Biomedical subjects

W T Godbey

Publications and source records attributed to W T Godbey.

9 recordsLinked to original sources

PubMed2003

Directed apoptosis in Cox-2-overexpressing cancer cells through expression-targeted gene delivery.

The principle of promoter-targeted gene delivery was used to direct the expression of reporter genes and inducible caspases to Cox-2-overexpressing cancer cells. The polycation poly(ethylenimine) was used in unmodified form to nonvirally deliver genes into cells, and targeting was achieved at the transcriptional level. Results demonstrated that reporter expression was reduced by an average of 89.8% in normal cells and cell lines not overexpressing Cox-2 when the strong cytomegalovirus promoter was replaced with the human Cox-2 promoter in delivered plasmids. Cocultures of normal and Cox-2-overexpressing cancer cells showed less than 0.5% reporter expression in normal fibroblast cells but over 35% reporter expression in PC3 prostate cancer cells. This targeting method was then used to direct the expression of inducible forms of caspases 3 and 9 to Cox-2-overexpressing cancer cells of the bladder and prostate. Following activation of the resulting caspase pro-forms, cells underwent apoptosis as evidenced by DNA fragmentation and cytoskeletal degradation. This result was also observed in cells resistant to apoptosis in terms of TNF-alpha initiation. Such directed apoptosis could eventually serve as a treatment for an entire class of Cox-2-overexpressing carcinomas.

Apoptosis↗

PubMed2002

In vitro systems for tissue engineering.

Tissue engineering, by necessity, encompasses a wide array of experimental directions and scientific disciplines. In vitro tissue engineering involves the manipulation of cells in vitro, prior to implantation into the in vivo environment. In contrast, in vivo tissue engineering relies on the body's natural ability to regenerate over non-cell-seeded biomaterials. Cells, biomaterials, and controlled incubation conditions all play important roles in the construction and use of modern in vitro systems for tissue engineering. Gene delivery is also an important factor for controlling or supporting the function of engineered cells both in vitro and post implantation, where appropriate. In this review, systems involved in the context of in vitro tissue engineering are addressed, including bioreactors, cell-seeded constructs, cell encapsulation, and gene delivery. Emphasis is placed upon investigations that are more directly linked to the treatment of clinical conditions.

Animals↗

PubMed2001

Recent progress in gene delivery using non-viral transfer complexes.

The delivery of genetic material into cells is a field that is expanding very rapidly. Non-viral delivery methods, especially ones that focus on the use of chemical agents complexed with genetic material, are the focus of this mini-review. More-recent uses of known transfection agents such as poly(ethylenimine), poly(L-lysine), and various liposomes are discussed, and some novel approaches (both chemical and methodical) are reviewed as well. A very brief look at how non-viral gene delivery research is being aimed at the clinic is also included.

Animals↗

PubMed2001

Poly(ethylenimine)-mediated gene delivery affects endothelial cell function and viability.

Poly(ethylenimine) (PEI) was used to transfect the endothelial cell line EA.hy 926, and the secreted levels of three gene products, tissue-type plasminogen activator (tPA), plasminogen activator inhibitor type 1 (PAI-1), and von Willebrand Factor (vWF), were assessed via ELISA. We found that the levels of these gene products in cell supernatants increased by factors up to 16.3 (tPA), 8.3 (PAI-1), or 6.7 (vWF) times the levels recorded for untreated cells, and roughly correlated with the percentage of cells that expressed the reporter plasmid. Transfections carried out using promotorless constructs of the same reporter plasmid also yielded increases in tPA, PAI-1, and vWF to similar extents. Additionally, data regarding cell viability were gathered and found to inversely relate to both the effectiveness of the PEI used for transfection and the secreted levels of the three mentioned products. There appeared to be two distinct types of cell death, resulting from the use of either free PEI (which acts within 2 h) or PEI/DNA complexes (which cause death 7-9 h after transfection). Cells were also transfected by poly(L-lysine) and liposomal carriers, and increases in secreted tPA similar to those seen with PEI-mediated transfection were observed for positively transfected cells. The results of these investigations indicate that non-viral gene delivery can induce a state of endothelial cell dysfunction, and that PEI-mediated transfection can lead to two distinct types of cell death.

Cell Line↗

PubMed2000

Poly(ethylenimine)-mediated transfection: a new paradigm for gene delivery.

Poly(ethylenimine) (PEI) is a synthetic polycation that has been used successfully for gene delivery both in vitro and in vivo due, in theory, to a form of protection that is afforded to the carried plasmids. In this study the stability of PEI/DNA complexes was demonstrated using deoxyribonuclease (DNase) 1 and DNase 2, various levels of pH, and increasing exposure times. DNA that was complexed with PEI was not degraded when exposed to at least 25 Units of either enzyme for 24 h while uncomplexed forms of the same plasmid were digested when exposed to 0.010 Units of DNase 1 for 0.05 h or 0. 003 Units of DNase 2 for 1 h. For further comparison, the stability of complexes made with poly(L-lysine) (PLL) and DNA was examined and found to be lower than that of PEI/DNA complexes; PLL-complexed DNA was digested on exposure to 1.25 Units of DNase 1 for 3 min. Cells were transfected with PEI/DNA complexes and, by using a pH indicator and optical recording techniques, it was found that the normal lysosomal pH value of 5.0 was not altered, bringing into question PEI's hypothesized lysosomal entry. Confocal microscopy showed that PEI/DNA complexes and lysosomes do not merge during transfection (although PLL/DNA complexes do). The lack of lysosomal involvement in PEI-mediated transfection is surprising because it goes against the conventional wisdom that has attempted to explain how PEI functions during transfection. PEI forms a stable complex with DNA, which moves from endocytosis to nuclear entry without significant cellular obstacles.

Animals↗

PubMed1999

Poly(ethylenimine) and its role in gene delivery.

Since the first published examination of poly(ethylenimine) (PEI) as a gene delivery vehicle, there has been a flurry of research aimed at this polycation and its role in gene therapy. Here we will briefly review PEI chemistry and the characterization of PEI/DNA complexes used for gene delivery. Additionally, we will note various PEI transfection considerations and examine findings involving other polycationic gene delivery vehicles used with cellular targeting ligands. The current state of our knowledge regarding the mechanism of PEI/DNA transfection will also be discussed. Finally, we will survey toxicity issues related to PEI transfection.

DNA Adducts↗

PubMed1999

Size matters: molecular weight affects the efficiency of poly(ethylenimine) as a gene delivery vehicle.

Poly(ethylenimine) (PEI) samples of various molecular weights and pHs were used to transfect endothelial cells to achieve levels of gene expression for comparison. PEIs with nominal molecular weights of 600, 1200, 1800, 10,000, and 70,000 Da were examined at pHs of 5. 0, 6.0, 7.0, and 8.0, and the results were recorded in terms of transfection efficiencies at 24, 48, 68, 92, and 120 h post-transfection. Trials were performed on the human endothelial cell-derived cell line EA.hy 926. We found that, for the polymers tested, transfection efficiency increased as the molecular weight of PEI increased. Representative values of PEIs at pH 6 and molecular weight 70,000 produced average transfection efficiencies of 25.6 +/- 7.9% (n = 8) at the greatest average expression levels, while PEI of molecular weight 10,000 yielded efficiencies of only 11.4 +/- 1.7% (n = 6). Transfection efficiencies for molecular weight 1,800 PEI were essentially zero, and PEIs of lower molecular weights produced no transfection at all. In contrast, the pH of the PEI solutions had no discernible effect on transfection. Optimal expression of the green fluorescent protein reporter occurred between 2 and 3 days post-transfection. The amount of reporter expression also was noted, as determined by the brightness of fluorescing cells under UV. The data obtained demonstrate that the molecular weight of the PEI carrier has an effect on transfection efficiency while the pH of the PEI solutions prior to DNA complexation has no such effect.

Chromatography, Gel↗

PubMed1999

Tracking the intracellular path of poly(ethylenimine)/DNA complexes for gene delivery.

Poly(ethylenimine) (PEI) is one of a number of polycations that has been used successfully to transfer genes into living cells. Although PEI shows promise in the field of gene therapy, to date no rigorous proof of mechanism has been published regarding the fate of PEI/DNA administered for transfection. Here we show, by using fluorescent labeling and confocal microscopy, the paths of PEI/DNA complexes from endocytosis to gene expression. We found that complexes attach to cell surfaces and migrate into clumps that are endocytosed. The endocytotic vesicles grow in number and size and are occasionally seen to lyse. Most interesting is the fact that endocytosed PEI, whether administered with or without DNA, undergoes nuclear localization in the form of ordered structures.

Cell Line↗

PubMed1999

Improved packing of poly(ethylenimine)/DNA complexes increases transfection efficiency.

We have developed a modified poly(ethylenimine) (PEI) transfection procedure that significantly increases PEI's transfection efficiency. While the basic transfection procedure had a transfection efficiency of 37%, our modified procedure yielded a 53% transfection efficiency. The altered procedure gives improved results because of two simultaneous actions: free polycations are removed from the transfecting solutions, and the composition of the PEI complexes that are administered to cells has been modified. The reduction in the amount of free polycations in transfecting solutions reduced the toxicity sometimes associated with the administration of polycations to cellular environments. The structural modification of PEI/DNA transfecting complexes involves improved PEI packing around the delivered plasmid to yield a greater buffering capacity without a change in the complex's surface charge concentration. These structural properties were confirmed by titration and zeta potential analyses. Whether the modified PEI/DNA complexes are more effective because of increased cellular uptake or an enhanced ability to escape from endolysosomes has been addressed. The increase in transfection efficiency was obtained when the buffering capacity of the PEI/DNA was increased without a change in surface charge concentration, which implies that it is the property of enhanced lysosomal buffering that is responsible for successful PEI transfection.

Cells, Cultured↗