Research paper Stabilising cubosomes with Tween 80 as a step towards targeting lipid nanocarriers to the blood–brain barrier Hanisah Azhari a , Mike Strauss b , Sarah Hook a , Ben J. Boyd c,d , Shakila B. Rizwan a,⇑ a School of Pharmacy, University of Otago, Adams Building, 18 Frederick Street, 9054 Dunedin, New Zealand b Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Munich, Germany c Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus), 381 Royal Pde, Parkville, VIC 3052, Australia d ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus), 381 Royal Parade, Parkville, VIC 3052, Australia article info Article history: Received 29 January 2016 Revised 5 April 2016 Accepted in revised form 3 May 2016 Available online 7 May 2016 Keywords: Cubosomes Tween 80 Pluronic F127 Phytantriol Blood–brain barrier Cryogenic electron tomography (cryo-ET) abstract Coating nanoparticles with the surfactant Tween 80 have been previously shown to enhance drug deliv- ery across the blood–brain barrier (BBB). The aim of this study was to investigate whether Tween 80 could be used to stabilise phytantriol-based cubosomes thereby enabling potential application in deliv- ering macromolecular therapeutics to the brain. Cubosome particles with their large internal and exter- nal surface area by virtue of their nanostructure are ideal for delivery of macromolecules. Phase behaviour studies were conducted using a combination of optical microscopy and small-angle X-ray scat- tering (SAXS) and the addition of Tween 80 to mixtures of phytantriol and water resulted in a rich array of lyotropic mesophases. In particular, a large cubic phase region and a two-phase region of readily dis- persed cubosomes is observed. Cubosomes with different concentrations of Tween 80 and phytantriol as the liquid crystal forming lipid were prepared using the solvent precursor method and their physical properties were investigated. A combination of dynamic light scattering, cryogenic electron tomography and SAXS shows formation of well-defined cubosomes with a narrow size distribution and the Im3m cubic structure. Collectively, the results confirm that Tween 80 can effectively stabilize phytantriol cubo- somes, opening the possibility for future application in drug delivery across the BBB. Moreover, well- defined, homogenous cubosome formulations prepared using the mild solvent precursor dilution method has significant implications for large-scale production of cubosomes, which currently is a major barrier to the application of cubosomes in the clinic. Ó 2016 Elsevier B.V. All rights reserved. 1. Introduction Biopharmaceutical therapeutics such as peptides, proteins and monoclonal antibodies have the potential to modify the disease course of neurodegenerative diseases. These macromolecular ther- apeutics offer the advantage of high specificity and potency com- pared to small molecule drugs [1]. However, a major challenge to their development, in addition to their susceptibility to physical and chemical degradation, is their limited permeability across the blood–brain barrier (BBB) [2]. Poor BBB permeability has led to the failure of many high profile biopharmaceuticals in late- stage clinical trials [3–7]. Formulation into nanoparticles can mask the unfavourable physicochemical properties of biopharmaceuticals and has been proposed as a strategy to smuggle these molecules into the brain at therapeutic concentrations [8,9]. To promote BBB transport, nanoparticles can be modified with targeting moieties to enable preferential binding to receptors expressed at the BBB [2,10,11]. At present, several nanoparticle systems, based on lipids and poly- mers, functionalised with targeting groups on the surface, are being explored for drug delivery across the BBB [9]. Cubosomes are liquid crystalline nanoparticles (LCNPs) that are typically 100–200 nm in size and have a highly ordered bicontinu- ous cubic or honeycombed nanostructure of water channels through the lipid matrix [12–15]. Their unique structure offers a large internal and external surface area that is theoretically ideal for drug delivery applications. Advantages that cubosomes may offer over other lipid-based systems, such as liposomes, are the potential to encapsulate a large drug-payload and for sustained release of the entrapped bioactive [12,15]. However cubosome par- ticles are not colloidally stable, and steric stabilisers are added to http://dx.doi.org/10.1016/j.ejpb.2016.05.001 0939-6411/Ó 2016 Elsevier B.V. All rights reserved. ⇑ Corresponding author. E-mail address: shakila.rizwan@otago.ac.nz (S.B. Rizwan). European Journal of Pharmaceutics and Biopharmaceutics 104 (2016) 148–155 Contents lists available at ScienceDirect European Journal of Pharmaceutics and Biopharmaceutics journal homepage: www.elsevier.com/locate/ejpb