Preparation of Curcumin-Liposomes using pH-Driven Method to Enhance Stability and Encapsulation Efficiency

Curcumin, a hydrophobic polyphenol, derived from turmeric, has numerous pharmacological activities, including hepatoprotective, antioxidant, anti-inflammatory, antimicrobial, and anticarcinogenic. However, the therapeutic efficacy of curcumin is limited due to its poor oral bioavailability and high susceptibility to degradation. The poor oral bioavailability of curcumin has been attributed to its poor aqueous solubility and extensive first past metabolism. Curcumin dissolves in alkaline conditions but it is highly unstable undergoing rapid hydrolytic degradation in neutral or alkaline condition. To solve these problems, liposome-based encapsulation technology is applied to improve the bioavailability of curcumin. The existing method to prepare curcumin-liposome, thin-film method, is complex and usually required the use of organic solvents. In this study, pH-driven method, an organic solvent-free and easily encapsulation technique utilizing the pH-dependent solubility of curcumin was proposed to improve the stability and bioavailability. Curcumin-liposomes were prepared using phospholipon 90 H and soya lecithin as phospholipid sources. The characterizations of curcuminliposomes include particle size, microstructure, encapsulation efficiency, infrared spectra, salt stability and storage stability. The particle size measured was 77.85 ± 0.39 nm. It was found that curcumin-liposomes prepared by the pH-driven method was stable during storage for 30 days and gave encapsulation efficiency of 60.71 ± 0.20%, higher as compared to the ones prepared by thin-film method. In conclusion, pH-driven is a promising method for the preparation of curcumin-liposomes to enhance stability and encapsulation efficiency.


INTRODUCTION
Curcumin, a natural hydrophobic polyphenol derived from rhizomes of Curcuma longa Linn. (Zingiberaceae), has  numerous  medicinal  benefit  effects,  including hepatoprotective, antioxidant,  antimicrobial, anti-inflammatory, and anticarcinogenic activities. 1 In spite of its efficacy, curcumin has not yet been approved as a therapeutic agent because of its low water solubility, low oral bioavailability, and high susceptibility to degradation. 2,3 The several strategies have been performed to overcome these drawbacks. Numerous researches have reported on the use of various curcumin formulations toward improving the solubility as well as bioavailability of curcumin in aqueous systems, including nanoparticles, cyclodextrins, micelles, liposomes, and emulsions. 4 Among these approaches, encapsulating curcumin in liposomes has become one of the most promising methods because of its excellent compatibility of hydrophilic and hydrophobic molecules. Feng et al. have succeeded to entrap curcumin in liposomes and the result showed that liposomal curcumin suppressed pancreatic carcinoma growth in murine xenograft models and inhibited tumor angiogenesis. 3 Liposome is a spherical vesicle with phospholipid bilayer which can encapsulate both hydrophilic and hydrophobic components in the inner aqueous phase and incorporating them into the lipid bilayers, respectively. 5,6 Curcumin-liposomes (CLs) have been prepared by the thin film method, as well as the properties and stability of these CLs have been widely studied by a number of groups. Nevertheless, the organic solvents used in this method is unacceptable for oral drug delivery. 5,7 Recently, an organic solvent-free encapsulation, the pH-driven method, was developed. This method is based on the deprotonation and dissolution of hydrophobic phytochemicals under alkaline conditions followed by neutralization to encapsulate the precipitated hydrophobic phytochemicals. 7 The previous study showed that curcumin-liposomes which were formed using a single compound of phospholipid with pH-driven method had the average diameter more than 200 nm (~217.5 nm) and encapsulation efficiency ~62,8% with relatively intermediate stability which little bit precipitation was observed after the stability test. 2 The stability of curcumin liposomes has potential to be improved by reducing the particle size and enhance the encapsulation efficiency.
In this research, curcumin was encapsulated in liposomes using two compounds of phospholipid with the pHdriven method to improve its stability and encapsulation efficiency for the further aim as a hepatoprotective agent. The encapsulation efficiency and particle size of curcumin-liposomes formed by the pHdriven method (PDM-CLs) were compared to curcumin-liposomes prepared using the thin film method (TFM-CLs). The stability study of the PDM-CLS and TFM-CLs after treatment with sodium chloride solution and storage for 30 days were also evaluated.

Preparation of curcumin-liposomes
Curcumin-liposomes were prepared by the pH-driven method and the thin film method. The phospholipid concentration was 10 mg/mL, and the curcumin feed concentration was 0.4 mg/mL. 2 The phospholipid ratio (w/w) between phospholipon 90 H and soya lecithin was 1:1.

pH-driven method
Curcumin and phospholipid were added to pure water grade II and stirred for 4 hours at room temperature. The pH of the solution was then adjusted to 12.0 with 4 M NaOH.
After stirring for 20 minutes, the pH of the solution was adjusted back to 5.3 with 4 M HCl. The curcumin-liposome suspension was further sonicated (on ice, 1 x 30 min) using a probe sonicator (Sonics, Vibra Cell, USA) and the PDM-CLs were then collected. 2,7 Thin-film method Briefly, curcumin and phospholipid were dissolved in chloroform and methanol (2:1 v/v), and the organic solvents were then evaporated using a rotary evaporator. The thin film was hydrated with phosphate buffer (pH 7.4) and prepared the curcuminliposome suspension. The curcuminliposome suspension was further sonicated (on ice, 1 x 30 min) using a probe sonicator (Sonic, Vibra Cell, USA) and the TFM-CLs were collected. 1,3,8,9 Characterization of curcumin-liposomes particle size The particle size of curcumin-liposomes (PDM-CLs and TFM-CLs) were measured using Partica LA-950V2 Laser Scattering Particle Size Analyzer (Horiba, Japan) with a fraction cell system. All data presented are the averages of at least triplicate measurements.

Microstructure
The microstructure of pH-driven method curcumin-liposomes (PDM-CLs) was analyzed using a transmission electron microscope (TEM). Briefly, a drop of PDM-CLs was placed onto a carbon-coated copper grid. After 3 minutes, the copper mesh grid was stained with acetic uranyl for 4 minutes and air-dried at room temperature after removing the excess liquid with filter paper. The sample was examined under a JEM-1400 transmission electron microscope (JEOL, Ltd., Tokyo, Japan) at a voltage of 120 kV.

Encapsulation efficiency (EE)
The curcumin-liposomes were centrifuged at 10,000 rpm for 30 min using a MIKRO 22 R centrifuge (Hettich Zentrifugen, Germany) to separate unencapsulated curcumin. The concentration of free curcumin (supernatant) was determined by UV-Vis spectrophotometry. At 421 nm, the absorbance of free curcumin was calculated from the calibration curve, and encapsulation efficiency was then calculated by the following equation 1,10 :

Fourier transform infrared spectroscopy (FTIR)
The infrared spectra of PDM-CLs, phospholipid and curcumin were separately obtained using Nicolet iS10 FTIR Spectrometer (Thermo Fisher Scientific Inc, Madison, USA). The samples were placed on Smart iTR™ Attenuated Total Reflectance (ATR) accessory composed of diamond crystal as sample handling technique at a controlled ambient temperature (25°C). The samples were scanned by an infrared spectrometer over the range from 4000 to 400 cm −1 . 11

Stability study Salt stability
The influence of ionic strength on the stabilities of the PDM-CLs and TFM-CLs was determined by treatment with several concentrations of NaCl solution (10, 50, 100, 200, 500, and 1000 mM) for 1 hour at room temperature. 2 The appearance of the samples was observed after incubation for 1 hour in room temperature and after 3 days at 4°C.

Storage stability
To evaluate the shelf life, the PDM-CLs and TFM-CLs were stored in a container at 4°C. 2 Changes in the particle size and encapsulation efficiency were investigated every five days during storage for one month.

Preparation and characterization of PDM-CLs
The process for preparing pH-driven method curcumin liposomes (PDM-CLs) is illustrated in Figure 1. The amounts of curcumin and phospholipid were mixed to pure water grade II and agitated for 4 hours at room temperature. After agitation for 4 hours, the phospholipid was fully dissolved and had self-assembled into liposomes with an average diameter of 11.58 μm. The encapsulation efficiency (EE) of curcumin into liposome was 12.91% ± 0.26%, which indicate the liposomes had been prepared by dissolving the phospholipid in water and the curcumin was loaded into liposomes but with low EE. The liposomes solution was then adjusted to pH 12.0 using 4 M NaOH to dissolve curcumin. Under alkaline conditions, the amount of curcumin loaded into liposomes decreased as well as deprotonated curcumin become unstable, deep red in color, and water-soluble. 2 This observation prompted us to examine the stability of curcumin-liposomes at pH 12.0. In the previous study, the curcumin was completely dissolved after 20 minutes, indicating that decomposition was negligible, thus the curcumin-liposomes could be safely prepared at pH 12.0. 7 After 20 minutes, the solution was adjusted back to pH 5.3 using 4 M HCl, and the PDM-CLs were collected. The average diameter and EE of the PDM-CLs were 77.85 nm ± 0.39 nm and 60.71% ± 0.20%, respectively.
There are two noticeable phenomena associated with the pH-driven method. First, the particle size of the liposomes decreased dramatically from 11.58 μm to 77.85 nm after adjusting the pH. This reduction diameter might due to the osmotic force generated by the ionic gradient across the membrane and lead to the evacuation of water from the inner core of the liposome. 7,12 Second, the EE of the curcumin-liposomes increased from 12.91% to 60.71% in the pHdriven process. This phenomenon can be explained by the different states of curcumin. Curcumin is insoluble (hydrophobic) at pH 5.3 and soluble (hydrophilic) at pH 12.0. When curcumin and phospholipid are mixed in water at low pH, the hydrophobic form of curcumin is not in contact with the liposomal membrane. 2 During the pH-driven process, curcumin was dissolved in water at pH 12.0 and made full contact with the liposomal membrane. When the pH decreased to 5.3, the curcumin was transformed from the hydrophilic form to the hydrophobic form and diffused into the liposomal membrane driven by hydrophobic forces. 2 The apparent microstructure of curcuminliposomes using TEM (Figure 2) showed that the PDM-CLs were spherical define shapes and multilamellar vesicles. In pH-driven method, the phospholipid is dissolved in water and self-assembles into liposomes. Since there is no ethanol present in the preparation process, the phospholipid molecules do not form bilayer fragments. 6,12 As a result, the liposomes prepared by the pH-driven method are more likely to assemble into multilamellar membrane structures. 13 FTIR was examined to further verify whether curcumin was successfully incorporated into the liposomes. The FTIR spectrum of curcumin powder, phospholipid and PDM-CLs are presented in Figure 3.
Meanwhile, the PDM-CLs represented peaks at 1627 cm -1 (aromatic moiety C=C stretching), 1499 cm -1 (C=O and C=C vibrations), 1426 cm -1 (olefinic C-H bending vibration), 1269 cm -1 (aromatic C-O stretching vibration) and 1024 cm -1 (C-O-C stretching vibration), which are the characteristic peaks of curcumin and indicated the existence of curcumin in the liposome. 11 The peaks of curcumin encapsulated in liposomes were shifted from 1503 to 1499 cm -1 which exhibited an interaction between curcumin and phospholipid, as well as the disappearance peak of 3505 cm -1 in PDM-CLs spectrum indicated the interaction between the phenolic -OH of curcumin and phospholipid, most likely through hydrogen bonding. 11,16 The absorption of curcumin main peak's wane and the peaks shifted when curcumin encapsulated in liposomes suggesting that the curcumin molecules were located inside the liposomes, consequently, the signature spectrum of curcumin was hidden. 16

Stability of PDM-CLs
The average diameter and encapsulation efficiency (EE) of curcuminliposomes were measured every five days during storage for one month at 4°C (Table  1 and Figure 4). The TFM-CLs were unstable during storage and displayed particle swelling, and there was leakage of curcumin which was marked by the precipitation after storage for 30 days ( Figure 5A). The amount of curcumin encapsulated by the TFM-CLs decreased from 43.64% to 24.94%, and the particle size increased dramatically from 88.82 nm to 7557.58 nm. This phenomenon is probably caused by the heating during the process of hydrating a thin film which makes curcumin becomes unstable and precipitates after several days of storage. On the other hand, the PDM-CLs were relatively stable during storage for 30 days and there was not any precipitation occurred ( Figure 5B). The amount of curcumin encapsulated by the PDM-CLs decreased slightly from 60.71% to 51.78%, as well as the particle size increased from 77.85 nm to 200.66 nm after 30 days at 4°C. These results indicate that PDM-CLs are stable during storage at 4°C with little leakage of curcumin.
The influence of ionic strength on the stability of the PDM-CLs and TFM-CLs was evaluated by observing the visual appearance of curcumin-liposomes after incubation with different concentration of NaCl for 1 hour at room temperature and after storage for 3 days at 4°C (Figure 6). Although NaCl was produced during the preparation of PDM-CLs, it presented a relatively PDM-CLs, it presented a relatively low concentration and did not influence the results of the stability study. 2  The transparency of the PDM-CLs decreased after treatment with 500 mM and 1000 mM NaCl for 1 hour until 3 days. Moreover, the TFM-CLs also become turbid after treatment with 500 mM and 1000 mM NaCl for 1 hour, and some precipitation were observed at high concentration of NaCl (> 200 mM) after 3 days treatment. These results indicate that TFM-CLs are sensitive to changes in ionic strength. 9 On the other hand, PDM-CLs have intermediate stability at low concentration of NaCl (≤ 200 mM) and less stable at higher concentration of NaCl solution (> 200 mM).
When the ionic gradient across the liposomal membrane is too high, an osmotic force is generated and water flows out of the liposomes to compensate for the high external ion concentration. 17 This causes a reduction in particle size until the maximum compaction of the bilayer is reached. 2 This phenomenon was not observed in the PDM-CLs because it had already occurred during liposome formation. Liposomal dispersions are thermodynamically unstable. According to the extended Derjaguin-Landau-Verwy-Overbeek theory, there are two main repulsive forces, the electrostatic force and hydration force. 2,7 At relatively low concentration of NaCl (< 200 mM), the electrostatic and hydration forces were sufficiently strong to overcome the attractive force, thus all of the two liposomal formulations were stable. At relatively high concentration of NaCl (> 500 mM), the counter-ions in NaCl screen the electrostatic repulsive force acting between the particles. 17 In this condition, the stability of liposomes decreases which the PDM-CLs were more stable than TFM-CLs.
Based on the result of this research, it observed that the use of two compounds phospholipid could reduce the particle size of curcumin-liposomes which made it stable during storage compared to the previous study which only used a single compound. The smaller of liposomal particle size will be resulting in better stability.
On the other hand, the use of two compounds of phospholipid does not increase the encapsulation efficiency of curcumin-liposomes. The result showed that encapsulation efficiency still stayed at around 60% as same as the uses of a single compound phospholipid. This probably depends on the type of phospholipid is used which will affect the character of the hydrophobic layer being occupied by curcumin.

CONCLUSION
The curcumin-liposomes could be prepared using the pH-driven method which based on the self-assembly behavior of phospholipid in water and the diffusion of curcumin into liposomal membranes by hydrophobic forces. The result showed that the PDM-CLs with two compounds of phospholipid have good encapsulation efficiency, nano-scale liposomal particle size, and high stability during storage. The pH-driven method is a promising method to prepare curcumin-liposomes with organic solvent-free, simple equipment and easy to control.