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. Conventional liposomes, originally used for studies in membrane biophysics and biochemistry, have been used in therapy for the past two decades. However, when applied to deliver drugs into cells, conventional liposomes proved inefficient and so novel unconventional or specialized liposomes are constantly being prepared to enhance cell-specific delivery in-vivo. One possible way of achieving better targeting is combination of the positive attributes of more than one specialized type of liposome into one vesicle. Although a limited number of studies has examined the combined effect of such dual-specialty liposomes, more studies are warranted using appropriate models. Liposomes are composed of one, a few, or many concentric bilayer membranes which alternate with aqueous spaces. The drugs are encapsulated within the aqueous internal volume if they are hydrophilic or in the lipid bilayers if they are hydrophobic (Kim 1993). Liposomes range in size from 25 nm to more than 20 microns (Sugarman & Perez-Soler 1992). Depending on their solubility and method of formulation antimicrobial, cytotoxic and other conventional drugs, hormones, antigens, enzymes, genetic material, viruses and bacteria can be incorporated in either the aqueous or hydrophobic phase. This review discusses the types and characteristics of non-conventional liposomes used in various modes of cancer therapy, mainly chemotherapy and gene therapy. It concludes with suggestions on improving these novel liposomal to effect better targeting to cancer cells.

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.

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.

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.

The basis for somatic gene therapy of cancer.

A decade of advances in understanding of the molecular basis of sporadic and familial cancers has combined with developments in mammalian gene transfer technology to stimulate intensive research into the potential applications of somatic gene therapy for cancer. Somatic gene immunotherapy is already in progress to stimulate and direct the natural targeting capabilities of the immune system against the threat of disseminated residual disease. The association of a plethora of mutated tumor suppressor genes (p53, p16 BRCA1, BRCA2) with diverse cancers has also highlighted the potential of somatic gene therapy with wild-type versions of suppressor genes as an anti-cancer therapeutic modality either in its own right or in synergistic association with traditional anti-cancer therapies. The methodologies for gene transfer technology range from direct intravenous injection of naked modified DNAs to intravenous injection of liposome-encapsulated DNAs or microsphere-bound DNAs. Recombinant retroviral and adenoviral vectors have natural transfection capabilities and display tropism for particular tissues that are of selective advantage against particular cancers. Liposomes display very high efficiencies of gene transfer with the advantages of successful transfer to a wide range of tissue types but their widespread systemic distribution offers problems in relation to selective targeting of tumor cells. The challenges to current gene transfer processes are much the same as that of other anti-cancer therapies: achieving selective targeting of cancer cells whilst optimizing dosages and minimizing the risk of collateral damage to healthy tissues.

Nomenclature relating to restriction of modified DNA in Escherichia coli.

At least three restriction systems that attack DNA containing naturally modified bases have been found in common Escherichia coli K-12 strains. These systems are McrA, McrBC, and Mrr. A brief summary of the genetic and phenotypic properties so far observed in laboratory strains is set forth, together with a proposed nomenclature for describing these properties.

Quantitative evaluation of Escherichia coli host strains for tolerance to cytosine methylation in plasmid and phage recombinants.

Many strains of E. coli K12 restrict DNA containing cytosine methylation such as that present in plant and animal genomes. Such restriction can severely inhibit the efficiency of cloning genomic DNAs. We have quantitatively evaluated a total of 39 E. coli strains for their tolerance to cytosine methylation in phage and plasmid cloning systems. Quantitative estimations of relative tolerance to methylation for these strains are presented, together with the evaluation of the most promising strains in practical recombinant cloning situations. Host strains are recommended for different recombinant cloning requirements. These data also provide a rational basis for future construction of 'ideal' hosts combining optimal methylation tolerance with additional advantageous mutations.