Tailored Design of Au Nanoparticle-siRNA Carriers Utilizing Reversible Addition-Fragmentation Chain Transfer Polymers | Stacey Kirkland-York, † Yilin Zhang, ‡ Adam E. Smith, † Adam W. York, † Faqing Huang, ‡ and Charles L. McCormick* ,†,‡ Departments of Polymer Science and Chemistry and Biochemistry, The University of Southern Mississippi, Hattiesburg, Mississippi 39406 Received January 7, 2010; Revised Manuscript Received March 1, 2010 The facile synthesis of polymer-stabilized Au nanoparticles (AuNPs) capable of forming neutral, sterically stable complexes with small interfering RNA (siRNA) is reported. The amine-containing cationic block of poly(N-2- hydroxypropyl methacrylamide 70 -block-N-[3-(dimethylamino)propyl] methacrylamide 24 ) [P(HPMA 70 -b-DMAPMA 24 )] was utilized to promote the in situ reduction of Au 3+ to AuNPs and subsequently bind small interfering RNA, while the nonimmunogenic, hydrophilic block provided steric stabilization. The ratio of [DMAPMA] 0 /[Au 3+ ] 0 utilized in the reduction reaction was found to be critical to the production of polymer-stabilized AuNPs capable of complexing siRNA. Significant protection (∼100 times) against nucleases was demonstrated by enzymatic tests, while gene down- regulation experiments indicated successful delivery of siRNA to cancerous cells. Introduction The manner in which small interfering RNA (siRNA) operates, down-regulation of a homologous target mRNA via the RNA interference pathway, provides a promising route for treatment of numerous diseases. 1,2 However, several issues must be addressed to make siRNA a viable therapeutic option, including in vivo stability and cell transfection. 3 Interpolyelec- trolyte complexes (IPECs), electrostatic complexes formed between polycations (typically amine containing) and oligo- nucleotides (anionic), are often used to enhance serum stability. 4 Based on the ratio of cationic (amine) and anionic (phosphate) groups (N/P ratio), the IPEC can be neutral, cationic, or anionic. In general, neutral complexes have limited aqueous solubility and thus charged IPECs are employed. 4 However, anionic complexes (N/P < 1) often exhibit poor transfection due to electrostatic repulsion from the negatively charged cell mem- brane, while cationic complexes (N/P > 1) interact strongly with cell membranes resulting in nonspecific transfection; charged complexes may also adsorb serum proteins resulting in clearance by phagocytic cells. 5–13 Recent advances in controlled radical polymerization (CRP) techniques 14–20 and other methods have allowed the synthesis of well-defined polymers capable of addressing solubility issues associated with neutral complexes. Specifically, hydrophilic-b- cationic copolymers (HbC) can form neutral (N/P ) 1), water- soluble complexes with oligonucleotides known as block ionomer complexes (BICs). 6,7,16,21–24 In contrast to IPECs, which lose solubility upon charge neutralization, BICs retain solubility due to a hydrophilic block. In 2006, Scales et al. in our laboratories demonstrated the controlled synthesis of a series of poly(N-2-hydroxypropyl methacrylamide-block-N-[3-(di- methylamino)propyl] methacrylamide) P(HPMA-b-DMAPMA) copolymers for complexation with siRNA. 21 The DMAPMA block provided cationic functionality necessary for binding siRNA, while the HPMA block served as a nonimmunogenic, hydrophilic, sterically stabilizing shell. Because a CRP technique (reversible addition-fragmentation chain transfer, RAFT) was utilized, both the HPMA and the DMAPMA block lengths could be controlled allowing the tailored design of the siRNA carrier. In subsequent work conducted by York et al., an HbC carrier containing multiple pendant folic acid groups along the hydro- philic block was synthesized. 23 Not only did this allow the formation of neutral, stable complexes as in earlier work, but it also allowed cell-specific delivery to cancerous cells overex- pressing folate receptors. 23 While synthetic and biological polymers have been widely explored as carriers, a number of research groups 25–31 have focused on preformed Au nanoparticles (AuNPs) as gene carriers. AuNPs have features that forecast utility in nanomedi- cine including availability in a wide variety of sizes, facile modification by a ligand-exchange process, 32–36 and potential as X-ray contrast agents. 37 Promising work has been reported in which preformed AuNPs are modified with thiolated oligo- nucleotides (SH-ONs) via a ligand exchange process. 28–31,38 However, the researchers point out that this method requires the use of modified oligonucleotides capable of chelating to the Au surface (i.e., thiol) and conditions that limit the number of unexchanged ligands (excess SH-ONs). Additionally, this method results in a highly anionic surface charge resulting in the nonspecific adsorption of serum proteins (opsonization) and, hence, clearance by the mononuclear phagocytic system (MPS). 5–13 In principle, these drawbacks can be circumvented by forming polymer-stabilized AuNPs via the in situ reduction of Au 3+ to Au 0 in the presence of a suitable block copolymer capable of complexing RNA. Several groups, 39–42 including ours, 43,44 have formed stabilized AuNPs by utilizing an amine- containing segment, which can serve as both reducing and stabilizing agent at appropriate pH. With recent advances in aqueous RAFT polymerization, 14–18 an opportunity exists to synthesize narrowly dispersed block copolymers with the requisite properties for AuNP stabilization and siRNA complexation. Herein we report the facile synthesis of a HbC-stabilized AuNP (HbC-AuNP) and subsequent deliv- * To whom correspondence should be addressed. E-mail: charles. mccormick@usm.edu; faqing.huang@usm.edu. | Paper number 145 in a series entitled “Water-Soluble Polymers”. † Department of Polymer Science. ‡ Department of Chemistry and Biochemistry. Biomacromolecules 2010, 11, 1052–1059 1052 10.1021/bm100020x  2010 American Chemical Society Published on Web 03/25/2010