(9a) Engineering Biomaterial-Associated Complement Activation for Immune Modulation

Accumulating evidence suggests that complement, which is better known for its role in innate immunity, is also an important regulator of adaptive immunity. Our group has recently reported that lymph node-targeting, complement-activating nanoparticles may be used as an effective vaccine platform. Here we explore nanoparticle (NP) surface chemistry (e.g. nucleophiles and charge) as a means to control NP activation and regulation of C3 such that we might exploit its associated immunological functions for use in material-based vaccines. We investigated NPs composed of the polymer poly(propylene sulfide) (PPS) to form the hydrophobic core, surface stabilized with Pluronic-OH (a block copolymer of poly(ethylene glycol) and poly(propylene glycol) terminated by α and ω hydroxyl groups) alone or in combination with carboyxyl (COOH)-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. We found that NPs activate complement via the alternative pathway as measured by C3a release after NP exposure to human serum relative to untreated serum as this process was EDTA-, but not EGTA-, sensitive. NP core thiols also enhanced complement activation. 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 become opsonized with C3 fragments. Our hypothesis was confirmed by Western blot analysis of supernatants from washed iron oxide-containing NPs after human serum exposure indicating that OH-, COOHOHSH-, and COOHOH-NPs were decorated with a 115 kDa C3 alpha chain-reactive species corresponding to C3b. However, OH-SH-NPs were not opsonized with C3b as a result of its degredation by serine protease factor I, as indicated by enzyme inhibition experiments using serine protease inhibitor suramin. NP surface charge was found by surface plasmon resonance measurements to furthermore control the relative affinity of NPs for complement regulatory molecule factor H that serves as a cofactor for factor I, with COOH-NPs having higher factor H affinities relative to OH-NPs. However, factor H affinity paradoxically did not correlate with observed patterns of NP opsonization with C3b. Next, as C3b is a potent immune stimulator, we hypothesized that 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 enhanced anti-OVA antibody titers and inflammatory cytokine IFN-γ production with ex vivo OVA restimulation of splenocytes day 21 post-immunization relative to controls, suggesting that NP surface chemistry design directs the immunomodulatory properties of biomaterials-based immunotherapeutics.