A New Frontier in Nanomedicine: The Promise of Cubosomes in Drug Delivery Systems

Nanotechnology has played a critical role in a number of research fields. Nanotechnology is slowly gaining significance in the nanomedicine field day by day. As regards this perspective, nanotechnology has been employed in formulating carriers in order to enhance drug delivery and thereby improve therapeutic outcomes [1, 2]. Cubosomes are biocompatible drug delivery carriers composed of nanostructured liquid crystals; they are based on a specific mixture of amphiphilic lipids in various ratio. Additionally, they exhibit a typical reversed bicontinuous cubic phase structure that grants them several of the desirable physical and chemical properties [3]. Cubosomes have been shown to release drugs from hydrophobic to hydrophilic via even amphiphilic (by way of oral, transdermal, ocular and chemotherapy) routes with yield of increased bioavailability and thereby ability to load more in a given mass. Thanks to their high pharmacological and biological potential to be alternative to liposomes, cubosomes have emerged to be one of the main options in the development of drug nanocarriers in a recent period. In particular, monoolein-water cubosomes belong to binary systems, spontaneously self-assemble into stable cubical crystalline structures [4].

Cubosomes and its components      

The vesicles (polymersomes), spheres, cylinders, ribbons, films, fibers, tubules, and multishaped nanoparticles can all be formed by the spontaneous self-assembly of amphiphilic bicontinuous cubic phases (BCPs) in specific solvents. These structures arise as the system maintains the deleterious contact between the polymer chains and solvent. Of them, bicontinuous mesostructures are of immense interest due to the complex 3D networked phase structures. Though bicontinuous structures are highly defined, they stabilize with a narrow range [5]. Cubosomes are self-assemblies of lipids with particle sizes between 50 nm and 500nm [6]. Ia3d, which is also known as G-surface; Pn3m, which is also known as D-surface; and Im3m, which is also known as P-surface, are the three morphologies. The Ia3d morphology has hydrophilic channels in sets of three. In contrast, its two paired water channels in the Pn3m structure adopt a tetrahedral arrangement and form a four-channel bond simultaneously. Last but not least, the Im3m structure forms an orthogonal network whose water channels are located in sets of six [7, 8].

Fig 1. Structure of cubosomes

Im3m (P-surface)

Three basic entities, on whose interaction the process of cubosome formation depends are a lipid fraction, a stabilizer and a molecule to be encapsulated in the particles. Cubosomes spontaneously form from their constituent elements by self-assemblization in to a bicontinuous cubic phase as similarly named with characterization as its constituent structural feature formed upon being formed together due to them direct assembling as structures. The majority of the amphiphilic molecules, like monoolein and phytantriol are applied during cubosome preparation [9, 10]. Stabilizers like Pluronic F127 are also required to inhibit the agglomeration of the nanoparticles when in water and maintain them in a dispersed condition. The third group consists of active pharmaceutical or bioactive compounds that may be incorporated within the hydrophilic or hydrophobic regions of the cubic structure and thus can provide systems for drug targeting/controlled drug release [11].

Phytantriol

Drug release mechanism of cubosomes

The gradient in drug concentration within the cubosome structure initiates diffusion, which is primarily responsible for the drug release from cubosomes. We see that the method used is in agreement with classic diffusion models for instance Higuchi or Fick equations. The rate of drug release is a function of drug’s solubility, diffusion and partition coefficients, geometrical drug’s which is the drug’s size in the cubic structure, pore size and distribution, interfacial curvature also plays a role along with the external phase conditions which includes the releasing medium's ionic strength, pH, and temperature. Studies on hydrophilic pharmaceutical compounds suggest that diffusion serves as the main release mechanism, with these medications demonstrating significantly faster release from cubic liquid-crystalline phases compared to reversed hexagonal phases. Additionally, our observations indicate that the in vivo liberation of 14C-labeled glucose from cubosomes and hexagonal phases accurately reflects in vitro release patterns, which subsequently influences how the cubosome nanostructure and lipid composition affect hydrophilic drug release dynamics [12]. For hydrophobic drugs release out of cubosomes is a different story. Due to the cubical phase’s natural affinity for hydrophobic areas these drugs do not diffused as well under basic in vitro conditions. In a study conducted that examined the release of hydrophobic drugs in purified water of pH 6.5 and in acidic digestive fluid (0.1 M HCl), it was seen that there was very great release in the acid media. Also in vivo use of Cubosomes which had Silymarin loaded into them did in fact outperform Legalon® the commercial capsule form of the drug. This is an indication that cubosomes have great delivery and release of hydrophilic and hydrophobic drugs especially under optimized formulation and environment [13].

Merits and demerits of cubosomes [14]     

Merits:

1. Cubosomes are stable, biocompatible, biodegradable and non-irritating systems with broad clinical applications in medicine.
2. Cubosomes can deliver any drug regardless of whether the drug is hydrophilic, lipophilic or a mix of both (amphiphilic) in nature.
3.  Because the surface area is extremely high inside of cubosomes, a greater amount of drugs can be contained in cubosomes.
4. Owing to the three-dimensional polymorphism of the unique crystal lattice arrangement in cubic liquid-crystalline phases, which comprise both hydrophilic and lipophilic domains, these structures serve as compelling prototype systems for pharmaceutical delivery in therapeutic contexts.
5. These lipids are biocompatible, adhere to the surface, are easily absorbed by the body, and because of the huge surface area they provide as a matrix, it offers an elaborate route for sustained and slow drug delivery of the drug.
6. These systems are easy to construct more solubilizing agents than other lipid-based carriers and also lead to better uptake of water-soluble peptides.

Demerits:

1. Drugs that dissolve in water are challenging targets because they contain a lot of water.
2. The high resistance of cubosomes increases their production difficulties making it harder to scale up. 
3. These particles pose storage and transport risks because they might break open.
4. Cubosomes undergo a phase change when exposed to external surface content.
5. Continuous long term storage of the particles without movement allows them to become larger.

Drug loading in cubosomes

Cubosomes are capable of carrying a broad range of therapeutic drugs including low molecular weight drugs, peptides, biologics and other bioactive compounds. These drugs can be incorporated into the cubosomes in three different ways: in the lipophilic bilayer, adsorbed onto lipid surface or inside the water pores of the cubic phase. Drug loading is achieved by fusion of the therapeutic compound to molten lipids, or co-lyophization of the therapeutic compound with lipid film before dispersion and addition of the therapeutic compound to pre-formed cubosomes during incubation. But for most low molecular weight compounds, polypeptides, and macromolecules, the lipid membrane is the main stage of loading. Cubosomes are mostly produced in single or binary lipid mixtures, i.e., containing phytantriol and monoolein, as the most suitable. Small-angle X-ray scattering (SAXS) is the most widely employed method for measuring drug encapsulation, however there are several other ways that may be adopted. Such findings reveal the heterogeneous nature of cubosomes as a drug carrier, especially in anticancer applications [15]. Cubosomes also have a significant advantage over other nanoparticles (e.g., liposomes) because of having a bigger hydrophobic surface area. This allows them to carry a greater load of hydrophobic drugs, and in doing so carry ionic molecules enclosed in a hydrophobicity shell. For example, it has been reported that phytantriol cubosomes can carry curcumin to a greater extent, than liposomes. In addition, the characteristic lattice of cubosomes can be independently regulated by membrane curvature scale, even with arbitrary size of cubosomes. Especially in this regard the possibility to replicate the strongly curved structures which have a more pronounced surface-area per volume ratio, since they contain more drug molecules on their membranes [16] can be seen as beneficial. In accordance with the Higuchi model kinetics principle, drugs may become trapped in the cubic shape of cubosomes and then released according to variations in molar mass and charge distribution.

Q = [D_mC_d (2A − C_d) t] ½

From the above equation, it is quite evident that diffusion or secretion of the agent out of the matrix is clock time squared dependent. Some of the variables found in the equation are drug release rate (Q) as rate normalized to the matrix surface area, agent diffusion coefficient (D_m) in the cubic matrix, agent solubility (C_d), and initial drug concentration (A) in the matrix per unit volume over time (t). From the relationship, the science of medicine release can be determined in terms of degree and speed [17].

Methods of preparation

1. Top-down technique:

For cubosome formation, the most often used technique is the top-down strategy, which usually involves two stages. Initially, a lipid is combined with a stabilizer that stops particles from clumping together to form a thick cubic phase. The cubic phase is subsequently crushed and dispersed in an aqueous solution by an intense process like high-pressure emulsification or sonication. This process ultimately generates cubosomes. Despite the fact that the characteristic cubic phase looks like a gel-like clear polymer, it is also quite peculiar in that it forms bulk liquid-crystalline order with periodic structure, and it turns out to be an unusual thermodynamic phase from this. The benefit of this method is that it produces stable for aggregation cubosomes for 1 year. Nevertheless, vesicles-nanoparticles of lamellar liquid-crystalline phases as well as vesicle-like structures-are always mixed with the cubosomes created with this method. Unexpectedly, when exposed to high oscillation frequency, cubic phases are compliant [18].

Top- down approach

2. Bottom-up technique:

In this way of synthesizing cubosomes, it starts with the construction of nanostructure components, which are subsequently integrated into the final product. This approach is a relatively new tool for cubosome fabrication. One method, known as crystallization from precursors, enables the formation of cubosomes at room temperature. Spicer and colleagues indicate that the terms solvent-based dilution approach and liquid precursor refer to the same method. In this type of combination, a hydrotrope, a polymer, and a lipid forming liquid crystal are mixed and dispersed in water with low energy consumption, thus forming discrete nanoparticles [19]. Hydrotropes are essential to bottom-up methods since they break down water-resistant lipids and stop liquid crystals from developing at high concentrations to generate liquid starting materials. Due to the absence of extensive fragmentation the dilution method works faster and consumes less energy. These methods produce finely dispersed particles without needing complex processing. The varying results from both methods depend on the specific formation process of cubosomes. Through top-down processing big particles get broken into smaller components while dilution-based methods assemble tiny particles into larger ones like methods used to make nanoparticles [20].

Bottom up approach

3. Spray drying method

The liquid proportion of the spray-drying process enters the uppermost part of the spray dryer through a dual-fluid nozzle having a 1 mm liquid orifice size, and air flows in from an annular air orifice 0.25 cm at 300 kPa pressure. Inside the nozzle, the liquid feed interacts with downflowing heated air at 200 °C throughout the drying operation. Because the liquid residence time is minimal, monoolein oxidation is minimized, even at the higher temperatures required for spray drying. Strong stresses are generated within the nozzle to disperse the highly viscous cubic gel when feed contains cubic LC materials like those in monoolein-starch-water. The resulting dispersion flows through the twin-fluid nozzle’s liquid conduit at 15 mL/min, making slight changes to guarantee the exit temperature for air is maintained at 90°C to 95°C. The dextran-monoolein-ethanol-water system experiences spray drying under identical process conditions, except that the feed rate is maintained at 4 mL/min, and the exit air temperature is raised to 130°C. The inclusive of this modification supports increased evaporation of both ethanol and water [21].

Evaluation of cubosomes [22]

1. Ultra-filtration method & UV spectrophotometer:

Ultrafiltration procedures can be employed to evaluate the efficiency of entrapment and drug loading in cubosomes. The unbound drug concentration is measured respectively and subtracted from the total drug amount injected. Drug content quantification sales are observed to do this using UV spectrophotometry or HPLC analytical techniques.

2. Photon correlation spectroscopy:

Another methodology used for the cubosome characterization purposes is represented by Photon correlation spectroscopy. The most important outcome resulting from dynamic laser light scattering, taken with a Zetasizer is particle size distribution. Later on, the sample is diluted using an applicable solvent, adjusted to approximately 300 Hz intensity, and analyzed three times at 25 °C. Results are usually given in terms of average volume- weighted dimensions. In addition, records of polydispersity index and zeta potential values are observed.

3. Polarized light microscopy:

The analysis of cubosomes can be performed using polarized light microscopy. It reveals a birefringent, potentially vesicle-like surface layer and distinguishes birefringence from isotropic materials.

4. X-ray scattering:

Small-angle X-ray scattering (SAXS) is a useful technique for determining the spatial arrangement of various groups inside a sample. The resultant patterns of diffraction are used to construct intensity versus q-value charts, which allow peak positions to be determined. These peaks may then be transformed into Miller indices, which can then be compared to values that are known for various space groups and liquid crystalline structures. This comparison aids in determining the sample's predominant internal nanostructure.

5. Transmission electron microscopy:

It is possible to see the cubosomes form using transmission electron microscopy. The suspensions of cubic phase nanoparticles were subjected to negative staining with freshly prepared 2% phosphotungstic acid solution (pH 6.8), then deposited on a 200-mesh formvar or carbon-coated grid and allowed to dry at room temperature, as described by Kim et al. Using an electron microscope, the electron microphotographs were captured. It is not possible to perform SEM analysis on cubosomes or certain vesicular systems because the formulation's resilience and integrity may be compromised during the electron array exposure process.

6. Visual Inspection:

The optical characteristics of the dispersions- such as appearance, clarity, uniformity, and absence of visible particles- were evaluated visually around a week following manufacture.

7. Light microscopy:

After being appropriately diluted with deionized water, samples of the produced cubosomes were seen using an optical microscope equipped with a micrometer slide at magnifications of x 400 and x 10007.

8. Entrapment efficiency:

The cubosome entrapment efficiency (EE) had to be defined by separating unencapsulated and cubosome-containing ALA. Spectrophotometric determination at wave length of 250 nm (λmax) was used to analyze free drug in the dispersion. This free drug quantity was deducted from the initially added drug amount to evaluate the amount of drug entrapped. In the analytical practice, the method used was a serial dilution of the dispersion. At first mixing one milliliter with four milliliters of deionized water, and after that taking one milliliter of this diluted solution and diluting it by mixing it with four milliliters of deionized water. Lastly, the final diluted dispersion was filtered with the use of a 0.1 μm pore size syringe filter. The concentration of the drug determined at 250 nm was adjusted by multiplying it by the dispersion’s total volume to account for the dilution factor. Drug quantity trapped inside the cubosome was determined by subtracting unencapsulated drug content (Cf) from the overall concentration of the drug (Ct) in the preparation. Triplicates samples were used to run each experiment in order to be accurate and reliable.

EE % = [(Ct – Cf)/Ct] x 100

Past work findings