title: Direct visualization of electroporation-assisted in vivo gene delivery to tumors using intravital microscopy - spatial and time dependent distribution.
abstract: BACKGROUND
  Electroporation is currently receiving much attention as a way to increase drug and DNA delivery. Recent studies demonstrated the feasibility of electrogene therapy using a range of therapeutic genes for the treatment of experimental tumors. However, the transfection efficiency of electroporation-assisted DNA delivery is still low compared to viral methods and there is a clear need to optimize this approach. In order to optimize treatment, knowledge about spatial and time dependency of gene expression following delivery is of utmost importance in order to improve gene delivery. Intravital microscopy of tumors growing in dorsal skin fold window chambers is a useful method for monitoring gene transfection, since it allows non-invasive dynamic monitoring of gene expression in tumors in a live animal.
  
  
  METHODS
  Intravital microscopy was used to monitor real time spatial distribution of the green fluorescent protein (GFP) and time dependence of transfection efficiency in syngeneic P22 rat tumor model. DNA alone, liposome-DNA complexes and electroporation-assisted DNA delivery using two different sets of electric pulse parameters were compared.
  
  
  RESULTS
  Electroporation-assisted DNA delivery using 8 pulses, 600 V/cm, 5 ms, 1 Hz was superior to other methods and resulted in 22% increase in fluorescence intensity in the tumors up to 6 days post-transfection, compared to the non-transfected area in granulation tissue. Functional GFP was detected within 5 h after transfection. Cells expressing GFP were detected throughout the tumor, but not in the surrounding tissue that was not exposed to electric pulses.
  
  
  CONCLUSIONS
  Intravital microscopy was demonstrated to be a suitable method for monitoring time and spatial distribution of gene expression in experimental tumors and provided evidence that electroporation-assisted gene delivery using 8 pulses, 600 V/cm, 5 ms, 1 Hz is an effective method, resulting in early onset and homogenous distribution of gene expression in the syngeneic P22 rat tumor model.
authors.affiliation: Department of Experimental Oncology, Institute of Oncology, Zaloska 2, SI-1000 Ljubljana, Slovenia. mcemazar@onko-i.si
authors.forename: Maja
authors.initials: M
authors.lastname: Cemazar
authors.forename: Ian
authors.initials: I
authors.lastname: Wilson
authors.forename: Gabi U
authors.initials: GU
authors.lastname: Dachs
authors.forename: Gillian M
authors.initials: GM
authors.lastname: Tozer
authors.forename: Gregor
authors.initials: G
authors.lastname: Sersa
mesh_terms: D000818:Animals; D002296:Carcinosarcoma / Q000235:genetics* / Q000378:metabolism / Q000628:therapy*; D004247:DNA / Q000008:administration & dosage* / Q000235:genetics; D018274:Electroporation / Q000379:methods*; D015316:Genetic Therapy / Q000379:methods; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D008081:Liposomes; D008297:Male; D008853:Microscopy / Q000379:methods; D010957:Plasmids / Q000008:administration & dosage / Q000235:genetics; D051381:Rats; D014162:Transfection / Q000379:methods*
chemical_list: D008081:Liposomes; D049452:Green Fluorescent Proteins; D004247:DNA