PEGylation of lipoplexes: The right balance between cytotoxicity and siRNA effectiveness

https://doi.org/10.1016/j.ejps.2016.08.058Get rights and content

Abstract

The delivery of small interfering RNA (siRNA) is an attractive therapeutic approach to treat several pathologies, such as viral infections or cancers. However, the stability and the efficacy of these biotherapies are still a major obstacle to their use. Cationic liposomes (DOTAP/Chol/DOPE 1/0.75/0.5 M ratio) have been complexed to siRNA (lipoplexes) in order to be administrated by the vaginal route, in the context of HPV16 induced cervical preneoplastic lesions. To overcome the constraint of the cervico-vaginal mucus, PEGylation is required to allow the diffusion of lipoplexes through it. Thereby, PEGylated lipoplexes coated with three types of polyethylene glycol (PEG) as DSPE-PEG2000, DSPE-PEG750 or C8-PEG2000-Ceramide (Ceramide-PEG2000) at different densities have been developed and characterized. PEGylated lipoplexes were successfully prepared and showed a hydrodynamic diameter around 200 nm, appropriate for vaginal application. In vitro assays on HPV16 positive cell lines revealed that a positive charge of PEGylated lipoplexes allows a higher mRNA knockdown by siRNA. However, the cationic property is also associated to cytotoxicity. The addition of a high percentage of PEG prevented this toxicity but seemed also to reduce siRNA endosomal escape, probably by steric hindrance. The decreasing of PEG density of Ceramide-PEG2000 to 20% allows the release of siRNA and in consequence, biological activities, contrarily to DSPE-PEG. These results suggest that Ceramide-PEG is more appropriate for siRNA delivery compared to DSPE-PEG. In conclusion, the right balance between cytotoxicity and siRNA effectiveness has been found with the transfection of lipoplexes coated with 20% of Ceramide-PEG2000. This new nanovector could have a high potential against multiple mucosal diseases, such as human papillomavirus-induced genital lesions.

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.

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