Cellular and Molecular Bioengineering

, Volume 10, Issue 5, pp 357–370 | Cite as

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

  • Fanfan Du
  • Yu-Gang Liu
  • Evan Alexander ScottEmail author



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.


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.


Modular self-assembly of tetrablock/diblock copolymers in aqueous solutions induced ππ 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.


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 ππ 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.


Polymersome Fluorescence Theranostics Perylene Macrophage Dendritic cell 



Antigen presenting cells


Cryogenic transmission electron microscopy


Dendritic cells


Dynamic light scattering


Fetal bovine serum


Gel permeation chromatography


Major histocompatibility complex I


Molecular weight


Natural killer


Perylene bisimides


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


Phosphate-buffered saline


Polydispersity index


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


Polymer-bound isothiocyanate


Reactive oxygen species




Toll like receptors



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.

Supplementary material

12195_2017_486_MOESM1_ESM.docx (473 kb)
Supplementary material 1 (DOCX 473 kb)


  1. 1.
    Belizaire, R., and E. R. Unanue. Targeting proteins to distinct subcellular compartments reveals unique requirements for MHC class I and II presentation. Proc Natl. Acad. Sci. U.S.A. 106(41):17463–17468, 2009. doi: 10.1073/pnas.0908583106.CrossRefGoogle Scholar
  2. 2.
    Berton, G., P. Bellavite, G. de Nicola, P. Dri, and F. Rossi. Plasma membrane and phagosome localisation of the activated NADPH oxidase in elicited peritoneal macrophages of the guinea-pig. J. Pathol. 136(3):241–252, 1982. doi: 10.1002/path.1711360307.CrossRefGoogle Scholar
  3. 3.
    Blander, J. M., and R. Medzhitov. On regulation of phagosome maturation and antigen presentation. Nat. Immunol. 7(10):1029–1035, 2006. doi: 10.1038/ni1006-1029.CrossRefGoogle Scholar
  4. 4.
    Cerritelli, S., C. P. O’Neil, D. Velluto, A. Fontana, M. Adrian, J. Dubochet, and J. A. Hubbell. Aggregation behavior of poly(ethylene glycol-bl-propylene sulfide) di- and triblock copolymers in aqueous solution. Langmuir 25(19):11328–11335, 2009. doi: 10.1021/la900649m.CrossRefGoogle Scholar
  5. 5.
    Chen, Z., V. Stepanenko, V. Dehm, P. Prins, L. D. Siebbeles, J. Seibt, P. Marquetand, V. Engel, and F. Wurthner. Photoluminescence and conductivity of self-assembled pi–pi stacks of perylene bisimide dyes. Chemistry 13(2):436–449, 2007. doi: 10.1002/chem.200600889.CrossRefGoogle Scholar
  6. 6.
    Cohn, L., and L. Delamarre. Dendritic cell-targeted vaccines. Front Immunol. 5:255, 2014. doi: 10.3389/fimmu.2014.00255.Google Scholar
  7. 7.
    Delamarre, L., M. Pack, H. Chang, I. Mellman, and E. S. Trombetta. Differential lysosomal proteolysis in antigen-presenting cells determines antigen fate. Science 307(5715):1630–1634, 2005. doi: 10.1126/science.1108003.CrossRefGoogle Scholar
  8. 8.
    Dexter, D. L., H. M. Kowalski, B. A. Blazar, Z. Fligiel, R. Vogel, and G. H. Heppner. Heterogeneity of tumor cells from a single mouse mammary tumor. Cancer Res. 38(10):3174–3181, 1978.Google Scholar
  9. 9.
    Dowling, D. J., E. A. Scott, A. Scheid, I. Bergelson, S. Joshi, C. Pietrasanta, S. Brightman, G. Sanchez-Schmitz, S. D. Van Haren, J. Ninkovic, D. Kats, C. Guiducci, A. de Titta, D. K. Bonner, S. Hirosue, M. A. Swartz, J. A. Hubbell, O. Levy. Toll-like receptor 8 agonist nanoparticles mimic immunomodulating effects of the live BCG vaccine and enhance neonatal innate and adaptive immune responses. J. Allergy Clin. Immunol. 2017. doi: 10.1016/j.jaci.2016.12.985.Google Scholar
  10. 10.
    Du, F. F., J. Tian, H. Wang, B. Liu, B. K. Jin, and R. K. Bai. Synthesis and luminescence of POSS-containing perylene bisimide-bridged amphiphilic polymers. Macromolecules 45(7):3086–3093, 2012. doi: 10.1021/ma300100s.CrossRefGoogle Scholar
  11. 11.
    Elliott, M. R., K. M. Koster, and P. S. Murphy. Efferocytosis signaling in the regulation of macrophage inflammatory responses. J. Immunol. 198(4):1387–1394, 2017. doi: 10.4049/jimmunol.1601520.CrossRefGoogle Scholar
  12. 12.
    Elliott, M. R., and K. S. Ravichandran. Clearance of apoptotic cells: implications in health and disease. J. Cell Biol. 189(7):1059–1070, 2010. doi: 10.1083/jcb.201004096.CrossRefGoogle Scholar
  13. 13.
    Gray, E. E., and J. G. Cyster. Lymph node macrophages. J Innate Immun. 4(5–6):424–436, 2012. doi: 10.1159/000337007.CrossRefGoogle Scholar
  14. 14.
    Gupta, M. K., T. A. Meyer, C. E. Nelson, and C. L. Duvall. Poly(PS-b-DMA) micelles for reactive oxygen species triggered drug release. J. Control. Release 162(3):591–598, 2012. doi: 10.1016/j.jconrel.2012.07.042.CrossRefGoogle Scholar
  15. 15.
    Gvishi, R., and R. Reisfeld. New stable tunable solid-state dye-laser in the red. Optoelectron. Appl. Ind. Med. 1972:390–399, 1993. doi: 10.1117/12.151121.Google Scholar
  16. 16.
    Gvishi, R., R. Reisfeld, and Z. Burshtein. Spectroscopy and laser action of the red perylimide dye in various solvents. Chem. Phys. Lett. 213(3–4):338–344, 1993. doi: 10.1016/0009-2614(93)85142-B.CrossRefGoogle Scholar
  17. 17.
    Joffre, O. P., E. Segura, A. Savina, and S. Amigorena. Cross-presentation by dendritic cells. Nat. Rev. Immunol. 12(8):557–569, 2012.CrossRefGoogle Scholar
  18. 18.
    Jouault, N., Y. J. Xiang, E. Moulin, G. Fuks, N. Giuseppone, and E. Buhler. Hierarchical supramolecular structuring and dynamical properties of water soluble polyethylene glycol-perylene self-assemblies. PCCP 14(16):5718–5728, 2012. doi: 10.1039/c2cp23786e.CrossRefGoogle Scholar
  19. 19.
    Kaiser, T. E., H. Wang, V. Stepanenko, and F. Wurthner. Supramolecular construction of fluorescent J-aggregates based on hydrogen-bonded perylene dyes. Angew. Chem. Int. Ed. Engl. 46(29):5541–5544, 2007. doi: 10.1002/anie.200701139.CrossRefGoogle Scholar
  20. 20.
    Kelkar, S. S., and T. M. Reineke. Theranostics: combining imaging and therapy. Bioconj Chem. 22(10):1879–1903, 2011. doi: 10.1021/bc200151q.CrossRefGoogle Scholar
  21. 21.
    Klebe, G., F. Graser, E. Hadicke, and J. Berndt. Crystallochromy as a solid-state effect—correlation of molecular-conformation, crystal packing and color in perylene-3,4-9,10-bis(dicarboximide) pigments. Acta Crystallogr. B 45:69–77, 1989. doi: 10.1107/S0108768188010407.CrossRefGoogle Scholar
  22. 22.
    Langhals, H., S. Demmig, and T. Potrawa. The relation between packing effects and solid-state fluorescence of dyes. J. Prakt. Chem. 333(5):733–748, 1991. doi: 10.1002/prac.19913330508.CrossRefGoogle Scholar
  23. 23.
    Langhals, H., W. Jona, F. Einsiedl, and S. Wohnlich. Self-dispersion: spontaneous formation of colloidal dyes in water. Adv. Mater. 10(13):1022–1024, 1998. doi: 10.1002/(Sici)1521-4095(199809)10:13<1022::Aid-Adma1022>3.3.Co;2-F.CrossRefGoogle Scholar
  24. 24.
    Lennon-Dumenil, A. M., A. H. Bakker, R. Maehr, E. Fiebiger, H. S. Overkleeft, M. Rosemblatt, H. L. Ploegh, and C. Lagaudriere-Gesbert. Analysis of protease activity in live antigen-presenting cells shows regulation of the phagosomal proteolytic contents during dendritic cell activation. J. Exp. Med. 196(4):529–540, 2002.CrossRefGoogle Scholar
  25. 25.
    Li, T. S., and E. Marban. Physiological levels of reactive oxygen species are required to maintain genomic stability in stem cells. Stem Cells 28(7):1178–1185, 2010. doi: 10.1002/stem.438.Google Scholar
  26. 26.
    Lim, E. K., T. Kim, S. Paik, S. Haam, Y. M. Huh, and K. Lee. Nanomaterials for theranostics: recent advances and future challenges. Chem. Rev. 115(1):327–394, 2015.CrossRefGoogle Scholar
  27. 27.
    Lukacs, G. L., O. D. Rotstein, and S. Grinstein. Phagosomal acidification is mediated by a vacuolar-type H(+)-ATPase in murine macrophages. J. Biol. Chem. 265(34):21099–21107, 1990.Google Scholar
  28. 28.
    Lukacs, G. L., O. D. Rotstein, and S. Grinstein. Determinants of the phagosomal pH in macrophages. In situ assessment of vacuolar H(+)-ATPase activity, counterion conductance, and H+ “leak”. J. Biol. Chem. 266(36):24540–24548, 1991.Google Scholar
  29. 29.
    Mais, S., J. Tittel, T. Basche, C. Brauchle, W. Gohde, H. Fuchs, G. Muller, and K. Mullen. Terrylenediimide: a novel fluorophore for single-molecule spectroscopy and microscopy from 1.4 K to room temperature. J. Phys. Chem. A 101(45):8435–8440, 1997. doi: 10.1021/jp9719063.CrossRefGoogle Scholar
  30. 30.
    Napoli, A., M. J. Boerakker, N. Tirelli, R. J. M. Nolte, N. A. J. M. Sommerdijk, and J. A. Hubbell. Glucose-oxidase based self-destructing polymeric vesicles. Langmuir 20(9):3487–3491, 2004. doi: 10.1021/La0357054.CrossRefGoogle Scholar
  31. 31.
    Napoli, A., M. Valentini, N. Tirelli, M. Muller, and J. A. Hubbell. Oxidation-responsive polymeric vesicles. Nat. Mater. 3(3):183–189, 2004. doi: 10.1038/nmat1081.CrossRefGoogle Scholar
  32. 32.
    Parker, J. S., and C. M. Perou. Tumor heterogeneity: focus on the leaves, the trees, or the forest? Cancer Cell 28(2):149–150, 2015. doi: 10.1016/j.ccell.2015.07.011.CrossRefGoogle Scholar
  33. 33.
    Postow, M. A., J. Chesney, A. C. Pavlick, C. Robert, K. Grossmann, D. McDermott, G. P. Linette, N. Meyer, J. K. Giguere, S. S. Agarwala, M. Shaheen, M. S. Ernstoff, D. Minor, A. K. Salama, M. Taylor, P. A. Ott, L. M. Rollin, C. Horak, P. Gagnier, J. D. Wolchok, and F. S. Hodi. Nivolumab and ipilimumab versus ipilimumab in untreated melanoma. N. Engl. J. Med. 372(21):2006–2017, 2015. doi: 10.1056/NEJMoa1414428.CrossRefGoogle Scholar
  34. 34.
    Savina, A., and S. Amigorena. Phagocytosis and antigen presentation in dendritic cells. Immunol. Rev. 219:143–156, 2007. doi: 10.1111/j.1600-065X.2007.00552.x.CrossRefGoogle Scholar
  35. 35.
    Savina, A., C. Jancic, S. Hugues, P. Guermonprez, P. Vargas, I. C. Moura, A. M. Lennon-Dumenil, M. C. Seabra, G. Raposo, and S. Amigorena. NOX2 controls phagosomal pH to regulate antigen processing during crosspresentation by dendritic cells. Cell 126(1):205–218, 2006. doi: 10.1016/j.cell.2006.05.035.CrossRefGoogle Scholar
  36. 36.
    Scott, E. A., A. Stano, M. Gillard, A. C. Maio-Liu, M. A. Swartz, and J. A. Hubbell. Dendritic cell activation and T cell priming with adjuvant- and antigen-loaded oxidation-sensitive polymersomes. Biomaterials 33(26):6211–6219, 2012. doi: 10.1016/j.biomaterials.2012.04.060.CrossRefGoogle Scholar
  37. 37.
    Stano, A., E. A. Scott, K. Y. Dane, M. A. Swartz, and J. A. Hubbell. Tunable T cell immunity towards a protein antigen using polymersomes vs. solid-core nanoparticles. Biomaterials 34(17):4339–4346, 2013. doi: 10.1016/j.biomaterials.2013.02.024.CrossRefGoogle Scholar
  38. 38.
    Vasdekis, A. E., E. A. Scott, C. P. O’Neil, D. Psaltis, and J. A. Hubbell. Precision intracellular delivery based on optofluidic polymersome rupture. ACS Nano 6(9):7850–7857, 2012. doi: 10.1021/nn302122h.CrossRefGoogle Scholar
  39. 39.
    Voigt, J., K. Hunniger, M. Bouzani, I. D. Jacobsen, D. Barz, B. Hube, J. Loffler, and O. Kurzai. Human natural killer cells acting as phagocytes against Candida albicans and mounting an inflammatory response that modulates neutrophil antifungal activity. J. Infect. Dis. 209(4):616–626, 2014. doi: 10.1093/infdis/jit574.CrossRefGoogle Scholar
  40. 40.
    Vulcano, M., S. Dusi, D. Lissandrini, R. Badolato, P. Mazzi, E. Riboldi, E. Borroni, A. Calleri, M. Donini, A. Plebani, L. Notarangelo, T. Musso, and S. Sozzani. Toll receptor-mediated regulation of NADPH oxidase in human dendritic cells. J. Immunol. 173(9):5749–5756, 2004.CrossRefGoogle Scholar
  41. 41.
    Wagner, C. S., J. Grotzke, and P. Cresswell. Intracellular regulation of cross-presentation during dendritic cell maturation. PLoS ONE 8(10):e76801, 2013. doi: 10.1371/journal.pone.0076801.CrossRefGoogle Scholar
  42. 42.
    Wurthner, F. Perylene bisimide dyes as versatile building blocks for functional supramolecular architectures. Chem. Commun. 14:1564–1579, 2004. doi: 10.1039/b401630k.CrossRefGoogle Scholar
  43. 43.
    Würthner, F., C. Bauer, V. Stepanenko, and S. Yagai. A black perylene bisimide super gelator with an unexpected J-type absorption band. Adv. Mater. 20(9):1695–1698, 2008. doi: 10.1002/adma.200702935.CrossRefGoogle Scholar
  44. 44.
    Yamaguchi, T., and M. Kaneda. Presence of cytochrome b-558 in guinea-pig alveolar macrophages-subcellular localization and relationship with NADPH oxidase. Biochim. Biophys. Acta 933(3):450–459, 1988.CrossRefGoogle Scholar
  45. 45.
    Yi, S., S. D. Allen, Y. G. Liu, B. Z. Ouyang, X. Li, P. Augsornworawat, E. B. Thorp, and E. A. Scott. Tailoring nanostructure morphology for enhanced targeting of dendritic cells in atherosclerosis. ACS Nano 10(12):11290–11303, 2016. doi: 10.1021/acsnano.6b06451.CrossRefGoogle Scholar
  46. 46.
    Zhang, X., Z. Chen, and F. Wurthner. Morphology control of fluorescent nanoaggregates by co-self-assembly of wedge- and dumbbell-shaped amphiphilic perylene bisimides. J. Am. Chem. Soc. 129(16):4886–4887, 2007. doi: 10.1021/ja070994u.CrossRefGoogle Scholar

Copyright information

© Biomedical Engineering Society 2017

Authors and Affiliations

  • Fanfan Du
    • 1
    • 4
  • Yu-Gang Liu
    • 1
  • Evan Alexander Scott
    • 1
    • 2
    • 3
    • 4
    • 5
    Email author
  1. 1.Department of Biomedical EngineeringNorthwestern UniversityEvanstonUSA
  2. 2.Chemistry of Life Processes InstituteNorthwestern UniversityEvanstonUSA
  3. 3.Interdisciplinary Biological Sciences ProgramNorthwestern UniversityEvanstonUSA
  4. 4.Simpson Querrey InstituteNorthwestern University Feinberg School of MedicineChicagoUSA
  5. 5.Robert H. Lurie Comprehensive Cancer CenterNorthwestern University Feinberg School of MedicineChicagoUSA

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