Fibroblasts transfection by electroporation in 3D reconstructed human dermal tissue.

The understanding of the mechanisms involved in DNA electrotransfer in human skin remains modest and limits the clinical development of various biomedical applications, such as DNA vaccination. To elucidate some mechanisms of DNA transfer in the skin following electroporation, we created a model of the dermis using a tissue engineering approach. This model allowed us to study the electrotransfection of fibroblasts in a three-dimensional environment that included multiple layers of fibroblasts as well as the self-secreted collagen matrix. With the aim of improving transfection yield, we applied electrical pulses with electric field lines perpendicular to the reconstructed model tissue. Our results indicate that the fibroblasts of the reconstructed skin tissue can be efficiently permeabilized by applied millisecond electrical pulses. However, despite efficient permeabilization, the transfected cells remain localized only on the surface of the microtissue, to which the plasmid was deposited. Second harmonic generation microscopy revealed the extensive extracellular collagen matrix around the fibroblasts, which might have affected the mobility of the plasmid into deeper layers of the skin tissue model. Our results show that the used skin tissue model reproduces the structural barriers that might be responsible for the limited gene electrotransfer in the skin.

Therapeutic perspectives of high pulse repetition rate electroporation.

Electroporation, a technique that uses electrical pulses to temporarily or permanently destabilize cell membranes, is increasingly used in cancer treatment, gene therapy, and cardiac tissue ablation. Although the technique is efficient, patients report discomfort and pain. Current strategies that aim to minimize pain and muscle contraction rely on the use of pharmacological agents. Nevertheless, technical improvements might be a valuable tool to minimize adverse events, which occur during the application of standard electroporation protocols. One recent technological strategy involves the use of high pulse repetition rate. The emerging technique, also referred as "high frequency" electroporation, employs short (micro to nanosecond) mono or bipolar pulses at repetition rate ranging from a few kHz to a few MHz. This review provides an overview of the historical background of electric field use and its development in therapies over time. With the aim to understand the rationale for novel electroporation protocols development, we briefly describe the physiological background of neuromuscular stimulation and pain caused by exposure to pulsed electric fields. Then, we summarize the current knowledge on electroporation protocols based on high pulse repetition rates. The advantages and limitations of these protocols are described from the perspective of their therapeutic application.

Effects of Nanosecond Pulsed Electric Field (nsPEF) on a Multicellular Spheroid Tumor Model: Influence of Pulse Duration, Pulse Repetition Rate, Absorbed Energy, and Temperature.

Cellular response upon nsPEF exposure depends on different parameters, such as pulse number and duration, the intensity of the electric field, pulse repetition rate (PRR), pulsing buffer composition, absorbed energy, and local temperature increase. Therefore, a deep insight into the impact of such parameters on cellular response is paramount to adaptively optimize nsPEF treatment. Herein, we examined the effects of nsPEF ≤ 10 ns on long-term cellular viability and growth as a function of pulse duration (2-10 ns), PRR (20 and 200 Hz), cumulative time duration (1-5 µs), and absorbed electrical energy density (up to 81 mJ/mm3 in sucrose-containing low-conductivity buffer and up to 700 mJ/mm3 in high-conductivity HBSS buffer). Our results show that the effectiveness of nsPEFs in ablating 3D-grown cancer cells depends on the medium to which the cells are exposed and the PRR. When a medium with low-conductivity is used, the pulses do not result in cell ablation. Conversely, when the same pulse parameters are applied in a high-conductivity HBSS buffer and high PRRs are applied, the local temperature rises and yields either cell sensitization to nsPEFs or thermal damage.

Gene Electrotransfer Efficiency in 2D and 3D Cancer Cell Models Using Different Electroporation Protocols: A Comparative Study.

Electroporation, a method relying on a pulsed electric field to induce transient cell membrane permeabilization, can be used as a non-viral method to transfer genes in vitro and in vivo. Such transfer holds great promise for cancer treatment, as it can induce or replace missing or non-functioning genes. Yet, while efficient in vitro, gene-electrotherapy remains challenging in tumors. To assess the differences of gene electrotransfer in respect to applied pulses in multi-dimensional (2D, 3D) cellular organizations, we herein compared pulsed electric field protocols applicable to electrochemotherapy and gene electrotherapy and different "High Voltage-Low Voltage" pulses. Our results show that all protocols can result in efficient permeabilization of 2D- and 3D-grown cells. However, their efficiency for gene delivery varies. The gene-electrotherapy protocol is the most efficient in cell suspensions, with a transfection rate of about 50%. Conversely, despite homogenous permeabilization of the entire 3D structure, none of the tested protocols allowed gene delivery beyond the rims of multicellular spheroids. Taken together, our findings highlight the importance of electric field intensity and the occurrence of cell permeabilization, and underline the significance of pulses' duration, impacting plasmids' electrophoretic drag. The latter is sterically hindered in 3D structures and prevents the delivery of genes into spheroids' core.

High Power Electromagnetic Waves Exposure of Healthy and Tumor Bearing Mice: Assessment of Effects on Mice Growth, Behavior, Tumor Growth, and Vessel Permeabilization.

High power radiofrequencies may transiently or permanently disrupt the functioning of electronic devices, but their effect on living systems remains unknown. With the aim to evaluate the safety and biological effects of narrow-band and wide-band high-power electromagnetic (HPEM) waves, we studied their effects upon exposure of healthy and tumor-bearing mice. In field experiments, the exposure to 1.5 GHz narrow-band electromagnetic fields with the incident amplitude peak value level in the range of 40 kV/m and 150 MHz wide-band electric fields with the amplitude peak value in the range of 200 kV/m, did not alter healthy and tumor-bearing animals' growth, nor it had any impact on cutaneous murine tumors' growth. While we did not observe any noticeable behavioral changes in mice during the exposure to narrow-band signals when wide-band HPEM signals were applied, mice could behave in a similar way as they respond to loud noise signals: namely, if a mouse was exploring the cage prior to signal application, it returned to companion mates when wide-band HPEM signals were applied. Moreover, the effect of wide-band signals was assessed on normal blood vessels permeability in real-time in dorsal-chamber-bearing mice exposed in a pilot study using wide-band signal applicators. Our pilot study conducted within the applicator and performed at the laboratory scale suggests that the exposure to wide-band signals with the amplitude of 47.5 kV/m does not result in increased vessel permeability.

A nanosecond pulsed electric field (nsPEF) can affect membrane permeabilization and cellular viability in a 3D spheroids tumor model.

Three-dimensional (3D) cellular models represent more realistically the complexity of in vivo tumors compared to 2D cultures. While 3D models were largely used in classical electroporation, the effects of nanosecond pulsed electric field (nsPEF) have been poorly investigated. In this study, we evaluated the biological effects induced by nsPEF on spheroid tumor model derived from the HCT-116 human colorectal carcinoma cell line. By varying the number of pulses (from 1 to 500) and the polarity (unipolar and bipolar), the response of nsPEF exposure (10 ns duration, 50 kV/cm) was assessed either immediately after the application of the pulses or over a period lasting up to 6 days. Membrane permeabilization and cellular death occurred following the application of at least 100 pulses. The extent of the response increased with the number of pulses, with a significant decrease of viability, 24 h post-exposure, when 250 and 500 pulses were applied. The effects were highly reduced when an equivalent number of bipolar pulses were delivered. This reduction was eliminated when a 100 ns interphase interval was introduced into the bipolar pulses. Altogether, our results show that nsPEF effects, previously observed at the single cell level, also occur in more realistic 3D tumor spheroids models.

Electric Field Based Therapies in Cancer Treatment.

Enormous progress has been made in pulsed electric field-based therapies since J [...].

Gold-based therapy: From past to present.

Despite an abundant literature on gold nanoparticles use for biomedicine, only a few of the gold-based nanodevices are currently tested in clinical trials, and none of them are approved by health agencies. Conversely, ionic gold has been used for decades to treat human rheumatoid arthritis and benefits from 70-y hindsight on medical use. With a view to open up new perspectives in gold nanoparticles research and medical use, we revisit here the literature on therapeutic gold salts. We first summarize the literature on gold salt pharmacokinetics, therapeutic effects, adverse reactions, and the present repurposing of these ancient drugs. Owing to these readings, we evidence the existence of a common metabolism of gold nanoparticles and gold ions and propose to use gold salts as a "shortcut" to assess the long-term effects of gold nanoparticles, such as their fate and toxicity, which remain challenging questions nowadays. Moreover, one of gold salts side effects (i.e., a blue discoloration of the skin exposed to light) leads us to propose a strategy to biosynthesize large gold nanoparticles from gold salts using light irradiation. These hypotheses, which will be further investigated in the near future, open up new avenues in the field of ionic gold and gold nanoparticles-based therapies.

Ever-Evolving Identity of Magnetic Nanoparticles within Human Cells: The Interplay of Endosomal Confinement, Degradation, Storage, and Neocrystallization.

Considerable knowledge has been acquired in inorganic nanoparticles' synthesis and nanoparticles' potential use in biomedical applications. Among different materials, iron oxide nanoparticles remain unrivaled for several reasons. Not only do they respond to multiple physical stimuli (e.g., magnetism, light) and exert multifunctional therapeutic and diagnostic actions but also they are biocompatible and integrate endogenous iron-related metabolic pathways. With the aim to optimize the use of (magnetic) iron oxide nanoparticles in biomedicine, different biophysical phenomena have been recently identified and studied. Among them, the concept of a "nanoparticle's identity" is of particular importance. Nanoparticles' identities evolve in distinct biological environments and over different periods of time. In this Account, we focus on the remodeling of magnetic nanoparticles' identities following their journey inside cells. For instance, nanoparticles' functions, such as heat generation or magnetic resonance imaging, can be highly impacted by endosomal confinement. Structural degradation of nanoparticles was also evidenced and quantified in cellulo and correlates with the loss of magnetic nanoparticle properties. Remarkably, in human stem cells, the nonmagnetic products of nanoparticles' degradation could be subsequently reassembled into neosynthesized, endogenous magnetic nanoparticles. This stunning occurrence might account for the natural presence of magnetic particles in human organs, especially the brain. However, mechanistic details and the implication of such phenomena in homeostasis and disease have yet to be completely unraveled.This Account aims to assess the short- and long-term transformations of magnetic iron oxide nanoparticles in living cells, particularly focusing on human stem cells. Precisely, we herein overview the multiple and ever-evolving chemical, physical, and biological magnetic nanoparticles' identities and emphasize the remarkable intracellular fate of these nanoparticles.

Magnetic Silica-Coated Iron Oxide Nanochains as Photothermal Agents, Disrupting the Extracellular Matrix, and Eradicating Cancer Cells.

Cancerous cells and the tumor microenvironment are among key elements involved in cancer development, progression, and resistance to treatment. In order to tackle the cells and the extracellular matrix, we herein propose the use of a class of silica-coated iron oxide nanochains, which have superior magnetic responsiveness and can act as efficient photothermal agents. When internalized by different cancer cell lines and normal (non-cancerous) cells, the nanochains are not toxic, as assessed on 2D and 3D cell culture models. Yet, upon irradiation with near infrared light, the nanochains become efficient cytotoxic photothermal agents. Besides, not only do they generate hyperthermia, which effectively eradicates tumor cells in vitro, but they also locally melt the collagen matrix, as we evidence in real-time, using engineered cell sheets with self-secreted extracellular matrix. By simultaneously acting as physical (magnetic and photothermal) effectors and chemical delivery systems, the nanochain-based platforms offer original multimodal possibilities for prospective cancer treatment, affecting both the cells and the extracellular matrix.

Evaluations of Acute and Sub-Acute Biological Effects of Narrowband and Moderate-Band High Power Electromagnetic Waves on Cellular Spheroids.

High power electromagnetic signals can disrupt the functioning of electronic devices. As electromagnetism plays a role in cells homeostasis, such electromagnetic signals could potentially also alter some physiological processes. Herein we report on distinct biological parameters assessment after cellular spheroids exposure to high power electromagnetic signals, such as the ones used for defense applications. Signals effects were assessed in tumor cells spheroids and in normal human dermal fibroblasts spheroids, where macroscopic aspect, growth, plasma membrane integrity, induction of apoptosis, ATP content, and mitochondrial potential were investigated after spheroids exposure to high power electromagnetic signals. No significant effects were observed, indicating that 1.5 GHz narrowband electromagnetic fields with incident amplitude level of 40 kV/m, and 150 MHz moderate-band electric fields with an amplitude of 72.5 to approximately 200 kV/m, do not cause any significant alterations of assessed parameters.

Pulsed Electric Field Treatment Enhances the Cytotoxicity of Plasma-Activated Liquids in a Three-Dimensional Human Colorectal Cancer Cell Model.

Cold atmospheric plasma and more recently, plasma-activated liquids (culture media, water or buffered solutions previously exposed to plasma), are gathering momentum in cancer cells treatment. Nevertheless, in vitro tests show that this novel approach is sometimes less efficient than expected. We here evaluate the mechanisms of action of the plasma-activated PBS and suggest to use electropermeabilization (EP) in combination with the plasma-activated phosphate-buffered saline (PBS), in order to potentiate the cytotoxic effect of the plasma activated liquid. Human multicellular tumor spheroids (MCTS), a three-dimensional cell model, which resembles small avascular tumors, was used to define the optimal treatment conditions for single and dual-mode treatments. MCTS growth, viability, and global morphological changes were assessed by live cell video-microscopy. In addition, the induction of caspases activation, the appearance of DNA damages, and cell membrane permeabilization, as well as the early modifications in the cellular ultrastructure, were examined by immunofluorescence, propidium iodide staining, confocal fluorescence microscopy and transmission electron microscopy, respectively. Altogether, our results show that a combined treatment resulted in an earlier onset of DNA damage and caspases activation, which completely abolished MCTS growth. This report is a proof of concept study evidencing that electropermeabilization greatly potentiates the cytotoxic effect of plasma-activated PBS in vitro in a three-dimensional cancer cell model.

Electric field-responsive nanoparticles and electric fields: physical, chemical, biological mechanisms and therapeutic prospects.

Electric fields are among physical stimuli that have revolutionized therapy. Occurring endogenously or exogenously, the electric field can be used as a trigger for controlled drug release from electroresponsive drug delivery systems, can stimulate wound healing and cell proliferation, may enhance endocytosis or guide stem cell differentiation. Electric field pulses may be applied to induce cell fusion, can increase the penetration of therapeutic agents into cells, or can be applied as a standalone therapy to ablate tumors. This review describes the main therapeutic trends and overviews the main physical, chemical and biological mechanisms underlying the actions of electric fields. Overall, the electric field can be used in therapeutic approaches in several ways. The electric field can act on drug carriers, cells and tissues. Understanding the multiple effects of this powerful tool will help harnessing its full therapeutic potential in an efficient and safe way.

Intracellular Biodegradation of Ag Nanoparticles, Storage in Ferritin, and Protection by a Au Shell for Enhanced Photothermal Therapy.

Despite their highly efficient plasmonic properties, gold nanoparticles are currently preferred to silver nanoparticles for biomedical applications such as photothermal therapy due to their high chemical stability in the biological environment. To confer protection while preserving their plasmonic properties, we allied the advantages of both materials and produced hybrid nanoparticles made of an anisotropic silver nanoplate core coated with a frame of gold. The efficiency of these hybrid nanoparticles (Ag@AuNPs) in photothermia was compared to monometallic silver nanoplates (AgNPs) or gold nanostars (AuNPs). The structural and functional properties of AuNPs, AgNPs, and Ag@AuNPs were investigated in environments of increasing complexity, in water suspensions, in cells, and in tumors in vivo. While AgNPs showed the greatest heating efficiency in suspension (followed by Ag@AuNPs and AuNPs), this trend was reversed intracellularly within a tissue-mimetic model. In this setup, AgNPs failed to provide consistent photothermal conversion over time, due to structural damage induced by the intracellular environment. Remarkably, the degraded Ag was found to be stored within the iron-storage ferritin protein. By contrast, the Au shell provided the Ag@AuNPs with total Ag biopersistence. As a result, photothermal therapy was successful with Ag@AuNPs in vivo in a mouse tumor model, providing the ultimate proof on Au shell's capability to shield the Ag core from the harsh biological environment and preserve its excellent heating properties.

High power electromagnetic pulse applicators for evaluation of biological effects induced by electromagnetic radiation waves.

The effects of electromagnetic radiation waves on health is one of the major public concerns. These waves are mainly produced at a large scale but it is important to evaluate these effects on biological samples at the laboratory scale. Here we developed a set of micro applicators, which allow evaluating the effect of electromagnetic fields on biological samples with volumes in the microliter range. The applicators can be coupled to an optical microscope and allow a real-time observation of potential structural and functional alterations of the tested sample induced by different waveforms. New design approaches are suggested to simultaneously achieve maximized electric field coupling effect and optimized electric field homogeneity in the tested sample, while minimizing the return loss when the applicators are loaded with the biological samples. These applicators allow studying the biological effect of a variety of different signals, due to their wide frequency bandwidth (beyond 1.5 GHz) and their high permissible power. In addition, different electromagnetic parameters such as the electromagnetic field magnitude, pulse repetitive factor, number of bursts or delay between bursts may be set. The efficacy of the applicators was addressed for three different signals: two types of electromagnetic waves - a damped sinusoid centered at 200 MHz (wide band signal), a radar-like signal at 1.5 GHz (the ultra-narrow band signal) and a train of millisecond square-wave monopolar electric field pulses (causing electroporation). The biological effects were thus assessed (at the microscopic scale) on two different biological models, the giant unilamellar vesicles, and tumor and normal human cells, as well as being compared to results obtained (at full scale) with signals generated by antennas.

Nanoparticle-based hyperthermia, a local treatment modulating the tumor extracellular matrix.

The structural complexity and physical properties of the tumor microenvironment negatively affect the penetration and efficiency of conventional anticancer drugs. While previously underestimated, the tumor microenvironment now becomes a potential target for cancer treatment. This microenvironment can be modulated either systemically by pharmacological means, or locally, through physical effects mediated by certain nanoparticles. Some of them, such as magnetic, plasmonic or carbon-based nanoparticles, can generate heat on demand in a spatially and temporally controlled manner. In addition, the nanoparticles can be either activated by light or magnetic stimuli. The impact of the resulting local heating can be observed on the ultrastructural level, as it strongly affects the organization of collagen fibers, and on the macroscopic level, since the thermal damages alter the mechanical properties of the tumor. Nanoparticle-based hyperthermia thus improves the effect of conventional anticancer drugs, as it allows their better penetration through the altered extracellular matrix. Here we suggest the use of nanoparticle-generated hyperthermia, obtained after magnetic or light activation, as an adjuvant treatment to prime the tumor microenvironment and improve the efficacy of chemotherapy.

Tumor Stiffening, a Key Determinant of Tumor Progression, is Reversed by Nanomaterial-Induced Photothermal Therapy.

Tumor stiffening, stemming from aberrant production and organization of extracellular matrix (ECM), has been considered a predictive marker of tumor malignancy, non-invasively assessed by ultrasound shear wave elastography (SWE). Being more than a passive marker, tumor stiffening restricts the delivery of diagnostic and therapeutic agents to the tumor and per se could modulate cellular mechano-signaling, tissue inflammation and tumor progression. Current strategies to modify the tumor extracellular matrix are based on ECM-targeting chemical agents but also showed deleterious systemic effects. On-demand excitable nanomaterials have shown their ability to perturb the tumor microenvironment in a spatiotemporal-controlled manner and synergistically with chemotherapy. Here, we investigated the evolution of tumor stiffness as well as tumor integrity and progression, under the effect of mild hyperthermia and thermal ablation generated by light-exposed multi-walled carbon nanotubes (MWCNTs) in an epidermoid carcinoma mouse xenograft. SWE was used for real-time mapping of the tumor stiffness, both during the two near infrared irradiation sessions and over the days after the treatment. We observed a transient and reversible stiffening of the tumor tissue during laser irradiation, which was lowered at the second session of mild hyperthermia or photoablation. In contrast, over the days following photothermal treatment, the treated tumors exhibited a significant softening together with volume reduction, whereas non-treated growing tumors showed an increase of tumor rigidity. The organization of the collagen matrix and the distribution of CNTs revealed a spatio-temporal correlation between the presence of nanoheaters and the damages on collagen and cells. This study highlights nanohyperthermia as a promising adjuvant strategy to reverse tumor stiffening and normalize the mechanical tumor environment.

Ferritin Protein Regulates the Degradation of Iron Oxide Nanoparticles.

Proteins implicated in iron homeostasis are assumed to be also involved in the cellular processing of iron oxide nanoparticles. In this work, the role of an endogenous iron storage protein-namely the ferritin-is examined in the remediation and biodegradation of magnetic iron oxide nanoparticles. Previous in vivo studies suggest the intracellular transfer of the iron ions released during the degradation of nanoparticles to endogenous protein cages within lysosomal compartments. Here, the capacity of ferritin cages to accommodate and store the degradation products of nanoparticles is investigated in vitro in the physiological acidic environment of the lysosomes. Moreover, it is questioned whether ferritin proteins can play an active role in the degradation of the nanoparticles. The magnetic, colloidal, and structural follow-up of iron oxide nanoparticles and proteins in lysosome-like medium confirms the efficient remediation of potentially harmful iron ions generated by nanoparticles within ferritins. The presence of ferritins, however, delays the degradation of particles due to a complex colloidal behavior of the mixture in acidic medium. This study exemplifies the important implications of intracellular proteins in processes of degradation and metabolization of iron oxide nanoparticles.