Cellular Delivery of Large Functional Proteins and Protein-Nucleic Acid Constructs via Localized Electroporation

Nibir Pathak, Cesar A. Patino, Namrata Ramani, Prithvijit Mukherjee, Devleena Samanta, Sasha B. Ebrahimi, Chad A. Mirkin, Horacio D. Espinosa*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

7 Scopus citations

Abstract

Delivery of proteins and protein-nucleic acid constructs into live cells enables a wide range of applications from gene editing to cell-based therapies and intracellular sensing. However, electroporation-based protein delivery remains challenging due to the large sizes of proteins, their low surface charge, and susceptibility to conformational changes that result in loss of function. Here, we use a nanochannel-based localized electroporation platform with multiplexing capabilities to optimize the intracellular delivery of large proteins (β-galactosidase, 472 kDa, 75.38% efficiency), protein-nucleic acid conjugates (protein spherical nucleic acids (ProSNA), 668 kDa, 80.25% efficiency), and Cas9-ribonucleoprotein complex (160 kDa, ∼60% knock-out and ∼24% knock-in) while retaining functionality post-delivery. Importantly, we delivered the largest protein to date using a localized electroporation platform and showed a nearly 2-fold improvement in gene editing efficiencies compared to previous reports. Furthermore, using confocal microscopy, we observed enhanced cytosolic delivery of ProSNAs, which may expand opportunities for detection and therapy.

Original languageEnglish (US)
Pages (from-to)3653-3660
Number of pages8
JournalNano letters
Volume23
Issue number8
DOIs
StatePublished - Apr 26 2023

Keywords

  • CRISPR gene editing
  • localized electroporation
  • protein delivery
  • spherical nucleic acids

ASJC Scopus subject areas

  • General Chemistry
  • Condensed Matter Physics
  • Mechanical Engineering
  • Bioengineering
  • General Materials Science

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