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

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Abstract

Purpose

Cytosolic delivery of proteins accesses intracellular targets for chemotherapy and immunomodulation. Current delivery systems utilize inefficient endosomal pathways of uptake and escape that lead to degradation of delivered cargo. Cationic poly(oxanorbornene)imide (PONI) polymers enable highly efficient cytosolic delivery of co-engineered proteins, but aggregation and denaturation in solution limits shelf life. In the present study we evaluate polymer-protein nanocomposite vehicles as candidates for lyophilization and point-of-care resuspension to provide a transferrable technology for cytosolic protein delivery.

Methods

Self-assembled nanocomposites of engineered poly(glutamate)-tagged (E-tagged) proteins and guanidinium-functionalized PONI homopolymers were generated, lyophilized, and stored for 2 weeks. After reconstitution and delivery, cytosolic access of E-tagged GFP cargo (GFPE15) was assessed through diffuse cytosolic and nuclear fluorescence, and cell killing with chemotherapeutic enzyme Granzyme A (GrAE10). Efficiency was quantified between freshly prepared and lyophilized samples.

Results

Reconstituted nanocomposites retained key structural features of freshly prepared assemblies, with minimal loss of material. Cytosolic delivery (> 80% efficiency of freshly prepared nanocomposites) of GFPE15 was validated in several cell lines, with intracellular access validated and quantified through diffusion into the nucleus. Delivery of GrAE10 elicited significant tumorigenic cell death. Intracellular access of cytotoxic protein was validated through cell viability.

Conclusion

Reconstituted nanocomposites achieved efficient cytosolic delivery of protein cargo and demonstrated therapeutic applicability with delivery of GrAE10. Overall, this strategy represents a versatile and highly translatable method for cytosolic delivery of proteins.

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Abbreviations

DLS:

Dynamic light scattering

DMEM:

Dulbecco’s Modified Eagle Medium

DPBS:

Dulbecco’s phosphate-buffered saline

EDTA:

Ethylenediaminetetraacetic acid

FBS:

Fetal bovine serum

GFP:

Green fluorescent protein

MFI:

Mean fluorescence intensity

MW:

Molecular weight

PONI:

Poly(oxanorbornene)imide

TCEP:

Tris(2-carboxyethyl)phosphine

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ACKNOWLEDGMENTS AND DISCLOSURE

The authors acknowledge Dr. James Chambers and the Light Microscopy Core Facility, and Dr. Amy Burnside and the Flow Cytometry Core Facility at UMass Amherst. The authors declare no competing financial interests.

Funding

This research was supported by the NIH (EB022641 and DK121351).

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Authors and Affiliations

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

    David C. Luther, Harini Nagaraj, Ritabrita Goswami, Yağız Anıl Çiçek, Taewon Jeon, Sanjana Gopalakrishnan & Vincent M. Rotello

  2. Molecular and Cellular Biology Graduate Program, University of Massachusetts, 710 North Pleasant St., Massachusetts, 01003, Amherst, USA

    Taewon Jeon

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  1. David C. Luther
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Correspondence to Vincent M. Rotello.

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Luther, D.C., Nagaraj, H., Goswami, R. et al. Direct Cytosolic Delivery of Proteins Using Lyophilized and Reconstituted Polymer-Protein Assemblies. Pharm Res 39, 1197–1204 (2022). https://doi.org/10.1007/s11095-022-03226-w

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