(347a) A Materials-Based Approach for Immunotherapeutics Design: Tailoring Nanoparticle Surface Chemistry-Associated Complement Activation for Immune Modulation

Introduction: Accumulating evidence suggests that complement, which is better known for its role in innate immunity, is also an important regulator of adaptive immunity. In complement's alternative pathway, tickover of complement component C3 leads to C3a release and generation of C3b with an exposed thioester bond. This thioester can then react with nearby nucleophiles such as hydroxyls or thiols to form a covalent bond that may lead to generation of further downstream complement activation products. In this way, cells in the blood may become covered with C3b that can trigger a multitude of immune cell responses such as internalization of C3b-opsonized pathogens by phagocytic cells. Our group has recently reported that lymph node-targeting, complement-activating nanoparticles may be used as an effective vaccine platform via direct maturation and activation of dendritic cells [1]. Here we explore surface chemistry (e.g. nucleophiles and charge) as a means to control nanoparticle activation of C3 such that we might exploit its associated immunological functions for use in material-based vaccines.

Methods: We used nanoparticles (NPs) composed of the polymer polypropylene sulfide (PPS) to form the hydrophobic core, surface stabilized with Pluronic-OH (a block copolymer of polyethylene glycol and polypropylene glycol terminated by α and ω hydroxyl groups) [2] alone or in combination with carboyxyl-terminated Pluronic. The hydrophobic core is stabilized by disulfide crosslinking of the linear PPS chains. However, not all the PPS chains become crosslinked, leaving free sulfhydryl groups on the NP surface. These free surface thiols can be irreversibly blocked by covalent attachment of the alkylating sulfhydryl reagent, iodoacetamide. Thus, we assessed the role of NP surface chemistry in the initiation of the complement cascade. Furthermore, by rendering these NPs magnetic by the addition of oleic acid-coated iron oxide to the hydrophobic NP core, the relative purity of NP-deposited complement C3 fragments, namely C3b, was determined.

Results: All NP formulations induced complement activation as measured by C3a release after NP exposure to human serum relative to untreated serum. As C3a release is indicative of the generation of C3b that can readily form covalent thiol ester bonds with available nucleophiles, we hypothesized that these complement-activating NPs are in fact opsonized with C3 fragments. Our hypothesis was confirmed by tandem mass spectrometry with peptide fragmentation analysis of supernatants from washed iron oxide-containing NPs after human serum exposure and subsequent overnight trypsin digestion, revealing peptide sequences corresponding to those of human C3. Furthermore, by Western blot analysis, we demonstrated that samples from washed iron oxide-containing OHSH-, OH-, COOHOHSH-, and COOHOH-NPs after serum exposure resolved by SDS-PAGE in non-reducing conditions do, in fact, possess C3 immunoreactivity. Furthermore, these C3-immunoreactive species migrate slower relative to C3-immunoreactive species in serum alone or to purified C3b. As the NPs do not migrate into the gel in SDS-PAGE analysis, this suggests that C3-reactive species are covalently bound to the NPs. Next, using Western blot analysis, we found that OH-, COOHOHSH-, and COOHOH-NPs were decorated with a 115 kDa C3 alpha chain-reactive species corresponding to C3b while OH-SH-NPs did not. As C3b is a potent immune stimulator, we hypothesized that this C3b deposition onto the NPs may influence the immunomodulatory properties of our NP vaccine platform. Immunization of mice with OVA-conjugated NPs with surface chemistries that induce C3b opsonization demonstrated reduced production of anti-inflammatory cytokine IL-10 with ex vivo OVA restimulation of draining lymph nodes day 6 post-immunization relative to controls, suggesting that NP surface chemistry design directs the immunomodulatory properties of biomaterials-based immunotherapeutics.

Conclusions: While the presence of NP surface nucleophiles induces complement activation, the type and quantity of nucleophiles and/or changes in surface charge on the NP controls the deposition of C3b onto the NPs and may direct the immunomodulatory properties of the NP vaccine platform. These findings motivate the further investigation of complement-activating biomaterials-based schemes for immunotherapeutic design.

References:

1 Reddy ST et al. Nat Biotech. 2007;25:1159-64.

2 Rehor A et al. Langmuir 2005; 21, 411-417.