Drug Delivery DOI: 10.1002/anie.201002221 Polyacrylate Dendrimer Nanoparticles: A Self-Adjuvanting Vaccine Delivery System** Mariusz Skwarczynski, Mehfuz Zaman, Carl N. Urbani, I-Chun Lin, Zhongfan Jia, Michael R. Batzloff, Michael F. Good, Michael J. Monteiro,* and Istvan Toth* Infection with group A streptococci (Streptococcus pyogenes , GAS), which is one of the most common and widespread human pathogens, can result in a broad range of diseases, including pharyngitis, with the potential of acute and post- infectious rheumatic fever (ARF) and rheumatic heart disease (RHD). [1] It is estimated that GAS infection is responsible for over half a million deaths per year world- wide. [2] The inability to effectively control GAS infections with antibiotics [3] has prompted extensive research into the development of a vaccine against this infection. However, to date, no prophylactic GAS vaccine is available on the market. Immunity to GAS relies on the production of opsonic antibodies specific to the hypervariable N-terminal and conserved C-terminal regions of the coiled-coil a-helical surface M protein, which is the major virulent factor in GAS. [1, 4] The development of an effective vaccine for GAS has been challenged by induced autoimmunity from epitopes derived from the C-terminal regions. [5] The minimal B-cell epitopes are believed to be safe but showed little or no immunogenicity. [6] It was recently hypothesized that self-assembled amphi- philic polymers can serve as nanoscale delivery systems for subunit vaccines, but no proof of concept has been demon- strated. [7] We report herein a polymer-based vaccine delivery system that offers several potential advantages over previ- ously reported strategies. It was expected that 1) an amphi- philic structure can self-assemble to produce particles with the desired size, 2) the hydrophobic polymer ensures pre- sentation of the peptide epitope on the surface of the nanoparticles, and 3) the dendritic structure and dense packing of epitopes on the surface of the nanoparticles induce native conformation of antigen which is essential for B-cell recognition. We have synthesized a dendritic structure consisting of a polyacrylate core and a peripheral generation of the minimal B-cell epitope (J14, KQAEDKVKASREAKKQVEKA- LEQLEDKVK; [8] Figure 1 a and Scheme S1 in the Support- ing Information). The dendrimer structure, which contains the antigen peptides, resulted in a self-assembled nanoparticle of 20 nm diameter in water. We chose the hydrophobic poly(tert-butyl acrylate) as the dendritic core as it had little or no toxicity, was shown to posses adjuvant properties when simply mixed with inactivated viral antigens, [9, 10] and has a high affinity for self-assembly into nanoparticles when the peripheral outer layer is hydrophilic. [11] The a-helical 20-mer epitope p145 (LRRDLDASREAKKQVEKALE), which is a peptide from the C-repeat region of the M protein, can elicit a protective antibody immune response when administered with an adjuvant or incorporated into the lipid core of a murine model. [8, 12] However, p145 was not a suitable vaccine candidate as T-cells specific to p145 were found to be cross- reactive with human heart tissue, [5] and may prompt the development of autoimmune disease (RHD). [8] Therefore, the J14 epitope used in this work was the minimal B-cell epitope (J14i, ASREAKKQVEKALE; derived from p145) incorpo- rated between two helix-promoting sequences from the yeast GCN4 protein to induce a native conformation. J14i alone had no native secondary helical conformation and produced little or no immunogenicity. [6] The J14 epitope was found to induce protective responses without stimulating cross-reac- tive antibodies but only when mixed with the toxic complete Freund’s adjuvant (CFA). [13] Previous work using J14 peptides covalently attached to linear polymers (with a broad molecular-weight distribution) generated an antibody response only when coadministered with CFA. [14] Such an imprecise and heterogeneous antigen display combined with CFA makes their mechanistic under- standing and regulatory approval challenging. The dendrimer structures described in this work are well-defined (Figure 1). The alkyne-functionalized four-arm star 1 was synthesized by successive atom-transfer radical polymerization (ATRP) [15] and copper-catalyzed alkyne–azide 1,3-dipolar cycloaddition (CuAAC) “click” reaction (Scheme S1 in the Supporting Information). [16] The “living” radical polymerization allowed us to obtain a core with a very narrow molecular-weight distribution (polydispersity index of less than 1.09). Unpro- tected J14 epitopes possessing an N-terminus azide moiety [*] Dr. M. Skwarczynski, M. Zaman, I-C. Lin, Prof. I. Toth School of Chemistry and Molecular Biosciences The University of Queensland, Brisbane QLD 4072 (Australia) Fax: (+ 61) 7-336-54273 E-mail: i.toth@uq.edu.au Dr. C. N. Urbani, Dr. Z. Jia, Prof. M. J. Monteiro Australian Institute for Bioengineering and Nanotechnology (AIBN) The University of Queensland, Brisbane QLD 4072 (Australia) Fax: (+ 61) 7-3346-3973 E-mail: m.monteiro@uq.edu.au Dr. M. R. Batzloff, Prof. M. F. Good The Queensland Institute of Medical Research, Brisbane (Australia) Prof. I. Toth School of Pharmacy The University of Queensland, Brisbane QLD 4072 (Australia) [**] This work was supported by the National Health and Medical Research Council. We thank George Blazak at the University of Queensland, Australia, for performing elemental analysis, and Dr. M. Georgousakis from QIMR for providing us with IgG J14 antibody. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.201002221. Communications 5742  2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2010, 49, 5742 –5745