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Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells
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Alcohol-containing protein transduction domain mimics.

Kayla C Koch1, Tamara M Bizmark2, Gregory N Tew3

  • 1Department of Polymer Science & Engineering, University of Massachusetts, Amherst, MA 01003, United States.

Journal of Controlled Release : Official Journal of the Controlled Release Society
|December 8, 2023
PubMed
Summary

This study introduces novel synthetic protein transduction domain mimics (PTDMs) with alcohol groups for enhanced biomacromolecule delivery. One PTDM (PTDM4) significantly improved antibody delivery and enzyme activity with reduced cationic density.

Keywords:
Antibody deliveryCation densityCre recombinaseRing opening metathesis polymerization

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Area of Science:

  • Biotechnology and Biomaterials Science
  • Polymer Chemistry
  • Drug Delivery Systems

Background:

  • Protein transduction domains (PTDs) and mimics (PTDMs) are crucial for intracellular biomacromolecule delivery.
  • Previous work synthesized block copolymer PTDMs using ring-opening metathesis polymerization (ROMP), optimizing hydrophobicity and cationic density.
  • Naturally occurring PTDs contain alcohol groups, but their impact in synthetic PTDMs is unexplored.

Purpose of the Study:

  • To investigate the effect of incorporating alcohol groups into synthetic PTDMs as a novel design parameter.
  • To synthesize and evaluate a library of novel PTDMs with varying alcohol group structures.
  • To assess the efficiency of these new PTDMs in the intracellular delivery of antibodies and active enzymes.

Main Methods:

  • Synthesis of nine novel PTDMs incorporating alcohol groups via ROMP.
  • Evaluation of PTDMs for intracellular delivery of fluorescently labeled antibodies.
  • Comparison of delivery efficiency and cationic density against a previously established PTDM control.
  • Assessment of active enzyme (TAT-Cre Recombinase) delivery and activity using the lead PTDM candidate.

Main Results:

  • A novel PTDM, PTDM4, incorporating alcohol groups in both hydrophobic and cationic blocks demonstrated superior performance.
  • PTDM4 achieved nearly double the median fluorescence intensity for antibody delivery compared to the control.
  • PTDM4 required only half the cationic density of the control for effective delivery.
  • The activity of TAT-Cre Recombinase delivered by PTDM4 was comparable to the control, also at half the cationic density.

Conclusions:

  • Incorporating alcohol groups is a viable and effective strategy for designing advanced PTDMs.
  • PTDM4 represents a significant advancement in PTDMs, offering enhanced delivery efficiency and reduced charge density.
  • This research opens new avenues for optimizing synthetic PTDMs for diverse intracellular delivery applications.