PEGylation of lipoplexes: The right balance between cytotoxicity and siRNA effectiveness
Graphical Abstract
Introduction
During the last 30 years, RNA interference (RNAi) turned out to be one of the most important part of biotherapies, according to the Food and Drug Administration (FDA) (Foldvari et al., 2015, Naldini, 2015). Among RNAi molecules, small interfering RNA (siRNA) that target messenger RNA (mRNA) in a sequence-specific way, emerged as the best therapeutic tools. However, siRNA has sensitive physico-chemical properties such as negative charge, high molecular weight or sensitivity to nuclease (Wang et al., 2015). Therefore, an efficient delivery vehicle is required to protect siRNA, to overcome biological barriers and reach the target cells (Conde et al., 2015). Viral vectors, such as adenovirus or lentivirus, have been proposed but are associated to many drawbacks (immunogenic responses, limited gene packaging, cytotoxicity, hard production process …) which explain the difficulty of this approach to reach the market and the small number of drugs available after more than 20 years of research (Hill et al., 2015). Thereby, many non-viral vectors composed of polymers or lipids have been proposed to address these issues (Kesharwani et al., 2012, Zhou et al., 2014). Actually, 10 clinical trials are underway concerning the vectorisation of siRNA for cancer treatment. Among them, 3 studies are based on polymeric nanoparticles and 7 on lipidic nanoparticles. Currently, cationic liposomes are one the most widely studied vector for siRNA delivery (Hirko et al., 2003, Shim et al., 2013). Key advantages of liposomes are the biodegradability due to the use of natural lipids, the design flexibility of their formulations, the cost-effective manufacturing and the possibility of cells targeting (Rodriguez-Gascon et al., 2015, Rodriguez-Gascon et al., 2014).
Cationic liposomes, composed of a mixture of phospholipids, are used to facilitate the complexation with siRNA to form lipoplexes. When the vector has reached its target cells, different steps are crucial for the siRNA effectiveness. First, cationic lipoplexes have to cross the anionic cellular membrane via endocytosis pathways. Once into cells, lipoplexes have to escape from the endosome before lysosomal acidic degradation. Finally, siRNA must be released from its carrier in order to target its complementary mRNA (Resnier et al., 2013). Lipoplexes are largely studied both for systemic or topical/mucosal administrations (Belletti et al., 2015, Buyens et al., 2012, Qu et al., 2014, Wu et al., 2011).
In this project, we focused on mucosal drug delivery and more precisely on vaginal delivery which offers many benefits, such as the ability to target local diseases decreasing therefore systemic side effects. However, the two main constraints of mucosal surfaces are the presence of a mucus gel layer and the multilayer epithelium (Netsomboon and Bernkop-Schnurch, 2015). Currently, the best strategy to cross this mucus layer and to reach the underlying epithelium is to coat lipoplexes with polyethylene glycol (PEG), called PEGylation. The addition of this hydrophilic polymer around lipoplexes can stabilize the positive surface charge and thereby, prevent the aggregation with mucin fibres of the mucus. Nowadays, it is well described that engineering of mucus-penetrating particles (MPP) is possible by the coating of conventional nanoparticles with a high density of low molecular weight PEG (Das Neves et al., 2013, Das Neves et al., 2012, Ensign et al., 2012, Lai et al., 2009, Suk et al., 2015). Moreover, the decrease of the surface charge due to the PEGylation could reduce cytotoxicity of cationic nanovectors (Masotti et al., 2009). On the other hand, PEGylation may also affect siRNA-loaded efficacy; this phenomenon is known as the PEG dilemma (Hama et al., 2015, Wang et al., 2012). The PEG surface coverage is essential to control the transport through biological matrices whereas increasing the PEG coating also reduces cellular uptake and endosomal escape of siRNA and consequently, siRNA efficacy. The PEG dosage is a current challenge and has to be adapted according to the pharmaceutical application (Rabanel et al., 2014, Xia et al., 2015).
In this work, we have developed lipoplexes post-PEGylated with three different types of PEG (the DSPE-PEG2000, DSPE-PEG750 and C8-PEG2000-Ceramide (Ceramide-PEG2000)) in different densities. In vitro studies were performed on human papillomavirus 16 (HPV16) positive cells that induced several diseases into the cervico-vaginal tract, as cervical cancer, vaginal cancer or genital warts (Doorbar et al., 2012, Stanley, 2012). Therefore, a mix of two siRNA efficient against the oncoprotein E7 (siE7) (Jiang and Milner, 2002) and against the anti-apoptotic protein MCL-1 (siMCL1) (Rajalingam et al., 2008) was used. These two targets are known to be implicated in the cancerisation induced by HPV16 infection and their expression is closely linked (Cheng et al., 2008, Ghittoni et al., 2010, Zhang et al., 2012). An efficient knockdown of these two genes may induce the reduction of HPV16 cell viability and the increasing of apoptosis and thereby represent an appropriate model to study siRNA activities.
We have investigated the impact of PEG type and densities coated around lipoplexes on physico-chemical properties, cytotoxicity, mRNA knockdown, as well as biological activities of siRNA. This report described a new PEGylated lipoplexes usable on mucosal surfaces.
Section snippets
Materials
1,2-Dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-750] (DSPE-PEG750) and C8-PEG2000-Ceramide (Ceramide-PEG2000) were purchased from Avanti Polar Lipids, Inc. (Alabaster, Alabama, USA). Quant-iT™ RiboGreen® RNA assay was obtained from
Post PEGylation of Lipoplexes With Different Types of PEG Reduces Differently Their Surface Charges
Different types and different amounts of PEG were added around lipoplexes using a stirring process. This technique was performed at 37 °C in order to increase the fluidity of the lipidic bilayer and therefore facilitate the insertion of the lipidic part of DSPE-PEG or of Ceramide-PEG into the liposome membrane (Abe et al., 2015, Briuglia et al., 2015). The quantity of PEG was calculated compared to the mole of DOTAP (%mol). 0, 10, 20, 30 and 50% of DSPE-PEG2000, DSPE-PEG750 and Ceramide-PEG2000
Conclusion
In this study, lipoplexes coated with different type of PEG have been developed and tested in vitro on two HPV16 positive cancer cell lines. We have found that the surface charge is a crucial parameter to develop an efficient and non-toxic lipidic nanocarrier. First, a positive surface charge seems to be necessary for a highest mRNA knockdown by siRNA. Secondly, we have demonstrated that the cytotoxicity is closely linked to the surface charge of the nanocarriers.
Since the PEGylation decreases
Acknowledgements
The authors thank the Belgium National Fund for Scientific Research (FNRS, http://www.frs-fnrs.be) and the Fonds Leon Fredericq (FNRS: no. 7.4558.14) for financial support, Patrick Roncarati for technical support, Etienne Hanon for statistical help and the Giga Cell Imaging and Flow Cytometry Platform for their collaboration.
References (61)
- et al.
Effects of the PEG molecular weight of a PEG-lipid and cholesterol on PEG chain flexibility on liposome surfaces
Colloids Surf., A
(2015) - et al.
Liposomes and MTT cell viability assay: an incompatible affair
Toxicol. in Vitro
(2015) - et al.
Liposome based systems for systemic siRNA delivery: stability in blood sets the requirements for optimal carrier design
J. Control. Release
(2012) - et al.
Structure and kinetics of lipid-nucleic acid complexes
Adv. Colloid Interf. Sci.
(2014) - et al.
The biology and life-cycle of human papillomaviruses
Vaccine
(2012) - et al.
Cell line-dependent internalization pathways and intracellular trafficking determine transfection efficiency of nanoparticle vectors
Eur. J. Pharm. Biopharm.
(2008) - et al.
Overcoming the polyethylene glycol dilemma via pathological environment-sensitive change of the surface property of nanoparticles for cellular entry
J. Control. Release
(2015) - et al.
Investigating the uptake and intracellular fate of pH-sensitive liposomes by flow cytometry and spectral bio-imaging
J. Control. Release
(2006) - et al.
Physicochemical characterization techniques for lipid based delivery systems for siRNA
Int. J. Pharm.
(2012) - et al.
A review of nanocarriers for the delivery of small interfering RNA
Biomaterials
(2012)
Mucus-penetrating nanoparticles for drug and gene delivery to mucosal tissues
Adv. Drug Deliv. Rev.
Development of anti-E6 pegylated lipoplexes for mucosal application in the context of cervical preneoplastic lesions
Int. J. Pharm.
Effective siRNA delivery to inflamed primary vascular endothelial cells by anti-E-selectin and anti-VCAM-1 PEGylated SAINT-based lipoplexes
Int. J. Pharm.
Analysis of relative gene expression data using real-time quantitative PCR and the 2(− delta delta C(T)) method
Methods
Comparison of different commercially available cationic liposome–DNA lipoplexes: parameters influencing toxicity and transfection efficiency. Colloids and surfaces
B, Biointerfaces
Stealth monoolein-based nanocarriers for delivery of siRNA to cancer cells
Acta Biomater.
Post-insertion into lipid nanocapsules (LNCs): from experimental aspects to mechanisms
Int. J. Pharm.
Liposome-based co-delivery of siRNA and docetaxel for the synergistic treatment of lung cancer
Int. J. Pharm.
Assessment of PEG on polymeric particles surface, a key step in drug carrier translation
J. Control. Release
Pegylation of liposomes favours the endosomal degradation of the delivered phosphodiester oligonucleotides
J. Control. Release
A review of the current status of siRNA nanomedicines in the treatment of cancer
Biomaterials
Vaginal gene therapy
Adv. Drug Deliv. Rev.
Application of cationic liposomes for delivery of nucleic acids
Asian J. Pharm. Sci.
Molecular shape of the cationic lipid controls the structure of cationic lipid/dioleylphosphatidylethanolamine-DNA complexes and the efficiency of gene delivery
J. Biol. Chem.
Mechanism of the uptake of cationic and anionic calcium phosphate nanoparticles by cells
Acta Biomater.
Targeted delivery of small interfering RNA to angiogenic endothelial cells with liposome–polycation–DNA particles
J. Control. Release
Design of multifunctional non-viral gene vectors to overcome physiological barriers: dilemmas and strategies
Int. J. Pharm.
Delivery of oligonucleotides with lipid nanoparticles
Adv. Drug Deliv. Rev.
Comparison of different hydrophobic anchors conjugated to poly(ethylene glycol): effects on the pharmacokinetics of liposomal vincristine
Biochim. Biophys. Acta
Vaginal delivery of siRNA using a novel PEGylated lipoplex-entrapped alginate scaffold system
J. Control. Release
Cited by (0)
- 1
Equal contribution.