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)
Graphical abstract
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 SigmaAldrich (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 SigmaAldrich
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 Governments 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
References (44)
- et al.
Treatment failure in hepatitis C: mechanisms of non-response
J. Hepatol.
(2009) - et al.
Inhibition of hepatitis C virus replication by intracellular delivery of multiple siRNAs by nanosomes
Mol. Ther.
(2012) - et al.
Solid lipid nanoparticles as non-viral vector for the treatment of chronic hepatitis C by RNA interference
Int. J. Pharm.
(2015) - et al.
Solid lipid nanoparticles: formulation factors affecting cell transfection capacity
Int. J. Pharm.
(2007) - et al.
Solid lipid nanoparticles for retinal gene therapy: transfection and intracellular trafficking in RPE cells
Int. J. Pharm.
(2008) - et al.
A novel gene therapy vector based on hyaluronic acid and solid lipid nanoparticles for ocular diseases
Int. J. Pharm.
(2014) - et al.
Self-assembled nanoparticles based on amphiphilic chitosan derivative and hyaluronic acid for gene delivery
Carbohydr. Polym.
(2013) - et al.
Dextran-protamine-solid lipid nanoparticles as a non-viral vector for gene therapy: in vitro characterization and in vivo transfection after intravenous administration to mice
Int. J. Pharm.
(2012) - et al.
Understanding the mechanism of protamine in solid lipid nanoparticle-based lipofection: the importance of the entry pathway
Eur. J. Pharm. Biopharm.
(2011) - et al.
Inducible macropinocytosis of hyaluronan in B16-F10 melanoma cells
Matrix Biol.
(2010)
CD44-mediated uptake and degradation of hyaluronan
Matrix Biol.
Hyaluronic acid controls the uptake pathway and intracellular trafficking of an octaarginine-modified gene vector in CD44 positive- and CD44 negative-cells
Biomaterials
A proline-rich peptide improves cell transfection of solid lipid nanoparticle-based non-viral vectors
J. Control. Release
Solid lipid nanoparticles as potential tools for gene therapy: in vivo protein expression after intravenous administration
Int. J. Pharm.
New gene delivery system based on oligochitosan and solid lipid nanoparticles: in vitro' and in vivo' evaluation
Eur. J. Pharm. Sci.
Multicenter experience using simeprevir and sofosbuvir with or without ribavirin to treat hepatitis C genotype 1 after liver transplant
Hepatology
In vivo therapeutic potential of dicer-hunting siRNAs targeting infectious hepatitis C virus
Sci. Rep.
The molecular basis of drug resistance against hepatitis C virus NS3/4A protease inhibitors
PLoS Pathog.
Targets and tools: recent advances in the development of anti HCV nucleic acids
Infect. Disord. Drug Targets
Hepatitis C virus translation inhibitors targeting the internal ribosomal entry site
J. Med. Chem.
Therapeutic targeting of HCV internal ribosomal entry site RNA
Antivir. Chem. Chemother.
Adenovirus vectors lacking virus-associated RNA expression enhance shRNA activity to suppress hepatitis C virus replication
Sci. Rep.
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