A structurally diverse library of safe-by-design citrem-phospholipid lamellar and non-lamellar liquid crystalline nano-assemblies
Graphical abstract
Introduction
Lyotropic non-lamellar liquid crystalline (LC) nanodispersions consisting of internally self-assembled dispersed nanoparticles (e.g., cubosomes and hexosomes), bring a wide range of advantages in terms of drug solubilisation and loading capacities, and therefore gaining popularity in nanomedicine [1]. Internally self-assembled nanodispersions of inverse bicontinuous cubic (V2) and discontinuous hexagonal (H2) LC phases are often formed in excess water through high-energy emulsification of lipids such as monounsaturated monoglycerides (e.g. monoolein), glycolipids, and phytantriol in the presence of an efficient stabilizer (a secondary emulsifier) such as Pluronic® block co-polymers (e.g., Pluronic® F127), polysorbate 80 (Tween 80) [1], [2] and phospholipid-poly(ethylene glycol) conjugates [3], [4]. The resultant preparations often exhibit poor hemocompatibility, and may induce cellular toxicity, which limits their application for intravenous route of injection and site-specific targeting within the vasculature [3], [4], [5], [6], [7], [8]. Indeed, Pluronic® F127, Tweens and poly(ethylene glycol)s incite complement (the first line of the innate defence system) in the blood, which in turn could initiate cardiopulmonary distress in sensitive patients [8], [9], [10], [11], whereas lipids such as phytantriol induce membrane disruption and oxidative stress [3], [7].
Naturally occurring phospholipids such as phosphatidylcholine (PC), however, may represent a promising safer amphiphile in lyotropic non-lamellar LC nano-engineering, but PC has a strong tendency to form lamellar mesophases with approximately zero-mean curvature [12]. Accordingly, formation of PC-based lyotropic non-lamellar LC delivery systems is challenging and scarcely reported [12], [13], [14], [15], [16], [17], since higher negative curvatures of the interface must be attained by adding a lipid or an apolar solvent modifier that may overcome the strong tendency of PC to form lamellae and enhance a structural transition to non-lamellar phases. However, the modifier must also be nontoxic, and should confer structural stability to the non-lamellar mesophases in the blood, which is important in the development of injectable nanocarriers intended for intravenous applications. Among the reported examples are mesophases of egg yolk PC and diolein, but these LC phases display limited structural diversity and stability for systemic administration [17]. For instance, at an egg yolk PC/diolein molar ratio of 8:2, a bicontinuous cubic phase of primitive type (Im3m) was assembled, whereas a mixing ratio of 7:3 yielded a bicontinuous cubic phase of diamond type, which coexisted with an inverted hexagonal H2 phase [17]. However, when dispersed by high-pressure emulsification with Pluronic® F127 to provide colloidal stability in excess water, no cubosomes were formed, and instead vesicles were obtained [17]. Another interesting example is polysorbate 80 (Tween 80)-stabilized non-lamellar nanodispersions of soy PC (SPC)/glycerol dioleate/glycerol monooleate [15]. Although this preparation showed low hemolytic activity, polysorbate 80 similar to other Tweens is a strong activator of the complement system [11]. Finally, a hydrotrope method [18] has also been introduced for preparation of PC-based non-lamellar LC nanodispersions. This process involves addition of a water-miscible solvent and heat treatments, and is therefore not suitable for encapsulation of biopharmaceuticals. Accordingly, it would be of pharmaceutical advantage if formation of biologically safe PC-based non-lamellar mesophases can be achieved without the use of solvents and through a low-energy input technique, which may even reduce vesicle formation.
In the present work, we report on a binary lipid system (citrem/SPC) that forms a tunable library of ISAsomes (internally self-assembled ‘somes’ or nanoparticles) in excess aqueous medium. Citrem, an anionic citric acid ester of monoglycerides, has anti-oxidative properties, and on hydrolysis generates citric acid and glycerol [19]. Citrem also confers stability to ISAsomes in plasma [8]. We show nanodispersions of citrem/SPC lamellar and non-lamellar mesophases neither induce hemolysis nor activate the terminal pathway of the complement system, but depending on citrem concentration, ISAsomes uptake by macrophages can be modulated (macrophage targeting versus macrophage avoidance). Our approach has also overcome the need for a secondary emulsifier, which conventionally is necessary to achieve colloidal stability of ISAsomes [3], [13], [14], [20], [21]. Finally, the aforementioned library of citrem/SPC ISAsomes can be produced with low energy input, and without the use of an organic hydrotropic solvent, which is advantageous for the development of ISAsome encapsulating temperature-sensitive or solvent-labile therapeutic agents [1], [18], [22], [23].
Section snippets
Materials
Soy phosphatidylcholine (SPC) with purity of 99.1% was purchased from Lipoid AG (Steinhausen, Switzerland). Grinsted® citrem LR10, which is a citric acid ester of monoglycerides made from sunflower oil, was received as a gift from Danisco A/S (Copenhagen, Denmark). 149 mM Dulbecco's Phosphate-buffered solution (PBS) at pH 7.4 (was purchased from Sigma Aldrich (Poole, UK). Human plasma for SAXS characterization was a gift from Rigshospitalet (Copenhagen, Denmark) with Citrate Phosphate Dextrose
ISAsomes Formation
We prepared six combinations of binary citrem/SPC (CS) mixtures (Table 1), and studied their internal nanostructure by synchrotron small-angle X-ray scattering (SAXS) at 25 °C (Fig. 1a). SPC in the presence of small fractions of citrem (e.g., CS1:4, CS2:3) formed LC lamellar (Lα) phases (Table 1). This observation was similar to the well-known tendency of SPC to form vesicles [28]. The first non-lamellar phase was observed after addition of 2.5 wt% citrem, resulting in a final citrem/SPC weight
Conclusions
In summary, we formed and characterized a diverse library of lyotropic lamellar and non-lamellar LC nanodispersions based on binary lipid mixtures of citrem and SPC. This is an attractive library for pharmaceutical applications, particularly for the development of injectable safe and hemocompatible nanocarriers with rich polymorphism. By augmenting citrem content, the internal nanostructure transforms from vesicular dispersions to ELPs via cubosomes and hexosomes. Colloidal and structural
Conflict of interest
The authors declare no competing financial interest.
Acknowledgments
Financial support by the Danish Council for Independent Research | Technology and Production Sciences, reference 1335-00150b (to AY and SMM) is gratefully acknowledged. AY further acknowledges financial support from the Danish Natural Sciences Research Council (DanScatt) for SAXS experiments. IDMA is a recipient of a PhD Scholarship Award from the Ministry of Higher Education of Malaysia (MOHE) and Universiti Putra Malaysia (UPM).
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Contributed equally.