Electric field-mediated transport of plasmid DNA in tumor interstitium in vivo

Department of Biomedical Engineering, Duke University, 136 Hudson Hall, Durham, NC 27708, USA.
Bioelectrochemistry (Impact Factor: 4.17). 12/2007; 71(2):233-42. DOI: 10.1016/j.bioelechem.2007.07.005
Source: PubMed


Local pulsed electric field application is a method for improving non-viral gene delivery. Mechanisms of the improvement include electroporation and electrophoresis. To understand how electrophoresis affects pDNA delivery in vivo, we quantified the magnitude of electric field-induced interstitial transport of pDNA in 4T1 and B16.F10 tumors implanted in mouse dorsal skin-fold chambers. Four different electric pulse sequences were used in this study, each consisted of 10 identical pulses that were 100 or 400 V/cm in strength and 20 or 50 ms in duration. The interval between consecutive pulses was 1 s. The largest distance of transport was obtained with the 400 V/cm and 50 ms pulse, and was 0.23 and 0.22 microm/pulse in 4T1 and B16.F10 tumors, respectively. There were no significant differences in transport distances between 4T1 and B16.F10 tumors. Results from in vivo mapping and numerical simulations revealed an approximately uniform intratumoral electric field that was predominantly in the direction of the applied field. The data in the study suggested that interstitial transport of pDNA induced by a sequence of ten electric pulses was ineffective for macroscopic delivery of genes in tumors. However, the induced transport was more efficient than passive diffusion.

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Available from: Fan Yuan, Aug 04, 2014
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    • "Second, EP also has other effects on the target tissue besides vascular effects, and very little is known about these effects. It is now known that application of electric pulses to tissues affects not only the permeability of cell membranes but also the distribution of DNA and other molecules in the tissues (Zaharoff et al. 2002; Henshaw et al. 2007, 2011). One of the hallmarks of DNA vaccination combined with EP is that EP significantly enhances the immunogenicity of DNA vaccines relative to DNA injection alone also in human clinical trials (Hirao et al. 2010; Livingston et al. 2010; Sardesai and Weiner 2011; Vasan et al. 2011). "
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    ABSTRACT: Electropermeabilization/electroporation (EP) is a physical method that by application of electric pulses to cells increases cell membrane permeability and enables the introduction of molecules into the cells. One of the uses of EP in vivo is plasmid DNA electrotransfer to the skin for DNA vaccination. EP of tissues induces reduction of blood flow and, in combination with plasmid DNA, induction of an immune response. One of the EP protocols for plasmid DNA electrotransfer to the skin is a combination of high-voltage (HV) and low-voltage (LV) pulses. However, the effects of this pulse combination on skin-vessel blood flow are not known. Therefore, using intravital microscopy in a dorsal window chamber in mice and fluorescently labeled dextrans, the effects of one HV and eight LV pulses on skin vasculature were investigated. In addition, a detailed histological analysis was performed. Image analysis of fluorescence intensity changes demonstrated that EP induces a transient constriction and increased permeability of blood vessels as well as a "vascular lock." Histological analysis revealed rounding up of endothelial cells and stacking up of erythrocytes at 1 h after EP. In addition, extravasation of erythrocytes and leukocyte infiltration accompanied by edema were determined up to 24 h after EP. In conclusion, our results show that blood flow modifying effects of EP in skin contribute to the infiltration of immune cells in the exposed area. When combined with plasmid DNA for vaccination, this could enable the initial and prolonged contact of immune cells with encoded therapeutic proteins.
    Journal of Membrane Biology 05/2012; 245(9):545-54. DOI:10.1007/s00232-012-9435-5 · 2.46 Impact Factor
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    • "B16.F10, a murine melanoma cell line [61], [62], [63], were cultured as monolayers in T75 flasks in high glucose Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with 10% bovine growth serum (Hyclone, Logan, UT) and penicillin/streptomycin (Invitrogen). The cells were incubated at 37°C in 5% CO2 and 95% air and passaged every 2–3 days. "
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    ABSTRACT: Electric field mediated gene delivery or electrotransfection is a widely used method in various studies ranging from basic cell biology research to clinical gene therapy. Yet, mechanisms of electrotransfection are still controversial. To this end, we investigated the dependence of electrotransfection efficiency (eTE) on binding of plasmid DNA (pDNA) to plasma membrane and how treatment of cells with three endocytic inhibitors (chlorpromazine, genistein, dynasore) or silencing of dynamin expression with specific, small interfering RNA (siRNA) would affect the eTE. Our data demonstrated that the presence of divalent cations (Ca(2+) and Mg(2+)) in electrotransfection buffer enhanced pDNA adsorption to cell membrane and consequently, this enhanced adsorption led to an increase in eTE, up to a certain threshold concentration for each cation. Trypsin treatment of cells at 10 min post electrotransfection stripped off membrane-bound pDNA and resulted in a significant reduction in eTE, indicating that the time period for complete cellular uptake of pDNA (between 10 and 40 min) far exceeded the lifetime of electric field-induced transient pores (∼10 msec) in the cell membrane. Furthermore, treatment of cells with the siRNA and all three pharmacological inhibitors yielded substantial and statistically significant reductions in the eTE. These findings suggest that electrotransfection depends on two mechanisms: (i) binding of pDNA to cell membrane and (ii) endocytosis of membrane-bound pDNA.
    PLoS ONE 06/2011; 6(6):e20923. DOI:10.1371/journal.pone.0020923 · 3.23 Impact Factor
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    • "DSCs were implanted in mice anesthetized with an i.p. injection of 80 mg ketamine and 10 mg xylazine per kg body weight. The procedure has been described in previous studies 13, 15, 31. In brief, 10 µl of cell suspension (~5 × 105 cells) was injected into the fascia layer at the center of the DSC, and then the DSC was sealed with a sterile glass coverslip. "
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    ABSTRACT: Pulsed electric fields can enhance interstitial transport of plasmid DNA (pDNA) in solid tumors. However, the extent of enhancement is still limited. To this end, the effects of cellular resistance to electric field-mediated gene delivery were investigated. The investigation used two tumor cell lines (4T1 (a murine mammary carcinoma) and B16.F10 (a metastatic subline of B16 murine melanoma)) either in suspensions or implanted in two in vivo models (dorsal skin-fold chamber (DSC) and hind leg). The volume fraction of cells was altered by pretreatment with a hyperosmotic mannitol solution (1 M). It was observed that the pretreatment reduced the volumes of 4T1 and B16.F10 cells, suspended in an agarose gel, by 50 and 46%, respectively, over a 20-min period, but did not cause significant changes ex vivo in volumes of hind-leg tumor tissues grown from the same cells in mice. The mannitol pretreatment in vivo improved electric field-mediated gene delivery in the hind-leg tumor models, in terms of reporter gene expression, but resulted in minimal enhancement in pDNA electrophoresis over a few microns distance in the DSC tumor models. These data demonstrated that hyperosmotic mannitol solution could effectively improve electric field-mediated gene delivery around individual cells in vivo by increasing the extracellular space.
    Cancer gene therapy 01/2011; 18(1):26-33. DOI:10.1038/cgt.2010.51 · 2.42 Impact Factor
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