1. MEA Preparation
<ol>
	<li>MEAs for use in electroporation experiments can either be fabricated using standard photolithography technique as described previously18 or purchased directly from vendors of MEA manufacturers such as Multi Channel Systems <a href="http://www.multichannelsystems.com" target="_blank">(http:/www.multichannelsystems.com/)</a> and Alpha MED Scientific, Inc. <a href="http://www.med64.com/" target="_blank">(http://www.MED64.com)</a>.The MEAs used in our experiments were fabricated in-house at the clean room facility in Arizona State University, which is administered by the Center for Solid State Electronics Research (CSSER). The MEAs used in our experiments had 32 indium tin oxide electrodes in an 8 by 4 array with electrode diameter sizes from 50-200 &mu;m within a 1 cm inner diameter glass well. The glass well was bonded on the MEA using polydimethylsiloxane (PDMS).</li>
	<li>Sterilize the MEA in an autoclave prior to cell seeding. Other methods of sterilization such as a soaking in 70% ethanol, UV treatment and hot water treatment as described elsewhere can also be used as long they do not compromise the integrity of the MEA.</li>
	<li>Transfer the autoclaved MEA to a laminar flow hood and place it in a standard sterile polystyrene Petri dish. Prior to seeding cells on the MEA, sterilize the laminar flow hood by turning on the UV light for 20 min. If the MEA is sensitive to UV light cover the Petri dish containing the MEA with sterile aluminum foil while the flow hood is under UV illumination.</li>
</ol>
2. Seeding Cells on the MEA
<ol>
	<li>Harvest cells from a running cell line of adherent mammalian cells using Trypsin/EDTA and raise them to a concentration of 100-200 cells/&mu;l in 1 ml cell media for plating. In our experiments we have been successful in culturing NIH 3T3 fibroblasts cell line, HeLa cell line and primary cortical and hippocampal neurons on the MEA.</li>
	<li>Place a 20-30 &mu;l drop of media with cells on the MEA. Transfer the Petri dish with the MEA to the incubator. It is important to ensure that the cell drop size is large enough to cover all the electrodes on the MEA.</li>
	<li>2 hr after seeding the cells transfer the Petri dish with the MEA from the incubator to laminar flow hood and gently add 400-500 &mu;l of pre warmed media (37 &deg;C) to the MEA well. Check under the microscope to ensure that cells are still attached and evenly spread on the MEA surface. Transfer the MEA back to the incubator. The 2 hr wait period before the addition of media ensures enough time for the cells to adhere with the MEA surface. Addition of cell media too soon after seeding the cells can wash away the cells. If cells do not adhere well on the MEA, its surface can be treated with cell attachment factors such aspolylysine, fibronectin, and laminin to improve cell adhesion. In our HeLa cell experiments we typically treat the MEA surface with fibronectin (10 &mu;g/ml) for 1-2 hr.</li>
	<li>8-12 hr after seeding the cells, perform 1-2 washes with PBS to remove any dead cells. Cell medium can replaced every 24-48 hr as needed. In a typical experiment, the cells are usually ready for transfection 24-48 hr after seeding. To maintain consistency in electroporation efficiency it is recommended to wait until the culture is at least 80% confluent before electroporation.</li>
</ol>
3. Site-specific Transfection of siRNA
<ol>
	<li>Prepare nucleic acid solution. For siRNA transfection, prepare a 1-2 &mu;M siRNA electroporation solution in ice cold electroporation buffer to attain a final volume of 200-400 &mu;l. In this video article we demonstrate the transfection of HeLa cells with Alexa 488 conjugated scrambled sequence of siRNA.</li>
	<li>Transfer the MEA with cells from the incubator to sterile laminar flow hood. Remove the cell media from the MEA and perform a wash with PBS. Thereafter, add 200-400 &mu;l of the ice cold 1 &mu;M siRNA electroporation solution to the well.</li>
	<li>Apply anodic square electric pulses to the targeted electrodes (Refer to step 4 for determining optimal parameters of the electric pulse). In our experiments we used a Pragmatic Instruments 2414A waveform generator to generate electric pulses and a dual-inline-package (DIP) 16 pin clip to make contact with the MEA bond pads. The waveform generator can be connected to the DIP clip via a demultiplexor printed circuit board to provide selectivity of individual electrodes. For the reference electrode, place a steel cathode in the cell media. While lowering the steel reference electrode in the media it is extremely important to ensure that it does not make contact with the MEA surface. Upon contact, it can cause damage to the cells and the MEA. It is recommended to lower the reference electrode to a position of 1 mm above the cells. A spacer with a height of 1 mm can be placed in the well to assist in placement of the reference electrode. An alternate approach to using an external reference electrode is to use neighboring electrodes as cathode-anode pairs.</li>
	<li>Immediately after applying the electric pulses to the targeted electrodes on the MEA, transfer the MEA back to the incubator. After an incubation period of 5 min, gently replace 75% of the electroporation buffer with pre-warmed (37 &deg;C) cell media. Later, fluorescent imaging can be done to confirm the delivery of fluorescently tagged siRNA to the cells.</li>
</ol>
4. Electroporation Parameter Optimization
<ol>
	<li>To determine the optimal electric pulse parameters for electroporation, design electroporation experiments for a range of values of pulse amplitude, pulse duration, and number of pulses. In our experience with HeLa cells, we observed that a single voltage pulse of amplitude from 3 V to 9 V, and duration from 1 msec to 10 msec for an electrode size of 100 &mu;m led to successful electroporation with minimal cell death. It is our recommendation that one parameter be varied at a time and others are kept constant. To quantitate the electroporation efficiency for each of the pulse parameters, we use propidium iodide (PI), a cell impermeant dye that stains nucleic material, and live assay based methodology, as described in steps 4.2-4.5.</li>
	<li>Establish a cell culture on the MEA following the directions outlined in steps 1 and 2.</li>
	<li>Prior to electroporation prepare a 300-500 &mu;l of 30 &mu;g/ml PI solution in ice cold electroporation buffer and 400 &mu;l of 4 &mu;M calcien AM solution in PBS.</li>
	<li>Transfer the MEA from the incubator to the laminar flow hood. Remove the cell media from the MEA and perform a wash with PBS. Thereafter, add 300-500 &mu;l of the ice cold electroporation buffer with PI.</li>
	<li>Apply electric pulses with desired pulse amplitude and duration to the targeted electrodes (The equipment used in our experiments for generating and applying the electric pulse to the MEA is described in step 3.3). For the reference electrode either lower a steel electrode into the electroporation buffer in the MEA well or use the neighboring electrode as reference. By selectively applying pulses of varying amplitudes or durations to different electrodes multiple experiments can be performed on the same device. For example, on a 32 electrode MEA, 8 experiments can be performed by splitting the 32 electrodes in 8 groups, each consisting of 4 electrodes. Each of the 8 groups can then be stimulated by a single rectangular voltage pulse of duration 5 msec with amplitudes varying in 3 V-6 V range in 0.5 V increments and one group can be used as a control.</li>
	<li>Immediately after applying the voltage pulses transfer the MEA to the incubator for 5 min. Afterwards, gently remove 75% of the electroporation buffer from the MEA well and replace it with warm cell media. Transfer the MEA back to the incubator.</li>
	<li>After an incubation period of 2-4 hr replace the cell media in the MEA well with 300-500 &mu;l of 4 &mu;M Calcein AM solution in PBS. Transfer the MEA back to incubator for another 30 min.</li>
	<li>Following the incubation of the cells with Calcein AM, replace the Calcein AM solution in the MEA well with PBS after two washes with PBS. Obtain fluorescent images of cells using with an optical microscope with filters for PI (Excitation/Emission - 535 nm/617 nm) and Calcein AM (Excitation/Emission - 490 nm/520 nm). The uptake of PI, due to electroporation, causes cells to fluoresce red and the Calcein AM causes cells that are alive to fluoresce green. Three possible scenarios that can be observed are 1) cell electroporated and alive (will fluoresce both red and green), 2) Cells alive but not electroporated (will fluoresce just green) and 3) Cells dead due to electroporation (will fluoresce just red). The cell viability was calculated as a percent of the total number of cells alive on the electrode and the electroporation efficiency was calculated as percent of the total number of cells loaded with PI/siRNA.</li>
</ol>
5. Representative Results
Figure 1 shows the site-specific loading of HeLA cells with PI. It can be observed that only cells on the electrode demonstrate an uptake of PI (Figure 1B). A live assay performed post electroporation was used to assess the viability of cells (Figure 1A). The electroporation efficiency and cell viability for the example shown in Figure 1 are 81.8 % and 96.1 % respectively. High cell viability and electroporation efficiency can be achieved by optimizing the electroporation pulse parameters. Site-specific transfection of HeLA cells with fluorescently tagged siRNA for an optimized electroporation pulse (8 V, 1 msec) is shown in Figure 2. The electroporation efficiency for the siRNA at 8 V, 1 msec was found to be 74.4 &plusmn; 16.0 % and cell viability was found to be 87.9 &plusmn; 8.4 % (all data represented as mean &plusmn; standard deviation, N=5).
<img alt="Figure 1" src="/files/ftp_upload/4415/4415fig1.jpg" /> 
Figure 1. Site-specific delivery of PI in HeLa cells on a 200 &mu;m electrode (6 V, 5 msec). A) Calcien based live assay performed 2 hr post electroporation. Alive cells are indicated by green fluorescence. B) Red fluorescence demonstrates the uptake of PI by cells on the electrode. C) A superimposed image of A and B. The white arrows indicate cells that are electroporated and alive. The black arrows indicate cells that are dead. The white circle in A, B and C indicates the outline of the electrode.
<img alt="Figure 2" src="/files/ftp_upload/4415/4415fig2.jpg" /> 
Figure 2. Transfection of HeLa cells with fluorescently tagged scramble sequence of siRNA. A) Image of HeLa cells on a 100 &mu;m electrode transfected with Alexa 488 tagged siRNA (8 V, 1 msec). B) Image of cell nuclei stained with DAPI. The white circle marks the edge of the electrode.

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No conflicts of interest declared.

In this video article we demonstrate the use of MEA for site-specific transfection of HeLa cells with scrambled sequence of siRNA. One of the advantages of this technology is its applicability to different cell lines including primary cell lines. Our lab has previously demonstrated the use of this technology for site-specific transfection of primary hippocampal neuronal culture from E18 day old rat and NIH-3T3 cells with scrambled siRNA sequences and GFP plasmid18, 19. We have also had success in delivering fu...

<table border="1">
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Cell media: Advanced MEM L-Glutamine 200 mM Penicillin/Streptomycin Fetal bovine serum</td>
 <td>Gibco/Invitrogen Himedia Laboratories/VWR Lonza group Ltd. Gibco/Invitrogen</td>
 <td>12492-013 95057-448 09-757F 16000-044</td>
 <td>Cell media composition: 2% FBS, 2%L-glutamine and 2% Pennstrep in Advance MEM</td>
 </tr>
 <tr>
 <td>Trypsin EDTA</td>
 <td>Mediatech, Inc.</td>
 <td>25-053-CL</td>
 <td></td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>Mediatech, Inc.</td>
 <td>21-040-CV</td>
 <td></td>
 </tr>
 <tr>
 <td>Alexa 488 and rhodamine tagged scrambled sequence siRNA</td>
 <td>Qiagen, Inc.</td>
 <td>1027292</td>
 <td></td>
 </tr>
 <tr>
 <td>Electroporation buffer</td>
 <td>Biorad Laboratories</td>
 <td>165-2677</td>
 <td></td>
 </tr>
 <tr>
 <td>Waveform generator</td>
 <td>Pragmatic</td>
 <td>2414A</td>
 <td>Any waveform/pulse generator that can deliver the desired pulses can be used.</td>
 </tr>
 </table>

high efficiency, site-specific transfection of adherent cells with sirna using microelectrode arrays (mea)

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for silencing specific genes1-8 for functional genomics and therapeutics. A major challenge involved in RNAi based studies is the delivery of RNAi agents to targeted cells. Traditional non-viral delivery techniques, such as bulk electroporation and chemical transfection methods often lack the necessary spatial control over delivery and afford poor transfection efficiencies9-12. Recent advances in chemical transfection methods such as cationic lipids, cationic polymers and nanoparticles have resulted in highly enhanced transfection efficiencies13. However, these techniques still fail to offer precise spatial control over delivery that can immensely benefit miniaturized high-throughput technologies, single cell studies and investigation of cell-cell interactions. 
Recent technological advances in gene delivery have enabled high-throughput transfection of adherent cells14-23, a majority of which use microscale electroporation. Microscale electroporation offers precise spatio-temporal control over delivery (up to single cells) and has been shown to achieve high efficiencies19, 24-26. Additionally, electroporation based approaches do not require a prolonged period of incubation (typically 4 hours) with siRNA and DNA complexes as necessary in chemical based transfection methods and lead to direct entry of naked siRNA and DNA molecules into the cell cytoplasm. As a consequence gene expression can be achieved as early as six hours after transfection27. Our lab has previously demonstrated the use of microelectrode arrays (MEA) for site-specific transfection in adherent mammalian cell cultures17-19. In the MEA based approach, delivery of genetic payload is achieved via localized micro-scale electroporation of cells. An application of electric pulse to selected electrodes generates local electric field that leads to electroporation of cells present in the region of the stimulated electrodes. The independent control of the micro-electrodes provides spatial and temporal control over transfection and also enables multiple transfection based experiments to be performed on the same culture increasing the experimental throughput and reducing culture-to-culture variability. 
Here we describe the experimental setup and the protocol for targeted transfection of adherent HeLa cells with a fluorescently tagged scrambled sequence siRNA using electroporation. The same protocol can also be used for transfection of plasmid vectors. Additionally, the protocol described here can be easily extended to a variety of mammalian cell lines with minor modifications. Commercial availability of MEAs with both pre-defined and custom electrode patterns make this technique accessible to most research labs with basic cell culture equipment.

Watch this Scientific Journal Video about High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA at JoVE.com

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA

The article details the protocol for site-specific transfection of scrambled sequence of siRNA in an adherent mammalian cell culture using a microelectrode array (MEA).

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays (MEA)

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for ...

high-efficiency-site-specific-transfection-adherent-cells-with-sirna

Research

JoVE Journal

Bioengineering

13.4K visualizações.  Arizona State University. Os detalhes do protocolo de artigo para site-specific transfecção de scrambled sequência de siRNA em uma cultura de células aderentes mamíferos usando uma rede de microeletrodos (MEA).

Vídeo: High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. 
Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect 6. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8
Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11. 
Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.

Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

Site-specific Bacterial Chromosome Engineering: &#934;C31 Integrase Mediated Cassette Exchange (IMCE)

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as plasmid replication, plasmid stability, plasmid incompatibility, and plasmid copy number variance. This method uses the site-specific integrase from the Streptomyces phage (&Phi;) C312,3. The &Phi;C31 integrase catalyzes a direct recombination between two specific DNA sites: attB and attP (34 and 39 bp, respectively)4. This recombination is stable and does not revert5. A "landing pad" (LP) sequence consisting of a spectinomycin- resistance gene, aadA (SpR), and the E. coli &szlig;-glucuronidase gene (uidA) flanked by attP sites has been integrated into the chromosomes of Sinorhizobium meliloti, Ochrobactrum anthropi, and Agrobacterium tumefaciens in an intergenic region, the ampC locus, and the tetA locus, respectively. S. meliloti is used in this protocol. Mobilizable donor vectors containing attB sites flanking a stuffer red fluorescent protein (rfp) gene and an antibiotic resistance gene have also been constructed. In this example the gentamicin resistant plasmid pJH110 is used. The rfp gene6 may be replaced with a desired construct using SphI and PstI. Alternatively a synthetic construct flanked by attB sites may be sub-cloned into a mobilizable vector such as pK19mob7. The expression of the &Phi;C31 integrase gene (cloned from pHS628) is driven by the lac promoter, on a mobilizable broad host range plasmid pRK78139.
A tetraparental mating protocol is used to transfer the donor cassette into the LP strain thereby replacing the markers in the LP sequence with the donor cassette. These cells are trans-integrants. Trans-integrants are formed with a typical efficiency of 0.5%. Trans-integrants are typically found within the first 500-1,000 colonies screened by antibiotic sensitivity or blue-white screening using 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (X-gluc). This protocol contains the mating and selection procedures for creating and isolating trans-integrants.

Site-specific Bacterial Chromosome Engineering: &amp;#934;C31 Integrase Mediated Cassette Exchange (IMCE)

A quick and efficient method to integrate foreign DNA of interest into pre-made acceptor strains, termed landing pad strains, is described. The method allows site-specific integration of a DNA cassette into the engineered landing pad locus of a given strain, through conjugation and expression of the &#934;C31 integrase.

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as ...

Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon

The method described here consists in redesigning E. coli adherence properties by assembling the minimum number of curli genes under the control of a strong and metal-overinducible promoter, and in visualizing and quantifying the resulting gain of bacterial adherence. This method applies appropriate engineering principles of abstraction and standardization of synthetic biology, and results in the BBa_K540000 Biobrick (Best new Biobrick device, engineered, iGEM 2011).
The first step consists in the design of the synthetic operon devoted to curli overproduction in response to metal, and therefore in increasing the adherence abilities of the wild type strain. The original curli operon was modified in silico in order to optimize transcriptional and translational signals and escape the "natural" regulation of curli. This approach allowed to test with success our current understanding of curli production. Moreover, simplifying the curli regulation by switching the endogenous complex promoter (more than 10 transcriptional regulators identified) to a simple metal-regulated promoter makes adherence much easier to control.
The second step includes qualitative and quantitative assessment of adherence abilities by implementation of simple methods. These methods are applicable to a large range of adherent bacteria regardless of biological structures involved in biofilm formation. Adherence test in 24-well polystyrene plates provides a quick preliminary visualization of the bacterial biofilm after crystal violet staining. This qualitative test can be sharpened by the quantification of the percentage of adherence. Such a method is very simple but more accurate than only crystal violet staining as described previously 1 with both a good repeatability and reproducibility. Visualization of GFP-tagged bacteria on glass slides by fluorescence or laser confocal microscopy allows to strengthen the results obtained with the 24-well plate test by direct observation of the phenomenon.

The design of a synthetic operon encoding both the secretory apparatus and the structural monomers of curli fibers is described. Overproduction of these amyloids and adherent polymers allows a measurable gain of adherence of the E. coli chassis1. Easy ways to visualize and quantify adherence are explained.

The  method described here consists in redesigning E. coli adherence properties by assembling the minimum number of  curli genes under the control of a ...

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening based on cells in such environments have therefore serious limitations: cell organelles show extreme phenotypes, cell morphologies and sizes are heterogeneous and/or specific cell organelles cannot be properly visualized. In addition, cells in vivo are located in a 3D environment; in this situation, cells show different phenotypes mainly because of their interaction with the surrounding extracellular matrix of the tissue. In order to standardize and generate order of single cells in a physiologically-relevant 3D environment for cell-based assays, we report here the microfabrication and applications of a device for in vitro 3D cell culture. This device consists of a 2D array of microcavities (typically 105 cavities/cm2), each filled with single cells or embryos. Cell position, shape, polarity and internal cell organization become then normalized showing a 3D architecture. We used replica molding to pattern an array of microcavities, &#8216;eggcups&#8217;, onto a thin polydimethylsiloxane (PDMS) layer adhered on a coverslip. Cavities were covered with fibronectin to facilitate adhesion. Cells were inserted by centrifugation. Filling percentage was optimized for each system allowing up to 80%. Cells and embryos viability was confirmed. We applied this methodology for the visualization of cellular organelles, such as nucleus and Golgi apparatus, and to study active processes, such as the closure of the cytokinetic ring during cell mitosis. This device allowed the identification of new features, such as periodic accumulations and inhomogeneities of myosin and actin during the cytokinetic ring closure and compacted phenotypes for Golgi and nucleus alignment. We characterized the method for mammalian cells, fission yeast, budding yeast, C. elegans with specific adaptation in each case. Finally, the characteristics of this device make it particularly interesting for drug screening assays and personalized medicine.

We report a device and a new method to study cells and embryos. Single cells are precisely ordered in microcavity arrays. Their 3D confinement is a step towards 3D environments encountered in physiological conditions and allows organelle orientation. By controlling cell shape, this setup minimizes variability reported in standard assays.

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the successful implementation of siRNA therapy requires the use of delivery platforms that can overcome the major challenges of siRNA delivery, such as enzymatic degradation, low intracellular uptake and lysosomal entrapment. Here, a protocol for the preparation and use of a biocompatible and effective siRNA delivery system is presented. This platform consists of polyethylenimine (PEI) and arginine (Arg)-grafted porous silicon microparticles, which can be loaded with siRNA by performing a simple mixing step. The silicon particles are gradually degraded over time, thereby triggering the formation of Arg-PEI/siRNA nanoparticles. This delivery vehicle provides a means for protecting and internalizing siRNA, without causing cytotoxicity. The major steps of polycation functionalization, particle characterization, and siRNA loading are outlined in detail. In addition, the procedures for determining particle uptake, cytotoxicity, and transfection efficacy are also described.

Delivery remains the main challenge for the therapeutic implementation of small interfering RNA (siRNA). This protocol involves the use of a multifunctional and biocompatible siRNA delivery platform, consisting of arginine and polyethylenimine grafted porous silicon microparticles.

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes. The emergence of prion diseases in wildlife populations and their increasing threat to human health has led to increased efforts to find a treatment for these diseases. Recent studies have found numerous anti-prion compounds that can either inhibit the infectious PrPRes isomer or down regulate the normal cellular prion protein. However, most of these compounds do not cross the blood brain barrier to effectively inhibit PrPRes formation in brain tissue, do not specifically target neuronal PrPC, and are often too toxic to use in animal or human subjects. 
We investigated whether siRNA delivered intravascularly and targeted towards neuronal PrPC is a safer and more effective anti-prion compound. This report outlines a protocol to produce two siRNA liposomal delivery vehicles, and to package and deliver PrP siRNA to neuronal cells. The two liposomal delivery vehicles are 1) complexed-siRNA liposome formulation using cationic liposomes (LSPCs), and 2) encapsulated-siRNA liposome formulation using cationic or anionic liposomes (PALETS). For the LSPCs, negatively charged siRNA is electrostatically bound to the cationic liposome. A positively charged peptide (RVG-9r [rabies virus glycoprotein]) is added to the complex, which specifically targets the liposome-siRNA-peptide complexes (LSPCs) across the blood brain barrier (BBB) to acetylcholine expressing neurons in the central nervous system (CNS). For the PALETS (peptide addressed liposome encapsulated therapeutic siRNA), the cationic and anionic lipids were rehydrated by the PrP siRNA. This procedure results in encapsulation of the siRNA within the cationic or anionic liposomes. Again, the RVG-9r neuropeptide was bound to the liposomes to target the siRNA/liposome complexes to the CNS. Using these formulations, we have successfully delivered PrP siRNA to AchR-expressing neurons, and decreased the PrPC expression of neurons in the CNS.

The goal of this protocol is to use cationic/anionic liposomes with a neuro-targeting peptide as a CNS delivery system to enable siRNA to cross the BBB. The optimization of a delivery system for treatments, like siRNA, would allow for more treatment options for prion and other neurodegenerative diseases.

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes.  The ...

Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to the placenta while minimizing fetal and maternal side effects remains challenging. Targeted nanoparticle carriers provide new opportunities to treat placental disorders. We recently demonstrated that a synthetic placental chondroitin sulfate A binding peptide (plCSA-BP) could be used to guide nanoparticles to deliver drugs to the placenta. In this protocol, we describe in detail a system for assessing the efficiency of drug delivery to the placenta by plCSA-BP that employs three separate methods used in combination: in vivo imaging, high-frequency ultrasound (HFUS), and high-performance liquid chromatography (HPLC). Using in vivo imaging, plCSA-BP-guided nanoparticles were visualized in the placentas of live animals, while HFUS and HPLC demonstrated that plCSA-BP-conjugated nanoparticles efficiently and specifically delivered methotrexate to the placenta. Thus, a combination of these methods can be used as an effective tool for the targeted delivery of drugs to the placenta and development of new treatment strategies for several pregnancy complications.

We describe a system that utilizes three methods to evaluate the safety and effectiveness of placenta-targeted drug delivery: in vivo imaging to monitor nanoparticle accumulation, high-frequency ultrasound to monitor placental and fetal development, and HPLC to quantify drug delivery to tissue.

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to ...

Interfacing Microfluidics with Microelectrode Arrays for Studying Neuronal Communication and Axonal Signal Propagation

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of electrophysiological activity for long periods. However, the property of sensing signals from all sources around every microelectrode can become unfavorable when trying to understand communication and signal propagation in neuronal circuits. In a neuronal network, several neurons can be simultaneously activated and can generate overlapping action potentials, making it difficult to discriminate and track signal propagation. Considering this limitation, we have established an in vitro setup focused on assessing electrophysiological communication, which is able to isolate and amplify axonal signals with high spatial and temporal resolution. By interfacing microfluidic devices and MEAs, we are able to compartmentalize neuronal cultures with a well-controlled alignment of the axons and microelectrodes. This setup allows recordings of spike propagation with a high signal-to-noise ratio over the course of several weeks. Combined with specialized data analysis algorithms, it provides detailed quantification of several communication related properties such as propagation velocity, conduction failure, firing rate, anterograde spikes, and coding mechanisms.
This protocol demonstrates how to create a compartmentalized neuronal culture setup over substrate-integrated MEAs, how to culture neurons in this setup, and how to successfully record, analyze and interpret the results from such experiments. Here, we show how the established setup simplifies the understanding of neuronal communication and axonal signal propagation. These platforms pave the way for new in vitro models with engineered and controllable neuronal network topographies. They can be used in the context of homogeneous neuronal cultures, or with co-culture configurations where, for example, communication between sensory neurons and other cell types is monitored and assessed. This setup provides very interesting conditions to study, for example, neurodevelopment, neuronal circuits, information coding, neurodegeneration and neuroregeneration approaches.

This protocol aims to demonstrate how to combine in vitro microelectrode arrays with microfluidic devices for studying action potential transmission in neuronal cultures. Data analysis, namely the detection and characterization of propagating action potentials, is performed using a new advanced, yet user-friendly and freely available, computational tool.

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of ...

Fabrication of Ti3C2 MXene Microelectrode Arrays for In Vivo Neural Recording

Implantable microelectrode technologies have been widely used to elucidate neural dynamics at the microscale to gain a deeper understanding of the neural underpinnings of brain disease and injury. As electrodes are miniaturized to the scale of individual cells, a corresponding rise in the interface impedance limits the quality of recorded signals. Additionally, conventional electrode materials are stiff, resulting in a significant mechanical mismatch between the electrode and the surrounding brain tissue, which elicits an inflammatory response that eventually leads to a degradation of the device performance. To address these challenges, we have developed a process to fabricate flexible microelectrodes based on Ti3C2 MXene, a recently discovered nanomaterial that possesses remarkably high volumetric capacitance, electrical conductivity, surface functionality, and processability in aqueous dispersions. Flexible arrays of Ti3C2 MXene microelectrodes have remarkably low impedance due to the high conductivity and high specific surface area of the Ti3C2 MXene films, and they have proven to be exquisitely sensitive for recording neuronal activity. In this protocol, we describe a novel method for micropatterning Ti3C2 MXene into microelectrode arrays on flexible polymeric substrates and outline their use for in vivo micro-electrocorticography recording. This method can easily be extended to create MXene electrode arrays of arbitrary size or geometry for a range of other applications in bioelectronics and it can also be adapted for use with other conductive inks besides Ti3C2 MXene. This protocol enables simple and scalable fabrication of microelectrodes from solution-based conductive inks, and specifically allows harnessing the unique properties of hydrophilic Ti3C2 MXene to overcome many of the barriers that have long hindered the widespread adoption of carbon-based nanomaterials for high-fidelity neural microelectrodes.

Fabrication of Ti3C2 MXene Microelectrode Arrays for In Vivo Neural Recording

We describe here a method for fabricating Ti3C2 MXene microelectrode arrays and utilizing them for in vivo neural recording.

Implantable microelectrode technologies have been widely used to elucidate neural dynamics at the microscale to gain a deeper understanding of the neural ...

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided a unique opportunity to study the complex interplay among different cardiovascular cell types that drives tissue development and disease. In the area of cardiac tissue models, several sophisticated three-dimensional (3D) approaches use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to mimic physiological relevance and native tissue environment with a combination of extracellular matrices and crosslinkers. However, these systems are complex to fabricate without microfabrication expertise and require several weeks to self-assemble. Most importantly, many of these systems lack vascular cells and cardiac fibroblasts that make up over 60% of the nonmyocytes in the human heart. Here we describe the derivation of all three cardiac cell types from iPSCs to fabricate cardiac microtissues. This facile replica molding technique allows cardiac microtissue culture in standard multi-well cell culture plates for several weeks. The platform allows user-defined control over microtissue sizes based on initial seeding density and requires less than 3 days for self-assembly to achieve observable cardiac microtissue contractions. Furthermore, the cardiac microtissues can be easily digested while maintaining high cell viability for single-cell interrogation with the use of flow cytometry and single-cell RNA sequencing (scRNA-seq). We envision that this in vitro model of cardiac microtissues will help accelerate validation studies in drug discovery and disease modeling.

Here, we describe an easy-to-use methodology to generate 3D self-assembled cardiac microtissue arrays composed of pre-differentiated human-induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells. This user-friendly and low cell requiring technique to generate cardiac microtissues can be implemented for disease modeling and early stages of drug development.

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided ...