The especial features and advanced characteristics of Lyotropic Liquid Crystalline (LLC) phases as potent nano materials for encapsulation and the development of novel delivery systems for nutraceuticals and other bioactive compounds are reviewed. Exemplary, a focus is set on the health benefits of flavonoids and their current restrictions in bioavailability. Accordingly, our visions for application of LLC phases in the engineering of enhanced flavonoid-based food supplements and correlated challenges to overcome are highlighted.

Keywords: lyotropic liquid crystals, isasomes, mesosomes, cubosomes, polyphenols, flavonoids, smart foods, nano structured functional foods

Nanostructures Formulation for Food Products: Advances in soft matter nanotechnology are increasingly being realised to offer smart and generic solutions in various production fields including food and agriculture industries.¹ In recent years there have been several studies reporting on nano medicinal applications of Lyotropic liquid crystalline (LLC) phases like in development of novel contrast enhancers for magnetic resonance imaging (MRI).^2,3 Within the food and pharmaceutical research, LLC nano materials have shown potentials in the development of novel delivery systems.^4‒6 These nanostructures are self-assemblies of naturally occurring surfactant-like lipid molecules which are closely related to those observed in biological membranes and empower the formation of efficient delivery matrices due to their impressive characteristics:⁶ (i) their great interfacial area per volume ratio allow high compound loads; (ii) sustained or controlled release of compounds is supported due to their special and tuneable narrow channel networks (typically, few nano meters thick water-channels); (iii) loading of bioactive molecules with different physicochemical properties is possible, e.g., highly water-insoluble nutraceuticals can be incorporated; (iv) their effective encapsulation capabilities enhance the stability, improve bioavailability and reduce possible toxicity of administered drugs, and (v) their good bio membrane absorption properties increase the compounds transdermal penetration.

Various types of LLCs form under different conditions. Stable inverse bicontinuous cubic (V₂) phases spontaneously form with glycerol monooleate or phytantriol at excess water conditions at ambient temperature. Discontinuous phases such as the inverse hexagonal (H₂) and the inverse micellar (L₂) phase form when increasing amounts of oil are added to the LLC systems. The latter inverse structures can be imagined as isolated water droplets (e.g.L₂ phase) or separated rod-like water channels (H₂ phase) in a continuous oil phase, whereas in the V₂ phases two intertwined, continuous water channel networks are separated by self-assembled lipid bilayers of about 3nm thickness (Figure 1). We note that bicontinuous cubic phases have very good temperature stability and are relatively insensitive to even extreme variations in pH and salinity, which are normally met within the gastrointestinal tract. Their aqueous dispersions were firstly reported in 1981 by Lindström et al. in a fat digestion study.⁷ Later in 1996, Landh and Larsson patented the preparation of particles comprising different types of liquid crystalline phases in their interiors.⁸ These stabilized particles with Internally Self-Assembled mesophases are sometimes referred to as ISAsomes^9‒11or as in this review simply termed as mesosomes.

Figure 1 Cryo-TEM observations taken for four different types of monoglyceride-based aqueous dispersions [12] and corresponding Small Angle X-ray Scattering (SAXS) patterns. Control measurements carried out on empty mesosomes demonstrate the different types of delivery systems that can be designed.

Applications of the LLC bicontinuous cubic phase containing mesosomes (cubosomes) and the inverted hexagonal phase based mesosomes (hexosomes) have received considerable attention in recent years.^12‒15 In particular various types of stabilizers have been tested in order to formulate mesosomes with improved physico-chemical characteristics, but also to assure food or pharmacological safety aspects, Table 1. Very recently, Pickering stabilisation, in which solid nano particles are attached to the oil-water interface¹⁶ rather than surfactant molecules, have received greater interest in the formulation of mesosomes due to the high stability and surfactant free approach offered by this methodology.^10,17,18

Alike bulk LLC phases, mesosomes offer loading capacities for both hydrophobic and hydrophilic bioactives, and further allow sustained release of loaded material as long as highly stable and appropriately sized particles are formulated. Alternatively, other studies have pointed out, how in-situ forming depots of LLC phases can be exploited for a sustained release usage.³³ Each of the underlying mesophases vary in their suitability for different administration routes or delivery of various types of bioactive compounds with respect to optimized encapsulation and release behavior.³⁴ Moreover, mesosomes have the potential to protect the incorporated molecule from oxidation and biodegradation, and structural LLC transitions can be triggered via a range of stimuli including temperature and pH. The later offers the opportunity to actively modulate the delivery system and control the compound release.³⁴ Further, also the release time can be tuned by varying the size of the carriers, however, submicron-sized particles are commonly favoured due to their high interfacial area.

Health benefits of plant based polyphenols: Plant based bioactives especially polyphenolic compounds, are biomaterials with great potentials in the development of food supplements. Flavonoids are one of the main classes of Polyphenols consisting of two phenyl rings and one heterocyclic group. Among flavonoids anthocyanins and catechins are two main groups having several reported health benefits. For example, anthocyanins from bilberry and blueberry have shown strong intracellular antioxidant activity³⁵ Epigallocatechin-3-gallate (EGCG), an abundant Polyphenol in green tea, has even shown to prevent tumour formation and growth. For example, colon cancer prevention activity of tea Polyphenols has been demonstrated in animal models.³⁶ Similarly, lemon verbena (containing Polyphenols from a popular herbal tea) has high antioxidant activity. While many phenolic compounds have shown anticancer and antibacterial activity, efficacy in reducing oxidative stress and other health benefits in vitro experiments, often their function in vivo, in particular in human, is limited due to their reduced bioavailability or bioactivity. Identified reasons are (i) their interactions with proteins or enzymes throughout the gastrointestinal (GI) tract, (ii) poor solubility in aqueous media and (iii) interactions with the food matrix. An overview of further plant based polyphenols with their health benefits and restrictions is given in Table 2.

LLCs as the basis for novel food supplements: The applications of LLCs in encapsulation and the delivery of pharmaceuticals have been widely considered in recent years. Camptothecin,¹⁵ protopanaxadiol,⁶³ dacarbazine⁶⁴ and fluorouracil⁶⁵ are the examples of nutraceuticals encapsulated into cubosomes in order to improve their delivery and hence their therapeutic behaviour. A comprehensive list of various bioactive molecules loaded into mesosomes have been reported recently in a review by Chong et al.,⁴ Nevertheless, the opportunities for enhancing the nutritional values of food by addition of flavonoids are largely unexploited. In particular, the interactions of flavonoids with lipid self-assemblies, formulation of polyphenol-rich mesosomes and eventually engineering of nano structured food supplements with proper taste, stability and digestibility behaviour are scarcely considered. In this respect, the challenges for highly stable and effective encapsulation of flavonoids through formulation of fully food-grade mesosomes which are able to protect bioactives throughout the different digestion stages and guarantee efficient delivery to the small intestine, still remains an essential task for the development of future functional food.

LLC mesosomes are potential nano materials to overcome the challenges concerning the restricted bioavailability, and thus, limited health benefits of current flavonoid-based food supplements. The flavonoids interactions with, and their release behaviour from various types of mesosomes have yet to be understood and further studies are required in order to provide intelligent formulations of mesosomes loaded with flavonoids as future smart food supplements.

None.

Author declares that there is no conflict of interest.

1. Sagalowicz L, Moccand C, Davidek T, et al. Lipid self-assembled structures for reactivity control in food. Philos Trans A Math Phys Eng Sci. 2016;374(2072).
2. Bye N, Hutt OE, Hinton TM, et al. Nitroxide-Loaded Hexosomes Provide MRI Contrast in vivo. Langmuir. 2014;30(29):8898‒8906.
3. Muir BW, Acharya DP, Kennedy DF, et al. Metal-free and MRI visible theranostic lyotropic liquid crystal Nitroxide-based nano particles. 2012;33(9):2723‒2773.
4. Chong JYT, Mulet X, Boyd BJ, et al. Chapter Five - Steric Stabilizers for Cubic Phase Lyotropic Liquid Crystal Nano dispersions (Cubosomes). IN: Iglič A, Kulkarni CV, Rappolt M, editors. Advances in Planar Lipid Bilayers and Liposomes: Academic Press; 2015;21:131‒187.
5. Kwon TK, Hong SK, Kim JC. In vitro skin permeation of cubosomes containing triclosan. Journal of Industrial and Engineering Chemistry. 2012;18(1):563‒567.
6. Rizwan SB, Boyd BJ, Rades T, et al. Bicontinuous cubic liquid crystals as sustained delivery systems for peptides and proteins. Expert Opin Drug Deliv. 2010;7(10):1133‒1144.
7. Lindstrom M, Ljusbergwahren H, Larsson K, et al. Aqueous Lipid Phases of Relevance to Intestinal Fat Digestion and Absorption. 1981;16(10):749‒754.
8. Landh T, Larsson K. Particles, method of preparing said particles and uses thereof. US; 1996.
9. Iglesias GR, Pirolt F, Sadeghpour A, et al. Lipid Transfer in Oil-in-Water Isasome Emulsions: Influence of Arrested Dynamics of the Emulsion Droplets Entrapped in a hydrogel. 2013;29(50):15496‒15502.
10. Sadeghpour A, Pirolt F, Iglesias GR, et al. Lipid Transfer between Sub micrometer Sized Pickering ISA some Emulsions and the Influence of Added hydrogel. 2014;30(10):2639‒2647.
11. Tomšič M, Guillot S, Sagalowicz L, et al. Internally Self-Assembled Thermo reversible Gelling Emulsions: ISAsomes in Methylcellulose, κ-Carrageenan, and Mixed hydrogel. 2009;25(16):9525‒9534.
12. Yaghmur A, Østergaard J, Larsen SW, et al. Drug formulations based on self-assembled liquid crystalline nanostructures. In: Pabst G, Katsaras J, Kucerka N, editors. Liposomes, Lipid Bilayers and Model Membranes: From Basic Research to Technology. Baco Raton: Taylor and Francis Group; 2013:341‒360.
13. Baskaran R, Madheswaran T, Sundaramoorthy P, et al. Entrapment of curcumin into monoolein-based liquid crystalline nano particle dispersion for enhancement of stability and anticancer activity. Int J Nano medicine. 2014;9:3119‒3130.
14. Sun W, Vallooran JJ, Zabara A, et al. Controlling enzymatic activity and kinetics in swollen mesophases by physical nano-confinement. Nano scale. 2014;6(12):6853‒6859.
15. Caltagirone C, Falchi AM, Lampis S, et al. Cancer-Cell-Targeted Theranostic Cubosomes. 2014;30(21):6228‒6236 .
16. Pickering SU. Emulsions. Journal of the Chemical Society, Transactions. 1907;91:2001‒2021.
17. Sadeghpour A, Pirolt F, Glatter O. Sub micrometer-Sized Pickering Emulsions Stabilized by Silica Nano particles with Adsorbed Oleic Acid. 2013;29(20):6004‒6012.
18. Muller F, Degousee T, Degrouard J, et al. Probing structure in submicronic aqueous assemblies of emulsified micro emulsions and charged spherical colloids using SANS and cryo-TEM. J Colloid Interface Sci. 2015;446:114‒121.
19. Guillot S, Bergaya F, de Azevedo C, et al. Internally structured pickering emulsions stabilized by clay mineral particles. J Colloid Interface Sci. 2009;333:563‒569.
20. Salonen A, Muller F, Glatter O. Dispersions of Internally Liquid Crystalline Systems Stabilized by Charged Disk like Particles as Pickering Emulsions: Basic Properties and Time-Resolved Behavior. 2008;24:5306‒5314.
21. Bhatt AB, Barnes TJ, Prestidge CA. Silica Nano particle Stabilization of Liquid Crystalline Lipid Dispersions: Impact on Enzymatic Digestion and Drug Solubilization. Curr Drug Deliv. 2015;12(1):47‒55.
22. Nakano M, Teshigawara T, Sugita A, et al. Dispersions of liquid crystalline phases of the monoolein/oleic acid/pluronic F127 system. 2002;18:9283‒9288.
23. Murgia S, Falchi AM, Meli V, et al. Cubosome formulations stabilized by a dansyl-conjugated block copolymer for possible nano medicine applications. Colloids Surf B Biointerfaces. 2015;129:87‒94.
24. Chong JY, Mulet X, Keddie DJ, et al. Novel Steric Stabilizers for Lyotropic Liquid Crystalline Nano particles: PEGylated-Phytanyl Copolymers. 2015;31(9):2615‒2629.
25. Tilley AJ, Drummond CJ, Boyd BJ. Disposition and association of the steric stabilizer Pluronic® F127 in lyotropic liquid crystalline nano structured particle dispersions. J Colloid Interface Sci. 2013;392:288‒296.
26. Sagalowicz L, Guillot S, Acquistapace S, et al. Influence of Vitamin E Acetate and Other Lipids on the Phase Behavior of Mesophases Based on Unsaturated Monoglyceride. 2013;29(26):8222‒8232.
27. Chong JYT, Mulet X, Waddington LJ, et al. Steric stabilisation of self-assembled cubic lyotropic liquid crystalline nano particles: high throughput evaluation of triblock polyethylene oxide-polypropylene oxide-polyethylene oxide copolymers. Soft Matter. 2011;7:4768‒4777.
28. Zhai JL, Waddington L, Wooster TJ, et al. Revisiting beta-Casein as a Stabilizer for Lipid Liquid Crystalline Nano structured Particles. 2011;27(4):14757‒1466.
29. Angelova A, Angelov B, Papahadjopoulos-Sternberg B, et al. Proteocubosomes: Nanoporous vehicles with tertiary organized fluid interfaces. 2005;21(9):4138‒4143.
30. Yaghmur A, Larsen SW, Schmitt M, et al. In situ characterization of lipidic bupivacaine-loaded formulations. Langmuir. 2011;28(5):8291‒8295.
31. Guo C, Wang J, Cao F, et al. Lyotropic liquid crystal systems in drug delivery. Drug Discov Today. 2010;15(23‒24):1032‒1040.
32. Bornsek SM, Ziberna L, Polak T, et al. Bilberry and blueberry anthocyanins act as powerful intracellular antioxidants in mammalian cells. Food Chem. 2012;134(4):1878‒1884.
33. Yang CS, Wang X, Lu G, et al. Cancer prevention by tea: animal studies, molecular mechanisms and human relevance. Nat Rev Cancer. 2009;9(6):429‒439.
34. Patil-Sen Y, Sadeghpour A, Rappolt M, et al. Facile Preparation of Internally Self-assembled Lipid Particles Stabilized by Carbon Nanotubes. J Vis Exp. 2016;(108):53489.
35. Uyama M, Nakano M, Yamashita J, et al. Useful Modified Cellulose Polymers as New Emulsifiers of Cubosomes. 2009;25(8):4336‒4338.
36. Spicer PT, Small WB, Lynch ML, et al. Dry powder precursors of cubic liquid crystalline nano particles (cubosomes). J Nanopart Res. 2002;4:297‒311.
37. Cavia-Saiz M, Busto MD, Pilar-Izquierdo MC, et al. Antioxidant properties, radical scavenging activity and biomolecule protection capacity of flavonoid naringenin and its glycoside naringin: a comparative study. J Sci Food Agric. 2010;90(7):1238‒1244.
38. Kanaze FI, Bounartzi MI, Georgarakis M, et al. Pharmacokinetics of the citrus flavanone aglycones hesperetin and naringenin after single oral administration in human subjects. Eur J Clin Nutr. 2006;61(4):472‒477.
39. Sarkar FH, Li Y, Wang Z, et al. Lesson Learned from Nature for the Development of Novel Anti-Cancer Agents: Implication of Isoflavone, Curcumin, and their Synthetic Analogs. Curr Pharm Des. 2010;16(16):1801‒1812.
40. Setchell KDR, Faughnan MS, Avades T, et al. Comparing the pharmacokinetics of daidzein and genistein with the use of 13C-labeled tracers in premenopausal women. Am J Clin Nutr. 2003;77(2):411‒419.
41. Aviram M, Dornfeld L, Rosenblat M, et al. Pomegranate juice consumption reduces oxidative stress, atherogenic modifications to LDL, and platelet aggregation: studies in humans and in atherosclerotic apolipoprotein E-deficient mice. Am J Clin Nutr. 2000;71(5):1062‒1076.
42. Qiu JF, Gao X, Wang BL, et al. Preparation and characterization of monomethoxy poly(ethylene glycol)-poly(epsilon-caprolactone) micelles for the solubilization and in vivo delivery of luteolin. Int J Nano medicine. 2013;8:3061‒3069.
43. Zhou P, Li LP, Luo SQ, et al. Intestinal Absorption of Luteolin from Peanut Hull Extract Is More Efficient than That from Individual Pure Luteolin. J Agric Food Chem. 2008;56(1):296‒300.
44. Shen LN, Zhang YT, Wang Q, et al. Enhanced in vitro and in vivo skin deposition of apigenin delivered using ethosomes. Int J Pharm. 2014;460(1‒2):280‒288.
45. Patel D, Shukla S, Gupta S. Apigenin and cancer chemoprevention: Progress, potential and promise (review). Int J Oncol. 2007;30(1):233‒245.
46. Nichenametla SN, Taruscio TG, Barney DL, et al. A review of the effects and mechanisms of polyphenolic in cancer. Crit Rev Food Sci Nutr. 2006;46(2):161‒183.
47. Rawel HM, Czajka D, Rohn S, et al. Interactions of different phenolic acids and flavonoids with soy proteins. Int J Biol Macromol. 2002;30(3‒4):137‒150.
48. Arts MJ, Haenen GR, Voss HP, et al. Masking of antioxidant capacity by the interaction of flavonoids with protein. Food Chem Toxicol. 2001;39(8):787‒791.
49. Calderón-Montaño JM, Burgos-Morón E, Pérez-Guerrero C, et al. A Review on the Dietary Flavonoid Kaempferol. Mini Rev Med Chem. 2011;11(4):298‒344.
50. Tzeng CW, Yen FL, Wu TH, et al. Enhancement of Dissolution and Antioxidant Activity of Kaempferol Using a Nano particle Engineering Process. J Agric Food Chem. 2011;59:5073‒5080.
51. Sala A, Recio MC, Schinella GR, et al. Assessment of the anti-inflammatory activity and free radical scavenger activity of tiliroside. Eur J Pharmacol. 2003;461(1):53‒61.
52. Velagapudi R, Aderogba M, Olajide OA. Tiliroside, a dietary glycosidic flavonoid, inhibits TRAF-6/NF-κB/p38-mediated neuro inflammation in activated BV2 microglia. Biochim Biophys Acta. 2014;1840(12):3311‒3319.
53. Qin N, Li CB, Jin MN, et al. Synthesis and biological activity of novel tiliroside derivants. Eur J Med Chem. 2011;46(10):5189‒5195.
54. Goto T, Horita M, Nagai H, et al. Tiliroside, a glycosidic flavonoid, inhibits carbohydrate digestion and glucose absorption in the gastrointestinal tract. Mol Nutr Food Res. 2012;56(3):435‒445.
55. Gundimeda U, McNeill TH, Barseghian BA, et al. Polyphenols from green tea prevent anti neuritogenic action of Nogo-A via 67-kDa laminin receptor and hydrogen peroxide. J Neurochem. 2015;132(1):70‒84.
56. Charlton AJ, Baxter NJ, Khan ML, et al. Polyphenols/Peptide Binding and Precipitation. J Agric Food Chem. 2002;50(6):1593‒1601.
57. Zanchi D, Poulain C, Konarev P, et al. Colloidal stability of tannins: astringency, wine tasting and beyond. Journal of Physics-Condensed Matter. 2008;20.
58. Nakagawa K, Okuda S, Miyazawa T. Dose-dependent incorporation of tea catechins, (-)-epigallocatechin-3-gallate and (-)-epigallocatechin, into human plasma. Biosci Biotechnol Biochem. 1997;61(12):1981‒1985.
59. Kang SY, Seeram NP, Nair MG, et al. Tart cherry anthocyanins inhibit tumor development in Apc (Min) mice and reduce proliferation of human colon cancer cells. Cancer Lett. 2003;194(1):13‒19.
60. Jayaprakasam B, Vareed SK, Olson LK, et al. Insulin Secretion by Bioactive Anthocyanins and Anthocyanidins Present in Fruits. J Agric Food Chem. 2005;53(1):28‒31.
61. Tsuda T, Horio F, Osawa T. Absorption and metabolism of cyanidin 3-O-β-D-glucoside in rats. FEBS Lett. 1999;449(2‒3):179‒82.
62. Ernst IMA, Wagner AE, Lipinski S, et al. Cellular uptake, stability, visualization by 'Naturstoff reagent A', and multidrug resistance protein 1 gene-regulatory activity of cyanidin in human keratinocytes. Pharmacol Res. 2010;61(3):253‒258.
63. Jin X, Zhang ZH, Sun E, et al. Enhanced oral absorption of 20(S)-protopanaxadiol by self-assembled liquid crystalline nano particles containing piperine: in vitro and in vivo Int J Nano medicine. 2013;8:641‒652.
64. Bei D, Marszalek J, Youan BB. Formulation of Dacarbazine-Loaded Cubosomes-Part I: Influence of Formulation Variables. Aaps PharmScitech. 2009;10(3):1032‒1039.
65. Nasr M, Ghorab MK, Abdelazem A. In vitro and in vivo evaluation of cubosomes containing 5-fluorouracil for liver targeting. Acta Pharm Sin B. 2015;5(1):79‒88.