Mark A. Burton

Analysis of the distribution of intra-arterial microspheres in human liver following hepatic yttrium-90 microsphere therapy

The microscopic distribution of microspheres in human liver following hepatic infusion of 32 microm diameter resin microspheres labelled with 90Y as treatment for an 80 millimetre diameter liver cancer has been investigated. Microspheres were found to deposit inhomogeneously in tissues, preferentially lodging in a region approximately 6 mm wide around the periphery of the tumour. A relative concentration of microspheres of 50 to 70 times that of normal hepatic parenchyma and 65 to 94 times that in the tumour centre was measured in this region. The deposition of spheres in the tumour periphery was not uniform, and cluster analysis showed that the spheres could be classified into clusters. The number of microspheres in a cluster was skewed towards low numbers and cluster sizes varied from 20 to 1500 microm. The observed deposition patterns indicate that the vascular tumour periphery will receive much greater radiation doses from radioactive microspheres than both normal tissue and the avascular tumour centre.

Tumour dosimetry in human liver following hepatic yttrium-90 microsphere therapy.

Radiation dose distributions arising from intrahepatic arterial infusion of 90Y microspheres have been investigated. Tissue samples from normal liver, the tumour periphery and tumour centre were taken from a patient following infusion of 3 GBq of 32 microm diameter resin microspheres labelled with 90Y as treatment for an 80 mm diameter metastatic liver tumour. The measured microsphere distributions in three dimensions were used to calculate radiation dose patterns. Although microspheres concentrated in the tumour periphery, heterogeneous doses were delivered to all tissues. Within the tumour periphery average doses ranged from 200 Gy to 600 Gy with minimum doses between 70 Gy and 190 Gy. The average and minimum doses for the tumour centre sample were 6.8 Gy and 3.7 Gy respectively. In the normal liver sample the average dose was 8.9 Gy with a minimum dose of 5 Gy. Less than 1% of the normal liver tissue volume received more than 30 Gy, the level above which complications have resulted for whole liver exposure using external beam radiotherapy. These calculations suggest that preferential deposition of microspheres in the well-vascularized periphery of large tumours will lead to a high proportion of the tumour volume receiving a therapeutic dose, with most of the normal liver tissue being spared substantial damage.

Enhanced Anticancer Therapy Mediated by Specialized Liposomes

It has been a central aim of experimental and clinical therapeutics to deliver therapeutic agents as close as possible to, or if possible within, a diseased cell. Such targeting achieves two major aims of drug delivery, the maximum dose of therapeutic agent to the diseased cell and avoidance of uptake by and, usually, accompanying side‐effects to normal, healthy cells.

Lipoplexes and tumours. A review

The need for genotherapy to refocus its attention on to laboratory evaluation of better methods rather than proceeding to the clinic with semi-apt tools for genetic transfer has been highlighted in clinical study reports documented to date. Quintessential for tumour genotherapy is the ability to target abnormal cells, hence reducing exposure of normal cells to genetic material whilst maximizing gene dosage to tumour cells. This becomes increasingly important as genotherapy establishes itself in the clinic alongside the older modes of treatment. This review has discussed the applicability of lipoplexes for genotherapy of solid tumours. Lipoplexes have been used extensively for gene transfer into cells, such as cancerous cells, deficient for a certain gene product. While cationic liposomes have many advantages over other forms of delivery mechanisms, several problems hinder their use in-vivo. A closer examination of the physical limitations of current lipoplex preparations, the development and testing of novel formulations, combined with more attention to the cellular processes of cell membrane breaching and nuclear entry, may enhance gene delivery. Essential for tumour genotherapy is the ability to target these lipoplexes into tumour sites whilst reducing gene dosage to other normal tissues. Development of a better lipofection agent may indeed require a collaboration of the fields of physiology, cell biology, molecular biology, biochemistry, chemistry and membrane physics.

Liposomes Containing Cationic Dimethyl Dioctadecyl Ammonium Bromide: Formulation, Quality Control, and Lipofection Efficiency

This article describes a novel, simple, and relatively inexpensive method to prepare cationic liposomes using an ethanol injection/pressure extrusion method. The study also demonstrated that binding erythrosine dye to cationic liposomes results in a shift of the absorption maximum of the dye from 528 nm to 549 nm at pH 4.25, allowing quantification and visualization of these vesicles. In addition, a relatively simple Ficoll-based gradient centrifugation method for separation of lipoplexes from unbound molecules is presented. Laboratory-formulated dimethyl dioctadecyl ammonium bromide (DDAB) containing liposomes were just as efficient in complexing nucleic acids as commercially available types, and binding increased as the positive to neutral lipid ratio was increased. Transfection efficiency of the DDAB-containing liposomes increased as the ratio of cationic to neutral lipid was increased from 1:1 to 4:1 with either PtdChol or DOPE as the neutral lipid. A concomitant increase in cytotoxicity of CSU-SA1 cancer cells was noted as the ratio of positive to neutral lipid of the liposomes was increased. Nevertheless, our present study showed that the 2:1 liposome is a good choice since it delivers functional plasmids at a comparable rate to commercial liposome formulations, has similar toxicities to the less harmful commercial liposomes, and is at least 1000-fold more economical to prepare inhouse, a major factor to be considered in preclinical and clinical studies with these carriers.

Tumour cells surviving in vivo cisplatin chemotherapy display elevated c-myc expression.

The c-myc oncogene has been extensively implicated in cell proliferation, cell differentiation and programmed cell death. Aberrant expression of the c-myc gene product has been observed in a range of tumours and has also been implicated in cisplatin (cis-dichlorodiammineplatinum)-mediated chemoresistance. A solid transplantable tumour model in syngeneic DA rats was subjected to treatment with cisplatin to determine the impact of such therapy on endogenous c-myc gene expression. Serially transplanted tumours were intravenously treated with a single cisplatin dose (1 mg/kg) and c-myc expression analysed 2 and 7 days after treatment. The surviving tumour cells display a significant 2-fold elevation in c-myc expression at 48 h and 7 days after treatment. Primary cell cultures have been derived from untreated in vivo tumours of the same model and subjected to treatment with a c-myc phosphorothioate antisense oligomer. Administration of 5 microM c-myc antisense oligomer directed at the initiation codon and first four codons of c-myc mRNA results in total inhibition of c-myc expression and coincident suspension of cell growth for a period of 4 days in culture. Antisense therapies directed at the c-myc gene may well prove an effective tool for treating tumours in conjunction with cisplatin as these findings show that tumour cells surviving cisplatin chemotherapy display elevated c-myc expression.

Respiratory function testing: The impact of respiratory scientists on the training and support of primary health care providers

Objective:  In rural Australia access to doctors is limited, access to respiratory physicians even more so and these are the traditional sources of lung function testing.The aim of this study was to assess the feasibility of training and supporting existing rural primary healthcare providers in lung function testing as a screening and monitoring mechanism due to the shortage of healthcare professionals capable of providing such a service.

Modified microplex vector enhances transfection of cells in culture while maintaining tumour‐selective gene delivery in‐vivo

A non‐commercial liposome (dimethyl dioctadecyl ammonium bromide: dioleoyl phosphatidyl‐ethanolamine) was compared with a commercial variety (Lipofectamine) for transfection of cultured rat adenocarcinoma cells and in an in‐vivo kidney tumour model. Transfection of the cells in culture and in tumours in‐vivo was variable with both types of liposomes. A high‐dose microplex (lipoplex–microsphere) vector enhanced liposome‐mediated transfection of cells in culture. When these high‐dose microplexes were tested in‐vivo, they were better than both microspherical and liposomal delivery modes in terms of transgene expression levels and the tumour‐to‐normal tissue ratio of gene delivery. Microplexes have been demonstrated to be capable of not only selective delivery of plasmids to solid tumours, but also of increasing transfection in cell culture, a finding that may be used in ex‐vivo transfection studies. It is hypothesized that microspheres anchored the combination vector closer to the cultured cells, allowing attached liposomes to gain easier access into cells. In‐vivo, microspheres permitted the microplexes to selectively deliver their genetic payload within the tumour tissue, from where the action of cationic liposomes on cellular membranes facilitated increased access of plasmids into the cytosol of target cells.

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...