Universal intracellular biomolecule delivery with precise dosage control

Cao, Yuhong; Chen, Haodong; Qiu, Ruoyi; Hanna, Mira; Ma, Enbo; Hjort, Martin; Zhang, A.; Lewis, Richard S.; Wu, Joseph C.; Melosh, Nicholas A.

doi:10.1126/sciadv.aat8131

Cited by 101 publications

(167 citation statements)

References 42 publications

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“…The capability of simultaneous delivery of multiple biomolecules into a single cell would advance cellular manipulation technology such as stem cell reprogramming, gene editing, and cancer therapy. [ 26 ] Given the observed SiNT‐mediated loading and intracellular delivery of IgGs and ssDNAs into GPE86 cells, we further examined whether such SiNT arrays can be used as a codelivery platform. First, we mixed IgG‐AF647 and IgG‐AF488, both at 200 µg mL −1 concentration but with different v/v ratios, and loaded the IgG mixture onto SiNTs.…”

Section: Figurementioning

confidence: 99%

“…After validating the successful delivery of Cas9 RNP into cells, we next studied the gene‐editing efficiency by delivering either a control (SiNT‐Neg) or Hprt‐targeted Cas9 RNP (SiNT‐Hprt) into GPE86 cells using SiNTs; cells transfected with Hprt Cas9 RNP using Lipofectamine 2000 (Lipo‐Hprt) served as the positive control. Using the T7 endonuclease 1 (T7E1) assay, where T7E1 cleaves DNAs at mismatched spots that can be generated by Cas9 cutting, [ 26 ] we observed the two cleavage bands at expected sizes (130 and 520 bp) for cells transfected with SiNT‐Hprt and Lipo‐Hprt. In contrast, SiNT‐Neg cells and those without T7E1 digestion did not show these two cleavage bands, but the intact DNA strand at size 650 bp (Figure S11d, Supporting Information).…”

Section: Figurementioning

confidence: 99%

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Silicon‐Nanotube‐Mediated Intracellular Delivery Enables Ex Vivo Gene Editing

Chen

Aslanoglou

Murayama³

et al. 2020

Advanced Materials

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Engineered nano–bio cellular interfaces driven by vertical nanostructured materials are set to spur transformative progress in modulating cellular processes and interrogations. In particular, the intracellular delivery—a core concept in fundamental and translational biomedical research—holds great promise for developing novel cell therapies based on gene modification. This study demonstrates the development of a mechanotransfection platform comprising vertically aligned silicon nanotube (VA‐SiNT) arrays for ex vivo gene editing. The internal hollow structure of SiNTs allows effective loading of various biomolecule cargoes; and SiNTs mediate delivery of those cargoes into GPE86 mouse embryonic fibroblasts without compromising their viability. Focused ion beam scanning electron microscopy (FIB‐SEM) and confocal microscopy results demonstrate localized membrane invaginations and accumulation of caveolin‐1 at the cell–NT interface, suggesting the presence of endocytic pits. Small‐molecule inhibition of endocytosis suggests that active endocytic process plays a role in the intracellular delivery of cargo from SiNTs. SiNT‐mediated siRNA intracellular delivery shows the capacity to reduce expression levels of F‐actin binding protein (Triobp) and alter the cellular morphology of GPE86. Finally, the successful delivery of Cas9 ribonucleoprotein (RNP) to specifically target mouse Hprt gene is achieved. This NT‐enhanced molecular delivery platform has strong potential to support gene editing technologies.

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Section: Figurementioning

confidence: 99%

Section: Figurementioning

confidence: 99%

Silicon‐Nanotube‐Mediated Intracellular Delivery Enables Ex Vivo Gene Editing

Chen

Aslanoglou

Murayama³

et al. 2020

Advanced Materials

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“…This allows the application of lower voltage without reducing the electric field strength. This approach minimizes heating and pH changes [55,56]; as a consequence cell viability after electroporation and transfection efficiency are both increased [57][58][59][60][61][62].…”

Section: Bulk and Localized Electroporationmentioning

confidence: 99%

“…NanoEP has been used to deliver a variety of cargoes, including mRNAs, DNA plasmids, CRISPR/Cas9 RNA-protein complexes and functional stromal interaction molecule 1 (STIM1) proteins into the cell cytoplasm [58]. The nanostraws electroporation system (NES) has essentially the same set-up as the nanostraws system described above, but the addition of electrodes in the liquid reservoir underneath the straws and in the cell culture medium reservoir allows localized electroporation to be performed [59,66]. This increases the delivery efficiency over that of fluidic delivery and has been used to deliver proteins, CRISPR/Cas9 RNA-protein complexes and STIM1, into cells [59,66].…”

Section: Bulk and Localized Electroporationmentioning

confidence: 99%

Methods for protein delivery into cells: from current approaches to future perspectives

Chau

Actis

Hewitt

2020

Biochemical Society Transactions

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The manipulation of cultured mammalian cells by the delivery of exogenous macromolecules is one of the cornerstones of experimental cell biology. Although the transfection of cells with DNA expressions constructs that encode proteins is routine and simple to perform, the direct delivery of proteins into cells has many advantages. For example, proteins can be chemically modified, assembled into defined complexes and subject to biophysical analyses prior to their delivery into cells. Here, we review new approaches to the injection and electroporation of proteins into cultured cells. In particular, we focus on how recent developments in nanoscale injection probes and localized electroporation devices enable proteins to be delivered whilst minimizing cellular damage. Moreover, we discuss how nanopore sensing may ultimately enable the quantification of protein delivery at single-molecule resolution.

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“…For example, nanoneedle arrays can be used to achieve highly efficient intracellular delivery with a wide variety of materials and cell types. [ 45,66 ] Direct physical membrane penetration circumvents conventional biochemical delivery pathways and may avoid accompanying degradation routes. In addition, artificial nanoprobes can be directly inserted into the cell to interact with cytosolic contents, which allows for recording of intracellular electrical signals or sensing of intracellular biochemical activities.…”

Section: Introductionmentioning

confidence: 99%

Nanoneedle Platforms: The Many Ways to Pierce the Cell Membrane

He¹,

Hu²,

et al. 2020

Adv Funct Materials

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Establishing techniques to efficiently and nondestructively access the intracellular milieu is essential for many biomedical and scientific applications, ranging from drug delivery, to electrical recording, to biochemical detection. Cell penetration using nanoneedle arrays is currently a research focus area because it not only meets the increasing therapeutic demands of cell modifications and genome editing, but also provides an ideal platform for tracking long-term intracellular information. Although the precise mechanism driving membrane penetration by nanoneedle arrays is still unclear, the low cytotoxicity, wide range of delivered materials, diverse cell type targets, and simple material structures of nanoneedle arrays make these splendid platforms for cell access. Here, the recent progress in this field is reviewed by examining device architectures and discussing mechanisms for nanoneedle penetration, and the major studies demonstrating the most general applicability of nanoneedle arrays, typical methodologies to access the intracellular environment using nanoneedles with spontaneous or assisted penetration modes, as well as biosafety aspects are presented. This review should be valuable for deeply understanding the materials fabrication principles, device designs, cell penetration methodologies, biosafety aspects, and application strategies of nanoneedle array-based systems that are of crucial importance for the development of future practical biomedical platforms.

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Universal intracellular biomolecule delivery with precise dosage control

Abstract: We demonstrate quantitatively controlled delivery of DNA, mRNA, and protein into primary cells using nanostraw electroporation.

Cited by 101 publications

References 42 publications

Silicon‐Nanotube‐Mediated Intracellular Delivery Enables Ex Vivo Gene Editing

Silicon‐Nanotube‐Mediated Intracellular Delivery Enables Ex Vivo Gene Editing

Methods for protein delivery into cells: from current approaches to future perspectives

Nanoneedle Platforms: The Many Ways to Pierce the Cell Membrane

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