Hyperbranched–hyperbranched polymeric nanoassembly to mediate controllable co-delivery of siRNA and drug for synergistic tumor therapy

Abstract

This study reported a flexible nanoplatform constructed on the pH-dependent self-assembly of two kinds of hyperbranched polymers, and then validated its potency as the controllable siRNA/drug co-delivery vehicle for the combination of chemotherapy with RNA interfering (RNAi) therapy. By virtue of pH-reversible phenylboronate linking, phenylboronic acid-tethered hyperbranched oligoethylenimine (OEI600-PBA) and 1,3-diol-rich hyperbranched polyglycerol (HBPO) can be spontaneously interlinked together into a core–corona nanoconstruction. The special buildup of compactly clustering OEI600-PBA units around hydrophobic HBPO aggregate offered significant advantages over parent OEI600-PBA, including strengthened affinity to siRNA, ability of further loading anticancer drug, easier cellular transport, and acidity-responsive release of payloads. To evaluate the co-delivery capability, Beclin1 siRNA and antitumor DOX were used as the therapeutic models in order to suppress the post-chemotherapy survival of tumor cells caused by drug-induced autophagy. The nanoassembly-mediated single delivery of DOX displayed even better anticancer effects than free DOX, demonstrating the superiority of our pH-responsive nano-design. The nanoassembly-mediated co-delivery of siRNA together with DOX can effectively silence Beclin1 gene, suppress DOX-induced autophagy, and consequently provide strong synergism with a significant enhancement of cell-killing effects in cultured cancerous cells. The in vivo combinational treatment was shown to make the tumor more sensitive to DOX chemotherapy while displaying substantially improved safety as compared with the monochemotherapy.

Introduction

The performance of tumor chemotherapy is yet far from satisfactory since its single antitumor mechanism fails to combat the heterogeneity of tumor environment and the complexity of signaling pathways that regulate cancer progression and metastasis [1], [2]. With increasing understanding of tumor development and progression, the combination of chemotherapy with gene therapy (using nucleic acid therapeutics, e.g. DNA, siRNA, shRNA) represents a promising strategy to achieve synergistic therapies for tumor diseases [1], [2], [3]. When threatened by antitumor agents, tumor cells always generate an autophagy response to counteract metabolic stress through cellular self-digestion that produces new building blocks and energies for cellular renovation and homeostasis [4], [5]. The autophagy process can support cell survival in the stressed, nutrient-limited environments experienced by many cancer cells. This evolutionarily conserved progress is believed to be one of the main resistance mechanisms closely related with the unsuccessful chemotherapy [6]. Genetic studies have identified that Beclin1 protein plays a crucial role in the autophagy activation by regulating the nucleation of autophagic vesicles [7]. When antitumor agents take action, therefore, concomitantly using siRNA therapeutics to block the Beclin1 protein-relevant signaling pathway offers an effective solution to overcome the antiapoptotic defense of cancer cells caused by drug-induced autophagy.

Combinational siRNA/drug tumor treatments need specific vehicles capable of simultaneously co-loading both the therapeutics into a delivery nanosystem with high efficiency. The nanosystem can enable the preferential accumulation of therapeutic payloads at tumor tissues via enhanced permeability and retention effect, which is often referred to as passive targeting [8]. In addition, an ideal co-delivery nanosystem should be able to readily enter into tumor cells, preferably offer a controlled release of therapeutics in a tumor-specific manner, and bear acceptable safety profiles [9], [10], [11], [12]. Polymeric vehicles have been extensively explored for the single delivery of either siRNA or drug therapeutics [13], [14]. However, the intrinsically different natures of macromolecular siRNA with small molecular drug impose rigorous challenges in the development of suitable co-delivery vehicles. Nowadays, polymeric micellar nanovehicles (NVs) have been intensively investigated to deal with the co-delivery of drugs and siRNA. The micellar NVs are generally nanoscopic core/shell structures formed by amphiphilic copolymers typically composed of two chemically linked functional moieties, of which one is cationic and hydrophilic for siRNA condensation while the other is a hydrophobic segment for physical encapsulation of drugs [15], [16]. Nevertheless, the screening and optimization of these NVs suffer tremendously from the failure to rapidly and finely regulate the NV's composition and function, due to the complicated chemistry involved in the preparation. Furthermore, it is difficult to endow the micellar nanovehicles (NVs) with the ability to release therapeutic payloads in a controlled manner. Flexible and adjustable nanoplatforms for the siRNA/drug co-delivery, particularly for the controlled co-delivery, are therefore urgently needed to meet the frequently varied demands in applications. To address the issues, dynamic assembly approaches may be a suitable option owing to the facile and efficient control over NP construction without parallel in chemical synthesis [17]. In our opinion, the integrative assembly of different functional building blocks derived from hyperbranched polymers are especially appealing when considering the unique hyperbranched architecture as well as the functional versatility arising from the numerous functional groups in the polymer surface. To our knowledge, the attempts in this field have been rarely reported.

Taking all those into account, the present study prepared a flexible nanoplatform based on the pH-dependent assembly of two hyperbranched polymers with predicted functions, and then validated its potency as the controlled co-delivery NV for the combinational siRNA/drug therapies. As illustrated in Fig. 1, driven by spontaneously phenylboronate linking, hydrophobic hyperbranched polyglycerol (HBPO) with a cis-diol-rich surface and hyperbranched oligoethylenimine (OEI600, Mw = 600 Da) tethered with phenylboronic acids (PBA) can be readily interlinked together at neutral conditions. By adjusting feeding ratios, OEI600-PBA units are arranged to adhere around HBPO aggregates, resulting in the core–corona nanoconstruction. Therein, the inner core of HBPO aggregates was responsible for the accommodation of antitumor drugs through hydrophobic interaction while the surface clustering of cationic OEI600-PBA units allows much enhanced affinity to siRNA over parent OEI600-PBA. Of special note, owing to the acid-labile feature of phenylboronate linkage [18], [19], [20], HBPO/OEI600-PBA nanoassembly is expected to undergo reversible disintegration upon the cellular endocytosis into the lysosome compartment featured with considerably low pH 4.0 – 5.5. This transition would facilitate the liberation inside tumor cells of the extracellularly protected siRNA/drug payloads, favoring the synchronization of two therapeutic mechanisms. Meanwhile, the decomposition into individual OEI600-PBA units would reduce the systematic toxicity of the nanoassembly. One predictable advantage of this nanoplatform lies in the expandability of function diversity by accommodating other functional components such as targeting groups via the same assembly pathway. Since non-therapeutic Beclin1 siRNA can impair the cell's ability of autophagy activation by down-regulating the expression of Beclin1 gene [21], [22], Beclin1 siRNA and antitumor doxorubicin (DOX) were chosen as the therapeutic models for the in vitro and in vivo evaluation of HBPO/OEI600-PBA nanoassembly for the combinational tumor treatments.