Vesicular nanocarrier formulations confer the ability to deliver hydrophobic and hydrophilic cargos simultaneously to cells of interest in vivo. While liposomal formulations reached the clinic long ago, younger technologies such as polymeric vesicles (polymersomes) have yet to make the transition to clinical approval and use, in part due to difficulties in ensuring their safe and scalable production. In this work, we demonstrate the scalable production of poly(ethylene glycol)-block-poly(propylene sulfide) (PEG-bl-PPS) polymersomes via flash nanoprecipitation, and further show the safe administration of these nanocarriers to mice and non-human primates. In mice, PEG-bl-PPS polymersomes were found to be well tolerated at up to 200 mg/(kg·week). Following the administration of a more relevant 20 mg/(kg·week) dosage in non-human primates, polymersomes were found to associate with numerous phagocytic immune cell populations, including a remarkable 68% of plasmacytoid dendritic cells and > 95% of macrophages in the spleen, while showing no toxicity or abnormalities in the liver, kidney, spleen, or blood. Despite the presence of a dense PEG corona, neither anti-PEG antibodies nor complement activation were detected. This work provides evidence of the translatability of PEG-bl-PPS polymersomes into the clinic for therapeutic applications in humans.
This is a preview of subscription content, access via your institution.
Buy single article
Instant access to the full article PDF.
Price excludes VAT (USA)
Tax calculation will be finalised during checkout.
Bobo, D.; Robinson, K. J.; Islam, J.; Thurecht, K. J.; Corrie, S. R. Nanoparticle-based medicines: A review of FDA-approved materials and clinical trials to date. Pharm. Res. 2016, 33, 2373–2387.
Torchilin, V. P. Multifunctional, stimuli-sensitive nanoparticulate systems for drug delivery. Nat. Rev. Drug. Discov. 2014, 13, 813–827.
Singh, R.; Lillard, J. W. Nanoparticle-based targeted drug delivery. Exp. Mol. Pathol. 2009, 86, 215–223.
Allen, S.; Osorio, O.; Liu, Y. G.; Scott, E. Facile assembly and loading of theranostic polymersomes via multi-impingement flash nanoprecipitation. J. Control. Release 2017, 262, 91–103.
Dowling, D. J.; Scott, E. A.; Scheid, A.; Bergelson, I.; Joshi, S.; Pietrasanta, C.; Brightman, S.; Sanchez-Schmitz, G.; Van Haren, S. D.; Ninkovic, J. et al. 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, 140, 1339–1350.
Discher, D. E.; Eisenberg, A. Polymer vesicles. Science 2002, 297, 967–973.
Cerritelli, S.; O'Neil, C. P.; Velluto, D.; Fontana, A.; Adrian, M.; Dubochet, J.; Hubbell, J. A. Aggregation behavior of poly(ethylene glycol-bl-propylene sulfide) Di-and triblock copolymers in aqueous solution. Langmuir 2009, 25, 11328–11335.
Yi, S. J.; Allen, S. D.; Liu, Y. G.; Ouyang, B. Z.; Li, X. M.; Augsornworawat, P.; Thorp, E. B.; Scott, E. A. Tailoring nanostructure morphology for enhanced targeting of dendritic cells in atherosclerosis. ACS Nano 2016, 10, 11290–11303.
Bobbala, S.; Allen, S. D.; Scott, E. A. Flash nanoprecipitation permits versatile assembly and loading of polymeric bicontinuous cubic nanospheres. Nanoscale 2018, 10, 5078–5088.
Karabin, N. B.; Allen, S.; Kwon, H. K.; Bobbala, S.; Firlar, E.; Shokuhfar, T.; Shull, K. R.; Scott, E. A. Sustained micellar delivery via inducible transitions in nanostructure morphology. Nat. Commun. 2018, 9, 624.
Napoli, A.; Valentini, M.; Tirelli, N.; Müller, M.; Hubbell, J. A. Oxidation-responsive polymeric vesicles. Nat. Mater. 2004, 3, 183–189.
Vasdekis, A. E.; Scott, E. A.; O'Neil, C. P.; Psaltis, D.; Hubbell, J. A. Precision intracellular delivery based on optofluidic polymersome rupture. ACS Nano 2012, 6, 7850–7857.
Scott, E. A.; Stano, A.; Gillard, M.; Maio-Liu, A. C.; Swartz, M. A.; Hubbell, J. A. Dendritic cell activation and T cell priming with adjuvant-and antigen-loaded oxidation-sensitive polymersomes. Biomaterials 2012, 33, 6211–6219.
Stano, A.; Scott, E. A.; Dane, K. Y.; Swartz, M. A.; Hubbell, J. A. Tunable T cell immunity towards a protein antigen using polymersomes vs. solid-core nanoparticles. Biomaterials 2013, 34, 4339–4346.
Eetezadi, S.; Ekdawi, S. N.; Allen, C. The challenges facing block copolymer micelles for cancer therapy: In vivo barriers and clinical translation. Adv. Drug. Deliver. Rev. 2015, 91, 7–22.
Anselmo, A. C.; Prabhakarpandian, B.; Pant, K.; Mitragotri, S. Clinical and commercial translation of advanced polymeric nanoparticle systems: Opportunities and material challenges. Transl. Mater. Res. 2017, 4, 014001.
O'Neil, C. P.; Suzuki, T.; Demurtas, D.; Finka, A.; Hubbell, J. A. A novel method for the encapsulation of biomolecules into polymersomes via direct hydration. Langmuir 2009, 25, 9025–9029.
Rameez, S.; Bamba, I.; Palmer, A. F. Large scale production of vesicles by hollow fiber extrusion: A novel method for generating polymersome encapsulated hemoglobin dispersions. Langmuir 2010, 26, 5279–5285.
Tang, C.; Amin, D.; Messersmith, P. B.; Anthony, J. E.; Prud'homme, R. K. Polymer directed self-assembly of pH-responsive antioxidant nanoparticles. Langmuir 2015, 31, 3612–3620.
Tang C.; Amin D.; Messersmith P.B.; Anthony J. E.; Prud'homme R. K. Polymer directed self-assembly of pH-responsive antioxidant nanoparticles. Langmuir 2015, 31, 3612–3620.
Johnson, B. K.; Prud'homme, R. K. Flash nano precipitation of organic actives and block copolymers using a confined impinging jets mixer. Aust. J. Chem. 2003, 56, 1021–1024.
Han, J.; Zhu, Z. X.; Qian, H. T.; Wohl, A. R.; Beaman, C. J.; Hoye, T. R.; Macosko, C. W. A simple confined impingement jets mixer for flash nanoprecipitation. J. Pharm. Sci. 2012, 101, 4018–4023.
Johnson, B. K.; Prud'homme, R. K. Chemical processing and micromixing in confined impinging jets. AIChE J 2003, 49, 2264–2282.
Saad, W. S.; Prud'homme, R. K. Principles of nanoparticle formation by flash nanoprecipitation. Nano Today 2016, 11, 212–227.
Liu, Y.; Cheng, C. Y.; Liu, Y.; Prud'homme, R. K.; Fox, R. O. Mixing in a multi-inlet vortex mixer (MIVM) for flash nano-precipitation. Chem. Eng. Sci. 2008, 63, 2829–2842.
Maecker, H. T.; McCoy, J. P.; Nussenblatt, R. Standardizing immunophenotyping for the Human Immunology Project. Nat. Rev. Immunol. 2012, 12, 471.
Guilliams, M.; Ginhoux, F.; Jakubzick, C.; Naik, S. H.; Onai, N.; Schraml, B. U.; Segura, E.; Tussiwand, R.; Yona, S. Dendritic cells, monocytes and macrophages: A unified nomenclature based on ontogeny. Nat. Rev. Immunol. 2014, 14, 571–578.
Malyala, P.; Singh, M. Endotoxin limits in formulations for preclinical research. J. Pharm. Sci. 2008, 97, 2041–2044.
Dane, K. Y.; Nembrini, C.; Tomei, A. A.; Eby, J. K.; O'Neil, C. P.; Velluto, D.; Swartz, M. A.; Inverardi, L.; Hubbell, J. A. Nano-sized drug-loaded micelles deliver payload to lymph node immune cells and prolong allograft survival. J. Control. Release 2011, 156, 154–160.
Hoffman, W. P.; Ness, D. K.; van Lier, R. B. Analysis of rodent growth data in toxicology studies. Toxicol. Sci. 2002, 66, 313–319.
Hall, A. P.; Elcombe, C. R.; Foster, J. R.; Harada, T.; Kaufmann, W.; Knippel, A.; Küttler, K.; Malarkey, D. E.; Maronpot, R. R.; Nishikawa, A. et al. Liver hypertrophy: A review of adaptive (adverse and non-adverse) changes—conclusions from the 3rd International ESTP Expert Workshop. Toxicol. Pathol. 2012, 40, 971–994.
Lake, B. G.; Evans, J. G.; Gray, T. J. B.; Körösi, S. A.; North, C. J. Comparative studies on nafenopin-induced hepatic peroxisome proliferation in the rat, Syrian hamster, guinea pig, and marmoset. Toxicol. Appl. Pharmacol. 1989, 99, 148–160.
Ray, K. Liver: Clearance of nanomaterials in the liver. Nat. Rev. Gastroenterol. Hepatol. 2016, 13, 560.
Armstrong, J. K.; Hempel, G.; Koling, S.; Chan, L. S.; Fisher, T.; Meiselman, H. J.; Garratty, G. Antibody against poly(ethylene glycol) adversely affects PEG-asparaginase therapy in acute lymphoblastic leukemia patients. Cancer 2007, 110, 103–111.
Richter, A. W.; Åkerblom, E. Polyethylene glycol reactive antibodies in man: Titer distribution in allergic patients treated with monomethoxy polyethylene glycol modified allergens or placebo, and in healthy blood donors. Int. Arch. Allergy Appl. Immunol. 1984, 74, 36–39.
Yang, Q.; Lai, S. K. Anti-PEG immunity: Emergence, characteristics, and unaddressed questions. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2015, 7, 655–677.
Judge, A.; McClintock, K.; Phelps, J. R.; Maclachlan, I. Hypersensitivity and loss of disease site targeting caused by antibody responses to PEGylated liposomes. Mol. Ther. 2006, 13, 328–337.
Wang, X. Y.; Ishida, T.; Kiwada, H. Anti-PEG IgM elicited by injection of liposomes is involved in the enhanced blood clearance of a subsequent dose of PEGylated liposomes. J. Control. Release 2007, 119, 236–244.
Yang, W.; Liu, S. J.; Bai, T.; Keefe, A. J.; Zhang, L.; Ella-Menye, J. R.; Li, Y. T.; Jiang, S. Y. Poly(carboxybetaine) nanomaterials enable long circulation and prevent polymerspecific antibody production. Nano Today 2014, 9, 10–16.
Szebeni, J.; Baranyi, L.; Savay, S.; Milosevits, J.; Bunger, R.; Laverman, P.; Metselaar, J. M.; Storm, G.; Chanan-Khan, A.; Liebes, L. et al. Role of complement activation in hypersensitivity reactions to doxil and hynic PEG liposomes: Experimental and clinical studies. J. Liposome Res. 2002, 12, 165–172.
Szebeni, J.; Baranyi, L.; Savay, S.; Bodo, M.; Morse, D. S.; Basta, M.; Stahl, G. L.; Bünger, R.; Alving, C. R. Liposome-induced pulmonary hypertension: Properties and mechanism of a complement-mediated pseudoallergic reaction. Am. J. Physiol. Heart Circ. Physiol. 2000, 279, H1319–H1328.
Park, J. K.; Utsumi, T.; Seo, Y. E.; Deng, Y.; Satoh, A.; Saltzman, W. M.; Iwakiri, Y. Cellular distribution of injected PLGA-nanoparticles in the liver. Nanomed.-Nanotechnol. Biol. Med. 2016, 12, 1365–1374.
Huh, Y.; Smith, D. E.; Feng, M. R. Interspecies scaling and prediction of human clearance: Comparison of small-and macro-molecule drugs. Xenobiotica 2011, 41, 972–987.
Vaure, C.; Liu, Y. Q. A comparative review of toll-like receptor 4 expression and functionality in different animal species. Front. Immunol. 2014, 5, 316.
Chiarelli, P. A.; Revia, R. A.; Stephen, Z. R.; Wang, K.; Jeon, M.; Nelson, V.; Kievit, F. M.; Sham, J.; Ellenbogen, R. G.; Kiem, H. P. et al. Nanoparticle biokinetics in mice and nonhuman primates. ACS Nano 2017, 11, 9514–9524.
Velásquez-Lopera, M. M.; Correa, L. A.; García, L. F. Human spleen contains different subsets of dendritic cells and regulatory T lymphocytes. Clin. Exp. Immunol. 2008, 154, 107–114.
Du, F. F.; Liu, Y. G.; Scott, E. A. Immunotheranostic polymersomes modularly assembled from tetrablock and diblock copolymers with oxidation-responsive fluorescence. Cell. Mol. Bioeng. 2017, 10, 357–370.
We acknowledge staff and instrumentation support from the Structural Biology Facility at Northwestern University, the Robert H Lurie Comprehensive Cancer Center of Northwestern University and NCI CCSG P30 CA060553. The Gatan K2 direct electron detector was purchased with funds provided by the Chicago Biomedical Consortium with support from the Searle Funds at The Chicago Community Trust. SAXS experiments were performed at the DuPont-Northwestern-Dow Collaborative Access Team (DND-CAT) located at Sector 5 of the Advanced Photon Source (APS). DND-CAT is supported by Northwestern University, E.I. DuPont de Nemours & Co., and The Dow Chemical Company. This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02- 06CH11357. This work made use of the EPIC facility of Northwestern University’s NUANCE Center, which has received support from the Soft and Hybrid Nanotechnology Experimental (SHyNE) Resource (NSF ECCS-1542205); the MRSEC program (NSF DMR-1121262) at the Materials Research Center; the International Institute for Nanotechnology (IIN); the Keck Foundation; and the State of Illinois, through the IIN. This work made use of the IMSERC at Northwestern University, which has received support from the NSF (CHE-1048773); Soft and Hybrid Nanotechnology Experimental (SHyNE) Resource (NSF NNCI-1542205); the State of Illinois and International Institute for Nanotechnology (IIN). This work was supported by the Northwestern University–Flow Cytometry Core Facility supported by Cancer Center Support Grant (NCI CA060553). Imaging work was performed at the Northwestern University Center for Advanced Molecular Imaging generously supported by NCI CCSG P30 CA060553 awarded to the Robert H Lurie Comprehensive Cancer Center. The authors acknowledge Jonathan Remis (Structural Biology Facility, NU) for his contribution to cryoTEM image acquisition. We acknowledge Sierra M. Paxton and Courtney R. Burkett for their excellent technical assistance with the NHP study.
About this article
Cite this article
Allen, S.D., Liu, YG., Bobbala, S. et al. Polymersomes scalably fabricated via flash nanoprecipitation are non-toxic in non-human primates and associate with leukocytes in the spleen and kidney following intravenous administration. Nano Res. 11, 5689–5703 (2018). https://doi.org/10.1007/s12274-018-2069-x