Todd L. Walker

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.

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.

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...

Tumor Gene Targeting Using Microspheres: Cell Culture and in vivo Studies

In a previous study, Dass et al. (Pharm. Sci. 2:401-405, 1996), it was shown that 1 mg of two types of ion-exchange microspheres was capable of binding and releasing plasmids in a continuous-flow system to the order of 10(11) copies in phosphate-buffered saline at 37°C. However, the functionality of the plasmids was not evaluated. In this study, one of the plasmids, pCMV-CAT, was bound to both microspheres and the functionality of the biomol-ecule was examined in cell culture and in vivo transfection studies. Rat tumor cells incubated with the hydroxyapatite (HA) plasmids were transfected 5.4-fold better than when incubated with free plasmids and 56.0-fold better than polystyrene divinylbenzene (PDB) microspheres. Cells incubated with PDB microspheres were transfected 10.4-fold less than cells incubated with free plasmids. However, HA microspheres were highly cytotoxic to the cells whereas PDB spheres had no effect on cell numbers compared with control cells. Based on expression levels of delivered plasmids in the in vivo study, delivery on microspheres to kidneys was 2.7-fold better than plasmids delivered free. Microspherical delivery of plasmids to tumors was 1.6-fold better than free delivery. However, these results were not significantly different (p >. 05). The tumor/normal tissue ratio of gene expression was 4.5:1 for free delivery and 2.6-fold when delivered on microspheres. Although the difference between deliveries in the two tissues was significant (p > 0.005) for free delivery, it was not so for micro-spherical delivery. Expression of the CAT gene was not noted in either liver or spleen of any of the animals. This present study has proved that ion-exchange microspheres have no detrimental effect on released plasmid DNA expression. In an in vivo setting, resistance to enzymatic degradation of plasmids that are bound to microspheres and subsequent release of plasmids once embolization has occurred in the tumor vascular bed effected better transfection than delivery of free plasmids.

Trichomonas vaginalis in Vanuatu

OBJECTIVE To assess the prevalence of Trichomonas vaginalis in two island populations of Vanuatu using the Pap smear as the screening technique. STUDY DESIGN Women were randomly selected from specific sites on the islands of Efate (urban setting) and Ambae (rural setting). Pap smears were collected, screened and reported. SETTING The first collection site was the Womens Health/Antenatal Care Clinic at Vila Central Hospital in Port Vila, the capital city located on the island of Efate, and the second collection site was a rural village on a sparsely populated inhabited northern island, Ambae. PARTICIPANTS A total of 905 Ni-Vanuatu women participants: Efate (n = 562) 62%, and Ambae (n = 343) 38%. The mean age was 35.8 years: Efate 32.6 years, and Ambae 40.8 years. MAIN OUTCOME MEASURE The presence or absence of T. vaginalis in these Pap smears was documented during the studys cervical screening process. RESULTS The overall prevalence of T. vaginalis within the study participants was 25.3%. Almost half of the infected sample group were in the age group of 30-39 years (43.8%). The prevalence of T. vaginalis in Efate was 14.7%, compared with 43.4% in Ambae. CONCLUSION The prevalence of T. vaginalis in Vanuatu women is significantly higher compared with developed countries. Women in rural settings are less likely to have access to sexually transmitted disease prevention and treatment programs, thus contributing to high infection rates compared with women in urban settings. Cultural and educational differences in the rural setting might also contribute to higher sexually transmitted disease rates among these women.

The efficacy of doxorubicin microspheres for hepatic micrometastases in a rat tumour model

The use of sustained-release microspheres is of potential benefit as an adjuvant treatment for patients with occult hepatic micrometastases. This study investigates the response of a model of implantable adenocarcinoma micrometastases in the livers of DA rats following the intraportal injection of doxorubicin-incorporated ion-exchange microspheres compared to free drug bolus administration. A point-counting technique was used to determine the percentage of liver consisting of tumour 13 days after treatment. This was used as an indicator of tumour response, as was the derived tumour mass. There was a significantly higher tumour response in animals treated with the microspheres compared to animals treated with free drug delivered at the same concentration. This effect, however, was shown to decrease with a delay in the time of treatment. The tumour response of the sustained-release microspheres was achieved in the absence of any detectable local or systemic toxicity. This study demonstrates the potential of sustained-release microspheres in the treatment of patients with hepatic micrometastases.

Estimates for cervical abnormalities in Vanuatu.

Objective: To use the Pap smear to establish a recent prevalence of cervical abnormalities within a select population in Vanuatu, a developing country.

Bronchoalveolar lavage: Comparison of nucleopore filters and direct smear preparations

To determine whether cytopreparation affects the diagnostic yield of bronchoalveolar lavage specimens, we compared 50 lavage samples by using two methods. One nucleopore filter preparation and four direct slides were prepared on each sample submitted. All slides were stained by the Papanicolaou method. Assessment of cellularity, cellular preservation, and an independent diagnosis were rendered on each sample for both preparatory methods. Statistical analysis showed no difference in cellularity between the two methods (P = .06). The degree of nuclear and cytoplasmic preservation was higher using the direct method, although this did not appear to affect diagnosis in this study. One major discrepancy in diagnosis was observed between the two methods. By prospectively comparing nucleopore filter and direct preparation of bronchoalveolar lavages, we found that there was minimal affect on either cellularity or diagnosis. We conclude that either method delivers reliable cytologic results. Diagn. Cytopathol. 17:160–164, 1997.