Actively-targeted polyion complex micelles stabilized by cholesterol and disulfide cross-linking for systemic delivery of siRNA to solid tumors

Abstract

For small interfering RNA (siRNA)-based cancer therapies, we report an actively-targeted and stabilized polyion complex micelle designed to improve tumor accumulation and cancer cell uptake of siRNA following systemic administration. Improvement in micelle stability was achieved using two stabilization mechanisms; covalent disulfide cross-linking and non-covalent hydrophobic interactions. The polymer component was designed to provide disulfide cross-linking and cancer cell-targeting cyclic RGD peptide ligands, while cholesterol-modified siRNA (Chol-siRNA) provided additional hydrophobic stabilization to the micelle structure. Dynamic light scattering confirmed formation of nano-sized disulfide cross-linked micelles (<50 nm in diameter) with a narrow size distribution. Improved stability of Chol-siRNA-loaded micelles (Chol-siRNA micelles) was demonstrated by resistance to both the dilution in serum-containing medium and counter polyion exchange with dextran sulfate, compared to control micelles prepared with Chol-free siRNA (Chol-free micelles). Improved stability resulted in prolonged blood circulation time of Chol-siRNA micelles compared to Chol-free micelles. Furthermore, introduction of cRGD ligands onto Chol-siRNA micelles significantly facilitated accumulation of siRNA in a subcutaneous cervical cancer model following systemic administration. Ultimately, systemically administered cRGD/Chol-siRNA micelles exhibited significant gene silencing activity in the tumor, presumably due to their active targeting ability combined with the enhanced stability through both hydrophobic interactions of cholesterol and disulfide cross-linking.

Introduction

Small interfering RNA (siRNA) inhibits expression of genes by a sequence-specific gene silencing effect, known as RNA interference (RNAi) [1], [2], [3]. This property has generated much interest for development of siRNA drugs that inhibit production of proteins associated with disease. However, low bioavailability of siRNA has hampered its translation into clinical use. Efforts to improve the efficacy of siRNA drugs have led to development of many types of siRNA-loaded nanoparticles to overcome biological hurdles associated with siRNA delivery, e.g., enzymatic degradation, accumulation in non-target organs/tissues and inefficient cellular uptake [4], [5], [6]. In particular, the ability to target specific cells has proven to be highly effective for enhanced accumulation of nanoparticles in solid tumors through systemic administration and has also been shown to facilitate cellular/subcellular delivery of siRNA [7], [8], [9], [10], [11], [12], [13], [14], [15], [16]. Thus, a variety of ligand molecules that bind to specific receptors on cancer cells have been installed on the surface of nanoparticles [7], [8], [9], [10], [11], [12], [13], [14], [15], [16]. In order to take full advantage of such targeting ligands, however, maintaining the nanoparticle structure in circulation is essential; targeting ligands can cooperatively function when distributed on the nanoparticle surface, allowing for avidity through multisite binding [13], [15], [17], [18]. Therefore, a highly effective siRNA delivery system should result from incorporating cellular surface-targeting ability to a nanoparticle platform resistant to destabilization (or dissociation), thus maximizing the ligand binding effect.

A promising platform for systemic siRNA delivery into solid tumors is the polyion complex (PIC) micelle, constructed with block copolymers of poly(ethylene glycol) (PEG) and a polycation as an siRNA binding segment [13], [16], [19], [20], [21], [22]. Charge neutralization between siRNA and the polycationic segment of the block copolymer in aqueous solution enables formation of PIC micelles, in which the siRNA-loaded PIC core is surrounded by a nonionic and hydrophilic PEG shell. This core–shell structure results in enhanced colloidal stability and reduced nonspecific interactions with charged biomacromolecules. To further increase micelle stability for in vivo delivery, several stabilizing approaches via hydrophobic interactions [16], [23] or disulfide cross-links [13], [24], [25], [26] have been investigated so far. Disulfide cross-links are noteworthy as they impart reversible stability to the micelle core upon cleavage (reduction) in the cell interior in response to the increased glutathione concentration, which is 100–1000 times higher than that in the cell exterior [27], [28]. Reversible micelle stability is an important feature for nucleic acid delivery vehicles, since siRNA release into the cytoplasm is required to access the RNAi pathway.

Meanwhile, our previous studies revealed that siRNA micelles could be disrupted even with disulfide cross-linking in the core, leading to undesirable release of siRNA payloads [26]. These results suggest that the cross-linking within the micelle core may be highly localized, incapable of stabilizing the whole core structure. Thus, an additional stabilizing mechanism may further reinforce the cross-linked siRNA micelle structure, leading to longer blood circulation and enhanced tumor accumulation. Herein, cholesterol-conjugated siRNA (Chol-siRNA) [29] was utilized to stabilize micelle core structures in addition to disulfide cross-linking. Hydrophobized siRNAs are expected to suppress micelle disruption and subsequent leakage of siRNA due to hydrophobic associations of cholesterol groups [16], [30]. Therefore, the combined use of a thiolated block copolymer and Chol-siRNA creates a stable, yet reversible, platform for improved systemic siRNA delivery.

In this work, we employed a functional block copolymer comprising PEG segment installed with cyclo-Arg-Gly-Asp (cRGD) peptide as the tumor-targeting hydrophilic block [13], [16], [31] and poly(l-lysine) (PLL) segment modified with dithiobispropionimidate (DTBP) as the cationic block [26]. DTBP modification was chosen for generating a single and stable side chain structure comprising an amidine and thiol functionality, making polyionic pairs/hydrogen bonds with siRNA phosphates in addition to disulfide cross-linking [26]. After examining the contribution of Chol-siRNA to micelle stability, the targeting ability of cRGD ligand was verified utilizing a luciferase-expressing cervical cancer (HeLa-Luc) cell line. Finally, the in vivo siRNA delivery efficacy of the actively-targeted/stabilized micelles was evaluated by luciferase gene silencing activity in the murine subcutaneous tumors after systemic administration, demonstrating strong potential for tumor-targeted systemic siRNA delivery.