of a new type of synthetic vector based on a linear reducible polycation (RPC) that can be cleaved by the intracellular environment to facilitate release of nucleic acids. METHODS: RPCs of molecular weight (mwt) 45 and 187 kDa were prepared by oxidative polycondensation of the peptide Cys-Lys(10)-Cys and used to condense nucleic acids. The stability of RPC-based polyplexes to reduction was determined using electrophoresis, dynamic light scattering and fluorescence techniques. Transfection activity was studied in several cancer cell lines (HeLa, LNCaP, PC-3 and B16-F10) using luciferase and green fluorescent protein (GFP) genes as reporter genes in the presence of chloroquine or the cationic lipid (N-(1-(2,3-dioleoyloxy)propyl)-N, N, N-trimethylammonium chloride) (DOTAP). A CMV-driven plasmid expressing the nitroreductase (ntr) gene was used to evaluate the therapeutic efficacy of RPC-based delivery vectors. RESULTS: A 187-fold higher level of gene expression indicated that intracellular delivery of DNA was more efficient using RPC/DOTAP compared with vectors based on non-reducible PLL. Analysis by flow cytometry also showed enhanced delivery of the GFP gene by RPC/DOTAP in HeLa (51.5 +/- 7.9%), LNCaP (55.2 +/- 6.7%) and PC-3 (66.1 +/- 3.7%) cells. Transfection with the ntr gene and treatment with the prodrug CB1954 resulted in significant cell killing, achieving IC(50) values similar to those previously attained with adenoviral vectors. Delivery of mRNA (20-75% of cells) was also more efficient using RPC/DOTAP than PLL/DOTAP (<5% of cells). CONCLUSIONS: These results demonstrate that lipid-mediated activation of RPC-based polyplexes is a useful strategy to enhance intracellular delivery of nucleic acids and potentiate therapeutic activity.

Gene Transfer Techniques↗

PubMed2003

Stimuli-responsive gene delivery vectors.

One of the major limitations to efficient gene delivery mediated by synthetic non-viral vectors is the presence of various barriers between the site of administration and localization in the cell nucleus. Each of the barriers often demands significantly different properties from the vector. Delivery vectors that can recognize changes in the environment and that are capable of actively responding by altering their properties or behavior are therefore being developed to address the challenge of integrating the sometimes contradictory characteristics necessary for effective gene delivery. This review discusses recent developments in the area of gene delivery vectors capable of responding to various endogenous and exogenous stimuli by changing their properties or behavior.

Acoustic Stimulation↗

PubMed2002

Physical properties and in vitro transfection efficiency of gene delivery vectors based on complexes of DNA with synthetic polycations.

Biophysical properties of polycation/DNA complexes designed for gene delivery were studied with respect to the conditions of their preparation, chemical structure and molecular weight of the polycations involved. The polycations used included a variety of cationic polymers and copolymers containing primary and tertiary amino or quaternary ammonium groups. It was found that the molecular weight and the size of these polyelectrolyte complexes (PECs) increase with increasing temperature and pH of the buffer. By decreasing the molecular weight of polycations used for PEC formation, the complexes become unstable towards coagulation in aqueous solution at lower pH. The self-assembly of DNA with low-molecular-weight polycations in water provides PECs with the lowest molecular weight, smallest size and the lowest density but their stability in NaCl solutions is very poor. Despite the complexity of the multistep transfection process, a direct correlation between the transfection efficiency in vitro and the stability of the complexes in NaCl solutions and coagulation in 0.15 M NaCl solution was found. DNA complexes with polycations containing primary amino groups showed the best stability in saline solutions and also the best transfection activity. PECs formed by polycations with quaternary ammonium groups were the least resistant to destruction by the added salt and provided the lowest activity in transfection assays. The highest transfection activity was found for DNA complexes formed with a statistical copolymer containing primary and tertiary amines.

Animals↗

PubMed2002

Laterally stabilized complexes of DNA with linear reducible polycations: strategy for triggered intracellular activation of DNA delivery vectors.

Target-specific DNA delivery requires vectors that combine stability in the biological milieu, receptor-mediated uptake into target cells, and intracellular activation to mediate transgene expression. This is achieved here using polymer-coated vectors based on plasmid DNA complexed with a reductively degradable polycation (RPC), designed for intercellular degradation. The RPC were prepared by oxidation of the terminal cysteinyl thiol groups of Cys(Lys)10Cys. The complexes were coated and surface-cross-linked using multivalent reactive copolymers of N-(2-hydroxypropyl)methacrylamide (PHPMA), providing a unique combination of steric and reversible lateral stabilization, known to promote extended circulation in the bloodstream. Coated complexes containing RPC exhibited lateral stabilization that was reversible by treatment with 2.5 mM dithiothreitol, releasing free DNA after incubation with a polyanion. In contrast, coated complexes containing nonreducible poly(l-lysine) (PLL) were not destabilized by reduction. The biological usefulness of this trigger mechanism was examined by measuring transfection activity in human retinoblast 911 cells of coated complexes, based on PLL or RPC, targeted to cell surface receptors by covalent linkage of basic fibroblast growth factor. The levels of transgene expression observed for RPC-based targeted vectors indicated efficient intracellular activation, authenticating the concept that lateral stabilization introduced by surface coating with PHPMA can be reversed by intracellular reduction.

Biotransformation↗

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

Multiblock reducible copolypeptides containing histidine-rich and nuclear localization sequences for gene delivery.

Polyplexes sensitive to redox potential gradients represent promising gene delivery vectors. High molecular weight polypeptides containing disulfide bonds in the backbone were synthesized by an oxidative copolymerization of a histidine-rich peptide (HRP) and a nuclear localization sequence (NLS) peptide. The synthetic approach allowed an easy synthesis of reducible copolypeptides (rCPP) with different relative contents of the HRP and NLS sequences. Cytotoxicity and transfection activity of rCPP-based DNA polyplexes were evaluated in vitro. In comparison with control polyethylenimine (PEI), only minimum toxic effects of rCPPs were observed on the metabolic activity and membrane integrity of human endothelial cells. These findings are predominantly ascribed to favorable structural features like lower charge density and higher chain rigidity of the rCPPs compared to PEI and also to a reductive intracellular and plasma membrane degradation. Transfection activity in all tested cell lines increased with increasing content of the HRP sequence in the rCPPs, while no clearly measurable effect of the NLS sequence was found.

Animals↗