Original Article
Analysis of human innate immune responses to PRINT fabricated nanoparticles with cross validation using a humanized mouse model

https://doi.org/10.1016/j.nano.2014.11.010Get rights and content

Abstract

Ideal nanoparticle (NP)-based drug and vaccine delivery vectors should be free of inherent cytotoxic or immunostimulatory properties. Therefore, determining baseline immune responses to nanomaterials is of utmost importance when designing human therapeutics. We characterized the response of human immune cells to hydrogel NPs fabricated using Particle Replication in Non-wetting Templates (PRINT) technology. We found preferential NP uptake by primary CD14+ monocytes, which was significantly reduced upon PEGylation of the NP surface. Multiplex cytokine analysis of NP treated primary human peripheral blood mononuclear cells suggests that PRINT based hydrogel NPs do not evoke significant inflammatory responses nor induce cytotoxicity or complement activation. We furthered these studies using an in vivo humanized mouse model and similarly found preferential NP uptake by human CD14+ monocytes without systemic inflammatory cytokine responses. These studies suggest that PRINT hydrogel particles form a desirable platform for vaccine and drug delivery as they neither induce inflammation nor toxicity.

From the Clinical Editor

The authors here fabricated hydrogel nanorods using the PRINT (Particle Replication In Nonwetting Templates) fabrication process. They tested the interaction of human immune cells with these particles and found no immunoreactivity. This finding would suggest that monodisperse PRINT particles of identical shape and size could serve a variety of clinical applications.

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.

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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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    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.

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