Photothermal nanoblades for delivery of large-sized cargo into mammalian cells at high throughput
01 Aug 2016-pp 47-50
TL;DR: This talk will present two new technologies, photothermal nanoblade and BLAST, that overcome the size limitation of cargo delivery into mammalian cells.
Abstract: Technologies for transferring large-sized cargo into mammalian cells are needed to advance key applications in cell engineering. However, reliable methodologies for introducing large-sized cargo into mammalian cells are nearly completely lacking. This talk will present two new technologies, photothermal nanoblade and BLAST, that overcome the size limitation of cargo delivery into mammalian cells.
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01 Jan 2020
TL;DR: This chapter mainly focuses on different physical drug-delivery techniques such as electroporation, optoporation, mechanopsoration, magnetoporation and hybrid techniques along with their working mechanisms, advantages, disadvantages, and limitations.
Abstract: Delivery of exogenous materials or cargo such as drugs, proteins, peptides, and nucleic acids into cells is a vital segment in molecular and cellular biology for potential cellular therapy and drug-discovery applications contributing toward personalization of medicine. Over the years, drug-delivery techniques have been developed in order to gain more control over the drug dosage, targeted delivery, and to minimize side effects. The major drug-delivery techniques can be classified as viral, chemical, and physical methods. Viral vectors are prominently used for gene therapy; however, they are cell-specific and have an immune response with high toxicity. Chemical methods are often limited by the low efficiency of plasmid delivery into different cell types due to plasmid degradation and toxicity. Considering these limitations, different physical methods such as photoporation, gene gun, hydrodynamic injection, electroporation, and mechanoporation, etc., are being widely developed for highly efficient cargo delivery with low toxicity. These methods are able to create transient hydrophilic membrane pores to deliver cargos into cells using different physical energies. Currently, ex vivo cargo delivery is widely studied while few in vivo applications have been developed. Concerning several obstacles to cargo delivery into cells, this chapter mainly focuses on different physical drug-delivery techniques such as electroporation, optoporation, mechanoporation, magnetoporation, and hybrid techniques along with their working mechanisms, advantages, disadvantages, and limitations. An insight into the future prospects and real-time applications of these techniques is also discussed.
18 citations
References
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TL;DR: Depending upon the cell line, lipofection is from 5- to greater than 100-fold more effective than either the calcium phosphate or the DEAE-dextran transfection technique.
Abstract: A DNA-transfection protocol has been developed that makes use of a synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). Small unilamellar liposomes containing DOTMA interact spontaneously with DNA to form lipid-DNA complexes with 100% entrapment of the DNA, DOTMA facilitates fusion of the complex with the plasma membrane of tissue culture cells, resulting in both uptake and expression of the DNA. The technique is simple, highly reproducible, and effective for both transient and stable expression of transfected DNA. Depending upon the cell line, lipofection is from 5- to greater than 100-fold more effective than either the calcium phosphate or the DEAE-dextran transfection technique.
5,434 citations
"Photothermal nanoblades for deliver..." refers background in this paper
...Chemical methods vary in delivery efficiency, are highly cell type dependent, and delivered cargo are frequently trapped in undesirable endosomes [2]....
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TL;DR: The ability of HIV-based viral vectors to deliver genes in vivo into nondividing cells could increase the applicability of retroviral vectors in human gene therapy.
Abstract: A retroviral vector system based on the human immunodeficiency virus (HIV) was developed that, in contrast to a murine leukemia virus-based counterpart, transduced heterologous sequences into HeLa cells and rat fibroblasts blocked in the cell cycle, as well as into human primary macrophages. Additionally, the HIV vector could mediate stable in vivo gene transfer into terminally differentiated neurons. The ability of HIV-based viral vectors to deliver genes in vivo into nondividing cells could increase the applicability of retroviral vectors in human gene therapy.
5,076 citations
"Photothermal nanoblades for deliver..." refers background in this paper
...Biological approaches are restricted to kbsized nucleic acids [1]....
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TL;DR: The data suggest that the primary means for intercellular spread involves cell fusion, as opposed to pseudopod engulfment and bacterial escape from double-membrane vacuoles, and that plaque formation represents MNGC death.
Abstract: Burkholderia pseudomallei and Burkholderia thailandensis are related pathogens that invade a variety of cell types, replicate in the cytoplasm, and spread to nearby cells. We have investigated temporal and spatial requirements for virulence determinants in the intracellular life cycle, using genetic dissection and photothermal nanoblade delivery, which allows efficient placement of bacterium-sized cargo into the cytoplasm of mammalian cells. The conserved Bsa type III secretion system (T3SSBsa) is dispensable for invasion, but is essential for escape from primary endosomes. By nanoblade delivery of B. thailandensis we demonstrate that all subsequent events in intercellular spread occur independently of T3SSBsa activity. Although intracellular movement was essential for cell–cell spread by B. pseudomallei and B. thailandensis, neither BimA-mediated actin polymerization nor the formation of membrane protrusions containing bacteria was required for B. thailandensis. Surprisingly, the cryptic (fla2) flagellar system encoded on chromosome 2 of B. thailandensis supported rapid intracellular motility and efficient cell–cell spread. Plaque formation by both pathogens was dependent on the activity of a type VI secretion system (T6SS-1) that functions downstream from T3SSBsa-mediated endosome escape. A remarkable feature of Burkholderia is their ability to induce the formation of multinucleate giant cells (MNGCs) in multiple cell types. By infection and nanoblade delivery, we observed complete correspondence between mutant phenotypes in assays for cell fusion and plaque formation, and time-course studies showed that plaque formation represents MNGC death. Our data suggest that the primary means for intercellular spread involves cell fusion, as opposed to pseudopod engulfment and bacterial escape from double-membrane vacuoles.
156 citations
"Photothermal nanoblades for deliver..." refers background in this paper
...This new device allows intracellular delivery of variably sized objects such as biomolecules, proteins, enzymes, quantum dots [7], beads, bacteria [8], to whole mitochondria [9], into soft mammalian somatic cells with high efficiency and cell viability....
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TL;DR: A high-throughput platform for delivering large cargo elements into 100,000 cells in 1 min and discovering that the iglC gene is unexpectedly required for intracellular replication even after phagosome escape into the cell cytosol is reported.
Abstract: We report a high-throughput platform for delivering large cargo elements into 100,000 cells in 1 min. Our biophotonic laser-assisted surgery tool (BLAST) generates an array of microcavitation bubbles that explode in response to laser pulsing, forming pores in adjacent cell membranes through which cargo is gently driven by pressurized flow. The platform delivers large items including bacteria, enzymes, antibodies and nanoparticles into diverse cell types with high efficiency and cell viability. We used this platform to explore the intracellular lifestyle of Francisella novicida and discovered that the iglC gene is unexpectedly required for intracellular replication even after phagosome escape into the cell cytosol.
149 citations
TL;DR: A new method for transferring isolated mitochondria into somatic mammalian cells using a photothermal nanoblade, which bypasses endocytosis and cell fusion is reported, which rescued the pyrimidine auxotroph phenotype and respiration of ρ0 cells that lack mtDNA.
Abstract: mtDNA sequence alterations are challenging to generate but desirable for basic studies and potential correction of mtDNA diseases. Here, we report a new method for transferring isolated mitochondria into somatic mammalian cells using a photothermal nanoblade, which bypasses endocytosis and cell fusion. The nanoblade rescued the pyrimidine auxotroph phenotype and respiration of ρ0 cells that lack mtDNA. Three stable isogenic nanoblade-rescued clones grown in uridine-free medium showed distinct bioenergetics profiles. Rescue lines 1 and 3 reestablished nucleus-encoded anapleurotic and catapleurotic enzyme gene expression patterns and had metabolite profiles similar to the parent cells from which the ρ0 recipient cells were derived. By contrast, rescue line 2 retained a ρ0 cell metabolic phenotype despite growth in uridine-free selection. The known influence of metabolite levels on cellular processes, including epigenome modifications and gene expression, suggests metabolite profiling can help assess the quality and function of mtDNA-modified cells.
77 citations
"Photothermal nanoblades for deliver..." refers background in this paper
...This new device allows intracellular delivery of variably sized objects such as biomolecules, proteins, enzymes, quantum dots [7], beads, bacteria [8], to whole mitochondria [9], into soft mammalian somatic cells with high efficiency and cell viability....
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