1. Introduction
Technological advancements in biomedicine are accelerating due to environmental and social changes, leading to substantial progress in therapeutic gene and drug delivery systems. Among these, emulsion systems have emerged as highly promising drug delivery platforms [1-3].
An emulsion is a mixture of two thermodynamically unstable, incompatible liquids in which one liquid is dispersed within the other. Emulsions consist of three primary components: the oil phase, the water phase, and an emulsifier. Emulsifiers are amphiphilic molecules containing lipophilic and hydrophilic groups that reside at the oil-water interface, reducing interfacial free energy and tension. This reduction in free energy enhances the stability of the dispersed particles, facilitating the mixing of the oil and water phases. The physicochemical prop-erties and size distribution of emulsions can be easily controlled, making them versatile for various applications. Notably, the oil phase can accommodate large quantities of therapeutic genes and poorly soluble drugs, including various hydrophobic anticancer agents, regardless of size[4-7].
Emulsions are mainly categorized as water-in-oil (W/O) or oil-in-water (O/W). In W/O emulsions, the oil phase is continuous, while the water phase is dispersed. These emulsions exhibit low electrostatic repulsion, leading to rapid phase separation and stability issues. Additionally, W/O emulsions require substantial amounts of oil and emulsifiers, increasing production costs. Conversely, O/W emulsions, with a dispersed oil phase within a continuous water phase, demonstrate high electrostatic repulsion and enhanced stability. This configuration effectively retards phase separation, making O/W emulsions efficient drug carriers[8-10].
Dequalinium, employed in this study, has been previously utilized as DQAsomes and extensively studied for various gene-delivery systems [11-13]. Recent studies have explored their potential as novel drugand gene-delivery systems[14]. Dequalinium is known for its antibacterial, anticancer, and antimalarial properties, and it effectively localizes to the mitochondria. Structurally, dequalinium has a bolaform conformation with two hydrophilic quinaldinium units linked by a hydrophobic ten-methylene chain. As a cationic bola-amphiphile, dequalinium can potentially be used as an emulsifier (Figure 1)[15-16].
We used vegetable castor oil, primarily composed of ricinoleic acid, as the oil phase. Castor oil, a light yellow and highly viscous vegetable oil, is classified as a safe and effective food additive by the United States Food and Drug Administration. It is widely used in pharmaceuticals, supporting its potential for development into an efficient emulsion system[17-18].
Curcumin, extracted from the roots of Curcuma longa Linn., is a hydrophobic polyphenolic compound containing ring-structured phenols and unsaturated carbonyl groups. Known for its antioxidant, anti-inflammatory, antitumor, and anti-amyloid activities, curcumin’s hydrophobic nature makes it insoluble in aqueous solutions but readily soluble in the oil phase, facilitating mitochondrial-targeted drug delivery [19].
The curcumin-loaded dequalinium emulsions (DQE) based on the O/W emulsion system were prepared using homogenization and sonication. The morphology and size distribution of the emulsion particles were characterized using nanoparticle analysis, atomic force microscopy (AFM), and transmission electron microscopy (TEM). The surface charge of the DQE particles was determined by measuring their zeta potentials. Dequalinium and curcumin in DQE were quantified using absorbance measurements, and stability was assessed by monitoring size changes over three months at room temperature. Confocal microscopy evaluated the mitochondrial-targeting efficiency of DQE in HeLa cells compared to controls.
2. Materials and methods
2.1. Materials
Dequalinium chloride, castor oil, N-(2-hydroxyethyl)piperazine-N'- (2-ethanesulfonic acid) (HEPES), and 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl-2H-tetrazolium bromide were purchased from Sigma Aldrich (Seoul, Korea). Curcumin was purchased from Alfa Aesar (Incheon, Korea). 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine-N-carboxyfluorescein ammonium salt (CF-PE) was purchased from Avanti Polar Lipids (Alabaster, AL, USA). Polyethylene glycol-8, caprylic/capric glyceride (LAS), isopropyl myristate (IPM), and lecithin were provided by Phytos (Anyang, Korea). HEPES buffer was prepared at a concentration of 5 mM and a pH of 7.4. Tertiary distilled water was prepared by autoclaving and filtering through a 0.22 μm filter. The 0.22 and 0.8 μm filtration units were purchased from Merck Millipore (Darmstadt, Germany). Dulbecco's modified Eagle’s medium (DMEM), antibiotic- antimycotic agent, Dulbecco’s phosphate-buffered saline, and fetal bovine serum (FBS) were purchased from Gibco (Gaithersburg, MD, USA).
2.2. Preparation of DQE
To investigate the effects of dequalinium chloride concentration and castor oil content on emulsion formation, emulsions were prepared with varying amounts of these components (Table 1). Dequalinium chloride, castor oil, and distilled water were combined in a 20 mL glass bottle. The mixtures were sonicated for 15~20 min, followed by homogenization at room temperature for at least 3 h using a homogenizer (Polytron-PT3100, KINEMATICA Inc., Bohemia, USA). The resulting DQE was subsequently filtered through a 0.8 μm filter and stored at room temperature (Figure 2).
2.3. Preparation of curcumin-loaded DQE
To investigate the impact of curcumin concentration on DQE formation, dequalinium chloride, castor oil, and curcumin were sequentially added to a 20 mL glass reagent bottle (Table 1). Distilled water was added, and the mixture was sonicated for 15~20 min. Following sonication, the emulsion was homogenized at room temperature for at least 3 h using a homogenizer. The resulting DQE was filtered through a 0.8 μm filter and stored at room temperature (Figure 2).
2.4. Preparation of control emulsion
The control emulsion was prepared by sequentially adding IPM, LAS, lecithin, and distilled water to a 20 mL glass reagent bottle. The mixture was sonicated for 15~20 min and homogenized at room temperature for at least 3 h. The prepared control emulsion was filtered through a 0.8 μm filter and stored at room temperature (Table 1).
2.5. Preparation of DQAsome
DQAsomes were used as controls for the DQE and were prepared using a previously described method. First, 5 mL of methanol and 0.0528 g of dequalinium were added to a round-bottomed flask to dissolve the dequalinium completely. Methanol was subsequently removed using a vacuum pump. Next, 5 mL of HEPES buffer (pH 7.4, 5 mM) was added, and the resulting mixture was sonicated at 25 °C or below for 1 h. After sonication, any undissolved dequalinium was removed by centrifugation at 10,000 rpm for 5 min. The prepared DQAsome was filtered through a 0.8 μm filter and stored in a glass reagent bottle at room temperature.
2.6 Preparation of fluorescently labeled DQE and DQAsome
2.6.1. Preparation of CF-DQE
To prepare fluorescently labeled DQE, 0.0528 g of dequalinium, 0.3 g of castor oil, 1.12 μg of CF-PE, and 9.848 g of distilled water were sequentially added to a 20 mL glass reagent bottle. The mixture was sonicated for 15~20 min and homogenized at room temperature for over 3 h using a homogenizer. The resulting CF-DQE was then filtered through a 0.8 μm filter and stored at room temperature.
2.6.2. Preparation of fluorescently labeled DQAsomes
To prepare fluorescently labeled DQAsomes, 5 mL of methanol and 0.0528 g of dequalinium were added to a round-bottomed flask to dissolve dequalinium completely. Subsequently, 1.12 μg of CF-PE was added, and the methanol solvent was evaporated using a vacuum pump. Then, 5 mL of HEPES buffer (pH 7.4, 5 mM) was added, and the mixture was sonicated for 1 h at or below 25 °C. After sonication, any undissolved dequalinium was removed using centrifugation at 10,000 rpm for 5 min. The prepared CF-DQAsome was filtered through a 0.8 μm filter and stored in a glass reagent bottle at room temperature.
2.7. Quantification of dequalinium and curcumin
The dequalinium content of the DQE was quantified using a UV/VIS spectrophotometer (Optizen POP, Mecasys, Korea). A standard curve was plotted using various concentrations of dequalinium dissolved in methanol, measured at a wavelength of 325 nm. The dequalinium content in the DQE was determined using this standard curve. Similarly, the curcumin content in the curcumin-loaded DQE was quantified using a spectrophotometer. A standard curve was prepared using various concentrations of curcumin dissolved in castor oil, and absorbance was measured at 420 nm. The curcumin content in the curcumin-loaded DQE was evaluated using this standard curve.
2.8. Particle size and zeta potential measurement of DQE
The particle size distribution of the DQE emulsions was determined using a Zetasizer Nano ZS (Malvern Instruments, UK) and ELS-Z (Otsuka Electronics, Otsuka, Japan). Prior to measurement, the DQE emulsions were diluted with deionized water to an appropriate concentration to avoid multiple scattering effects. The measurements were conducted at a fixed temperature of 25 °C, with each sample being measured in triplicate to ensure accuracy and reproducibility. The zeta potential was measured using a Zetasizer Nano ZS (Malvern Instruments, UK). The samples were placed in cuvettes, and measurements were conducted at 25 °C. Each sample was measured in triplicate, and the results were reported as the average ± standard deviation (mV).
2.9. Image measurement of DQE particles
2.9.1. AFM
The morphology and size of the DQE emulsion particles were characterized using Atomic Force Microscopy (AFM). A small drop of the DQE emulsion was carefully placed onto a clean glass plate to prepare the samples. The sample was allowed to dry slowly at room temperature to ensure adequate adhesion of the particles to the substrate without altering their native structure. Once dried, the sample was mounted on the AFM stage, and imaging was performed in tapping mode to minimize damage to the emulsion particles.
2.9.2. TEM
Transmission electron microscopy (TEM) was employed to examine further the internal structure and detailed morphology of the emulsions and DQAsome. The samples were prepared by placing a drop of the emulsion onto a grid, followed by drying at room temperature. Once dried, the samples were stained with a contrast agent to enhance the visibility of the particle structures under the electron beam. TEM analysis was conducted using a high-resolution TEM instrument.
2.10. Stability evaluation of DQE
The long-term stability of the DQE emulsion was evaluated by monitoring changes in particle size over 3 months. The emulsion samples were stored at a constant temperature of 25 °C, simulating typical storage conditions. At predetermined intervals, the particle size of the emulsion was measured using the ELS-Z (Otsuka Electronics, Otsuka, Japan), which utilizes dynamic light scattering to track any variations in size distribution. The stability of the emulsion was assessed by comparing the initial particle size measurements to those obtained after the indicated days of storage.
2.11. Cell culture and confocal microscopy
HeLa cells were cultured at 37 °C with 5% CO2 in a 90% DMEM medium, 10% FBS, and 1% antibiotics. Confocal wells (μ-slide 8 well, ibidi, Germany) were seeded with 5,000 cells per well and incubated at 37 °C with 5% CO2 for 24 h. After the incubation period, CF-DQAsome and CF-DQE10-3% emulsions were prepared at a concentration of 0.25 μg/mL, added to the cells, and incubated at 37 °C with 5% CO2 for 4 h. Nuclear staining was performed using bisbenzimide H 33342 trihydrochloride for 5 min at room temperature, and mitochondrial staining was performed using MitoTracker for 5 min at room temperature. The cells were then observed under a confocal microscope (LSM5 live configuration with two VRGB).
3. Results and discussion
3.1. Confirmation of DQE formation and measurement of particle size as a function of dequalinium concentration and castor oil content
The formation of homogeneous emulsions was investigated based on dequalinium concentration. Homogeneous emulsions were not successfully formed at concentrations exceeding 8 mM. However, when the dequalinium concentration was 10 mM or higher, emulsions with particles approximately 200 nm in size were successfully obtained. To further explore the influence of castor oil content, emulsions were prepared using a fixed dequalinium concentration of 10 mM. The results indicated that the particle size increased with higher castor oil content. Specifically, emulsions with particle sizes around 200 nm were consistently produced when the castor oil content was maintained at 3% or less. These results suggest that the DQE10-3% formulation, containing 10 mM dequalinium and 3% castor oil, yielded the most homogeneous particles (Figure 3).
3.2. Zeta potential measurement of DQE
The DQE10-3% emulsion demonstrated optimal particle homogeneity and exhibited favorable surface charge characteristics, crucial for its potential applications. The zeta potential measurement for DQE10-3% was recorded at 42.7 mV, significantly higher than the control emulsion (7.6 mV) but slightly lower than the DQAsome reference material (55.0 mV). This positive zeta potential confirms that the DQE10-3% emulsion maintains a cationic nature, which is advantageous for interactions with negatively charged cell membranes. This property is significant for biomedical applications, where effective particle uptake by cells is critical (Figure 4).
3.3. Evaluation of physical stability of DQE
The physical stability of DQE emulsions was assessed over time, revealing that emulsions with 8 mM or lower dequalinium concentrations exhibited significant morphological changes with a large size distribution, indicating particle instability. In contrast, emulsions with concentrations of 10 mM or higher demonstrated enhanced stability, with particle sizes consistently below 200 nm and no visible precipitate formation. The addition of castor oil further improved stability, with the best results observed at a castor oil concentration of 3%. Thus, the formulation containing 10 mM dequalinium and 3% castor oil produced the most homogeneous and physically stable emulsion (Figure 5). However, after 90 days, the DQE emulsion exhibited an increase in particle size. This result may be attributed to several factors. First, smaller emulsion droplets might have dissolved and redeposited onto larger ones through a process called Ostwald ripening, resulting in an overall increase in particle size. Second, the gradual merging of droplets, likely due to weakened repulsive forces or changes in interfacial tension, could have led to coalescence and the formation of larger particles. Third, partial phase separation may have occurred over time, causing the dispersed phase to coalesce into larger droplets. To improve the stability of the DQE emulsion, incorporating additional emulsifiers, such as Tween 80 or Span 60, could be advantageous.
3.4. Characterization of DQE particles
The morphology and size of the DQE emulsion particles were characterized using Atomic Force Microscopy (AFM) and Transmission Electron Microscopy (TEM). AFM analysis revealed that both DQE10-1% and DQE10-3% emulsions formed uniform particles, with the DQE10-3% emulsion showing enhanced uniformity, with particle sizes ranging from 100 to 200 nm (Figure 6). TEM analysis corroborated these findings, confirming the presence of spherical particles within the same size range of 100 to 200 nm in the DQE10-3% emulsion (Figure 7). The consistency of results across these complementary imaging techniques validates the physical characteristics of the optimized DQE formulation.
3.5. Mitochondrial-targeting ability of DQE
The ability of the DQE10-3% formulation to target mitochondria was evaluated using confocal microscopy in HeLa cells. The results demonstrated a significant enhancement in the localization of the DQE10-3% emulsion to the mitochondria compared to the control DQAsome formulation. This enhanced targeting indicates the superior mitochondrial-targeting capability of the DQE formulation, which is promising for delivering therapeutics directly to the mitochondria. Such targeted delivery is crucial for maximizing therapeutic efficacy while minimizing off-target effects (Figure 8). The observed localization pattern in confocal microscopy images provides strong evidence for the potential of the DQE formulation in developing targeted therapies for mitochondria-related diseases.
3.6. Curcumin-loaded DQE particles
In addition to mitochondrial targeting, the DQE formulation was assessed for its ability to encapsulate curcumin, a compound known for its therapeutic properties. The particle sizes of the curcumin-loaded DQE emulsions ranged from 100~200 nm. Interestingly, particle size decreased as the curcumin content increased, suggesting that curcumin may play a stabilizing role in the emulsion. (Figure 9a). This size reduction is beneficial for drug delivery applications, as smaller, more stable particles can enhance bioavailability and improve the pharmacokinetic profile of the encapsulated drug. These findings highlight the potential of DQE formulations in developing effective drug delivery systems for curcumin and other therapeutic agents.
3.7. Encapsulation efficiency of curcumin in DQE
The encapsulation efficiency of curcumin within the DQE emulsion was assessed by comparing the absorbance values to a predetermined standard curve. This approach enabled precise measurement of the effectiveness of curcumin incorporation into the emulsion matrix. The results indicated that while encapsulation efficiency slightly decreased with increasing curcumin concentration, nearly 100% efficiency was achieved at a low curcumin concentration of 1 mg (Figure 9b). This observation indicates that the DQE emulsion effectively encapsulates curcumin, especially at low concentrations. The observed decrease in efficiency at higher concentrations may be due to saturation effects or interactions between curcumin molecules, which could hinder the encapsulation process. Overall, these findings exhibit the potential of the DQE formulation as a promising vehicle for curcumin delivery, particularly at optimized concentrations, which is crucial for maximizing therapeutic outcomes.
4. Conclusion
This study developed a novel oil-in-water emulsion system (DQE) designed explicitly for mitochondria-targeted drug delivery. By systematically varying dequalinium concentrations and castor oil content and employing sonication and homogenization techniques, we optimized the DQE formulation. Comprehensive characterization using nanoparticle analysis, AFM, and TEM confirmed the formation of homogeneous emulsion particles, with sizes consistently ranging from 100 to 200 nm. Zeta potential measurements revealed a stable cationic surface charge of approximately +42 mV, critical for effective interaction with negatively charged cell membranes. Stability assessments over three months at room temperature demonstrated that the emulsion containing 10 mM dequalinium and 3% castor oil exhibited superior physical stability. Confocal microscopy studies in HeLa cells further validated the DQE formulation's enhanced mitochondrial localization. Additionally, the DQE system demonstrated an impressive loading efficiency of nearly 100% for the model drug curcumin at a concentration of 1 mg, highlighting its potential as an effective drug delivery vehicle. These findings suggest that the DQE system holds significant promise as a mitochondria-targeted drug delivery platform. The inherent targeting capability of dequalinium, combined with its ability to encapsulate therapeutic agents like curcumin efficiently, positions the DQE system as a strong candidate for further research and development. With continued optimization and validation, the DQE system could pave the way for innovative therapies to treat mitochondria-related diseases and enhance the precision of drug delivery in clinical applications.
