title: Soluble biodegradable polymer-based cytokine gene delivery for cancer treatment.
abstract: Transgene expression and tumor regression after direct injection of plasmid DNA encoding cytokine genes, such as mIL-12 and mIFN-gamma, remain very low. The objective of this study is to develop nontoxic biodegradable polymer-based cytokine gene delivery systems, which should enhance mIL-12 expression, increasing the likelihood of complete tumor elimination. We synthesized poly[alpha-(4-aminobutyl)-l-glycolic acid] (PAGA), a biodegradable nontoxic polymer, by melting condensation. Plasmids used in this study encoded luciferase (pLuc) and murine interleukin-12 (pmIL-12) genes. PAGA/plasmid complexes were prepared at different (+/-) charge ratios and characterized in terms of particle size, zeta potential, osmolality, surface morphology, and cytotoxicity. Polyplexes prepared by complexing PAGA with pmIL-12 as well as pLuc were used for transfection into cultured CT-26 colon adenocarcinoma cells as well as into CT-26 tumor-bearing BALB/c mice. The in vitro and in vivo transfection efficiency was determined by luciferase assay (for pLuc), enzyme-linked immunosorbent assay (for mIL-12, p70, and p40), and reverse transcriptase-polymerase chain reaction (RT-PCR) (for Luc and mIL-12 p35). PAGA condensed and protected plasmids from nuclease degradation. The mean particle size and zeta potential of the polyplexes prepared in 5% (w/v) glucose at 3:1 (+/-) charge ratio were approximately 100 nm and 20 mV, respectively. The surface characterization of polyplexes as determined by atomic force microscopy showed complete condensation of DNA with an ellipsoidal structure in Z direction. The levels of mIL-12 p40, mIL-12 p70, and mIFN-gamma were significantly higher for PAGA/pmIL-12 complexes compared to that of naked pmIL-12. This is in good agreement with RT-PCR data, which showed significant levels of mIL-12 p35 expression. The PAGA/pmIL-12 complexes did not induce any cytotoxicity in CT-26 cells as evidenced by 3-¿4, 5-dimethylthiazol-2-yl¿-2,5-diphenyltetrazolium bromide assay and showed enhanced antitumor activity in vivo compared to naked pmIL-12. PAGA/pmIL-12 complexes are nontoxic and significantly enhance mIL-12 expression at mRNA and protein levels both in vitro and in vivo.
authors.affiliation: Center for Controlled Chemical Delivery, University of Utah, Salt Lake City, Utah 84112-5820, USA.
authors.forename: A
authors.initials: A
authors.lastname: Maheshwari
authors.forename: R I
authors.initials: RI
authors.lastname: Mahato
authors.forename: J
authors.initials: J
authors.lastname: McGregor
authors.forename: S o
authors.initials: So
authors.lastname: Han
authors.forename: W E
authors.initials: WE
authors.lastname: Samlowski
authors.forename: J S
authors.initials: JS
authors.lastname: Park
authors.forename: S W
authors.initials: SW
authors.lastname: Kim
mesh_terms: D020341:Absorbable Implants; D000818:Animals; D016923:Cell Death / Q000187:drug effects / Q000235:genetics; D016207:Cytokines / Q000235:genetics* / Q000627:therapeutic use*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006016:Glycolates / Q000138:chemical synthesis; D006801:Humans; D018664:Interleukin-12 / Q000235:genetics / Q000627:therapeutic use; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D018625:Microscopy, Atomic Force; D009368:Neoplasm Transplantation; D009369:Neoplasms / Q000628:therapy*; D010957:Plasmids / Q000235:genetics; D011100:Polyglycolic Acid / Q000031:analogs & derivatives / Q000138:chemical synthesis; D011108:Polymers; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D016207:Cytokines; D006016:Glycolates; D011108:Polymers; C414262:poly((4-aminobutyl)glycolic acid); C031149:glycolic acid; D018664:Interleukin-12; D011100:Polyglycolic Acid; D008156:Luciferases