Rapid Generation of Therapeutic Nanoparticles Using Cell-Free Expression Systems

Justin A. Peruzzi, Timothy Q. Vu, Taylor F. Gunnels, Neha P. Kamat*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

Abstract

The surface modification of membrane-based nanoparticles, such as liposomes, polymersomes, and lipid nanoparticles, with targeting molecules, such as binding proteins, is an important step in the design of therapeutic materials. However, this modification can be costly and time-consuming, requiring cellular hosts for protein expression and lengthy purification and conjugation steps to attach proteins to the surface of nanocarriers, which ultimately limits the development of effective protein-conjugated nanocarriers. Here, the use of cell-free protein synthesis systems to rapidly create protein-conjugated membrane-based nanocarriers is demonstrated. Using this approach, multiple types of functional binding proteins, including affibodies, computationally designed proteins, and scFvs, can be cell-free expressed and conjugated to liposomes in one-pot. The technique can be expanded further to other nanoparticles, including polymersomes and lipid nanoparticles, and is amenable to multiple conjugation strategies, including surface attachment to and integration into nanoparticle membranes. Leveraging these methods, rapid design of bispecific artificial antigen presenting cells and enhanced delivery of lipid nanoparticle cargo in vitro is demonstrated. It is envisioned that this workflow will enable the rapid generation of membrane-based delivery systems and bolster our ability to create cell-mimetic therapeutics.

Original languageEnglish (US)
Article number2201718
JournalSmall Methods
Volume7
Issue number12
DOIs
StatePublished - Dec 15 2023

Funding

J.A.P. and T.Q.V. contributed equally to this work. The authors thank C. Hilburger for helpful discussions regarding the design of lipid nanoparticle experiments, and the Kamat lab for providing feedback and proofreading the manuscript. This work was supported in part by the McCormick Research Catalyst Program at Northwestern University. This work was supported by the Northwestern University – Flow Cytometry Core Facility supported by Cancer Center Support Grant (NCI CA060553). The authors also thank the financial support of the CBC-NU Cell-free Biomanufacturing Institute funded by the U.S. Army Contracting Command Award W52P1J-21-9-3023 (J.A.P.). J.A.P. and T.F.G. were supported by an NSF Graduate Research Fellowship. J.A.P. gratefully acknowledges support from the Ryan Fellowship, and the International Institute for Nanotechnology at Northwestern University. T.Q.V. was supported by the National Institutes of Health Training Grant (T32GM008449) through Northwestern University's Biotechnology Training Program. J.A.P. and T.Q.V. contributed equally to this work. The authors thank C. Hilburger for helpful discussions regarding the design of lipid nanoparticle experiments, and the Kamat lab for providing feedback and proofreading the manuscript. This work was supported in part by the McCormick Research Catalyst Program at Northwestern University. This work was supported by the Northwestern University – Flow Cytometry Core Facility supported by Cancer Center Support Grant (NCI CA060553). The authors also thank the financial support of the CBC‐NU Cell‐free Biomanufacturing Institute funded by the U.S. Army Contracting Command Award W52P1J‐21‐9‐3023 (J.A.P.). J.A.P. and T.F.G. were supported by an NSF Graduate Research Fellowship. J.A.P. gratefully acknowledges support from the Ryan Fellowship, and the International Institute for Nanotechnology at Northwestern University. T.Q.V. was supported by the National Institutes of Health Training Grant (T32GM008449) through Northwestern University's Biotechnology Training Program.

Keywords

  • cell-free expression
  • functionalization
  • lipid nanoparticles
  • liposomes
  • vesicles

ASJC Scopus subject areas

  • General Chemistry
  • General Materials Science

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