- Nano Express
- Open Access
Effects of Micro-environmental pH of Liposome on Chemical Stability of Loaded Drug
- Xiao-Ru Shao†1,
- Xue-Qin Wei†1,
- Shu Zhang1,
- Na Fu1,
- Yun-Feng Lin1,
- Xiao-Xiao Cai1Email author and
- Qiang Peng1Email authorView ORCID ID profile
© The Author(s). 2017
- Received: 8 June 2017
- Accepted: 28 July 2017
- Published: 23 August 2017
Liposome is a promising carrier system for delivering bioactive molecules. However, the successful delivery of pH-sensitive molecules is still limited by the intrinsic instability of payloads in physiological environment. Herein, we developed a special liposome system that possesses an acidic micro-environment in the internal aqueous chamber to improve the chemical stability of pH-sensitive payloads. Curcumin-loaded liposomes (Cur-LPs) with varied internal pH values (pH 2.5, 5.0, or 7.4) were prepared. These Cur-LPs have similar particle size of 300 nm, comparable physical stabilities and analogous in vitro release profiles. Interestingly, the chemical stability of liposomal curcumin in 50% fetal bovine serum and its anticancer efficacy in vitro are both micro-environmental pH-dependent (Cur-LP-2.5 > Cur-LP-5.0 > Cur-LP-7.4). This serum stability still has space to be further enhanced to improve the applicability of Cur-LP. In conclusion, creating an acidic micro-environment in the internal chamber of liposome is feasible and efficient to improve the chemical stability of pH-sensitive payloads.
- Drug delivery
- Controlled release
Liposome, an artificial membrane vehicle, has shown great potentials in drug delivery due to its drug loading capacity, biodegradability, and biocompatibility [1–4]. The classic liposome is similar with living cells in structure, typically consisting of a phospholipid bilayer and an aqueous inner chamber [5–7]. Due to this structure, liposome is able to solubilize the insoluble drug molecules and prevent the loaded drug from the harsh physiological environment [8–10]. In addition, the surface of liposome can be modified to prolong the blood circulation time and/or target specific tissues [11–15]. With these abovementioned advantages, various liposome systems have been clinically approved [8, 9, 16].
Although the delivery of many drugs has been improved by incorporation into liposome, the delivery of some pH-sensitive drugs is still limited by the instability of drug molecule itself in physiological environment (neutral pH values). Generally, liposome is prepared in a neutral buffer solution and thus the loaded drug molecules are also in a neutral environment after incorporation into liposome. Accordingly, those molecules which are only stable in acidic environment would be still instable even in the form of liposome. Therefore, development of a novel approach for enhancing the stability of pH-sensitive drugs is of great importance for successful delivery of these payloads by liposome.
Phospholipids (soybean lecithin for injection use) were purchased from Shanghai Tai-Wei Pharmaceutical Co., Ltd., (Shanghai, China). Cholesterol was obtained from Amresco (Solon, OH, USA). Poloxamer 188 (F68) was kindly donated by BASF (China) Co., Ltd., (Shanghai, China). Curcumin was supplied by Sigma (St. Louis, MO, USA). Fetal bovine serum (FBS) was purchased from HyClone (Logan, UT, USA). All other chemical reagents used in this study were of analytical grade or better.
Preparation of Curcumin-Loaded Liposomes (Cur-LPs)
The liposomes with varied micro-environmental pH values were prepared using evaporation method according to previous works with some modifications [27, 28]. Briefly, phospholipids (75 mg) and cholesterol (5 mg) were dissolved in 0.5 ml ethanol containing 2 mg/ml curcumin. The ethanol solution was mixed with 5 ml 0.001 M PBS containing 1% (w/v) F68 that served as a surfactant to narrow the size distribution. After magnetically stirring for 1 min (constant temperature magnetic mixer, DF-101S, Zhengzhou Greatwall Scientific Industrial and Trade Co., Ltd., Zhengzhou, China), the resultant emulsion was evaporated under vacuum and dark for 30 min at 35 °C to remove ethanol. The acidity in the inner chamber of Cur-LP was adjusted via using PBS with varied pH values of 2.5, 5.0, or 7.4 during preparation. The resultant suspension was centrifuged at a low speed (3000 rpm, 5 min) to precipitate free curcumin. The supernatant was then centrifuged at a high speed (16 krpm, 10 min), and the pellets were re-suspended in PBS (pH 7.4) before further use. This procedure provided these LPs with an identical external environment. The obtained liposomes with different micro-environmental pH values were presented as Cur-LP-2.5, Cur-LP-5.0, and Cur-LP-7.4, respectively. Blank liposomes were also fabricated as above.
Characterization of Liposome
The hydrodynamic size, size distribution, and zeta potential are the three basic parameters for liposome systems. The size and zeta potential of LP were determined by dynamic light scattering (DLS) and electrophoretic light scattering (ELS), respectively, using ZetasizerNano ZS90 (Malvern Instruments Ltd., Malvern, UK) at 25 °C . The measurement cycle was automatically determined by the instrument system. The particle size was presented by intensity distribution, and the size distribution was evaluated by polydispersity index (PDI).
Encapsulation Efficiency (EE) Determination
EE, an important parameter for quality control, is of great significance in developing liposome-based delivery systems. The EE determination was based on the high speed centrifugation method. Briefly, 100 μl Cur-LPs was centrifuged at the low speed (3000 rpm, 5 min) to precipitate non-dissolved free curcumin, and 50 μl supernatant was subjected to high-speed centrifugation (16 krpm, 10 min) to separate Cur-LPs from the tiny dissolved curcumin. The pellets were re-suspended in 500 μl PBS (i.e., 10-fold dilution), an aliquot of 10 μl of which was mixed with 300 μl ethanol by vortex and sonication for 30 s. The fluorescent intensity of curcumin in the resultant solution was determined (excitation wavelength (Ex), 458 nm; emission wavelength (Em), 548 nm) and presented as F e , i.e., the fluorescent intensity of encapsulated curcumin. Another 50 μl of fresh Cur-LP containing encapsulated and free curcumin was also diluted by 10-fold with PBS, and 10 μl of the diluted solution were mixed with 300 μl ethanol. The fluorescent intensity of the resultant solution was measured and presented as F t , i.e., the fluorescent intensity of total curcumin. The EE was therefore calculated with the following equation: EE = F e /F t.
Scanning Electron Microscopy (SEM)
The morphology of LP was observed by the scanning electron microscopy (SEM, INSPECT F, FEI, Netherlands) . Briefly, the LP suspension was 100-fold diluted with distilled water, and one drop of the diluted suspension was placed on a clean glass sheet. After air-drying, the sample was coated with gold right before SEM.
Physical Stability of Liposomes
Physical stability is a very meaningful parameter for storage and transportation of a colloidal system. The physical stability of liposome was presented by colloidal stability and investigated according to a previous method . Briefly, 100 μl of LP was added to tubes and kept at 37 °C. At different time intervals, the LP size was measured and compared to the initial size so as to indicate the thermodynamic stability. In addition, another 300 μl of LP were also added to tubes and kept at 37 °C. At the same time intervals, 100 μl of the upper layer liquid were collected. The transmittance of the collected specimens was measured at 550 nm and compared to the initial value so as to indicate the kinetic stability.
In Vitro Release
The release profile of liposome plays an important role in predicting in vivo fate and efficacy of liposome. The in vitro release of curcumin from Cur-LP was studied using the dynamic dialysis method . Briefly, 1 ml of each Cur-LP was added into a dialysis bag (molecular weight cutoff, 10 kD), which was used to retain liposome but keep the released curcumin molecules permeable. The specimen-loaded dialysis bag was soaked in 4 ml release medium (0.001 M PBS containing 0.1% Tween 80, pH 7.4), and the release study was conducted away from light (37 °C, 100 rpm). At each fixed time interval, the release medium was collected and replaced with 4 ml fresh medium so as to simulate sink conditions. The collected medium was diluted to 5 ml with PBS and further diluted by 15-fold with ethanol. The curcumin in the resultant solution was quantified by fluorescence spectrophotometry (Ex 458 nm, Em 548 nm). In addition, curcumin powder was dissolved in the above release medium, and the release of curcumin solution was conducted at pH 7.4 to investigate whether the dialysis bag would retain curcumin molecules.
Chemical Stability of Liposomal Curcumin
Chemical stability is a key parameter for predicting drug metabolism, efficacy, and toxicity. The chemical stability of Cur-LPs was examined in 50% FBS. Briefly, 100 μl of Cur-LPs were diluted by 10-fold with PBS (pH 7.4) and then mixed with 1 ml FBS. The specimens were shaken on a horizontal shaker away from light (37 °C, 100 rpm). At fixed time intervals, an aliquot of 10 μl of specimen was collected and mixed with 300 μl ethanol immediately followed by centrifugation (16 krpm, 5 min). The remained curcumin in the supernatant was quantified as above.
In Vitro Anticancer Efficacy
The preliminary anticancer efficacy of the three Cur-LPs was investigated using human liver hepatocellular carcinoma HepG2 cells. Briefly, HepG2 cells were plated onto the 96-well cell culture plates at a density of 10,000 cells per well and cultured under standard conditions (37 °C/5% CO2) for 24 h in PRIM-1640 culture medium supplemented with 10% FBS. Subsequently, the culture medium was removed and cells were washed with PBS. The Cur-LPs were diluted in serum-free culture medium (4 μg/ml curcumin) and added to cells, followed by continuous incubation for 1 and 3 days at 37 °C. The OD value of viable cells was measured by cck-8 assay. The cells treated with blank culture medium served as control and cell viability (%) was the OD value percentage of specimens relative to control.
All the data are presented as mean ± sd (standard deviations). The differences between two groups, analyzed by the Student’s t test, were considered to be statistically significant when the p value was less than 0.05.
Characterization of Liposome
The micro-environmental pH of liposome refers to the acidity in the inner aqueous chamber of liposome (Fig. 1), which is different from the pH in the external environment. In this work, the external environmental pH of all liposome suspensions was 7.4 unless otherwise stated.
EE is of concern during liposome development. Usually, increasing EE is important for reducing cost and enhancing efficacy. In this work, the EE of Cur-LP-2.5 is 74% (Fig. 2c), which is the highest among Cur-LP-5.0 (45%) and Cur-LP-7.4 (64%), indicating that Cur-LP-2.5 is the best formulation for delivering curcumin from the point of EE. The reasons for the variety in EE at different pH values are not very clear but may be related to the solubility of curcumin which is soluble in alkali or extremely acidic solvents .
Physical Stability of Liposome
Liposome is a colloidal system and its physical stability can be presented by colloidal stability, which has substantial impacts on liposome storage and further applications [34, 35]. Particle aggregation (thermodynamic instability) and sedimentation (kinetic instability) are the two essential aspects of colloidal instability. Aggregation leads to a larger apparent size and sedimentation leads to changes in transmittance of suspension. More importantly, size increase can directly affect the efficacy of nano-systems since particle size has been shown to have great impacts on cellular uptake, cytotoxicity, pharmacokinetic profile, and tissue distribution [36, 37].
In Vitro Release
Linear fitting of the in vitro release profiles of Cur-LPs with varied micro-environmental pH values
Fitting degree (r)
y = 0.424x + 0.608
y = 0.403x + 0.800
y = 0.449x + 0.885
Effect of Micro-environmental pH on Chemical Stability of Cur-LP
Liposomes consist of two parts in structure: one is the hydrophobic lipid bilayer and the other is the hydrophilic inner aqueous chamber. It is easy to understand that a pH-sensitive hydrophilic drug would be located in the inner aqueous chamber, and its stability would be significantly affected by the micro-environmental pH in the aqueous chamber, where the buffering volume and buffering capacity would be much higher than that in the lipid bilayer. In contrast, curcumin is a hydrophobic molecule and would be located in the lipid bilayer. For this reason, it is quite interesting to find the micro-environmental pH-dependent chemical stability of liposomal curcumin. It is assumed that the space in lipid bilayer would not be absolutely anhydrous although it is hydrophobic. As we know, the living cell membrane is not absolutely anhydrous in its lipid bilayer. Instead, it contains a certain small volume of aqueous solution for transport of water-soluble molecules and ions. Likewise, a certain small volume of buffer solution with the same components as the inner chamber would also exist in the hydrophobic lipid bilayer after successful preparation of liposome. Thus, the hydrophobic drug located in the lipid bilayer can be directly affected by the micro-environmental pH of liposome. In addition, the acidic micro-environment may reduce the activities of some enzymes which show the best activity in normal physiological condition. This also contributes to the higher chemical stability of liposomal curcumin in the lower micro-environmental pH. It has been reported that liposomes composed of egg phosphatidylcholine (EPC) rapidly lost their internal pH-gradient in buffer (pH 7.4), and the pH-gradient maintaining ability was substantially enhanced by substituting EPC (phase transition temperature (T m ) ≈ −5 °C) with the high T m (41 °C) lipid DPPC (dipalmitoyl phosphatidylcholine) and by addition of cholesterol . In this present work, the liposome is composed of soybean lecithin (T m is around 238.2 °C ) and cholesterol. Hence, the micro-environmental pH-gradient of liposomes prepared in this work can be expected to maintain for a long period. This is a strong support to the results and assumptions shown above.
In Vitro Anticancer Efficacy
Liposome, as a widely used drug delivery system, is capable of improving the solubility of water-insoluble drugs, protecting the drug payloads from the harsh physiological environment and delivering the payloads to a targeted tissue. However, the delivery of pH-sensitive drugs is still limited by their natural instability in physiological conditions (neutral environment). In this present work, we propose a novel approach for enhancing the chemical stability of pH-sensitive drug payloads by regulating the micro-environmental acidity of liposome. The findings show that the chemical stability and in vitro efficacy of the model pH-sensitive drug curcumin is significantly enhanced by acidifying the micro-environment of liposome. In conclusion, regulation of micro-environmental pH of liposome is feasible to enhance the chemical stability of pH-sensitive drug payloads, even for the hydrophobic drugs which are located in the lipid bilayer.
This work was supported by the National Natural Science Foundation of China (nos. 81402860, 81470721), the Excellent Young Scientist Foundation of Sichuan University to Q. Peng (no. 2082604194312), and Sichuan Science and Technology Innovation Team (no. 2014TD0001).
XRS and XQW performed the experiments and collected the data. SZ and NF performed the experiments. YFL and XXC analyzed the data. QP conceived and designed the study, analyzed the data, and wrote the paper. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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