Stealth monoolein-based nanocarriers for delivery of siRNA to cancer cells

Abstract

While the delivery of small interfering RNAs (siRNAs) is an attractive strategy to treat several clinical conditions, siRNA-nanocarriers’ stability after intravenous administration is still a major obstacle for the development of RNA-interference based therapies. But, although the need for stability is well recognized, the notion that strong stabilization can decrease nanocarriers’ efficiency is sometimes neglected. In this work we evaluated two stealth functionalization strategies to stabilize the previously validated dioctadecyldimethylammonium bromide (DODAB):monoolein (MO) siRNA-lipoplexes. The nanocarriers were pre- and post-pegylated, forming vectors with different stabilities in biological fluids. The stealth nanocarriers’ behavior was tested under biological mimetic conditions, as the production of stable siRNA-lipoplexes is determinant to achieve efficient intravenous siRNA delivery to cancer cells. Upon incubation in human serum for 2 h, by fluorescence Single Particle Tracking microscopy, PEG-coated lipoplexes were found to have better colloidal stability as they could maintain a relatively stable size. In addition, using fluorescence fluctuation spectroscopy, post-pegylation also proved to avoid siRNA dissociation from the nanocarriers in human serum. Concomitantly it was found that PEG-coated lipoplexes improved cellular uptake and transfection efficiency in H1299 cells, and had the ability to silence BCR-ABL, affecting the survival of K562 cells.

Based on an efficient cellular internalization, good silencing effect, good siRNA retention and good colloidal stability in human serum, DODAB:MO (2:1) siRNA-lipoplexes coated with PEG-Cer are considered promising nanocarriers for further in vivo validation.

Statement of Significance

This work describes two stealth functionalization strategies for the stabilization of the previously validated dioctadecyldimethylammonium bromide (DODAB):monoolein (MO) siRNA-lipoplexes. These nanocarriers are capable of efficiently incorporating and delivering siRNA molecules to cells in order to silence genes whose expression is implicated in a pathological condition. The main objective was to functionalize these nanocarriers with a coating conferring protection to siRNA in blood without compromising its efficient delivery to cancer cells, validating the potential of DODAB:MO (2:1) siRNA-lipoplexes as therapeutic vectors. We show that the stealth strategy is determinant to achieve a stable and efficient nanocarrier, and that DODAB:MO mixtures have a very promising potential for systemic siRNA delivery to leukemic cells.

Introduction

RNA interference (RNAi) is a natural mechanism by which short interfering RNAs (siRNAs) specifically inhibit protein synthesis [1]. The remarkable potential of this silencing mechanism has made siRNA therapy an important area in the biomedical field over the past years [2], [3], [4], [5], [6], [7]. The scientific focus has not only been devoted to designing efficient siRNA molecules [8] but also to develop methods to deliver siRNAs to their site of action [9]. The development of nanocarriers has been the strategy of choice to provide protection, improve internalization by target cells and to optimize the intracellular trafficking of therapeutic siRNA molecules [9], [10], [11], [12].

The design of nanocarriers for siRNA delivery must take into account properties such as small and uniform size distribution to allow internalization by endocytosis, and the ability to protect the encapsulated siRNA from enzymatic degradation. Biocompatibility and stability are additional requirements [2], [9], [13], [14]. Systemic administration poses additional challenges to the nanocarriers, as interaction with serum proteins might induce premature release of siRNA and prompt aggregation [15], [16], [17], [18], [19]. Extended circulation times of stabilized delivery systems in the bloodstream has been mainly achieved by coating their surface with poly(ethylene glycol) (PEG). PEG is a highly soluble neutral polymer that forms a protective hydrophilic layer at the nanocarriers surface, providing a “steric stabilization” effect that changes the surface properties, reduces opsonization by blood proteins, and decreases uptake by phagocytic cell populations [20], [21]. Steric stabilization can be achieved by other polymers besides PEG [22], but pegylation is still the most popular strategy to increase the circulation time of nanocarriers. Yet, steric stabilization provided by PEG was found to be undesirable during certain phases of the drug/nucleic acid delivery process. Pegylated nanocarriers might not satisfactorily protect nucleic acids against enzymatic degradation, and PEG can prevent nanocarriers cellular uptake or hinder siRNA endosomal escape [16], [23], [24], [25], [26]. To overcome these issues, exchangeable PEG-derivatives such as PEG-ceramides (PEG-Cer) have been proposed as a semi-stable coating, to provide good initial protection and stability in blood, while maintaining good cellular uptake and transfection [27]. Dissociation of the PEG-Cer chains from the siRNA nanocarrier will improve interaction with the cell membrane, cellular internalization and escape from endosomal compartments. However, it is not only the type of PEG that influences the transfection efficiency outcome, but also the pegylation method. Peeters et al. [23] prepared stealth nanocarriers by pre- and post-pegylation with PEG-Cer. The authors found that, even though the same type of PEG was used in both strategies, pre-pegylation of lipoplexes inhibited transfection efficiency due to slower internalization and entrapment inside endosomes, while post-pegylation improved transfection efficiency. Moreover, these and other authors have also shown that post-pegylation with DSPE–PEG leads to very low transfection efficiencies [23], [25].

Recent work from our group has shown that liposomes prepared with dioctadecyldimethylammonium bromide (DODAB) and monoolein (MO) (DODAB:MO) are promising carriers for pDNA delivery [28], [29], [30], [31]. The presence of MO on the formulation was found to be advantageous due to an increased fluidification of the nanocarrier membranes and stabilization of the liposomal structures [29], [32], [33]. DODAB:MO liposomes were also found to be suitable for siRNA delivery, as they efficiently encapsulated siRNA, were extensively taken up by the cells and were able to silence GFP expression without inducing cytotoxicity [34]. With this work we aimed to optimize the previously developed DODAB:MO (2:1) formulation for siRNA systemic delivery, by improving the nanocarriers’ performance in physiological conditions. Taking previous reports into account [23], [25], we have compared pre- and post-pegylation strategies in terms of stability in serum and capability to transfect cells. For pre-pegylation, DSPE–PEG was added to the liposomes before complexation with siRNA. For post-pegylation, PEG-Cer was inserted into the already formed siRNA lipoplexes.

Advanced fluorescence microscopy techniques such as fluorescence Single Particle Tracking (fSPT) and fluorescence fluctuation spectroscopy (FFS) were used to characterize the stability of the nanocarriers and protection of siRNA in human serum [15], [16], [18]. Fluorescence spectroscopy was additionally used to assess if the presence of PEG influences the binding of human serum proteins to the liposomes. Gene silencing efficiency and cellular internalization were also evaluated and correlated with the nanocarriers’ properties. The therapeutic potential of the siRNA-lipoplexes was further determined in a leukemia cell line model.