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International Journal of Pharmaceutics 478 (2015) 569–587

Contents lists available at ScienceDirect

International Journal of Pharmaceutics


journal homepage: www.elsevier.com/locate/ijpharm

Review

Glycerol monooleate liquid crystalline phases used in drug delivery


systems
Spomenka Milak, Andreas Zimmer *
University of Graz, Institute of Pharmaceutical Sciences, Department of Pharmaceutical Technology, NAWI Graz, Universitätsplatz 1, 8010 Graz, Austria

A R T I C L E I N F O A B S T R A C T

Article history: During the last few decades, both scientific and applied research communities have shown increased
Received 17 July 2014 attention to self-assembled lyotropic liquid crystalline phases of polar lipids, due to their remarkable
Received in revised form 20 November 2014 structural complexity and usefulness in diverse applications.
Accepted 29 November 2014
Amphiphilic properties of polar lipids in relation to water are the driving force for self-assemblies
Available online 3 December 2014
following an extraordinary polymorphism. This polymorphism is an interesting phenomenon in which
lipids combine short-range disorder and long-range order. The most widely investigated liquid
Keywords:
crystalline phases are the lamellar, the cubic and the hexagonal.
Glycerol monooleate
Liquid crystalline phase
Such phases have high solubilization capacity for hydrophilic, lipophilic and amphiphilic guest
Cubic phase molecules and the ability to protect molecules against hydrolysis or oxidation. So, they can be used as an
Hexagonal phase interesting drug delivery matrix for drugs, amino acids, peptides, proteins and vitamins in various food,
Drug delivery pharmaceutical and biotechnical applications.
This review presents recent progress in glycerol monooleate liquid crystalline phases used as drug
delivery vehicles.
ã 2014 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
2. Glycerol monooleate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
3. The structure of liquid crystalline phases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3.1. The reversed cubic liquid crystalline phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3.2. The reversed hexagonal liquid crystalline phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3.3. The lamellar liquid crystalline phase (La) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3.4. Intermediate phases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
4. Phase diagram of glycerol monooleate/water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
5. Introduction of guest molecules in the liquid crystalline phases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
5.1. Introduction of guest molecules in the reversed cubic phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
5.2. Introduction of guest molecules in the reversed hexagonal phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
5.3. Introduction of guest molecules in the reversed micellar cubic phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
5.4. Introduction of guest molecules in the bicontinuous sponge phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
6. Characterization of liquid crystalline phases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
6.1. Polarized light microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
6.2. X-ray diffraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
6.3. Differential scanning calorimetry (DSC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
6.4. Rheology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Abbreviations: SAXS, small-angle X-ray scattering; DSC, differential scanning calorimetry; NMR, nuclear magnetic resonance; FTIR, Fourier transform infrared
spectroscopy; MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5-dyphenyl tetrazolium bromide; TGA, thermogravimetry analysis; PEG, polyethylene glycol; M, molar; mM, millimolar;
Mr, molecular mass.
* Corresponding author at: University of Graz, Institute of Pharmaceutical Sciences, Department of Pharmaceutical Technology, NAWI Graz, Universitätsplatz 1, 8010 Graz,
member of: BioTechMed Graz, Austria. Tel.: +43 316 380 8881; fax: +43 316 389 9100.
E-mail address: andreas.zimmer@uni-graz.at (A. Zimmer).

http://dx.doi.org/10.1016/j.ijpharm.2014.11.072
0378-5173/ ã 2014 Elsevier B.V. All rights reserved.
570 S. Milak, A. Zimmer / International Journal of Pharmaceutics 478 (2015) 569–587

6.5. Low frequency dielectric spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15


6.6. Spectroscopic methods (NMR, IR- and Raman spectroscopy) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
7. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

1. Introduction structures. In the solid state, the general structure of lipids is a


stack of planar molecular bilayers. The hydrocarbon chains are
Liquid crystalline phases are frequently encountered in every- close-packed in these bilayers, and the polar heads form the outer
day life. For example, the cell membranes in the body are the result surfaces. The hydrogen-bond system in the sheets formed by the
of the lyotropic liquid crystalline phase that is generated from the polar head groups is strong compared to the weak van der Waals
dissolution of phospholipids in water (Collings and Hird, 1997; interaction between the hydrocarbon chains (Collings and Hird,
Seddon and Templer, 1995). Probably, microsomes’ membranes, 1997; Larsson, 1989, 2000; Seddon and Templer, 1995). During the
mitochondria and tight junctions between cells are formed from melting of this structure, first the hydrocarbon chains become
non-lamellar liquid crystalline structures. Etiolated chloroplasts, disordered into a liquid-like structure, with the overlying gross
which consists of six-fold or four-fold interconnected tubular structure remaining intact, then, at a higher temperature the
membrane structures, are strikingly similar to the structure complete melting occurs. In solids, planar zigzag conformations of
elements of the inverse bicontinuous cubic phases. The structures the carbon–carbon bonds exist (all-trans), whereas in disordered
of certain membranous organelles in cells, for example in states, occurring in liquid crystals and in melts, gauche con-
endoplasmic reticulum, bear a quite striking similarity to the formations form dynamically along the chain. The combination of
sponge phase. disorder on the atomic scale with the long-range order in layers is
It has been assumed that liquid crystalline phases, possibly the characteristic property of liquid crystalline phases of lipids.
including cubic phases, play a role in the process of fat digestion in Several review articles about lipid liquid crystalline phases have
vivo (Collings and Hird, 1997; Lynch and Spicer, 2005; Seddon and been published to give insights into their structure and the
Templer, 1995). During this process, triglyceride is hydrolyzed first diversity of applications. The purpose of this review is to
to diacylglycerol plus fatty acid, then to monoacylglycerol plus two summarize the data about drug delivery systems based on glycerol
fatty acid molecules. Research on phase equilibria of lipid mixtures monooleate liquid crystalline phases (Amar-Yuli et al., 2009; Caboi
similar to those found in the intestine showed that liquid crystalline et al., 2001; Chernik, 1999; Drummond and Fong, 1999; Engström,
phases, as well as an inverse micellar solution, were formed and it 1990; Fong et al., 2012; Garti et al., 2012; Guo et al., 2010; Hitesh
was suggested that the latter phase may coexist with mixed micelles et al., 2011; Kaasgaard and Drummond, 2006; Kulkarni et al., 2011;
in the human intestine. These phases have an important property Larsson, 2009; Leser et al., 2006,b; Sagalowicz et al., 2006a,b; Shah
that all reactants and products, whether polar, non-polar or et al., 2001).
amphiphilic can diffuse freely across the structure. Accordingly,
life itself critically depends upon liquid crystalline phases. 2. Glycerol monooleate
Liquid crystals show properties between those of conventional
liquid and solid crystals (Seddon and Templer, 1995). For instance, Glycerol monooleate is one of the most widely studied
a liquid crystal may flow like a liquid but have the molecules in the amphiphilic lipid used in the formation of various liquid crystalline
liquid arranged and oriented in a crystal-like way. drug formulations (Ganem-Quintanar et al., 2000).
The type of molecular structure that generates liquid crystalline Glycerol monooleate (Fig. 1) is a glycerol fatty acid ester. It has a
phases is amphiphilic (Collings and Hird, 1997; Seddon and cis double bond at C9. From the molecular point of view, glycerol
Templer, 1995). The amphiphilicity implies the dualistic properties monooleate has the acyl chain which is by an ester bond attached
of the molecules in relation to water, with flexible hydrocarbon to the glycerol backbone (Ganem-Quintanar et al., 2000; Kulkarni
chains avoiding water contact and a polar head group that tends to et al., 2011). The two remaining carbons of the glycerol moiety are
orient towards water. Amphiphilic molecules form aggregates free, giving polar characteristics to this part of the molecule. This
through a self-assembly process that is driven by the “hydrophobic hydrophilic part can form hydrogen bonds with water in an
effect” when they are mixed with a solvent (usually water). The aqueous environment (the headgroup). The hydrocarbon chain
aggregates formed by amphiphilic molecules are characterized by (the tail) gives hydrophobic properties to glycerol monooleate.
structures in which the hydrophilic head-groups shield the Glycerol monooleate is a lipophilic substance, HLB = 3–4, almost
hydrophobic chains from contact with water. For most lyotropic insoluble in an aqueous phase. Its solubility in water is ffi106 M
systems aggregation occurs only when the concentration of the and it forms micellar solution with water above its critical
amphiphile exceeds a CMC (critical micelle concentration) or the aggregation concentration, approx. 4  106 M (Barauskas et al.,
CAC (critical aggregation concentration). Above the CMC the self- 2010).
assembled amphiphile aggregates exist as independent entities, in
equilibrium with monomeric amphiphiles in solution, and with no
[(Fig._1)TD$IG]
long ranged orientational or positional (translational) order. These
dispersions are micellar solutions (its constituent aggregates are
micelles, generating isotropic phases). The lyotropic liquid
crystalline phases are formed as the concentration of amphiphile
in water is increased beyond the point where the micellar
aggregates are forced to be disposed regularly in space. For
amphiphiles that consist of a single hydrocarbon chain the
concentration at which the first liquid crystalline phases are
formed is typically in the range 25–30 w/w%.
In the same way, polar lipids, as amphiphilic molecules, have a
remarkable ability to self-assembly in water to form different Fig. 1. Chemical structure of glycerol monooleate.
S. Milak, A. Zimmer / International Journal of Pharmaceutics 478 (2015) 569–587 571

Furthermore, glycerol monooleate is a nontoxic, biodegradable In the bicontinuous cubic phase, a minimal surface has an
and biocompatible material, classified as GRAS (generally recog- average curvature equal to zero everywhere (Seddon and Templer,
nized as safe) and it is included in the FDA Inactive Ingredients 1995), which means that the pressure gradient is equal to zero. This
Guide (Bode et al., 2013; Ganem-Quintanar et al., 2000; Matschke unique curvature of the bilayer in the cubic phase is associated
et al., 2002). One important aspect of the use of glycerol with the curvature elastic energy which determines the stability of
monooleate as a safe parenteral material is the necessity to the cubic phase as a function of composition. Spontaneous
confirm its biological tolerance. Although glycerol monooleate formation and thermodynamic stability of a cubic phase, consist-
disappears after in vivo subcutaneous and intramuscular injection, ing of bicontinuous bilayers arranged in geometries of periodic
principally by lipase activity, its nonirritant effect on the tissues minimal surfaces found in many lipid/water systems, are due to a
has not been entirely confirmed. Glycerol monooleate has competition between the two free energy terms: the curvature
hemolytic properties and therefore it is not suitable for intrave- energy of each monolayer versus the stretching energy of the lipid
nous administration. chains.
Glycerol monooleate was first used in 1930 in the margarine To date three cubic bicontinuous phases have been identified
production (Ganem-Quintanar et al., 2000). Today, it is used as a (Collings and Hird, 1997; Larsson et al., 2006; Lynch and Spicer,
processing aid in the production and stabilization of emulsions and 2005; Seddon and Templer, 1995), based on these three surfaces:
foams in bread, cakes, margarine, ice creams and chewing gums. Its the P (Im3m), the D (diamond, Pn3m) and most frequently, the G
major functions are absorption at interface or on solids, promotion (gyroid, Ia3d) bicontinuous liquid crystalline phases (Fig. 2). Such
of wetting phenomena, co-crystallization, complex formation surfaces describe the mid-surface of the amphiphilic bilayer (Hyde,
(with proteins or starch components) and self-association. 2001). Probably there are many other examples of cubic phases,
In the pharmaceutical area, glycerol monooleate was first used that have not yet definitely been identified, so more cubic phases
as an emulsifier and an absorption enhancer in combination with remain to be discovered. In summary, Garti et al. (2012), described
bile salts (Dash et al., 1999; Ganem-Quintanar et al., 2000; Herai the seven cubic structures which were discovered so far.
et al., 2007). As an absorption enhancer, glycerol monooleate Glycerol monooleate at low water content forms the bicontin-
probably acts by causing a temporary and reversible disruption of uous cubic phase with space group Ia3d (proposed to be a
the lamellar structure of the lipid bilayer in the stratum corneum G-surface) and then, at higher water content, the Pn3m phase is
and, in this way, increasing intercellular lipid fluidity. As a obtained (Larsson et al., 2006; Lynch and Spicer, 2005; Seddon and
biocompatible encapsulating material, glycerol monooleate was Templer, 1995). In terms of minimal surfaces, the inverse
first proposed in 1984. Since then many papers were released and bicontinuous cubic phases Ia3d,Pn3m and Im3m are formed by
new applications were proposed. Extremely helpful for all new dripping a continuous lipid bilayer onto the gyroid, F- and
applications was the significant advance in understanding the P-minimal surfaces, respectively. These three surfaces constitute
physico-chemical properties of glycerol monooleate. a family of infinite periodic minimal surfaces which are related to
each other by the Bonnet transformation. This means that one
3. The structure of liquid crystalline phases surface can be transformed into either of the others by bending,
which leaves the Gaussian curvature at all points unchanged, and
Liquid crystalline phases, having long-ranged orientational preserves all angles, distances and areas on the surface. These
order, are induced by the addition of a solvent (lyotropic liquid transitions between bicontinuous liquid crystalline phases are
crystalline phases) or by temperature (thermotropic liquid [(Fig._2)TD$IG]
crystalline phases) (Chernik, 1999; Larsson et al., 2006; Larsson,
2009). Three main classes of lyotropic liquid crystalline phase
structures are: the lamellar, the hexagonal and the cubic phases,
and their structures have been classified by X-ray diffraction
techniques. The most widely used nomenclature for lyotropic
phases (Collings and Hird, 1997; Hyde, 2001; Lynch and Spicer,
2005; Seddon and Templer, 1995) is that proposed by Luzzati and
this is denoted by a capital letter, e.g., L for lamellar, H for
hexagonal, Q for cubic. Subscripts I and II are used to denote normal
(oil in water) or reversed (water in oil) topology phases. A Greek
subscript is used to denote the chain conformation: c for
crystalline, b for ordered gel-like, a for liquid-like, ab for
coexisting gel- and liquid-like regions.

3.1. The reversed cubic liquid crystalline phase

The name cubic comes from the fact that its X-ray diffraction
pattern shows cubic symmetry (Larsson et al., 2006; Seddon and
Templer, 1995). The cubic liquid crystalline phases exhibit the most
complex spatial organization of all known liquid crystalline phases.
There are two distinct families of cubic phases: bicontinuous
(based on underlying periodic minimal surfaces) and micellar
(based on complex packings of discrete micellar aggregates). Both
types may be normal (oil-in-water) or inverse (water-in-oil).
The general structure principle of the bilayer curvature in
bicontinuous cubic phases could be described by the infinite periodic
minimal surface (IPMS), whereas its dynamic structure could be Fig. 2. Schematic representations of the various crystals, liquid crystal and fluid
better described by the nodal surface. The bilayer in a cubic phase is phases identified in the temperature–composition phase diagram of glycerol
far from being static, as a minimal surface (Larsson, 2000). monooleate. Adopted from (Qiu and Caffrey, 2000).
572 S. Milak, A. Zimmer / International Journal of Pharmaceutics 478 (2015) 569–587

common, often requiring only small changes in composition (or Larsson et al., 2006; Seddon and Templer, 1995). This bilayer
temperature). conformation is formed when layers of water interpenetrate polar
The gyroid (G), Ia3d structure consists of two interwoven yet heads groups. The bilayer thickness is 10–30% less than twice the
unconnected chiral networks of water/lipid cylinders, connected length of an “all-trans” non-polar chain, and the water layer
coplanarly three by three and separated by the G-minimal surface. thickness is between 1 and 10 nm if the water content is between
As in the other minimal surface structures, the two continuous 10 and 50% in weight. Lamellar liquid crystalline phases are less
water compartments, separated by one continuous lipid bilayer, viscous than the hexagonal liquid crystalline phases despite the
are congruent and have no contact with one another. In the G- fact that they contain less water. This is described by the parallel
surface type of bilayer structure, the water channels between the layers which slide over each other with relative ease during shear.
structure units follow a helical network, with three connections/ The a subscript refers to the molten chains in this phase. Like all
openings between each unit. anisotropic phases, lamellar liquid crystalline phases exhibit
The structure of diamond (D), Pn3m consists of two interwoven distinct optical textures, when confined in thin slabs between
tetrahedral networks of water channels arranged on a double- crossed polarizers and viewed through an optical microscope.
diamond lattice, separated by the F-minimal surface. Typically, the texture is “streaky” or mosaic-like and resembles the
The third bicontinuous cubic phase, Im3m has orthogonal marbling in freshly cut steak. Alternatively, lamellae can eradicate
network of water channels connected six-by-six, and separated by all edges by folding into vesicles – essentially spherical globules.
the P-minimal surface. With six connectivities, the P-surface These are typically multiwalled (liposomes), exhibiting character-
structure in the glycerol monooleate system is formed only when istic “maltese cross” textures in the optical microscope.
the water compartments are expanded by an amphiphile, whereas
successive reduction of the aqueous volume forms first the D- 3.4. Intermediate phases
surface with four connectivities, and finally the G-surface structure
with three connectivities. A variety of other intermediate phases have been proposed in the
The first well-established example of a cubic phase composed scientific literature over the years such as novel arrays of meshes,
of a packing of discrete inverse micelles is the phase Fd3m. The sponges and hybrids (Hyde, 2001; Seddon and Templer, 1995).
Fd3m structure has two types of aggregates, both are quasi- Certain surfactant systems form highly swollen lamellar phases,
spherical but of different sizes. There are 8 of the larger and 16 of which may transform upon dilution to a sponge phase (L3)
the smaller inverse micelles per unit cell. (Drummond and Fong, 1999; Engström et al., 1998; Kulkarni
Cubic liquid crystalline phases are extremely viscous (some- et al., 2011). This phase is essentially a disordered version of the
times termed “ringing gels” in the older literature). They are even bicontinuous cubic phases whose interface is highly flexible. Also,
more viscous than the hexagonal phases. The high viscosity is a thermal excitations lead to the breakdown of the long range order of
result of the lack of shear planes within the structure that would the channel network so that the interface is no longer arranged on a
allow a sliding movement. The arrangement of the molecular lattice. Sponge phases are characterized by flow of birefringence
aggregates in cubic phases (cubic symmetries) means that they are (giving anisotropic optical textures), yet they are isotropic at rest.
optically isotropic and do not display optical textures (Collings and They often form at high (water) dilution, usually in regions of the
Hird, 1997; Lynch and Spicer, 2005). As with other liquid crystalline phase diagram intermediate to lamellar and bicontinuous cubic
systems, e.g., reversed hexagonal phase and reversed micellar mesophases. Among many intermediate phases reported, more
cubic phase, the cubic phase under full hydration conditions is work needs to be done in order to sort out these phases.
physically stable upon contact with excess water (de Campo et al.,
2004). Except glycerol monooleate, this was shown on the example 4. Phase diagram of glycerol monooleate/water
of other lipids, such as monolinolein (de Campo et al., 2004) and
monoeladin (having the same molecular weight as glycerol A phase diagram presents the system behavior in thermody-
monooleate, but different molecular shape) (Yaghmur et al., namic equilibrium, when the system is in the lowest state of free
2008, 2012a). energy (Chernik, 1999; Larsson, 2009; Larsson et al., 2006; Leser
et al., 2006; Qiu and Caffrey, 2000).
3.2. The reversed hexagonal liquid crystalline phase There is a “natural” sequence in which the various possible fluid
phases occur, determined by the average mean curvature of the
The reversed hexagonal liquid crystalline phase has a molecular polar–nonpolar interface (Seddon and Templer, 1995). Water
aggregate ordering which corresponds to a hexagonal arrange- content and temperature present the primary variables for binary
ments (Collings and Hird, 1997; Hyde, 2001). It consists of a dense lipid/water systems. The phase diagram of glycerol monooleate/
packing of cylindrical micelles, arranged on a 2D hexagonal lattice. water shows that at 37  C (pure monoolein melts at 36  C) and in
Water is contained within the cylindrical reversed micelles which the presence of a small amount of water, glycerol monooleate
have a diameter of 1–2 nm. The remaining space is occupied by the forms reversed micelles (L2), characterized by an oily texture
non-polar chains which overlap to leave the cylinders much closer (Fig. 3). Adding more water, a mucous like system is formed that
together than in the normal hexagonal phase. Usually they contain corresponds to the lamellar phase. In general, there will be a
30–60% water by weight, and despite this high water content the tendency for the monolayer to curve, either towards the water
phase is very viscous. This anisotropic phase is of intermediate region or towards the hydrocarbon chain region, depending on
viscosity to discrete micellar and bicontinuous cubic phases. By whether the interfacial tension is balanced primarily by the chain
optical polarizing microscopy, it is identified by a characteristic pressure or the headgroup pressure, respectively. By adding more
“fan” textures, due to focal conic domains of columns. water (20–40%), a large isotropic cubic liquid crystalline phase G
(gyroid, Ia3d) is formed. At more than 40% of water, the cubic liquid
3.3. The lamellar liquid crystalline phase (La) crystalline phase D (diamond, Pn3m) exists which is in equilibrium
with pure water. Glycerol monooleate is a unique lipid in
The lamellar structure makes the basic building block of all exhibiting a wide region with cubic structures in the phase
biological membranes (Garti et al., 2012). Lamellar phase consists diagram, both in composition and in temperature (including the
of planar lipid bilayers stacked in a one-dimensional lattice room temperature). At the temperature above 80  C the hexagonal
separated by layers of water (Collings and Hird, 1997; Hyde, 2001; phase appears, which exists in equilibrium with water as well.
S. Milak, A. Zimmer / International Journal of Pharmaceutics 478 (2015) 569–587 573
[(Fig._3)TD$IG]
parameter, cpp (or critical packing parameter) (Engström, 1990;
Engström and Engström, 1992; Hyde, 2001; Kaasgaard and
Drummond, 2006).
V
cpp ¼ (1)
al
where V: molecular volume; l: molecular length; a: effective (or
hydrated) cross-sectional area of the polar head group.
If an amphiphile is an aggregate and can be mimicked by a
cylinder, then cpp = 1. The amphiphiles with cpp near 1 aggregate
with a planar interface, as in a lamellar phase. If cpp is far from 1,
then 2 cases are possible. For the amphiphilic molecules with a
large polar head group area, cpp is less than 1 (al > V) and these
molecules will aggregate to the normal type in water, such as
micelles of spherical and cylindrical shape. If the polar head group
area of the amphiphile molecule is small, the cpp is larger than 1
(al < V) and inverted micelles are formed.

Fig. 3. Temperature–composition phase diagram of the monoolein/water system


(up to 50 wt% water). The phases are: Lc – crystal lamellar, La – lamellar, Ia3d – 5.1. Introduction of guest molecules in the reversed cubic phase
gyroid reversed bicontinuous cubic, Pn3m – primitive reversed bicontinuous cubic,
HII – reversed hexagonal and FI – reverse micelles isotropic fluid phase. Adopted One of the first substances investigated in the glycerol
from (Qiu and Caffrey, 2000).
monooleate cubic liquid crystalline phase was lysozyme (Ericsson
et al., 1983). The same research group revealed that lysozyme was
A compilation of lipid phase diagrams has been published and located in the water channel system. Lysozyme kept its native
databases of lipid transition temperatures and enthalpies are given structure and increased the lattice dimensions. Investigating other
by Caffrey et al. (1991). Qiu and Caffrey (2000), described the proteins, such as a-lactalbumin, bovine serum albumin, myoglo-
glycerol monooleate–water phase diagram at temperatures below bin, pepsin to form the cubic phase, Ericsson et al. (1983), found
20  C, where there exists a complicated behavior involving out that high apparent net charge repulsion between the protein
metastable phases. molecules favors the creation of the cubic glycerol monooleate–
Glycerol monooleate can be obtained in a highly pure form. protein–water phase. Formation of the cubic phase is favored by an
However, in most studies in the literature, commercial mono- isoelectric point far from pH 7 in a salt-free solution (by high
glycerides are used, such as Dimodan U, Monomuls, Myverol. electrostatic repulsive forces). Liquid crystalline lipid–protein–
Commercial monoglycerides contain different monoglycerides, water phases are formed only when there is a possibility for ionic
diglycerides and free fatty acids, with the dominant component – interaction between lipid and protein molecules. In addition, the
monooleate. The phase diagram of such a mixture behaves like that same research group also investigated the incorporation of
of a pure component. different peptides and aminoacids: desmopressin, lysine, vaso-
pressin, somatostatin and renin inhibitor into the cubic phase of
5. Introduction of guest molecules in the liquid crystalline glycerol monooleate/water (Ericsson et al., 1991). They found out
phases that these peptides could be incorporated into the cubic phase up
to 5–10% w/w. Above the certain concentrations of the water
During the last few decades glycerol monooleate liquid soluble oligopeptides, the phase transformation was noted from
crystalline bulk phases have been investigated as an interesting the cubic phase to the lamellar phase which was explained by
drug delivery matrix for both conventional and peptide/protein electrostatic repulsion at the glycerol monooleate–water interface
drugs (Table 1). This is connected with their high solubilization caused by the peptide. For desmopressin, they found that its
capacities for hydrophilic, lipophilic and amphiphilic guest diffusion coefficient in the cubic phase at 40  C, D = 0.24  1010
molecules, abilities to protect molecules against hydrolysis or m2 s1, is about a factor 9 smaller than in water at 25  C,
oxidation and to keep peptide/protein in their native conforma- D = 2.25  1010 m2 s1.
tion. Engström (1990) and Engström and Engström (1992), investi-
All additives, more or less significantly, influence the phase gated the glycerol monooleate/water system (cubic phase) with
behavior. After solubilization of a certain “critical” amount of lidocaine HCl. In the example of lidocaine HCl, the cubic phase was
additive, a phase transition is induced. Each additive affects the transformed into the lamellar liquid crystalline phase. However,
interfacial curvature of the lipid bilayer in a different way. when the base form of lidocaine is added, transition of the cubic
Generally, lipophilic additives induce a transition from a reversed phase to reversed hexagonal liquid crystalline phase occurs. With
bicontinuous cubic to reversed hexagonal phase, i.e., the mean the same amount of the salt and lidocaine base, the cubic phase
interfacial curvature becomes more negative. However, more persisted. Lidocaine in both forms and the interfacial curvature
hydrophilic additives induce the transition from the reversed changes in the opposite direction in relation to the curvature of the
bicontinuous cubic to the lamellar, i.e., the mean interfacial cubic phase participate in the lipid aggregation. This was explained
curvature changes towards zero. The changes in the molecular by the packing concept of amphiphilic molecules (cpp). Engström
geometry are used to explain phase transitions as well as intercubic and Engström (1992), assumed that cpp for lidocaine HCl is less
transformations, in lipid–water systems. The self-assembly of lipid than 1.2 and for the base form is larger than 1.2. The cubic phase is
molecules can be described by the use of a dimensionless packing preserved by cancelling the deviations of the cpp valued from
parameter (Engström, 1990; Engström and Engström, 1992; Fong 1.2 when the base and salt forms are mixed in roughly equal
et al., 2012; Hyde, 2001; Kaasgaard and Drummond, 2006). The amounts.
concept of the packing parameter is very useful for qualitative/ Wyatt (1992), incorporated drugs of different size (Mr) and
semiquantitative considerations. According to this concept, the solubility (vitamin E TPGS, hydroxychloroquine sulfate, diclofenac
shape of molecules is defined as the dimensionless packing sodium, aspirin), into the cubic phase (glycerol monooleate, water
574 S. Milak, A. Zimmer / International Journal of Pharmaceutics 478 (2015) 569–587

Table 1
Studies done incorporating different molecules in glycerol monooleate liquid crystalline phases.

No. System investigated Parameters studied Characterization Refs.


Techniques/analyses
1 Glycerol monooleate/water/lysozyme Ternary phase diagram, thermal stability of the Polarized light microscopy, small- (Ericsson
protein structure in a cubic glycerol monooleate/ angle X-ray scattering (SAXS), et al., 1983)
water phase differential scanning calorimetry
(DSC)
2 Glycerol monooleate and monoeladin/NaCl solution Dynamics and mechanism of the various Static X-ray diffraction, timeresolved (Caffrey,
(0–5 M), at 20–120  C thermotropic phase transitions X-ray diffraction 1987)
3 Glycerol monooleate/water/desmopressin, lysine Interaction of peptide with the cubic phase, self Polarized light microscopy, nuclear (Ericsson
vasopressin, somatostatoin, renin inhibitor diffusion, in vitro and in vivo release, enzymatic magnetic resonance (NMR) et al., 1991)
(subcutaneous or intramuscular depot for extended degradation in simulated intestinal fluid
peptide release)
4 Glycerol monooleate/water/lidocaine, lidocaine HCl Effect of lidocaine on the phase behavior Polarized light microscopy, SAXS (Engström
and
Engström,
1992)
5 Glycerol monooleate/sesame oil/metronidazole Phase diagram, in vitro drug release DSC, viscosimetry, SAXS, polarized (Norling et al.,
benzoate – suspension upon contact with gingival light microscopy 1992)
fluid transforms into reversed hexagonal phase
(periodontal)
6 Glycerol monooleate/water(35% w/w)/vitamin E In vitro drug release / (Wyatt, 1992)
TPGS, hydroxychloroquine sulfate, diclofenac
sodium, aspirin
7 Glycerol monooleate/glycerin/AG337 In vitro drug release DSC (Longer et al.,
(antitumorogenic substance); oral administration 1996)
8 Glycerol monooleate/water/cytochrome c Interaction between cytochrome c and glycerol SAXS, Fourier transform infrared (Razumas
monooleate cubic phase spectroscopy (FTIR), DSC, et al., 1996a)
electrochemical studies
9 Glycerol monooleate/water introducing Phase diagram DSC, SAXS, Raman scattering (Razumas
distearoylphosphatidylglycerol and lysozyme (5%, et al., 1996b)
7%, 8% w/w)
10 Glycerol monooleate/water Temperature–composition phase diagram SAXS (Briggs et al.,
1996)
11 Glycerol monooleate/water/glucose oxidase, Evaluation of glycerol monooleate as biosensor, Holographic laser interferometry, (Nylander
ceruloplasmin diffusion coefficient NMR, chronoamperometry et al., 1996)
12 Glycerol monooleate/water/chlorpheniramine Swelling kinetics, in vitro drug release Polarized light microscopy, DSC (Chang and
maleate, pseudoephedrine HCl, propranolol HCl Bodmeier,
1997c)
13 Glycerol monooleate/water/chlorpheniramin In vitro drug release, surface tension measurements, Polarized light microscopy (Chang and
maleate, diltiazem HCl, propranolol HCl, absorption of drug to the glycerol monooleate Bodmeier,
pseudoephedrin HCl, phenylpropanolamine HCl, 1997b)
theophylline anhydrous
14 Glycerol monooleate/water/chlorpheniramin Effect of dissolution media and additives in the Polarized light microscopy (Chang and
maleate, guaifanesin, propranolol HCl (oral) monoolein phases on the water uptake, in vitro drug Bodmeier,
release 1997a)
15 Glycerol monooleate/water/propantheline bromide, Phase diagram, swelling, in vitro drug release Polarized light microscopy (Geraghty
oxybutynin HCl (vaginal application) et al., 1996)
16 Salbutamol sulphate, nicotine (transdermal Passive and electrically assisted transport / (Carr et al.,
delivery) 1997)
17 Glycerol monooleate/water/chlorpheniramine Transformation from the lower viscosity phases Polarized light microscopy (Chang and
maleate, propranolol HCl (parenteral application) (lamellar or isotropic solution) to cubic–phase Bodmeier,
diagram, in vitro drug release 1998)
18 Glycerol monooleate and monolinolein/water/ In vitro mucoadhesive properties, influence of drug Mucoadhesion: “flushing” (Nielsen
miconazole isosorbide mononitrate, indometacin, on mucoadhesion bioadhesion test system and et al., 1998)
prochlorperazine tensiometric method; polarized light
microscopy
19 Glycerol monooleate/water/cefazolin, cefuroxim Stability (against hydrolysis and oxidation), assay / (Sadhale and
and degradation products at 22  C, 37  C and 50  C Shah, 1998)
20 Glycerol monooleate/water/insulin Integrity of the secondary structure, physical Circular dichroism (CD) (Sadhale and
stability, optical density, aggregation profile Shah, 1999b)
21 Glycerol monooleate/water/insulin In vitro release, biological activity of insulin (rat) Polarized light microscopy (Sadhale and
Shah, 1999a)
22 Glycerol monooleate/water/ubiquinone-10 Electrochemical investigations SAXS, FTIR, cyclic voltammetry (Razumas
technique et al., 1998)
23 Lipid–water phases Crystallographic study of cubic lipid systems Freeze-fracture electron microscopy, (Delacroix,
quantitative image processing 1998)
24 Glycerol monooleate/water liquid crystal systems Phase behavior, dielectric behavior Hot stage polarizing microscopy, (He and Craig,
with 15%, 20%, 30%, 35% w/w water dielectric spectroscopy 1998b)
25 Glycerol monooleate/water liquid crystal systems Phase behavior, dielectric behavior Hot stage polarizing microscopy, (He and Craig,
with 10%, 22%, 30% w/w water dielectric spectroscopy 1998a)
26 Glycerol monooleate and polymerizable 1,2- Phase investigation – structure, diffusion coefficient Polarized light microscopy, NMR, (Srisiri et al.,
diacylglycerol SAXS 1998)
27 Glycerol monooleate/water Phase behavior with polar solvents: propylene Polarized light microscopy, SAXS (Engström
glycol, dimethylsulfoxide, polyethylene glycol, et al., 1998)
ethanol
S. Milak, A. Zimmer / International Journal of Pharmaceutics 478 (2015) 569–587 575

Table 1 (Continued)
No. System investigated Parameters studied Characterization Refs.
Techniques/analyses
28 Glycerol monooleate/water/lidocain HCl Max. loading, drug compatibility in the sponge / (Alfons and
phase, diffusion coefficient, in vitro drug release Engstrom,
1998)
29 Glycerol monooleate/water/ubiquinone-10 Solubilization of ubiquinone-10 in the glycerol SAXS, polarized light microscopy (Barauskas
monooleate/water system (86% glycerol et al., 1999)
monooleate)
30 Glycerol monooleate/nicotine Effect of glycerol monooleate as bioadhesive DSC, SAXS, MTT toxicity assay (Dash et al.,
substance in the suppositories release/flux/rectal 1999)
delivery, in vitro drug release, caco-2 cell
permeability studies
31 Glycerol monooleate/water/clomethiazole, Drug partition into the lipid bilayers SAXS (Engström
lidocaine, prilocaine, 4-phenylbutylamine et al., 1999)
32 Glycerol monooleate/water/[D-Ala2, D-leu5] Swelling of matrices, in vitro drug release Polarized light microscopy (Lee and
enkephalin (buccal-adhesive peptide delivery Kellaway,
system) 2000)
33 Glycerol monooleate/water with oleic acid, In vitro drug release, in vivo drug release (rat) / (Malonne
propylene glycol, phospholipids or surfactants/ et al., 2000)
tramadol HCl (subcutaneous, intramuscular or
intrathecal injections)
13
34 Glycerol monooleate/diglycerol monooleate/water Microstructure C NMR, polarized light microscopy, (Pitzalis et al.,
SAXS, rheology 2000)
35 Glycerol monooleate/water/tricosane Curvature elastic properties of glycerol monooleate SAXS, volume measurements by DMA (Vacklin et al.,
in water (bending elasticity), structural and 2000)
energetic analysis
36 Glycerol monooleate/water Structure, stability and transformation of the Pn3m Synchrotron X-ray diffraction (Pisani et al.,
phase under pressure 2001)
37 Glycerol monooleate/water/sodium decanoat, Influence of drug of different polarity on phase SAXS, NMR, polarized light (Caboi et al.,
decanoic acid, dodecanoic acid, acetyl salicylic acid, behavior, stability microscopy 2001)
retinol, 1-adamantanamine
38 Fluorine labeled poly(amidoamine) dendrimer, Diffusion of globular macromolecule-synthesized NMR (Jeong et al.,
synthesized by Michael reaction water-soluble dendrimer in cubic phase 2002)
39 Glycerol monooleate/maleimide(triethylene glycol) Mechanism of formation under physiological Synchrotron SAXS (Angelova
ether lipid/water system loaded by immunoglobulin hydration conditions (excess water and appropriate et al., 2003)
Fab fragments, whole IgG, albumin, human salt concentration and pH) and structural
transferrin, fibrinogen parameters
40 Glycerol monooleate/n-octyl-b-D-glucopyranoside/ Thermal behavior SAXS (Persson et al.,
water 2003)
41 Glycerol monooleate/water/pro drug 5- Photodynamic activity, stability Fluorescence spectroscopy (Turchiello
aminolevulonic acid, its ester derivatives, chlorine et al., 2003)
compounds (m-THPC) (topical application in
photodynamic therapy)
42 Incorporation of cytochrome c into glycerol Topology of the mesophase, phase behavior of the SAXS, FTIR (Lendermann
monooleate cubic mesophases dependent on lipid system, the lipid conformational states, and Winter,
concentration, temperature and pressure hydration properties 2003)
43 Concentrated emulsions and cubic phases based on In vitro drug release, partition coefficient, phase SAXS, rheology, interfacial tension (Fa et al.,
fluorinated and hydrogenated oils and surfactants diagram, interfacial tension measurements 2004)
with caffeine
44 Glycerol monooleate, sponge phase with 2-methyl- Influence of detergents, drugs, lipids on the sponge Polarized light microscopy (Ridell et al.,
2,4-pentanediol phase 2003)
45 Lauric acid, monolaurin, simulated endogenous In vitro drug release, absorption in rat model SAXS, polarized light microscopy (Kossena
intestinal fluid et al., 2004)
46 Floating gastroretentive system loaded by Effect of PEG 4000, PEG 10000 and stearic acid on Gamma scintigraphy (Kumar et al.,
chlorpheniramine maleate, diazepam floatability, in vitro drug release 2004)
47 H2O2/glycerol monooleate (topical disinfected gel In vitro release, adhesiveness of cubic phase DSC, polarized light microscopy, SAXS (Kim et al.,
for a wounded skin) 2004)
48 Glycerol monooleate/water liquid crystal systems Influence of cyclosporine A on the phase structure Rheology, dielectric spectroscopy (Bonacucina
with 10%, 22%, 30% w/w water, incorporated et al., 2005)
cyclosporine A (1% w/w)
49 Floated system loaded by acid-labile enzyme – Water uptake, in vitro drug release, proteolytic Polarized light microscopy, gamma (Shah and
serratiopeptidase activity scintigraphy Paradkar,
2005)
50 Glycerol monooleate/water and different Phase diagram, morphology, topology of liquid Polarized light microscopy, SAXS, (Mezzenga
hydrophilic mono-, oligo-, and polysaccharides crystalline structures oscillatory shear rheometry, shear et al., 2005)
rheology, molecular dynamics
simulations
51 Glycerol monooleate/water (30%)/iopamidol Viscoelastic properties/structure Rheology, SAXS (Shui et al.,
2005)
52 Glycerol monooleate/water (20% w/w)/cytochrome Phase behavior as a function of temperature, Synchrotron X-ray small-angle (Kraineva
c (0.2–10% w/w) pressure and protein conc., kinetics of various lipid diffraction, time-resolved SAXS et al., 2005)
mesophase transformations
53 Glycerol monooleate/water/salicylic acid Influence of drug on the liquid crystalline phase, Polarized light microscopy (Lara et al.,
effects of swelling and drug loading on the release 2005)
mechanism; structure morphology, swelling
kinetics, in vitro drug release
54 Glycerol monooleate and tryptophan/lysozyme/ In vitro release, partition coefficient SAXS, CD, electrophoresis (Clogston and
ovalbumin, apo-ferritin, cytochrome c, DNA Caffrey, 2005)
576 S. Milak, A. Zimmer / International Journal of Pharmaceutics 478 (2015) 569–587

Table 1 (Continued)
No. System investigated Parameters studied Characterization Refs.
Techniques/analyses
55 Glycerol monooleate used as penetration enhancer In vitro permeation studies / (Lopes et al.,
for the topical delivery of cyclosporine A 2005)
56 Cubic phase (glycerol monooleate and phytantriol) Photodynamic activity Polarized light microscopy, (Bender et al.,
incorporated d-aminoluvulinic acid and its methyl fluorescence spectroscopy 2005)
ester, topical administration
57 Glycerol monooleate/water Transformation from cubic gyroid to cubic diamond SAXS (Rappolt,
phase 2006)
58 Glycerol monooleate (63% w/w)/aqueous solution Hydration dynamics of confined water in aqueous Femtosecond-resolved fluorescence (Kim et al.,
containing 4 mM tryptophan–alkyl ester (37% w/w) nanochannels dynamics 2006)
59 Glycerol monooleate/limonene oil/water ternary Structure and viscoelastic properties of the Fd3m Polarized light microscopy, SAXS, (Pouzot
system phase shear rheology, turbidimetry, density et al., 2007)
measurements
60 Biocathodes and bioanodes for electrochemical Applicability of the matrix for holding enzymes: / (Nazaruk and
sensing and biofuel cells glucose oxidase, pyranose oxidase, lactase Bilewicz,
2007)
61 Glycerol monooleate/water/anionic and cationic Effect of anionic and cationic peptides on the SAXS (Yaghmur
peptides stability of Pn3m phase of the glycerol monooleate/ et al., 2007)
water system
62 Glycerol monooleate/oleyl glyceride/0.1 M HCl and In vitro drug release, oral bioavailability in rats Polarized light microscopy (Boyd et al.,
cinnarizine 2007)
63 Vit. K in: glycerol monooleate hexagonal phase, Phase diagram, in vitro skin penetration and / (Lopes et al.,
liquid vaseline, glycerol monooleate nanodispersion transdermal delivery 2007)
of hexagonal phase
64 Sodium salicylate in cubic phase: glycerol Surface tension measurement, in vitro drug release Polarized light microscopy, DSC (Choi et al.,
monooleate and hydrophobic polymer poly(N- 2007)
isopropylacrylamide)
65 Gentamicin sulphate/glycerol monooleate/water Moisture content, in vitro drug release, stability Spectrophotometry, GC, rheology, hot (Ouedraogo
cubic phase, bioresorbable bone implant for chronic stage microscopy, TGA, DSC, et al., 2008)
osteomyelitis determination of moisture content
(Karl Fischer), SAXS
66 Gentamicin sulphate/glycerol monooleate/water Biocompatibility and preliminary toxicity in mice Mira Plus analyses (clinical chemistry (Henschel
cubic phase, bioresorbable bone implant for chronic assays) et al., 2008)
osteomyelitis
67 Preconcentrate: metronidazole benzoate/ Water absorption mechanism (Enslin method), in Polarized light microscopy, rheology (Fehér et al.,
Cremophor EL(or RH40)/Miglyol vitro drug release 2008)
68 Cyclosporine A in glycerol monooleate/tricaprylin/ Effect of 3 dermal penetration enhancers: Polarized light microscopy, SAXS, (Libster et al.,
water phosphatidylcholine, ethanol, Labrasol DSC, rheology 2007)
69 Vit. E, glucose, Allura Red and FITC-dextran in cubic Diffusion coefficient, in vitro release, in vivo oral Polarized light microscopy, SAXS (Lee et al.,
and hexagonal phase prepared from glycerol absorption in rat 2009)
monooleate and phytantriol
70 Vit. E acetate, glucose in glycerol monooleate and Respond to different temperature, in vitro release Polarized light microscopy, SAXS (Fong et al.,
phytantriol cubic and hexagonal phase study, in vivo subcutaneous absorption study 2009)
71 Low viscosity precursor for cubic phase: In vitro drug release, viscosity, bioburden, sterility Polarized light microscopy, (Ahmed et al.,
oligonucleotides in glycerol monooleate/water/ sterilization methods 2010)
cosolvent
72 Radio-labelled glucose in glycerol monooleate cubic, In vitro drug release SAXS (Phan et al.,
hexagonal, micellar cubic crystalline phases and 2011)
inverse micelle
73 Bupivacaine/glycerol monooleate/medium chain Diffusion coefficient, effect of varying the lipid SAXS, HPLC (Yaghmur
triglycerides composition on nanostructure, in vitro drug release et al., 2012b)
74 Clonidine/glycerol monooleate/hyaluronic acid for Syringeability, in vitro drug release Rheology, DSC, TGA, polarized light (Réeff et al.,
parenteral administration (intraarticular) microscopy 2013a)
75 Clonidine/glycerol monooleate/hyaluronic acid/ Influence of sodium oleate and soybean oil in Rheology (Réeff et al.,
sodium oleate (or soybean oil) for parenteral formulation on the release properties (in vitro drug 2013b)
administration (intraarticular) release), syringeability
76 d-Aminolevulinic acid or methylaminolevulinate in In vivo study in mice Polarized light microscopy, SAXS, (Evenbratt
1-glyceryl monooleyl ether/aprotic solvent/water fluorescence spectrophotometry et al., 2013)

35%). All drugs (highly water soluble drugs of smaller molecular been reached where the glycerol monooleate intrinsically controls
weight such as hydroxychloroquine sulfate, diclofenac sodium, and the AG337 release through phase transitions (these are dependent
large molecules such as vitamin E TPGS) showed sustained release upon the dynamics of water uptake, 73% released in 24 h).
properties after incorporation into the cubic phase and were The effect of the lipid composition on the long-term stability of
compared with their solution. A small increase of the dissolution the enzymes, glucose oxidase and ceruloplasmin, entrapped in the
rate of hydroxychloroquine sulfate (chosen as example for a small glycerol monooleate/water cubic liquid crystalline phase was
molecule) was found and indicates that the size of the molecule investigated by Nylander et al. (1996). The lipid mixture, glycerol
influences the release rate. monooleate and phosphatidylcholine, 4:1 w/w, stabilizes the
The feasibility study of using the cubic liquid crystalline phases glucose oxidase incorporated (30% of its activity after 80 days
made of glycerol monooleate as a matrix for controlled release of for this mixture compared to only some rest activities after 20 days
an antitumorogenic substance, AG337, was done by Longer et al. for glycerol monooleate alone). Furthermore, they showed that
(1996). There was an initial release at the beginning (40% released dimensions of the water channels in the bicontinuous cubic phases
in the first 15 min. during the initial contact between the are in the same range as the size of a typical globular protein. The
formulation and an acidic release medium) until a condition has entrapment of protein itself depends on the interactions with the
S. Milak, A. Zimmer / International Journal of Pharmaceutics 478 (2015) 569–587 577

lipid bilayer and the physical dimensions of the space, i.e., the partition mostly at the lipid–water interface. Both drugs investi-
water channels available for protein. The important factors for the gated promoted the formation of the lamellar phase and showed
guest molecules release are the degree of swelling, the curvature of the sustained release over a period of 18 and 20 h. Geraghty et al.
the interfaces and the type of cubic liquid crystalline phases. (1996), concluded that the drug release profiles depend upon the
In addition the group of Bodmeier demonstrated the sustained solubility of the drug in the lipid base and the extent of partitioning
release of chlorpheniramine maleate and pseudoephedrine into the lipid bilayer.
hydrochloride from glycerol monooleate/water systems (Chang Furthermore, also the release and swelling behavior of glycerol
and Bodmeier, 1997b,c, 1998). The drug release follows the square- monooleate-based drug delivery systems were studied by Chang
root of time relationship during the first 12 h, indicating a and Bodmeier (1997a). The drug release and swelling behavior
diffusion-controlled drug release mechanism. Within 24 h, pseu- depended on the composition and purity of the lipid matrix and
doephedrine hydrochloride was completely released, however, the components of the gastrointestinal tract. In the same study it
some other water soluble drugs (chlorpheniramin maleate, was shown that the water uptake of a glycerol monooleate based
diltiazem HCl, propranolol HCl, phenylpropanolamine HCl, the- system is rapid and levelled off after 4 h (at both pH investigated
ophylline anhydrous) showed an incomplete release from the 1.2 and 7.4). Thus, the pH of the medium will not affect the type of
cubic phase. Similarly, Wyatt (1992), also investigated the release the swollen liquid crystalline phase.
properties of hydrophilic drugs incorporated into the glycerol The glycerol monooleate cubic phase is too viscous to be
monooleate matrix, and again, an incomplete release was obtained injected directly, either intramuscularly or subcutaneously. Con-
for drugs such as vitamin E, TPGS or aspirin. Considering the drug sequently, Chang and Bodmeier (1998), developed a low viscosity
absorption to the monoglycerides, and surface tension, Chang and glycerol monooleate parenteral delivery systems by addition of a
Bodmeier (1997b,c, 1998),),), concluded that an incomplete release third component, the drug itself, or by the addition of organic
of hydrophilic drugs from the glycerol monooleate systems was solvents. Chlorpheniramine maleate and propranolol HCl showed
due to the binding of the drug molecules to the glycerol that upon increasing the drug content, the cubic phase was
monooleate liquid crystalline phases. However, later in 2001, it transformed into the lamellar phase. Furthermore, chlorphenir-
was suggested that the binding of drug molecules to the glycerol amine maleate showed that by further increasing the drug content,
monooleate liquid crystalline phases is incorrectly referred instead a transformation into an isotropic phase was observed. However,
of a bilayer participation (Shah et al., 2001). Hence, the drug this seems to be not a general law, i.e., propranolol hydrochloride
incorporated into bilayers is a more appropriate explanation for did not show an isotropic solution phase with such further
the above mentioned incomplete drug release phenomenon. This increasing drug content (Chang and Bodmeier, 1998).
was attributed mainly to the specific drug interaction with The mucoadhesive properties of glyceryl monooleate and
surfactant components which generally could also be used for glyceryl monolinoleate were investigated in vitro by “flushing”
fine tuning of the release profiles (Drummond and Fong, 1999). bioadhesive test and a tensiometric method (Texture Analyzer) by
Investigating the drug release from glycerol monooleate (Nielsen et al., 1998). Tensiometric measurements showed that the
matrices containing the same amount of drug but different unswollen monoglycerides have the largest mucoadhesion,
amount of water (10–30%), Chang and Bodmeier (1997c), showed followed by the partly swollen lamellar phase and the fully
an increased drug release with increasing water content (10–30%). swollen cubic liquid crystalline phase. The values for the work of
Under these conditions the hydrophilic domain also increased, and adhesion were in the range 0.007–0.048 mJ cm2. Probably the
the drug release increased despite the rise in matrix viscosity. mechanism of mucoadhesion is unspecific and involves dehydra-
These differences occurred only within the initial dissolution tion of the mucosa.
phase, while for longer time periods nearly parallel drug release Incorporating cefazolin in the glycerol monooleate cubic phase,
curves were obtained. This is due to the cubic phase created during Sadhale and Shah (1998), presented that cefazolin was six-fold
the release later, irrespective of the initial water content. As the more stable in the cubic phase than in solution. For cefazolin
main parameters which are able to control the drug release from 50 mg/g incorporated in the cubic phase, the energy of activation
glycerol monooleate matrices, Chang and Bodmeier (1997c), was found to be 23.4 kcal/mol, whereas in solution, the energy of
emphasized: the surface-to-volume ratio, the drug loading and activation was 4.2 kcal/mol. Similarly, they showed that cefuroxim
the water content of the lipid matrix. Furthermore, the group of incorporated in cubic phase degraded significantly slower at 22  C
Bodmeier also demonstrated that there were no drug release (kd = 0.0013  0.00003 h1) than in solution conc. 200 mg/g (kd =
differences between glycerol monooleate matrices prepared from 0.0029  0.00003 h1), resulting a 2.2-fold slower degradation.
different sources (Myverol 18-99 produced by Eastman Chemical Thus, it was demonstrated that the cubic phase is able to be used as
Co., Kingsport, TN, GMOrphic-80 produced by Eastman Chemical stabilizer against hydrolysis and oxidation at RT (22  C) and at
Co., Kingsport, TN and Dimodan DGMO produced by Grindsted 37  C. The enhanced stability was connected with the lower
Products, Brabrand, Denmark). The release of the drug from mobility and reactivity of uniquely structured water in the cubic
glycerol monooleate bases is governed primarily by the type of liquid crystalline phase (Sadhale and Shah, 1998). Furthermore, the
swollen phase and the main component of the monoglyceride (also same authors investigated the glycerol monooleate self-assembly
affecting the melting point) (Chang and Bodmeier, 1997c). structure to prevent insulin from aggregating (Sadhale and Shah,
The addition of drugs into the glycerol monooleate matrices 1999a,b,b) In this study it was demonstrated that the cubic phase is
affected their phase and release behavior (Engström, 1990; able to protect insulin from agitation induced aggregation
Engström and Engström, 1992). It was shown that above a certain successfully with little effect on its biological activity (Sadhale
concentration, hydrophilic drugs (chloropheniramin maleate, and Shah, 1999a).
propranolol HCl) transformed the cubic phase into a lamellar A further investigation used ubiquinone-10 which was entrapped
phase, whereas lipophilic drugs (ibuprofen, propranolol) trans- in the reversed bicontinuous cubic phase of aqueous glycerol
formed the cubic phase into a reversed hexagonal phase (Chang monooleate and the cyclic voltammetry technique was used to
and Bodmeier, 1997b). investigate the redox activity of the ubiquinone-10 in such a system
Developing a potential vaginal delivery system based on (38.7% of water, 0.5% of ubiquinone w/w) (Barauskas et al., 1999;
glycerol monooleate/water liquid crystalline phases, Geraghty Razumas et al., 1998). Using the electrochemical method, the
et al. (1996), incorporated two amphiphilic drugs: propantheline diffusion coefficient of ubiquinone-10 in such a system was
bromide and oxybutynin HCl in this system, and proposed their determined to be 1.9  108 cm2/s. Ubiquinone-10 (in concentration
578 S. Milak, A. Zimmer / International Journal of Pharmaceutics 478 (2015) 569–587

lower than 0.5% w/w) has no effect on the glycerol monooleate surface activity and intermolecular interactions. Further, it was
bilayer thickness and swelling behavior of phases, but it promotes shown that the blood proteins do not destabilize the structural
thermotropic transition into the reversed hexagonal phase at a lower organization of the bicontinuous cubic media prepared with
concentration. This is due to the ubiquinone-10 partitioning into the glycerol monooleate and maleimide(triethyleneglycol) ether at
reversed hexagonal region where the glycerol monooleate chains room temperature and higher temperature as well, as they
probably should be stressed upon the phase transition. At the marginally influence the structural parameters.
concentration of ubiquinone-10 above 0.5% w/w, inside the initially A potentially new application of glycerol monooleate/water
homogenous phases a solid ubiquinone-10 phase appeared which system was investigated by Turchiello et al. (2003), to deliver pro-
could be due to ubiquinone-10 lateral diffusion in the glycerol drugs and photosensitizers such as 5-aminolevulinic acid for
monooleate bilayer (Barauskas et al., 1999). topical applications in photodynamic therapy. The gel formulation
Glycerol monooleate was also investigated as bioadhesiveness prepared (glycerol monooleate/water, 70/30, w/w) was able to
in the design of rectal delivery systems of nicotine (Dash et al., maintain the stability and photodynamic activity of the incorpo-
1999). Using a Caco-2 cell permeation test it was shown that the rated molecules.
nicotine flux in the formulation with glycerol monooleate is lower Further structural changes of the cubic system was reported by
in comparison to a nicotine solution. In this study this observation Persson et al. (2003). This study demonstrated that the cubic liquid
was explained by an additional diffusion barrier created by the crystalline phases are stable with small fractions of n-octyl-b-D-
adhesion of glycerol monooleate to the cell monolayer (Dash et al., glycopiranoside, while higher n-octyl-b-D-glycopiranoside con-
1999). centrations trigger a cubic-to-lamellar phase transitions. Interest-
A pH-dependent partitioning of a drug into a lipid bilayer in a ingly, both the Ia3d and Pn3m cubic structures could be in
cubic liquid crystalline phase was demonstrated by Engström et al. equilibrium with excess water in this ternary system (Persson
(1999). The equilibration time for such an experiment is controlled et al., 2003).
by experimental conditions such as agitation, temperature and Kraineva et al. (2005), and Lendermann and Winter (2003),
interfacial area between the cubic phase and the aqueous bulk. A demonstrated that the incorporation of cytochrome c into the
shorter equilibration time is obtained by enhancing drug diffusion glycerol monooleate cubic phase has significant effects on the
by heating, by increasing the interface between the cubic phase, structure and pressure stability of this system. At low concentra-
and the aqueous bulk, as well as by decreasing the amount of the tion of cytochrome c (<0.2% w/w), changes were found only in the
cubic phase (Engström et al., 1999). phase transition temperature and pressures. At 20% w/w water, the
Malonne et al. (2000), showed that glycerol monooleate–water small protein cytochrome c forms the cubic Ia3d with glycerol
and quaternary (oleic acid–phospholipid–glycerol monooleate– monooleate/water spontaneously. Upon pressurization, the
water) formulations have controlled drug release profiles which P4332 phase undergoes a phase transition to a cubic Pn3m phase,
were accelerated by surfactant adjunction. Pluronic1 and the pressure limit of the P4332 phase stability is shifted to
F68 accelerated the release in glycerol monooleate/water for- higher values with increasing protein content. In general, it was
mulations. Partial substitution of lipid with oleic acid showed a concluded that the physical properties of the cubic phase can be
slower drug release, but while increasing the concentration of oleic tuned by changing the water content, by adding charged lipids or
acid again a similar release profile of the glycerol monooleate/ surfactant, or by application of temperature and pressure
water system was observed. These changes are connected with the (Kraineva et al., 2005).
structural changes in the arrangement of the lipids (Malonne et al., Comparative caffeine release trials from concentrated emul-
2000). sions and cubic liquid crystalline phases based on fluorinated and
Further studies investigated the diffusion of large hydrophilic hydrogenated oils and surfactants were done by Fa et al. (2004).
molecules. As one example a fluorine labelled poly(amidoamine) The most controlled release pattern was obtained with the
dendrimer was encapsulated into the cubic phase (Ia3d symmetry) fluorinated-concentrated emulsion, whereas the rate of release
and its diffusion in the water channels was investigated by NMR was faster with the hydrogenated-concentrated emulsions. The
(Jeong et al., 2002). The hydrodynamic diameter of the fluorinated cubic phases showed an intermediary behavior.
dendrimer was determined with 32.6 Å and the diffusion coeffi- Glycerol monooleate matrices were also considered as potential
cient in the water channels of the cubic phase was found to be drug delivery system with gastroretentivity by Kumar et al. (2004),
1 1012 m2/s at 25  C, which was compared to the free diffusion because a glycerol monooleate system reduces its density upon
coefficient of the dendrimer in water with 1.42  1010 m2/s. From swelling and floats. This study presented that it is possible to
this value it was estimated that small globular proteins or modulate the floatability and the release profile from glycerol
molecules similar to the above mentioned dendrimer can diffuse monooleate matrices depending on the drug polarity and suitable
sufficiently enough within the stabilized cubic phase to achieve excipients (PEG, stearic acid) (Kumar et al., 2004).
modified release properties (Jeong et al., 2002). H2O2 was chosen as small polar compound and was incorpo-
More detailed in terms of the partition of large hydrophilic rated into the cubic phase as 12% solution (30% water in total) and
molecules Angelova et al. (2003), encapsulated proteins (immu- showed the prolonged release over 50 h (50% released in 10 h, more
noglobulin Fab fragments, whole IgG, albumin, transferrin, than 80% released in 55 h) (Kim et al., 2004). Furthermore, this
fibrinogen) in the glycerol monooleate matrix and investigated publication showed that the hydrolysis and the oxidation of
their structure under physiological conditions (excess water, glycerol monooleate by H2O2 lead to the breakdown of the cubic
appropriate salt concentration and pH) by SAXS. Glycerol phase (cubic phase stable for 56 days at 45  C when H2O2 was
monooleate was the main component of such a system and it incorporated) demonstrating a different approach to modulate the
was mixed with a second uncharged amphiphilic component, a release mechanism.
maleimide(triethylene glycol) ether lipid comprising a polar An acid-labile enzyme, seratiopeptidase, was used as a model
terminal group which is reactive to free SH groups. Despite of drug to design the glycerol monooleate cubic phase precursor in
many authors who previously assumed that water-soluble proteins stomach (Shah and Paradkar, 2005). The release from such in situ
are mainly located in the water channels of the bicontinuous cubic cubic phase transforming system was altered depending on
lattices, in this study, Angelova et al. (2003), assumed that the microenvironment and water uptake by the matrix. Magnesium
proteins are most likely located at the interface or associated to the trisilicate improved stability of the model drug and controlled its
surface of the interconnected cubosomal entities, due to their release from the glycerol monooleate matrix, while Gelucire
S. Milak, A. Zimmer / International Journal of Pharmaceutics 478 (2015) 569–587 579

incorporated into the matrix provided a delayed lag time (Shah and thermosensitive polymer is included in the water channels of the
Paradkar, 2005). gel and additionally hinders the diffusion of sodium salicylate.
Mezzenga et al. (2005), investigated the order-to-order Depending on the concentration there was no influence of the
transitions of lyotropic liquid crystalline phases formed by self- polymer on the temperature-dependent release at low concen-
assembled glycerol monooleate–water–polysaccharide systems. trations (i.e., 0.17%), however, induced by temperature, at higher
The presence of hydrophilic mono-, oligo-, and polysaccharides in values (40  C) an improved release resulted due to the thermally
the water domains of liquid crystalline phases resulted in a general enhanced diffusion of sodium salicylate (Choi et al., 2007).
decrease of the cubic-to-hexagonal transition temperature. A gentamycin sulfate implant (gentamicin–glycerol monoo-
Assuming that the sugar could fit within the water channels, this leate–water, 5–80–15) was prepared and its in vitro characteriza-
decrease was observed to be dependent on the (poly)saccharide tion was reported by Ouedraogo et al. (2008). Suitable drug release
concentration but independent of its molecular weight. For (over 3 weeks) and also acceptable drug stability (min. 10 months
isotropic bicontinuous cubic phases, monomeric sugars, such as at 2–8  C) were obtained. The same group confirmed the
glucose, shrink the lattice parameter of the structure without gentamycin implant’s potential as bioresorbable implant for the
inducing phase transitions. However, when a polymeric form of local treatment of chronic osteomyelitis (Henschel et al., 2008).
glucose was used, such as dextran, transitions from the gyroidal Other hydrophilic drugs, such as glucose, Allura Red and FITC-
Ia3d cubic phase to double diamond Pn3m cubic phases were dextrans, incorporated into the glycerol monooleate and phytan-
observed at well-defined molecular weights of the polysaccharide. triol cubic liquid crystalline phase (75/25% w/w) and hexagonal
These results were interpreted in terms of polymer sugars size liquid crystalline phase (prepared by phytantriol and vitamin E)
exclusion by the liquid crystalline phases water domains, as well as also showed a diffusion controlled release mechanism (Lee et al.,
different topologies of water channels (Mezzenga et al., 2005). 2009). For each drug, the release rate decreased as the matrix was
Investigating the release properties of salicylic acid from changed from glycerol monooleate cubic liquid crystalline phase to
glycerol monooleate/water matrices, Lara et al. (2005), demon- phytantriol cubic liquid crystal phase and, finally, to hexagonal
strated that the rate of drug release is controlled by the diffusion of liquid crystal phase. In addition, in vivo absorption rate of glucose
molecules through the system and it decreases with time due to an followed the same trend observed in the corresponding in vitro
extended distance that the drug must diffuse through the matrix to studies. It was demonstrated that there is an ability to tailor the
reach the exterior shell. It was demonstrated that salicylic absorption kinetics of hydrophilic drugs in vivo by the nanostruc-
acid incorporated into the glycerol monooleate/water matrices ture of the matrix materials used (Lee et al., 2009).
in the range up to 20% w/w has no effect on the phase behavior Further studies including large hydrophilic drugs described in
(Lara et al., 2005). situ cubic phase-forming monoglyceride drug delivery systems as
Significant efforts in understanding the kinetics and mecha- an interesting approach to achieve an extended release matrix for
nism of drug release from the cubic phase were undertaken by oligonucleotide drugs (Ahmed et al., 2010; Hatefi and Amsden,
Clogston and Caffrey (2005). This research group investigated the 2002). Ahmed et al. (2010) investigated mixtures of monoglycer-
transport properties of the cubic phase and described the diffusion ide, water and water–miscible cosolvents (ethanol, PEG 300, 2-
processes. The diffusion from mesophases was quantified with pyrrolidone, DMSO) as injectable in situ cubic phase-forming
respect to the size (from single amino acids to large proteins), formulations for parenteral administration with low viscosity. An
shape and charge of the diffusing species. Aqueous channel extended drug release was obtained, which followed the square
dimensions were adjusted by using lipids with different chain root of time kinetics. They investigated the bioburden of
characteristics (glycerol monooleate, monopalmitoolein and monoglyceride and precursor formulation, and suitable steriliza-
monovaccenin). Consequently it was shown that this will affect tion methods (Ahmed et al., 2010). The bioburden of commercially
the diffusion rate. Furthermore, they showed that interactions available monoglyceride and of the prepared formulations met
between the diffusing species and the lipid wall of the cubic phase USPXXIII requirements. Gamma irradiation and autoclaving were
are adjustable over a wide range and in a way that allowed a very shown as successful sterilization methods for monoglycerides,
precise release control. while autoclaving and aseptic filtration were the successful
The structural mechanisms were reported during the gyroid sterilization methods for in situ cubic phase-forming formulations.
Ia3d to diamond Pn3m cubic liquid crystal phase transition by In most cases the drug release from the inverted-type liquid
Rappolt (2006). While an unknown transient phase coexists with crystalline phases was investigated keeping the surface area
the cubic phase under non-equilibrium conditions, it vanishes as between the inverted-type crystalline phase and the release
the system approaches near equilibrium and the transition medium constant. To mimic the drug release and transport from in
proceeds by a direct, nearly isoareal, transformation. situ formed non-lamellar systems, the surface area between the
An application in the field of bioanalytics and engineering self-assembled system and the release medium should be variable.
science was presented by Nazaruk and Bilewicz (2007), who Yaghmur et al. (2012b), kept this surface variable by injecting low
revealed that the glycerol monooleate cubic liquid crystalline viscous stimulus-responsive precursors to a buffer in a dialysis cell.
phase is a convenient matrix for enzyme entrapment (glucose or Furthermore, the same research group also emphasized the
pyranose oxidases) and for its application in biocathodes and important role of pH and lipid composition on the self-assembled
bioanodes for electrochemical sensing and biofuel cells. In nanostructure and on the in vitro drug release profile.
addition, functionalization of bicontinuous cubic phases by the Described by a feasibility study performed to develop a clonidine
addition of charged short peptides was presented by Yaghmur et al. sustained release formulation for intraarticular administration,
(2007). These peptides are able to be used as anchors in the water/ Réeff et al. (2013a), found the most suitable formulation with 10%
lipid interface and they allow enhancing the loading capacity of w/w ethanol, 20% w/w propylene glycol, 7.5 mg of hyaluronic acid,
charged active molecules. 15% w/w water and 55% w/w glycerol monooleate. This solution upon
Glycerol monooleate cubic liquid crystalline phases containing a contact with a synovial fluid formed a viscous crystalline phase and
hydrophobically modified thermosensitive polymer poly(N-isopro- showed an in vitro release of clonidine for about 1 week (similar to a
pylacrylamide) were prepared by hydrating glycerol monooleate cubic reference formulation). Different excipients (sodium hyalur-
using a polymer solution (Choi et al., 2007). Sodium salicylate, used onate, propylene glycol, sodium oleate, purified soybean oil) were
as a model drug, showed a slower release than the cubic phase made incorporated in the same clonidine solution, in an attempt to sustain
from glycerol monooleate alone. It is assumed that this further the clonidine release (Réeff et al., 2013b). From these
580 S. Milak, A. Zimmer / International Journal of Pharmaceutics 478 (2015) 569–587

formulations the clonidine release was substantially reduced using Different conc. of glycerol monooleate in the formulation of
sodium oleate or purified soybean oil (50% release after 10 days) due cyclosporine A with propylene glycol have a different influence on
to the increased hydrophobicity of the system. This leads to a slower the delivery of cyclosporine A (Lopes et al., 2005). At 5% w/w,
and reduced water uptake and reduced clonidine mobility. glycerol monooleate increased only the transdermal delivery, at
Surprisingly, Fick’s second law of diffusion was used to describe 10% both the topical and the transdermal delivery were increased.
the clonidine release quantitatively. At higher concentration of glycerol monooleate (20–70%), the
Dermal photodynamic therapy using a glycerol monooleate topical delivery of cyclosporine A was still increased but its
cubic system was studied by Turchiello et al. (2003) and Bender transdermal delivery was substantially reduced. Due to their
et al. (2005), previously. Both authors demonstrated the lipophilic nature, glycerol monooleate and cyclosporine A are
advantages of a glycerol monooleate system: (a) stable system, likely to have a high affinity to each other. The interaction between
(b) improved release properties of d-aminolevulinic acid or the glycerol monooleate (at high concentration) and cyclosporine
methylaminolevulinate (comparing with standard ointments) A may result in drug retention in the skin (where glycerol
and (c) improved penetration into skin. To further evaluate its monooleate is better partitioned) (Lopes et al., 2005).
accurate penetration depth into skin, the same group, Evenbratt Oleyl glycerate has a very close structural relationship to
et al. (2013), prepared an instantly formed cubic formulation glycerol monooleate and forms a reverse hexagonal phase in excess
based on 1-glyceryl monooleyl ether and glycerol monooleate and water, providing an interesting point of differentiation with
investigated their opportunities for transdermal drug delivery. glycerol monooleate (which forms a bicontinuous cubic phase
Interestingly, both formulations, demonstrated the same effi- instead). According to its structure, oleyl glycerate (with no ester
ciency. bond) would be more resistant to lipolysis in comparison to
glycerol monooleate, and consequently, to form a more persistent
5.2. Introduction of guest molecules in the reversed hexagonal phase matrix for sustained release in the gastrointestinal tract. Boyd et al.
(2007), did oral bioavailability studies in rats using cinnarizin as a
To prepare physically and chemically stable formulations of model drug and glycerol monooleate and oleyl glycerate aqueous
metronidazole benzoate during storage, Norling et al. (1992), suspension, expecting that the oleyl glycerate and glycerol
developed a metronidazole benzoate suspension (water free monooleate formulations would form a reverse hexagonal phase
formulation) with sesame oil for periodontal administration. On and a bicontinuous cubic phase on exposure to excess fluids in the
contact with gingival fluid, this suspension undergoes transforma- gastrointestinal tract. It was confirmed that oleyl glycerate is less
tion to reversed hexagonal phase which has favorable sustained susceptible to hydrolysis by pancreatic lipase than glycerol
release properties compared to those from the cubic phase. Norling monooleate, and that the liquid crystal structures formed in
et al. (1992), also used sesame oil to improve the flow properties of excess water may have application as an oral sustained release
the glycerol monooleate and metronidazole mixtures by reducing delivery system (Boyd et al., 2007).
the crystallinity of glycerol monooleate or by adding sesame oil to Vitamin K chosen as an additional compound was incorporat-
lower the melting point. By the addition of 4% of sesame oil, the ed into a glycerol monooleate/water system (the reversed
melting point of the mixture decreases by 4  C to reach an eutectic hexagonal phase). This study showed an improvement in its in
temperature. As mentioned before, the release of metronidazole vitro skin penetration and transdermal delivery (porcine ear skin
from reversed hexagonal phase was slower (46.3% after 6 h) than mounted in a Franz diffusion cell) (Lopes et al., 2007). The
from the cubic phase (66.7% after 6 h). This is due to closed water hexagonal phase gel delivered 2 times more vitamin K to the
channels in the reversed hexagonal phase and more obstructed stratum corneum and 2.0–3.7 times more vitamin K to the
diffusion pathways than in the cubic phase. Thus the hexagonal (epidermis + dermis) than the control (vaseline solution of vitamin
phase was preferred because it slows down the diffusion of K) (Lopes et al., 2007).
dissolved drugs through the matrix. In addition to these results the reversed hexagonal phase was
Similarly to Norling et al. (1992), Malonne et al. (2000), revealed studied as a delivery system for cyclosporine A, (Libster et al.,
that oleic acid and phospholipids added to the mixture of 2007) revealed that solubilization of cyclosporine A alone has a
tramadol–glycerol monooleate–water decrease the drug release, destabilizing effect on the reversed hexagonal phase. This caused a
but further substitution with oleic acid and phospholipids resulted deterioration of the elastic properties of the system, leading to
in a drug release profile close to that of the native formulation. more liquid-like behavior and resulting in very short relaxation
These changes were attributed to the structural changes in the time. Trying to see the influence of drug permeation enhancers on
arrangement of the lipids. Interestingly, Pluronic F 68 proportion- the mesophases integrity, labrasol and ethanol showed a
ally accelerated the tramadol release (Malonne et al., 2000). destabilizing effect on the reversed hexagonal phase as well.
Small-angle X-ray scattering was used to determine the However, 20% of phosphatidylcholine improved the elastic
bending energy of glycerol monooleate in the hexagonal phase properties of these systems and increased the thermal stability
which is formed by tricosene in glycerol monooleate/water system of the liquid crystalline phases enabling to solubilize up to 6% w/w
(Vacklin et al., 2000). The transition to the hexagonal phase occurs cyclosporine A (Libster et al., 2007).
because of the packing stress in the hydrophobic regions of the Similar to Norling et al. (1992), a water free-lyotropic liquid
hexagonal phase which is reduced to levels where this phase is at a preconcentrate of metronidazole benzoate was prepared by Fehér
lower free energy level than the cubic phase. et al. (2008), but in the present study consisting of Miglyol 810 and
Incorporating different drugs (sodium decanoate, 1-adaman- different surfactants (Cremophor EL or Cremophor RH40) (1:4)
tanamine, decanoic acid, dodecanoic acid, acetyl salicylic acid, which spontaneously form lyotropic liquid crystalline phases in
retinol) into glycerol monooleate/water systems (Caboi et al., the aqueous environment during the administration into peri-
2001), the transition from cubic to reversed hexagonal phase was odontal pocket. Lamellar, hexagonal and isotropic gel phase were
noted by sodium decanoat, decanoic acid, dodecanoic acid. The identified with the addition of water. Cremophor EL absorbed
drugs affects the interfacial curvature of the lipid layer differently, significantly more water than Cremophor RH40 (during 2 h of
which in turn triggers transition to disparate phases. The glycerol trials) and showed lower inhibition zones (in vitro drug release by
monooleate hydrolysis plays a crucial role in the transition to the modified Kirby–Bauer disk diffusion method). The water absorp-
cubic-to-hexagonal phase with the release of free oleic acid that tion and the stiffness of the phase structure are evaluated as the
favors the reverse interfacial curvature (Caboi et al., 2001). most important factors in drug release (Fehér et al., 2008).
S. Milak, A. Zimmer / International Journal of Pharmaceutics 478 (2015) 569–587 581

The phase structure of liquid crystalline phases could be altered phase than the from the other liquid crystalline phases. The state of
by modifying the lipid packing, by differences in the lipids used water compartments, whether open or closed, has a great
and external factors such as pH, ionic strength, temperature, influence on the rate of drug release. The slowest release was
pressure and the presence of further additives (Fong et al., 2009; observed from the reversed micellar cubic phase, followed by
Yaghmur et al., 2009). This is used to design a stimuli responsive hexagonal and then by the inverse micellar phase (Phan et al.,
drug delivery system. The most commonly studied stimuli are 2011). The glucose release followed first-order diffusion kinetics
those that switch phases in response to changes in temperature and the diffusion coefficients for glucose were determined in the
and pH. Using glycerol monooleate and phytantriol systems, Fong bicontinuous cubic phase: D = 36  108 cm2 s1, in the reverse
et al. (2009) used the temperature to switch glucose release “off” in hexagonal phase: D = 1.7  108 cm2 s1, in the inverse micellar
the inverse hexagonal phase and “on” in the reversed cubic phase phase: D = 2.95  108 cm2 s1, and finally, in the micellar cubic
after subcutaneous injection of the matrix into rats. In this study phase: D = 0.12  108 cm2 s1. Different liquid crystalline phase
Fong et al. (2009), reported that the glucose diffusion is reversible structure contributes to the different release properties. Therefore,
when switching between the reversed hexagonal and cubic liquid the open channels in the reversed bicontinuous cubic phase
crystalline phases at temperatures above and below the physio- contribute to the fast release, while in the reversed hexagonal
logical temperature, demonstrating the potential of this system as phase a slow release is related to closed water channels (Phan et al.,
a “on demand” drug release delivery system (Fong et al., 2009). 2011).
Stimuli responsive to drug delivery systems could be useful in The inverse micellar cubic phase was investigated in the system
therapeutic situations when continuous drug absorption is not containing monoelaidin by synchrotron SAXS and WAXS (Yaghmur
desirable e.g., for toxicity reasons and pulsatile drug release on et al., 2012a). Investigating the effects of variations in lipids
demand. (monoelaidin/glycerol monooleate, monoelaidin/oleic acid, mono-
elaidin/elaidic acid) and temperature on the self-assembled
5.3. Introduction of guest molecules in the reversed micellar cubic nanostructure of monoelaidin in excess water, it was shown that
phase between oleic acid and elaidic acid oleic acid is more efficient to
form an inverse micellar cubic phase (in perturbing the mono-
An inverse micellar cubic phase was discovered in 1992 by elaidin bilayer and inducing negative membrane curvature)
Luzzati’s group in a lipid extract from Pseudomonas fluorescence (Yaghmur et al., 2012a).
(Luzzati et al., 1992). The formation of the Fd3m cubic phase usually
needs the presence of at least two lipid components, one should be 5.4. Introduction of guest molecules in the bicontinuous sponge phase
very weakly hydrophilic (e.g., fatty acid, diglyceride, etc.). This
permits a partial segregation of the two lipid components between The phase diagrams of glycerol monooleate/water system with
the two types of micelles, with the less hydrophilic species locating differentpolarsolvents,suchasdimethylsulfoxide,propyleneglycol,
preferentially in the smaller, more strongly negatively curved polyethylene glycol and ethanol were prepared by Engström et al.
inverse micelles. (1998). All four systems showed one long but narrow region of
In the aqueous dispersion containing glycerol monooleate, oleic isotropic liquid sponge phase (L3). The extension of the narrow one-
acid and Pluronic F127, Nakano et al. (2002), revealed the phaseregionvarieswiththesolventbeingmuchlessforethanolthanfor
successive formation of the primitive type cubic, inversed theotherthreesolvents.Thebicontinuousspongephaseisformedasa
hexagonal and inversed micellar cubic phase by the increasing consequence of a subtle partitioning of the solvent between the
oleic acid fraction. This is ascribed to a change in the membrane glycerolmonooleateandaqueousdomaininthesystem,whichinturn
spontaneous curvature. Oleic acid provides a negative curvature at leadstotheslightlynegativeinterfacialmeancurvature.Thestructure
higher fractions, which is responsible for decreasing the lattice ofthespongephaseissimilartothereversedcubicphaseconsistingofa
constant in the primitive cubic phase and inducing the phase congruentlipidbilayerwithaslightlynegativelycurvedinterface.The
transition to reversed hexagonal and reversed micellar cubic phase differencebetweenthecubicphaseandthespongephaseisthestrong
(Nakano et al., 2002). This inverse micellar cubic phase was bilayer–bilayer correlation in the former, which gives rise to a long-
investigated by SAXS and cryo-TEM by Yaghmur et al. (2005), in the range order. In the sponge phase, this correlation has decreased
system containing monolinolein, water and tetradecane. The becauseofalargerseparationduetotheaddedsolvent(Engströmetal.,
inverse micellar cubic phase appears at a specific tetradecane/ 1998). It was found that water alone can not form the sponge phase.
monolinolein weight ratio and is situated between the inversed Consequently,aspongephaseoccursonlyinthepresenceofasolvent,
hexagonal and the isotropic liquid phase. The inverse micellar salt (Drummond and Fong, 1999; Engström et al., 1998) or dioleoyl-
cubic phase’s scattering curve shows more than seven peaks in the phosphatidylglycerol (DOPG) (Yaghmur et al., 2011a) with a distinct
characteristic ratio for a discontinuous micellar cubic phase tendency to form the interfacial and bilayer domain. These additives
(Yaghmur et al., 2005). will create a less negativelycurved interfacial regionand increase the
A bulk inverse micellar cubic phase of Fd3m structure was bendingflexibilityofthebilayer,whichbothareimportantparameters
obtained by adding a hydrophobic component, limonene oil, to the for the formation of the highly dynamical disordered sponge phase
binary system glycerol monooleate/water (the ratio of limonene oil (Yaghmur et al., 2011a).
to total lipids = 0.4) (Pouzot et al., 2007). The viscoelastic properties In the sponge phase prepared from propylene glycol and poly
of the Fd3m are similar to those of the bicontinuous cubic phases: (ethylene glycol) (water content 40 and 30% w/w), glycerol
diamond Pn3m and gyroid Ia3d. However, the Fd3m phase showed monooleate and lidocain, lidocaine as an amphiphilic drug
a complex set of slower relaxation mechanism (Pouzot et al., 2007). participates in the bilayer and acts on the interfacial curvature
More recently, Phan et al. (2011), investigated glycerol according to the amphiphilic packing concept (Alfons and
monooleate and phytantriol with an increasing hexadecane Engstrom, 1998). The interfacial curvature increases/decreases
content of excess water. At 4–25% w/w hexadecane glycerol depending on the salt/base status of the molecules. The quantity of
monooleate forms inverse micelles and at higher hexadecane the drug incorporated in the sponge phase depends not only on the
concentrations, 25–40% w/w hexadecane, glycerol monooleate form of lidocaine but also on the lipid content and the solvent used
forms the micellar cubic phase. Using glucose as a model in the sponge phase. The in vitro release of lidocaine from this
hydrophilic drug, it was presented that the drug release is sponge phase was significantly faster than from the cubic liquid
significantly faster from bicontinuous cubic liquid crystalline crystalline phase (Alfons and Engstrom, 1998).
582 S. Milak, A. Zimmer / International Journal of Pharmaceutics 478 (2015) 569–587

Further more, Ridell et al. (2003), demonstrated a reasonably Some other investigations of “optical textures” in a polarizing
good correlation between the water content of the sponge phase and optical microscope could be useful as, for example, lamellar liquid
the octanol/water partition coefficient for the solvent. The water crystalline phases can often be distinguished from hexagonal
content of the sponge phase can be increased considerably (up to liquid crystalline phase by microscopic techniques (both are
50%) by adding ionic compounds (such as sodium dodecyl sulphate, anisotropic).
cetyl trimethyl ammonium bromide) and the salts of amphiphilic
drugs (Ridell et al., 2003). It was also reported that the methyl ester of 6.2. X-ray diffraction
the d-aminolevulinic acid transformed the isotropic bicontinuous
sponge phase (made of glycerol monooleate/water/propylene X-ray diffraction is considered as the most powerful method for
glycol) partly (4–10% w/w methyl ester of d-aminolevulinic acid) structure determination in the solid state and lipid phase
or completely (13 and 16% w/w methyl ester of d-aminolevulinic identification (Dong and Boyd, 2011; Müller-Goymann, 2004).
acid) into an anisotropic lamellar phase, indicating that the methyl One of the difficulties encountered is preparing single crystals that
ester of d-aminolevulinic acid has a flattening effect on the bilayer are perfect enough to allow resolution of the reflections from
curvature (Bender et al., 2008). However, the addition of 16% (w/w) of different crystal planes. As long as these prerequisites are achieved
d-aminolevulinic acid did not show any effect on the sponge phase. this method is able to determine the exact position of all atoms
The water diffusion coefficient in the sponge phase was found to be except hydrogen atoms in the cell unit. Even when single-crystal
approximately 3.1–3.9  1010 m2 s1, compared to 5.3–5.7  1010 data are not available, it is still possible to derive the main feature
m2 s1 in relevant water/propylene glycol solutions or 2.3  109 of the molecular packing from the X-ray diffraction curves, which
m2 s1 in pure water. This reduction in the diffusion coefficient is a can always be obtained. This is due to the arrangement of the lipid
consequence of the microstructure (congruent glycerol monooleate molecules into layers, where the distance between hydrocarbon
bilayer) of the sponge phase and of the viscosity effect caused by chains can be derived from one region of the diffraction pattern
propylene glycol and the methyl ester of d-aminolevulinic acid (“short-spacings”) and the thickness of the layers can be obtained
(Bender et al., 2008). from another region (“long-spacings”). As the repetition distances
in the hydrocarbon chain direction are very long, small diffraction
6. Characterization of liquid crystalline phases angles should be recorded. So the small angle region identifies the
symmetry and long range organization of the phases, whereas the
The techniques mostly used in characterization of liquid wide angle region gives information on the molecular packing, or
crystalline phases (Demetzos, 2008; Dong and Boyd, 2011; Lynch short range organization of the phase.
and Spicer, 2005; Müller-Goymann, 2004,b; Sagalowicz et al., The enormous increase in X-ray intensity achieved by
2006a,b) are polarized light microscopy, small angle X-ray synchrotron sources has made time-resolved X-ray diffraction
scattering (SAXS) and differential scanning calorimetry studies analysis possible. This is particularly important in order to follow
(DSC) (Table 2). Some other known techniques which are getting the dynamics of the lipid self-assembly in water.
more and more interesting in liquid crystalline phases characteri- To gather more information on the energetic status and stability
zation are showing some significant advantages. For example, of lipid liquid crystalline phases, pressure was used as a suitable
nuclear magnetic resonance (NMR), Raman and Fourier transform thermodynamic variable during X-ray diffraction. Therefore, the
infrared spectroscopic studies (FT-IR) are able to resolve the inter- pressure effects on the structure and stability of the Pn3m
and intramolecular interactions in the three-dimensional liquid bicontinuous cubic phase in the glycerol monooleate–water
crystalline phases. system have been investigated using synchrotron X-ray diffraction
since more then 15 years (Pisani et al., 2001). In this study was
6.1. Polarized light microscopy observed a different phase behavior for the Pn3m sample prepared
in excess water and for the Pn3m samples prepared in less hydrated
The polarized light microscopy is the easiest way of character- conditions. In excess water, a phase transition from the Pn3m cubic
izing single-crystal quality and screening the phase properties of to the lamellar La phase at about 0.7 kbar was determined, whereas
lipid–water systems. Lyotropic liquid crystalline phases show a Pn3m to Ia3d cubic-to-cubic phase transition occurred when the
birefringence like real crystals (Müller-Goymann, 2004). Lack of external pressure increases up to about 1–1.2 kbar what was
birefringence means that a phase is cubic or a true liquid, whereas detected in the less hydrated samples. In a less dry glycerol
the birefringence texture is a characteristic of other liquid monooleate sample, a cubic Ia3d–lamellar La phase transition was
crystalline phases (lamellar, hexagonal). Birefringence can be detected at about 1.5 kbar. All destabilizing effects induced by
checked by observing whether the sample rotates the plane of pressure are strongly concentration dependent. The structure
polarized light polarization or not. If crossed polarization analysis of the Ia3d and Pn3m pressure dependence showed that
occurs which do not transmit the light, some transmission will the cross-sectional area at the lipid–water interface decreases as a
be visualized when the sample is placed between the polarizers function of pressure, whereas the monolayer thickness corre-
and the sample will be considered to be anisotropic and shows a spondingly increases. The crucial term describing the stability of
birefringence. However, as long as there is no transmission bicontinuous cubic phase is the curvature elastic energy. Pisani
under these conditions, the sample is isotropic and shows no et al. (2001), also showed that the glycerol monooleate interface is
birefringence. bending and stretching simultaneously as a function of pressure.

Table 2
Advantages and disadvantages of some techniques used in characterization of liquid crystalline phases.

Technique Analytical application Disadvantage


Polarized light microscopy Phase identification and macroscopic textures No differences between the optically isotropic phases
Small-angle X-ray diffraction Molecular dimensions of the liquid crystals Difficult to interpret for complex or heterogeneous
systems
Differential scanning Temperature and enthalpies of phase transitions during melting and Cannot identify the different phases between
calorimetry crystallization transitions
S. Milak, A. Zimmer / International Journal of Pharmaceutics 478 (2015) 569–587 583

By increasing the pressure, the spontaneous curvature of the The uncommon feature with these systems is the virtual absence
glycerol monooleate tends to move to 0. The curvature elastic of entanglements between hydrophobic chains which makes the
energy is reduced progressively as a function of pressure indicating relaxation phenomena to occur in short time scales, typically in the
that in these conditions the curvature elasticity does not dominate order of seconds. This happens in the typical time scale of the tasting
the total free energy. and release process, so rheology appears to be one of the most
SAXS was used to study the dynamics of lipidic nanostructures relevant techniques that allows to obtain an insight in the perception
formed via self-assembly mimicking the biological environment to or release features of liquid crystalline phase materials.
understand their structural formation mechanism (Yaghmur and The interpretation rheological phenomena of liquid crystalline
Rappolt, 2012; Yaghmur et al., 2011b). Furthermore, it is important phases has been tacked by studying the storage (G0 ) and loss
that this study is done under non-equilibrium conditions, which moduli (G00 ). Changes in the slope of G0 and G00 versus temperature
are mostly present in the biological environment (Yaghmur and and composition are a valuable tool to detect order–order and
Rappolt, 2012). For example, a bupivacaine loaded inverted type order–disorder transitions. In such a way, cubic to hexagonal, cubic
micellar solution and a hexagonal phase were investigated upon to cubic and, hexagonal to isotropic fluid transitions were
exposure to phosphate buffer (pH 7.4 and 6.0) (Yaghmur et al., observed. t MAX is the longest relaxation time, obtained from data
2011b). Both precursors, the micellar solution and hexagonal phase of G0 and G00 versus frequency, and it is defined as the inverse of the
responded similar. First an intermediate bicontinuous cubic phase frequency at which the crossover of G0 and G00 takes place. t MAX
appeared, followed by the appearance of a neat hexagonal phase at provides data an order of magnitude typical for many diffusion
the pH of 7.4 (lower degree of bupivacaine ionization and higher processes taking place in liquid crystalline phases and can be used
partitioning into the lipid bilayer) or a mixture of Pn3m and to parameterize the release kinetics of active molecules through
hexagonal phases at the pH of 6.0 (increased level of ionization, the hydrophilic/hydrophobic interface (Sagalowicz et al., 2006b).
higher amount of drug is accommodate in the hydrophilic Studies of t MAX variations on temperature and composition are the
nanochannels, no complete transition to hexagonal phase). most reliable rheological methods to detect order–order and
order–disorder transitions in liquid crystalline phases. Alterna-
6.3. Differential scanning calorimetry (DSC) tively, data from rheology measurements can be used to establish
the phase diagrams, since changes in the slope of t MAX versus
The phase transitions of different liquid crystalline phases could temperature and composition capture all phase transitions, the
be investigated by DSC in order to identify possible transitions as a regions of phase coexistence, and also order–order transitions
function of storage temperature (Demetzos, 2008; Müller-Goy- induced by the presence of guest molecules in either hydrophilic or
mann, 2004). Chang and Bodmeier (1997b), noted a broad hydrophobic phases.
endothermic peak in the temperature range from 5 to 25  C in The rheological method (steady flow properties and dynamic
the case of swollen Myverol 18-99 (glycerol monooleate). This viscoelastic properties) was also used in the evaluation of glycerol
phase transition decreased with increasing water content and no monooleate/water system with different concentration of iopa-
other phase transitions was observed, suggesting that the swollen midol (Shui et al., 2005). The rheological properties are a function
glycerol monooleate liquid crystalline phases should remain stable of the structural arrangement of particles and various interaction
in the studied temperature range (5 to 35  C) without phase forces that operate in the system. Viscoelastic properties go
transformation. These liquid crystalline phases could also be stored through three regions to increase the concentration of iopamidol.
and preserved at 25  C. When the concentration of iopamidol was higher than 3.97% w/w
Further, Qiu and Caffrey (2000), provided the more reliable and the viscoelastic properties were increased, whereas the phase
detailed temperature–composition of the glycerol monooleate/ structure parameter was decreased which led to the more compact
water phase diagram. They highlighted the caution which is structure. Finally, the lipid bilayer will be attenuated or disrupted
necessary to prepare the phase diagram with liquid crystalline when the concentration of iopamidol reaches 99.8% w/w, and the
phases at low temperature. Such liquid crystal phases are usually viscoelastic properties of the system are decreased (Shui et al.,
undercooled, therefore special care should be taken to ensure that 2005).
the system is set into its equilibrium state before making phase Moreover, measuring the rheological properties of the bulk
characterization measurements in the heating direction. Usually liquid crystalline phases as a function of temperature is an elegant
preincubation at 10  C for 1 h is sufficient to destabilize the way to distinguish between different self-assembly structures. The
undercooled liquid crystalline phase in favor of the equilibrium storage moduli, loss moduli and relaxation time contain sufficient
phase (Qiu and Caffrey, 2000). information to discriminate between the different liquid crystal-
line structures.
6.4. Rheology
6.5. Low frequency dielectric spectroscopy
Rheology is able to determine the crystallographic structure of
the lipid crystalline phases and dynamic processes occurring Dielectric analysis involves the application of an oscillating
during relaxation of liquid crystalline phases, which are directly electrical field to a sample and the subsequent measurement of the
affecting diffusion properties in heterogeneous phases (Müller- real and imaginary components of the response over a range of
Goymann, 2004; Sagalowicz et al., 2006b). The crystallographic frequencies, i.e., 103–106 Hz (He and Craig, 1998a,b, 1999). This
structure and the dynamic properties of the liquid crystalline approach allows interpretation of the dielectric data using
phase account and control the release pattern and the rate of guest parameters with specific structural meaning. The low-frequency
molecules from such structures. dielectric analysis has a useful role in the range of complex
By studying the storage modulus (G0 ) and loss modulus (G00 ) semisolid materials structural characterization. This technique is
dependence on shear frequency, the rheological signatures of able to identify the different phases of liquid crystalline systems
individual liquid crystalline phases have been established. The and to monitor discrete regions within such samples, for example,
bicontinuous cubic phases are the most rigid liquid crystalline how a drug molecule may alter different regions within the
phases, followed by the reversed hexagonal phase, which is a systems.
moderately viscoelastic fluid, and the lamellar phase, which could He and Craig (1998a), showed that the dielectric responses of
be described as a plastic fluid undergoing yielding. the lamellar, cubic and hexagonal phases exhibited by these
584 S. Milak, A. Zimmer / International Journal of Pharmaceutics 478 (2015) 569–587

systems could be described in terms of equivalent circuit models, organization of the cubic Pn3m glycerol monooleate–water phase
which could in turn be related to specific structural features of the upon introducing low amounts of distearoylphosphatidylglycerol
sample. The low-frequency dielectric analysis is highly sensitive to and lysozyme. X-ray measurements indicated that distearoyl-
the phase behavior of glycerol monooleate–water systems, phosphatidylglycerol and lysozyme induced the phase transition
particularly in terms of the resistance which may change up to Pn3m–Im3m, as well as distearoylphosphatidylglycerol induced
4 orders of magnitude on transformation from the lamellar or formation of the lamellar phase. This is due to the decrease of the
hexagonal phase to the cubic phase. This resistance is associated hydrocarbon chain mobility (effect of distearoylphosphatidylgly-
with charge movement across the lipid bilayer. In an isotropic cerol) and increase in the relative number of hydrogen-bonded
system, such as the cubic phase, there are continuous charge C¼O groups of glycerol monooleate (effects of both distearoyl-
transport routes. In the lamellar or hexagonal phases, charge phosphatidylglycerol and lysozyme). Lysozyme is responsible for
transport is blocked due to discontinuities in the lipid crystalline the decrease of the lipid packing parameter and it induces the
structure, necessitating the movement of charge through the intercubic Pn3m–Im3m phase transition. Lysozyme (59% of apolar
bilayer itself. Furthermore, He and Craig (1998a) showed that the surface, isoelectric point 11.1) is also capable to penetrate into the
dielectric response may be effectively modelled by a comparatively hydrophobic interior of the bilayer formed by acidic lipids. The
simple circuit diagram, components of which may be related to initial electrostatic interaction between distearoylphosphatidyl-
specific features within the sample. glycerol and lysozyme, and the subsequent penetration of the
The same research group, (He and Craig, 1998b), presented that protein into the mixed lipid bilayer, break the integrity of the
the dielectric technique is able to identify the phase transition bilayer and retard the formation of the bicontinuous cubic phase.
temperatures and ranges in a quantifiable manner which may be In addition raman spectra showed that distearoylphosphatidyl-
difficult to be identified using other techniques. They also glycerol decreased the mobility of acyl chains and increased the
investigated the effects of drug addition on the formation and number of hydrogen-bonded C¼O groups of glycerol monooleate.
structure of liquid crystalline phases by dielectric spectroscopy. It Consequently, lysozyme was located in the water channel system
was presented that the liquid crystalline systems containing 0 and of the cubic phase.
5% propantheline bromide have typical spectra of the cubic and For example, Nylander et al. (1996), showed that the diffusion
lamellar phases, while the gels containing 1% and 3% propantheline coefficient of glucose is about five times smaller in a cubic phase,
bromide showed features of both systems which are an 1.2  1010 m2 s1, than in a bulk phase, 6.7  1010 m2 s1. This
intermediate between the two phases. The circuit modelling is confirmed that the diffusion is not disturbed by macroscopic
able to provide information on the location of the drug within the defects in the cubic phase, but it is likely to be controlled by
gel as well as to identify structures which are intermediate transport through the water channels.
between the lamellar and cubic phases. This is important for In summary, infrared and raman spectra provide an information
understanding the drug release properties of these gels and their on the molecular conformation, for example whether the
mechanical and bioadhesive behavior. hydrocarbon chains of lipid crystalline phases have their zigzag
Bonacucina et al. (2005) used the dielectric spectroscopy in planes parallel or not.
combination with a complementary technique, oscillatory rheolo-
gy, to investigate the inclusion of a model peptide cyclosporine A 7. Conclusion
on the gel properties. Both techniques showed that the systems
with the 10% and 30% (w/w) water correspond to the lamellar and Among lipid liquid crystalline phases investigated, in the majority
cubic phase at 20  C. At the 22% w/w of water such a system of cases, glycerol monooleate cubic liquid crystalline phases were
showed intermediate behavior. Incorporation of cyclosporine A studied during the last decades. In many examples, it was
resulted in marked changes in a rheological and dielectric response demonstrated that the glycerol monooleate cubic liquid crystalline
of the 10% w/w water system, causing a decrease in both elasticity phase is able to incorporate a variety of active substances, from small
and permittivity values, while a less pronounced effect was to large molecules, and to achieve different applications. Transitions
observed for the 22% and, in particular, the 30% w/w systems. They between phases may be induced by addition of a drug, changes in
showed that rheological and dielectric measurements give distinct hydration, temperature, pressure, pH, salt concentration etc. After
yet complementary information. The inclusion of a model peptide, solubilization of a certain “critical” amount of additive, a phase
cyclosporine A, may alter the properties of the gel, the extent of the transition is induced. Generally, lipophilic additives induce a
effect being dependent on the phase composition of the system transition from a reversed bicontinuous cubic to reversed hexagonal
(Bonacucina et al., 2005). Rheology allows the determination of phase, i.e., the mean interfacial curvature will be more negative.
both, viscous and elastic properties (mechanical properties of the More hydrophilic additives induce the transition from the reversed
gels indicating changes in moduli between phases), while bicontinuous cubic to the lamellar phase, i.e., the mean interfacial
dielectric analysis gives information concerning charge movement curvature changes towards zero.
within the samples. Most authors reported a sustained drug release from glycerol
monooleate cubic liquid crystalline phase up to one week. The
6.6. Spectroscopic methods (NMR, IR- and Raman spectroscopy) release mechanism is typical for a matrix-type delivery system and
dissolution of the active substance is described by diffusion out of
NMR (nuclear magnetic resonance) provides structural infor- the cubic phase gel matrix.
mation for the identification of liquid and liquid crystalline phases During the last decades there were significant advances
(Lynch and Spicer, 2005). NMR has been used by certain authors for published in the structural analyses of liquid crystalline phases,
phase identification (and phase transitions), (Caboi et al., 2001; especially by SAXS and spectroscopic techniques, which contrib-
Ericsson et al., 1991; Jeong et al., 2002; Nylander et al., 1996; uted to a better understanding of polar lipid liquid crystalline
Pitzalis et al., 2000; Srisiri et al., 1998) although this can lead under phase structure and hence their function, and release pattern.
certain circumstances to incorrect assignments. Furthermore, by Recent SAXS and NMR results have significantly triggered
NMR diffusion measurements, it is possible to determine the investigations of advanced liquid crystalline formulations, e.g.,
continuity of polar or apolar domains. stimuli responsive formulations, formulations using charged lipids
Razumas et al. (1996b) used X-ray and Raman scattering (to modulate aqueous channel dimensions and to manipulate with
spectroscopy to study phase transitions and changes in a molecular drug loading, drug release).
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