Crispin R. Dass

Transcription factor Egr-1 supports FGF-dependent angiogenesis during neovascularization and tumor growth

Current understanding of key transcription factors regulating angiogenesis is limited. Here we show that RNA-cleaving phosphodiester-linked DNA-based enzymes (DNAzymes), targeting a specific motif in the 5′ untranslated region of early growth response (Egr-1) mRNA, inhibit Egr-1 protein expression, microvascular endothelial cell replication and migration, and microtubule network formation on basement membrane matrices. Egr-1 DNAzymes blocked angiogenesis in subcutaneous Matrigel plugs in mice, an observation that was independently confirmed by plug analysis in Egr-1-deficient animals, and inhibited MCF-7 human breast carcinoma growth in nude mice. Egr-1 DNAzymes suppressed tumor growth without influencing body weight, wound healing, blood coagulation or other hematological parameters. These agents inhibited endothelial expression of fibroblast growth factor (FGF)-2, a proangiogenic factor downstream of Egr-1, but not that of vascular endothelial growth factor (VEGF). Egr-1 DNAzymes also repressed neovascularization of rat cornea. Thus, microvascular endothelial cell growth, neovascularization, tumor angiogenesis and tumor growth are processes that are critically dependent on Egr-1.

Cytotoxicity issues pertinent to lipoplex-mediated gene therapy in-vivo

Cationic liposomes bind with nucleic acids such as plasmids and oligodeoxynucleotides to form complexes known as lipoplexes. Although these lipoplexes have several advantages over other forms of nucleic acid transfer methods in cell culture and in‐vivo, toxicity remains a problem, especially in‐vivo. Nevertheless, these carriers have been used in clinical trials against cystic fibrosis and cancer and their usage is attributed mainly to their versatility, especially when it comes to the range of routes available for administration of nucleic‐acid‐based drugs in‐vivo.

Vehicles for oligonucleotide delivery to tumours

The vasculature of a tumour provides the most effective route by which neoplastic cells may be reached and eradicated by drugs. The fact that a tumours vasculature is relatively more permeable than healthy host tissue should enable selective delivery of drugs to tumour tissue. Such delivery is relevant to carrier‐mediated delivery of genetic medicine to tumours. This review discusses the potential of delivering therapeutic oligonucleotides (ONs) to tumours using cationic liposomes and cyclodextrins (CyDs), and the major hindrances posed by the tumour itself on such delivery. Cationic liposomes are generally 100–200 nm in diameter, whereas CyDs typically span 1.5 nm across. Cationic liposomes have been used for the introduction of nucleic acids into mammalian cells for more than a decade. CyD molecules are routinely used as agents that engender cholesterol efflux from lipid‐laden cells, thus having an efficacious potential in the management of atherosclerosis. A recent trend is to employ these oligosaccharide molecules for delivering nucleic acids in cells both in‐vitro and in‐vivo. Comparisons are made with other ON delivery agents, such as porphyrin derivatives (< 1 nm), branched chain dendrimers (≈ 10 nm), polyethylenimine polymers (≈ 10 nm), nanoparticles (20–1000 nm) and microspheres (> 1 μm), in the context of delivery to solid tumours. A discourse on how the chemical and physical properties of these carriers may affect the uptake of ONs into cells, particularly in‐vivo, forms a major basis of this review.

Biochemical and biophysical characteristics of lipoplexes pertinent to solid tumour gene therapy

Cationic liposomes have become the reagent of choice for transfer of nucleic acids such as plasmids and oligodeoxynucleotides to cells in culture and in vivo. Whilst these reagents have several advantages over other forms of nucleic acid transfer methods, toxicity remains a significant problem, especially in vivo. Recent studies have also highlighted the immunostimulatory nature of these cationic vesicles when complexed to plasmid DNA, a phenomenon that may be harnessed for efficacious usage against tumours. Current research in this dynamic technological field is aimed at the development of cationic lipids that have negligible toxic effects and enhanced transfection capabilities.

Liposome-Mediated Delivery of Oligodeoxynucleotides In Vivo

Oligodeoxynuclotides (ODNs) are deoxyribonucleic-acid-based sequences showing therapeutic potential against such diseases as cancer, arteriosclerosis, arthritis, viral infections, and inflammation. Administration of these chemical entities using carriers has certain advantages over administration of free nucleic acid strands. Liposomes, one class of carriers, have been proven to be a popular choice for ODN delivery in vivo because of their potential to increase resistance of ODNs against nucleases and enhance circulation half-life. This result increases ODN uptake in target cells. Several liposomal formulations with proven potential for ODN delivery in vivo are discussed in this article.

Apolipoprotein A-I, Cyclodextrins and Liposomes as Potential Drugs for the Reversal of Atherosclerosis. A Review

Several studies have revealed that high‐density lipoprotein (HDL) is the most reliable predictor for susceptibility to cardiovascular disease. Since apolipoprotein A‐I (apoA‐I) is the major protein of HDL, it is worthwhile evaluating the potential of this protein to reduce the lipid burden of lesions observed in the clinic. Indeed, apoA‐I is used extensively in cell culture to induce cholesterol efflux. However, while there is a large body of data emanating from in‐vitro and cell‐culture studies with apoA‐I, little animal data and scant clinical trials examining the potential of this apolipoprotein to induce cholesterol (and other lipid) efflux exists. Importantly, the effects of oxysterols, such as 7‐ketocholesterol (7KC), on cholesterol and other lipid efflux by apoA‐I needs to be investigated in any attempt to utilise apoA‐I as an agent to stimulate efflux of lipids.

Particle-mediated intravascular delivery of oligonucleotides to tumors: associated biology and lessons from genotherapy.

For a solid tumor to become life-threatening, an adequate blood supply has to be established. Although neovascularization has dire consequences for the host, it furnishes a common route through which tumors may be accessed and eradicated by drugs. The fact that a tumors vasculature is relatively more permeable than that of healthy host tissue means selective delivery of drugs may be achieved. The role played by the cells making up the tumor vascular bed, vascular endothelial cells (VECs), has to be evaluated closely in attempts to design ways for enhancing drug delivery to solid tumors via the vasculature. The two major roles of VECs in the body, as barrier and as transport, are both highly pertinent to drug delivery. Our review examines how VECs may be manipulated in vivo to improve the selective delivery of carriers for oligonucleotide constructs to solid tumors. It also discusses how oligonucleotide drugs may be targeted against tumor VECs on the premise that by killing these cells, the tumor itself will perish. Cationic liposomes and microspheres are the major delivery vehicles discussed, with added analyses of such other nucleic acid carriers as nanospheres, dendrimers, and polyethyleneimine.

Delivery of Lipoplexes for Genotherapy of Solid Tumours: Role of Vascular Endothelial Cells

The cells constituting a solid tumour may vary considerably due to biological disparities, but for a solid tumour to pose as a threat to its host, an adequate blood supply has to be established. Although neovascularisation may have dire consequences for the host, it provides a common route by which tumours in general may be reached and eradicated by drugs. The fact that a tumours vasculature is relatively more permeable than healthy host tissue means that selective delivery of drugs may be achieved. A closer examination of the role played by the cells making up the tumour vascular bed, vascular endothelial cells (VECs), is required to facilitate design of ways for enhancing drug delivery to solid tumours via the vascular route.

A Microsphere-Lipoplex (Microplex) Vector for Targeted Gene Therapy of Cancer. I. Construction and In Vitro Evaluation

Plasmid DNA binding to cationic liposomes and the ability to bind these liposomes, both with and without complexed plasmid DNA, to cation-exchange microspheres were examined. The two plasmids tested were pCMV-CAT and pRcCMV-p53. Commercial Lipofectin, Lipofectace, Lipofectamine, and three formulation ratios of dimethyldioctadecyl ammonium bromide (DDAB):phosphatidylcholine and DDAB:dioleoylphosphatidyl ethanolamine liposomes were evaluated. The binding of empty liposomes onto microspheres increased and the release from microspheres decreased with increasing ratio of cationic:neutral lipid. Of all liposomes, Lipofectamine bound the most copy numbers of both plasmids. The amount of plasmid bound on the laboratory-formulated liposomes increased as the ratio of cationic:neutral lipid was increased. The amount of plasmid bound to the formulated liposomes was not affected by the type of neutral lipid used. On average, in terms of copy numbers, binding with pCMV-CAT was 1.38-fold higher than pRcCMV-p53. However,...

Cationic Liposomes and Gene Therapy for Solid Tumors

AbstractEfforts in treatment have concentrated on the development of an ideal carrier for effective delivery of therapeutic agents into affected regions of a human body. Ideally, drugs would have to be delivered as close as possible, if not within, the affected cell. From its seminal production 30 years ago for the purpose of membrane research, the liposome has passed through various stages of development to become a vehicle of choice for numerous therapeutic applications. One category of these vesicles, positively charged or cationic liposomes, are commonly used for transfer of reporter and therapeutic genes into both mammalian and nonmammalian cells both in vitro and in vivo. While cationic liposomes have many advantages over other forms of delivery mechanisms, several problems hinder their efficient use. Development of a better liposomal transfection agent may indeed require a closer look at the present cationic vesicles, the biological milieu to which they are exposed, mechanisms of membrane breaching...