Tissue nanotransfection (TNT) is an electroporation-based technique capable of gene and drug cargo delivery or transfection at the nanoscale. Furthermore, TNT is a scaffold-less tissue engineering (TE) technique that can be considered cell-only or tissue inducing depending on cellular or tissue level applications. The transfection method makes use of nanochannels to deliver cargo to tissues topically.
Cargo delivery methods rely on carriers, for example nanoparticles, viral vectors, or physical approaches such as gene guns, microinjection, or electroporation.The various methods can be limited by size constraints or their ability to efficiently deliver cargo without damaging tissue. Electroporation is a physical method which harnesses an electric field to open pores in the normally semi-permeable cell membrane through which cargo can enter. In this process, the charges can be used to drive cargo in a specific direction.
Bulk electroporation (BEP) is the most conventional electroporation method. Benefits come in the form of high throughput and minimal set-up times.The downside of BEP is that the cell membrane experiences an uneven distribution of the electric field and many membranes receive irreversible damage from which they can no longer close, thus leading to low cell viability.
Attempts have been made to miniaturize electroporation such as microelectroporation (MEP)and nanochannel electroporation (NEP) which uses electroporation approached to deliver cargo through micro/nanochannels respectively. These techniques have shown to have higher efficiency of delivery, increased uniform transfection, and increased cell viability compared to BEP.
Tissue nanotransfection uses custom fabricated nanochannel arrays for nanoscale delivery of genetic cargo directly onto the surface of the skin. The postage stamp-sized chip is placed directly on the skin and an electric current is induced lasting for milliseconds to deliver the gene cargo with precise control. This approach delivers ample amounts of reprogramming factors to single-cells, creating potential for a powerful gene transfection and reprogramming method.The delivered cargo then transforms the affected cells into a desired cell type without first transforming them to stem cells. TNT is a novel technique and has been used on mice models to successfully transfect fibroblasts into neuron-like cells along with rescue of ischemia in mice models with induced vasculature and perfusion. Current methods require the fabricated TNT chip to be placed on the skin and the loading reservoir filled with a gene solution. An electrode (cathode) is placed into the well with a counter electrode (anode) placed under the chip intradermally (into the skin). The electric field generated delivers the genes.
Initial TNT experiments showed that genes could be delivered to the skin of mice.Once this was confirmed, a cocktail of gene factors (ABM) used by Vierbuchen and collaborators to reprogram fibroblast into neurons was used. Delivery of these factors demonstrated successful reprogramming in-vivo and signals propagated from the epidermis to the dermis skin layers. This phenomenon is believed to be mediated by extracellular vesicles and potentially other factors . Successful reprogramming was determined by performing histology and electrophysiological tests to confirm the tissue behaved as functional neurons.
Beyond inducing neurons, Gallego-Perez et al. also set out to induce endothelial cells in an ischemic mouse limb that, without proper blood flow, becomes necrotic and decays. Using a patented cocktail of plasmids (Etv2, Fli1, Foxc2, or EFF), these factors were delivered to the tissue above the surgery site. Using various methods, including histology and laser speckle imaging, perfusion and the establishment of new vasculature was verified as early as 7 days post-treatment.
The technique was developed to combat the limitations of current approaches, such as a shortage in donors to supply cell sources and the need to induce pluripotency.Reprogramming cells in vivo takes advantage of readily available cells, bypassing the need for pre-processing. Most reprogramming methods have a heavy reliance on viral transfection. TNT allows for implementation of a non-viral approach which is able to overcome issues of capsid size, increase safety, and increase deterministic reprogramming.
The tissue nanotransfection technique was developed as a method to efficiently and benignly deliver cargo to living tissues. This technique builds on the high-throughput nanoelectroporation methods developed for cell reprogramming applications by Lee and Gallego-Perez of Ohio State's Chemical and Biomolecular Engineering department. Development was a joint effort between OSU's College of Engineering and College of Medicine led by Gallego-Perez (Ph.D), Lee (Ph.D), and Sen (Ph.D).
This technology was fabricated using cleanroom techniques and photolithography and deep reactive ion etching (DRIE) of silicon wafers to create nanochannels with backside etching of a reservoir for loading desired factors as described in Gallego-Perez et al 2017.This chip is then connected to an electrical source capable of delivering an electrical field to drive the factors from the reservoir into the nanochannels, and onto the contacted tissue.
Electroporation, or electropermeabilization, is a technique in which an electrical field is applied to cells in order to increase the permeability of the cell membrane. This may allow chemicals, drugs, electrode arrays or DNA to be introduced into the cell.
Transdifferentiation, also known as lineage reprogramming, is the process in which one mature somatic cell is transformed into another mature somatic cell without undergoing an intermediate pluripotent state or progenitor cell type. It is a type of metaplasia, which includes all cell fate switches, including the interconversion of stem cells. Current uses of transdifferentiation include disease modeling and drug discovery and in the future may include gene therapy and regenerative medicine. The term 'transdifferentiation' was originally coined by Selman and Kafatos in 1974 to describe a change in cell properties as cuticle producing cells became salt-secreting cells in silk moths undergoing metamorphosis.
In molecular biology and genetics, transformation is the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material from its surroundings through the cell membrane(s). For transformation to take place, the recipient bacterium must be in a state of competence, which might occur in nature as a time-limited response to environmental conditions such as starvation and cell density, and may also be induced in a laboratory.
Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded RNA at first non-coding RNA molecules, typically 20–24 base pairs in length, similar to miRNA, and operating within the RNA interference (RNAi) pathway. It interferes with the expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription, preventing translation.
Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. It may also refer to other methods and cell types, although other terms are often preferred: "transformation" is typically used to describe non-viral DNA transfer in bacteria and non-animal eukaryotic cells, including plant cells. In animal cells, transfection is the preferred term as transformation is also used to refer to progression to a cancerous state (carcinogenesis) in these cells. Transduction is often used to describe virus-mediated gene transfer into eukaryotic cells.
In biology, reprogramming refers to erasure and remodeling of epigenetic marks, such as DNA methylation, during mammalian development or in cell culture. Such control is also often associated with alternative covalent modifications of histones.
A short hairpin RNA or small hairpin RNA is an artificial RNA molecule with a tight hairpin turn that can be used to silence target gene expression via RNA interference (RNAi). Expression of shRNA in cells is typically accomplished by delivery of plasmids or through viral or bacterial vectors. shRNA is an advantageous mediator of RNAi in that it has a relatively low rate of degradation and turnover. However, it requires use of an expression vector, which has the potential to cause side effects in medicinal applications.
Sonophoresis also known as phonophoresis, is a method that utilizes ultrasound to enhance the delivery of topical medications through the stratum corneum, to the epidermis and dermis. Sonophoresis allows for the enhancement of the permeability of the skin along with other modalities, such as iontophoresis, to deliver drugs with lesser side effects. Currently, sonophoresis is used widely in transdermal drug delivery, but has potential applications in other sectors of drug delivery, such as the delivery of drugs to the eye and brain.
Gene delivery is the process of introducing foreign genetic material, such as DNA or RNA, into host cells. Gene delivery must reach the genome of the host cell to induce gene expression. Successful gene delivery requires the foreign gene delivery to remain stable within the host cell and can either integrate into the genome or replicate independently of it. This requires foreign DNA to be synthesized as part of a vector, which is designed to enter the desired host cell and deliver the transgene to that cell's genome. Vectors utilized as the method for gene delivery can be divided into two categories, recombinant viruses and synthetic vectors.
Magnetofection is a transfection method that uses magnetic fields to concentrate particles containing vectors to target cells in the body. Magnetofection has been adapted to a variety of vectors, including nucleic acids, non-viral transfection systems, and viruses. This method offers advantages such as high transfection efficiency and biocompatibility which are balanced with limitations.
Induced pluripotent stem cells are a type of pluripotent stem cell that can be generated directly from a somatic cell. The iPSC technology was pioneered by Shinya Yamanaka and Kazutoshi Takahashi in Kyoto, Japan, who together showed in 2006 that the introduction of four specific genes, collectively known as Yamanaka factors, encoding transcription factors could convert somatic cells into pluripotent stem cells. Shinya Yamanaka was awarded the 2012 Nobel Prize along with Sir John Gurdon "for the discovery that mature cells can be reprogrammed to become pluripotent."
Magnet-assisted transfection is a transfection method which uses magnetic interactions to deliver DNA into target cells. Nucleic acids are associated with magnetic nanoparticles, and magnetic fields drive the nucleic acid-particle complexes into target cells, where the nucleic acids are released.
Gene therapy utilizes the delivery of DNA into cells, which can be accomplished by several methods, summarized below. The two major classes of methods are those that use recombinant viruses and those that use naked DNA or DNA complexes.
A list of examples of transdifferentiation:
A list of examples of in vivo transdifferentiation through transfection:
Induced stem cells (iSC) are stem cells derived from somatic, reproductive, pluripotent or other cell types by deliberate epigenetic reprogramming. They are classified as either totipotent (iTC), pluripotent (iPSC) or progenitor or unipotent – (iUSC) according to their developmental potential and degree of dedifferentiation. Progenitors are obtained by so-called direct reprogramming or directed differentiation and are also called induced somatic stem cells.
Genetic engineering techniques allow the modification of animal and plant genomes. Techniques have been devised to insert, delete, and modify DNA at multiple levels, ranging from a specific base pair in a specific gene to entire genes. There are a number of steps that are followed before a genetically modified organism (GMO) is created. Genetic engineers must first choose what gene they wish to insert, modify, or delete. The gene must then be isolated and incorporated, along with other genetic elements, into a suitable vector. This vector is then used to insert the gene into the host genome, creating a transgenic or edited organism.
Mark Robert Prausnitz is an American chemical engineer, currently Regents’ Professor and J. Erskine Love, Jr. Chair in Chemical & Biomolecular Engineering at the Georgia Institute of Technology. He also serves as adjunct professor of biomedical engineering at Emory University and Adjunct Professor of Chemical & Biomolecular Engineering at the Korea Advanced Institute of Science and Technology. He is known for pioneering microneedle technology for minimally invasive drug and vaccine administration, which has found applications in transdermal, ocular, oral, and sustained release delivery systems.
Intracellular delivery is the process of introducing external materials into living cells. Materials that are delivered into cells include nucleic acids, proteins, peptides, impermeable small molecules, synthetic nanomaterials, organelles, and micron-scale tracers, devices and objects. Such molecules and materials can be used to investigate cellular behavior, engineer cell operations or correct a pathological function.
Hydrodynamic Delivery (HD) is a method of DNA insertion in rodent models. Genes are delivered via injection into the bloodstream of the animal, and are expressed in the liver. This protocol is helpful to determine gene function, regulate gene expression, and develop pharmaceuticals in vivo.