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. 2023 Sep 19;20(10):5066–5077. doi: 10.1021/acs.molpharmaceut.3c00378

Preparation of Cubosomes with Improved Colloidal and Structural Stability Using a Gemini Surfactant

Masao Nagao †,‡,*, Abdul-Hackam Ranneh §, Yasunori Iwao ∥, Katsuhiko Yamamoto †,§, Yukihiro Ikeda †,§
PMCID: PMC10548465  PMID: 37726201

Abstract

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Cubosomes are nanoparticles with bicontinuous cubic internal nanostructures that have been considered for use in drug delivery systems (DDS). However, their low structural stability is a crucial concern for medical applications. Herein, we investigated the use of a gemini surfactant, sodium dilauramidoglutamide lysine (DLGL), which is composed of two monomeric surfactants linked with a spacer to improve the structural stability of cubosomes prepared with phytantriol (PHY). Uniform nanosuspensions comprising a specific mixing ratio of DLGL and PHY in water prepared via ultrasonication were confirmed by using dynamic light scattering. Small-angle X-ray scattering and cryo-transmission electron microscopy revealed the formation of Pn3̅m cubosomes in a range of DLGL/PHY solid ratios between 1 and 3% w/w. By contrast, cubosome formation was not observed at DLGL/PHY solid ratios of 5% w/w or higher, suggesting that excess DLGL interfered with cubosome formation and caused them to transform into small unilamellar vesicles. The addition of phosphate-buffered saline to the nanosuspension caused aggregation when the solid ratio of DLGL/PHY was less than 5% w/w. However, Im3̅m cubosomes were obtained at solid ratios of DLGL/PHY of 6, 7.5, and 10% w/w. The lattice parameters of the Pn3̅m and Im3̅m cubosomes were approximately 7 and 11–13 nm, respectively. The lattice parameters of Im3̅m cubosomes were affected by the concentration of DLGL. Pn3̅m cubosomes were surprisingly stable for 4 weeks at both 25 and 5 °C. In conclusion, DLGL, a gemini surfactant, was found to act as a new stabilizer for PHY cubosomes at specific concentrations. Cubosomes composed of DLGL are stable under low-temperature storage conditions, such as in refrigerators, making them a viable option for heat-sensitive DDS.

Keywords: cubosomes, gemini surfactant, drug delivery systems, stability improvement, lipid-based liquid crystalline nanoparticles

Introduction

Self-assembled lipid-based liquid crystalline nanoparticles, including cubosomes, hexosomes, and micellarsomes, have been extensively studied in recent decades because of their distinctive structures and morphologies.1 Cubosomes are liquid crystalline nanoparticles that can be used for drug delivery because of their highly ordered internal structure, high lipid content, and large surface area.1,2 Cubosomes are widely used to enhance the solubility and chemical stability of various drugs, such as 7-ethyl-10-hydroxycamptothecin (SN38),3,4 provide sustained release of docetaxel,5 and improve the cutaneous penetration of cyclosporin A.6 Therefore, many additional applications are expected based on their unique properties.

The standard composition of cubosomes typically includes lipids, stabilizers, and loaded compounds.1 Owing to their chemical stability and purity, monoolein (GMO) and phytantriol (PHY) are the standard amphiphilic lipids used for preparing cubosomes in aqueous solutions, making them ideal for drug delivery applications.7 Steric stabilizers are essential for the stable dispersion of cubosomes to prevent aggregation by coating the outer cubosome surface. Examples of such stabilizers include poly(ethylene oxide)–poly(propylene oxide)–poly(ethylene oxide) (Pluronic F127), PEGylated polymers, and Tween 80.8 Although the structural stability of cubosomes has been extensively studied at room temperature (25 °C),2,4,9,10 limited research has investigated their stability under refrigerated conditions (2–8 °C), which is crucial for drug delivery applications. Moreover, the definition of room temperature varies among different pharmacopeia: for example, controlled room temperature is defined as 20–25 °C by the United States Pharmacopeia,11 while room temperature is defined as 1–30 °C by the Japanese Pharmacopoeia.12 This suggests that the structural stability of the formulation over a wide range of temperatures is a crucial property affecting medical use because it allows for flexible handling at the site of use. When cubosomes, which are stable at both room temperature and refrigerator conditions, are manufactured, they are expected to improve commercial productivity not only for resolving the poor stability of lipid-based drug delivery system (DDS) carriers but also for their application as DDS carriers for thermolabile compounds. Therefore, the identification of new stabilizers that can stabilize cubosomes even under refrigerated conditions would pave the way for future medical use.

The steric effect of a long hydrophilic group of stabilizers has been reported as the mechanism of stabilizers.1 However, increases of particle size and polydispersity were observed by decreasing the temperature when Pluronic F127 was used as a stabilizer for PHY cubosomes,13 suggesting that Pluronic F127 is not sufficient for stabilizing cubosomes over a wide range of temperatures for medical applications. Therefore, a new stabilizer with a stabilization mechanism different from steric effects is desired. In this study, we focused on gemini surfactants, which are more efficient than monomeric surfactants owing to their unique structure, which consists of two monomeric surfactants linked by a spacer.14 Gemini surfactants have an extremely low critical micelle concentration (CMC) and are much more efficient at reducing surface tension compared to conventional monomeric surfactants.14,15 They have been applied in various fields, such as cosmetics,15 solubility improvement,16 and pharmaceuticals, owing to their unique characteristics.14 Gemini surfactants are widely recognized as key components for creating particles, such as liposomes,17 micelles,18 and multilamellar vehicles.19 In DDS research, a variety of gemini surfactants have been synthesized and evaluated for gene therapy;20,21 here, lipoplexes created by gemini surfactants and DNA were found to interact with the lipid membrane and penetrate the cell membrane.21 Additionally, gemini surfactants reportedly affect the physicochemical properties of phosphatidylcholine-based liposomes,17 suggesting that they can also improve the physicochemical properties of cubosomes. We hypothesized that gemini surfactants exert a stabilizing effect on nanoparticles by inserting hydrophobic moieties into the interspaces of lipids in nanosuspensions with two hydrophobic chains as anchors. This mechanism is potentially different from the steric effects of Pluronic F127.

Sodium dilauramidoglutamide lysine (DLGL; Figure 1), a recently developed gemini surfactant, is expected to be useful for drug delivery. Its safety and applicability in medicine as an intestinal absorption enhancer22,23 and in local skin therapy24 have been confirmed. Additionally, the stabilization effect of DLGL was confirmed in stable amorphous microcapsules composed of a mixture of DLGL and ceramide.25 Thus, DLGL is considered a new stabilizer for cubosomes with several unique characteristics, such as low CMC (0.01% w/w),15 biodegradability, and a potentially different anchor mechanism from those of typical stabilizers, such as Pluronic.

Figure 1.

Figure 1

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Chemical structures of (A) sodium dilauramidoglutamide lysine (DLGL) and (B) phytantriol (PHY).

In this study, nanosuspensions were prepared with phytantriol and DLGL, and the effect of DLGL on phytantriol cubosome formation was evaluated using dynamic light scattering (DLS), small-angle X-ray scattering (SAXS), and cryo-transmission electron microscopy (Cryo-TEM). The structural stability of the nanoparticles prepared with different mixing ratios of phytantriol and DLGL was also evaluated by using DLS and SAXS.

Materials and Methods

Materials

Phytantriol (PHY, Figure 1) was kindly provided by DSM Nutritional Products Ltd. (Basel, Switzerland). Pellicer L-30 was kindly supplied by Asahi Kasei Chemical (Tokyo, Japan). Pellicer L-30 contains 29 w/v % of sodium dilauramidoglutamide lysine (DLGL, Figure 1) and 71 w/v % of water; therefore, the amount of DLGL was calculated as a solid base in this paper. Phosphate-buffered saline (PBS) tablet was purchased from Sigma-Aldrich (MO) and one tablet was dissolved in 20 mL of in-house prepared Milli-Q water for use as 10-times concentrated PBS.

Preparation of Mixed Dispersions

PHY (200 mg) and specific weighted DLGL were weighed in a 10 mL glass test tube. In-house prepared Milli-Q (Merck Millipore, MA) water (5 mL) was added and dissolved at 70 °C in a water bath for 30 min. The resulting mixture was probe-sonicated by Sonifier 450 (Branson Ultrasonics Corp., CT) in pulse mode for 120 s (duty cycle 50%, output 2). Additional Milli-Q water was added up to 7.2 mL and probe-sonicated in pulse mode for 120 s. 800 μL of Milli-Q water or 10-times concentrated PBS was added to the sonicated solution. The final PBS solution contained NaCl, KCl, Na2HPO4, and KH2PO4 with the concentrations of 137, 2.7, 10, and 1.8 mM, respectively.

Particle Size Distribution Measurement

The mean particle size and polydispersity index (PDI) were determined with a Zetasizer Ultra (Malvern Instruments, Worcestershire, U.K.). The dispersion samples were diluted 20-fold (v/v) with Milli-Q water, and the measurements were performed at 25 °C, three times for each sample.

Small-Angle X-ray Scattering (SAXS) Measurement

SAXS measurements were performed to characterize the liquid crystalline phase of the cubosomes by using a SAXSpace (Anton Paar GmbH, Graz, Austria). 35 μL sample was loaded into a sample tube with 1 mm in diameter. The temperature, exposure time, and repeat measurement were set to 25 °C, 20 min, and 3 times, respectively. The tube voltage and current were adjusted to 40 kV and 50 mA, respectively. The 1D scattering patterns were recorded with a Mythen microstrip X-ray detector (Dectris Ltd., Baden, Switzerland).

Cryo-Transmission Electron Microscopy (Cryo-TEM) Observation

Cryo-TEM observations were performed to visualize the created nanoparticles by NISSAN ARC, Ltd. Specimens were quickly frozen on a thin film by an EM GP plunge freezer (Leica Microsystems GmbH, Wetzlar, Germany). The frozen thin films were imaged at −269 °C with a JEM-F200 (JEOL Co., Ltd., Tokyo, Japan) incorporating a cryospecimen stage cooled by liquid helium. Samples were operated at accelerating voltage of 200 kV.

Structural Stability Studies of the Cubosomes

The cubosomes were stored at laboratory ambient conditions (25 °C) or in a refrigerator (5 °C) for the evaluation of the structural stability. The appearance, particle properties, and SAXS were determined using the methods described above.

Results and Discussion

Appearance and Particle Size of Prepared Nanosuspensions

An aqueous mixture containing PHY and DLGL was prepared, and nanosuspensions were prepared by using probe sonication. The appearance and particle sizes of the samples were evaluated immediately after preparation. The formulated solution appeared as a translucent or milky white solution for all DLGL/PHY formulations (Figure 2). A milky white solution was observed when the solid ratio of DLGL/PHY was between 1 and 5% w/w, while the solution with a solid ratio exceeding 6% w/w appeared to be translucent white (Figure 2A). The presence of undissolved PHY residue at 0% w/w DLGL/PHY solid ratio indicates the importance of DLGL in forming a uniform dispersion in this composition and manufacturing process. PBS induces a conformational change from liposome vesicles composed of PHY to cubosomes;26 therefore, we also evaluated the effect of the addition of PBS on the DLGL/PHY nanosuspensions. When PBS was added to the formulated solution with a solid ratio of DLGL/PHY between 1 and 5% w/w, immediate aggregation was observed (Figure 2B). When PBS was added to the formulated solution with a solid ratio of DLGL/PHY between 6 and 15% w/w, the solution turned milky white (Figure 2B). The nanosuspensions of DLGL/PHY at 6% w/w were unstable, partially aggregating within a few minutes of preparation. The formulated solution with the solid ratio of DLGL/PHY of 30% w/w did not show any changes in the appearance even after the addition of PBS (Figure 2B).

Figure 2.

Figure 2

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Optical images of DLGL/PHY dispersions with different compositions from 0 to 30% w/w in glass containers. (A) DLGL/PHY dispersion system and (B) DLGL/PHY with the PBS dispersion system.

The characteristics of nanosuspensions with and without PBS are shown in Figure 3. The particle size of the DLGL/PHY nanosuspensions was less than 200 nm and gradually decreased as the amount of DLGL increased (Figure 3A). The particle sizes of nanosuspensions, which appeared milky white in the range of solid ratios of DLGL/PHY between 1 and 5% w/w, were 170–200 nm (Figure 3A). Meanwhile, the particle size of nanosuspensions appearing translucent white at solid ratios of DLGL/PHY exceeding 6% w/w quickly decreased from 170 nm to finally 57 nm at DLGL/PHY 30% w/w (Figure 3A). Therefore, some conformational changes were expected to occur in DLGL/PHY nanosuspensions.

Figure 3.

Figure 3

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Mean particle size and PDI of created nanoparticles at different DLGL/PHY ratios. (A) Mean particle size (nm), (B) PDI, (C) mean particle size (nm) with PBS, and (D) PDI with PBS. The size measurements were repeated for three independent batches.

The polydispersity index (PDI) values of the prepared nanosuspensions ranged from 0.15 to 0.24, and no clear relationship with the amount of DLGL was observed (Figure 3B). In general, a PDI of 0.3 or below is considered to be acceptable and indicates homogeneous vesicles in drug delivery applications using lipid-based carriers, such as liposomes and nanoliposomes.27 Therefore, the variation in our nanosuspensions was comparable to that of well-known nanocarriers. However, PDI tended to increase within the range of solid ratio of DLGL/PHY between 10 and 30% w/w, while the size of nanosuspensions decreased.

The particle size of the nanosuspensions decreased with increasing amounts of DLGL after the addition of PBS (Figure 3C). The mean hydrodynamic size of the nanosuspension with a solid ratio of DLGL/PHY at 6% w/w was 373 nm. The nanosuspensions were unstable, aggregating within a few minutes. The notably high standard deviations of the particle size and PDI indicated instability during sample preparation and measurement (Figure 3C,D). The size of nanosuspensions at DLGL/PHY ratios exceeding 7.5% w/w gradually decreased and finally reached 52 nm at a DLGL/PHY ratio of 30% w/w (Figure 3C). The PDI values of nanoparticles of DLGL/PHY 7.5% w/w were 0.32; by contrast, those of DLGL/PHY greater than 10% w/w were approximately 0.2 and remained constant, regardless of the amount of DLGL (Figure 3D). Interestingly, the appearance, particle size, and PDI values of nanoparticles for DLGL/PHY 30% w/w both with or without PBS were almost the same, namely, translucent white, approximately 50 nm and approximately 0.2, respectively.

Structure of Prepared Nanosuspensions

Cryo-TEM and SAXS were used to investigate the differences in the appearance and particle size of DLGL/PHY nanosuspensions and their conformational changes upon the addition of PBS, respectively. The internal structures of the prepared samples were characterized by SAXS (Figure 4). The typical peak of cubosomes was confirmed in the formed nanoparticles in a range of DLGL/PHY solid ratios between 1 and 3% w/w (Figure 4A). The diffraction patterns of the cubosomes show three Bragg peaks with relative positions at a spacing ratio of √2:√3:√4. These peaks were indexed according to the Miller indices (hkl) of (110), (111), and (200) for the cubic space group Pn3̅m28 No peak was observed at the solid DLGL/PHY ratio of 0% w/w; therefore, the cubic phase could be prepared in the presence of DLGL under these conditions. Quite high concentration of PHY is required to create cubosomes comprising only PHY.29−31 Therefore, DLGL is a key component for forming PHY cubosomes at low concentration of PHY and is equivalent to other stabilizers, such as amphiphilic block copolymers or PEGylated lipids. No diffraction peak was observed at the solid ratio of DLGL/PHY of 5% w/w; however, the peak pattern was completely different from that of DLGL/PHY of 0% w/w (Figure 4A). Several noise peaks were observed around the cubosome-specific peaks of Pn3̅m cubosomes, and a similar broad peak pattern was confirmed at solid ratios of DLGL/PHY of 5 or 6% w/w (Figure 4A). Excess DLGL presumably disrupts the formation of cubosomes and transforms them into SUVs, as observed in cubosomes composed of PHY and 1,2-dipalmitoylphosphatidylserine (DPPS).13 A similar transformation was reported for cubosomes composed of an ionic surfactant and PHY.32 An increase in the ionic surfactant content increased the curvature of the self-assembled system toward the hydrophobic region, resulting in a phase transition from the cubic phase to the lamellar phase.

Figure 4.

Figure 4

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SAXS Profiles of created cubosomes at different DLGL/PHY ratios. The internal structure data of the formulations with peaks showing corresponding Miller indices were obtained by SAXS: (A) without PBS and (B) with PBS.

A clear, typical peak of cubosomes was observed in the formed nanoparticles with PBS in the range of solid DLGL/PHY ratios between 6 and 10% w/w (Figure 4B). The diffraction patterns of the nanoparticles show three Bragg peaks with relative positions at a spacing ratio of √2:√4:√6. These peaks were indexed according to the Miller indices (hkl) of (110), (200), and (211) for the cubic space group Im3̅m.10 Aggregation occurred rapidly at solid ratios of DLGL/PHY of 1, 2, 3, and 5% w/w when PBS was added; therefore, these measurements were not performed. SAXS measurement was completed at a solid ratio of DLGL/PHY of 6% w/w with PBS even though partial aggregation was observed within a few minutes after preparation. The SAXS scattering peaks of cubosomes became broad at a solid ratio of DLGL/PHY of 15% w/w with PBS and disappeared at a solid ratio of DLGL/PHY of 30% w/w with PBS (Figure 4B). When the structure of the nanosuspensions for DLGL/PHY of 30% w/w was measured, the peak pattern of SAXS remained the same before and after the addition of PBS, and the Bragg peaks were not identified (Figure 4A,B). This finding suggests that excess DLGL interferes with the cubic phase transition owing to its surfactant properties.

Cubosomes were prepared at specific concentrations of DLGL with or without PBS (Figure 4A,B). Structural changes occurred upon the addition of DLGL in the presence or absence of PBS. This phenomenon was not observed when Pluronic F127 was used as a stabilizer for cubosomes.33 Therefore, different mechanisms of interaction may occur between PHY and DLGL.

The lattice parameters of each cubosome are shown in Figure 5. The lattice parameters of the Pn3̅m cubosomes showed no differences at the solid ratios of DLGL/PHY of 1, 2, and 3% w/w and were constant at approximately 7 nm. The mean particle sizes were similar: 190, 188, and 185 nm (Figure 3A). Therefore, cubosomes of similar dimensions were suggested to form in this range. The lattice parameters of Im3̅m cubosomes at solid ratios of DLGL/PHY of 6, 7.5, and 10% w/w with PBS were larger than those of Pn3̅m cubosomes. The lattice parameters gradually increased with increasing DLGL content. This tendency was unique to cubosomes composed of DLGL because the lattice parameter of Im3̅m remained constant when Pluronic F127 was added to GMO cubosomes.33 Particle size decreased with an increasing DLGL (Figure 3C). Therefore, the internal size of the cubosomes increased, despite the decrease in the size of the nanoparticles. The peaks of cubosomes at solid ratios of DLGL/PHY of 7.5 and 10% w/w with PBS indicated Im3̅m; however, different peaks from Im3̅m were also observed and were mixed with the Im3̅m peak. This suggests that the peaks are a mixture of Im3̅m and other conformations, which may be the lamellar phase since the portion of lamellar phase was increased according to the increase of the ratio of DLGL in the components. Consequently, this possibly affected the change in the lattice parameter at each solid ratio of DLGL/PHY with PBS (Figure 5).

Figure 5.

Figure 5

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Effect of DLGL/PHY ratio on the space group and lattice parameter of created cubosomes with and without PBS that were obtained by SAXS.

The morphology of the cubosomes and their internal structure were observed by using cryo-TEM (Figure 6). Cubosomes prepared at DLGL/PHY of 2% w/w were selected based on SAXS results as a representative of Pn3̅m cubosomes and subjected to cryo-TEM. Similarly, cubosomes formed at DLGL/PHY of 10% w/w with PBS were selected as a representative of Im3̅m cubosomes. Cubosomes of approximately 200 nm in size were confirmed to have a cubic phase at the solid ratio of DLGL/PHY of 2% w/w (Figure 6A). This observation corresponded to the results of the particle size (Figure 3A) and the internal structure confirmed by SAXS (Figure 4A). Cubosomes in a range of particle sizes from 100 to 200 nm were confirmed to have a cubic phase at a solid ratio of DLGL/PHY of 10% w/w with PBS (Figure 6B). These findings corresponded with the results of the particle size (Figure 3C) and internal structure confirmed by SAXS (Figure 4B). Nanosuspensions formed at DLGL/PHY ratios of 30% w/w and 30% w/w with PBS were selected based on SAXS results as a representative of noncubic conformation. Cubic phase was not identified, and the lamellar phase was confirmed for both samples (Figure 6C,D). The particle size was around 50 nm for both samples. This observation corresponds to the results of particle size (Figure 3A,C) and internal structure confirmed by SAXS (Figure 4A,B). This conformation change would be one of the causes for the significant reduction of particle size at a DLGL/PHY ratio of 30% w/w. Similarly, this conformation change would be one of the causes for the increased trend of PDI within the range of solid ratio of DLGL/PHY between 10 and 30% w/w (Figure 3B). A similar PDI trend was reported in a study investigating the effect of DPPS on PHY nanosuspensions,13 suggesting that the conformation change from cubosomes to lamellar phase would affect PDI.

Figure 6.

Figure 6

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Cryo-TEM images of formulated nanosuspensions. (A) Solid ratio of DLGL/PHY of 2% w/w, (B) solid ratio of DLGL/PHY of 10% w/w with PBS, (C) solid ratio of DLGL/PHY of 30% w/w, and (D) solid ratio of DLGL/PHY of 30% w/w with PBS. The arrows indicate typical cubosomes.

Structural Stability of Prepared Cubosomes

The structural stability of cubosomes at solid ratios of DLGL/PHY of 1, 2, and 3% w/w and that at 7, 8, and 10% w/w with PBS at 25 and 5 °C were evaluated for appearance, particle size (Figure 7), and internal structure by SAXS (Figure 8). Cubosomes produced at DLGL/PHY of 1 and 2% w/w were stable in appearance (milky white) and particle size for 4 weeks at both 25 and 5 °C (Figure 7A,C). The particle size and PDI of DLGL/PHY 3% w/w varied during storage at 25 °C (Figure 7A,B), and aggregation occurred during storage at 5 °C (Figure 7C,D). Although similar cubosomes appeared to have formed at DLGL/PHY of 3% w/w and DLGL/PHY of 1 and 2% w/w (Figure 4A), their stabilities apparently varied. The SAXS data profile measured at 5 °C was similar to the data measured at 25 °C for DLGL/PHY 1 and 2% w/w (Supporting Information Figure S1). It suggests that cubosomes at DLGL/PHY 1 and 2% w/w maintained Pn3̅m cubosomes and resulted in stable state at 5 °C. On the other hand, the typical peak of Pn3̅m cubosomes disappeared for DLGL/PHY 3% w/w measured at 5 °C (Supporting Information Figure S1), suggesting that cubosomes at DLGL/PHY 3% w/w could not maintain the cubic phase and resulted unstable at 5 °C. Consequently, the damage by temperature change had a bigger impact to cubosomes at DLGL/PHY 3% w/w. DLGL/PHY of 1 and 2% w/w were the optimum concentrations for creating the stable Pn3̅m cubosomes.

Figure 7.

Figure 7

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Results of structural stability study of formulated cubosomes for 4 weeks. (A) Mean particle size and (B) PDI at the solid ratio of DLGL/PHY of 1, 2, and 3% w/w without PBS after the storage at 25 °C, (C) mean particle size and (D) PDI at the solid ratio of DLGL/PHY of 1, 2, and 3% w/w without PBS after the storage at 5 °C, (E) mean particle size and (F) PDI at the solid ratio of DLGL/PHY of 7, 8, and 10% w/w with PBS after the storage at 25 °C, and (G) mean particle size and (H) PDI at the solid ratio of DLGL/PHY of 7, 8, and 10% w/w with PBS after the storage at 5 °C. #: Discontinued the evaluation by aggregation.

Figure 8.

Figure 8

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SAXS profiles of formulated cubosomes after the storage at 25 or 5 °C. The internal structure data of the formulations with peaks showing corresponding Miller indices were obtained by SAXS. (A) the solid ratio of DLGL/PHY of 2% w/w without PBS, (B) the solid ratio of DLGL/PHY of 10% w/w with PBS.

Cubosomes prepared at DLGL/PHY of 10% w/w with PBS were visibly stable for 4 weeks at 5 °C. However, aggregation occurred during storage at 25 °C for 1 week (Figure 7E,F). The particle size and PDI of the DLGL/PHY of 10% w/w cubosomes with PBS gradually increased during storage at 5 °C (Figure 7G,H); however, aggregation did not occur. Therefore, we confirmed that stable Im3̅m cubosomes at 5 °C were successfully prepared at DLGL/PHY of 10% w/w with PBS. The structural stability of cubosomes at solid ratios of DLGL/PHY 7 and 8% w/w with PBS was also evaluated. Notably, aggregation occurred at 25 °C for 1 week and at 5 °C for 1 or 2 weeks (Figure 7E–H), respectively. Therefore, DLGL/PHY of 10% w/w was the optimal concentration for producing stable Im3̅m cubosomes. The SAXS data profile for DLGL/PHY of 10% w/w measured at 50 °C showed much more obvious typical peak of Im3̅m cubosomes than that measured at 25 °C, suggesting that the mixture components other than Im3̅m cubosomes could not maintain the structure at high temperatures (Supporting Information Figure S2). As a result, such unstable mixture components would affect the structural stability of the whole nanodispersion system, including Im3̅m cubosomes. On the other hand, the SAXS data profile measured at 50 °C was similar to the data measured at 25 °C for Pn3̅m cubosomes (Supporting Information Figure S1), suggesting that Pn3̅m cubosomes were not damaged by temperature change. These properties resulted in a difference in the structural stability between Pn3̅m cubosomes and Im3̅m cubosomes.

The internal structures of the stored samples were also evaluated by using SAXS (Figure 8). Samples of DLGL/PHY of 2 and 10% w/w with PBS were selected for SAXS measurements based on the structural stability results of particle size. No changes of structure were observed for Pn3̅m cubosomes at a solid ratio of DLGL/PHY 2% w/w both at 25 and 5 °C for 4 weeks (Figure 8A). Meanwhile, no change of structure was observed for Im3̅m cubosomes at the solid ratio of DLGL/PHY 10% w/w with PBS at 5 °C for 4 weeks (Figure 8B), although the particle size gradually increased. This unique structural stability is expected to contribute to stable drug release from cubosomes and stabilization for incorporated thermolabile drugs because of their nonchangeable internal structure. This property will be the subject of future investigations.

The proposed mechanism explaining the observed conformational differences along the amount of added DLGL is shown in Figure 9. In the absence of DLGL, PHY is not well dispersed because of its high viscosity and low solubility in water (Figure 9A). However, the addition of DLGL, which has a low CMC,15 tends to create a spherical structure owing to its self-assembling attributes.18,25 PHY was dispersed, causing the self-assembly of the mixture. The resulting particles became reverse micelles and created cubosomes through the lyotropic phase behavior of PHY, which was the major component in the mixture at the solid ratios of DLGL/PHY of 1, 2, and 3% w/w (Figure 9B).18 Further increase in DLGL in the mixture with PHY decreased the negative curvature of the vesicle because of repulsion between the hydrophilic headgroups of DLGL. Consequently, the cubic vesicles transformed into a lamellar phase at a solid ratio of DLGL/PHY of 5% w/w or more in salt-free conditions without PBS (Figure 9C).

Figure 9.

Figure 9

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Schematic diagram of phase transition of DLGL/PHY dispersions driven by the amount of DLGL and the existence of PBS.

When PBS was added to the cubosome solution, salting-out was induced, and aggregation was observed at solid ratios of DLGL/PHY of 1, 2, 3, and 5% w/w (Figure 9D). The ionic salts screened the repulsion between the hydrophilic headgroups by the addition of the ion salt of PBS, thereby increasing the negative curvature of the DLGL/PHY mixture. At the solid ratios of DLGL/PHY of 6, 7.5, and 10% w/w, the negative curvature was well balanced with PHY, and the lamellar phase transitioned to the cubic phase with the addition of PBS (Figure 9E). The effective hydrophilic headgroup area of DLGL became smaller because of the interaction with ionic salts, requiring a larger minimum amount of DLGL (6% w/w) to create cubosomes than that required in the absence of PBS (1% w/w). At a solid ratio of DLGL/PHY of 15% w/w, a broad peak was observed in the area, where the Im3̅m peak appeared (Figure 4B). Therefore, the conformational change may have been partially induced by the addition of PBS; however, the majority of the lamellar phase was maintained, and cubosomes were not clearly detected in the SAXS measurements. A further increase in DLGL to 30% w/w did not induce a conformational change, and the lamellar phase was maintained (Figure 9F). Presumably, the excess DLGL solubilized cubosomes and changed to the lamellar phase.13

The structural differences between cubosomes with and without PBS may be attributed to the size of the effective headgroup of DLGL. Compared to Pn3̅m, the Im3̅m cubic phase has a wider lattice parameter. The smaller hydrophilic headgroup of DLGL created in the presence of PBS required a large volume of DLGL to produce cubosomes. Thus, the amount of DLGL in the cubosomes was larger than that produced without PBS, and the volume of DLGL in the internal cubic nanostructures increased. Consequently, Im3̅m was formed in the presence of PBS, which has a wider lattice parameter than Pn3̅m. A similar conversion of Pn3̅m to Im3̅m has been reported for GMO and Pluronic F127 cubosomes.33 The space group of the cubosomes changed from Pn3̅m to Im3̅m upon the addition of Pluronic F127. Pluronic F127 is located in an internal cubic nanostructure and causes an increase in lattice parameter. A similar phenomenon is considered to occur for the difference in size of the hydrophilic headgroup.

Our hypothesis for the stabilization mechanism of gemini surfactants for cubosomes is their effective insertion into the lipid membrane of PHY (Figure 9B,E). However, the location of DLGL in the cubosomal membrane was not determined in the present study. Due to its lack of steric properties because of its structural formula, DLGL has a structural stability mechanism that deviates from the steric effect as in Pluronic or PEGylated lipids. The instability of cubosomes at low temperatures is suggested to occur because of decreased steric effects resulting from the reduced molecular mobility at low temperatures. This can cause the release of stabilizers from the cubosomes, leading to their collapse. DLGL has two hydrophobic tails linked to three hydrophilic peptides, which allow it to be strictly anchored to the lipid membrane through the hydrophobic tails. The configurational degrees of freedom of the hydrophilic group provide flexibility for molecular mobility and maintain the anchoring effect in cubosomes at low temperatures. Further studies are required to elucidate the mechanism of DLGL stabilization.

The ability to prepare stable cubosomes at refrigerator conditions (5 °C) offers a significant advantage in their application as DDS carriers, not only for overcoming the poor stability of lipid-based DDS carriers but also for their use in heat-sensitive compounds. Furthermore, the creation of stable cubosomes with PBS offers an advantage in their pharmaceutical applications owing to their stability under physiological conditions after administration.

Conclusions

This study demonstrates that the gemini surfactant DLGL has the potential to stabilize PHY cubosomes. Here, an optimal amount range of DLGL for preparing the cubosomes in the absence or presence of PBS was determined, that is, solid ratios of DLGL/PHY from 1 to 3% w/w or from 6 to 10% w/w, respectively. This suggests that excess DLGL induces conformational changes in cubosomes. The internal structure of the cubosomes differed in the absence and presence of PBS. SAXS data showed Pn3̅m in the absence of PBS and Im3̅m in the presence of PBS. The structural stability of the cubosomes formed differed between Pn3̅m and Im3̅m. Pn3̅m cubosomes at the solid ratio of DLGL/PHY of 2% were stable for 4 weeks at 25 °C, as well as for 4 weeks at 5 °C, surprisingly. This study shows that the gemini surfactant DLGL can function as a new stabilizer for cubosomes that are stable at 5 °C. Cubosomes composed of DLGL can be used not only to improve the poor stability of lipid-based DDS carriers but also to construct DDS carriers for heat-sensitive active compounds.

Acknowledgments

The authors acknowledge Takeda Pharmaceutical Company Limited for using the laboratory, general reagents and all the equipment we used.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.molpharmaceut.3c00378.

  • SAXS profiles measured at different temperatures for prepared cubosomes (PDF)

The authors declare no competing financial interest.

Special Issue

Published as part of the Molecular Pharmaceuticsvirtual special issue “Research Frontiers in Industrial Drug Delivery and Formulation Science”.

Supplementary Material

mp3c00378_si_001.pdf (131.6KB, pdf)

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Supplementary Materials

mp3c00378_si_001.pdf (131.6KB, pdf)

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