Activation of innate immune responses in a pathogen-mimicking manner by amphiphilic polyanhydride nanoparticle adjuvants
Introduction
Successful vaccines induce protective immune responses that mimic those induced by a natural infection but do not elicit the negative effects associated with disease [1]. To do so, they must signal to the innate immune system using mechanisms similar to those employed by pathogens. Upon infection, the innate immune system is activated by pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS) found on the surface of gram negative bacteria, which interact with Toll-like receptors (TLRs) and other pattern recognition receptors (PRRs) on the surface of antigen presenting cells (APCs), including dendritic cells (DCs) [2], [3]. PAMPs contain repetitive molecular patterns that are recognized as “danger signals” by the host [3]. Moreover, PAMPs are often comprised of insoluble, hydrophobic moieties and are presumed to interact with PRRs, thereby providing signals that activate the innate immune system [4], [5]. In contrast, the soluble antigens and adjuvants found in current vaccine formulations cannot provide the same degree of continued stimulation. An opportunity exists to exploit the material properties of biodegradable polymers in order to rationally design vaccine adjuvants that mimic the behavior of pathogens, including prolonged in vivo residence times capable of providing extended immune activation and continued stimulation of APCs, without inducing the deleterious effects of disease.
Polyanhydrides are biodegradable materials that have been well documented to provide sustained delivery of proteins and stabilization of vaccine antigens [6], [7], [8], [9], [10], [11], [12]. Copolymers based upon sebacic acid (SA), 1,6-bis-(p-carboxyphenoxy)hexane (CPH), and 1,8-bis-(p-carboxyphenoxy)-3,6-dioxaocatane (CPTEG) have been studied as antigen carriers and adjuvants. These copolymers degrade into non-toxic, non-mutagenic degradation products and have demonstrated biocompatibility both in vivo and in vitro at concentrations expected for human use [13], [14], [15], [16]. Some amphiphilic polyanhydrides have also been reported to exhibit adjuvant characteristics capable of enhancing the adaptive immune response [13], [16].
When designing vaccine adjuvants, it is integral to understand the molecular properties of the adjuvant responsible for immune cell activation that would facilitate the induction of long-lived, protective immunity. In this work, we investigated the molecular properties of polyanhydride nanoparticles responsible for their robust stimulation of DCs. A simultaneous investigation of nanoparticle internalization and activation of DCs was complemented by informatics analysis to identify important polymer descriptors responsible for mimicking the adjuvant capabilities of the PAMP, LPS. While many previous studies have investigated the expression of cell surface markers and production of cytokines caused by stimulation with polymeric adjuvants [12], [17], [18], this study reports on the complex relationship between nanoparticle internalization, DC activation, and polymer chemistry (i.e., through polymer descriptors). This study also presents a direct comparison of cellular activation between DCs that have engulfed the nanoparticles and those that have not in the same culture.
Section snippets
Materials
The chemicals utilized in the monomer synthesis include: 1,6-dibromohexane, tri-ethylene glycol, 4-hydroxybenzoic acid, 1-methyl-2-pyrrolidinone, 4-p- and 1,6-dibromohexane; these were purchased from Sigma Aldrich (St. Louis, MO); 4-p-fluorobenzonitrile was purchased from Apollo Scientific (Cheshire, UK); and sulfuric acid, acetonitrile, dimethyl formamide (DMF), toluene, and potassium carbonate were obtained from Fisher Scientific (Fairlawn, NJ). Chemicals for the polymerization and
Importance of nanoparticle internalization for DC activation
Bacterial internalization by APCs is an important step in cellular activation and immune signaling [27], [28], [29]. The CPH:SA and CPTEG:CPH nanoparticles were, therefore, tested for their ability to be internalized by DCs. Incubation of CPTEG- and SA-rich chemistries with DCs resulted in an average of ∼6% and ∼30% nanoparticle-positive cells, respectively (Fig. 1). The observed positive correlation between nanoparticle internalization and decreasing hydrophobicity is consistent with that
Discussion
In this work, nanoparticle chemistry and hydrophobicity were found to play an integral role in particle internalization by DCs (Fig. 1). Once internalized, these properties continued to influence cell surface marker expression (Fig. 2) and cytokine production (Fig. 3). The least hydrophobic polymer chemistries, poly(SA) and 60:40 CPTEG:CPH, exhibited the greatest internalization by DCs (Fig. 1); however, it is unlikely that hydrophobicity alone dictated this cellular response. These results are
Conclusions
The combinatorial approach developed in this work enabled the rapid investigation of the effects of polymer chemistry on nanoparticle internalization and activation of DCs. Our studies identified amphiphilic polyanhydride nanoparticles that possess pathogen-mimicking properties with respect to their capacity to activate DCs. Chemistry-dependent trends were observed, with the least hydrophobic chemistries (SA- and CPTEG-rich) promoting the greatest internalization of nanoparticles and the
Acknowledgments
The authors would like to thank the ONR-MURI Award (NN00014-06-1-1176) and the U.S. Army Medical Research and Materiel Command (Grant No. W81XWH-10-1-0806) for financial support. This material is based upon the work supported by the National Science Foundation under Grant No. EEC 0851519. We would also like to thank Dr. Aaron Clapp of the Department of Chemical and Biological Engineering (CBE) at Iowa State University for providing cadmium selenide quantum dots and Ms. Ashley Yeager of the CBE
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