Nanostructured liquid-crystalline particles for drug delivery.

Review

Nanostructured liquid-crystalline particles for drug delivery Alexandre Lancelot, Teresa Sierra & Jose´ Luis Serrano† †

University of Zaragoza, Department of Organic Chemistry, Zaragoza, Spain

1.

Introduction

2.

Composition, preparation and characterization of the LLC NPs used in drug delivery

3.

Biomedical applications of LLC

Expert Opin. Drug Deliv. Downloaded from informahealthcare.com by Ondokuz Mayis Univ. on 05/17/14 For personal use only.

NPs 4.

Conclusion

5.

Expert opinion

Introduction: Nanostructured lyotropic liquid crystal particles (LLC NPs) have proven to be extremely useful tools for applications in drug delivery. These structured nanoparticles are formed by amphiphilic molecules and contain internal water channels, which are not in contact with external water, and where polar drugs can situate; on the other hand, apolar drugs can be loaded in the lipophilic part of the structure and the amphiphilic drugs can locate at the polar/apolar interfaces. Areas covered: A revision of the most relevant results published in the field of LLC NPs has been made. The first section discusses the most common compounds used in these nanoparticles and their preparation and characterization. A summary of recent and relevant results including the composition and type of nanoparticles used, the illness treated, the administration via and some special features in each case have been summarized in a table. Expert opinion: LLC NPs are highly versatile drug delivery systems, which can be applied by topical, oral and intravenous treatments. Especially relevant is their use for the release of anticancer drugs, biomolecules and vaccines. Nevertheless a number of critical points need to be solved in order to attain practical applications. Keywords: biomedical applications, cubosomes, drug delivery, hexosomes, lyotropic liquid crystal nanoparticles Expert Opin. Drug Deliv. (2014) 11(4):547-564

1.

Introduction

In 2013, 125 years have gone since the official birth of liquid crystals (LC) [1]. In 1888, at the German Technical University in Prague, Friedrich Reinitzer, Professor of Botany and Technical Microscopy, sent a letter to professor Otto Lehmann, an expert in the physical isomerism of crystals and lecturer in Dresden, in which he described the unusual melting process he had observed in a cholesterol derivative [2]. Reinitzer measured a first melting point of cholesteryl benzoate at 145.5 C; however, he found a second melting point at 178.5 C. Furthermore, between these two temperatures, he could observe a milky liquid phase, which turned a clear phase at 178.5 C. Reinitzer is known as the discoverer of thermotropic LC. To date, the principal applications of thermotropic LC are displays [3] and high-performance LC polymers [4]. Nevertheless, some years before the famous letter of Reinitzer, another class of fluid materials, namely, lyotropic liquid crystals (LLC) were also observed to exhibit similar properties as anisotropy and optical birefringence. In this case, the disruptive effect on the crystal lattice involves a solvent coupled with thermal changes. These first observations were described by Virchow and Mettenheimer and involved biological molecules derived of nervous tissues as myelin, which behaves as a birefringent fluid in water sodium oleate solution [5,6]. In spite of these early studies, the lack of reproducibility and the complexity of these biological systems hampered the progress of LLC. Important developments began in the 1970s with the publication of the works of Windsor [7] and Friberg [8] and with the practical applications of these materials in 10.1517/17425247.2014.884556 © 2014 Informa UK, Ltd. ISSN 1742-5247, e-ISSN 1744-7593 All rights reserved: reproduction in whole or in part not permitted

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The intersection between lyotropic liquid crystals and nanotechnology has provided lyotropic liquid-crystalline nanoparticles (LLC NPs), which have been investigated in numerous examples in drug delivery. Most of the LLC NPs used to date are cubosomes formed from the inverse bicontinuous cubic phase, micellar cubosomes formed from the discontinuous cubic phase and hexosomes formed from inversed hexagonal phases. Only a few amphiphilic molecules exhibiting lyotropic behavior have been used to form LLC NPs for drug delivery. Glyceryl monooleate (GMO) and phytantiol are the most used lipids. Nanoparticle dispersions need to be stabilized to avoid aggregation across time. The nature and the concentration of stabilizers, as pluronic, are not meaningless as they can affect important parameters like the mesophase formation, the toxicity or the protection against the hydrolysis. Given their extensive water channel networks, LLC NPs have been reported to entrap different therapeutic agents, ranging from small drug molecules to biomacromolecules, both water soluble and poorly water soluble. In this way, LLC NPs incorporate different types of therapeutic agent focused on a variety of diseases and used through different types of administration vias. The use of LLC NPs for the delivery of biomacromolecules has deserved extensive attention due to the current interest on protein-based and nucleic acid-based therapeutics. Current investigations on strategies to improve the immunogenicity of protein--peptide-based vaccines propose LLC NPs as promising candidates as vehicles for the delivery and release of vaccines. One major issue of these systems is related to the sustained drug release. Release rates depend on the internal structure of the particles as well as on the nature and size of the cargo. LLC NPs that present discontinuous water compartments, micellar cubosomes and hexosomes, show lower release rates than the bicontinuous cubic phase, cubosomes, which forms continuous water channels. Multifunctionality is a current challenge of the investigation on drug-containing LLC NPs to attain imaging and targeting functions for these systems.

This box summarizes key points contained in the article.

the oil, detergent and food industries in order to understand and control the behavior of the amphiphile/water/oil systems. Additionally, an important number of papers are devoted to the role played by the lyotropic LC concepts in biological systems as cell membranes, ribosomes, Golgi apparatus, etc [9,10]. Generally, lyotropic phases described to date appear in mixtures formed by a combination of surfactants (surface active agents), which are usually natural or synthetic lipid molecules, some stabilizers and a solvent. The key role is played by the surfactants, usually amphiphilic molecules bearing two main 548

parts: a head group formed by a polar (hydrophilic) part that can be anionic, cationic, zwitterionic or nonionic, and an apolar (hydrophobic) part, usually formed by aliphatic chains and more rarely by semi or perfluorocarbon chains or dimethylsiloxane derivatives. In biological applications, water is the most common solvent. At certain concentrations and temperatures, in order to minimize unfavorable interactions between the apolar part and the polar external medium, the amphiphilic molecules can self-assemble in aqueous medium to form aggregates, named micelles. These micelles appear when concentration and temperature are superior to limited values: the critical micelle concentration and the Krafft point. The critical micelle concentration is the lowest concentration of amphiphilic molecules required to observe the formation of micelles [11]. The Krafft point, also called critical micelle temperature, is the minimal temperature at which the amphiphilic molecules form micelles [12]. Below the Kraft temperature the amphiphile remains in the crystalline state even in aqueous solution. Commonly, micelles will act as building blocks of macroscopic ordered structures leading to lyotropic mesophases. Classically, six different types of lyotropic liquidcrystalline phases have been described (Figure 1): lamellar, hexagonal, cubic, nematic, gel and intermediate [13]. Lamellar phases, also called neat phases, are formed by amphiphiles’ bilayers extended at a large distance and separated by water layers (Figure 1A). Hexagonal phases are formed by extended cylindrical micelles packed together in a hexagonal lattice. Depending on concentration, two different hexagonal phases can be formed. The normal hexagonal phase (H1) is water continuous, whereas the reversed hexagonal phase (H2) is alkyl chain continuous (Figure 1-B-1) and (Figure 1-B-2). In 2009, Shearman et al. discovered a third lyotropic liquid-crystalline phase based on three dimensional (3D) hexagonal closepacked arrangement of inverse micelles P63/mmc [14]. A first type of cubic phases (I type, also named discontinuous) consist of three different cubic lattices in which globular micelles (with the same or with different sizes) are situated in the vertex of a cube (primitive), in the vertex and in the center of the faces (face centered) and in the vertex and in the center of the cube (body centered) (Figure 1-C-1). Also in this case, depending on concentration, either normal phases, I1 (Pm3n-primitive, Fm3m-face centered and Im3m-body centered), or reversed ones, I2 (Fd3m), are formed. A second type of cubic phases (V type) consists on curved bicontinuous lipid bilayers extended in 3D. The aggregates structure form a network having curvatures toward water, V1, or toward aliphatic chains, V2. Three main types of structures with different space group have been described: Pn3m (primitive lattice), Ia3d (body-centered lattice) and Im3m (body-centered lattice) (Figure 1-C-2). Pn3m consists on a double diamond structure formed by linear micellar aggregates connecting four by four in a tetrahedral way. The Ia3d gyroid structure consists of two independent networks formed

Expert Opin. Drug Deliv. (2014) 11(4)

Nanostructured liquid-crystalline particles for drug delivery

1-B-1. Normal hexagonal 1-B-2. Reverse hexagonal phase (H1) phase (H2)


1A. Lamellar phases

Pm3n

Fm3m

Im3m

Fd3n

1-C-1. Discontinuous cubic phases (I1 and I2)

Ia3d

Pn3m

Im3m

1-C-2. Bicontinuous cubic phases (V1 and V2)

1-D. Lyotropic nematic phases

Lc

Lβ′

Lβ

Lα

Rippled

1-F. Mesh phases

1-E. Gel phases

Figure 1. Schematic representations of lamellar phase (1-A), normal hexagonal phase(1-B-1), reverse hexagonal phase (1-B-2), discontinuous cubic phases (1-C-1), bicontinuous cubic phases (1-C-2), lyotropic nematic phases (1-D), gel phases (1-E) and mesh phases (1-F). Adapted with permission from [135-137].


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by linear aggregates joined three by three. Im3m consists on a network of water channels connected six by six. Lyotropic nematic phases, which are very unusual, are formed by rod-like or disk-like micelles oriented in a preferred direction (Figure 1-D). Gel phases are structurally very similar to the lamellar ones but they exhibit high viscosity due to very regular intermolecular arrangements, usually observed in phospholipids bilayers. In these phases, amphiphiles usually bear one or two alkyl chains in all-trans conformation and form very rigid layers. Four different lyotropic gel phases are distinguished [15]: the subgel (Lc), the lamellar gel phases (Lb or Lb¢), the rippled phases (Pb¢) and the liquid crystalline or fluid phase (La) (Figure 1-E). Finally, lyotropic intermediate phases are equivalent to some previously described phases but with structural modifications. Thus for example, ribbon phases as distorted hexagonal phases, mesh phases as distorted lamellar phases presenting defects in the continuous bilayers (Figure 1-F) and distorted cubic structures have been described. Most common applications of LLC are based on the amphiphilic nature of the constituent molecules, which leads to phases that can absorb a given amount of other molecules without altering their structure [16-18]. Thus, polar molecules can be present in water channels or water layers interacting with the polar part of the surfactant, whereas hydrophobic molecules can interact with the apolar part (usually aliphatic chains). In this way, amphiphilic self-assembled materials can be used to enhance different product formulations [19]. These properties have been widely used in Pharmacy, Cosmetics or Food Industries. Indeed, since self-assembled amphiphiles allow solving and protecting drugs from adverse external environments, the application of these systems as drug delivery vectors has been the most relevant one [20-22]. Lyotropic phases are highly versatile and can be modified, not only by components’ concentration and temperature, but also with other external physical agents, such as pH, which can be used to induce the specific release of encapsulated molecules [23]. Most of the applications described in the literature for drug-containing LLC are focused on their topical use on skin or mucous membranes [24-26]. However, they have been also used to improve drug delivery efficiency through other administration routes [27,28]. Meanwhile, since the 1980s, the emergence of Nanoscience and Nanotechnologies revolutionized Applied and Basic Science [29]. Nanotechnology has been defined as the manipulation of matter with at least one dimension sized between 1 and 100 nm. Nanotechnology may be able to create many new materials with a huge number of applications in both Nanomedicine [30] and Biomedicine [31,32]. Basically, two different types of nanomaterials can be defined, the hard ones and the soft ones with, in both cases, biological applications. LC are logically related to the soft ones. To date, soft nanostructured materials based on organic molecules of both high and low molecular weight have proven 550

themselves as excellent candidates for biological applications [33]. Organic nanostructures are found in different applications like gene transfection agents [34], magnetic resonance imaging, using metal-organic derivatives [35] and drug delivery [36,37]. As a curiosity, it is worth mentioning that some examples of these nanostructures deal with the cholesterol molecule, which, as explained before, was the first LC described [38]. Also interestingly, amphiphilic molecules play an important role in nanotechnology [39,40]. The intersection between LLC and nanotechnology has provided a type of nanoparticles of special relevance. Indeed, nanostructured LLC particles (LLC NPs) have been investigated in the last years and numerous examples can be found in drug delivery. Generally, the various mesophases described above present high viscosity and, as we explain before, therapeutic applications are restricted to gels for topical applications and oral solutions. Some of these lyomesophases, when consisting on amphiphiles with very low water solubility, can be dispersed to form nanoparticles which, in general, retain the internal phase structure of the bulk. This process makes them easier to handle, broadening their applications’ field [41-43]. Numerous examples of these soft nanomaterials have been reported in the last years, which mainly consist on micelles and three types of lyotropic mesophase: lamellar, cubic and hexagonal. Micellar particles, in spite of having a nanometric size and formed by promesogenic molecules (molecules able to generate LC phases) but do not exhibit internal nanostructured organizations. However, they can encapsulate active molecules, and this has been widely exploited in drug delivery to improve drugs internalization using so-called mixed micelles (i.e., phospholipid and drug) [44-46] or bicellar systems (i.e., bilayered disk-shaped nanoaggregates) [47,48] Most of the nanoparticles coming from lamellar phases consist on vesicular systems formed by either anionic or non-ionic (niosomes) surfactants. If a vesicular system is formed by lipid molecules, the particles are named liposomes [49-51]. In ‘stricto senso’ neither micelles, nor vesicles can be considered as nanostructured liquid-crystalline particles because they do not exhibit internal molecular organization, and, for this reason, both types of nanoparticles are out of the scope of this review. Most of the LLC NPs used to date are formed from four types of mesophases that are thermodynamically stable in excess of water: 3D inverse bicontinuous and discontinuous cubic phases and 2D and 3D inverse hexagonal phases. These phases can be dispersed into LLC NPs, which are named as cubosomes when formed from the inverse bicontinuous cubic phase, micellar cubosomes when are formed from the discontinuous cubic phase and hexosomes when formed from inversed hexagonal phases. These structured nanoparticles contain internal water channels, which are not in contact with external water and where polar drugs can be situated [52]. Apolar drugs can be loaded in the lipophilic part of the structure and the amphiphilic drugs can be located at the polar/ apolar interfaces.



120 FI + water

Temperature (ºC)

HII + water

HII

FI

Pn3m

80 Pn3m + water Ia3d

40

Lα

Pn3m + Ia3d Lα + Ia3d


Lc + Pn3m Lc + water

0 Lc

0

Lc + ice

10

20

30

40

50

Composition, % (w/w) water

Figure 2. Glyceryl monooleate (GMO) phase diagram. Reproduced with permission from [62].

Due to the interest of these last materials in drug delivery, a numerous number of papers dealing with this subject have been published during the last years, including some specialized revisions [19,43,53-61].

Composition, preparation and characterization of the LLC NPs used in drug delivery

2.

Composition of the LLC NPs Amphiphilic molecules self-assemble in aqueous medium in order to minimize unfavorable interactions between their hydrophobic part and water. Three parameters are crucial to form the lyotropic phases: the amphiphilic molecules, the appropriate solvent (water for biological applications) and the temperature. Figure 2 represents the phase diagram of a common amphiphilic molecule with lyotropic liquid-crystalline properties, glyceryl monooleate (GMO) [62]. In addition to temperature, other external factors as pressure, pH or salt concentrations can modify the properties of the mesophases [19]. Many amphiphilic molecules exhibiting lyotropic liquidcrystalline behavior in water have been described to date, nevertheless only a few have been used as LLC NPs in drug delivery. Their stability in physiological media is the major restricting issue. GMO, molecules analogous to GMO (such as monolinolein) and mixtures of GMO with other lipids are some examples of these amphiphilic molecules (Figure 3). These compounds were a part of the first molecules investigated to form structured nanoparticles [63]. The presence of an ester bond and of one or several unsaturations in the aliphatic chain of the GMO analogs decreases their stability versus enzymes. The partial degradation of GMO at physiological temperature induces the transformation of the reverse cubic lattice into a reverse hexagonal one. To overcome this 2.1

issue, phytantriol (Figure 3) was investigated. It also forms cubic mesophases at physiological temperatures [64,65]. The aliphatic chain of phytantriol is methyl branched and fully saturated, and this increases its resistance to enzymes’ degradation [66]. Inverse hexagonal mesophase (H2) can be reached in a more controlled way by addition of small molecules, like tetradecane or vitamin E; this strategy decreases the temperature of formation of (H2) mesophases to physiological temperature [41,67]. Another solution is to modify the classic GMO head by a glycerate group (Figure 3). These subtle modifications allow the formation of (H2) mesophases at physiological conditions [68]. Nanoparticle dispersions need to be stabilized to avoid aggregation across time. The nature and the concentration of stabilizers are not meaningless as they can affect important parameters like the mesophase formation, the toxicity or the protection against hydrolysis. Pluronic copolymers are good candidates to avoid nanoparticles flocculation [69]. They are tri-block copolymers composed of two external parts of ethylene oxide units (EO) and one central part of propylene oxide units (PO), EOx--POy--EOx. The two EO blocks act as steric agents and inhibit flocculation, whereas the PO block is responsible of the adhesion of the polymer on the nanoparticle. Both parts are required, and the hydrophilic/lipophilic balance is a key parameter. The most common pluronic stabilizer is the triblock copolymer EO99--PO67--EO99, known as Pluronic F127 or Poloxamer 407 (Figure 3). Lower quantity of Pluronic F127 promotes bigger nanoparticles, whereas higher Pluronic F127 concentration promotes smaller ones [70]. Pluronic F127 can well disperse GMO-based nanoparticles at 1.0% wt and phytantriol ones at 0.5% wt. Nevertheless, it is not the best Pluronic to stabilize these nanoparticles, and F108, EO132--PO50--EO132 (Figure 3), showed a better ability to


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Amphiphiles

Stabilizers

OH HO

O O

OH HO

(1) HO

O O

OH

O 99

(2) HO

HO

O 132

(3) HO

HO

O

O

O 67 O 50

O H n

H 99

(5)

(6) H 132 (7)


OH HO

O

(4)

O

Figure 3. Structures of glyceryl monooleate (1), monolinolein (2), phytantriol (3), oleyl glycerate (4), Pluronic F127 (5), Pluronic F108 (6) and PEG (7).

stabilize cubosomes with less modification of the internal mesophase structure [71]. Other materials based on EO units are described in order to stabilize the nanoparticles at lower concentration than Pluronic F127 like ethoxylated phytosterol with 30 oxyethylene units [72], a commercial hydrophobically modified cellulose ether [73] or Mjry 59 a commercial poly(ethylene oxide) stearate with an average of 100 units of EO that can stabilize phytantriol-based nanoparticles at 0.1% wt [74]. b-Casein was also used as stabilizer as it increases the domain of existence of H2 mesophase for phytantriol and GMO [75]. Additionally, some non-organic structures are described as specific LLC NPs stabilizers, disk-like particles of Laponite clay for GMO/tetradecane/water systems [76], spherical silica colloids for phytantriol/tetradecane/water systems [77,78]. Preparation of LLC NPs for drug delivery Lipophilic and hydrophilic drugs have been loaded inside the LLC NPs. Typically, the drug is dissolved in the bulk mesophase before its dispersion. Lipophilic molecules are located in the hydrophobic parts of the nanostructures. Sometimes these drugs are situated near the hydrophilic domains inducing modifications of the curvature toward the water channels [79]. Hydrophilic drugs remain in the water channels after dispersion [68]. Two techniques are commonly used to prepare drugloaded LLC NPs for applications in drug delivery. The first one consists on a top-down approach. A mixture of the amphiphile and stabilizers is first hydrated to allow them to self-assemble in a really viscous bulk. The bulk is then dispersed in aqueous solution inputting high level of energy. High-pressure homogenization [80] and ultra-sonication [81] are commonly used. It has to be noticed that all these techniques heat the samples and this can seriously damage the amphiphilic molecules or the encapsulated drugs. This damage has been avoided using a shear device based on a Couette 2.2

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cell to disperse the bulk mesophase that contains the drug and the stabilizers [82]. This process has the advantages of limiting the heating energy income and producing monodisperse LLC NPs with high concentration of lipophilic molecules (up to 70% wt) in a fast way. This method was also applied to hydrophilic cargos especially large proteins [83]. Polyglycerol ester was added to the monoglycerol/stabilizer mixture to increase the size of the internal channels allowing them to accept large molecules such as proteins bovine serum albumin and cytochrome C. The second preparation technique consists on a bottom-up approach. In this technique, amphiphiles are mixed with hydrotropes, prior to dilution. The presence of hydrotropes is an important issue since they are used to improve nanoparticle solubility but might induce toxicity. Controlled addition of aqueous medium into the amphiphiles/hydrotrope solution reduces rapidly the lipid solubility inducing formation of dispersed LLC NPs. This technique, more accessible, has the advantage to produce nanoparticles with higher stability [84]. Both techniques produce not only LLC NPs but also vesicles that are speculated to interfere during the drug release process. Three treatments are available to purify LLC NP solutions. The first one is the heat treatment (usually above 120 C). It is used as a way to purify cubosomes, producing ordered cubic particles from smaller vesicles. In fact, increasing the temperature, the solubility of the surfactant decreases and vesicles fusion occurs, producing new nanoparticles. It has been demonstrated that a combination of shear energy homogenization and heat treatment allows GMO-based cubosomes to be produced in a scalable and reproducible way with narrow particle size and well-defined inner structure [85]. Nevertheless, as an increase of temperature is required, this purification technique is really restricted to specific cases and not always applicable in drug delivery applications [86]. The second treatment is called modified dialysis



A. Cubosomes

q B.


Hexosomes

q SAXS

Cryo-TEM

Cryo-FESEM

Figure 4. A. SAXS sprectrum and cryo-TEM, cryo-FESEM images of cubosomes made from phytantriol. B. SAXS sprectrum and cryo-TEM, cryo-FESEM images of hexosomes made from a mixture of phytantriol and vitamine E. Reproduced with permision from [138].

technique and was introduced by Abraham et al. in 2004 and avoids high-energy processes. It is a combination of the bottom-up approach and dialysis. The bulk phase of lipid is mixed with water, hydrotropes and Pluronic and the mixture is dialyzed against water to obtain a solution free of hydrotropes and micelles [87]. A third treatment is called premix membrane emulsification and it is a process where a predispersed emulsion is extruded repetitively through a membrane with desired pores’ size forming smaller emulsion droplets. This technique allows the formation of a monodisperse solution of nanoparticles [88]. More recently, it has been demonstrated that the formation and the stability of cubosomes or hexosomes can be induced or modified changing different solution characteristics such as pH or ionic strength. These processes allow the formation of desired nanostructure without high-energy input. Dispersion of cubosomes has been reached by adding phosphate buffer to an aqueous solution of cationic vesicles [89]. A mixture of phytantriol and two anionic surfactants was described to be able to shift from cubic mesophases to lamellar ones when the contents of ionic surfactants or the ionic strength of the solution is modified, and this leads to the visual detection of specific toxic ions, for example, Cr(III) [90]. Characterization of LLC nanostructures To evaluate the potential of LLC NPs in drug delivery, parameters like the type of mesophase, the quality of the dispersions and their stability, drug diffusion, etc., must be controlled. The best technique to study the mesophases is small-angle X-ray scattering (SAXS) (Figure 4). Briefly, the periodic structure of the nanoparticles diffracts incident X-rays to 2.3

specific areas, called Bragg peaks. Comparing obtained Bragg peaks with databases allows the determination of the repetitive assemblies (double diamond, gyroid, primitive cubic, discontinuous cubic or hexagonal). Due to the facts that LLC are made with low electron density atoms (i.e., H, C, N, O) and that X-ray sources in laboratory only provide small X-ray flux, data acquisition is slow and of limited quality in common laboratory devices. To obtain diffractograms of higher quality and reduce acquisition times, synchrotron X-ray source has to be used. Using this technique, it has been possible to study phenomena such as the transformation from lamellar mesophases to cubic ones [91], or the transformation from cubic to hexagonal mesophases by adding vitamin E [92]. Phase transitions can be studied using differential scanning calorimetry (DSC) and polarized light microscopy [93]. DSC can give other type of information like the interactions between encapsulated drug and the mesophase [94]. Cryogenic transmission electron microscopy (cryoTEM) is principally used to determine the size and morphology of the nanoparticles. The sample must be vitrified in order to not alter nanoparticle structures. Time of acquisition is slow and only few particles can be observed. Moreover, cryo-TEM can be also used to determine the mesophase using fast Fourier transform analysis (Figure 4) [95,96]. Cryo field emission scanning electron microscopy (cryo-FESEM) is another alternative technique to study the structure of the nanoparticles. Like cryo-TEM, it allows to observe the particle in solution. Cryo-FESEM provides more superficial information of the materials than cryo-TEM but cannot be used to determine the type of mesophase (Figure 4) [97]. Atomic force microscopy is used as well to obtain better knowledge upon the morphology of the LLC NPs [98].


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Dynamic light scattering (DLS) gives information about the size over a larger number of nanoparticles [99]. The colloidal stability of the LLC NP dispersion can be controlled repeating size measurements across time. The time of experiment can be reduced using centrifugation. The centrifugation process accelerates the migration of the dispersed particles; 300 rpm corresponds to multiply by 12 the gravimetric factor. There are equipments that permit to centrifuge the sample while light transmission is being analyzed, and this gives information about local concentration of nanoparticles and demixing phenomena [72]. Diffusion into and out of the nanoparticles’ water channels is generally studied using 13C nuclear magnetic resonance (13CNMR). Local intermolecular interactions can modify the chemical shift of one specific 13C. Relaxation experiments give information about the diffusion speed of small and large molecules in solution. For example, the migration of Eu3+ 23 [100] or Na [101] from the external medium to the inner part of cubosomes has been studied. These experiments also give information about the presence of cubic or hexagonal mesophases, the stability across time (especially resistance to hydrolysis) and the internal structure of the nanoparticle [102]. Infrared (IR) spectroscopy is a suitable technique to study interactions between molecules. Attenuated total reflectance gives information on the interactions between the nanoparticles, the surfactant, the external medium and the loaded drug. As the technique is simple to handle, it permits to study the stability of the interactions along with the variation of different factors like temperature, pH and concentration of stabilizers [18] and the adsorption of nanoparticles on surfaces [103]. 3.

Biomedical applications of LLC NPs

LLC NPs have been reported to entrap different therapeutic agents, ranging from small-molecule drug to biomacromolecules. Table 1 gathers most recently reported drug delivery systems based on LLC NPs, which incorporate different types of therapeutic agent focused on a variety of diseases and used through different types of administration vias. The references herein selected have demonstrated bioavailability enhancement with respect to other delivery systems, such as raw-drug solutions or drug-containing liposomes. Cancer therapy Given their extensive water channel networks, cubosomes and hexosomes can load both water-soluble and poorly watersoluble anticancer drugs. Thus, as an example, irinotecan is a water-soluble camptothecin that shows activity against colorectal cancer; its active lactone form is stable at pH 3.5. This drug has been entrapped in glycerate-based Hexosomes in which the drug maintains its active form even at neutral pH, and this should permit formulations of this drug for intravenous administration [104]. On the other hand, paclitaxel (PTX) is a poorly water-soluble drug with low permeability 3.1

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across the intestinal barrier, which is used in cancer treatment under the trade name of Taxol via intravenous administration. This drug is frequently used as model of poorly water-soluble drug for the development of drug carrier systems with increased efficacy in cancer treatment. For intravenous administration, issues related with circulation time and hemolysis activity have been investigated with LLC NPs [105]. Improved bioavailability with respect to Taxol has been described for PTX-containing LLC NPs based on glyceryl dioleate (GDO) stabilized with soy phosphatidylcholine (SPC) and incorporating polysorbate 80 (P80), containing three poly(ethylene glycol) (PEG) chains. These NPs had been formerly described to increase circulation time [106]. With this objective, Zeng et al. proposed PEG-grafted 1,2-distearoylsn-glycero-3-phosphatidylethanolamine (DSPE--PEG) as an alternative to P80 [107]. The so formulated LLC system shows non-lamellar H2 and I2 phases coexistence, and this makes them appropriate for the construction of nanoparticles with organized inner architectures. Furthermore, it provides suitable nanocarriers for sustained release of hydrophobic drugs with enhanced circulation times. The incorporation of DSPE--PEG to provide PEG-shielding onto the LLC NPs has been also proposed by Jain et al., who reported an efficient PEGylated delivery system based on GMO, and stabilized with pluronic F127 [108]. Non-PEGylated LLC NPs based on GMO/pluronic F127 show a P-type cubic phase (Im3m) according to X-ray diffraction data, and the incorporation of DSPE--PEG-OCH3 and PTX leads to the development of hexagonal phases. In any case, the carrier system shows increased circulation time and better tumor accumulation compared non-PEGylated LLC NPs based on GMO. In general, PEGylation enhances the safety and efficacy of GMO or GDO delivery systems to be used as intravenous delivery vehicles for PTX. Cancer therapies based on oral administration of systems with high oral absorption are receiving increasing attention nowadays [109]. Anticancer drugs such as PTX and 20(S)-protopanaxadiol (PPD) are poorly water-soluble but also show low permeability across the intestinal barrier. Drug delivery systems based on LLC NPs are being investigated as an efficient alternative for increasing oral bioavailability of such drugs, while simultaneously providing sustained release at the tumor site. Previously described SPC/GDO/P80 nanoparticles have also been investigated by Zeng et al. as carrier systems for oral delivery of PTX [110]. An in vivo pharmacokinetic study of these PTX-loaded LLC NPs concludes that oral bioavailability can be enhanced in 2.1 times that of the commercial formulation of PTX (i.e., Taxol). LLC NPs are proposed to protect the drug from the recognition by the P-glycoprotein and also improve intestinal endothelial permeability by increasing membrane fluidity. Likewise, Jin et al. have recently described formulations based on cubosomes of GMO that display improved oral bioavailability for PPD [111]. The study demonstrated the increase of bioavailability with respect to raw PPD, which is attributed to better absorption rather than improved release. The incorporation of


Phytanyl derivative of 5-fluorocytosine (5-FCPhy), pro-drug of 5-fluoruracyl GMO Pluronic F108 GMO Pluronic F127

Cubosomes/ hexosomes


Quercetin + fluorescent probes Simvastatin

20(S)-protopanaxadiol/ piperine (1:3)

20(S)-protopanaxadiol

Paclitaxel

Cancer theranostic applications Hypercholesterolemia

Cancer

Cancer

Cancer (breast cancer and solid tumors)

Cancer

Cancer

Cancer

Cancer

Paclitaxel

Paclitaxel

Cancer (metastasis of colorectal cancer) Cancer

Illness

Irinotecan

Drug

Oral

Oral

Oral

Oral

Oral

Oral

Oral

Intravenous

Intravenous

Intravenous

Administration

Enhancement of oral bioavailability 2.42 times compared to micronized simvastatin crystal powder

Increased absorption of PPD with respect to raw PPD Piperine inhibits the rapid metabolism of PPD: Increased absorption The amphiphile is the pro-drug that undergoes enzymatic reactions to give the bioactive 5-fluoroacyl. decreased release rates The amphiphile is the pro-drug that undergoes enzymatic reactions to give the bioactive 5-fluoroacyl. decreased release rates Slow release and viable alternative to pro-drug capecitabine, showing similar toxicity levels In vitro experiments

Oral bioavailability 2.1 times that of commercial Taxol

Increased hemocompatibility. Release control: 44% in 24 h

Irinotecan maintains its active lactone form within hexosomes, even at neutral pH Increased circulation time

Some special features

[116]

[115]

[93]

[114]

[113]

[112]

[111]

[110]

[108]

[107]

[104]

Ref.

FITC: Fluorescein isothiocyanate; GMO: Glyceryl monooleate; MPL: Monophosphoryl lipid A; OA: Oleic acid; OAM: Oleyl amine; PEI: Polyethylenimine; PG: Propylene glycol; PEG: Poly(ethylene glycol); PHY: Phytantriol; PPD: 20(S)-Protopanaxadiol.

Cubosomes

Cubosomes

Cubosomes

C18 alkyl or alkenyl derivatives of 5-fluorocytosine (5-FCC18), pro-drugs of 5-fluorouracyl 5-fluorocytosine derivatives, pro-drugs of 5-fluorouracyl

Oleyl glycerate or phytanyl glycerate Pluronic F127 SPC/GDO DSPE-mPEG/ethanol GMO Pluronic F127, DSPE-mPEG/ethanol SPC/GDO polysorbate 80 (P80)/ ethanol GMO Pluronic F127 GMO Pluronic F127

Amphiphile stabilizer/hydrotrope

Cubosomes

Cubosomes

Cubosomes

Cubosomes + hexosomes

Cubosomes + hexosomes Hexosomes

Hexosomes

LLC NP

Table 1. Recently reported drug delivery systems based on LLC NPs, which incorporate different types of therapeutic agent focused on a variety of diseases and used through different types of administration vias.



555

556

GMO Pluronic F127/glycerol GMO Pluronic F127/glycerol GMO Pluronic F127

GMO/OA/OAM GMO/OA/PEI Pluronic F127 GMO/MAL-PEG--OA Pluronic F127

Cubosomes

Hexosomes


Phytantriol Pluronic F127/ propylene glycol

Phytantriol Pluronic F127/ propylene glycol

Cubosomes

Cubosomes

Ovalbumin adjuvant: Quil A

FITC--ovalbumin adjuvants: imiquimod or MPL

FITC--ovalbumin

S14G-HN adjuvant: Odorranalectin

siRNA

Cyclosporine A

Flurbiprofen

Dexamethasone

Cinnarizine

Ibuprofen

Cinnarizine

Drug

Vaccine

Vaccine

Vaccine

Central nervous system disorders, Alzheimer’s disease

Gene-caused diseases

Autoimmune disorders

Posterior segment eyes diseases Cataract surgery

See thickness

Anti-inflammatory drug

See thickness

Illness

Intravenous

Intravenous

Intravenous

Intranasal

Topical

Oral

Ophthalmic

Ophthalmic

Oral

Oral

Oral

Administration

Targeting effect of Odorranalectin: Increased mucoadhesive ability for efficient brain delivery Sustained release after initial burst. Release slower from PHY cubosomes than from GMO cubosomes (In vitro studies) Increased immune cellular response with respect to liposomes containing ova + adjuvants Cubosomes are embedded in a thermosensitive hydrogel, chitosan, for slow subunit antigen release

Stable LLC NPs and enhanced bioavailability Improved trans-corneal permeation Non-irritating flurbiprofen preparation Enhanced bioavailability with respect to commercial microemulsion of CyA: Neoral Higher penetration into the skin without irritation

Phytantriol cubosomes lead to enhanced oral bioavailability Improved absorption of the drug

Some special features

[130]

[129]

[128]

[126]

[127]

[125]

[122]

[121]

[119]

[118]

[117]

Ref.

FITC: Fluorescein isothiocyanate; GMO: Glyceryl monooleate; MPL: Monophosphoryl lipid A; OA: Oleic acid; OAM: Oleyl amine; PEI: Polyethylenimine; PG: Propylene glycol; PEG: Poly(ethylene glycol); PHY: Phytantriol; PPD: 20(S)-Protopanaxadiol.

Phytantriol Pluronic F127/ PEG200 or propylene glycol

Cubosomes

Cubosomes

Cubosomes

Cubosomes

Hexosomes

Cubosomes

Phytantriol Pluronic F127 Phytantriol Pluronic F127 Selachyl alcohol

Amphiphile stabilizer/hydrotrope

Cubosomes

LLC NP

Table 1. Recently reported drug delivery systems based on LLC NPs, which incorporate different types of therapeutic agent focused on a variety of diseases and used through different types of administration vias (continued).


A. Lancelot et al.



piperine, which has inherent anticancer activity, to these LLC NP systems has been described by the same authors to increase further the bioavailability with respect to raw PPD [112]. Indeed, piperine acts as inhibitor of the cytochrome P450, which is responsible for the rapid metabolism of PPD that decreases its anticancer efficacy. An important issue to take into account to attain sustained delivery of anticancer drugs is the control of release rates. A strategy proposed to decrease release rates is based on the formation of LLC NPs from amphiphiles that are themselves pro-drugs. Thus, these amphiphiles are both the nanocontainers and the active agents. Drummond et al. have worked with amphiphilic pro-drugs of 5-fluoroacil (5-FU), which is the oldest chemotherapeutic agent for the treatment of breast cancer and other solid tumors [113]. The amphiphilic prodrugs consist on C18-alkyl derivatives of 5-fluorocytosine, with different numbers of unsaturations on the C18-alkyl chain. The resulting structured NPs form lamellar architectures at room temperature that evolve to cubic phases at 37 C, thus providing cubosomes that enable the controlled release of the bioactive molecule, 5-FU. Furthermore, improved efficacy of these systems against the mouse 4TI breast tumors has been observed when compared with the analog capecitabine, which does not have the capacity to self-assemble into nanostructures. Likewise, a phytanyl derivative of 5-fluorocytosine, which can also undergo enzymatic hydrolysis toward 5-fluorouracyl, has been studied by these authors to form nanostructured LLC particles [114]. The NPs display a bicontinuous cubic phase that upon equilibration transforms partially into an inverse hexagonal phase giving rise to multiphase NPs, cubosomes/hexosomes, formed by the pro-drug. As a result, the pro-drug is released in a sustained manner providing higher efficacy than the model pro-drug capecitabine [93]. In addition to host anticancer drugs, cubosomes have been recently shown as possible systems for simultaneous drug delivery and imaging, what is named theranostic applications. In vitro studies have demonstrated that the incorporation of a fluorescent probe provides cubosomes that have the ability to image living cells together with the delivery of an anticancer drug, quercetin, while maintaining the inner structure [115]. Other therapies Other poorly water-soluble drugs for other applications different than anticancer activity have been incorporated to LLC NPs providing systems for oral delivery. Entrapment of drugs, such as simvastatin, cinnarizine or ibuprofen, within LLC NPs have shed light into the influence of the inner NP structure on the release rate and drug bioavailability. Simvastatin was entrapped into GMO cubosomes with high encapsulation efficiency [116]. In vivo studies showed enhanced bioavailability likely due to increase absorption rather than improved release. Phytantriol-based cubosomes loaded with cinnarizine [117] and ibuprofen [118] also provided drug delivery systems with significant enhancement of oral bioavailability with respect to the 3.2

corresponding drug suspension. Furthermore, in contrast to GMO-cubosomes, phytantriol cubosomes maintain their LC structure and can be retained in the stomach unaltered, what facilitates the slow release of poorly water-soluble drugs [117]. Similar observations of stability and bioavailability enhancement have been reported for cinnarizine-containing hexosomes based on selachyl alcohol [119]. These studies provide useful information about the capability of non-digestable LC materials such as phytantriol or selachyl alcohol for sustained oral delivery of poorly water-soluble drugs. Ophthalmic delivery is also a topic of interest for which LLC NPs are also being considered as effective drug vehicles [120]. Recent studies reported by Gan et al. show that aspects such as improved preocular retention, reduction of ocular irritancy and enhanced bioavailability can be attained using cubosomes as nanocarriers for ocular drugs. The trans-corneal permeation of dexamethasone (DEX) [121] and flurbiprofen [122] is enhanced when these drugs are formulated in cubosomes. Indeed, the drug-loaded cubosomes are retained in the preocular region much longer than that of the corresponding solutions administered through eye drops, and this enhances their ocular bioavailability. Moreover, the DEX-cubosomes formulation is confirmed not to affect the corneal structure and tissue integrity. For flurbiprofen, cubosomes formulation reduces its inherent irritancy. Therapies based on biomacromolecules The use of LLC NPs for the delivery of biomacromolecules has deserved extensive attention due to the current interest on protein-based and nucleic acid-based therapeutics. Bicontinuous cubic LLC phases [123] as well as reverse hexagonal phases [17,124] have been studied as matrixes for encapsulation and delivery of proteins, peptides or nucleic acids. One drawback about protein-based therapy is that proteins and peptides are susceptible of hydrolysis by enzymes, and this decreases their bioavailability and hence their efficacy as therapeutic agents. Also in these cases, the previous cited cubosomes made of GMO/pluronic F127, used by anticancer applications, have provided improved oral absorption of cyclosporine A, a highly lipophilic cyclic polypeptide immunosuppressant, as compared with a commercial self-microemulsifying concentrate [125]. Proteins related with treatment of central nervous system diseases, which must cross the brain blood barrier, have also been tested for efficient delivery through LLC NPs. For example, cubosomes encapsulating S14G-HN, a very promising anti-Alzheimer peptide, have been prepared and studied [126]. In order to favor intra-brain delivery, the GMO cubosomes have been functionalized with odorranalectin (OL), a protein that acts as a delivery adjuvant improving the mucoadhesion of the nanoparticles during nasal delivery. These OLsurface-decorated cubosomes have led to S14G-HN carriers with enhanced therapeutic effect via intranasal administration, as well as sustained release with 30% of the peptide drug liberated during in 2 weeks. 3.3


557


A. Lancelot et al.

With respect to nucleic acid-based therapies, recent experiments have been performed aimed at delivering siRNA into the skin in an efficient way [127]. Indeed, a delivery system consisting on GMO-based hexosomes has been reported containing either polyethylenimine or oleylamine in their cationic form that allow them complexing siRNA. This formulation gives rise to an efficient topical non-viral vehicle that promotes higher penetration into the skin without causing skin irritation. Current investigations on strategies to improve the immunogenicity of protein--peptide-based vaccines propose LLC NPs as promising candidates as vehicles for the delivery and release of vaccines. Indeed, modern approaches to vaccine design are based on the use of antigenic fragments of pathogens (proteins, peptides or DNA). The challenge remains in potentiating the immune response, which is low when these moieties are directly administered. Ovalbumin has been employed as a model subunit antigen to investigate the potential of cubosomes for vaccine delivery with increased immunogenicity. Phytantriol and GMO cubosomes, with different proportions of the stabilizer, pluronic F127, and a hydrotrope, ethanol, PEG or propylene glycol, that helps their solubilization in water, have been prepared and studied as for their capacity to incorporate and release ovalbumin in a sustained manner [128]. Recent approaches toward efficient cubosomes-based vaccine delivery systems have consisted on the incorporation of adjuvants, such as the Toll-like receptor antagonists imiquimod and monophosphoryl lipid A, to the nanoparticulate system [129]. In general, it is concluded that cubosomes improve antigen-specific immune responses when compared to liposomes. In order to enhance immunogenicity further, the design of slow release systems has been investigated. In this respect, ovalbumin-loaded cubosomes have been entrapped within chitosan thermogels [130]. It is proposed a tight binding between the antigen-containing cubosomes and the chitosan gel matrix due to their opposite surface charges, and this helps to slow down the release of the antigen.

4.

Conclusion

The interest and versatility of LLC NPs can be deduced from all studies mentioned above and others. It is concluded that LLC NPs are able to entrap different therapeutic agents, ranging from small drug molecules to biomacromolecules, focused on a variety of diseases and used through different types of administration vias. Current investigations on strategies to improve the immunogenicity of protein-peptide-based vaccines propose LLC NPs as promising candidates as vehicles for the delivery and release of vaccines. Nevertheless, some important issues still remain unsolved and they need to be approached by multidisciplinary teams with tight collaboration between physicians, chemists, biologists and physicists. 558

5.

Expert opinion

In general, LLC NPs have proven to be extremely useful tools for applications in drug delivery. Their versatility allows transporting therapeutic agents of different nature and administrating them through different ways: oral, topical (skin, ophthalmic) or intravenous. In addition, their easy preparation together with the low toxicity of molecules employed for their production makes them promising candidates for pharmaceutical applications. Nevertheless, as any other chemical system developed for drug delivery applications, LLC NPs have issues to be tackled, such as adjustment of release rates, but have many beneficial features that strengthen their potentiality. One major issue of these systems is related to the sustained drug release. While it has been reported the sustained release of small-molecule drugs form bulk LLC phases [131], the increase of contact surface area with aqueous surrounding medium seems to lead to fast release of small-molecule drugs when encapsulated within LLC NPs. Release rates depend on the internal structure of the particles. Those that present discontinuous water compartments, that is, cubic micellar and hexagonal phase, show lower release rates than the bicontinuous cubic phase, that is, cubosomes, which forms continuous water channels. For the former, cylindrical micelles forming the hexosomes may permit faster release than spherical micelles forming cubic micellar phase due to longer distances for diffusion of small molecules. Burst release has been generally observed for cubosomes, and especially for small drug molecules encapsulated, the release of which seems to be only limited by diffusion. It is a general opinion that small poor-water-soluble molecules are released from cubosomes during the first minute after injection, and this can be even faster if measurements are done under sink conditions [132]. For example, cubosomes encapsulating diazepam and chloramphenicol burst release the drug regardless the nanoparticle inner structure [133]. In contrast, highly lipophilic drugs such as amphotericin B [134] or PPD [112] show low release rates from cubosomes. This may be due to the high affinity of these water-insoluble drugs with the hydrophobic domain in the cubic phase that makes the release from the nanoparticles difficult. In order to attain sustained delivery of small-molecule drugs, modifications on the formulation of LLC NPs have been successfully assayed. Hydrophilic PEG barriers have been employed to retain further a lipophilic drug within the nanoparticle [107,108]. Also, the incorporation of pro-drugs with amphiphilic nature makes the drug itself to form part of the structure of the nanoparticles, and this makes its release and transformation into the active drug more prolonged in time [114]. Contrary to the release of small molecules, the release of proteins is not governed by the partition coefficient between channeled water and the hydrophobic region within the nanoparticle [83], and this helps to provide sustained release,




making LLC NPs very convenient drug delivery systems for protein-based therapies and vaccines. Bovine serum albumine (BSA) and cytochrome C release in a sustained manner from cubosomes. Also, ovalbumin showed sustained release from cubosomes [128], and cyclosporine A release from cubosomes was measured as

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Development of multicore hybrid particles for drug delivery through the precipitation of CO2 saturated emulsions.

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Alginate Particles as Platform for Drug Delivery by the Oral Route: State-of-the-Art.

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