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Survival rate of eukaryotic cells following electrophoretic nanoinjection.

ABSTRACT: Insertion of foreign molecules such as functionalized fluorescent probes, antibodies, or plasmid DNA to living cells requires overcoming the plasma membrane barrier without harming the cell during the staining process. Many techniques such as electroporation, lipofection or microinjection have been developed to overcome the cellular plasma membrane, but they all result in reduced cell viability. A novel approach is the injection of cells with a nanopipette and using electrophoretic forces for the delivery of molecules. The tip size of these pipettes is approximately ten times smaller than typical microinjection pipettes and rather than pressure pulses as delivery method, moderate DC electric fields are used to drive charged molecules out of the tip. Here, we show that this approach leads to a significantly higher survival rate of nanoinjected cells and that injection with nanopipettes has a significantly lower impact on the proliferation behavior of injected cells. Thus, we propose that injection with nanopipettes using electrophoretic delivery is an excellent alternative when working with valuable and rare living cells, such as primary cells or stem cells.

SUBMITTER: Simonis M

PROVIDER: S-EPMC5264641 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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Survival rate of eukaryotic cells following electrophoretic nanoinjection.

Simonis Matthias M Hübner Wolfgang W Wilking Alice A Huser Thomas T Hennig Simon S

Scientific reports 20170125

Insertion of foreign molecules such as functionalized fluorescent probes, antibodies, or plasmid DNA to living cells requires overcoming the plasma membrane barrier without harming the cell during the staining process. Many techniques such as electroporation, lipofection or microinjection have been developed to overcome the cellular plasma membrane, but they all result in reduced cell viability. A novel approach is the injection of cells with a nanopipette and using electrophoretic forces for th ...[more]

PMID: 28120926

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Project description:BackgroundNanoinjection-the process of intracellular delivery using vertically configured nanostructures-is a physical route that efficiently negotiates the plasma membrane, with minimal perturbation and toxicity to the cells. Nanoinjection, as a physical membrane-disruption-mediated approach, overcomes challenges associated with conventional carrier-mediated approaches such as safety issues (with viral carriers), genotoxicity, limited packaging capacity, low levels of endosomal escape, and poor versatility for cell and cargo types. Yet, despite the implementation of nanoinjection tools and their assisted analogues in diverse cellular manipulations, there are still substantial challenges in harnessing these platforms to gain access into cell interiors with much greater precision without damaging the cell's intricate structure. Here, we propose a non-viral, low-voltage, and reusable electroactive nanoinjection (ENI) platform based on vertically configured conductive nanotubes (NTs) that allows for rapid influx of targeted biomolecular cargos into the intracellular environment, and for successful gene silencing. The localization of electric fields at the tight interface between conductive NTs and the cell membrane drastically lowers the voltage required for cargo delivery into the cells, from kilovolts (for bulk electroporation) to only ≤ 10 V; this enhances the fine control over membrane disruption and mitigates the problem of high cell mortality experienced by conventional electroporation.ResultsThrough both theoretical simulations and experiments, we demonstrate the capability of the ENI platform to locally perforate GPE-86 mouse fibroblast cells and efficiently inject a diverse range of membrane-impermeable biomolecules with efficacy of 62.5% (antibody), 55.5% (mRNA), and 51.8% (plasmid DNA), with minimal impact on cells' viability post nanoscale-EP (> 90%). We also show gene silencing through the delivery of siRNA that targets TRIOBP, yielding gene knockdown efficiency of 41.3%.ConclusionsWe anticipate that our non-viral and low-voltage ENI platform is set to offer a new safe path to intracellular delivery with broader selection of cargo and cell types, and will open opportunities for advanced ex vivo cell engineering and gene silencing.

Project description:The rehabilitation of wildlife can contribute directly to the conservation of threatened species by helping to maintain wild populations. This study focused on determining the post-rehabilitation survival and spatial ecology of sea turtles and on comparing the movements of individuals with flipper amputations (amputees) to non-amputee animals. Our aims were to assess whether rehabilitated sea turtles survive after release, to compare and contrast the movement characteristics of the different species of sea turtles we tracked, and to examine whether amputees and non-amputees within species behaved similarly post-release. Twenty-six rehabilitated sea turtles from four species, including hawksbill Eretmochelys imbricata (n = 12), loggerhead Caretta caretta (n = 11), green Chelonia mydas (n = 2), and olive ridley Lepidochelys olivacea (n = 1) sea turtles from the United Arab Emirates were fitted with satellite tags before release. Rehabilitation times ranged from 89 to 817 days (mean 353 ± 237 days). Post-release movements and survival were monitored for 8 to 387 days (mean 155 ± 95 days) through satellite tracking. Tag data suggested that three tracked sea turtles died within four days of release, one after 27 days, and one after 192 days from what are thought to be anthropogenic factors unrelated to their pre-rehabilitation ailments. We then compared habitat use and movement characteristics among the different sea turtle species. Although half of all turtles crossed one or more international boundaries, dispersal varied among species. Loggerhead turtles had a high dispersal, with 80% crossing an international boundary, while hawksbill turtles displayed higher post-release residency, with 66% remaining within UAE territorial waters. Amputee turtles moved similarly to non-amputee animals of the same species. Loggerhead turtles travelled faster (mean ± sd = 15.3 ± 8 km/day) than hawksbill turtles (9 ± 7 km/day). Both amputee and non-amputee sea turtles within a species moved similarly. Our tracking results highlight that rehabilitated sea turtles, including amputees, can successfully survive in the wild following release for up to our ~one-year monitoring time therefore supporting the suitability for release of sea turtles that have recovered from major injuries such as amputations. However, more broadly, the high mortality from anthropogenic factors in the Arabian Gulf region is clearly a serious issue and conservation challenge.

Dataset Information

Survival rate of eukaryotic cells following electrophoretic nanoinjection.

Publications

Survival rate of eukaryotic cells following electrophoretic nanoinjection.

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