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Immunotheranostic Polymersomes Modularly Assembled from Tetrablock and Diblock Copolymers with Oxidation-Responsive Fluorescence

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Abstract

Introduction

Intracellular delivery is a key step for many applications in medicine and for investigations into cellular function. This is particularly true for immunotherapy, which often requires controlled delivery of antigen and adjuvants to the cytoplasm of immune cells. Due to the complex responses generated by the stimulation of diverse immune cell populations, it is critical to monitor which cells are targeted during treatment. To address this issue, we have engineered an immunotheranostic polymersome delivery system that fluorescently marks immune cells following intracellular delivery.

Methods

Amine functionalized poly(ethylene glycol)-bl-poly(propylene sulfide) (PEG-PPS-NH2) was synthesized by anionic ring opening polymerization and bridged via perylene bisimide (PBI) to form a tetrablock copolymer (PEG-PPS-PBI-PPS-PEG). Block copolymers were assembled into polymersomes by thin film hydration in phosphate buffered saline and characterized by dynamic light scattering, cryogenic electron microscopy and fluorescence spectroscopy. Polymersomes were injected subcutaneously into the backs of mice, and draining lymph nodes were extracted for flow cytometric analysis of cellular uptake and disassembly.

Results

Modular self-assembly of tetrablock/diblock copolymers in aqueous solutions induced π–π stacking of the PBI linker that both red-shifted and quenched the PBI fluorescence. Reactive oxygen species within the endosomes of phagocytic immune cell populations oxidized the PPS blocks, which disassembled the polymersomes for dequenching and shifting of the PBI fluorescence from 640 to 550 nm emission. Lymph node resident macrophages and dendritic cells were found to increase in 550 nm emission over the course of 3 days by flow cytometry.

Conclusions

Immunotheranostic polymersomes present a versatile platform to probe the contributions of specific cell populations during the elicitation of controlled immune responses. Flanking PBI with two oxidation-sensitive hydrophobic PPS blocks enhanced π stacking and introduced a mechanism for disrupting π–π interactions to shift PBI fluorescence in response to oxidative conditions. Shifts from red (640 nm) to green (550 nm) fluorescence occurred in the presence of physiologically relevant concentrations of reactive oxygen species and could be observed within phagocytic cells both in vitro and in vivo.

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Abbreviations

APCs:

Antigen presenting cells

CryoTEM:

Cryogenic transmission electron microscopy

DCs:

Dendritic cells

DLS:

Dynamic light scattering

FBS:

Fetal bovine serum

GPC:

Gel permeation chromatography

MHCI:

Major histocompatibility complex I

MW:

Molecular weight

NK:

Natural killer

PBI:

Perylene bisimides

PTCDA:

Perylene-3,4,9,10-tetracarboxylic dianhydride

PBS:

Phosphate-buffered saline

PDI:

Polydispersity index

PEG-bl-PPS:

Poly(ethylene glycol)-bl-poly(propylene sulfide)

PITC:

Polymer-bound isothiocyanate

ROS:

Reactive oxygen species

SC:

Subcutaneous

TLRs:

Toll like receptors

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Acknowledgments

We would like to thank J. Remis for CryoTEM assistance and the following facilities at Northwestern University: Robert H. Lurie Comprehensive Cancer Center Flow Cytometry Core; Center for Advanced Molecular Imaging; Biological imaging facility; Mouse Histology and Phenotyping Laboratory; and the Keck Interdisciplinary Surface Science Facility. This work was supported by the National Institutes of Health Director’s New Innovator Award (grant no. 1DP2HL132390-01), the Louis A. Simpson & Kimberly K. Querrey Center for Regenerative Nanomedicine Regenerative Nanomedicine Catalyst Award.

Conflict of Interest

Fanfan Du, Yu-Gang Liu, and Evan A. Scott declare that they have no conflicts of interest.

Ethical Standards

No human studies were carried out by the authors for this article. All institutional and national guidelines for the care and use of laboratory animals were followed and approved by the Northwestern University Institutional Animal Care and Use Committee.

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Authors and Affiliations

  1. Department of Biomedical Engineering, Northwestern University, Evanston, IL, USA

    Fanfan Du, Yu-Gang Liu & Evan Alexander Scott

  2. Chemistry of Life Processes Institute, Northwestern University, Evanston, IL, USA

    Evan Alexander Scott

  3. Interdisciplinary Biological Sciences Program, Northwestern University, Evanston, IL, USA

    Evan Alexander Scott

  4. Simpson Querrey Institute, Northwestern University Feinberg School of Medicine, Chicago, IL, USA

    Fanfan Du & Evan Alexander Scott

  5. Robert H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine, Chicago, IL, USA

    Evan Alexander Scott

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  1. Fanfan Du
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Correspondence to Evan Alexander Scott.

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Associate Editor Michael R. King oversaw the review of this article.

Evan Alexander Scott completed his undergraduate degree in Biomedical Engineering at Brown University in 2002. After working for a year as a chemical and biological defense engineer at the Battelle Memorial Institute in Aberdeen MD, he obtained a Ph.D. in Biomedical Engineering in 2009 from Washington University in St. Louis. His dissertation work was performed in the laboratory of Prof. Donald Elbert, where he developed methods based in proteomics and polymer chemistry to both analyze and control the interactions between blood and the material surfaces of cardiovascular devices. As a Whitaker International Scholar, he performed postdoctoral research in Switzerland in the laboratories of Prof. Jeffrey Hubbell and Prof. Melody Swartz at the École Polytechnique Fédérale de Lausanne (EPFL) from 2009 to 2013. There he focused on the development of nanomaterial-based formulations and strategies for both neonatal vaccination and cancer immunotherapy. Dr. Scott joined Northwestern University as a tenure-track Assistant Professor of Biomedical Engineering in the fall of 2013. His immunoengineering laboratory applies principles from biomaterials science, nanotechnology and tissue engineering towards the development of translational immunotherapies for heart disease and the rational design of vaccines for cancer and infectious diseases.

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Du, F., Liu, YG. & Scott, E.A. Immunotheranostic Polymersomes Modularly Assembled from Tetrablock and Diblock Copolymers with Oxidation-Responsive Fluorescence. Cel. Mol. Bioeng. 10, 357–370 (2017). https://doi.org/10.1007/s12195-017-0486-7

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