Development of plasmid and oligonucleotide nanometric particles

Nucleic acids delivery vectors have shown promising therapeutic potential in model systems. However, comparable clinical success is delayed essentially because of their poor biodistribution and of their ineffective intracellular trafficking. The size of condensed DNA particles is a key determinant for in vivo diffusion, as well as for gene delivery to the cell nucleus. Towards this goal, we have developed cationic thiol-detergents that individually compact plasmid DNA molecules into anionic particles. These particles are then ‘stabilized’ by air-induced dimerization of the detergent into a disulfide lipid on the template DNA. The particles all measure approximately 30 nm, which corresponds to the volume of a single molecule of plasmid DNA. The gel electrophoretic mobility of the anionic particles was found to be higher than that of the plasmid DNA itself. Similarly, particles formed with a 31-mer oligonucleotide measured 19 nm. Improved in vivo diffusion, as well as improved intracellular trafficking may be inferred from the faster migration of the complexes. Moreover, the size of the particles remains compatible with nuclear pore crossing. Finally, in an attempt to improve the biodistribution of these particles, we have coated the monomolecular particles with a poly(ethylene glycol) corona

Recycling High-Density Polyethylene (HDPE) into construction materials as a key step in plastic waste reduction: case of Kigali City

Plastic wastes are increasing at an alarming rate and adversely affecting environment as they are not easily degraded. Worse still, roads along with parking yards have significantly shorter design life and so do other alternative road paving techniques such as reinforced concrete slabs and concrete paving blocks (developing countries). Owing to these challenges, this research aimed to minimize the quantity of plastic wastes dumped in Kigali landfills by recycling it into construction materials while considering both economic and environmental benefits. Specific objectives included to maximize the use of post-consumer plastic waste and produce alternative eco-friendly building products.Similarly, the research aimed at determining the melting point and subsequent effects of temperature on high density polyethylene (HDPE) as well as determining the mix ratio of the material components that gives the highest compressive strength. Finally, the research aimed at assessing the performance of paving material made of sand and plastic wastes.In order to achieve the stated objectives, three samples per data point were prepared and quantity of sand content was varying by 1:3; 1:4; 1:5 respectively while keeping plastic content constant. The samples were placed in mould of dimension 100,65 mm in diameter and height respectively where they cooled and set. Compaction was done using standard method. Afterwards, the respective samples were tested for compressive strength and water absorption. Compressive strength test showed the values of 21.73 N/mm2, 26.15 N/mm2, 4.79 N/mm2 before heat exposure and 17.79 N/mm2, 22.37 N/mm2, 3.52 N/mm2 after exposure to 350C for 12 hours for the pavers in HDPE and sand mix ratio of 1:3, 1:4 and 1:5 respectively. Water absorption test showed an average value of 0.052% which is lower compared to the cement concrete made pavers.The research concluded that if made and put into use, these pavers will not only reduce construction costs especially those for repairs, but also assist in environmental conservation. Roads and parking yards will be cheaply constructed, and with the increased durability, accessibility will be improved and economic growth boostered.Keywords: HDPE, wastes, recycle, paver, compressive strengt

Characterization and isolation of a T-DNA tagged banana promoter active during in vitro culture and low temperature stress

<p>Abstract</p> <p>Background</p> <p>Next-generation transgenic plants will require a more precise regulation of transgene expression, preferably under the control of native promoters. A genome-wide T-DNA tagging strategy was therefore performed for the identification and characterization of novel banana promoters. Embryogenic cell suspensions of a plantain-type banana were transformed with a promoterless, codon-optimized luciferase (<it>luc</it>⁺) gene and low temperature-responsive luciferase activation was monitored in real time.</p> <p>Results</p> <p>Around 16,000 transgenic cell colonies were screened for baseline luciferase activity at room temperature 2 months after transformation. After discarding positive colonies, cultures were re-screened in real-time at 26°C followed by a gradual decrease to 8°C. The baseline activation frequency was 0.98%, while the frequency of low temperature-responsive luciferase activity was 0.61% in the same population of cell cultures. Transgenic colonies with luciferase activity responsive to low temperature were regenerated to plantlets and luciferase expression patterns monitored during different regeneration stages. Twenty four banana DNA sequences flanking the right T-DNA borders in seven independent lines were cloned <it>via </it>PCR walking. RT-PCR analysis in one line containing five inserts allowed the identification of the sequence that had activated luciferase expression under low temperature stress in a developmentally regulated manner. This activating sequence was fused to the <it>uidA </it>reporter gene and back-transformed into a commercial dessert banana cultivar, in which its original expression pattern was confirmed.</p> <p>Conclusion</p> <p>This promoter tagging and real-time screening platform proved valuable for the identification of novel promoters and genes in banana and for monitoring expression patterns throughout <it>in vitro </it>development and low temperature treatment. Combination of PCR walking techniques was efficient for the isolation of candidate promoters even in a multicopy T-DNA line. Qualitative and quantitative GUS expression analyses of one tagged promoter in a commercial cultivar demonstrated a reproducible promoter activity pattern during <it>in vitro </it>culture. Thus, this promoter could be used during <it>in vitro </it>selection and generation of commercial transgenic plants.</p

Dimerizable Cationic Detergents with a Low cmc Condense Plasmid DNA into Nanometric Particles and Transfect Cells in Culture

The size of condensed DNA particles is a key determinant for in vivo diffusion and gene delivery to cells. Gene molecules can be individually compacted by cationic thiol detergents into nanometric particles that are stabilized by oxidative conversion of the detergent into a gemini lipid. To reach the other goal, gene delivery, a series of cationic thiol detergents with various chain lengths (C12−C16) and headgroups (ornithine or spermine) was prepared, using a versatile polymer-supported synthetic strategy. Critical micelle concentrations and thiol oxidation rates of the detergents were measured. The formation and stability of complexes formed with plasmid DNA, as well as the size, ξ-potential, morphology, and transfection efficiency of the particles were investigated. Using the tetradecane/ornithine detergent, a solution of 5.5 Kpb plasmid DNA molecules was converted into a homogeneous population of 35 nm particles. The same detergent, once oxidized, exhibited a typical lipid phase internal structure and was capable of effective cell transfection. The particle size did not increase with time. Surprisingly, the gel electrophoretic mobility of the DNA complexes was found to be higher than that of plasmid DNA itself. Favorable in vivo diffusion and intracellular trafficking properties may thus be expected for these complexes

Intracellular Delivery of Nanometric DNA Particles via the Folate Receptor

The size of condensed DNA particles is a key determinant for both diffusion to target cells in vivo and intracellular trafficking. The smallest complexes are obtained when each DNA molecule collapses individually. This was achieved using a designed cationic thiol-detergent, tetradecyl-cysteinyl-ornithine (C14COrn). The resulting particles were subsequently stabilized by air-induced dimerization of the detergent into a disulfide lipid on the DNA template. Particles are anionic (zeta potential = −45 mV), and their size (30 nm) corresponds to the volume of a single plasmid DNA molecule. The electrophoretic mobility of the condensed DNA, though quasi-neutralized, was found higher than that of the extended DNA. Moreover, the dimerized (C14COrn)2 lipid was found to be an efficient transfection reagent for various cell lines. In an attempt to achieve extended circulation times and to target tumors by systemic delivery, we have coated the particles with PEG−folate residues. Plasmid DNA was condensed into monomolecular particles as described above and coated by simple mixing with DPPE−PEG−folate. Physicochemical measurements showed particles coated with 2% of DPPE−PEG3400−folate remain monomolecular and are stable in the cell-culture medium. Caveolae-mediated cell entry was demonstrated by ligand-dependence, by competition with excess folic acid as well as by confocal microscopy

Systemic linear polyethylenimine (L‐PEI)‐mediated gene delivery in the mouse

Background Several nonviral vectors including linear polyethylenimine(L‐PEI) confer a pronounced lung tropism to plasmid DNA when injected into the mouse tail vein in a nonionic solution. Methods and results We have optimized this route by injecting 50 µg DNA with excess L‐PEI (PEI nitrogen/DNA phosphate=10) in a large volume of 5% glucose (0.4 ml). In these conditions, 1–5% of lung cells were transfected (corresponding to 2 ng luciferase/mg protein), the other organs remaining essentially refractory to transfection (1–10 pg luciferase/mg protein).β‐Galactosidase histochemistry confirmed alveolar cells, including pneumocytes, to be the main target, thus leading to the puzzling observation that the lung microvasculature must be permeable to cationic L‐PEI/DNA particles of ca 60 nm. A smaller injected volume, premixing of the complexes with autologous mouse serum, as well as removal of excess free L‐PEI, all severely decreased transgene expression in the lung. Arterial or portal vein delivery did not increase transgene expression in other organs. Conclusions These observations suggest that effective lung transfection primarily depends on the injection conditions: the large nonionic glucose bolus prevents aggregation as well as mixing of the cationic complexes and excess free L‐PEI with blood. This may favour vascular leakage in the region where the vasculature is dense and fragile, i.e. around the lung alveoli. Cationic particles can thus reach the epithelium from the basolateral side where their receptors (heparan sulphate proteoglycans) are abundant

The p21 cip1/waf1 cyclin-dependent kinase inhibitor enhances the cytotoxic effect of cisplatin in human ovarian carcinoma cells

The seriousness of ovarian cancer, which is related to the observed link between recurrency and cell cycle control defect, prompted us to explore the effect of ectopic expression of the cdk inhibitor p21cip1/waf1 on ovarian carcinoma chemosensitivity. The transfection of p21cip1/waf1 cDNA into SKOV3 and OVCAR3 cells led to reduction of tumor cell growth, enhanced susceptibility to cisplatin-induced apoptosis, and abolition of recurrency after cisplatin exposure. p21cip1/waf1 gene transfer allowed a marked reduction of the cisplatin concentration needed to erradicate the tumor cell population. These results suggest exploring the possible use of p21cip1/waf1 as an adjunctive to conventional chemotherapy

Protective copolymers for nonviral gene vectors: synthesis, vector characterization and application in gene delivery

Uncontrolled interactions of gene vectors and drug carriers in and with an in vivo environment pose serious limitations to their applicability. In order to reduce such interactions we have designed, synthesized and applied novel copolymers of poly(ethylene glycol) and reactive linkers which are derivatized with anionic peptides after copolymerization. The anionic copolymer derivatives are used to coat positively charged nonviral gene vectors by electrostatic interactions. The copolymer coat confers to polyelectrolyte colloids of DNA and polycations steric stabilization in their minimal size and prevents salt- and serum albumin-induced aggregation. Furthermore, complement activation and the interaction with serum proteins are drastically reduced or abolished in contrast to unprotected DNA complexes. The designed vectors are compatible with the intracellular steps of gene delivery and can even enhance transfection efficiency as demonstrated with various adherent and nonadherent cell lines in culture. The synthetic concept is amenable to the principles of combinatorial chemistry and the copolymeric products may be applicable beyond gene delivery in targeted drug delivery