The endothelin B (ETB) receptor agonist IRL 1620 is highly vasoconstrictive in two syngeneic rat tumour lines: potential for selective tumour blood flow modification

The vascular effects of the endothelin B (ETB) receptor agonist IRL 1620 were investigated in the rat P22 carcinosarcoma and a range of normal tissues in BDIX rats. Tissue blood flow rate was calculated from measurements of tissue uptake of radiolabelled iodoantipyrine. A comparison of vascular effects in the P22 tumour and the HSN sarcoma growing in CBH/CBi rats was made using laser Doppler flowmetry, showing similar effects of IRL 1620, with red cell flux rapidly decreasing by 50–60% and then returning to control levels within approximately 30 min. This corresponded to similar levels but different spatial organisation of ETB binding sites in the two tumours, as measured by autoradiography. The decrease in tumour blood flow and an increase in vascular resistance suggest that the vascular component of ETB receptors in the P22 tumour is localised on contractile elements rather than on endothelial cells. ETA receptors were also identified. Vasoconstriction occurred uniformly throughout the P22 tumour mass, consistent with a measured homogeneous distribution of ETB receptors. IRL 1620 caused vasoconstriction in normal skeletal muscle, kidney and small intestine of the BDIX rat as well as in tumour, but did not affect blood flow in other tissues. These effects could be useful for limiting toxicity of certain chemotherapeutic agents. Fully functional ETB receptors are clearly expressed on tumour vasculature and IRL 1620 shows promise for short-term modification of tumour blood flow. Expression levels of ETB receptors on the tumour vasculature could be useful for predicting which tumours are likely to respond to IRL 1620

Electroporation of human microvascular endothelial cells: evidence for an anti-vascular mechanism of electrochemotherapy

Recent studies have indicated that the antitumour effectiveness of electrochemotherapy, a combination of chemotherapeutic drugs with application of high voltage electric pulses applied to the tumour nodule (electroporation), result in a significant reduction in tumour blood flow and may therefore be mediated by an anti-vascular mechanism. The aim of this study was to evaluate the cytotoxicity of electroporation with bleomycin or cisplatin on cultured human microvascular endothelial cells (HMEC-1). The sensitivity of HMEC-1 cells to a 5 min treatment by electroporation with bleomycin or cisplatin (8 electric pulses, pulse duration 100 μs, frequency 1 Hz, electric field intensity 1400 V cm–1) was compared to the sensitivity of cells treated continuously for 3 days with drugs alone. HMEC-1 cells were moderately sensitive to continuous exposure to cisplatin, but showed greater sensitivity to bleomycin. Combination of a 5 min drug exposure with electric pulses increased cytotoxicity of cisplatin by ∼10-fold for cisplatin and ∼5000-fold for bleomycin. The electroporation of HMEC-1 cells with bleomycin for a 5 min exposure was ∼250-fold better than a continuous exposure to the drug alone. The results of this study indicate that the anti-tumour action of electrochemotherapy is likely to be due, in part, to the highly sensitive response of vascular endothelial cells. Further studies are necessary to identify the determinants of endothelial response and its relationship to the anti-vascular action of electrochemotherapy in vivo. © 2001 Cancer Research Campaign http://www.bjcancer.co

Direct visualization of electroporation-assisted in vivo gene delivery to tumors using intravital microscopy – spatial and time dependent distribution

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

A Hydrogel/Carbon-Nanotube Needle-Free Device for Electrostimulated Skin Drug Delivery

The permeability of skin allows passive diffusion across the epidermis to reach blood vessels but this is possible only for small molecules such as nicotine. In order to achieve transdermal delivery of large molecules such as insulin or plasmid DNA, permeability of the skin and mainly the permeability of stratum corneum has to be increased. Moreover, alternative routes that avoid the use of needles will improve the quality of life of patients. A method named “electropermeabilization” has been shown to increase skin permeability. Here, we report the fabrication of an innovative hydrogel, made of nanocomposite material. This nanocomposite device aims at permeabilizing the skin and delivering drug molecules at the same time. It includes a biocompatible polymer matrix (hydrogel) and double walled-carbon-nanotubes (DWCNTs) in order to bring electrical conductivity and improve mechanical properties. Carbon nanotubes and especially DWCNTs are ideal candidates, combining high electrical conductivity with a very high specific surface area together with a good biocompatibility when included into a material. The preparation and characterization of the nanocomposite hydrogel as well as first results of electro stimulated transdermal delivery using an ex vivo mouse skin model are presented

Efficacy and safety of electrochemotherapy combined with peritumoral IL-12 gene electrotransfer of canine mast cell tumours

Electrochemotherapy combined with peritumoral interleukin-12 (IL-12) gene electrotransfer was used for treatment of mast cell tumours in 18 client-owned dogs. Local tumour control, recurrence rate, as well as safety of combined therapy were evaluated. One month after the therapy, no side effects were recorded and good local tumour control was observed with high complete responses rate which even increased during the observation period to 72%. IL-12 gene electrotransfer resulted in 78% of patients with detectable serum IFN- and/or IL-12 levels. In the treated tumours vascular changes as well as minimal T-lymphocytes infiltration was observed. After 1week, the plasmid DNA was not detected intra- or peritumorally and no horizontal gene transfer was observed. In summary, our study demonstrates high antitumour efficacy of electrochemotherapy combined with IL-12 electrotransfer, which also prevented recurrences or distant metastases, as well as its safety and feasibility in treatment of canine mast cell tumours

\u3ci\u3eIn Vitro and In Vivo\u3c/i\u3e Correlation of Skin and Cellular Responses to Nucleic Acid Delivery

Skin, the largest organ in the body, provides a passive physical barrier against infection and contains elements of the innate and adaptive immune systems. Skin consists of various cells, including keratinocytes, fibroblasts, endothelial cells and immune cells. This diversity of cell types could be important to gene therapies because DNA transfection could elicit different responses in different cell types. Previously, we observed the upregulation and activation of cytosolic DNA sensing pathways in several non-tumor and tumor cell types as well in tumors after the electroporation (electrotransfer) of plasmid DNA (pDNA). Based on this research and the innate immunogenicity of skin, we correlated the effects of pDNA electrotransfer to fibroblasts and keratinocytes to mouse skin using reverse transcription real-time PCR (RT-qPCR) and several types of protein quantification. After pDNA electrotransfer, the mRNAs of the putative DNA sensors DEAD (AspGlu-Ala-Asp) box polypeptide 60 (Ddx60), absent in melanoma 2 (Aim2), Z-DNA binding protein 1 (Zbp1), interferon activated gene 202 (Ifi202), and interferon-inducible protein 204 (Ifi204) were upregulated in keratinocytes, while Ddx60, Zbp1 and Ifi204 were upregulated in fibroblasts. Increased levels of the mRNAs and proteins of several cytokines and chemokines were detected and varied based on cell type. Mouse skin experiments in vivo confirmed our in vitro results with increased expression of putative DNA sensor mRNAs and of the mRNAs and proteins of several cytokines and chemokines. Finally, with immunofluorescent staining, we demonstrated that skin keratinocytes, fibroblasts and macrophages contribute to the immune response observed after pDNA electrotransfer

Vascular disrupting action of electroporation and electrochemotherapy with bleomycin in murine sarcoma

Electrochemotherapy has a direct cytotoxic effect on tumour cells, and presumably, a vascular disrupting effect. In this study, on the basis of the prediction of the mathematical model, histological evaluation and physiological measurements of the tumours were carried out to confirm that electroporation and electrochemotherapy of tumours have a vascular disrupting action. In the study, SA-1 solid subcutaneous sarcoma tumours in A/J mice were treated by bleomycin (BLM) given intravenously (1 mg kg−1), application of electric pulses (8 pulses, 1040 V, 100 μs, 1 Hz) or a combination of both – electrochemotherapy. The vascular effect was determined by laser Doppler flowmetry, power Doppler ultrasonographic imaging and Patent blue staining. The extent of tumour hypoxia was determined immunohistochemically by hypoxia marker pimonidazole and partial pressure of oxygen (pO2) in tumours by electron paramagnetic resonance oximetry. Electrochemotherapy with BLM induced good antitumour effect with 22 days, tumour growth delay and 38% tumour cures. The application of electric pulses to the tumours induced instant but transient tumour blood flow reduction (for 70%) that was recovered in 24 h. During this tumour blood flow reduction, we determined an increase in hypoxic tumour area for up to 30%, which was also reflected in reduced tumour oxygenation (for 70%). According to the described mathematical model, endothelial cells lining in tumour blood vessels are exposed to a ∼40% higher electric field than the surrounding tumour cells, and therefore easily electroporated, allowing access of high BLM concentration to the cytosol. Consequently, electrochemotherapy has, besides the immediate vascular disrupting action, also a delayed one (after 24 h), as a consequence of endothelial cell swelling and apoptosis demonstrated by extensive tumour necrosis, tumour hypoxia, prolonged reduction of tumour blood flow and significant tumour growth delay, and tumour cures. Our results demonstrate that in addition to the well-established direct cytotoxic effect on tumour cells, electrochemotherapy also has an indirect vascular disrupting action resulting altogether in extensive tumour cell necrosis leading to complete regression of tumours

Radiosensitising effect of electrochemotherapy with bleomycin in LPB sarcoma cells and tumors in mice

BACKGROUND: Bleomycin is poorly permeant but potent cytotoxic and radiosensitizing drug. The aim of the study was to evaluate whether a physical drug delivery system – electroporation can increase radiosensitising effect of bleomycin in vitro and in vivo. METHODS: LPB sarcoma cells and tumors were treated either with bleomycin, electroporation or ionizing radiation, and combination of these treatments. In vitro, response to different treatments was determined by colony forming assay, while in vivo, treatment effectiveness was determined by local tumor control (TCD(50)). Time dependence of partial oxygen pressure in LPB tumors after application of electric pulses was measured by electron paramagnetic oxyimetry. RESULTS: Electroporation of cells in vitro increased radiosensitising effect of bleomycin for 1.5 times, in vivo radiation response of tumors was enhanced by 1.9 fold compared to response of tumors that were irradiated only. Neither treatment of tumors with bleomycin nor application of electric pulses only, affected radiation response of tumors. Application of electric pulses to the tumors induced profound but transient reduction of tumor oxygenation. Although tumor oxygenation after electroporation partially restored at the time of irradiation, it was still reduced at the level of radiobiologically relevant hypoxia. CONCLUSION: Our study shows that application of electric pulses to cells and tumors increases radiosensitising effect of bleomycin. Furthermore, our results demonstrate that the radiobiologically relevant hypoxia induced by electroporation of tumors did not counteract the pronounced radiosensitising effect of electrochemotherapy with bleomycin