Original ArticleAnalysis of human innate immune responses to PRINT fabricated nanoparticles with cross validation using a humanized mouse model
Graphical Abstract
Nanoparticle formulations intended for human vaccine and therapeutics need to be tested for their capacity to induce cytotoxicity, inflammation and passive immune cell targeting in primary human immune cells. We employed Particle Replication in Non-wetting Templates (PRINT) technology - which allows for precise fabrication of nanoparticles with specified size, charge and composition - to test whether PRINT fabricated hydrogel 80 × 320 nm rods elicit cytotoxic or inflammatory immune responses from primary human immune cells, humanized mice and human serum. We failed to detect inflammation by primary human peripheral blood mononuclear cells (PBMC) ex vivo or using the translational humanized mouse model, while identifying CD14+ human immune cells as the target of these particles in both systems.
Section snippets
Background
Biocompatible particles at the nano/micron scale are emerging tools for biological programming with the capacity to induce specific cellular responses in a variety of disease contexts. This has been demonstrated using mouse models of anti-microbial vaccines, cancer immunotherapy, tolerance induction during autoimmunity and siRNA-mediated gene knockdown.1, 2, 3, 4, 5, 6, 7, 8, 9 Concomitant with advances in nanotechnology there is a widespread appreciation for ensuring nanoparticle safety and
Particle materials
Poly(ethylene glycol) diacrylate (Mn 700) (PEG700DA), 2-aminoetheyl methacrylate hydrochloride (AEM), and diphenyl (2,4,6-trimethylbenzoyl)-phosphine oxide (TPO) were from Sigma-Aldrich. Tetraethylene glycol monoacrylate (HP4A) was synthesized in-house as previously described.34 Thermo Scientific maleimide-terminated Dylight 650 and Dylight 488, PTFE syringe filters (13 mm membrane, 0.220 μm pore size), dimethylformamide (DMF), triethanolamine (TEA), pyridine, sterile water, borate buffer (pH
Characterization of monodisperse and homogenous PRINT nanoparticles
The PRINT fabrication process was used to generate rod shaped 80 × 320 nm hydrogel particles with a base component of HP4A. Aliquots of the 80 × 320 nm HP4A-NPs were modified by covalent attachment of short PEG5k chains to yield PEGylated HP4A-NPs (HP4A-PEG). Particle uptake was tracked using fluorescent dyes (Dylight 650 or 488) that were covalently incorporated during particle fabrication. NPs were characterized by DLS to measure size, surface charge (zeta potential) and particle homogeneity using
Discussion
The PRINT fabrication process yields NPs with defined shape and composition making it a leading candidate platform for diagnostic, therapeutic and preventative treatments in human disease. The hydrogel particles used in these studies were fabricated using PRINT and composed of HP4A based PEG. Select formulations of PEG are F.D.A approved for human use and we sought to test how these particles interact with the human immune system. A variety of particulate molecules (alum, asbestos, silica,
Acknowledgments
We thank Kristina Riebe of the Duke University Human Vaccine Institute/Regional Biocontainment Laboratory Host Response Monitoring facility for her assistance with multiplex cytokine assays.
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2015, BiomaterialsCitation Excerpt :We chose to fabricate 80 × 320 nm PLGA nanorods and 1 micron PLGA cylinders using the PRINT platform with and without encapsulated PS. We have previously published PRINT particles in this size range passively target both murine and human innate immune cells in a manner that does not significantly activate immune responses [40,41]. The PRINT process was used for fabrication of both 80 × 320 nm and 1 μm PS-loaded PLGA particles with a pre-particle solution composed of 10 wt% PS.
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Conflicts of interest: Joseph M. DeSimone is a founder and maintains a financial interest in Liquidia Technologies. PRINT and Fluorocur are registered trademarks of Liquidia Technologies, Inc.
Statements of funding:
Ting: NC TRACS 100K1202, UCRF UNC Internal Grants and NIH U19AI109784
DeSimone: NIH 8-VP1-CA174425-04 and NIH U19AI109784
Sempowski: NIH UC6-AI058607 and NIH U19AI109784
Su: NIH R01AI095097 and AI080432 and NIH U19AI109784
Robbins: 1F32A1108159-01 and T-32-CA009156-37
Roberts: T-32-GM008719 and T32-AI007273-25
Select studies were performed in the Regional Biocontainment Laboratory at Duke University, which received partial support for construction from the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH) (grant UC6-AI058607). This work was also supported by NC TRACS 100K1202 (Ting and DeSimone), UCRF UNC Internal Grant (Ting), NMSS Cooperative Center Grant (Ting), NIH 8-VP1-CA174425-04 (DeSimone), NIH UC6-AI058607 (Sempowski), NIH R01AI095097/AI080432 (Su) and NIH U19-AI109784 9 (Ting, DeSimone, Sempowski and Su).
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Authors contributed equally.