A plethora of research has been carried out on lyotropic liquid crystalline nanostructures and their advantageous applications as drug delivery vehicles. Among the different lyotropic liquid crystalline mesophases, the non-lamellar ones, consisting of well-defined networks of aqueous channels and lipid bilayer membranes, namely, the bicontinuous cubic and inverse hexagonal mesophases, exhibit significant scientific research and experience rapid technological developments in the last decades. They can be applied either as bulk phases or as fabricated colloidal nanocarriers, e.g., cubosomes and hexosomes.

Non-lamellar lyotropic liquid crystalline nanosystems (LLCN) can be utilized as drug, protein, peptide, or nucleic acid delivery nanosystems, being able to host a wide variety of hydrophilic, hydrophobic, and amphiphilic small molecules and biomacromolecules. They exhibit major advantages, including the ability of controlled release, providing that a careful design has been carried out to manipulate the formation of their internal organization. They can be administrated by various routes, resulting in enhanced drug bioavailability and therapeutic efficiency. Moreover, they can be easily functionalized by the incorporation of smart biomaterials, e.g., stimuli responsive molecules, surfactants, or polymers, towards the development of “on-demand” drug release delivery vehicles.

2. Self-Assembly and Formation of Liquid Crystalline Nanostructures

Amphiphilic molecules, such as the lipids, being dispersed into water, have the ability for self-assembly into different mesophases, as a function of concentration and temperature, eventually forming lyotropic liquid crystalline mesophases ^[1][2]. The molecules position themselves in such a way in order to minimize the free energy of the system, by exposing the hydrophilic regions to the aqueous environment and tightly packing their hydrophobic domains in the interior as an effort to minimize the interface with the existing solvent. The type of the self-assembled nanostructure is mainly governed by the surfactant molecular shape that can be predicted by the critical packing parameter (CPP) (CPP = v/(A ∗ l)). The CPP is a geometrical value consisting of the ratio between the volume of the hydrophobic lipid tail (v) and the product of the cross-sectional lipid head area (A) and the lipid chain length (l). Taking into account the changes of the CPP, order–order transitions associated with the change in the curvature of the water–lipid interface can be forecasted. Cylindrical shaped surfactant molecules (CPP ≈ 1) tend to form planar membranes (fluid lamellar (L_a) phase), and conversely, cone-shaped (CPP < 1) and wedge-shaped surfactant molecules (CPP > 1) prefer the formation of convex (type 1/normal type, oil-in-water (O/W) phases) and concave (type 2/inverted type, water-in-oil (W/O) phases) interfaces, respectively. The nanostructures of inverted-type/type 2 liquid crystalline phases and micellar solutions are independent of water content under full hydration conditions and therefore are stable against water dilution and particularly attractive in the development of drug delivery systems ^[1][3][4]. The bicontinuous cubic (Q_II) phase is a very interesting, complex structure, consisting of a 3D network, separating two distinct, continuous but nonintersecting, hydrophilic sections/water channels. Three types of the bicontinuous cubic (Q_II) phases with different space group symmetry have been identified in various lipid systems, named as Im3m (primitive type), Pn3m (double-diamond type), and Ia3d (gyroid type) assemblies. Regarding the inverse hexagonal (H_II) phase, it is a 2D structure consisting of water-filled cylindrical rods (hydrophilic nanochannels), each surrounded by a lipidic bilayer and following a long-range two-dimensional order, being hexagonally close densely packed in a two-dimensional continuous hydrophobic medium ^{[1][5][6][7][8][9][10]}. Moreover, the “melted” cubic mesophase, called as sponge phase (L₃), has also been identified, containing a bicontinuous bilayer, but without a long-range order structure ^[11]. Finally, the Fd3m micellar cubic phase is composed of two discrete inverse micelle populations organized into a double diamond network that is isolated from the external environment, enabling the lowest diffusion coefficient among the studied mesophases.

3. Non-Lamellar Lyotropic Liquid Crystals as Drug Delivery Nanosystems

Lyotropic liquid crystals, focusing on the non-lamellar ones, have gained increased research interest in the field of pharmaceutics, being ideal as therapeutic drug delivery nanosystems. They can be used as protein, peptide, and nucleic acid delivery nanosystems, and are administrated either as colloidal nanoparticles or as bulk phases. Their utility to carry diagnostic probes for biomedical imaging has also been well investigated. Some of their most important advantages that make them ideal drug delivery nanosystems are the following: great structural variety, nanostructure versatility, high grade of internal organization, and tunable morphological characteristics. Due to these advantages, they are able to carry great volumes of cargo, even greater than liposomes. They can also be loaded with different kinds of agents such as amphiphilic, hydrophobic, and hydrophilic agents, also providing sustained release of their content. Colloidal dispersions of cubosomes and hexosomes possess low viscosity that ensures flexible preparation and handling, particularly in the engineering of parenteral dosage forms. These carriers can also be easily functionalized through an array of surface engineering strategies. Moreover, they are characterized by great bioavailability and lower toxicity in comparison to other systems. Some basic invaluable properties that they provide as therapeutic nanosystems are the following: (1) improving drug bioavailability and reducing drug toxicity, (2) enhancing the stability of drugs, (3) exhibiting sustained or controlled drug release, (4) increasing the penetration of drugs, and (5) providing efficient solubilization of poorly water-soluble drugs. There are many different routes of administration, varying from oral, intravenous, intraperitoneal, ophthalmic, subcutaneous, intra-periodontal, topical, to transdermal, targeting a diverse range of sites, including internal organs, brain, eyes, and skin ^{[1][6][12][13][14][15]}.

3.1. Controlling Drug Release Kinetic

A significant advantage of the liquid crystalline nanosystems is their ability to control the release kinetics of the contained drug. For example, Lee et al. ^[16] studied the in vitro sustained release behavior of a number of model hydrophilic drugs with various molecular weights. According to the obtained results, the cumulative amount of drug diffusion through the matrix followed a linear relationship with the square root of time, which represented a Higuchi diffusion controlled release profile, as was also previously confirmed by Boyd et al. ^[17] by testing a series of model hydrophobic and hydrophilic drugs (paclitaxel, irinotecan, glucose, histidine, and octreotide). Generally, hydrophilic drugs are located close to the polar head or in the water channels, while hydrophobic drugs are loaded in the lipid layer, and amphiphilic drugs at the interface. For example, Esposito et al. ^[18] studied the performance of cubosomes as sustained percutaneous delivery systems with the model hydrophobic drug indomethacin. A prolonged release of indomethacin was observed by the cubosomes, thus exhibiting a significant long-lasting anti-inflammatory activity. The authors suggested that the content GMO lipid interacted with the stratum corneum lipids, causing the formation of a cubosomes depot in stratum corneum, from which the indomethacin was released in a controlled manner. The release of hydrophobic drugs is primarily controlled by the partition coefficient of the drug, its diffusion into the lipid bilayer, and its diffusion into the surrounded aqueous environment. For example, when Clogston et al. ^[19] increased the hydrophobicity of hydrophilic model drug tryptophan by alkylation, they observed a delayed release from GMO cubic phase that was attributed to an increase of its partitioning within the lipid layer.

3.2. Non-Lamellar Lyotropic Liquid Crystalline Nanosystems (LLCN) for Anticancer Therapy

There are various examples in the literature regarding the successful application of liquid crystalline nanosystems in anticancer therapy. There are several examples in the literature describing the intravenous or subcutaneous in vivo administration of different anti-tumor drugs, such as docetaxel ^[20], paclitaxel ^[21][22][23], and 5-fluorouracil ^[24]. Another reported strategy to decrease release rates and attain sustained release during anticancer therapy is the formation of liquid crystalline nanoparticles from amphiphiles that are themselves pro-drugs, acting simultaneously as nanocontainers and active agents. Gong et al. ^[25] worked with amphiphilic pro-drugs of 5-fluoroacil (5-FU) against breast cancer and other solid tumors. For example, amphiphilic prodrugs consisting of C18-alkyl derivatives of 5-fluorocytosine, with different numbers of unsaturations on the C18-alkyl chain, have been reported ^[25].

3.3. Non-Lamellar LLCN Improving Oral Bioavailability

Liquid crystalline dispersions and especially the cubic ones are considered to exhibit significant advantages towards the oral delivery, because their structure can protect the drug against degradation in the gastrointestinal (GI) tract. In the physiological environment of the GI tract, liquid crystals consist of an oil phase and a solubilized micellar phase during lipid digestion, which enhances drug solubility and bioavailability in the lumen. Another common advantage of liquid crystals is the reduction in systemic toxicity of high-toxicity drugs, such as the antibiotics and chemotherapeutics, thus improving therapeutic efficacy. Moreover, they increase the possibility of the drug to penetrate across the endothelial cell membrane, exhibiting improved uptake mechanisms, while their large hydrophilic surface, due to the presence of the water channels, allows for easy contact with the endothelial cell layer and can cross the water layer. Their internal nanostructure increases the absorption of hydrophilic drugs, because it enhances their bioavailability by prolongation of gastric residence time, as well as increasing the absorption of hydrophobic drugs, due to the increased stability and improved membrane penetration ^[26][27].

3.4. Non-Lamellar LLCN for Skin Administration

The topical administration of drugs with liquid crystals, for example though the skin, can be successfully utilized, because liquid crystals can localize the drug within the stratum corneum and improve drug penetration by increased transdermal permeability. The cubic phase may interact with the stratum corneum structure, leading to the formation of a cubosomal mixture from GMO and the native lipids of stratum corneum that acts as a cubosome depot, where a controlled release has taken place. GMO can interact with the stratum corneum, being actually an absorption enhancer that boosts the intercellular lipid fluidity. The cubic phase can also form a biological membrane-like structure with a strong bioadhesive property to the skin, while it also exhibits the proper viscosity and mucoadhesiveness for topical applications. Apart from the liquid crystalline bulk phases that possess proper viscosity and mucoadhesiveness for topical applications, the liquid crystalline nanoparticles are also used due to their greater ease of handling, reduced viscosity, and the ability to deliver higher drug payloads than the bulk analogues. Tissue hydration is an extra advantage of the water included in the liquid crystalline nanostructure ^[26][28][29].

3.5. Non-Lamellar LLCN for Ocular, Brain, and Pulmonary Delivery

Non-lamellar liquid crystalline nanosystems have also been investigated for ocular topical delivery. Eye drops exhibit key problems including corneal permeability, retention times, and low solubility of some drugs, resulting in poor drug bioavailability that liquid crystalline formulations overcome. Several cases in the literature report liquid crystalline nanosystems formulated as eye drops, being in vivo tested in rabbit models, delivering anti-inflammatory drugs, such as dexamethazone ^[30], flubiprofen ^[31], and glaucoma treatment, such as pilocarpine ^[32] and brinzolamide ^[33]. For example, cyclosporine A was incorporated in GMO nanoparticles, stabilized by Poloxamer P407, resulting in a decreased ocular irritancy and improved corneal penetration, when compared with a control cyclosporine A formulation ^[34]. Liu et al. ^[35] developed a liquid crystalline nanosystem, owning a H_II nanostructure, prepared by GMO, wherein they incorporated the absorption enhancer Gelucire 44/14 and octadecyl-quaternized carboxymethyl chitosan adjuvants for the ocular delivery of tetrandrine, an agent against chronic keratitis, cataracts, retinopathy, and glaucoma, which exhibited enhanced transcorneal penetration of tetrandrine in a rabbit model. Similarly, GMO cubosomes of Pn3m internal symmetry stabilized with Poloxamer P407 and loaded with Timolol maleate, a beta blocker commonly used to treat glaucoma, exhibited higher penetration ex vivo than the commercially available product, increased retention times in vivo, and had an enhanced ocular pressure lowering effect, while neither cytotoxicity nor histological impairment in the rabbit corneas were observed ^[36]. More recently, Alharbi et al. ^[37] developed a ciprofloxacin-cubosomal in situ gel in order to improve eye permeation, prolong the ocular retention time, and enhance the antimicrobial activity of the antibiotic, compared with commercial drops.

3.6. Injectable Non-Lamellar Liquid Crystalline Depot Systems for Sustained Delivery

Sustained-release injections are designed to release a drug substance at a predetermined rate to maintain its effective plasma concentration for months. Although injectable polymeric microspheres (for example, from poly(lactic-co-glycolic acid (PLGA)) or implants have been developed as injectable sustained release systems in various studies, they are difficult to be prepared and can reduce the stability of protein drugs. These disadvantages can be overcome by injectable liquid crystal-forming systems (LCFS). The liquid crystalline mesophase is spontaneously formed from the LCFS in an aqueous fluid. The formed tortuous networks of their aqueous nano-channels are able to sustain the drug release. For example, for the efficient therapy of hepatitis B using entecavir, the drug must be taken consistently every day, due to disease re-occurrence in cases of discontinuation. Kim et al. ^[38] developed a novel LCFS of hexagonal phase for the sustained delivery of entecavir. A pharmacokinetic study in rats was carried out, showing sustained release of entecavir for 3–5 days from LCFS formulation. In another study ^[39], LCFS containing sorbitan monooleate (SMO) was investigated for sustained release injections of leuprolide acetate. The LCFS formed the hexagonal liquid crystalline phase. Both in vitro release test and in vivo pharmacokinetic and pharmacodynamic studies showed a sustained release of leuprolide. When compared with a commercial depot formulation of leuprolide, the LCFS exhibited a significantly reduced initial burst with sufficient suppression of testosterone. Later, Báez-Santos et al. ^[40] showed that tocopherol acetate can play a major role in mitigating drug release by altering the physicochemical properties of the liquid crystalline matrix, indicating the use of tocopherol acetate as a tailoring agent. More specifically, formulations with low amounts of tocopherol acetate and higher water uptake capacities had a higher propensity towards erodibility and thus in vivo biodegradability.

3.7. Non-Lamellar LLCN as Vaccines

Cubosomes and hexosomes have been referred to as vaccine adjuvants that carry both immune enhancers and antigens to regulate the immune response of the body. It has already been reported that nonlamellar structures (e.g., cubic and hexagonal) show fusogenic properties that are able to deliver antigens directly to the cytosol of antigen-presenting cells (APCs), stimulating cytotoxic T lymphocyte (CTL) immune responses. Most protein antigens are negatively charged at neutral pH, as are cubosomes. To link antigens on cubosomes, a cationic surfactant is usually utilized. Moreover, the supramolecular structure of the lyotropic liquid crystalline phase is considered to influence the immunostimulatory activity of lipid-based nanocarriers. Rodrigues et al. ^[41] designed PHYT hexosomes with the immunopotentiator monomycoloyl glycerol-1 (MMG-1). The effect of the nanostructure on the adjuvant activity was studied by comparing the immunogenicity of phytantriol/MMG-1 hexosomes with MMG-1-containing liposomes in mice. According to the results, the MMG-1-based hexosomes potentiated significantly superior MOMP-specific humoral responses in comparison with liposomes. The authors suggested that hexosomes exhibit great adjuvant potential, and engineering of the supramolecular structure can be used to design adjuvants with customized immunological properties. Another novel self-adjuvanting hexosome-based PHYT nanocarrier with mannide monooleate (MaMo), which is an emulsifier applied in several adjuvant systems, was developed by Rodrigues et al. ^[42].

4. Development of Stimuli-Responsive Non-Lamellar LLCN

Stimuli-responsive nanosystems are designed to exploit in a “smart” way the altered conditions (for example temperature, pH, and enzyme concentration) that take place in pathological tissues, causing triggered content release in the targeted tissue and resulting in enhanced bioavailability, prolonged blood circulation time, and overall increased therapeutic efficacy. Although the P407 is a commercially available and biocompatible polymer, having been proven to be an excellent stabilizer, it lacks functional targeting groups or stimuli-responsive groups. Thus, the upgrade of the conventional liquid crystalline nanosystems to stimuli-responsive ones by adding the suitable biomaterials may elicit on-demand drug delivery ^[43].

4.1. pH-Responsive Non-Lamellar LLCN

pH-responsive systems are able to exploit well-characterized pH differences inside the human body. pH differences, where the pH-responsive nanosystems usually target, exist between normal blood and pathological tissues (e.g., due to infection, inflammation, and cancer tending to acidic pH), between certain intracellular compartments (i.e., cytosol, endosomes, and lysosomes), as well as along the gastrointestinal track. “Smart” molecules such as polymers, lipids, and peptides that are biocompatible and responsive to particular pH conditions, due to their functional ionizable groups, are utilized for this scope ^[44][45].

4.2. Thermoresponsive Non-Lamellar LLCN

The “smartness” of the thermoresponsive nanosystems is owned to the ability of their biomaterials to undergo temperature-dependent phase and conformational transitions that cause structural changes in the resultant systems, triggering the controlled release of their content. It should also be taken into account that pathological tissues (40–42 °C), such as tumors, exhibit elevated temperatures compared to physiological ones. As for the lyotropic liquid crystalline nanosystems, their phase transition is strictly influenced by the temperature change and can be further monitored by accurate design of their formulation. The most commonly utilized GMO lipid has been proven to perform typical liquid crystal thermal expansivity, while the lattice parameter of all GMO mesophases decreases with increasing temperature ^[10][46]. By examining a similar lipid-forming non-lamellar liquid crystalline mesophase, namely, the monolinolein lipid (MLO), it was found that MLO particles have a cubic internal structure at 25 °C that is transformed to inverse hexagonal after heating at 58 °C and to inverse micellar phase upon further heating to 87 °C, in a reversible manner. MLO-based particles also expel water upon heating (deswelling/shrinkage) and take up water again upon cooling (swelling) also in a reversible way, termed as the “breathing mode” ^[47]. In the case of P407-stabilized PHYT particles, an inverse micellar solution is observed above 50 °C, while the cubic structure of Pn3m symmetry re-appears with cooling down ^[48].

5. Conclusions

Non-lamellar lyotropic liquid crystalline nanosystems have gained increasing interest recently in the pharmaceutical nanotechnology field. The present review focused on the cubic and hexagonal nanosystems, whose high level of internal organization provides great opportunities. The cubic and hexagonal nanostructures display high solubilization and encapsulation capacities for a variety of guest molecules, including drugs, peptides, proteins, and nucleic acids, as well as the ability to protect the active molecules, improve their bioavailability, and control their release kinetics profile. The lyotropic liquid crystalline nanoparticles are able to demonstrate superior in vitro and in vivo performance, enhancing the therapeutic efficiency in a diverse range of applications, being administrated by various routes. The studied liquid crystalline nanosystems also exhibit great versatility and are strictly influenced by the environmental and formulation parameters, a characteristic that people can exploit towards the “on-demand” controlled drug delivery, as well as the development of stimuli responsive systems. There is an emerging need for continuous development of smart innovative biomaterials (surfactants, polymers, etc.) that will upgrade and further functionalize the already existing systems. Moreover, there is a great challenge regarding the careful and rational design that should be carried out during the development process of such systems by taking into account all the different interactions that may take place among the constituting biomaterials or the effects of external parameters, such as the environment. Finally, the application of robust and standardized characterization techniques is absolutely necessary in order to precisely evaluate the characteristics of such complex structures.