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Direct Cytosolic Delivery of Proteins Using Lyophilized and Reconstituted Polymer-Protein Assemblies

David C Luther1, Harini Nagaraj1, Ritabrita Goswami1

  • 1Department of Chemistry, University of Massachusetts, 379A LGRT Tower A, 710 North Pleasant St., Massachusetts, 01003, Amherst, USA.

Pharmaceutical Research
|March 17, 2022
PubMed
Abstract

Insights

Lyophilized polymer-protein nanocomposites offer stable, efficient cytosolic delivery of therapeutic proteins. This versatile technology enables point-of-care applications for chemotherapy and immunomodulation.

Area of Science:

  • Biotechnology
  • Nanotechnology
  • Drug Delivery

Background:

  • Cytosolic delivery of proteins is crucial for intracellular therapies like chemotherapy and immunomodulation.
  • Current protein delivery systems often fail due to inefficient endosomal pathways, leading to cargo degradation.
  • Cationic poly(oxanorbornene)imide (PONI) polymers facilitate efficient cytosolic protein delivery but suffer from limited shelf life due to aggregation.

Purpose of the Study:

  • To evaluate polymer-protein nanocomposite vehicles for lyophilization and point-of-care resuspension.
  • To establish a transferable technology for stable and efficient cytosolic protein delivery.
  • To overcome the shelf-life limitations of PONI polymers for protein delivery.

Main Methods:

  • Self-assembled nanocomposites of engineered poly(glutamate)-tagged (E-tagged) proteins and guanidinium-functionalized PONI homopolymers were created.
  • Nanocomposites were lyophilized, stored for two weeks, and then reconstituted for delivery.
  • Cytosolic delivery efficiency of E-tagged GFP (GFPE15) and therapeutic enzyme Granzyme A (GrAE10) was assessed in cell lines.

Main Results:

  • Reconstituted nanocomposites maintained structural integrity with minimal material loss.
  • High efficiency (>80%) of cytosolic delivery for GFPE15 was achieved, including nuclear diffusion.
  • Delivery of GrAE10 resulted in significant cancer cell death, validated by cell viability assays.

Conclusions:

  • Reconstituted polymer-protein nanocomposites provide efficient cytosolic protein delivery.
  • The technology demonstrated therapeutic potential through the delivery of cytotoxic Granzyme A.
  • This strategy offers a versatile and highly translatable method for protein-based therapeutics.
Keywords:
cytosolic deliverylyophilizationpoly(oxanorbornene)imideprotein deliverysupramolecular

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