Towards a clinical application of freeze-dried squalene-based nanomedicines

Abstract

Squalene-adenosine (SQAd) nanoparticles (NPs) were found to display promising pharmacological activity similar to many other nanomedicines, but their long-term stability was still limited, and their preparation required specific know-how and material. These drawbacks represented important restrictions for their potential use in the clinic. Freeze-drying nanoparticles is commonly presented as a solution to allow colloidal stability, but this process needs to be adapted to each nanoformulation. Hence, we aimed at developing a specific protocol for freeze-drying SQAd NPs while preserving their structural features. NPs were lyophilised, resuspended and analysed by dynamic light scattering, atomic force microscopy and small-angle scattering. Among four different cryoprotectants, trehalose was found to be the most efficient in preserving NPs physico-chemical characteristics. Interestingly, we identified residual ethanol in NP suspensions as a key parameter which could severely affect the freeze-drying outcome, leading to NPs aggregation. Long-term stability was also assessed. No significant change in size distribution or zeta potential could be detected after three-month storage at 4 °C. Finally, freeze-dried NPs innocuity was checked in vitro on cultured hepatocytes and in vivo on mice. In conclusion, optimisation of freeze-drying conditions resulted in safe lyophilised SQAd NPs that can be easily stored, shipped and simply reconstituted into an injectable form.

Keywords:

• Nanomedicines
• nanoparticles
• squalenoylation
• freeze-drying
• lyophilisation
• nanoprecipitation

Acknowledgements

Special thanks to Professor Patrick Couvreur who has been greatly involved in this work. The authors also acknowledge the support provided by Patrick Guenoun (IRAMIS, CEA) and Isabelle Grillo (Institut Laue-Langevin) in SANS experiments, and Javier Perez (Synchrotron SOLEIL) in SAXS experiments. Finally, authors are deeply grateful to Anne Beilvert (Occlugel) for her assistance with the freeze-dryer.

Disclosure statement

No potential conflict of interest was reported by the authors.