Cubosomes in Drug Delivery: Exploring Their Potential for Advanced Therapeutic Applications

1. INTRODUCTION

The discipline of nanomedicine has greatly benefited from the emergence of nanotechnology, which has become a major actor in many areas of research. Its influence on medication distribution is particularly noteworthy. For example, nanotechnology has enabled the development of carriers that increase the potency of drug delivery, improving the overall success rate of therapies [1].

Cubosomes are distinguished by the fact that they are liquid crystals with bicontinuous cubic phase, exhibiting innate qualities that position them as a promising universal medium for transporting various drug actives [2]. These nanoparticles, like classic controlled drug delivery methods, use surfactant and polymeric systems, resulting in supramolecular assemblies known as active transport vesicles [3]. The surfactants produce three dimensional bilayers arranged limited surface-forming, periodic, tightly packed structures with bicontinuous lipid and water zones, suggesting a "honeycomb" shape [2]. Cubosomes are typically produced through labor-intensive processes, often involving high-energy input [4]. Initially, they are formed by disrupting the cubic,lipid-water phase inside a three-phase area that includes a dispersion of liposomes. These particles, however, are distinguished from ordinary liposomes by structural differences and their capacity to accept amphiphilic, lipid-soluble, and water-soluble actives [5].

Cubosomes are the particles composed of 2 internal aqueous channels, which are formed by curved bicontinuous lipid bilayers structured in three-dimensional honeycombed patterns. These channels can be effectively utilized for the incorporation of various bioactive ingredients, including peptides, proteins and chemical [6]. Due to their distinctive features such as stable thermodynamics, bio adhesion, the capability to entrap hydrophilic, lipophilic, and amphiphilic substances, as well as they have the capacity for controlling the release utilizing the specific functionalization. Cubosomes are recognized as a promising approach for multiple routes of administration [7]. Cubosomes are specifically liquid-crystalline nanostructured particles composed of various proportions of amphiphilic lipids. They are recognised as biocompatible carriers in medicine delivery. The physical and chemical features of these reversed bicontinuous cubic phases are distinct [8]. The basic structure of cubosomes is represented below Figure 1

Figure 1. Structure of a Cubosome [9].

This review particularly highlights the fresh prospective of the potential use of cubosomes for targeted therapies, while previous reviews only cover the properties and general uses of cubosomes. It provides data regarding the stability and suitable method of formulation for controlled release mechanism, this prospective has not been explored in depth. This review also provides the list of successful formulations which have shown better results as compared to conventional formulations using top-up, top down and heating method. It also provides an insight of characterization methods including DLS, NMR, photon correlation which provides a very clear understanding to access the physiochemical properties of cubosomes.

Cubosomes are distinguished from liposomes by their ability to simultaneously retain lipid-soluble, water-soluble, and amphiphilic molecules [10]. Cubosomes are eternal and have thermodynamic stability. When loaded proteins, stabilizers, or the required chemical or medicine are combined with cubosomes, a lipid cubic bi-continous phase is formed by the self-assembly of lipid mixture [11]. Polymers can be used to stabilize cubosome colloidal dispersions. Diffusion is mediated by the tortuous diffusion of actives through the cubic phase's "regular" channel structure, making cubosomes an attractive choice for regulated active delivery. At high concentrations of amphiphiles in aqueous surfactant systems, these structures develop, with enough aligned molecules to be identified geometrically. Differential geometry allows for the distinction between two types of cubosome structures: an open cubosome, in which both water channels are exposed to the outside world, and a closed cubosome, in which only one water channel is present [12].

2. MATERIAL USED IN CUBOSOMES

For the formation of bicontinuous cubic phases the potential demonstration is represented by the natural lipids, nonionic surfactants such as Lynch ML18 and cationic Boretta-M17 [13]. While monoolein monoglyceride is typically utilized to produce bicontinuous cubic phases, other monoglycerides show spontaneous development of such phases when diluted. Monoglycerides are typically insoluble and resistant to the changes in temperature. Monoolein, also called glyceryl monooleate, is the principal precursor for cubosome synthesis. It is a complex made up primarily of the glycerides of oleic acid and other fatty acids, with the monooleate being the dominating component [14]. 

To simplify large-scale manufacture of cubosomes, the liquid precursor approach is employed, which minimizes the dangers associated with working with bulk solids and decreases the destructive potential of high-energy operations. This strategy provides a less complicated and more productive way to prepare cubosomes for industrial use [15].

Lipid-made cubosomes have a characteristic waxy, sticky solidity texture. Waxy lipid coating is useful in preventing agglomeration and controlling particle size of water-soluble, non-cohesive starch. Spray drying stands out as a great method for accomplishing this goal [16].

Aggregation in various solvents produces morphologies as diverse as cylinders, vesicles (polymersomes), ribbons, spheres, films, tubules, fibers, and multi-geometry nanoparticles. These shapes form when unfavorable segment/solvent interactions are diminished. Bicontinuous mesostructured are an interesting subtype of these structures due to the percolating nature of their 3D phase structure [16]. Despite its notoriety, bicontinuous phase structures often have a constrained stability range. Low molecular weight surfactants and synthetic particles called cubosomes, which include bicontinuous cubic liquid-crystalline nanostructures, have been used to overcome this constraint. These nanostructured entities have the same cubic crystallographic symmetry as the parent phase, but with a much greater surface area and less viscosity thanks to their size and shape

3. PREPARATION OF CUBOSOMES

This approach, which entails two main phases, is by far the most commonly used methodology for manufacturing cubosomes. First, the lipid that forms cubosomes is added with a suitable stabilizer to produce large, viscous cubes. Next, high-energy methods, such as high-pressure homogenization or sonication, are used to disperse the resulting viscous cubic aggregates in aqueous fluids, which ultimately results in the formation of cubosomes [17].

To create cubosomes, a mixture of the lipid, stabilizer, and hydrotrope is diluted in excess water using the solvent dilution method, also known as the bottom-up strategy. The hydrotrope is crucial to this strategy because it dissolves water-insoluble lipids, creates lipid precursors, and stops liquid crystals from forming at high concentrations [18]. Urea, sodium alginate, and sodium benzoate are all examples of hydrotropes that see regular application. The solubilization mechanism involves forming complexes between the hydrotrope and the hydrophobic substance. This technique has a number of advantages as that of the top-down technique, including lower overall energy expenditure. This allows for the creation of cubosomes that can safely house temperature-sensitive substances [19].

Heat treatment stands out as a practical option in this setting. Heat treatment is not, strictly speaking, the only process involved in making Cubosomes; rather, it aids in the transition from disordered vesicles to well-ordered cubic particles [20].

Cubosomes can be produced using a number of dispersion processes, including as sonication, spray drying, high-pressure homogenization, and spontaneous emulsification [21]. Their significant characteristics and advantages, including as a multicompartmental structure, high drug loading capacity, uncomplicated and convenient manufacturing procedures, and the utilization of biodegradable lipids like glycerol monooleate, position them as adaptable carriers for bioactive compounds. To facilitate targeted and controlled release, cubosomes allow for the encapsulation of hydrophilic, hydrophobic, and amphiphilic components [22]. Details about nano cubosomes are presented in Figure 2.

Figure 2. Scheme for Drug Entrapment in Cubosome [7].

Efficient drug distribution is accomplished by using significant amounts of drugs and employing a simple method of preparation. Cubosomes have the capability to encapsulate lipophilic, hydrophilic, and amphiphilic medicines. Lipids have the potential to degrade naturally, this enables the precise and regulated delivery of bioactive substances [23].

Cubosomes' high viscosity makes large-scale manufacturing difficult, requiring high-energy processes that can be detrimental to the temperature sensitivity of active components [24].

4. CUBOSOME CHARACTERIZATION

The physicochemical features of drug or gene delivery systems are essential, regardless of the production method used. Parameters such as particle size and structure are particularly important in this regard. Cubosomes are characterized by a variety of methods and can be categorized as following: direct techniques, which identify the phases, and indirect procedures, where measurements help describe the phases [17].

4.1.1. Nuclear Magnetic Resonance (NMR). It has the ability to give extensive information on the structure, dynamics, reaction state, and chemical environment of molecules. NMR may be used to identify the physical and chemical characteristics of atoms and molecules. Pieter Cullis deserves special recognition for his noteworthy and inventive contributions to the field of NMR characterization of biological and synthetic lipidic systems [25, 26]. Rajesh et al. investigated the potential of substituting lipidic poly(2-methyl-2-oxazoline) (PMeOx) for F-127 in cubosome stabilization. The two lipopolymers, PMeOx80-OA: PMeOx40-OA were synthesized and their end-group efficiency evaluated using 1H NMR. These lipopolymers had different levels of polymerization. The 1H-NMR investigation indicated that  polymers reflected a remarkably high degree of terminal-group functionalization approximately  ≥95 percent, qualifying them for the production of PMeOx equilibrated cubosomes [27].

4.1.2. Photon Correlation Spectroscopy (PCS). The Zeta sizer is mostly utilized in dynamic laser light scattering to ascertain the particle size distributions of cubosomes. Three measurements are made by diluting the sample at a temperature of 25 °C after it has been adjusted to a light scattering intensity of around 300 Hz [28]. The data can be gathered and often displayed by utilizing the mean of volume, size, and weight. Also, the zeta potential and polydispersity index measurements can be verified [29].

4.1.3. Polarized Light Microscopy. Cubosomes' outer layer, which could show optical birefringence and a possible vesicular structure, can be recognized using this technique. Additionally, it is able to distinguish between substances that are anisotropic and isotropic [30].

4.1.4. Small Angle X-ray Scattering (SAXS). This methodology is utilized to ascertain the spatial configurations of distinct groups within the sample. The acquired diffraction patterns are transformed into plots displaying intensity as a function of the q value. These charts make it easier to identify peak positions and convert them into Miller Indices [31].

4.1.5. Transmission Electron Microscopy (TEM). It may be used to see how cubosomes are shaped. According to Kim et al., a technique involves transferring suspensions of cubic phase nanoparticles on negatively stained formvar/carbon-coated grid using freshly made phosphor tungstic acid solution (2%, pH 6.8) using 200 mesh [32]. The samples were then air dried at ambient temp. and microphotographs were employed using an electron microscope. However, SEM analysis for cubosomes or vesicular systems might not be suitable, as the integrity of the formulation and its robustness may be affected during the procedure when exposed to the electron beam [33].

4.1.6. Pressure via Ultrafiltration Method. The rate of drug release from cubosomes can be measured by applying the pressure through ultrafiltration technique. It nearly adheres to the proposal made by Magenheim et al. Employing an Amicon pressure ultrafiltration cell equipped with a Millipore membrane at the prevailing temperature of 22±2 degrees Celsius [34].

4.1.7. Dynamic Light Scattering (DLS) and Zeta Potential. This is a widely used technique for analyzing the size and movement of particles in colloidal systems. This method is straightforward and does not require any intrusive procedures to analyze particles in a liquid or gas [16]. When a particle solution is exposed to a single-color, synchronized light, the light is dispersed in various directions, resulting in the formation of a mottling. The particles are not stationary in a solution. They show Brownian motion, causing their proximal density to change over time. This procedure is frequently employed to quantify the dimensions of the manufactured cubosomes. A significant limitation of DLS measurements is that the particles which are larger and heavier have a greater impact on the average decomposition rate of a polydisperse solution, usually resulting in an amplification of these huge particles [35]. The dimensions of cubosomes with a honeycombed structure have been quantified for a wide range of diverse systems intended for various purposes, spanning from 10 to 500 nm in diameter [36].

4.1.8. Differential Scanning Calorimetry (DSC). DSC is highly beneficial for investigating the thermotropic properties of various systems because it offers helpful information on transition temperatures and respective transition enthalpies. Liquid crystalline system refers to a state of thermodynamic equilibrium. DSC can be used to determine the endothermic or exothermic energy changes that typically occur during a phase transition.  Mansour et al. conducted DSC experiments on dexamethasone-loaded cubosomes to examine how processing parameters and excipients influence the particles' physical stability. The experiments demonstrated that the characteristic dexamethasone’s endothermic peak was no longer present in the lyophilized-cubosomes. This indicates that the dexamethasone was trapped within the cubosomes and existed in an amorphous state [37].

4.1.9. Entrapment Efficiency. Cubosomes do have a lot of potential as drug delivery systems since they efficiently store large amounts of tiny drug molecules, peptides, biologics, or bioactive compounds. Various methods exist for incorporating the medication into cubosomes, all of which have been found to be successful in terms of entrapment-efficiency. The EE and loaded drug in the cubosomes are to be determined by employing chromatographic techniques, ultra-filtration procedures or small-angle X-ray scattering, dialysis [38]. The quantity of free drug can be assessed by using several methods such as UV spectrophotometry, fluorescence, HPLC analysis, Fluorescence Correlation Spectroscopy, or radioactivity. These techniques are employed to obtain a drug release profile, for instance.

4.1.10. Gel Permeation Chromatography. The utilization of ultrafiltration methods and UV spectrophotometer or by using HPLC analysis   the entrapment efficiency and drug loaded in cubosomes, might be assessed through the utilization of gel permeation chromatography technique or other ultrafiltration methods. The later procedure involves determining the unentrapped drug concentration, which is then eliminated from the overall amount of drug that was added before. The quantity of drug is determined through the utilization of UV spectrophotometer or HPLC analysis [39].

4.1.11. Stability Studies. The examination of organoleptic and morphological aspects over time allows for the study of the physical stability of cubosomes. This includes assessing particle-size distribution and drug-content at different times to find any possible variations. Essentially, the methods described above can be employed to determine these aspects, including the confirmation of the distinct internal structural trait of cubosomes at various times. This comprehensive approach allows to determine the changes in particle size distribution and drug content over time, providing insights into the potential variations in the formulation's stability [40].

Table 1. Application of Cubosomes Through Different Routes for drug Delivery

Hyalcubosomes and cubosomes containing tenoxicam were developed by Elakkad et al. Cubosomes formulation and hyalcubosomes formulation were both put through a stability analysis, which compared their particle size, EE and zeta-potential before or after being stored for 3 months at 4 °C ± 1 [41]. Based on the results of this investigation, we can say that storing the manufactured vesicles in airtight amber glass containers at 4 °C has no ill effects on particle size and PDI but does have an effect on zeta potential and entrapment efficiency percentage. Phytantriol cubosomes loaded with Latanoprost were also produced by Bessone et al. for the treatment of glaucoma. Amber glass vials containing the processed samples were kept for 30 days in a thermostatically regulated humid chamber (25 degrees Celsius). Thus, the stability investigations showed that the latanoprost concentration in the cubosomes and the physicochemical parameters were stable for a full month [42].

5. APPLICATION OF CUBOSOMES

The predominant application of cubosomes lies in the controlled release of solubilized substances. The cubic phase is particularly well-suited for controlled release due to its diminutive pore size (5-10nm), capability to solubilize hydrophilic, hydrophobic, and amphiphilic molecules, as well as its biodegradability through an enzymatic processes [73].

Figure 3. Graphical Representation of Cubosomes Application [74]

Recently, several anticancer drugs have been effectively enclosed within cubosomes and subjected to thorough physicochemical characterization. The unique approach shows promising nanocarrier formation and indicates its potential application in the realm of cancer therapy. (Figure 3)

Figure 4. Worldwide Distribution of the Estimated New Cases of Cancer in 2020 [75]

 Cubosomes present a viable solution to the multifaceted challenges with respect to oral delivery of with a list of potential drugs, like poor aqueous solubility, inadequate absorption, and considerable molecular size [76]. These entities, available in liquid and powder forms both within capsule products, embody self-emulsifying liquid crystalline nanoparticles (LCNP)technology [77]. An alternative application involves the encapsulation of large proteins for localized gastrointestinal tract function. LCNP technology carriers can be strategically released at distinct sites of absorption, such as the upper or lower intestine, a critical consideration for the drugs with narrow absorption windows (NAW) [78].

Lipid based nanoparticles, which consist of curved lipid membranes with internal liquid crystal formations, used to dissolve, encapsulate, and transport drugs to specific illness sites inside the body [79]. Emulsions and liposomes have been used as carriers for drug products administered intravenously. However, liquid crystal nanoparticle structures have shown to enhance the amount of peptides, proteins, and insoluble small molecules that can be delivered, making them optimal carriers for injection or infusion of various active substances [80].

Cubosomes have the potential to encapsulate antigens and/or adjuvants and then be transported to the desired location. An early example suggests that cubosomes have the potential to be utilized for protein-based vaccinations. The solvent precursor dilution approach was used to generate cubosomes containing monoolein and phytantriol [81]. The stratum corneum layer of skin can be a significant obstacle to this approach. Microneedles loaded with antigen and adjuvant were used to photograph skin penetration in piglets in vitro and in mice in vivo in one study [4].

Lipid-based colloidal systems can facilitate the fluidization of epidermal lipids, hence promoting the enhanced penetration of molecules. Cubosomes have the ability to serve as a viable carrier for the delivery and transportation of pharmaceuticals over the skin [82].

6. CONCLUSION

This review highlights the importance of cubosomes as a drug delivery system for various routes. Their unique self-assembled structures make them favorable for the encapsulation of both hydrophilic and hydrophobic drugs leading to enhanced stability and better controlled release properties. These properties are crucial for improving the therapeutic efficacy and minimizing side effects. This review also provides an in-depth sight of the method of formulation and characterization including NMR, PCS, and DLS which is essential to determine the physiochemical properties of cubosomes for optimal drug delivery. However, ongoing research into innovative materials and alternative methods continuous to make progress in overcoming the barriers, leading to enhanced feasibility of cubosomes technology for clinical applications. Cubosomes are the major advancement in nanomedicine, they have an immense potential to revolutionize drug delivery systems. Continued research into their preparation, characterization, and applications will be having significant improvements in targeted therapies, paving the way for better therapeutic outcomes across various diseases.

Author Contribution

Saman Ali: data curation, formal analysis, writing – original draft. Nauman Tarar: data curation, writing – original draft.  Sabi Ur Rehman: conceptualization, supervision, formal analysis. Fazal-ur-Rehman: writing – original draft, writing – review & editing.

Conflict of Interest

The authors of the manuscript have no financial or non-financial conflict of interest in the subject matter or materials discussed in this manuscript.

Data Availability Statement

This is a review article. No data was generated or analyzed. All the findings in this review are based on already published data which has been appropriately cited.

Funding Details

No funding has been received for this work.

Generative AI Disclosure Statement

The authors did not used any type of generative artificial intelligence software for this research.