Fabrication and use of silicon hollow-needle arrays to achieve tissue nanotransfection in mouse tissue in vivo.

Tissue nanotransfection (TNT) is an electromotive gene transfer technology that was developed to achieve tissue reprogramming in vivo. This protocol describes how to fabricate the required hardware, commonly referred to as a TNT chip, and use it for in vivo TNT. Silicon hollow-needle arrays for TNT applications are fabricated in a standardized and reproducible way. In <1 s, these silicon hollow-needle arrays can be used to deliver plasmids to a predetermined specific depth in murine skin in response to pulsed nanoporation. Tissue nanotransfection eliminates the need to use viral vectors, minimizing the risk of genomic integration or cell transformation. The TNT chip fabrication process typically takes 5-6 d, and in vivo TNT takes 30 min. This protocol does not require specific expertise beyond a clean room equipped for basic nanofabrication processes.

Development of Delivery Systems Enhances the Potency of Cell-Based HIV-1 Therapeutic Vaccine Candidates.

An effective therapeutic vaccine to eradicate HIV-1 infection does not exist yet. Among different vaccination strategies, cell-based vaccines could achieve in clinical trials. Cell viability and low nucleic acid expression are the problems related to dendritic cells (DCs) and mesenchymal stem cells (MSCs), which are transfected with plasmid DNA. Thus, novel in vitro strategies are needed to improve DNA transfection into these cells. The recent study assessed immune responses generated by MSCs and DCs, which were derived from mouse bone marrow and modified with Nef antigen using novel methods in mice. For this purpose, an excellent gene transfection approach by mechanical methods was used. Our data revealed that the transfection efficacy of Nef DNA into the immature MSCs and DCs was improved by the combination of chemical and mechanical (causing equiaxial cyclic stretch) approaches. Also, chemical transfection performed two times with 48-hour intervals further increased gene expression in both cells. The groups immunized with Nef DC prime/rNef protein boost and then Nef MSC prime/rNef protein boost were able to stimulate high levels of IFN-γ, IgG2b, IgG2a, and Granzyme B directed toward Th1 responses in mice. Furthermore, the mesenchymal or dendritic cell-based immunizations were more effective compared to protein immunization for enhancement of the Nef-specific T-cell responses in mice. Hence, the use of chemical reagent and mechanical loading simultaneously can be an excellent method in delivering cargoes into DCs and MSCs. Moreover, DC- and MSC-based immunizations can be considered as promising approaches for protection against HIV-1 infections.

Clarification of electrical current importance in plasma gene transfection by equivalent circuit analysis.

We have been developing a method of plasma gene transfection that uses microdischarge plasma (MDP) and is highly efficient, minimally invasive, and safe. Using this technique, electrical factors (such as the electrical current and electric field created through processing discharge plasma) and the chemical factors of active species and other substances focusing on radicals are supplied to the cells and then collectively work to introduce nucleic acids in the cell. In this paper, we focus on the electrical factors to identify whether the electric field or electrical current is the major factor acting on the cells. More specifically, we built a spatial distribution model that uses an electrical network to represent the buffer solution and cells separately, as a substitute for the previously reported uniform medium model (based on the finite element method), calculated the voltage and electrical current acting on cells, and examined their intensity. Although equivalent circuit models of single cells are widely used, this study was a novel attempt to build a model wherein adherent cells distributed in two dimensions were represented as a group of equivalent cell circuits and analyzed as an electrical network that included a buffer solution and a 96-well plate. Using this model, we could demonstrate the feasibility of applying equivalent circuit network analysis to calculate electrical factors using fewer components than those required for the finite element method, with regard to electrical processing systems targeting organisms. The results obtained through this equivalent circuit network analysis revealed for the first time that the distribution of voltage and current applied to a cellular membrane matched the spatial distribution of experimentally determined gene transfection efficiency and that the electrical current is the major factor contributing to introduction.

Impact of photobiomodulation using four diode laser wavelengths of on cationic liposome gene transfection into pre-osteoblast cells.

Gene therapy can be an effective treatment modality for some severe genetic diseases. Despite efforts to improve their performance, non-viral gene delivery methods remain inefficient and costly. As an alternative to viral vectors, cationic liposomes have a good safety profile and low immunogenicity, but relatively low transfection efficiency. They may also be toxic to cells at high concentrations. Given these challenges, the present study explored the impact of photobiomodulation (PBM) on cationic liposome plasmid DNA transfection in terms of its efficiency and toxicity, using Lipofectamine 2000 to carry green fluorescent protein (GFP) encoding plasmid DNA, with the pre-osteoblast MC3T3-E1 cell line as the target. Cultures were irradiated using diode lasers (445, 685, 810, or 970 nm) at 200 mW using pulsed mode (50 Hz), with a power density of 104.64 mW/cm2, and irradiance from 6 to 18 joules. To determine transfection efficiency, expression of GFP was assessed using confocal laser scanning microscopy and flow cytometry. Cell viability was evaluated using the MTT assay. PBM using 810 nm and 970 nm lasers significantly enhanced transfection efficiency for GFP, indicating more efficient uptake of plasmid DNA. Conversely, laser irradiation at 445 nm and 685 nm wavelengths reduced the GFP transfection efficiency. Treatment using 685, 810, and 970 nm lasers at 12 J maintained cell viability and prevented toxicity of cationic liposomes. Overall, these findings support the concept that PBM using near infrared laser wavelengths can enhance transfection efficiency and support cell viability when cationic liposomes are used as the vector in gene therapy.

Utilising Automated Electrophysiological Platforms in Epilepsy Research.

Genetic mutations have long been implicated in epilepsy, particularly in genes that encode ion channels and neurotransmitter receptors. Among some of those identified are voltage-gated sodium, potassium and calcium channels, and ligand-gated gamma-aminobutyric acid (GABA), neuronal nicotinic acetylcholine (CHRN), and glutamate receptors, making them key therapeutic targets. In this chapter we discuss the use of automated electrophysiological technologies to examine the impact of gene defects in two potassium channels associated with different epilepsy syndromes. The hKCNC1 gene encodes the voltage-gated potassium channel hKV3.1, and mutations in this gene cause progressive myoclonus epilepsy (PME) and ataxia due to a potassium channel mutation (MEAK). The hKCNT1 gene encodes the weakly voltage-dependent sodium-activated potassium channel hKCNT1, and mutations in this gene cause a wide spectrum of seizure disorders, including severe autosomal dominant sleep-related hypermotor epilepsy (ADSHE) and epilepsy of infancy with migrating focal seizures (EIMFS), both conditions associated with drug-resistance. Importantly, both of these potassium channels play vital roles in regulating neuronal excitability. Since its discovery in the late nineteen seventies, the patch-clamp technique has been regarded as the bench-mark technology for exploring ion channel characteristics. In more recent times, innovations in automated patch-clamp technologies, of which there are many, are enabling the study of ion channels with much greater productivity that manual systems are capable of. Here we describe aspects of Nanion NPC-16 Patchliner, examining the effects of temperature on stably and transiently transfected mammalian cells, the latter of which for most automated systems on the market is quite challenging. Remarkable breakthroughs in the development of other automated electrophysiological technologies, such as multielectrode arrays that support extracellular signal recordings, provide additional features to examine network activity in the area of ion channel research, particularly epilepsy. Both of these automated technologies enable the acquisition of consistent, robust, and reproducible data. Numerous systems have been developed with very similar capabilities, however, not all the systems on the market are adapted to work with primary cells, particularly neurons that can be problematic. This chapter also showcases methods that demonstrate the versatility of Nanion NPC-16 Patchliner and the Multi Channel Systems (MCS) multielectrode array (MEA) assay for acutely dissociated murine primary cortical neurons, enabling the study of potassium channel mutations implicated in severe refractory epilepsies.

The Inoue Method for Preparation and Transformation of Competent Escherichia coli: "Ultracompetent" Cells.

This protocol differs from other transformation procedures in that the bacterial culture is grown at 18°C rather than the conventional 37°C. Otherwise, the protocol is unremarkable and follows a fairly standard course. Under standard laboratory conditions, efficiencies of 1 × 108 to 3 × 108 transformed colonies/µg of plasmid DNA are typical.

Microfluidic Electroporation Coupling Pulses of Nanoseconds and Milliseconds to Facilitate Rapid Uptake and Enhanced Expression of DNA in Cell Therapy.

Standard electroporation with pulses in milliseconds has been used as an effective tool to deliver drugs or genetic probes into cells, while irreversible electroporation with nanosecond pulses is explored to alter intracellular activities for pulse-induced apoptosis. A combination treatment, long nanosecond pulses followed by standard millisecond pulses, is adopted in this work to help facilitate DNA plasmids to cross both cell plasma membrane and nuclear membrane quickly to promote the transgene expression level and kinetics in both adherent and suspension cells. Nanosecond pulses with 400-800 ns duration are found effective on disrupting nuclear membrane to advance nuclear delivery of plasmid DNA. The additional microfluidic operation further helps suppress the negative impacts such as Joule heating and gas bubble evolution from common nanosecond pulse treatment that lead to high toxicity and/or ineffective transfection. Having appropriate order and little delay between the two types of treatment with different pulse duration is critical to guarantee the effectiveness: 2 folds or higher transfection efficiency enhancement and rapid transgene expression kinetics of GFP plasmids at no compromise of cell viability. The implementation of this new electroporation approach may benefit many biology studies and clinical practice that needs efficient delivery of exogenous probes.

Tissue suction-mediated gene transfer to the beating heart in mice.

We previously developed an in vivo site-specific transfection method using a suction device in mice; namely, a tissue suction-mediated transfection method (tissue suction method). The aim of this study was to apply the tissue suction method for cardiac gene transfer. Naked plasmid DNA (pDNA) was intravenously injected in mice, followed by direct suction on the beating heart by using a suction device made of polydimethylsiloxane. We first examined the effects of suction conditions on transgene expression and toxicity. Subsequently, we analyzed transgene-expressing cells and the transfected region of the heart. We found that heart suction induced transgene expression, and that -75 kPa and -90 kPa of suction achieved high transgene expression. In addition, the inner diameter of the suction device was correlated with transgene expression, but the pressure hold time did not change transgene expression. Although the tissue suction method at -75 kPa induced a transient increase in the serum cardiac toxicity markers at 6 h after transfection, these markers returned to normal at 24 h. The cardiac damage was also analyzed through the measurement of hypertrophic gene expression, but no significant differences were found. In addition, the cardiac function monitored by echocardiography remained normal at 11 days after transfection. Immunohistochemical analysis revealed that CD31-positive endothelial cells co-expressed the ZsGreen1-N1 reporter gene. In conclusion, the tissue suction method can achieve an efficient and safe gene transfer to the beating heart in mice.

Fe3+-Coordinated Multifunctional Elastic Nanoplatform for Effective in Vivo Gene Transfection.

The common phenomenon that the nonviral vectors have much lower transfection efficiency in vivo than in vitro greatly restricts their further developments and applications. Possible reasons are lacking targeting ability, elimination by the reticuloendothelial system (RES), and insufficient nuclear transport. Here, a novel, flexible, and deformable polymer Fe@PEI-R12 (tLyp-1-NLS) is reported for shortening the gap between in vitro and in vivo gene transfection efficiency. The amorphous network structure Fe@PEI with deformation ability acquired by coordination cross-linking of Fe3+ and low-molecular-weight polyethylenimine (LMW-PEI) constructs the core and serves as the gene reservoir, and it can squeeze out through RES filter holes when trapped in the spleen. The bifunctional peptide R12 provided tumor targeting and enhanced nuclear delivery ability. Additionally, the Fe3+ from Fe@PEI-R12 could trigger endogenous hydrogen peroxide (H2O2) decomposition to produce O2, thereby reducing the adverse effects of tumor hypoxia. It is demonstrated that the Fe@PEI-R12/pDNA complexes could pass through membrane filters, subsequently achieving long circulation time, and Fe@PEI-R12 had a tendency to accumulate in tumor tissue and mediate pGL3-control expression. Therefore, the multifunctional nanoplatform has the potential for effective in vivo gene delivery.

pH-responsive polydopamine nanoparticles for photothermally promoted gene delivery.

Recently, stimuli-responsive gene carriers have been widely studied to overcome the extra- and intracellular barriers in cancer treatment. In this study, we modified polydopamine nanoparticles with low-molecular weight polyethylenimine (PEI1.8k) and polyethylene glycol-phenylboronic acid (PEG-PBA) to prepare pH-responsive gene carrier PDANP-PEI-rPEG. PBA and polydopamine could form pH-responsive boronate ester bonds. Non-responsive PDANP-PEI-nPEG and non-PEGylated PDANP-PEI were also studied as control. Both PDANP-PEI-rPEG/DNA and PDANP-PEI-nPEG/DNA complexes remained stable in the pH environment of blood circulation or extracellular delivery (pH 7.4) owing to the PEG modification. And after being internalized into endosomes, the boronate ester bonds could be cleaved. The pH responsive ability of PDANP-PEI-rPEG might facilitate complexes dissociation and gene release inside cells. The transfection level of PDANP-PEI-rPEG/DNA complexes was about 100 times higher than that of PDANP-PEI-nPEG/DNA complexes with the same mass ratios. Moreover, after NIR light irradiation at the power density of 2.6 W/cm2 for 20 min, the good photothermal conversion ability of PDANP resulted in quick endosomal escape. The transfection level of PDANP-PEI-rPEG/DNA complexes doubled, even higher than that of lipofectamine 2000/DNA complexes. This was also confirmed by Bafilomycin A1 inhibition test and CLSM observation. In response to the acidic pH within cancer cells and the NIR light irradiation, the PDANP-PEI-rPEG carrier could overcome multiple obstacles in gene delivery, which was promising for further application in gene therapy.

Photothermally Activated Electrospun Nanofiber Mats for High-Efficiency Surface-Mediated Gene Transfection.

Although electrospun nanofibers have been used to deliver functional genes into cells attached to the surface of the nanofibers, the controllable release of genes from nanofibers and the subsequent gene transfection with high efficiency remain challenging. Herein, photothermally activated electrospun hybrid nanofibers are developed for high-efficiency surface-mediated gene transfection. Nanofibers with a core-sheath structure are fabricated using coaxial electrospinning. Plasmid DNA (pDNA) encoding basic fibroblast growth factor is encapsulated in the fiber core, and gold nanorods with photothermal properties are embedded in the fiber sheath composed of poly(l-lactic acid) and gelatin. The nanofiber mats show excellent and controllable photothermal response under near-infrared irradiation. The permeability of the nanofibers is thereby enhanced to allow the rapid release of pDNA. In addition, transient holes are formed in the membranes of NIH-3T3 fibroblasts attached to the mat, thus facilitating delivery and transfection with pDNA and leading to increased proliferation and migration of the transfected cells in vitro. This work offers a facile and reliable method for the regulation of cell function and cell behavior via localized gene transfection, showing great potential for application in tissue engineering and cell-based therapy.

Cell Size-Specific Transfection by Micropillar Array Electroporation.

In this study, we reported a new micropillar array electroporation (MAE) platform to accomplish large-scale, size-specific DNA and RNA delivery to mammalian cells for nanomedicine. By introducing well-patterned micropillar array on the electrode surface, the number of micropillars each cell faces varies with the surface area of cell membrane or the size of cells. In this way, cell size-specific electroporation is conveniently done on a large population of cells in despite of their random locations between the two electrodes. The enhancement of this MAE system on the delivery of DNA and RNA probes without sacrifice of cell viability is demonstrated with an average increase of 2.5 to 3-fold on the transfection efficiency of DNA plasmids and additional knockdown of the targeted protein 10-55% more in siRNA delivery when compared to that using a commercial electroporation system. This MAE system works like many single cell electroporation are carried out in parallel, showing potential to bridge the gap between single cell electrophysiology study and in vitro electroporation to a large population of cells.

Nanofountain Probe Electroporation for Monoclonal Cell Line Generation.

In the field of genetic engineering, the modification of genes to produce stable cell lines has a variety of applications ranging from the development of novel therapeutics to patient specific treatments. To successfully generate a cell line, the gene of interest must be delivered into the cell and integrated into the genome. The efficiency of cell line generation systems therefore depends on the efficiency of delivery of genetically modifying molecules such as plasmids and CRISPR/CAS9 complexes. In this work, we describe a localized electroporation-based system to generate stable monoclonal cell lines. By employing the nanofountain probe electroporation (NFP-E) system, single cells in patterned cultures are selectively transfected with plasmids, grown, and harvested to obtain stably expressing cell lines. Methods for microcontact printing, cell culture, electroporation, and harvesting are detailed in this chapter.

3D Nanochannel Electroporation for Macromolecular Nucleotide Delivery.

Delivery of macromolecular nucleotides into the living cells holds a great promise for the development of new therapeutics. However, its abilities for adoptive immunotherapy, cell reprogramming, and primary cell transfection have been long-term hindered by the lack of a system that can locally deliver engineered therapeutic nucleotides (e.g., plasmids, siRNAs, miRNAs) without causing any side effects. In this chapter, the performance of a novel 3D nanoelectroporation system (3D NEP) is highlighted in three scenarios-adoptive immunotherapy, cell reprogramming, and adult mouse primary cardiomyocyte transfection. Detailed protocols were given to introduce the 3D NEP system assembly, as well as their applications in (1) natural killer (NK) cells transfection by delivery of chimeric antigen receptor (CAR) plasmids; (2) mouse embryonic fibroblasts transfection with OSKM factors; and (3) miR-29b molecular beacon (BMs) delivery into primary cardiomyocytes for interrogating the side effect of miR-29b-assisted treatment.

Nanoelectroporation and Collection of Genetically Modified Exosomes in Primary Cultures of Dendritic Cells.

Dendritic cells (DCs) are cells of the immune system that behave as antigen presenters and assist in T cell activation. DCs have recently been used in cell-based immunotherapies for the treatment of different diseases due to the lack of adverse nonspecific immune responses, typically elicited by other approaches. Genetically modified DCs, for example, have been used to stimulate CD4/CD8 antigen presenting immune responses. However, genetic manipulation of primary DCs remains a challenge. Here we describe a protocol for nonviral, benign transfection of primary DCs using nanochannel-based electroporation, and the subsequent collection of genetically modified exosomes for downstream applications.

Electroporation of CRISPR-Cas9 into Malignant B Cells for Loss-of-Function Studies of Target Gene Via Knockout.

CRISPR-Cas9 is a unique technology that enables geneticists and medical researchers to edit genomic DNA for studying biology, pathogenesis, and molecular basis of treatment in malignant B cells. Unfortunately, malignant B cells are extremely difficult to transfect by most traditional methods. In this chapter, we describe the use of the Nucleofector™ Technology-based electroporation system with optimized transfection conditions for generating a malignant B cell model, JEKO-1, with ROR1-gene knockout via CRISPR-Cas9 technology.

Microfluidic Device for Localized Electroporation.

Electroporation is a common method of transfection due to its relatively low risk and high transfection efficiency. The most common method of electroporation is bulk electroporation which is easily performed on large quantities of cells yet results in variable levels of viability and transfection efficiency across the population. Localized electroporation is an alternative that can be administered on a similar scale but results in much more consistent with higher quality transfection and higher cell viability. This chapter discusses the creation and use of a simple and cost-effective device using porous membrane for performing localized electroporation.

Targeted In Vivo Electroporation Using Nanoengineered Microelectrodes.

Targeted electroporation by using glass microelectrodes is a popular and versatile tool allowing for easy manipulation of single cells and cell ensembles in living tissue. Because of the highly focal distribution of the electric field, however, the range of reversible electroporation without causing irreversible damage is tight-especially when aiming for larger electroporation volumes. In this chapter, we describe the production of nanoengineered electroporation microelectrodes (NEMs), a practicable way to prepare glass microelectrodes providing a more even distribution around the tip of a pipette by using nanotechnological methods.

Transformation of the Model Microalga Chlamydomonas reinhardtii Without Cell-Wall Removal.

The green alga Chlamydomonas reinhardtii has been widely used to study many biological processes, including photosynthesis, flagellar motility, sexual reproduction, metabolism, and genetics. Here, we describe a step-by-step protocol of rapid and efficient transformation method for wild type cell-walled Chlamydomonas strains without cell-wall removal using a square electric pulses-generating electroporator. This method could be applied to the transformation of other industrially useful algae including diatom by optimizing the electric conditions.

Genetic Manipulation of Cryptosporidium parvum with CRISPR/Cas9.

Cryptosporidium parvum can be reliably genetically manipulated using CRISPR/Cas9-driven homologous repair coupled to in vivo propagation within immunodeficient mice. Recent modifications have simplified the initial protocol significantly. This chapter will guide through procedures for excystation, transfection, infection, collection, and purification of transgenic Cryptosporidium parvum.