A Phenylboronate‐Functionalized Polyion Complex Micelle for ATP‐Triggered Release of siRNA

Abstract

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.[2] 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. [3][4][5] 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, [3,[6][7][8] introduction of hydrophobic moieties to reinforce the core-aggregation, [9][10][11] and crosslinking the core aggregate by disulfide bridging. [5,12] 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 1,2-or 1,3-cis-diols including on a ribose ring, [13][14][15] 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.[16] 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; [17] it is strongly hydrophobic when uncharged but it becomes hydrophilic when negatively charged at pH values above its pK a . As shown in Figure 1, the binding between PBA and siRNAs is essentially a ...

Recent progress in development of siRNA delivery vehicles for cancer therapy

“…Similar to the aforementioned environment-responsive delivery vehicles, delivery vehicles can be designed to release payload siRNA into the ATP-abundant cytosol by utilizing phenylboronic acid (PBA) chemistry. PBA can form reversible covalent esters with 1,2-cis-diols on a ribose ring, which is present at the 3´end of siRNA and ATP [139]. As a representative, 3-fluoro-4-carboxyphenylboronic acid (FPBA) was conjugated into primary amino groups in the side chain in PEG-polycations and complexed with the ribose-terminated siRNA as an intermolecular crosslinker [ Fig.…”

Clustering Small Dendrimers into Nanoaggregates for Efficient DNA and siRNA Delivery with Minimal Toxicity

“…[32][33][34] In addition, PBA is capable of forming reversible covalent esters with the ribose ring of DNA or siRNA, thus cationic polymers modifi ed with PBA can bind with nucleic acid via the boronic ester linkage except ionic interactions. [ 35 ] PBA containing polymers are also used as gene vectors based on these promising properties. [36][37][38] Furthermore, PBA has a…”

Phenylboronic Acids-based Diagnostic and Therapeutic Applications

“…4). 56 PBA is able to reversibly bind with ribose, the binding constant of which is quite high relative to those for other common sugars. We found that the binding between the pendent PBA and 3′end ribose at both ends of double stranded siRNA along with hydrophobic interaction contribute to stability of the complex in quasi-extracellular conditions.…”