title: Magnetic nanoparticle hyperthermia induced cytosine deaminase expression in microencapsulated E. coli for enzyme-prodrug therapy.
abstract: Engineered bacterial cells that are designed to express therapeutic enzymes under the transcriptional control of remotely inducible promoters can mediate the de novo conversion of non-toxic prodrugs to their cytotoxic forms. In situ cellular expression of enzymes provides increased stability and control of enzyme activity as compared to isolated enzymes. We have engineered Escherichia coli (E. coli), designed to express cytosine deaminase at elevated temperatures, under the transcriptional control of thermo-regulatory λpL-cI857 promoter cassette which provides a thermal switch to trigger enzyme synthesis. Enhanced cytosine deaminase expression was observed in cultures incubated at 42°C as compared to 30°C, and enzyme expression was further substantiated by spectrophotometric assays indicating enhanced conversion of 5-fluorocytosine to 5-fluorouracil. The engineered cells were subsequently co-encapsulated with magnetic iron oxide nanoparticles in immunoprotective alginate microcapsules, and cytosine deaminase expression was triggered remotely by alternating magnetic field-induced hyperthermia. The combination of 5-fluorocytosine with AMF-activated microcapsules demonstrated tumor cell cytotoxicity comparable to direct treatment with 5-fluorouracil chemotherapy. Such enzyme-prodrug therapy, based on engineered and immunoisolated E. coli, may ultimately yield an improved therapeutic index relative to monotherapy, as AMF mediated hyperthermia might be expected to pre-sensitize tumors to chemotherapy under appropriate conditions.
authors.affiliation: Department of Radiology, Geisel School of Medicine at Dartmouth, Hanover, NH, USA.
authors.forename: Krishnamurthy V
authors.initials: KV
authors.lastname: Nemani
authors.affiliation: Dartmouth College, Hanover, NH, USA.
authors.forename: Riley C
authors.initials: RC
authors.lastname: Ennis
authors.affiliation: Thayer School of Engineering, Dartmouth, Hanover, NH, USA; Department of Biological Sciences, Dartmouth, Hanover, NH, USA; Program in Molecular and Cellular Biology, Dartmouth, Hanover, NH, USA.
authors.forename: Karl E
authors.initials: KE
authors.lastname: Griswold
authors.affiliation: Department of Radiology, Geisel School of Medicine at Dartmouth, Hanover, NH, USA; Thayer School of Engineering, Dartmouth, Hanover, NH, USA; Department of Medicine, Geisel School of Medicine at Dartmouth, Hanover, NH, USA. Electronic address: Barjor.Gimi@Dartmouth.edu.
authors.forename: Barjor
authors.initials: B
authors.lastname: Gimi
mesh_terms: D000464:Alginates / Q000737:chemistry; D000818:Animals; D000970:Antineoplastic Agents / Q000008:administration & dosage; D002214:Capsules; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D043525:Cytosine Deaminase / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005290:Ferric Compounds / Q000008:administration & dosage* / Q000737:chemistry; D005437:Flucytosine / Q000008:administration & dosage*; D020723:Glucuronic Acid / Q000737:chemistry; D006603:Hexuronic Acids / Q000737:chemistry; D006358:Hot Temperature; D006801:Humans; D060526:Magnetic Fields; D060328:Magnetic Phenomena; D053768:Metal Nanoparticles / Q000008:administration & dosage* / Q000737:chemistry; D011355:Prodrugs / Q000008:administration & dosage*; D051381:Rats
chemical_list: D000464:Alginates; D000970:Antineoplastic Agents; D002214:Capsules; D005290:Ferric Compounds; D006603:Hexuronic Acids; D011355:Prodrugs; C000499:ferric oxide; D020723:Glucuronic Acid; D005437:Flucytosine; D043525:Cytosine Deaminase
keywords: Enzyme–prodrug therapy; Magnetic nanoparticle hyperthermia; Microencapsulation; Recombinant E. coli; Thermoregulated protein expression
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