Lyotropic liquid crystals for parenteral drug delivery

The necessity for long-term treatments of chronic diseases has encouraged the development of novel long-acting parenteral formulations intending to improve drug pharmacokinetics and therapeutic efficacy. Lately, one of the novel approaches has been developed based on lipid-based liquid crystals. The lyotropic liquid crystal (LLC) systems consist of amphiphilic molecules and are formed in presence of solvents with the most common types being cubic, hexagonal and lamellar mesophases. LC injectables have been recently developed based on polar lipids that spontaneously form liquid crystal nanoparticles in aqueous tissue environments to create the in-situ long-acting sustained-release depot to provide treatment efficacy over extended periods. In this manuscript, we have consolidated and summarized the various type of liquid crystals, recent formulation advancements, analytical evaluation, and therapeutic application of lyotropic liquid crystals in the field of parenteral sustained release drug delivery.


Introduction
The enormous progress in nano-drug delivery has led to the reemergence of the parenteral route as one of the most crucial forms of drug delivery [1,2].Attaining controlled release via depot systems or by utilizing specialized drug carrier platforms is on the rise and plays a major role in the ever-emerging field of pharmaceuticals and biopharmaceuticals [3,4].These systems serve as potential alternatives to the conventional drug delivery vehicles in terms of their design to modulate drug release kinetics, safety, efficacy, and achievement of desired bioavailability due to their innovative nature [5][6][7].Intending to utilize the intrinsic physicochemical properties of nanomaterials, lyotropic liquid crystals (LLC) evolved and have garnered immense interest due to their vast applicability in parenteral, transdermal, topical as well as oral drug delivery systems.
Liquid crystals (LC) are a unique category of drug delivery platform that possesses the orientation orders of both solid and liquid substances.They are found to imbibe the stable orientation order of solids and a flowy orientation of the liquids which puts them in a condensed state of matter [8].Out of the two liquid crystals, namely thermotropic and lyotropic, the lyotropic type of liquid crystals are the ones that are used for the fabrication of drug delivery systems due to their ability to exhibit liquid crystallinity as a function of concentration [9].This property further enables them to solubilize drugs, especially biomolecules, in injection solvents.The LLC consists of amphiphilic molecules and is formed in presence of solvents with the most common types being cubic, hexagonal and lamellar mesophases [10,11].These mesophases possess interesting viscoelastic properties that are beneficial for dispersion technology and also impart thermodynamic stability along with an ordered internal nano-structure that serves as a drug release matrix [12].LLCs garner immense attention owing to their distinctive microstructures and versatile physicochemical features.They are famous for their ability to deliver biomolecules like peptides and proteins by imparting protection from oxidation as well as hydrolysis.
Over the last decade, self-assembled LLCs of polar lipids have acquired a higher appeal owing to remarkable structural features, versatile applications, and amphiphilic nature [13].Their amphiphilic nature allows them to form a self-assembled system, and the hydrocarbon chains in the lipids, upon melting, are disordered and have a long-range order in their layers and are the characteristic features of these lipids [13,14].Lipid-based LLCs are also referred to as 'reversed liquid crystalline mesophases' and can be cubic, hexagonal or micellar cubic systems that exhibit a special thermodynamic equilibrium in the presence of excess water [15].These inverse forms of cubic and hexagonal phases result from the liquid crystal-forming system (LCFS) in aqueous fluids, and they possess nano-channels that can act as passages for the sustained release of drugs.A variety of amphiphilic molecules have been explored for their LCFS ability including glycerol monooleate, glycerol dioleate, oleyl glycerate, phytantriol, and phytanyl glycerate, to name a few [16].Sustained release of leuprolide, for one month, was achieved using LCFS made using sorbitan mono-oleate as the hexagonal liquid crystal former for the injectable formulation [17].These systems lacked in-depth explorations in the past and are now being rigorously studied, which has paved the way for the fabrication of in-situ forming hexagonal LLC matrices using drug release tailoring agents like tocopherol acetate [18].
The injectable in-situ gel forming LLC-based sustained release system of the local anesthetic bupivacaine hydrochloride was also developed as a strategy for long-acting postoperative analgesia [19].Researchers are also investigating the influence of additives on the LLC architecture.A group of researchers used molecular dynamic simulations and demonstrated that the polarity of additives is responsible for variability in critical packing parameter (CPP) that further leads to the formation of different types of LLC structures [20].This parameter can be used as a guiding factor in determining the LLC self-assembly and LLC structure outcome.An approach used for tailoring the LLCs to accommodate macromolecule delivery is the addition of hydration-modulating agents like sucrose stearate.Sucrose stearate was added to phytantriol cubic phase along with oleic acid as a pH trigger, both of which increased the size of internal water channels and enabled control over the swellingde-swelling phenomenon, respectively [21].This has been proposed as a potential approach to control the loading and release of large macromolecules like antibodies from LLC systems.
Cubic and hexagonal nanosystems have immense potential and cubosomes, in particular, have been shown to potentiate the adjuvants and enhance their immunostimulatory character making them a system of interest in vaccine development [22,23].There have also been comparisons between LLCs and self-micro emulsifying drug delivery systems (SMEDDS) wherein, SMEDDS are said to form liquid crystals at specific compositions (ratios) of oil, water, and surfactants used.LLC hexagonal phase was observed using small-angle X-ray scattering (SAXS) in SMEDDS composed of fatty acid derivatives of propylene glycol, where aqueous dilution with 40% water resulted in a change of surfactant geometry leading to LLC formation [24].Another study based on SAXS analysis was performed to study in situ gel-forming sustained-release microemulsion (ME) for bufalin (anti-tumor drug) [25].A phase transition of the ME system into LC was observed upon progressive dilution, where the drug got encapsulated in the low viscosity ME but got released from the in situ generated lamellar LC structure enabling a sustained release of bufalin [11,25].The ability to deliver complex molecules along with their tailor-friendly structure and potential for enabling the desired release via phase transition phenomena make LC novel carriers and demonstrates a need for their extensive research, especially towards parenteral product development.

Types of liquid crystals
LCs are classified based on the subatomic structure of particles resulting in positional order in lattice and orientation order where particles are directed in the same manner [26].They are classified in the following ways:

Thermotropic liquid crystal
Thermotropic word is coined from "thermo" which means temperature and "tropic" is forms i.e the molecules which change their form due to temperature are thermotropic liquid crystals [27].Generally, the crystals on heating lose their long-range positional and orientation order thereby converting to the isotropic liquid phase.They are classified as nematic, smectic, and cholesteric.

Smectic (Sm) phase
In the smectic phase, the molecular arrangement is seen with interlayer spacing where the layers are arranged with their long axes.In chronological order of their detection, they are referred to as smectic A, smectic B, smectic C, etc [28].Moreover, these phases are either tilted or normal to the plan.The smectic A and smectic B phases are orthogonal (non-tilted) phases whereas smectic C, smectic F, and smectic I are tilted phases [29].

Nematic (N) phase
The nematic phase is the least ordered simplest mesophase structure [30].The nematic phase is characterized by the 1-D orientational order of the molecules but lacks the positional order.The "director" (n) parameter refers to the average orientation of molecules within the phase, while the degree of order is quantified by an order parameter, S [27,31].The equation is as follows.
where θ is the angle between an individual molecule and the director, and this is summed over all molecules.Generally, order parameter S for a nematic phase ranges from 0.4 to 0.7.They are anisotropic due to the parallel orientation of the molecules along their long axes.Their major area of application includes electronic display [32].

Cholesteric phase
It is also known as "chiral nematic liquid crystal" [33].The molecules are arranged in helical order in the successive layers.When planepolarized light interacts with such chiral structures, the rotation of light in the helical direction is observed.If the pitch of these helical structures corresponds to ~ 400-800 nm wavelength i.e visible region of the light then the phase appears to be colored [34].The pitch of the helix is dependent on the temperature which simultaneously affects the reflected wavelength.Additionally, the helix can get unwound by the change in the electric field which can be employed for a stimuliresponsive drug delivery system [34,35].

Lyotropic liquid crystals
The lyotropic term is coined from lyo means 'solvent' and tropic is 'forms'.The self-assembly of the structure depends on the concentration of water and other solvents.These are further categorized as lamellar, hexagonal, and cubic phases.

Lamellar phase
The lamellar phase is bilayers of amphiphilic molecules adjacent to oil and water phases [36].The bilayer structure minimizes the interaction of oil with an aqueous phase.This can be useful for the encapsulation of drugs [37].The thickness of the bilayer increases with an increase in the concentration of water or drug depending on the partitioning in particular oil or water phase [38].

Hexagonal liquid crystals
The surfactant micelles transform into a higher hierarchy of cylindrical micelles.These cylinders further aggregate to form higher-order 2-D structures in the hexagonal phase [39].Depending on the aqueous V.P. Chavda et al. condition, they form a normal hexagonal phase or reverse hexagonal phase.Under normal hexagonal phase, hydrophobic acid chain inward and polar head projects towards the aqueous medium [40].Whereas, in anhydrous or low aqueous conditions, the reverse structure is formed.The hydrophilic tail forms the inner core and hydrocarbon chains are facing the external non-aqueous media forming a reversed hexagonal phase.Hexagonal liquid crystals are characteristic of fan-like texture under polarizing light microscopy.It has enormous pharmaceutical application due to increased drug loading, greater in-vivo stability, and potential for delivery of macromolecules like peptides and proteins.

Cubic liquid crystals
The cubic liquid crystal is formed by a continuous curved bilayer of lipid (~ 3.5 nm thickness) with non-contacting aqueous channels providing a greater interfacial area of the cubic channel (~ 400 m2/g).The cubic phase is further divided into three classes: "the body-centered cubic lattice phase, gyroid lattice cubic lattice phase, and double diamond lattice cubic phase."These are stiffer compared to other mesophases thus providing controlled drug release [41,42].The advantage of this phase is higher drug loading capacity.Additionally, cubic phases are more stable to dilution compared to other LC phases.However, attributed to the prevalence of a quantity of water, it is unable to integrate highly water-soluble drugs [43,44].

Importance of lyotropic liquid crystals
In the last two decades, prolonged-release injectable formulations such as implants and microspheres have gained a significant market share.These include polymeric compact tablets of steroids (Norplant®), PLGA microspheres [Lupron Depot® (leuprolide), Nutropin® (somatropin), and Risperal® Consta® (risperidone)], and preformed implants (Zoladex® implant] [45].The limitations of microspheres and preformed implants are difficult as well as expensive manufacturing steps.Furthermore, time-consuming and complex administration procedures with the problem of unfinished mixing of the microparticles, and syringe blockage resulting in dispensing of an insufficient dose are often seen [46].To overcome the limitations of technologies including microparticles and preformed implants, enormous attempts have been done to generate substitute simple injectable formulations that exhibit characteristics such as immediate, pain-free, ease in injection via minute needle sizes, and low production cost. LLCs have been investigated as drug carriers and can be administered through parenteral, oral, topical, nasal, transdermal, and ophthalmic routes [43,47,48].LLCs can incorporate and extend the release of various drugs including hydrophilic, hydrophobic molecules, proteins, peptides, and nucleic acid [49,50].The lyotropic liquid phase (LCP) is a self-assembled system, commonly consisting of amphiphilic molecules (polar lipids and surfactants) and solvents.In the lyotropic phases, amphiphilic molecules are surrounded by solvent molecules to offer fluidity to the system [51].The presence of solvent and water molecules modify the self-assembly of microstructures.The particles are randomly arranged without any structure at low concentrations of amphiphilic molecules.As the concentration increases, amphiphilic molecules instantaneously arrange into vesicular structures known as micelles.In micellar structures, the hydrophobic tail of the amphiphilic molecules hides inside the core of the micelle, while the hydrophilic head groups are exposed to an aqueous solution [52].The arrangements would become ordered at elevated concentrations.A hexagonal columnar phase is a typical phase in which the amphiphiles create large cylinders (again with a hydrophilic surface) that aligned individuals into the hexagonal lattice.Additional increases in concentrations generate ordered structures i.e. hexagonal columnar phase which is a long cylindrical lattice and lamellar phase that lengthens sheets of amphiphilic molecules detached by an aqueous layer.In between these hexagonal and lamellar phases, the viscous isotropic phase may exist known as the cubic phase [10].For sustained release action, non-lamellar phases i.e. hexagonal and cubic play an imperative role [9].
One of the important ingredients of LLCs is amphiphilic molecules i. e. polar lipids or surfactants.Polar lipids are amphiphilic and can incorporate lipophilic molecules easily in the liquid crystalline phase [53].They have been studied for a variety of drug molecules viz.oxybutynin hydrochloride, hydroxychloroquine sulfate, vitamin E, acetylsalicylic acid, propanolol hydrochloride, chlorpheniramine, insulin, timolol maleate, cefazolin, hemoglobin, and pyrimethamine [53].However, major challenges of these LLCs to be explored as parenteral were poor injectability and their high formulation viscosity which might limit their application in vivo.This can be tuned by the addition of a minor amount of co-solvents like propylene glycol and ethanol.Thus, the feature of phase transition from liquid to LC permits the insertion of a low viscosity matrix that converts into a viscous fluid crystalline structure following dilution in bodily fluids.Furthermore, a transition of microemulsion into LCP has been considered [54].Importantly such LCP-based in situ gels generally comprise surfactants with or without other lipophilic excipients enabling easy incorporation of lipophilic drugs.Several molecules were incorporated in these phase transformation LLCs by varying co-solvents and lipids concentration to improve their release period and clinical efficacy.Examples of small drugs are testosterone enanthate, fentanyl, bupivacaine, buprenorphine, benzydamine, granisetron, and testosterone undecanoate.Whereas examples of peptides and proteins consist of leuprorelin (leuprolide), calcitonin, octreotide, somatostatin, histrelin, glucagon, triptorelin, interferons, antibody fragments, glucagon-like peptide-1 & analogs, and different enzymes [53].Some of the products based on this technology in the pipeline contain APIs octreotide chloride, buprenorphine, and leuprolide acetate (http://www.camurus.com).
The biocompatible and biodegradable polar lipids such as glycerol monooleate, as well as its derivatives, have been explored for the delivery of peptides and proteins and it could help to enhance stability [13].For the efficient management of orthotropic prostate cancer, an innovative injectable lyotropic preparation containing poly (ester anhydride) of sebacic acid and hydroxy oleic acid has been developed for targeted sustained delivery of paclitaxel [55].The surfactants with glycerate head groups were studied for the formation of LLCs in excess water for extended-release of octreotide peptide [56].LLCs-based phase separation gel was studied and evaluated for extended-release of octreotide peptide after subcutaneous injection (SC) [19].In situ hexagonal gel was developed post-SC injection of fluid lipid precursor formulations for prolonged release of naltrexone [57].In general, these preparations have proven beneficial offering lipase biodegradability and cost-effectiveness.Nevertheless, variability in lyotropic liquid crystalline structures after administration due to inconsistency in surrounding biological fluid.

In situ phase transition to the lyotropic liquid crystal
Parenteral injectable formulations are generally preferred as a liquid formulation with optimized stability and solubility of the drug molecule however it is of utmost importance to optimize the desired injectability parameters and tissue tolerability [58].Conventional injectable formulation with the use of various solubilization approaches like the use of cosolvent, salt preparation, pH adjustment, complex formation, use of surfactant and co-surfactant, etc. leads to a rapid onset of action [59].However, there is a need for the parenteral sustained release formulation as these rapid release formulations have postadministration precipitation issues which are caused by the poorly soluble drug's rapid release into the body's aqueous environment [60].In the recent past, there are many such aqueous-based sustained-release injectable gel formulation emerges that has better patient compliance with reduced dosing frequency however they are facing the problem of limited drug solubilizing capacity.
In situ is a Latin terminology meaning "In position."The unique characteristic of in-situ gelation is that they are less viscous or polymeric V.P. Chavda et al. fluid injectable systems even before injection.Once administered these low viscous or polymeric fluid formulations solidify into a semisolid or solid system triggered by a change in various factors (temperature or ions or solvent removal) [61,62].In-situ forming gel-based injectables are attractive to many pharmaceutical scientists as it offers numerous advantages over conventional parenteral formulations.
The in-situ forming gel has the following advantages compared to the preformed biodegradable depots and microparticles: (i) simplicity of administration, (ii) less stressful, and less complex designing and inexpensive manufacturing process [19,63].On the other side from the ease of administration, these preparations offer sustained release, decreased drug dosage resulting in a reduction in undesirable adverse effects that is common with conventional injectables, increased patient comfort, less invasive and less painful, high delivery efficiency, and precise control of dose and reduced toxicity [64].
Most often, in-situ sustained-release parenteral comprises a solvent with a synthetic or natural polymer [65].In-situ gel formation can be accomplished via many strategies such as temperature/pH-responsive polymers, cross-linking, and solvent removal.The interested readers can gain insights from reviews on the stimuli-triggered in situ gelation and phase inversion in situ forming implants [62,63,66].Solvent removal is concomitant with the procedures of polymeric membrane creation after phase separation triggered by non-solvent [63].Crosslinking can be achieved by ionic interaction or covalent bonds; the earlier one is facilitated by small cations, and the latter is usually mediated by photo and thermal-induced free radical reactions [67].In many thermally induced gel and thermoplastic paste formulations, temperature-sensitive polymers are the main ingredients [68].The improvement of these technologies has been raised by the development of novel materials and research.
In recent years, LLCs have gained a lot of attention from researchers for prolonged-release parenteral due to their excellent unique property of transition into gel after parenteral administration [67].As compared to other in-situ gelling techniques mentioned in the above paragraph, the in-situ gel formation due to phase transition into LCP requires a novel trigger i.e. aqueous environment (Fig. 1).Thus, upon contact with biological fluids inside the tissue, lipid or surfactant-based preconcentrates solutions convert into a highly viscous liquid crystalline gel that can provide prolonged release over days or months.The reason behind this may be is that fluidity of these lyotropic liquid crystals hindered by a tangled 3-dimensional gel network [69].
The development mechanism of lipid-based and surfactant-based LLC gel is distinctive (Fig. 1).A lipid-based LLC gel is formed by the interplay between polar lipids and aqueous solvents.The fascinating aspect of amphiphilic lipids is that they spontaneously interact with solvent and self-assemble into different LLC structures.Due to this instant conversion, polar lipids have been investigated for prolongedrelease application, examples are monoglycerides, phospholipids, and glycolipids.Mezzenga et al. reviewed in detail about design and application of lipid-based LLC gel [56].The advantages and rationale for in situ phase transition to liquid crystals are summarized in Fig. 2.
The formation of surfactant-based LLC gel can be achieved by several approaches.Steck and co-workers summarized the fabrication strategies of surfactant-based LLCgel.They discussed three different mechanisms that are based on the existence of gelators in the LLCphase; (i) The first strategy is the inclusion of less molecular weight organogelators in LLC.The concept assumes that the LCP act as a solvent phase for the gelator, which creates a 3D network in the anisotropic matrix.The formation of lyotropic liquid crystalline Fig. 1.In situ phase transition to liquid crystal for the sustained parenteral drug delivery.
V.P. Chavda et al. gels through this approach is based on the concept of orthogonal self-assembly.The phenomena of "orthogonal self-assembly" is usually based on the co-occurrence of two individually selfassembled structures confined in a similar system [69].(ii) Another approach to forming LLC-based gel is by incorporating hydrophilic polymer for example polyacrylamide.Gong et al., developed a system comprised of alternating stacks of soft hydrogel sheets and stiff surfactant-based anisotropic bilayers.However, this approach is only appropriate for the lamellar type of LLCs [70].(iii) In addition, to the above two discussed strategies, decoration of surfactant-based anisotropic bilayers by amphiphilic block copolymers namely short poly (ethylene glycol) also used.This approach was used by Warriner et al. to obtain a gel structure that is a highly defected microstructure.The distinguished 3D network gel was not observed as compared to the previously discussed two strategies.The lamellar domains are randomly arranged and tricked for stabilization by PEG-lipids [71].
Furthermore, thermodynamically stable microemulsions, the transparent colloidal system consisting of water, oil, surfactants, and cosurfactants also exhibit the potential to transform into liquid crystals [54,72].Nevertheless, the combination and ratio of water, oil, surfactants, and co-surfactants is very crucial to constructing LCP.Several studies have been reported based on the conversion of microemulsion into LCP and explored as an extended-release parenteral drug delivery system.After diluting a SMEDDS formulation, LCP was developed that demonstrated sustained release of hydrophobic drug UC 781 [24].The transition of microemulsions to liquid crystals was also employed to develop a prolonged release parenteral [54].In another study, SMEDDSbased in situ gel demonstrated extended-release of rifampicin after IM injection [73].
Numerous studies have been reported based on hydration triggered in situ gelling of LCP to achieve sustained release via the parenteral route.Yaghmur et al., designed preconcentrates that after intra-articular injection administration converts into the liquid crystalline depot.They performed real-time monitoring of hydration-triggered structural arrangements by using synchrotron SAXS [74].In another interesting work, in situ injectable self-assembled gel formation by paclitaxel, was investigated for sustained release of drug and anti-tumor activity [75].The transition of liquid crystalline preconcentrates into LCP after intramuscular injection that provides sustained release of artemether with lumefantrine was studied [76].The vascular endothelial growth factor (VEGF) prolonged-release was achieved up to 7 days post subcutaneous injection due to the formation of LLC-based gel at the site of injection for tissue regeneration [77].Chantadee et al., constructed vancomycin HCl loaded-lauric acid in situ gel for the management of periodontitis [78].Mei et al., developed a bupivacaine-loaded injectable precursor solution which, following administration, converted into an LLC-based gel for postoperative analgesia.The prolonged-release with enhanced plasma concentration was obtained for bupivacaine [19].Borgheti-Cardoso and co-workers fabricated LLC-based in situ gel encapsulating siRNA after subcutaneous administration for gene therapy [79].Zhang et al., compared three different in situ gels composed of glycerol trioleate (GTO), glycerol dioleate (GDO), and glycerol monooleate (GMO) incorporating model drug octreotide and they investigated its physicochemical properties [80].

Characterization of lyotropic liquid crystals
LLCs possess idiosyncratic physicochemical properties and microstructures that make them a system of choice to incorporate drugs for biocompatible parenteral delivery [81].LLC phases are developed by chirality, molecular shape, and micro-segregation effects [8,82].Table 1 summarizes various characterization methods for LLCs and their application.

Chemical characterization 5.1.1. X-ray diffraction
These studies were regularly performed on lipidic and oily dispersion systems to understand the physicochemical property [106].It is well reported that the morphological structure of lipidic carriers directly impacts the encapsulation efficiency and release of the drugs.Hence, an analysis of the internal structural arrangement of the LLC nanosystem becomes very important.The X-ray diffraction analysis is the most studied tool to accomplish this task.Generally, in LLC formulations, lipids can aggregate into many LLC structures through interplay factors, including water concentration, temperature, or the presence of different components.LLC phases are most often identified in drug delivery such as lamellar, hexagonal, and cubic phases [107].
X-ray scatterings result in characteristic interferences based on different ordered microstructures.Interferences utilizing methods are bifurcated into two parts namely, scintillation counters and positionsensitive detectors.Here, a long-range structural direction in liquid crystals is responsible for the development of diffraction patterns and electromagnetic radiation of respected wavelength causes turn-in interaction [106].The X-ray diffraction method is divided into two types: small-angle and wide-angle X-ray diffraction [108].Different tools, namely cubic, lamellar, and hexagonal are used in the characterization of LLCs.

Nuclear magnetic resonance (NMR) spectroscopy
NMR spectroscopy is an indispensable technique to investigate LLCs.The molecular construct and dynamics of LLCs are better understood by NMR, particularly various lyotropic mesophases [109].Therefore, this method is employed to find information on the supramolecular organization, conformations of the molecules, and orientation of LLCs [110].
Deuterium NMR spectroscopy is another powerful tool to look into different types of LC phases [111].Dynamic properties, like translational self-diffusion, molecular reorientation diffusion, and collective motions are deeply understood by NMR approaches through specific models [112].NMR-based approaches are also useful to study the temperature impact on LLCs and their molecular interactions [113].

Polarizing microscopy
All types of lyotropic liquid crystals except cubic mesophase are identified clearly with the polarized light microscope.To begin with, the anisotropic liquid crystalline phases have an outcome according to their molecular ordering like the lamellar, the reversed hexagonal phase along with the hexagonal phase are typically fragmented [114].LLCs are generally normally analyzed by the polarised light microscopy method, using liquid crystals fine-film stuck between a microscope slide and coverslip.In addition, in the microscope, two polarizers are present which are arranged at the right angle.One is above and the other one is below in the cross position of the objective and polarised light is below to above polarizers of the objective [115].These lamellar phases and hexagonal phases normally show the mosaic pattern and nongeometric texture, respectively, moreover the reversed micellar solutions, nonbirefringent micellar, and cubic phase yield in dark background.The smectic mesophases of the thermotropic LC appear in various types of textures [87].It is a beneficial technique, but on the flip side, the interaction of other imaging systems may cause deterioration of the sample [116].The liquid crystal orientation is bifurcated into two parts; one is parallel to the glass plain (homogeneous orientation), and the second one is perpendicular to the glass pain (homeotropic orientation) [117,118].

Transmission electron microscopy (TEM)
The microstructure of liquid crystals is visualized using TEM because the high vacuum and high magnification power of an electron microscope can depict inherent variations in the molecular structure of LLCs.The freeze-fracture microscopy method has been successfully used in this area.In the freeze-facture method, a replica of the sample is generated which preserves the original microstructure.K Nishijima et al., reveal electron microscopic observation that microcapsules develop hard carbon films that were fabricated in a liquid-like structure [87,119].

Differential scanning calorimetry (DSC)
DSC works on the principle of enthalpy phase transition where transitional changes occur in the content of the respective system [120].DSC is an accurate method for heat capacities and enthalpy changes.In solid and liquid states, molecules lose orientational and positional order in transition form.Still, in LCs, there are one or more states which have a different degree of orientational and translational order [121].Here enthalpy fluctuation determination is directly proportional to the transitions and according to this determination, it is possible to find that the liquid crystal phase is nearer to the solid or liquid phase [122].
Murgia S et al. reported that the consumption of energy released by the emission of energy depends on recrystallization, exothermic or endothermic reaction, and the consumption of crystalline and amorphous LCs [123,124].During the phase transition, changes in specific heat capacity and baseline slope are an indicator of entropy change.As a result, phase transformation of liquid crystalline polymers eluted from entropic areas are monitored as second-order changes together with glass transitions, and they may be superimposed by an enthalpic effect, making identification difficult [124].

Rheology
Various forms of LCs have different rheological properties.LCs consistency increases and flow becomes more viscous due to the microstructural arrangements of LLCs.The LLC possesses a coefficient of dynamic viscosity along with viscous Newtonian flow behavior.The cubic and hexagonal liquid crystals depict comparatively more viscosity than the lamellar phase.In addition, a less viscous nature is seen in  hexagonal and cubic crystals, but it is also observed in the lamellar phase, and it is the main element to differentiate between the two classes.The thermotropic liquid crystals possess the viscosity in the order: nematic<smectic A<smectic C. The elevated viscosity of LLCs with cubic mesophase is responsible for 2-D order while hexagonal mesophase is responsible for 3-D order.Lamellar mesophases with 1-D long-range order have a reasonably low viscosity.Because of their gel character, hexagonal and cubic mesophases result in yield values, after which flow occurs [125].

Morphology of the particles
Liquid crystal nanoparticles (LCNP) are differentiated with the help of their morphology as hexosomes and cubosomes.In addition, drug entrapment efficiency is dependent on the morphology of the nanodispersions.The drug release, stability, and effective bio-distribution are also governed by particle morphology.Various techniques such as optical or light microscopy are utilized, to evaluate the external curvature and the morphology of the dispersed particles.However, more successful techniques are Cryo-TEM and TEM.Kim et al. reported Cryo-TEM to identify the morphologies of the inner structures of the lipid nanoparticles [87].Gan et al., reported nano-dispersions structural determination by small-angle X-ray (SAXR) diffraction technique, and this SAXR technique was used to differentiate hexosomes and cubosomes.A novel method was able to correlate the SAXR and Cryo-TEM performed on particle morphology [126].On the other hand, the temperaturedependent SAXR approaches for the liquid crystal phase dispersion have been developed to observe the temperature-based structural changes [127].Here, in some samples, the degree of crystallinity can be detected by the microcalorimetry technique, whereby changes or shifts in the peak temperature.Every sample shows some degree of crystallinity and it also pinpoints the stability of nanoparticles with various compositions, based on their enthalpy and the shift of peak temperature.The phase transition in the LLC phase of drug-loaded and drug-free LC was observed utilizing polarized light microscopy [128].

Entrapment efficiency
The entrapment efficiency depicts the percentage of drug that is successfully adsorbed or entrapped into nanoparticles.Many drugs fall into BCS class 4; these types of drugs are not dissolved in an aqueous medium and LCNPs have been depicted to give an effective solution to resolve this problem.Abdelrahman et al., performed drug entrapment studies on LCNPs and developed cubosomes, where every sample was centrifuged utilizing at 1400rpm for half an hour, and aliquots of supernatant were further diluted in PBS utilizing 5% Triton-X solutions [49].

Manufacturing challenges
The foremost challenge for LLC-based drug delivery is the robust manufacturing process development.As we have observed throughout this manuscript that such LLCs are formed with a suitable combination of the lipidic agents along with surfactant molecules.The driving factor here includes temperature, concentration, and amount & ratio of lipid to surfactant [129,130].Apart from that the magnitude of repulsive forces, conformation, and interfacial activity with water impact the creation of stable LLCs [131].It causes serendipitous phase transitions, leading through lamellar, hexagonal, and cubic phases, based on the water content of the surroundings.In comparison to their dispersions, the formulation of liquid crystals is quite straightforward.The liquid crystal can be created by constant stirring at high speeds and blending lipid with an aqueous phase [132].Although it looks very simple operation for manufacturing it demands a lot of skill as well as formulation optimization during the early stage of development.To acquire the liquid crystal, the blend is equilibrated at normal room temperature for 48 hours.The complexity of the formulation and process may get increase at the stage where there is more than one lipidic component blended with water; some may require prior melting of the lipidic phase.In addition to that, other formulation components like stabilizers, complexing agents, preservatives, etc. add further complication to the process as they impact the physicochemical properties of lipid and surfactants and also change the HLB value of the blend leading to the structural transitions of the LLCs [133][134][135].The pH of the prepared formulation also has an impact on the stability of the LLCs as well as its transition behaviour; these will not imply the in situ transition system as they are the simple optimized blend of lipid/oil and surfactant with minimum numbers of formulation additives [136].Similarly, the concentration of the salt has an impact on the transition behavior of such LLCs [137].
Once the formulation optimization is done, the next step is the process design and optimization.Many process-related factors impact the overall stability of the LLC formulation like temperature, pressure, power input during mixing, and viscosity [138,139].Various methods of preparations for LLC are summarized in Fig. 3. To prepare LLC by lipid dispersion, two approaches are used.The first is known as the 'Top-Down' approach, in which the lipid and stabilizer are first wetted to form a viscous bulk, and afterward, the bulk is distributed through an aqueous phase employing high-pressure homogenization and ultrasonication [140].The second approach is known as the 'Bottom-Up' approach, in which the existence of hydrotrope tends to play a critical role in the establishment of liquid crystal structure.The creation of its dispersion is caused by the governed addition of an aqueous medium to the mixture [141].In the dilution-based method, fragmentation is not required.To make the manufacturing process cost-effective scientists have started using a combination of both methods which requires fewer energy inputs [142].Apart from that for thermolabile drugs a new form of platform approach that is liquid and powdered precursor techniques are devised [143,144].To eliminate the danger that organic solvents may break down and harm sensitive medications, several authors have modified the bottom-up approach by replacing ethanol and propylene glycol with biocompatible solvents or solvents that can be removed from the final heterogeneous mixture [145][146][147][148]. Shan X et al., have described various improved methods for the preparation of lipid-based LLCs i.e. inverse LLCs, along with their advantages and disadvantages [96].
In situ phase transition from an emulsion-based system to LLC is the simplest and more industry-friendly method for parenteral sustained release delivery that can be produced on a large scale and is easily scalable.

Advantages of LLCs for parenteral drug delivery
Designing formulations that cater to problems associated with drug solubility and bioavailability is of prime importance for successful applications [149].The scope of LCs for parenteral delivery is expanding because of their ability to exhibit properties of both liquid and solid states.LCs have the potential to modulate the drug release rates, impart reduced toxicity and offer versatile applications throughout numerous routes of administration [150].LLCs are a highly ordered system with increased thermodynamic stability due to internal nanostructure enabling the construction of a sustained release matrix [39].The advantage of non-lamellar liquid crystals apart from enhancing drug solubility, includes maintaining the stability of peptide and protein structures [151].The tunable properties of LLC materials concerning their size, morphology, and internal organization are deemed to aid in the predictable loading and release of therapeutic biomolecules, including peptides, proteins, and nucleic acids [152].Antimicrobial peptides are one such category of actives that possess a tendency to get degraded via enzymatic and chemical routes.LC structures have performed well as carriers for antimicrobial peptides, especially the ones fabricated with lipids like glycerol monooleate, oleic acid, etc., which make them biocompatible and biodegradable [153].The different liquid crystal phases of LC are characterized by distinct domains having hydrophobicity from lipids and hydrophilicity from aqueous components.This arrangement makes LC systems amphiphilic and garners immense interest from a drug delivery viewpoint citing the possibility of inclusion of hydrophobic, hydrophilic as well as amphiphilic active moieties that can either be dispersed or dissolved [154].Diffusion governs the release of actives from the hydrophilic phase via its water channels whereas partition coefficient phenomena help release the drug from the lipid phase [155].Cubic and hexagonal LCs have been of prime interest for the development of sustained-release parenteral depot-type formulations [156].By tailoring the composition of the LC, the system can be made to release drugs upon stimuli like change in pH (eg.phytantriol and sucrose stearate) [21] or temperature (eg.phytantriol and glycerol monooleate based system) [157].While designing lipid-based nanocarriers like MEs and SMEDDs, it was observed that these systems tend to transform into LLCs at specific surfactant concentrations and dilution ratios which ultimately aid in sustained release of actives [25,48].Thus, the release of both micro and macromolecules, increased bioavailability, better stability, and control over release properties via both internal and external stimuli demonstrates huge potential of LC based formulations, especially now because of the huge global share of biopharmaceutical industry.LC nanosystems are novel and demand extensive research in order to be exploited to their full potential in the realm of parenteral drug delivery systems [158].

Applications of lyotropic liquid crystals in the parenteral drug delivery
LLCs are suitable drug delivery carriers for the delivery of small molecular drugs but it is also an efficient drug delivery platform for the parenteral delivery of large/biological molecules due to their chirality, different forms, and non-uniformity in composition (Microsegregation).Zhai et al., have prepared the LLC-based parenteral delivery of phytantriol using 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) conjugated with PEG [159].The structural hierarchy was the function of PEG chain length, as the DSPE-PEG with PEG chains of 2000, 3400, and 5000 Da resulted in a structural transformation from liposome Lα to cubosome (Q II P and Q II D), respectively [159,160].
Réeff et al., developed a cubic phase with "glyceryl monooleate 1-(cis-9-octadecenoyl)-rac-glycerol (GMO)" and water containing clonidine and hyaluronic acid as the polymer for intraarticular administration [161].The addition of sodium oleate or soybean oil resulted in a significantly reduced drug release of approximately 50% in 10 days.This sustained-release effect was observed due to increases in the hydrophobicity of the system which results in slower drug release due to low water uptake and low drug mobility [161].
Trevizan et al., developed liquid crystalline nanodispersions of docetaxel (DTX) and cetuximab for prostate cancer.In prostate cancer, EGFR is overexpressed, thus Cetuximab (CTX) was included in the formulation as an anti-EGFR monoclonal antibody [102].The formulation contained oleic acid and soy phosphatidylcholine (oil), Proventil ® (surfactant), DSPE-PEG-MAL (anchor for CTX binding), and an aqueous phase of 1.5 % poloxamer in PBS buffer.The formulation showed a hexagonal phase at 80% aqueous content which transformed into the lamellar liquid crystal with an increase in the water content.The cytotoxic efficacy of liquid crystal containing DTX and CTX resulted in a significant reduction (p < 0.05) of in vitro cell viability at the lowest concentration of 0.1 nM [102].The parenteral administration of these viscous phases poses a problem during the administration of the drug.Thus, in situ phase forming formulations are gaining more interest which is low viscosity injectable which, upon contact with the physiological media, converts to the liquid crystalline phase.Ahmed et al., investigated the mixtures of mono-glyceride, water, and cosolvents like ethanol, PEG 300, 2-pyrrolidone, DMSO for in situ phase transition.The drug release was decreased with increasing water, increasing monoglyceride, and decreasing cosolvent content because of an increase in viscosity.The extended release of an oligonucleotide is observed with fully swollen [162].
Li et al., studied the in situ transition of parenteral injection of bufalin (an antitumor drug) from microemulsions to LLC [25].Bufalin is a poorly water-soluble drug that exhibits cardiotoxicity.The Pharmacokinetic of bufalin-loaded liquid crystal formulation on rats showed prolonged retention time in the blood.Similarly, Ren et al., reported the in-situ liquid crystal of progesterone for prolonged parenteral delivery using Miglyol 812N (oil), Solutol HS15, and span 80 (mixture of surfactant), and ethanol [163].On intravenous administration of microemulsion, the diffusion of water occurs from the surrounding tissue into microemulsion which causes the transition to a liquid crystal.
Aleandri et al., developed biotinylated Cubosomes for codelivery of paclitaxel and fluorescein (lipid dye) [164].According to them, "Cubosomes with a unit cell parameter of approximately 100 Å, were prepared using monoolein and biotinylated pluronic F108 at 1.65 mg/ mL in PBS at pH 7.4.Compared to paclitaxel or the nontargeted cubosomes, biotinylated cubosomes displaying a high degree of active functional biotin on their surface markedly increased the antitumor activity of PX at a concentration of 1 μ g/mL [164]."Likewise, Meli et al., developed monoolein-based cubosomes of docetaxel using F108 (PF108), and rhodamine-and folate-conjugated PF108 for cancer theranostic application [105].The degradation of these lipid-based materials is another major concern in drug delivery systems.The degradation of the two-component "soy phosphatidylcholine (SPC)/ glycerol dioleate (GDO)" system by triacylglycerol lipase (TCL) under physiological conditions was investigated [165].The 50/50 (w/w) ratio of SPC/GDO in excess water resulted in reversed cubic micellar (I 2) phase with Fd3m structure.This was dispersed in 5-10 wt % of Polysorbate 80, which aids in improved loading and stability, preserving its Fd3m structure.The degradation with TCL resulted in transitions from the reversed I2 structure to structures of less negative curvature such as 2D hexagonal, bicontinuous cubic, sponge, and finally multilamellar vesicles.This opens up the avenues for parenteral delivery due to improved loading, stability, and biocompatibility [166].
Chen and co-workers have developed in situ liquid crystal-forming parenteral formulations for intra-articular delivery of sinomenine hydrochloride for the treatment of rheumatoid arthritis [167].Phytantriol is utilized as the lipidic component of the emulsion mixed with water and ethanol as the other component of the system.Polarized light microscopy and small-angle X-ray scattering are used to confirm the liquid crystalline forming emulsion composition from the pseudo-ternary phase diagram.The in situ liquid crystalline phases were found to be cubic, and it is further studied for drug release by membrane diffusion method.A composition of phytantriol/ethanol/water, 64:16:20, w/w/w along with 6 mg/g of the drug was concluded as the optimized formulation with drug release up to 6 days.To increase the drug release up to 10 days, they incorporated vitamin E acetate in the emulsion mixture at 5 % w/w concentration.
Fonseca-Santos B et al., have resolved the solubility issue of resveratrol to increase its therapeutic acceptability by engineering transresveratrol-loaded nonionic LLCs [89].Similarly, Forys A and colleagues have proved that certain amphiphilic block copolymers [poly (ethylene oxide)-b-poly(lactic acid) (PEO-b-PLA) and poly (ethyleneoxide)-b-poly(5-methyl-5-ethyloxycarbonyl-1,3-dioxan-2one) (PEO-b-PMEC)] stabilizes the non-lamellar LLC nanoparticles made up of GMO colloidal dispersions loaded with resveratrol as model drug [168].As noticed by authors, the intersection of nanotechnology and LLC has created LC nanoparticles and its unique advantages can be exploited by the application of QbD that will save time and costs, and aid in scalability [169].
Yaghmur and colleagues [74] engineered an LLC-based depot formulation for intra-articular drug delivery by combining synchrotron SAXS.Recently HosseinKamali et al., have performed a comparative study for LLC and Vivitrol® for sustained release of Naltrexone [170].There are several research studies conducted on the parenteral delivery of LLCs in the last decade that are summarized in Table 2. Apart from that, Lyotropic Chromonic Liquid Crystals (LCLC) constitute a particular category of soft matter with distinctive attributes differentiated from those of conventional LLCs, and LCLCs have shown promise for new applications, such as the preparation of optically anisotropic films, micro-patterning, biosensing, and functional materials for nanofabrication [171][172][173].

Concluding remarks and future prospects
This review aims to demonstrate the underlying principles of liquid crystalline systems in terms of their potential for improved solubility and sustained release of parenteral formulations.Temperature, the framework of the drug, and the water/surfactant ratio are the driving forces behind the emergence of lyotropic mesophases.In situ phase transition from a Microemulsion to the LLC phases are providing an attractive niche in the recent past for the sustained delivery of drugs via parenteral route for systemic as well as localized delivery.
There is a lot of advancement in the characterization of the LLC, which accelerates the development process however, there is a need for robust platform processes that can be easily scalable and establish proper safety and efficacy of this delivery system.It has wide application for a range of drugs, including biologics and nucleic acid-based vaccines and therapeutics.Lipids that form the LLC phase are typically non-toxic, biocompatible, and biodegradable and are safe enough for various  • The siRNA was used to reduce the interleukin-6 (IL-6) levels in psoriasis.• The hexagonal phase was obtained using monoolein: oleic acid: polyethylenimine: Tris-HCl buffer.• siRNA containing IL-6 was able to reduce extracellular IL-6 levels by 3.3-fold compared with control treatment in the psoriasis skin model.[190] siRNA Lipid/Oil Phase: monoglycerides, Stabilizer: poloxamer 407, Gene carrier: polyethylenimine • The in situ gelling of LC system for local delivery of siRNA was attempted.• The cubic phase was developed, which converts to a reversed hexagonal phase in contact with water.• The subcutaneous injection in mice showed in situ gelling that provided sustained release and the gel was degraded in 30 days.[191] Vitamin C Lipid/Oil Phase: Mixed cetyl alcohol Stabilizer: Polysorbate 60 • The stability of Vitamin C was improved using LC.
• The complex structure containing lamellar liquid crystalline (Lα) and crystalline lamellar gel (Lβ) phases was observed by TEM, SAXS.[192] (continued on next page) V.P. Chavda et al. modes of administration [86].Numerous issues remain despite recent advances in material science and engineering.Some framework materials, such as GMO and PHYT, have outperformed conventional materials in terms of properties.However, due to the time-consuming task of synthesizing, purifying, and characterization, these components are hard to procure and extremely costly.This is, indeed, a significant challenge for the formation of a stable and orderly LLC in the development of drugs.Ample amount of further investigations need to be done to establish the safety and efficacy of such a system before it becomes commercially viable.LLC-based sustained-release parenteral formulations require extensive research to widen their spectra in the formulation development arena.Recent advancements in LLC for efficient sustained, and targeted delivery of anticancer agents, nucleic acid therapeutics, and vaccine delivery systems (especially mRNA) have opened the potential avenue for this drug delivery platform [203].• The lyotropic liquid crystalline nanoparticles were formed by ultrasonication method to enhance the solubility of berberine.• The IC50 value was 10-fold lower with berberine LC and was 55 times lower with LC prepared using PEG and Transcutol HP compared to berberine suspension.[193] siRNA Lipid/Oil Phase: monoglycerides Gene carrier: polyethylenimine • LC delivery of siRNA was attempted to prevent degradation of siRNA and obtain targeted delivery.• The precoursor fluid in presence of excess water gets converted to hexagonal and cubic phases.• The intratumoral injection of siRNA maintained its integrity for 14 days in tumor tissue.[194] mRNA Lipid/Oil Phase: 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) • Lamellar LC for controlled release of mRNA was developed.
• The d-spacing was found to increase with an increase in the concentration of mRNA.• The mRNA activity was evaluated using luciferase expression.[195] Imatinib mesylate (IMS) Lipid/Oil Phase: Glyceryl monooleate Stabilizer: Poloxamer 407 • IMS-loaded lactoferrin modified PEGylated LC targeting asialoglycoprotein receptor for hepatocellular carcinoma was developed.• The cubosomes were formed by using sonication method.
• The cytotoxicity study showed no toxicity for 13 μg/ml for phytantriol and 100 μg/ml for GMO-based LC. [197] Rapamycin Lipid/Oil Phase: Glyceryl monooleate; Stabilizer: Poloxamer-407 (P407) Polymer: Chitosan and Hyaluronic acid • Layer-by-layer-coated LC nanoparticles of rapamycin were developed to increase solubility and bioavailability.• The optimized formulation showed a 3.35-fold increase in bioavailability compared to free Rapamycin.• Moreover, LC reduced the adverse effects like nephrotoxic and hyperglycemic of Rapamycin.[198] Vascular endothelial growth factor (VEGF) Lipid/Oil Phase: Glyceryl monooleate Hydration modulating agent: Octyl glucoside • Octyl glucoside enlarges the water channel by a 2-fold increase in water channel and VEGF release was increased by 7-fold.• The sustained release inverse cubic phase of VEGF for 7 days was observed and angiogenesis was induced 14 days after subcutaneous injection. [77] Paclitaxel, irinotecan, glucose, histidine, and octreotide Lipid/Oil Phase: Glyceryl monooleate; Surfactant: Oleyl glycerate and phytanyl glycerate • The reverse hexagonal phase was formed in presence of excess water for sustained release of the drugs.• The LC formed with oleyl glycerate showed faster release compared to phytanyl glycerate matrix.[199] Naltrexone Lipid/Oil Phase: Soy phosphatidylcholine Stabilizer: Sorbitan monooleate • The injection depot of Naltrexone was attempted for sustained drug delivery.• LC of Naltrexone exhibited hexagonal phase resulting in invitro sustained release for 35 days. [170] Risperidone Lipid/Oil Phase: phosphatidylcholine and sorbitol monooleate Surfactant: Tween 80 • The sustained-release formulation of risperidone was attempted using lipid liquid crystal.• The formulation exhibited reverse hexagonal phase.
• The optimum formulation showed 100% drug release in two months and pharmacokinetic datashowed consistent serum level for 60 days.[200] Risperidone Lipid/Oil Phase: glycerol monooleate, glycerol dioleate, and glycerol trioleate • The in-situ gel of risperidone for the sustained delivery to schizophrenic patients was developed.• The formulation showed cubic and hexagonal phases and intermediate phases.
• The optimal LC at 2.2:1ratio of GDO/PC and 30% N-methyl-2pyrrolidone resulted in consistent release for 60 days.• As vaccine platform technology for sustained release of antigen • The intrinsic proinflammatory activity of Coa-ASC16 [202]

Fig. 2 .
Fig. 2. Advantages and rationale for in situ phase transition to liquid crystals.

Fig. 3 .
Fig. 3. Various methods of preparations for LLCs. A. Pseudo ternary phase diagram showing liquid crystalline region and B. Various methods of preparation for LLCs.

Table 1
Instrumental techniques utilized for characterization of LLC-based drug delivery systems.

Table 2
Research summary of Lyotropic liquid crystal-based formulations for the parenteral drug and vaccine delivery.Replacement of heavy metals like gadolinium that is toxic.•High water solubility, less toxicity, and stability suggests in vitro and in vivo applicability.• Effective in vivo performance of a T1 enhancing.

Table 2
(continued )The stability of Vitamin C was increased to 4 months by encapsulating it inside droplets of the α-gel phase of LC.