A phenylboronate-functionalized polyion complex micelle for ATP-triggered release of siRNA.

Therapeutics based on small interfering RNA (siRNA) offer an attractive clinical option because of its ability to silence genes in a highly sequence-specific manner. [1] A key challenge lies in developing a delivery system that helps protect the siRNAs from endogenous RNase degradation while allowing for controlled pharmacokinetics. One promising approach is a formulation of polyion complex (PIC) micelles that spontaneously form in an aqueous environment simply through electrostatic interactions between the anionic siRNA and cationic polymers. With versatile designs of the counterpart cationic polymers, representative poly(ethylene glycol) based block co-polymers, many creative PIC-based strategies have emerged, some of which have shown encouraging in vitro gene silencing abilities. However, in general, these PIC-based carriers suffer from instability under physiological conditions, primarily because of the relatively short chain length of the siRNA, that is 20–25 nucleotides, which results in poor thermodynamic stability. Therefore, stabilization of the PIC-based carriers so that programmed destabilization upon arrival at the site of intracellular targets (to release siRNA) has been of interest. Current efforts have focused on either one or combinations of the following three representative approaches: covalent conjugation of siRNAs to a homing polymer, introduction of hydrophobic moieties to reinforce the core-aggregation, and crosslinking the core aggregate by disulfide bridging. 12] As such, the combination of these approaches often results in a highly complex structure and method of preparation. Herein, we describe a sophisticated solution that can remarkably simplify the synthesis of PICs. It uses a phenylboronate functionality, which incorporates all of the aforementioned methods of stabilization (Scheme 1) while maintaining a wide window of control for environmental sensitivity. Phenylboronic acid (PBA) is a synthetic molecule capable of forming reversible covalent esters with 1,2or 1,3-cis-diols including on a ribose ring, a structure which is present at the 3’ end of RNAs and several kinds of ribonucleotides. Because of this property, PBA has historically been used as a ligand for RNA in affinity chromatography. Therefore, this binding property offers a facile route for chemical conjugation of siRNAs to the pendant PBA groups. Once electrostatically condensed into the PIC, the chances of equilibrium binding are increased, in which intermolecular cross-links could also form because of the bis-bidentate ribose arrangement at the 3’ end of the double-stranded siRNA, thereby further stabilizing the complex. Furthermore, PBA is unique in that it undergoes a dramatic inversion in its level of hydrophobicity depending on the degree of acid disassociation; it is strongly hydrophobic when uncharged but it becomes hydrophilic when negatively charged at pH values above its pKa. As shown in Figure 1, the binding between PBA and siRNAs is essentially a reversible equilibrium process dependent on the concentrations of each species. These features can be used to fine-tune or switch the stability of the complex, which is relevant to creating a system that is sensitive to the interand intracellular environments. Herein, we demonstrate that the PBA-assisted PIC micelles can be tailored to exhibit a dramatic disruption accompanied by the release of siRNAs in response to a change in the ribose concentration (which parallels events in the intracellular environment). A platform cationic polymer poly(ethylene glycol)-blockpoly(l-lysine) (PEG-b-PLys) was first prepared, the lysine residues of which were quantitatively modified with 3-fluoro4-carboxyphenylboronic acid (FPBA) to different extents. The weight-average molecular weight (Mw) of PEG and the mean degree of polymerization of PLys were determined to be 12000 Da and 42, respectively (Supporting Information, Table S1). The synthesized polymers are referred to as PEGb-P(Lys/FPBAX)42, where X denotes number of FPBA units introduced per polymer chain. According to the scattered light intensity of the polymer solutions, polymers were soluble (at 5 mgmL ) in HEPES buffered solution (HBS, pH 7.3) up to 55% FPBA modification, that is (PEG-b-P(Lys/FPBA23)42, however, those with 66 % (that is (PEG-b-P(Lys/FPBA28)42) or higher degrees of FPBA modification were partially insoluble because of the strong hydrophobicity of FPBA (data not shown). The HBS-soluble series of polymers, that is PEG-b-P(Lys/FPBA0,10,19,23)42, were allowed to complex with siRNA at various N/P ratios, which is defined as the molar [*] M. Naito, Dr. K. Kataoka Department of Materials Engineering, The University of Tokyo Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8656 (Japan) E-mail: kataoka@bmw.t.u-tokyo.ac.jp Homepage: http://www.bmw.t.u-tokyo.ac.jp/