Trends in Musculoskeletal Ablation: Emerging Indications and Techniques.

Image-guided percutaneous thermal ablation plays an increasingly important role in the multidisciplinary management of musculoskeletal lesions. Established indications for ablation in this setting include the treatment of osteoid osteomas, palliation of painful skeletal metastases, local control of oligometastatic disease, and consolidation of bone tumors at risk for fracture. Emerging indications include the treatment of symptomatic soft tissue masses such as extra-abdominal desmoid tumors and abdominal wall endometriosis. This review will discuss considerations in patient selection and preprocedural workup, ablation technology and techniques, strategies to avoid complications, and expected outcomes of ablation in the musculoskeletal system.

Methods for protein delivery into cells: from current approaches to future perspectives.

The manipulation of cultured mammalian cells by the delivery of exogenous macromolecules is one of the cornerstones of experimental cell biology. Although the transfection of cells with DNA expressions constructs that encode proteins is routine and simple to perform, the direct delivery of proteins into cells has many advantages. For example, proteins can be chemically modified, assembled into defined complexes and subject to biophysical analyses prior to their delivery into cells. Here, we review new approaches to the injection and electroporation of proteins into cultured cells. In particular, we focus on how recent developments in nanoscale injection probes and localized electroporation devices enable proteins to be delivered whilst minimizing cellular damage. Moreover, we discuss how nanopore sensing may ultimately enable the quantification of protein delivery at single-molecule resolution.

Electroquimioterapia para el tratamiento del melanoma cutáneo locorregionalmente avanzado irresecable. Revisión sistemática / Electrochemotherapy for the treatment of unresectable locoregionally advanced cutaneous melanoma: a systematic review

La electroquimioterapia (EQT) es una modalidad de tratamiento de lesiones cutáneas y subcutáneas originadas por melanoma u otros tumores. El procedimiento consiste en la administración de agentes antineoplásicos, seguido de impulsos eléctricos locales, para conseguir un efecto conocido como electroporación, que permite la entrada al citosol de medicamentos que no difunden a través de la membrana celular. El objetivo de esta revisión es establecer la evidencia que sustenta la incorporación de la EQT como estrategia terapéutica en el melanoma. Además, se ha llevado a cabo una revisión sistemática de la literatura con síntesis cualitativa. Se ha realizado una búsqueda cualificada de la literatura en bases de datos referenciales y a texto completo. Fueron seleccionados 7 estudios: 3 revisiones sistemáticas y 4 series de casos. La calidad de la evidencia encontrada no es buena, pero la coincidencia de sus resultados en algunas las variables le da consistencia. Los metaanálisis muestran resultados a favor de la EQT frente a la quimioterapia. La EQT parece un procedimiento efectivo en el tratamiento local de nódulos tumorales malignos (nivel medio o bajo de calidad de la evidencia). Es un tratamiento fácil de administrar, económico y bien tolerado con el que se consigue respuesta objetiva en circunstancias determinadas. No hay evidencia de que pueda afectar el curso natural de la enfermedad, por lo que debe considerarse un tratamiento paliativo. Con un nivel de la evidencia 1- (1 menos), puede recomendarse la incorporación de la EQT para el tratamiento paliativo del melanoma locorregionalmente avanzado irresecable (fuerza de la recomendación: B)

Electrochemotherapy is a therapeutic option for the treatment of cutaneous and subcutaneous metastases from melanoma and other tumors. The procedure consists of the administration of anticancer drugs followed by locally applied electrical impulses to achieve an effect known as electroporation, which facilitates entry into the cytosol of drugs that cannot cross the cell membrane. The aim of this review is to evaluate the evidence that supports the use of electrochemotherapy as a therapeutic strategy in melanoma. We conducted a qualitative systematic review of the literature using advanced searches of bibliographic databases and full text reviews. Seven studies (3 systematic reviews and 4 cases series) were selected. The quality of the evidence was not good, but the coincidence of results for certain variables supports their consistency. Results of the meta-analyses favored electrochemotherapy over chemotherapy. Electrochemotherapy appears to be an effective procedure for the local treatment of malignant tumor nodules (evidence of intermediate or low quality). This inexpensive method is simple to apply, well tolerated, and achieves objective responses under certain circumstances. There is no evidence that electrochemotherapy alters the natural course of the disease and it should therefore be considered a palliative treatment. With an evidence level of 1- (minus), electrochemotherapy can be recommended for the palliative treatment of unresectable, locoregionally advanced melanoma (grade B recommendation)

Recent advances in physical delivery enhancement of topical drugs.

The skin has evolved to resist the penetration of foreign substances and particles. Effective topical drug delivery into and/or through the skin is hindered by these epidermal barriers. A range of physical enhancement methods has been developed to selectively overcome this barrier. This review discusses recent advances in physical drug delivery by broadly separating the techniques into two main areas; indirect and direct approaches. Indirect approaches consist of electrical, vibrational or laser instrumentation that creates pores in the skin followed by application of the drug. Direct approaches consist of mechanical disruption of the epidermis using techniques such as microdermabrasion, biolistic injectors and microneedles. Although, in general, physical techniques are yet to be established in a clinical setting, the potential gains of enhancing delivery of compounds through the skin is of great significance and will no doubt continue to receive much attention.

Strategies for skeletal muscle targeting in drug discovery.

The targeting of drugs to skeletal muscle is an emerging area of research. Driven by the need for new therapies to treat a range of muscle-associated diseases, these strategies aim to provide improved drug exposure at the site of action in skeletal muscle with reduced concentration in other tissues where unwanted side effects could occur. By interacting with muscle-specific cell surface recognition elements, both tissue localization and selective uptake into skeletal muscle cells can be achieved. The design of molecules that are substrates for muscle uptake transporters can provide concentration in m uscle tissue. For example, drug conjugates with carnitine can provide improved muscle uptake via OCTN2 transport. Binding to muscle surface recognition elements followed by endocytosis can allow even large molecules such as antibodies to enter muscle cells. Monoclonal antibody 3E10 demonstrated selective uptake into skeletal muscle in vivo. Hybrid adeno-associated viral vectors have recently shown promise for high skeletal muscle selectivity in gene transfer applications. Delivery technology methods, including electroporation of DNA plasmids, have also been investigated for selective muscle uptake. This review discusses challenges and opportunities for skeletal muscle targeting, highlighting specific examples and areas in need of additional research.

Current developments and clinical applications of bubble technology in Japan: a report from 85th Annual Scientific Meeting of The Japan Society of Ultrasonic in Medicine, Tokyo, 25-27 May, 2012.

The potentials of bubble technology in ultrasound has been investigated thoroughly in the last decade. Japan has entered as one of the leaders in bubble technology in ultrasound since Sonazoid (Daiichi Sankyo & GE Healthcare) was marketed in 2007. The 85th Annual Scientific Meeting of The Japan Society of Ultrasonics in Medicine held in Tokyo from May 25 to 27, 2012 is where researchers and clinicians from all over Japan presented recent advances and new developments in ultrasound in both the medical and the engineering aspects of this science. Even though bubble technology was originally developed simply to improve the conventional ultrasound imaging, recent discoveries have opened up powerful emerging applications. Bubble technology is the particular topic to be reviewed in this report, including its mechanical advances for molecular imaging, drug/gene delivery device and sonoporation up to its current clinical application for liver cancers and other liver, gastrointestinal, kidney and breast diseases.

Therapeutic potential of irreversible electroporation in sarcoma.

Irreversible electroporation is a newly developed nonthermal tissue ablation technique in which certain short-duration electrical fields are used to permanently permeabilize the cell membrane to disrupt cellular homeostasis. This disruption of cellular homeostasis initiates apoptosis, which leads to permanent cell death. Sarcomas are generally divided into soft-tissue and bone sarcomas based on their different mesenchymal origins and anatomical locations. Each of these sarcomas present in different ways, exhibit different behaviors and prognoses, and present unique therapeutic challenges. In this article, a series of recently conducted irreversible electroporation treatment for sarcomas based on local nonthermal ablation are summarized, and the therapeutic potential of this newly developed technique is assessed.

Towards the mechanisms for efficient gene transfer into cells and tissues by means of cell electroporation.

INTRODUCTION: Intracellular gene electrotransfer by means of electroporation has been on the increase during the past decade. Significant progress has been achieved both in characterizing mechanisms of gene electrotransfer and in optimizing the protocol in many preclinical trials. Recently this has led to initiation of clinical trials of gene electrotransfer to treat metastatic melanomas. Further progress with the method in various clinical trials requires better understanding of mechanisms of gene electrotransfer. AREAS COVERED: A summary of recent progress in understanding mechanisms of gene electrotransfer, imparting general knowledge of cell electroporation and intracellular molecule electrotransfer. EXPERT OPINION: Gene electrotransfer into cells and tissues is a complex process involving multiple steps that lead to plasmid DNA passage from the extracellular region to the cell nucleus crossing the barriers of the plasma membrane, cytoplasm and nucleus membrane. Electrical parameters of pulses used for gene electrotransfer affect the initial steps of DNA translocation through the plasma membrane and play a crucial role in determining the transfection efficiency. When considering gene electrotransfer into tissues it becomes clear that other nonelectrical conditions are also of primary importance.

Electroporation based gene therapy--from the bench to the bedside.

A critical aspect of gene transfer is effective delivery of the transgene to the appropriate target. Electrically mediated delivery (electroporation) of plasmid DNA has been accepted as a viable approach to achieve effective delivery. One promising area is delivering plasmid DNA to skin. Gene transfer to the skin with electroporation is currently being evaluated for its potential for inducing angiogenesis for wound healing and for delivering DNA vaccines to the skin. Experiments utilizing a plasmid encoding for vascular endothelial growth factor has demonstrated how wound healing could be accelerated. In another study, delivery of a plasmid encoding Hepatitis B surface antigen have demonstrated that high antibody titers can be induced after two applications (prime/boost). Our laboratory has also examined the use of electroporation to delivery plasmid DNA encoding various cytokines as a potential therapy for melanoma. The plasmid is injected directly into the tumor followed by the administration of electroporation. Extensive preclinical work provided the rationale for a Phase I proof of concept first in human trial in patients with accessible cutaneous melanoma metastases. Biopsies of treated lesions showed significant necrosis of melanoma cells within the tumor as well as IL-12 expression. Lymphocytic infiltrate was observed in biopsies from patients in several cohorts. Clinical evidence of responses in untreated lesions suggested there was a systemic response following therapy was observed. Since this trial several other clinical studies utilizing electroporation to deliver plasmid DNA have been initiated. It is clear that this delivery approach has tremendous potential to facilitate the translation of gene transfer protocols from the bench to the bedside.

Basic principles and clinical advancements of muscle electrotransfer.

Muscle electrotransfer covers the delivery of molecules to muscle tissue by means of electric pulses. This method has proven highly efficient in transferring, in particular, plasmid DNA to muscles, resulting in long-term expression of the transferred genes. DNA electrotransfer to muscle tissue has clinical potential within DNA vaccination, systemic delivery of therapeutic proteins and correction of gene defects in muscles. In the recent years, DNA electrotransfer to muscle tissue has reached clinical advancement with 8 on-going clinical trials. In the present review, I will draw on the experiences obtained from the clinical studies, in understanding the mechanistic and practical advantages and limits of muscle electrotransfer. The effect of applying electric pulses to muscle tissue will be described in details, while present and future clinical applications are reviewed.

Sonoporation, drug delivery, and gene therapy.

Ultrasound is a very effective modality for drug delivery and gene therapy because energy that is non-invasively transmitted through the skin can be focused deeply into the human body in a specific location and employed to release drugs at that site. Ultrasound cavitation, enhanced by injected microbubbles, perturbs cell membrane structures to cause sonoporation and increases the permeability to bioactive materials. Cavitation events also increase the rate of drug transport in general by augmenting the slow diffusion process with convective transport processes. Drugs and genes can be incorporated into microbubbles, which in turn can target a specific disease site using ligands such as the antibody. Drugs can be released ultrasonically from microbubbles that are sufficiently robust to circulate in the blood and retain their cargo of drugs until they enter an insonated volume of tissue. Local drug delivery ensures sufficient drug concentration at the diseased region while limiting toxicity for healthy tissues. Ultrasound-mediated gene delivery has been applied to heart, blood vessel, lung, kidney, muscle, brain, and tumour with enhanced gene transfection efficiency, which depends on the ultrasonic parameters such as acoustic pressure, pulse length, duty cycle, repetition rate, and exposure duration, as well as microbubble properties such as size, gas species, shell material, interfacial tension, and surface rigidity. Microbubble-augmented sonothrombolysis can be enhanced further by using targeting microbubbles.

Electroporation: an avenue for transdermal drug delivery.

The stratum corneum (SC) is a primary rate limiting barrier to permeation of drug molecules through the skin. Small molecular weight lipophilic drugs that are effective at low doses can be effectively delivered by passive transdermal delivery. The SC does not permit passage of polar/hydrophilic and macromolecules. Passive and physical penetration enhancements strategies are used to overcome this barrier property of the SC. Passive penetration enhancement techniques include use of supersaturated solutions and penetration enhancers. In general, the drug delivery potential of chemical modalities is limited. Therefore, physical permeation enhancement techniques gained a lot of focus in the recent past. Physical penetration enhancement techniques include iontophoresis, electroporation and sonophoresis. Electroporation utilizes high voltage pulses that are applied for a very short time to permeabilize the skin to facilitate transport of macromolecules and hydrophilic compounds. Several drugs have been administered via this system successfully. This review presents an overview of in-vitro and in-vivo studies demonstrating therapeutic benefits offered by electroporation assisted permeation. Factors affecting electroporation, synergism between electroporation and other penetration enhancing strategies are also discussed.

Microscale electroporation: challenges and perspectives for clinical applications.

Microscale engineering plays a significant role in developing tools for biological applications by miniaturizing devices and providing controllable microenvironments for in vitro cell research. Miniaturized devices offer numerous benefits in comparison to their macroscale counterparts, such as lower use of expensive reagents, biomimetic environments, and the ability to manipulate single cells. Microscale electroporation is one of the main beneficiaries of microscale engineering as it provides spatial and temporal control of various electrical parameters. Microscale electroporation devices can be used to reduce limitations associated with the conventional electroporation approaches such as variations in the local pH, electric field distortion, sample contamination, and the difficulties in transfecting and maintaining the viability of desired cell types. Here, we present an overview of recent advances of the microscale electroporation methods and their applications in biology, as well as current challenges for its use for clinical applications. We categorize microscale electroporation into microchannel and microcapillary electroporation. Microchannel-based electroporation can be used for transfecting cells within microchannels under dynamic flow conditions in a controlled and high-throughput fashion. In contrast, microcapillary-based electroporation can be used for transfecting cells within controlled reaction chambers under static flow conditions. Using these categories we examine the use of microscale electroporation for clinical applications related to HIV-1, stem cells, cancer and other diseases and discuss the challenges in further advancing this technology for use in clinical medicine and biology.

Nucleic acids electrotransfer-based gene therapy (electrogenetherapy): past, current, and future.

About 25 years after the publication of the first report on gene transfer in vitro in cultured cells by the means of electric pulses delivery, reversible cell electroporation for gene transfer and gene therapy (DNA electrotransfer) is at a cross in its development. Present knowledge on the effects of cell exposure to appropriate electric field pulses, particularly at the level of the cell membrane, is reported here. The importance of the models of electric field distribution in tissues and of the correct choice of electrodes and applied voltages is highlighted. The mechanisms involved in DNA electrotransfer, which include cell electropermeabilization and DNA electrophoresis, are also surveyed. This knowledge has allowed developing new nucleic acids electrotransfer conditions using combinations of permeabilizing pulses of high voltage and short duration, and of electrophoretic pulses of low voltage and long duration, which are very efficient and safer. Feasibility of electric pulses delivery for gene transfer in humans is discussed taking into account that electric pulses delivery is already regularly used for localized drug delivery in the treatment of cutaneous and subcutaneous solid tumors by electrochemotherapy. Because recent technological developments made DNA electrotransfer more and more efficient and safer, this non-viral gene therapy approach is now ready to reach the clinical stage. A good understanding of DNA electrotransfer principles and the respect of safe procedures will be key elements for a successful future transfer DNA electrotransfer into the clinics.

Transdermoterapia por electroporación en la lipólisis abdominal / Transdermotherapy by electroporation in abdominal lipolysis

Este estudio presenta los efectos conseguidos por la electroporación en la lipólisis abdominal utilizando la vehiculación de la fosfatidilcolina (fosfolípido que facilita la absorción de las grasas). Se llevó a cabo en 10 mujeres voluntarias, nulíparas, sedentarias, con una edad media de 25,10 años e índice de masa corporal entre 18,5 y 25 kg/m2. Recibieron 15 sesiones de tratamiento fisioterápico, constando esta de una aplicación tópica de fosfatidilcolina al 10% liposomada en el abdomen con electroporación. La técnica consistió en colocar el transductor del equipo en la pared abdominal emitiendo ondas electromagnéticas con un voltaje de 500 mV y una frecuencia de 50 Hz durante 30 minutos. Este tratamiento alcanzó una reducción del tejido adiposo subcutáneo de la pared abdominal, comprobada por medida perimétrica del abdomen, medida del pliegue cutáneo infraumbilical y por ultrasonografía. En la perimetría, la reducción media fue de 4,75 cm, en la plicometría de 2,43 mm y la ultrasonografía demostró una disminución del grosor del tejido adiposo que pasó de una media de 2,21 cm a 1,65 cm. Sin embargo, no se halló ninguna reducción ponderal significativa, aunque la disminución presentada en los tres métodos de evaluación sugiere que la utilización de la fosfatidilcolina con la electroporación puede desencadenar efectos lipolíticos(AU)

The present study presents the effects achieved with electroporation in abdominallipolysis using vehiculization of phosphatidylcholine (phospholipide that facilitates fatabsorption). The study was conducted in 10 voluntary women, nulliparous, sedentary women, with a mean age of 25.10 years and body mass index between 18.5 and 25 kg/m2.They were administered 15 sessions of physiotherapy, this being made up of a topicalapplication of 10% liposomal phosphatidylcholine in the abdomen with electroporation.The technique consisted in placing the equipment transductor on the abdominal wall,emitting electromagnetic waves with a 500mV voltage and 50 Hz frequency for 30 minutes.This treatment achieved a reduction of the subcutaneous adipose tissue of the abdominalwall, verified by perimetric measurement of the abdomen, measurement of infraumbilicalskin fold and by ultrasound. In the perimetry, the mean reduction was 4.75 cm, in theplicometry 2.43mm and the ultrasonograph showed a decrease of adipose tissue thicknessthat went from a mean of 2.21 cm to 1.65 cm. However, no significant weight reduction wasfound, although the decrease found in the three evaluation methods suggests that the useof phosphatidylcholine with electroporation may precipitate lipolitic effects(AU)

Application of electroporation gene therapy: past, current, and future.

Twenty-five years after the publication of the first report on gene transfer in vitro in cultured cells by the means of electric pulse delivery, reversible cell electroporation for gene transfer and gene therapy (DNA electrotransfer) is at a crossroad in its development. Present knowledge on the effects of cell exposure to appropriate electric field pulses, particularly at the level of the cell membrane, is reported here as an introduction to the large range of applications described in this book. The importance of the models of electric field distribution in tissues and of the correct choice of electrodes and applied voltages is highlighted. The mechanisms involved in DNA electrotransfer, which include cell electropermeabilization and DNA electrophoresis, are also surveyed. The feasibility of electric pulse for gene transfer in humans is discussed taking into account that electric pulse delivery is already regularly used for localized drug delivery in the treatment of cutaneous and subcutaneous solid tumors by electrochemotherapy. Because recent technological developments have made DNA electrotransfer more efficient and safer, this nonviral gene therapy approach is now ready to reach the clinical stage. A good understanding of DNA electrotransfer principles and a respect for safe procedures will be key elements for the successful future transition of DNA electrotransfer to the clinics.

Applicator and electrode design for in vivo DNA delivery by electroporation.

As in vivo electroporation advances from the preclinical phase to clinical studies and eventually to routine medical practice, the design of electroporation devices becomes increasingly important. Achieving safety and efficacy levels that meet regulatory requirements, as well as user and patient friendliness, are major design considerations. In addition, the devices will have to be economical to manufacture. This chapter will focus on the design of applicators and electrodes, the pieces of hardware in direct contact with the user and the patient, and thus key elements responsible for the safety and efficacy of the procedure. The two major foreseeable applications of the technology in the DNA field are for gene therapy and DNA vaccination. Design requirements differ considerably for these applications and for the diseases to be treated or prevented. In addition to the trend of device differentiation, there is also a trend to build devices capable of performing both the step of delivering the DNA to the target tissue and the subsequent step of electroporation. This chapter presents the electrical and biological principles underlying applicator and electrode design, gives an overview of existing devices, and discusses their advantages and disadvantages. The chapter also outlines major design considerations, including regulatory pathways, and points out potential future developments.

Electrotransfer as a non viral method of gene delivery.

Over the last few decades, various vectors have been developed in the field of gene therapy. There still exist a number of important unresolved problems associated with the use of viral as well as non viral vectors. These techniques can suffer from secondary toxicity or low gene transfer efficiency. Therefore an efficient and safe method of DNA delivery still needs to be found for medical applications. DNA electrotransfer is a physical method that consists of the local application of electric pulses after the introduction of DNA into the extra cellular medium. As electrotransfer has proven to be one of the most efficient and simple non viral methods of delivery, it may provide an important alternative technique in the field of gene therapy. The present review focuses on questions related to the mechanism of DNA electrotransfer, i.e. the basic physical processes responsible for the electropermeabilisation of lipid membranes. It also addresses the current limitations of the method as applied to DNA transfer, in particular its efficiency in achieving in vitro gene expression in cells and also its potential use for in vivo gene delivery.

Electrotransfer into skeletal muscle for protein expression.

An efficient and safe method to deliver DNA in vivo is a requirement for several purposes, such as study of gene function and gene therapy applications. Among the different non-viral delivery methods currently under investigation, in vivo DNA electrotransfer has proven to be one of the most efficient and simple. This technique is a physical method of gene delivery consisting in local application of electric pulses after DNA injection. Although this technique can be applied to almost any tissue of a living animal, including tumors, skin, liver, kidney, artery, retina, cornea or even brain, this review will focus on electrotransfer of plasmid DNA into skeletal muscle and its possible uses in gene therapy, vaccination, or functional studies. Skeletal muscle is a good target for electrotransfer of DNA as it is: a large volume easily accessible, an endocrine organ capable of expressing several local and systemic factors, and muscle fibres as post-mitotic cells have a long lifespan that allows long-term gene expression. In this review, we describe the mechanism of DNA electrotransfer, we assess toxicity and safety considerations related to this technique, and we focus on important therapeutic applications of electrotransfer demonstrated in animal models in recent years.

New insights in the visualization of membrane permeabilization and DNA/membrane interaction of cells submitted to electric pulses.

Electropermeabilization designates the use of electric pulses to overcome the barrier of the cell membrane. This physical method is used to transfer anticancer drugs or genes into living cells. Its mechanism remains to be elucidated. A position-dependent modulation of the membrane potential difference is induced, leading to a transient and reversible local membrane alteration. Electropermeabilization allows a fast exchange of small hydrophilic molecules across the membrane. It occurs at the positions of the cell facing the two electrodes on an asymmetrical way. In the case of DNA transfer, a complex process is present, involving a key step of electrophoretically driven association of DNA only with the destabilized membrane facing the cathode. We report here at the membrane level, by using fluorescence microscopy, the visualization of the effect of the polarity and the orientation of electric pulses on membrane permeabilization and gene transfer. Membrane permeabilization depends on electric field orientation. Moreover, at a given electric field orientation, it becomes symmetrical for pulses of reversed polarities. The area of cell membrane where DNA interacts is increased by applying electric pulses with different orientations and polarities, leading to an increase in gene expression. Interestingly, under reversed polarity conditions, part of the DNA associated with the membrane can be removed, showing some evidence for two states of DNA in interaction with the membrane: DNA reversibly associated and DNA irreversibly inserted.