Silencing of hepatitis C virus replication by a non-viral vector based on solid lipid nanoparticles containing a shRNA targeted to the internal ribosome entry site (IRES)

https://doi.org/10.1016/j.colsurfb.2016.07.026Get rights and content

Highlights

  • HA-SLN induced inhibition of a subgenomic HCV replicon in a dose dependent manner.

  • The vector was quickly and efficiently internalized by Huh-7 cells.

  • Endocytosis was the most productive uptake mechanism for silencing.

  • The vector did not induce either hemolysis or agglutination of red cells.

Abstract

Gene silencing mediated by RNAi has gained increasing interest as an alternative for the treatment of infectious diseases such as refractory hepatitis C virus (HCV) infection. In this work we have designed and evaluated a non-viral vector based on solid lipid nanoparticles (SLN) bearing hyaluronic acid, protamine and a short hairpin RNA (shRNA74) targeted to the Internal Ribosome Entry Site (IRES) of the HCV. The vector was able to inhibit the expression of the HCV IRES in Huh-7 cells, with the inhibition level dependent on the shRNA74 to SLN ratio and on the shRNA74 dose added to the culture cells. The nanocarrier was also able to inhibit the replication in human hepatoma cells supporting a subgenomic HCV replicon (Huh-7 NS3-3⿲). The vector was quickly and efficiently internalized by the cells, and endocytosis was the most productive uptake mechanism for silencing. Clathrin-mediated endocytosis and to a lesser extent caveolae/lipid raft-mediated endocytosis were identified as endocytic mechanisms involved in the cell uptake. Internalization via the CD44 receptor was also involved, although this entry route seems to be less productive for silencing than endocytosis. The vector did not induce either hemolysis or agglutination of red cells in vitro, which was indicative of good biocompatibility. In summary, we have shown for the first time the ability of a non-viral SLN-based vector to silence a HCV replicon.

Introduction

Hepatitis C Virus (HCV) infection is a major health challenge worldwide because approximately 80% of infections persist to chronicity and can lead to hepatic cirrhosis and hepatocellular carcinoma and, ultimately, to liver failure and death [1]. The new protease inhibitors, such as simeprevir (Olysio®) or sofosvubir (Sovaldi®), have successfully improved sustained antiviral responses against HCV infection [2]. However, resistance to these inhibitors is expected to emerge in response to the pharmacological selection pressure [3], [4]. The use of interference RNA (RNAi) as an anti-HCV agent has been extensively reported as a promising strategy in therapeutics [5], [6]. HCV, which possesses a genome of 9.6 kb long single-stranded RNA molecule and has a replication cycle that occurs into the cytoplasm, is an ideal candidate for this therapeutic approach. Because of its important roles in translation and replication as well as its high sequence conservation across all HCV genotypes, HCV IRES (Internal Ribosome Entry Site) is considered as a promising target for RNAi-mediated antiviral therapy [7], [8]. Additionally, IRES is one of the most sequence-conserved region of the HCV genome, suggesting that clinical resistance against IRES inhibitors might be slow to develop [8]. Among the different types of commonly used RNAi molecules, short-hairpin RNA (shRNA) is considered to be an attractive strategy against HCV [9]. shRNA, also called expressed RNAi activators, is a plasmid-coded RNA that needs to be transcribed in the nucleus to down-regulate the expression of a desired gene. Since shRNA is constantly synthesized in host cells, more durable gene silencing is achieved in comparison to other forms of RNAi [10], [11]. shRNA requires entering the cell and must reach the nucleus to be effective, and a major challenge for its therapeutic use is the development of a suitable delivery system. In this sense, solid lipid nanoparticles (SLNs) have been increasingly recognized as one of the most promising non-viral vectors for gene therapy due to their biocompatibility and the ease of large-scale production [12], [13], [14]. In a previous study [15], we demonstrated the capacity of non-viral vectors composed by SLNs, protamine, and hyaluronic acid (HA) or dextran (DX) to silence HCV IRES in HepG2 cells. The efficacy of the nanocarriers prepared with HA resulted to be higher than those prepared with DX. In order to confirm the capacity of these HA vectors to inhibit HCV replication, the objective of the present work was to evaluate the silencing efficacy in the human hepatoma cell line Huh-7 NS3-3⿲ supporting a subgenomic HCV replicon [16]. Due to the importance of cell uptake and intracellular trafficking for transfection efficacy, we studied the mechanisms of cell internalization and the intracellular disposition of the vectors. Additionally, we also studied in vitro the hemagglutination capacity and the haemolytic activity of the vector, as indicative of biocompatibility.

Section snippets

Materials

Precirol® ATO 5 was generously provided by Gattefossé (Madrid, Spain). 1,2-Dioleoyl-3-trimethylammonium-propane chloride salt (DOTAP) was purchased from Avanti Polar Lipids (AL, USA). Tween 80, dichloromethane and paraformaldehyde (PFA) were obtained from Panreac (Madrid, Spain) and the fluorescent dye Nile Red from Sigma⿿Aldrich (Madrid, Spain). For the preparation of the vectors, protamine sulfate salt Grade X (P) and the Select-HA⿢ Hyaluronan 150 kDa (HA) were purchased from Sigma⿿Aldrich

Characterization of the nanocarriers

Fig. 1 shows a TEM photograph of the HA-SLN2 vector and the particle size (around 240 nm), zeta potential (about 30 mV), and polydispersity index of HA-SLN2 and HA-SLN5. The image shows the spherical shape of the nanocarrier, with an external layer or corona on the surface due to the HA. No significant difference in particle size and zeta potential was detected. In a previous study, we reported a more detailed characterization of the vectors, including the capacity to completely bind the shRNA74

Discussion

In recent years, SLN-based carriers have become one of the most interesting non-viral gene delivery vectors due to safe and cost-effective concerns [25], [28], [29]. In this study, SLNs have been combined with protamine (P), a cationic peptide which condenses DNA, presents nuclear localization signals and improves the transcription [30], and with hyaluronic acid (HA), a biocompatible, biodegradable and nontoxic polyanion used for pharmaceutical and biomedical applications [31]. The resulting

Conclusion

Our results indicate that the shRNA74 incorporated in a delivery system based on SLN, HA, and P is a promising therapeutic strategy for the treatment of chronic HCV infection. The silencing rate was related to the capacity of the vectors to enter the cells, with the endocytic mechanisms being the most productive. The vectors turned out to be biocompatible, a property that is a requisite to further address the ability of the system to clear HCV infection using a small animal model, and to assess

Acknowledgments

This work was supported by the Basque Government⿿s Department of Education, Universities and Investigation (IT-341-10), by the Department of Industry (Saiotek) S-PE11UN035 to A. Rodríguez-Gascón and by the Spanish Ministerio de Economía y Competitividad (BFU2012-31213) to A. Berzal-Herranz. The work is partially supported by FEDER funds from the EU. Technical and human support for TEM, flow cytometry and CLSM provided by SGIker (UPV/EHU, MINECO, GV/EJ, ERDF and ESF) is gratefully acknowledged

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