Stephanie David

Non-viral nanosystems for systemic siRNA delivery

To use siRNA (small interfering ribonucleic acids) for systemic administration, a delivery system is often necessary to overcome barriers between administration and the target sites. These delivery systems require different properties to be efficient. On the one hand, they have to protect siRNA from degradation and/or inactivation and, on the other hand, they have themselves to be stable in blood and possess stealth properties to avoid elimination and degradation. Active and/or passive targeting should help the delivery system to reach the desired cell type or tissue, to be internalised, and to deliver siRNA to the cytoplasm so that siRNA can act by RNA interference and inhibit protein synthesis. This review presents an overview of different non-viral delivery systems, which have been evaluated in vivo or entered in clinical trials, with a focus on their physicochemical properties in order to help the development of new and efficient siRNA delivery systems, as the therapeutic solutions of tomorrow.

Nature as a source of inspiration for cationic lipid synthesis

Synthetic gene delivery systems represent an attractive alternative to viral vectors for DNA transfection. Cationic lipids are one of the most widely used non-viral vectors for the delivery of DNA into cultured cells and are easily synthesized, leading to a large variety of well-characterized molecules. This review discusses strategies for the design of efficient cationic lipids that overcome the critical barriers of in vitro transfection. A particular focus is placed on natural hydrophilic headgroups and lipophilic tails that have been used to synthesize biocompatible and non-toxic cationic lipids. We also present chemical features that have been investigated to enhance the transfection efficiency of cationic lipids by promoting the escape of lipoplexes from the endosomal compartment and DNA release from DNA-liposome complexes. Transfection efficiency studies using these strategies are likely to improve the understanding of the mechanism of cationic lipid-mediated gene delivery and to help the rational design of novel cationic lipids.

siRNA LNCs – A novel platform of lipid nanocapsules for systemic siRNA administration

Several siRNA (small interfering RNA) therapeutics are undergoing clinical trials for cancer, respiratory diseases or macular degeneration, but most are administrated locally. In order to overcome the different barriers to attain an efficient siRNA action after systemic administration, nanocarriers able to carry and protect siRNA are awaited. With this aim, we developed a new platform of siRNA lipid nanocapsules (LNCs) using different cationic lipids, combining the properties of LNCs (siRNA protection and targeting) and lipoplexes (efficient siRNA delivery into the cell). The formulation was revealed to contain different compartments. A siRNA quantification method based on UV spectroscopy was developed to locate and quantify siRNA in each compartment. All in all, these novel siRNA LNCs presented sizes of about 55 nm with a neutral surface charge and siRNA encapsulation efficiencies up to 65% representing appropriate characteristics for systemic administration.

In vivo imaging of DNA lipid nanocapsules after systemic administration in a melanoma mouse model.

The biodistribution of intravenously injected DNA lipid nanocapsules (DNA LNCs), encapsulating pHSV-tk, was analysed by in vivo imaging on an orthotopic melanoma mouse model and by a subsequent treatment with ganciclovir (GCV), using the gene-directed enzyme prodrug therapy (GDEPT) approach. Luminescent melanoma cells, implanted subcutaneously in the right flank of the mice, allowed us to follow tumour growth and tumour localisation with in vivo bioluminescence imaging (BLI). In parallel, DNA LNCs or PEG DNA LNCs (DNA LNCs recovered with PEG(2000)) encapsulating a fluorescent probe, DiD, allowed us to follow their biodistribution with in vivo biofluorescence imaging (BFI). The BF-images confirmed a prolonged circulation-time for PEG DNA LNCs as was previously observed on an ectotopic model of glioma; comparison with BL-images evidenced the colocalisation of PEG DNA LNCs and melanoma cells. After these promising results, treatment with PEG DNA LNCs and GCV on a few animals was performed and the treatment efficacy measured by BLI. The first results showed tumour growth reduction tendency and, once optimised, this therapy strategy could become a new option for melanoma treatment.

DNA nanocarriers for systemic administration: characterization and in vivo bioimaging in healthy mice.

We hereby present different DNA nanocarriers consisting of new multimodular systems (MMS), containing the cationic lipid dioleylaminesuccinylparomomycin (DNA MMS DOSP), or bis (guanidinium)-tren-cholesterol (DNA MMS BGTC), and DNA lipid nanocapsules (DNA LNCs). Active targeting of the asialoglycoprotein receptor (ASGP-R) using galactose as a ligand for DNA MMS (GAL DNA MMS) and passive targeting using a polyethylene glycol coating for DNA LNCs (PEG DNA LNCs) should improve the properties of these DNA nanocarriers. All systems were characterized via physicochemical methods and the DNA payload of DNA LNCs was quantified for the first time. Afterwards, their biodistribution in healthy mice was analyzed after encapsulation of a fluorescent dye via in vivo biofluorescence imaging (BFI), revealing various distribution profiles depending on the cationic lipid used and their surface characteristics. Furthermore, the two vectors with the best prolonged circulation profile were administered twice in healthy mice revealing that the new DNA MMS DOSP vectors showed no toxicity and the same distribution profile for both injections, contrary to PEG DNA LNCs which showed a rapid clearance after the second injection, certainly due to the accelerated blood clearance phenomenon.

EGFR siRNA lipid nanocapsules efficiently transfect glioma cells in vitro.

Glioma are the most common malignant tumors of the central nervous system and remain associated with poor prognosis, despite the combination of chemotherapy and radiotherapy. EGFR targeting represents an interesting strategy to treat glioma. Indeed, a high level of endothelial growth factor receptors expression (EGFR), involved in the malignancy of the tumor, has been observed in glioma. Our strategy consisted in using EGFR siRNA entrapped into lipid nanocapsules (LNCs) via cationic liposomes. In vitro analyses on U87MG human glioma cells were performed to evaluate firstly the capacity of LNCs to efficiently deliver the siRNA and secondly the effect of EGFR siRNA targeting on U87MG proliferation. Then, the complement protein consumption was evaluated by CH50 assays to verify the suitability of the siRNA LNCs for systemic administration. The EGFR siRNA LNCs exhibited an adequate size lower than 150 nm as well as a neutral surface charge. The IC50 profile together with the 63% of protein extinction demonstrated the significant action of EGFR siRNA LNCs compared to scrambled LNCs. Dose and time-dependent survival assays showed a decrease of U87MG growth evaluated at 38%. Finally, low complement consumption demonstrated the suitability of EGFR siRNA LNCs for intravenous injection. In conclusion, EGFR siRNA LNCs demonstrated their capacity to efficiently encapsulate and deliver siRNA into U87MG human glioma cells, and will therefore be usable in the future for in vivo evaluation.

Novel alginate-based nanocarriers as a strategy to include high concentrations of hydrophobic compounds in hydrogels for topical application

The cutaneous penetration of hydrophobic active molecules is of foremost concern in the dermatology and cosmetic formulation fields. The poor solubility in water of those molecules limits their use in hydrophilic forms such as gels, which are favored by patients with chronic skin disease. The aim of this work is to design a novel nanocarrier of hydrophobic active molecules and to determine its potential as an ingredient of a topical form. The nanocarrier consists of an oily core surrounded by a protective shell of alginate, a natural polysaccharide isolated from brown algae. These calcium alginate-based nanocarriers (CaANCs) were prepared at room temperature and without the use of organic solvent by an accelerated nanoemulsification-polymer crosslinking method. The size (hydrodynamic diameter ~200 nm) and surface charge (zeta potential ~ - 30 mV) of the CaANCs are both compatible with their application on skin. CaANCs loaded with a fluorescent label were stable in model hydrophilic galenic forms under different storage conditions. Curcumin was encapsulated in CaANCs with an efficiency of ~95%, fully retaining its antioxidant activity. The application of the curcumin-loaded CaANCs on excised human skin led to a significant accumulation of the active molecules in the upper layers of the skin, asserting the potential of these nanocarriers in active pharmaceutical and cosmetic ingredients topical delivery.

Treatment efficacy of DNA lipid nanocapsules and DNA multimodular systems after systemic administration in a human glioma model.

We previously developed different types of DNA nanocarriers for systemic administration. Recently, the biodistribution profiles of these intravenously administered nanocarriers, DNA lipid nanocapsules (LNCs) and different multimodular systems (MMS), were analysed in healthy mice using in vivo biofluorescence imaging.

Use of experimental design methodology for the development of new magnetic siRNA nanovectors (MSN)

Short interfering RNAs (siRNAs) can downregulate the synthesis of proteins and thus be used to treat certain diseases where the protein synthesis is upregulated, such as cancer. The challenge is to deliver siRNAs in the target cell as they are rapidly degraded by nucleases and have difficulties to cross the cellular membranes. Superparamagnetic iron oxide nanoparticles (SPIONs) are widely studied as platforms for smart biocompatible nanosystems which can be used for magnetic drug targeting and magnetic resonance imaging. The aim of this work was to combine siRNAs, SPIONs, and chitosan, to develop new magnetic siRNA nanovectors suitable for systemic administration. In a first time, the one factor at a time (OFAT) methodology was used to adjust different formulation parameters and to test the feasibility of such a formulation. In a second time, design of experiment (DOE) methodology was used to analyze the influence of these formulation parameters on the physicochemical characteristics hydrodynamic diameter (DH) and ζ-potential. Finally, four MSNs suitable for systemic administration could be identified using the OFAT method. The DOE method showed a significant effect of CR and [NaNO3] on the DH and a significant effect of MR and [siRNA] on the ζ-potential of the nanocarriers.

siRNA delivery system based on magnetic nanovectors: Characterization and stability evaluation

Abstract Gene therapy and particularly small interfering RNA (siRNA) is a promising therapeutic method for treatment of various human diseases, especially cancer. However the lack of an ideal delivery system limits its clinical applications. Effective anticancer drug development represents the key for translation of research advances into medicines. Previously we reported, the optimization of magnetic siRNA nanovectors (MSN) formulation based on superparamagnetic iron oxide nanoparticles (SPION) and chitosan for systemic administration. This work aimed at using rational design to further optimize and develop MSN. Therefore, formulated MSN were first purified, then their physical and chemical properties were studied mainly through capillary electrophoresis. 95% of siRNA was found enclosed within the purified MSN (pMSN). pMSN showed colloidal stability at pH 7.4, effective protection of siRNA against ribonuclease degradation up to 24 hours and few siRNA release (less than 10%) at pH 7.4. These findings push toward further evaluation studies in vitro and/or in vivo, indicating the appropriateness of pMSN for cancer theranostics. Graphical abstract Figure. No Caption available.