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Overview of Transfection

TRANSFECTION IS...

the delivery of nucleic acids into living cells through nonviral methods.

Transfection, the delivery of nucleic acids into mammalian cells through nonviral methods, has origins as far back as the 1950s . The ultimate goal of transfection is to deliver nucleic acids into cells so as to investigate gene function. This goal can be accomplished by expression of exogenous genes or by knockdown of endogenous genes. Manipulation of gene expression is a core technique in research areas such as drug development, cancer research, gene therapy, and tissue engineering. Additionally, in vivo gene therapy applications have created a need to develop safer and more efficient techniques for delivering nucleic acids to different organs and tissues. This in vivo research requires proof of concept, which is made possible through in vitro transfection experiments.

Timeline1973 In 1973, F. L. Graham’s and A. J. van der Eb’s landmark study established the use of calcium phosphate coprecipitation for DNA transfection. This method relies on the use of positively charged calcium ions to bind to the negatively charged phosphate backbone of DNA and form a co-precipitation complex that can then be added to cells for intracellular uptake. Although transfection technology has advanced considerably since that time, most chemical transfection reagents still employ the same basic underlying principle of forming transfection complexes through electrostatic interactions. Other gene delivery methods that were developed after the introduction of calcium phosphate methods include direct delivery via injection into the cell nucleus (microinjection), use of viral vectors, electrical currents and lipid mediated techniques.

Microinjection is a method for physically transferring nucleic acid or other material (e.g., organelles, stem cells or embryos) into a cell (i.e., cytoplasm or nucleus) via the use of a glass micropipette. With regards to nucleic acid, one can bypass the cellular membrane or the nuclear envelope for direct injection into the cytoplasm or nucleus, respectively. Although microinjection provides a direct method for delivery of nucleic acid for cells that are difficult to transfect, it is low-throughput and a technique that is difficult to master. An alternative and more efficient method for delivering nucleic acid into cells is the use of viral vectors.

The use of viral vectors is a commonplace methodology for efficiently delivering nucleic acid, especially in hard-to-transfect cells. Generation of recombinant viruses requires the packaging of exogenous DNA within the viral genome and subsequent delivery through infection of the target cell. The use of viral vectors was employed as early as the late 1970’s to express functional mRNA and protein. Although viral transduction is an efficient and effective option, some disadvantages include viral recombination, off-target effects, immune response induction, and possible oncogenic effects.

SUCCESSFUL DELIVERY...

is affected by several common factors... type of nucleic acid and target cell... cell density, confluency, and passage number...

Timeline1982 An alternative method for nucleic acid delivery into hard-to-transfect cells was developed that relied on the use of an electrical charge to deliver nucleic acid into cells. Electroporation relies on an electrical field to transiently increase cell permeability, a phenomenon known as electropermeabilization. The first use of electroporation for DNA delivery was demonstrated in 1982 by Eberhard Neumann. Neumann’s work showed that electrical impulses at certain field strengths led to the passage of DNA across the cell membrane and accumulation within the cell. Electroporation proved to be very effective in delivering nucleic acids to a wide variety of cells, including hard-to-transfect cells. However, the application of an electrical field causes substantial cytotoxicity. Despite this limitation, electroporation serves as an efficient method for delivering nucleic acids to cell types that are refractory to chemical transfection and to primary cells, which do not actively divide.

Although electroporation can be a very effective means for transfection, chemical means of transfection were expanded to provide an alternative to the undesired effects from viral transduction and the toxic, time consuming nature of electroporation. Since chemical delivery typically relies on the use of electrostatic interactions between cationic compounds and the negatively charged backbone of nucleic acids, transfection reagents are commonly composed of cationic lipids and/or cationic polymers that form electrostatic complexes with the nucleic acids.

An early example that employed lipids for transfection is a technique known as lipofection, which uses artificial lipid bilayers known as liposomes created by using cationic and helper lipids, to help deliver nucleic acid to cells. Cationic lipids (lipids with positively charged head groups) can combine on their own or with neutral or helper lipids to form small unilamellar vesicles. DNA can interact spontaneously with these vesicles to form lipid-DNA complexes from the positively charged group on the cationic lipid with the negatively charged phosphate groups on the DNA.

Lipofection is an effective gene delivery method for a variety of cell types; however, this method is often associated with significant cellular toxicity. Liposomal formulations can also be unstable in media containing serum typically required for prolific cell growth. Exploration of less toxic gene delivery approaches led to the use of other chemical based approaches that alleviate unwanted cytotoxic effects and in turn increased the success of nucleic acid delivery.

Alternatively, cationic polymers can form nucleic acid and polymer complexes (i.e., polyplexes) via electrostatic interactions between the positively charged polymer and the negatively charged nucleic acid. This interplay between the polycations of the polymer afforded by nitrogen residues and the negatively charged phosphate groups of the nucleic acid is referred to as the N/P ratio.

Varying N/P ratios can affect condensation of the nucleic acid and the net surface charge, or zeta potential. Condensation provides protection to the nucleic acid from degradation via nucleases. A net positive zeta potential will direct the complex to the negatively charged cell membrane, which leads to cellular uptake of the transfection complex via endocytosis.

To avoid intracellular degradation within the endocytic machinery, these transfection complexes must escape from the endosome and deliver the nucleic acid to the cytoplasm (mRNA, siRNA/miRNA) or the nucleus (plasmid DNA or shRNA enconding vectors). Since the cytoplasm is the native site of function for RNA molecules such as mRNA/siRNA/miRNA, delivery to the cytoplasm ensures functionality; however, plasmid DNA needs to enter the nucleus for effective function. Actively dividing cells transfect more efficiently than non-dividing cells as the nuclear envelope breakdown during cell division promotes nuclear transport of DNA. Therefore, primary cells (i.e., cells taken from living tissue) that do not actively divide are very recalcitrant to transfection.

Successful delivery of nucleic acids is affected by several common factors. Depending on the nature of the experiment, some aspects are not amenable to change (e.g., the type of nucleic acid and target cell). However, other factors—such as cell density, confluency, and passage number—can be more easily controlled, readily tested, and optimized to improve efficiency. Additional considerations include cellular toxicity, off-target effects, and immunological responses that may affect experimental results. The nature of the delivery method (chemical or physical) will have a profound effect on gene expression due to the efficiency of delivery and effects on cell health and viability. Therefore, it is imperative to choose a delivery method that causes the least perturbation.

Research directed toward increasing transfection efficiency calls upon comprehensive and detailed studies of cell biology and, in particular, the intracellular processes that affect exogenous nucleic acids. For chemical transfection, these interactions are dictated through receptors on the cell surface, electrostatic charges between the cationic compounds and the anionic membrane components, passage across the membrane through endosomal pathways, and ultimate escape from the endosome to avoid degradation before reaching the appropriate subcellular location.

Insights that yield higher electroporation efficiencies include an understanding of membrane structure coupled with optimal pulse parameters for each unique cell type. In addition, to ensure safe passage of nucleic acids through the cytosol and intracellular vesicles, protection from hydrolytic enzymes and escape from the endosomal compartments must be considered.

As the repertoire of cell types that serve as ideal research models continues to expand toward hard-to-transfect cell types, including primary cells and stem cells, more effective nucleic acid delivery reagents and methods are needed. Changes in the genetic background of these cell types can mimic several disease states and can serve as models to determine the mechanisms behind these diseases. Better transfection methods can enable a thorough investigation of gene function and can become a step on the pathway to gene therapy in personalized medicine.

Transfection Methods

Transfection Schematic

Transfection Schematic

Liposome

Liposome

Cell Confluency

Confluency

Transfection Methods

The two main avenues for non-viral delivery of nucleic acids are chemical transfection and electroporation. An increased knowledge of cell and membrane biology, expansion of chemical libraries, and the introduction of new instruments will also aid the development of novel methods for transfection.

Chemical Methods for Transfection

Chemical Methods for Transfection

Chemical transfection methods typically rely on electrostatic interactions to bind nucleic acid and to target cell membranes. These methods utilize compounds such as calcium phosphate, polycations (e.g., DEAE-dextran), and liposomes, as well as more current technologies such as cationic lipids, polymers, dendrimers, and nanoparticles, to name a few.

Calcium phosphate represents the oldest and most inexpensive chemical method for transfecting nucleic acids. This technique utilizes a solution of HEPES-buffered saline (HeBS) that contains phosphate ions (e.g., Na3PO4), which, upon addition of CaCl2, forms a fine precipitate that binds DNA. The resulting complexes are added to cells, attach to the cell surface, and are taken up through endocytosis. Additional modifications to the calcium phosphate method that can achieve higher efficiencies include glycerol shock and/or chloroquine treatment. Despite the simple and cost-effective nature of the calcium phosphate method, it is ineffective for hard-to-transfect cells, is very sensitive to changes in pH, often lacks reproducibility, and requires large quantities of DNA. These disadvantages necessitate more robust methods for nucleic acid delivery.

More efficient transfection methods use organic compounds such as liposomes, charged lipids, and polymers. Liposomes can encapsulate DNA and requires extrusion, a method for forming lipid bilayers by forcing lipids through a membrane filter, or lipsomes can coat the nucleic acid and trap it between positively charged liposomes and does not require extrusion. Charged lipids and polymers form complexes with nucleic acids to generate lipoplexes and polyplexes, respectively. Additionally, lipid, polymer, and nucleic acids can combine to form a lipopolyplex. All of these complexes exhibit a net positive charge after binding to DNA, which enables association with the cell membrane through electrostatic interactions. Cellular uptake occurs via endocytosis.

Timeline1987 Liposome-mediated techniques were first introduced by Felgner and have become some of the most widely studied non-viral approaches for nucleic acid delivery. The spherical liposome structures contain hydrophilic and hydrophobic regions imparted by the lipid polar head and hydrophobic tail groups, respectively. The amphipathic properties of these lipids lead to the spontaneous formation of bilayer structures that make up the basic structure of liposomes. For recruitment to the cell, liposomes must have a slight net positive charge. Their sizes can vary from smaller unilamellar vesicles of 20–200 nm to larger vesicles of 200 nm–1 µm, and upwards of 1 µm for giant unilamellar vesicles. The latter can also exist as multilamellar vesicles, consisting of multiple lipid bilayers. Ultimately, size is dictated by the thermodynamic properties of the lipid used in the system.

Types of Vesicles

Types of Vesicles

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Despite the proven utility of cationic liposomes as nucleic acid delivery vehicles, lipofection can be quite toxic to cells, an obvious deterrent for many researchers. Lipofection-mediated toxicity has been demonstrated through cytotoxicity and cell proliferation assays, including those that measure cell morphology, mitochondrial activity, DNA synthesis, and cell viability. Transfection-mediated toxicity usually leads to changes in gene expression, even in the absence of any visual toxicity. Therefore, it is important to choose a transfection method that can mitigate both observed as well as non-apparent cellular toxicity.

Cationic lipids and polymers also form complexes with DNA through electrostatic interactions. However, lipoplexes and polyplexes cause less toxicity than liposomal formulations. A few well-characterized cationic lipids used to form lipoplexes include:

Dioleoylphosphatidylethanolamine (DOPE) is a neutral helper lipid often used in conjunction with cationic lipids because of its membrane-destabilizing effects at low pH, which facilitates endosomal escape.

Cationic polymers commonly used for transfection include polyethylenimine (PEI) and polylysine (ε-poly-L-lysine). These polymers condense DNA into positively charged particles that can bind to the anionic cell surface. Subsequent uptake into the cell also occurs via endocytosis.

After cellular uptake, successful transfection requires release of the nucleic acid from the endosome to avoid degradation, and localization to the cytoplasm (for RNA) or to the nucleus (for DNA). Various strategies have been used to promote endosomal release. Chemicals, such as chloroquine, or the intrinsic ability of certain cationic polymers can prevent acidification of the endosome by absorbing protons through a process known as the ‘proton sponge’ effect. As protons are absorbed, an influx of chloride ions and water causes osmotic swelling and eventual lysis of the endosome, which promotes the release of its contents.

Following endosomal release, the final localization of the transfected nucleic acid depends on properties of both the nucleic acid and the transfection reagent. Cytoplasmic delivery is sufficient for mRNA and siRNA or miRNA. In the cytoplasm, mRNA can access ribosomes for translation, and siRNA or miRNA can associate with the RNA-induced silencing complex (RISC) in order to target endogenous RNA sequences for gene silencing. In contrast to RNA molecules, plasmid DNA must enter the nucleus in order for transcription to occur. The nuclear envelope presents a huge barrier that may be circumvented by natural nuclear envelope breakdown and reformation during cell division. Nuclear localization sequences (NLS) or targeting ligands have been used to promote localization to the nucleus but without consistent, reproducible efficacy.

DEAE-dextran

Calcium Phosphate Method

Calcium Phosphate Method
DOTMA DOTAP DC-chol DOGS DOPE PEI PLL

Electroporation

Electroporation

Electroporation is a physical transfection method. It uses short electrical pulses to generate an electrical field that overcomes cell membrane capacitance and creates transient membrane pores through which small particles can pass. Electrical fields can be controlled via pulse settings that depend on the instrument and other experimental conditions, such as the composition of the resuspension solution. Optimization of these pulse parameters for each cell type can enhance electropermeabilization to promote passage of nucleic acids through the cell membrane.

This technology was initially developed for in vitro DNA delivery but has expanded to include transfection of other nucleic acids (e.g., oligos, mRNA, siRNA, and miRNA), drug delivery, cell-cell fusion (i.e., electrofusion), and membrane protein insertion (i.e., electroinsertion). Since electroporation can be effective regardless of the type of molecule delivered or the target source, this technique can provide a robust and universal approach for multiple applications.

Delivery of nucleic acids is typically conducted using an electroporator with exponential decay, square wave, or time-constant pulse conditions. Cells are suspended in an inert solution along with the nucleic acid to be transfected and then the mixture is placed within a container (e.g., a cuvette) containing electrodes that will allow the passage of an electrical current. In exponential-decay pulse conditions, a set voltage is released from the capacitor and decays exponentially. A square-wave pulse is defined by pulse duration, number of pulses, length of interval between pulses, and voltage delivered. In a time-constant pulse, a continuous pulse is applied for a set period of time at a set voltage.

Electroporation process

Electroporation can be an effective and efficient alternative to chemical transfection. Although this method can be toxic to cells, toxicity can be minimized through experimental optimization and may be counterbalanced by increased transfection efficiencies. Conditions similar to chemical transfection (e.g., nucleic acid amount and cell density) or parameters unique to electrical methods (e.g., voltage and pulse types) can be changed to decrease cell mortality. Despite overall higher toxicity, electroporation may be a viable option for cell types that are not responsive to chemical transfection. With this in mind, electroporation provides a robust and universal approach for transfecting various cell types—including bacterial, mammalian, yeast, and plant cells—with any type of nucleic acid.

Electrofusion & Electroinsertion

The disruption of cell membranes with brief electrical pulses has many in vitro and in vivo applications, some of which are discussed below.

The use of electrical pulses to disrupt cell membranes induces fusion of cells, i.e., electrofusion. Electrofusion can combine both different cell types and cytoplasmic contents, including organelles and nuclei. This technique involves alignment and compression of cells, membrane fusion, maturation of fused membranes, and cell recovery and growth. Applications of this technique include fusion of tumor and dendritic cells for immunotherapy, antibody production via hybridomas, and fertility or animal propagation via nuclear transfer. Another application is somatic cell nuclear transfer between an oocyte and donor cell for the production of stem cells.

Electroinsertion uses electrical charges to transiently break down membranes to allow implantation of foreign proteins into the cell membrane. Potential applications range from antibody binding to cellular detection. Insertion of extraneous proteins via electroporation has been reported in red blood cells, multilamellar and giant unilamellar vesicles, and mammalian cells. The success of this technique depends on the compatibility of hydrophobic regions between the protein of interest, the cell membrane and on potential for autoimmune response. However, with effective application, fully functional and stable proteins may be integrated into a novel membrane environment.

Drug delivery is also possible via electroporation through skin. This technique bypasses the use of needles and improper absorption of oral medication. The efficacy of chemotherapy increases when used in combination with electroporation of tumor cells. Some gene therapy strategies use electroporation across skin to deliver nucleic acids for regulating gene expression within a given target tissue.

Novel Transfection Methods

Advances in the field of transfection are spurred by the need to increase efficiency, to broaden the range of transfectable target cell or tissue types, and to address specific workflow requirements. Improvements have been made to chemical and electrical approaches to address these concerns. A few examples are highlighted in this section.

One modification to chemical transfection is the implementation of magnetic-mediated delivery to increase transfection efficiency. Nucleic acid complexes are formed with magnetic nanoparticles, and a magnetic field is applied to bring the complexes into close proximity with the target cell. The complexes are then taken up via endocytosis as in chemical transfection. This approach is thought to increase transfection efficiency by bringing complexes closer to their cellular targets.

Another interesting approach is the biolistic particle delivery system, more commonly referred to as a gene gun. This method was first introduced for transfection of plant cells but has since been expanded for use in various organisms, such as bacteria, yeasts, and mammalian cells—particularly hard-to-transfect cells, e.g., primary cells. In addition, specific organelles (e.g., chloroplasts and mitochondria) can also be targeted. Biolistic delivery works by binding complexes of nucleic acids to metal atoms, such as tungsten or gold, and accelerating the nucleic acid/metal complex through a vacuum for delivery to a target cell or tissue. This approach can also deliver DNA vaccines in vivo for gene therapy. This biolistic delivery approach can save time by circumventing cellular endosomal processes and can make the gene product readily available within the cell.

Continuous flow electroporation (CFE) was developed for optimal process workflow and high-throughput applications. In CFE, cells are mixed with the compound to be delivered (e.g., nucleic acid or therapeutic agent) and allowed to flow through an electroporation chamber. As the mixture of cells and compound pass through the chamber in a continuous stream, they are uniformly subjected to an electrical field, which provides for consistent delivery of the compound and allows for uniform transfection of a large number of cells.

With a burgeoning interest in transfection, particularly for therapeutic purposes, a wide variety of new transfection methods and developments are expected. These advances will most certainly be prompted by a need to address new targets, increase transfection efficiency, and expand workflow. Ultimately, these developments will provide better avenues to study existing and new disease states, leading to improved treatments.

Magnetic-Mediated Delivery

Magnetic Transfection