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An end-fire dielectric rod antenna is proposed as a heating device for integration with an array of electroporation electrodes in order to enable efficient delivery of DNA into the cells that comprise subcutaneous tumors. A 5-7 °C temperature elevation of tumors that are located near the fat-muscle boundary and 3-7 mm below the skin surface can be achieved in a short period of time without damaging surrounding tissues. This capability is demonstrated through a combination of a directional antenna applicator operating at 8 GHz, and utilization of forced air cooling of the outer surface (skin layer). The directionality of the antenna is improved by cladding its high permittivity core with 3D printed, low permittivity dielectric material. Experimental data using a pork skin-fat-muscle tissue show that the desired temperature elevation at the tumor location is obtained after 2.5 W RF illumination for 3 minutes, which is in good agreement with electro-thermal simulations. With the addition of realistic human body model parameters to the same simulation setup, the results indicate that tumors can be uniformly heated with 3 minutes of illumination at 2.5 W input power while keeping the surrounding healthy tissues at a safe temperature.

Planar diverging lenses attached to the front end of a dielectric rod antenna are proposed to control the heating area on the human body to assist the electroporation treatment of different tumor sizes. The desired 5–7 °C temperature elevation of the tumors is achieved in 3 minutes by applying 2.1-3.6 W input power depending on the size of tumor. In order to treat the tumors located 3–7 mm below the skin surface, the applicator is operated at 8 GHz. The experimental results obtained by measuring a pork muscle tissue show good agreement with electro-thermal simulations.

Electric field mediated gene delivery methods have the ability to efficiently transfect cells in vivo with an excellent safety profile. The method has historically used a fixed number of electric pulses with identical characteristics in induce delivery. Electrical treatment does not typically compensate for subject-to-subject variation and other differences. This study was designed to investigate if delivery/expression could be increased using a novel electropulsation method that compensated for variation using real-time electrical impedance measurements. The method involved delivering plasmid DNA encoding luciferase to murine skin. Tissue impedance in a 1–3 KHz range was measured before electric pulses were applied. Impedance was also measured after each successive pulse. Pulsation was stopped when impedance values were reduced by either 80% or 95% relative to prepulse values. Standard/fixed pulsing parameters were also used for comparison. The results indicated that up to 15-fold increases in luciferase expression could be obtained when electrical treatment was ceased based upon impedance reductions. Furthermore, peak expression levels of all treatment groups pulsed using the novel pulsing method were statistically higher than those that employed standard pulsing. These results strongly suggest that applying pulses until a defined impedance-based endpoint results in higher expression.

In vivo gene electro transfer technology has been very successful both in animal models and in clinical trials over the past 20years. However, variable transfection efficiencies can produce inconsistent outcomes. This can be due to differences in tissue architecture and/or chemical composition which may effectively create unique biological environments from subject to subject that may respond differently to the identical electric pulses. This study investigates the integration of impedance spectroscopy into the gene electro transfer process to measure murine skin impedance spectra before, during (after pulse delivery), and after gene electro transfer pulse application to determine if changes in impedance correlate with reporter gene expression. Both post-treatment impedance spectra and gene expression were dependent upon the applied electric field strength. These results indicate that alterations in tissue impedance produced by the applied electric field represent an excellent parameter to predict degrees of transfection and gene expression. These results could ultimately be used to alter pulsing parameters in order to optimize delivery/expression.

The use of electric fields in vivo to deliver DNA, called electroporation, has the potential to broadly impact vaccination and disease treatment. The evidence for this has emerged from a large number of recently completed and ongoing clinical trials. The methods for applying electric fields to tissues traditionally involve contact between metal electrodes and the tissue. In this study, we investigated the use of helium plasma as a noncontact method for electrically treating tissue in a manner that results in the uptake and expression of foreign DNA in murine skin. More specifically, our goal was to demonstrate that DNA encoding a model-secreted protein could be delivered, detected in the blood, and remain functional to produce its known biological effect. Murine erythropoietin (EPO) was the model-secreted protein. Results clearly demonstrated that an intradermal DNA injection followed by plasma treatment for 2 min resulted in elevated levels of EPO in the blood and corresponding hemoglobin increases that were statistically significant relative to DNA injection alone.

Plasma-based methods have recently emerged as a technique for augmenting plasmid DNA delivery to skin. This delivery modality relies on the deposition of ionized gas molecules on to targeted cells or tissue to establish an electric field. It is hypothesized that this electric field results in the dielectric breakdown of cell membranes, making cells permeable to exogenous molecules. This in vivo investigation sought to optimize the intradermal delivery of a luciferase expressing plasmid DNA by modulating the total exposure to the plasma source and the plasmid DNA dose. Varying the plasma exposure time from 2, 5, 10, and 20 minutes allowed the conditions resulting in the highest expression of luciferase to be found. These conditions correlated to the 10 minute exposure time for a plasma derived from either +8 kV or −8 kV, when the generator was operated 3 cm from the epidermal tissue surface with a helium flow rate of 15 L/min. Exposing the injected flank skin for 10 minutes resulted in a rise of 37.3-fold for a plasma created with +8 kV and 27.1-fold for a plasma created with −8 kV. When using this treatment time with 50, 100, or 200 μg of a luciferase expressing plasmid, it was found that 100 μg resulted in the highest peak luminescence.

A fluorometric assay is described that allows adjustment for non-viable cells that result during electroporation. The technique, unlike others, relies on only one dye, requires a single instrument, and eliminates the need for a separate cell counting step. Murine melanoma (B16-F10) cells were electroporated using electric fields ranging from 400 to 2500 V/cm in the presence of SYTOX(®)-green. Compensation for the fluorescence resulting from non-viable cells was facilitated by a correction curve established by lysing a known number of cells in the presence of SYTOX(®)-green. In uncorrected data, an applied electric field of 2500 V/cm increased dye delivery but also reduced cell viability significantly. Compensating for the fluorescence of non-viable cells showed that changing the field strength to 800 V/cm or 2500 V/cm from 400 V/cm had only marginal effects on membrane pore formation. The fluorometric assay was used to compare electroporation in high conductivity (PBS) and a low conductivity medium (LC-PBS). Statistically significant increases of 10 to 30-fold were observed for cells electroporated at 400 V/cm and 800 V/cm in LC-PBS.

We report the design, development and characterization of new atmospheric plasma-based protocols currently being examined in vivo using small animal models. Previously developed electric field mediated drug and gene delivery protocols use a pulsed DC voltage applied between electrodes on or in tissue to generate the required electric field to induce delivery of molecules. Plasma sources deliver roughly a million times less charge to skin compared with direct electrode-driven delivery methods, yet the efficiency of the process is dramatically similar with respect to the introduction of molecules to skin cells.

Non-viral in vivo administration of plasmid DNA for vaccines and immunotherapeutics has been hampered by inefficient delivery. Methods to enhance delivery such as in vivo electroporation (EP) have demonstrated effectiveness in circumventing this difficulty. However, the contact-dependent nature of EP has resulting side effects in animals and humans. Noncontact delivery methods should, in principle, overcome some of these obstacles. This report describes a helium plasma–based delivery system that enhanced humoral and cellular antigen-specific immune responses in mice against an intradermally administered HIV gp120-expressing plasmid vaccine (pJRFLgp120). The most efficient plasma delivery parameters investigated resulted in the generation of geometric mean antibody-binding titers that were 19-fold higher than plasmid delivery alone. Plasma mediated delivery of pJRFLgp120 also resulted in a 17-fold increase in the number of interferon-gamma spot-forming cells, a measure of CD8+ cytotoxic T cells, compared with non-facilitated plasmid delivery. This is the first report demonstrating the ability of this contact-independent delivery method to enhance antigen-specific immune responses against a protein generated by a DNA vaccine.

Non-viral in vivo delivery of DNA, encoding for specific proteins, has traditionally relied on chemical or physical forces applied directly to tissues. Physical methods typically involve contact between an applicator/electrode and tissue and often results in transient subject discomfort. To overcome these limitations of contact-dependent delivery, a helium plasma source was utilized to deposit ionized gasses to treatment/vaccination sites without direct contact between the applicator and the tissues. The study reported here evaluated the efficacy of this strategy as an effective method to administer DNA vaccines. Balb/C mice were vaccinated with a DNA plasmid expressing an HIV gp120 envelope glycoprotein either with or without co-administration of helium plasma or electroporation. The results indicated, for the first time, the potential efficacy of helium plasma delivery for the induction and enhancement of antigen specific immune responses following DNA vaccination.

Low energy ion deposition to create electric fields in tissue provides an attractive means to electrically mediate and induce delivery of normally non-permeant molecules to tissue cells in vivo. Electrically mediated therapies have progressed to clinical demonstration yet require anesthetic to minimize patient discomfort. Ions generated at atmospheric pressure may be applied to the skin surface to produce an electric field in the tissue leading to an effective delivery of molecules to cells. Such processes are charge limited and therefore do not produce high current in the tissue.

Gene therapy is an attractive method for the treatment of cardiovascular disease. However, using current strategies, induction of gene expression at therapeutic levels is often inefficient. In this study, we demonstrate a novel electroporation method to enhance delivery of a plasmid expressing an angiogenic growth factor (vascular endothelial growth factor, i.e. VEGF), which previously documented to stimulate revascularization in coronary artery disease. DNA expression plasmids were delivered in vivo to the porcine heart with or without co-administered electroporation in order to determine the potential effect of electrically mediated delivery. The results demonstrated that plasmid delivery through electroporation significantly increased cardiac expression of VEGF compared to injection of plasmid alone. This is the first report demonstrating successful intracardiac delivery, through in vivo electroporation, of a protein expressing plasmid in a large animal.

Non-viral delivery of cell-impermeant drugs and DNA in vivo has traditionally relied upon either chemical or physical stress applied directly to target tissues. Physical methods typically use contact between an applicator, or electrode, and the target tissue and may involve patient discomfort. To overcome contact-dependent limitations of such delivery methodologies, an atmospheric helium plasma source was developed to deposit plasma products onto localized treatment sites. Experiments performed in murine skin showed that samples injected with plasmid DNA encoding luciferase and treated with plasma demonstrated increased levels of expression relative to skin samples that received injections of DNA alone. Increased response relative to injection alone was observed when either positive or negative voltage was used to generate the helium plasma. Quantitative results over a 26-day follow-up period showed that luciferase levels as high as 19-fold greater than the levels obtained by DNA injection alone could be achieved. These findings indicate that plasmas may compete with other physical delivery methodologies when skin is the target tissue.

Electroporation is commonly performed to deliver drugs and genes to cells comprising tissues. A possible way to control and confirm delivery is through the use of impedance spectroscopy. Ideally, this tool should not interfere with delivery and should incorporate the use of electrodes, applicators, used for delivery. This work examines impedance spectra obtained with non-penetrating surface applicators commonly used for skin electroporation. After collecting and processing over 9,000 spectra from three animal models it was determined that the electrode systems tested would have no significant effect on the obtained spectra. Therefore, electroporation applicators currently in use could be used concurrently for the collection of impedance spectra.

Electrostrictive forces on the plasma membrane of a lipid bilayer vesicle that result as a consequence of an applied electric field and differential dielectric material properties can be calculated via the Maxwell stress tensor. In this situation, the plasma membrane is proposed as a barrier that separates compartments of a system with different conductivity and relative permittivity values. A numerical model of this case is presented. Model force calculations compare with analytical equation results and were used to validate published experimental work. The model also was used to study electrostatic forces in a simple vesicle system contrasting such forces to frequency dependent deformations. Model results for vesicles in variable conductivity and relative permittivity environments are analyzed to build a framework with the potential to become a tool to study more complex problems with multiple compartments such as cells and tissues. Impedance spectroscopy is also explored as a potential experimental method to predict cell and tissue system behavior in the presence of electric fields.

Brain machine interface (BMI) devices offer a platform that can be used to assist people with extreme disabilities, such as amyotrophic lateral sclerosis (ALS) and Parkinson's disease. Silicon (Si) has been the material of choice used for the manufacture of BMI devices due to its mechanical strength, its electrical properties and multiple fabrication techniques; however, chronically implanted BMI devices have usually failed within months of implantation due to biocompatibility issues and the fact that Si does not withstand the harsh environment of the body. Single crystal cubic silicon carbide (3C-SiC) and nanocrystalline diamond (NCD) are semiconductor materials that have previously shown good biocompatibility with skin and bone cells. Like Si, these materials have excellent physical characteristics, good electrical properties, but unlike Si, they are chemically inert. We have performed a study to evaluate the general biocompatibility levels of all of these materials through the use of in vitro techniques. H4 human neuroglioma and PC12 rat pheochromocytoma cell lines were used for the study, and polystyrene (PSt) and amorphous glass were used as controls or for morphological comparison. MTT [3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide] assays were performed to determine general cell viability with each substrate and atomic force microscopy (AFM) was used to quantify the general cell morphology on the substrate surface along with the substrate permissiveness to lamellipodia extension. 3C-SiC was the only substrate tested to have good viability and superior lamellipodia permissiveness with both cell lines, while NCD showed a good level of viability with the neural H4 line but a poor viability with the PC12 line and lower permissiveness than 3C-SiC. Explanations pertaining to the performance of each substrate with both cell lines are presented and discussed along with future work that must be performed to further evaluate specific cell reactions on these substrates. Copyright © 2009 John Wiley & Sons, Ltd.

This research demonstrates that use of Nanocrystalline Diamond (NCD) microspikes in a microfluidic chamber increases the efficiency of mechanical cell lysis, as compared to a standard microfluidic surface such as glass. Microspikes made of nanocrystalline diamond were fabricated using standard MEMS techniques, and were incorporated in microfluidic chamber developed as part of a lab on a chip system. Mechanical cell lysis was performed on B16-F10 (ATCC CRL-6475) murine melanoma cells using ultrasonic vibration and the efficiency of cell lysis was determined. The microspikes puncture the cell membranes on collision greatly increasing the efficiency of cell lysis (about 400% as per fluorescence measurements) as compared to a non-textured glass surface. The effect of using cell disruption glass beads during ultrasonic lyses was also explored. This methodology of cell disruption could potentially make mechanical cell lysis a viable and preferred lysis option for lab-on-a-chip applications.

Nanocrystalline diamond (NCD) is an attractive building material for lab-on-a-chip devices due to its excellent biochemical and physical properties. In this paper we demonstrate that diamond exhibited the most resistance to non-specific DNA binding when compared to six other commonly used lab-on-a-chip building materials. This property is of critical importance to applications requiring high levels of purity and sensitivity.

The easy accessibility of skin makes it an excellent target for gene transfer protocols. To take advantage of skin as a target for gene transfer, it is important to establish an efficient and reproducible delivery system. Electroporation is an established technique for enhancing plasmid delivery to many tissues in vivo. A critical component of this technique is the electrode configuration. Electroporation parameters were optimized for transgene expression with minimal tissue damage with a novel electrode. The highest transgene expression and efficiency of individual cell transformation with minimal damage was produced with eight 150 ms pulses at field strength of 100 V/cm. This electrode design offers the potential for easier and more reproducible electrically mediated cutaneous plasmid delivery than the simple electrodes currently commercially available. This electrode can be a valuable tool in determining the applicability of electrically mediated cutaneous gene transfer.

The efficient delivery of drugs to tumors is an important tool for the treatment of a variety of cancers. Electric pulses have been shown to facilitate the uptake of molecules through the cell membrane. This procedure has been successful in increasing the effectiveness of antitumor agents (electrochemotherapy; ECT). Response rates of >80% have been obtained in both animal and human trials for several types of skin malignancies. The study reported here examined the effect of ECT on normal tissue. The hind limbs of Sprague Dawley rats were treated with 1–3 electroporation sequences in the presence or absence of the drug (bleomycin) which was administered at 4, 8 or 16 units/ml. The treated sites were examined histologically 3, 14 and 56 days later. Limb function was not affected by the treatment and skin and muscle necrosis was only seen at the higher doses.

Electric field enhanced molecular delivery for cancer research and treatment is a new technology that has demonstrated its effectiveness in clinical trials using bleomycin or cisplatin (Heller, R., Gilbert, R., Jaroszeski, M. J. Clinical applications of electrocemotherapy. Advanced Drug Delivery Reviews 35, 119–129 (1999)), as chemotherapeutic agents. The technology is being investigated in research applications for applicability as a method to enhance gene expression in a target tumor. Success is predicated on an appropriate effective electric field mediated delivery protocol that triggers significant appropriate gene expression duration and levels. An electric field mediated delivery protocol includes a set of conditions associated with the electric field, the electroporation signature, as well as parameters associated with the plasmid and the electric field applicator. Manipulation of the electrical parameters within the electroporation signature generates different gene expression levels in liver hepatocellular carcinomas. Statistically significant gene expression levels were obtained that differed by an order of magnitude when two different electric field strength and duration conditions were employed.

In vivo electroporation is currently accomplished by one of two types of common waveforms: exponential decay or square-wave pulses. The purpose of this report is to present a new electroporation waveform, the exponentially enhanced pulse (EEP). Pulsing protocols including the EEP resulted in high levels of luciferase expression in muscle and skin, equal to or greater than expression resulting from low-voltage, millisecond square-wave pulses. This high level of expression requires fewer pulses when using an EEP protocol. Therefore, similar or greater plasmid DNA expression levels are obtained using fewer pulses with the EEP protocol than with current protocols. This is the first report of this new waveform and shows the success of using protocols employing the EEP to deliver plasmid DNA to various tissue types.

The effectiveness of antineoplastic agents has been augmented by applying pulsed electric fields directly to tumours after the administration of the drug. This treatment, known as electrochemotherapy (ECT), has been successful for cutaneous malignancies in animal models and in recent clinical trials. This study was aimed at investigating the applicability of ECT in a surgical setting for hepatocellular carcinomas induced in the livers of rats. Established tumours were injected with bleomycin, and electric pulses were then administered locally. Animals were followed based on tumour volumes and histological samples. Dose response data were obtained for both electric field intensity and bleomycin. Complete response rates for animals treated with electrochemotherapy ranged from 26.67% to 93.33 and were durable. In contrast, tumours that received no treatment, pulses only or drug only responded minimally. This supports the feasibility of using a ECT as a modality for treating hepatocellular carcinoma.

Preliminary studies that used electric pulses in vivo to facilitate entry of chemotherapeutic agents into tumour cells resulted in a 69% complete response rate for hepatocellular carcinoma in rats. This success motivated a focused investigation to define the adverse effects of this treatment on normal liver tissue. Bleomycin doses ranging from 0.5 to 2.5 U and electric fields from 500 to 2250 V/cm were investigated. Electrical treatment was administered using an array of six needles arranged in a circular pattern. Necrosis and four other histological parameters were examined 14 and 56 days after treatment. Results indicated that treatment effects were localised to the volume of treated tissue. These parameters, at both time points, were not significantly altered for liver tissue that was treated with all drug doses and electric fields of 1250 V/cm and below. Only the combination of more intense electric pulses with bleomycin produced adverse histological events in the form of localised liver necrosis at day 14. These effects were not visible at day 56. Liver function was normal through all of the treatment except for an elevation of several enzymes 1 day post-treatment.

Gene therapy depends on safe and efficient gene delivery. The skin is an attractive target for gene delivery because of its accessibility. Recently, in vivo electroporation has been shown to enhance expression after injection of plasmid DNA. In this study, we examined the use of electroporation to deliver plasmid DNA to cells of the skin in order to demonstrate that localized delivery can result in increased serum concentrations of a specific protein. Intradermal injection of a plasmid encoding luciferase resulted in low levels of expression. However, when injection was combined with electroporation, expression was significantly increased. When performing this procedure with a plasmid encoding interleukin-12, the induced serum concentrations of gamma-interferon were as much as 10 fold higher when electroporation was used. The results presented here demonstrate that electroporation can be used to augment the efficiency of direct injection of plasmid DNA to skin.

When cancer cells, including melanoma cells, are genetically altered to secrete cytokines, irradiated and injected into subjects, long-term antitumour immunity is induced. Optimally, existing melanomas induced to produce cytokines in vivo could stimulate this same immune response. Although in vivo electroporation enhances plasmid expression, electroporation of plasmids encoding granulocyte-monocyte colony stimulating factor (GM-CSF) and interleukin-2 (IL2) into B16 mouse melanomas did not significantly alter tumour growth at the concentration tested. Electrochemotherapy, which causes short-term, complete regressions of treated tumour but no resistance to challenge, was combined with plasmid delivery. The combination treatment resulted in the induction of long-term immunity to recurrence and resistance to challenge in up to 25% of mice.

Gene therapy by direct delivery of plasmid DNA has several advantages over viral gene transfer, but plasmid delivery is less efficient. In vivo electroporation has been used to enhance delivery of chemotherapeutic agents to tumors in both animal and human studies. Recently, this delivery technique has been extended to large molecules such as plasmid DNA. Here, the successful delivery of plasmids encoding reporter genes to rat hepatocellular carcinomas by in vivo electroporation is demonstrated.

Electroporation is a physical event that temporarily reduces cell membrane barrier properties. Diminished membrane barrier properties are achieved by exposing cells to pulsed electric fields. When a cell has been treated with electric fields it is possible for extracellular agents to gain access to the cell interior. This process has been used in vivo to increase the uptake of chemotherapeutic agents by tumor cells which results in dramatically higher response rates than when drug is used alone. This type of treatment is called electrochemotherapy (ECT); bleomycin is most often used as the drug for this type of treatment. It was hypothesized that electroporation could be used to augment the cytotoxicity of other anticancer agents. Therefore, this study was performed in order to screen 44 different combinations of drug and cell type in vitro to identify drugs that may have higher cytotoxicity when combined with electroporation. Results from seven cell types indicate that the IC50 of bleomycin can be reduced by a factor of 100-5000 when electroporation is used to facilitate internalization. The IC50 values of cisplatin and carboplatin could be reduced by factors ranging from 3 to 13 in six different cell lines as a result of electroporation. These IC50 reductions in multiple cell lines suggest that cisplatin and carboplatin may be effective in vivo as part of ECT treatment.

Flow cytometers are instruments that are used primarily to measure the physical and biochemical characteristics of biological particles. This technology is used to perform measurements on whole cells as well as prepared cellular constituents, such as nuclei and organelles. Flow cytometers are investigative tools for a broad range of scientific disciplines because they make measurements on thousands of individual cells/particles in a matter of seconds. This is a unique advantage relative to other detection instruments that provide bulk particle measurements. Flow cytometry is a complex and highly technical field; therefore, a basic understanding of the technology is essential for all users. The purpose of this article is to provide fundamental information about the instrumentation used for flow cytometry as well as the methods used to analyze and interpret data. This information will provide a foundation to use flow cytometry effectively as a research tool.

BACKGROUND Pulsed electric fields have been shown to increase the effectiveness of antineoplastic agents by temporarily increasing the permeability of cell membranes. This type of drug delivery is called electrochemotherapy, and it has been successful in the treatment of patients with cutaneous malignancies in clinical trials. This study focused on determining the applicability of electrochemotherapy to the treatment of soft tissue sarcoma, using an animal model bearing human sarcomas. The antitumor effects of single and multiple electrochemotherapy treatments were investigated using small (250 mm3) and large (4000 mm3) tumors. METHODS Established tumors were injected with bleomycin, then electric pulses were administered to the tumor site. Animals were followed based on periodic tumor volume determinations, which were used to categorize treatment of each tumor as a complete response, a partial response, stable disease, or progressive disease. Histologic analysis was used to confirm response data. RESULTS Animals were randomly assigned to one of four different treatment groups. These groups received no treatment, drug only, electric pulses only, or drug combined with electric pulses. A single electrochemotherapy treatment protocol for small tumors resulted in a 100% complete response rate and a 41.7% cure rate. Multiple treatments of small and large tumors resulted in complete response rates of 83.3% and 100%, respectively. These responses were identical to the cure rates. In contrast, tumors in the groups that received no treatment, electric pulses only, and drug only progressed for both single treatment and multiple treatment scenarios, regardless of tumor size. CONCLUSIONS In this study, a single electrochemotherapy treatment had a strong cytoreductive effect on small tumors that lasted approximately 35 days, until recurrences began. Multiple treatment of small and large tumors resulted in high complete response rates that lasted at least 100 days after treatment. This indicates the feasibility of electrochemotherapy as a modality of limb-preserving treatment for patients with sarcoma of the extremities. Cancer 1999;85:409–17. © 1999 American Cancer Society.

BACKGROUND Electrochemotherapy (ECT) is performed by locally administering a chemotherapeutic agent in combination with electric pulses. Previous clinical studies have demonstrated the effectiveness of ECT. In these initial trials, the drug was administered intravenously, followed by administration of electric pulses directly to the tumor. This study was initiated to determine whether an intralesional injection of the drug in combination with electric pulses could provide an improved result. A group of 34 patients was studied. METHODS The dose of intralesional bleomycin was based on tumor volume. This was followed 10 minutes later by 6 or 8 99-μsec pulses of electricity at an amplitude of 1.3 kV/cm. Both the bleomycin and the electric pulses were administered after 1% lidocaine with ephinephrine solution was injected around the treatment site. RESULTS All patients responded to the treatment. Responses were observed in 142 (99%) of 143 metastatic nodules or primary tumors within 12 weeks, with complete responses observed in 130 (91%) of the nodules. No complete responses were observed in nodules treated with bleomycin only or electric pulses only. Random biopsies confirmed the clinical findings. All patients tolerated the procedure well, and no significant side effects were noted. Muscle contraction was evident during administration of each electric pulse but promptly subsided after the pulse. CONCLUSIONS ECT was shown to be an effective local treatment for cutaneous malignancies. The results suggest that ECT may have a tissue-sparing effect and result in minimal scarring. ECT may be a suitable alternative therapy for the treatment of basal cell carcinoma, local or regional recurrent melanoma, and other skin cancers. Cancer 1998;83:148-157. © 1998 American Cancer Society.

Background: A new anticancer therapy, electrochemotherapy (ECT), has been introduced that entails exposing cancerous tissues to short pulses of electricity during chemotherapy. This enhances cell membrane permeability and has been shown to have potent antitumor effects in vitro in animal models and in several clinical trials, including nevoid basal cell carcinoma (BCC). Objective: We report the effects of ECT on 20 patients with primary BCC. Methods: Electrical pulses were delivered to 54 tumors after administration of intralesional bleomycin sulfate. Results: Complete responses were observed in 53 (98%), and in the majority of these (94%) after a single treatment. No recurrences have been recorded with a mean of 18 months of observation. Conclusion: Although these are preliminary results, ECT appears to be an effective alternative to surgical excision for the treatment of primary BCC. (J Am Acad Dermatol 1997;37:596-9.)

The combined treatment consisting of a chemotherapeutic agent and pulsed electric fields has been termed electrochemotherapy. This relatively new treatment modality relies on the physical effects of locally applied electric fields to destabilize cell membranes in the presence of a drug. Membrane destabilization, electroporation, allows increased movement of molecules into the cytosol. Thus, the pulses are used to locally deliver drugs to the interior of cells. This type of treatment has principally been used to deliver bleomycin to tumor cells in vitro and in vivo. Marked antitumor effects have been reported in preclinical studies. In addition, electrochemotherapy clinical trials have been conducted for the treatment of head and neck squamous cell carcinoma, melanoma, and basal cell carcinoma. Objective response rates ranging from 72 to 100% have been reported from these trials. A review of the preclinical and clinical data for this novel drug delivery method is presented.

Electrochemotherapy (ECT), chemotherapy administered in combination with electric fields, has the potential to be an effective localized treatment for cutaneous malignancies. Bleomycin's cytotoxicity was enhanced by exposing tumour cells to electrical fields following intravenous injection of the chemotherapeutic agent. Two issues associated with this procedure are the existence of a narrow but optimal time-window for effective treatment and the fact that a systemic drug dose is administered for a localized therapy. In order to address these issues, a study was initiated to examine the effectiveness of administering bleomycin by intratumoural injection. A dose-response relationship for intratumoural injection was determined. In addition, the minimal effective field strength necessary for ECT was established. Results of this study indicated drastic reductions in tumour volume for ECT-treated groups. In addition, ECT-treated groups showed increased survival over control groups. The minimum effective dose for the ECT intratumour bleomycin group was 0.025 units. The minimum effective field strength was found to be 1250 V/cm. The results demonstrate that intratumoural injection of bleomycin in combination with electric pulses is effective, and this information will be used to initiate clinical trials.

Direct current pulses for electrochemotherapy treatment are typically administered using two parallel plate electrodes that are placed on either side of the tumor. This simple design has produced high response rates (70 to 85%) in animal studies and in clinical trials. However, parallel plate electrodes are not suitable for all situations. This study describes five novel electrode designs and compares their effectiveness to a parallel plate design for treating melanoma tumors in mice. Results for the 2 x 2 needle array design showed 50% increases in doubling time and in complete response rate compared to the standard parallel plate electrode.

The use of in vivo electroporation in combination with a chemotherapeutic agent (electrochemotherapy) for the treatment of liver tumors was examined. Induced rat hepatomas were treated with a 0.5 unit intratumor bleomycin dose followed by rectangular direct current pulses. Six pulses were administered during treatment using a needle array electrode that rotated the applied electric field around the tumors. A 84.6% objective response rate resulted for tumors that received both bleomycin and electric pulses. Control groups that received partial or no treatment showed less than 10% objective response rates. These results indicate that electrochemotherapy can be translated from the treatment of cutaneous cancers to the treatment of liver tumors and other internal tumors.

BACKGROUND Electroporation is a process that causes a transient increase in the permeability of cell membranes. It can be used to increase the intracellular concentration of chemotherapeutic agents in tumor cells (electrochemotherapy; ECT). A clinical study was initiated to determine if this mode of treatment would be effective against certain primary and metastatic cutaneous malignancies. A group of six patients, three with malignant melanoma, two with basal cell carcinoma, and one with metastatic adenocarcinoma, were enrolled in the study. The treatment was administered in a two-step process. METHODS Each patient received a 10 unit/m2 dose of bleomycin administered intravenously at 1 to 1.5 units/minute. This was followed by eight 99 μsec pulses at an amplitude of 1.3 kV/cm administered directly to the tumors 5 to 15 minutes after the bleomycin was completely infused. Pulses were administered after the injection of 1% lidocaine solution around the treatment site. RESULTS Two of three melanoma patients had objective responses. In these two patients, five of six treated tumors decreased in size, and three completely responded. Untreated tumors displayed continued growth. Objective responses were observed in both basal cell carcinoma (BCC) patients. One patient had partial responses in both treated tumors. The other patient had one of four primary BCCs respond completely, and the remaining three respond partially. Patients with metastatic breast adenocarcinoma showed complete responses in both treated nodules after ECT. All patients tolerated the treatment well with no residual effects from the electric pulses. CONCLUSIONS ECT was an effective local treatment in the majority of nodules treated. The results thus far are very encouraging and the study is being continued. Cancer 1996;964-71.

Augmented delivery of cytokine-expressing DNA plasmids to subcutaneous tumors has been demonstrated to result in a level of enhanced anti-tumor activity. One delivery enhancement method which has been evaluated is in vivo electroporation (EP), a contact-dependent delivery technique where electric pulses are hypothesized to augment the transfer of DNA into cells and tissues through the induction of temporary cell membrane pores. Previous work by members of our group, as well as others, has demonstrated the anti-tumor effects of DNA plasmids expressing the cytokines IL-12 and IL-15. In this report the potential anti-tumor activity of a relatively newly-described cytokine, IL-28, was measured when administered intratumorally as a DNA expression plasmid (designated pIL28) to established murine (B16.F10) melanoma tumors. The administration of the IL-28 expressing plasmid was performed through enhanced delivery methods. One method was EP and the other a non-contact dependent technique using a helium plasma stream. IL-28 is a member of the type III interferon family of cytokines that has been characterized as possessing potent anti-viral activity. This cytokine has been demonstrated to function as an adjuvant in small animal model vaccination protocols and stimulates CD8+ CTL responses. In addition, stimulation of anti-tumor activity has been demonstrated in several studies using IL-28. Based on these activities, it was hypothesized that this cytokine could, when delivered through a DNA expression plasmid, mediate anti-tumor activity. The results of this study indicated that enhanced delivery of pIL-28 resulted in attenuation of tumor growth, compared with non-enhanced delivery. Of note, this is the first proof-of-concept experiment, of our knowledge, documenting the ability of a non-contact dependent helium plasma-based delivery method to mediate the enhancement of an anti-tumor effect by a cytokine-expressing DNA plasmid. This suggests the use of the helium plasma delivery method as an alternative or adjunctive method to EP for the effective delivery of agents that possess potential anti-tumor activity.

Silicon Carbide (SiC), has been shown to be a bio- and hema-compatible substrate that could potentially be used in biosensor applications. The development of a viable biorecognition interface using SiC as the substrate material for bio-detection is described. Surface modification with 3-aminopropyltriethoxysilane (APTES) and immobilization via covalent conjugation of antimyoglobin (anti-Myo) on the modified surfaces is achieved, which are initial steps for immunosensing based devices. Successful formation of APTES layers and antibody immobilization were identified with surface water contact angle (SWCA), X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM).

In this work we describe the characterization of anti-myoglobin immobilization on 3C-SiC (100) by means of surface modification with 3-aminopropyltriethoxysilane (APTES). Surface water contact angle measurements were used to compare the wettability of 3C-SiC (100) before (16 ±3°) and after APTES layer formation (61 ±1°). Atomic force microscopy (AFM) was used to confirm the homogenous formation of APTES and anti-myoglobin immobilization with EDC-sulfo NHS coupling. For the APTES surfaces no significant change in the surface roughness was obtained whereas with anti-myoglobin surfaces, particles on the order of ∼60 nm in diameter with a globular shape were observed.

Cell–semiconductor hybrid systems are a potential centerpiece in the scenery of biotechnological applications. The selection and study of suitable semiconductor materials are at the basis of the development of such hybrid systems. This work introduces crystalline SiC as an extremely promising biomaterial. To this end, epidermal and connective tissue cells are cultured directly on single-crystal SiC substrates. The resulting cell adhesion quality and proliferation indicate that SiC is indeed a candidate material for bioapplications, with better performance than crystalline Si, the leading semiconductor in biotechnology. Since a material’s biocompatibility is influenced by chemical, morphological, and electrostatic factors, the effect that different chemistries, morphologies, and wettabilities of SiC surfaces have on cell adhesion and proliferation is also explored. Moreover, the importance of using an appropriate cleaning procedure for the SiC samples before their use as substrates for cell cultures is discussed.

Publisher Summary This chapter describes the study of the hemocompatibility of single-crystal SiC, which was conducted at the University of South Florida. It begins with an overview of biocompatibility vis a-vis the cardiovascular system. The research is focused along three complementary tracks—blood platelet adhesion, protein attachment, and endothelial cell adhesion. Biocompatibility is the required property of materials and systems that are intended to be in contact with biological matter and to perform according to the system design, and without interfering with the integrity of the tissue(s) and biological matter that such material is in contact with and without generating any adverse reactions. Silicon (Si) has long been used in the construction of microelectromechanical systems (MEMS) devices. The limitations of how compatible a material is when in contact with tissue(s) are determined by the application, such as tissue engineering, implantable sensors, and drug delivery. To date, few crystalline semiconductor materials have been assessed for their biocompatibility. Crystalline semiconductor materials are of special interest for the development of biomedical sensors that can be in intimate contact with the human body. Therefore biocompatible sensing materials are critical to the development of implantable biomedical devices and this research was dedicated to evaluating the biocompatibility of such materials for use in the cardiovascular system.

Ion-based strategies have recently emerged as a method to facilitate molecular delivery. These methods are attractive as they separate the applicator from the treatment site avoiding some issues encountered with other electrically driven methods. Current literature on plasma delivery has shown utility in vitro and in vivo for both drugs and genes. To advance this technology more information must become available on the mechanism responsible for delivery and the effects of ion exposure on eukaryotic cells. This in vitro investigation found that molecular delivery facilitated by a DC-based plasma follows a dose-response behavior, with optimum uptake of Sytox Green occurring in two cell lines after 600 s of exposure. In both cell lines exposure to the discharge caused no adverse effects in viability for exposure times up to 600 s. It was also found that membranes treated with ions remained permeabilized for several minutes following plasma treatment and that membrane resealing exhibited first order kinetics.

The easy accessibility of skin makes it an excellent target for gene transfer protocols. To take full advantage of skin as a target for gene transfer, it is important to establish an efficient and reproducible delivery system. Electroporation is a strong candidate to meet this delivery criterion. Electroporation of the skin is a simple, direct, in vivo method to deliver genes for therapy. Previously, delivery to the skin was performed by means of applicators with relatively large distances between electrodes, resulting in significant muscle stimulation and pain. These applicators also had limitations in controlling the directionality of the applied field. To resolve this issue, a system consisting of an array of electrodes that decreased the distance between them and that were independently addressable for directional control of the field was developed. This new multielectrode array (MEA) was compared with an established electrode. In a rat model, comparable reporter expression was seen after delivery with each electrode. Delivery was also evaluated in a guinea pig model to determine the potential of this approach in an animal model with skin thickness and structure similar to human skin. The results clearly showed that effective delivery was related to both the electrode and the parameters chosen. With the MEA, the muscle twitching associated with application of electric fields was notably reduced compared with conventional electrode systems. This is important, as it will facilitate the translation of electroporation-mediated gene delivery to skin for clinical use with DNA vaccines or for therapies for cancer or protein deficiencies.

The promoter elements and transcription factors necessary for triiodothyronine (T{sub 3}) induction of hepatic HMG-CoA reductase (HMGR) were investigated by transfecting rat livers with wild type and mutant HMGR promoter-luciferase constructs using in vivo electroporation. Mutations in the sterol response element (SRE), nuclear factor-y (NF-Y) site, and the newly identified upstream transcription factor-2 (USF-2) site essentially abolished the T{sub 3} response. Chromatin immunoprecipitation (ChIP) analysis demonstrated that T{sub 3} treatment caused a 4-fold increase in in vivo binding of USF-2 to the HMGR promoter. Co-transfection of the wild type HMGR promoter with siRNAs to USF-2, SREBP-2, or NF-Y nearly abolished the T{sub 3} induction, as measured by promoter activity. These data provide in vivo evidence for functional roles for USF-2, SREBP-2, and NF-Y in mediating the T{sub 3}-induction of hepatic HMGR transcription.

The easy accessibility of skin makes it an excellent target for gene transfer protocols. To fully take advantage of skin as a target for gene transfer, it is important to establish an efficient and reproducible delivery system. Electroporation as a tool for delivery of plasmid DNA is a strong candidate to meet these delivery criteria. Electroporation of the skin is a simple, direct, in vivo method to deliver genes for therapy. Efficient delivery to the skin can be utilized for both prophylactic and therapeutic approaches. In previous work, we evaluated delivery conditions to the skin utilizing a plasmid encoding luciferase. These studies were performed in various animal models. Most recently, the work has been performed in a guinea pig model to evaluate the potential of this approach in an animal model with skin thickness and structure similar to human. Results clearly showed that effective delivery was related to both the electrode and the parameters chosen. For delivery of DNA vaccines, a multielectrode array was utilized to deliver a plasmid encoding hepatitis b surface antigen resulted in high antibody levels. Use of this array reduced muscle twitches associated with administration of pulses. This approach was also assessed for delivery of angiogenic growth factors to the skin in areas of ischemia. For this work a 4-plate electrode was utilized with parameters for therapeutic delivery of angiogenic growth factors such as vascular endothelial growth factor (VEGF). Delivery resulted in accelerated wound healing when compared to no treatment or injection of plasmid without electroporation. These results further demonstrate that electroporation can be used to augment the efficiency of direct injection of plasmid DNA to skin and may have utility in several therapeutic applications.

A novel method of inducing the delivery of nonpermeant molecules to the cytosol of cells is presented in this paper. Corona discharge in air was utilized to produce ions that in turn were deposited onto the liquid surface of media containing cultured cells. Murine B16 melanoma cells were used to demonstrate the molecular delivery of fluorescent dye calcein, the drug bleomycin, and a nucleic acid stain SYTOX-green. None of these molecules penetrate cells with intact membranes. Following the corona treatment, cells were observed to admit significant quantities of these molecules from the culture media, relative to control samples. Further, greater than 95% viability of treated cells was observed by Trypan Blue assay. This method may provide an attractive alternative to electroporation where a physical contact between electrodes and cells is needed to deliver molecules to the cytosol.

Electric field mediated drug and gene delivery is a novel method that uses pulsed electric fields to improve permeability of cell membranes and therefore desired agent uptake by tissues. In this paper, we describe the modeling and experimental proof of concept of a method to direct electric fields to subsequently focus drug or gene uptake at a desired site. The in vitro experimental results presented are consistent with simulation models and could be scaled into different in vivo applications that can concentrate the effects of electroporation and overcome several problems related to localized effects near the electrodes.