A nano-delivery system for bioactive ingredients using supercritical carbon dioxide and its release behaviors

Food Chemistry 228 (2017) 219–225

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A nano-delivery system for bioactive ingredients using supercritical carbon dioxide and its release behaviors Wenbei Situ ⇑, Xianliang Song, Shucan Luo, Yan Liang College of Food Science, South China Agricultural University, Guangzhou 510640, China

a r t i c l e

i n f o

Article history: Received 12 July 2016 Received in revised form 7 January 2017 Accepted 12 January 2017 Available online 16 January 2017 Chemical compounds studied in this article: Melatonin (PubChem CID: 896) Phosphatidylcholine (PubChem CID: 45266626) Cholesterol (PubChem CID: 5997) Keywords: Liposome Supercritical carbon dioxide technique Thin-film hydration Bioactive ingredients Controlled release Nano-delivery system

a b s t r a c t For the purpose of ensuring the bioavailability of bioactive ingredients, a nano-delivery system with low toxicity was developed using supercritical carbon dioxide (SC-CO2). Compared to thin-film hydration (TFH), obtaining nano-scale liposomes is easier using SC-CO2. The characteristic of these liposomes was also demonstrated by the analysis of particle size and morphology. An in vitro release study showed that liposomes produced using SC-CO2 were resistant to low pH in simulated gastric conditions. In a simulated intestinal environment, enteric solubility of these liposomes was enhanced, which are important properties for controlled releasing bioactive ingredient. Furthermore, SC-CO2-produced liposomes had a higher storage stability than those produced using TFH. Analysis of the organic solvent residue in the liposomes by gas chromatography–mass spectrometry (GC–MS) indicated that SC-CO2-produced liposomes had lower toxicity than those produced by TFH. A chemical free nano-delivery system using SCCO2 has been revealed for storage and controlled release of bioactive ingredients. Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction With the increasing public perception of there being a strong correlation between food and disease prevention, bioactive ingredients with special physiological effects have gained attention as substances that can prevent disease. The enormous challenges facing the successful use of bioactive ingredients include not only determining the appropriate food processing and storage conditions (temperature, oxygen exposure and light), but also finding ways to sustain the stability of the ingredients in the gastrointestinal environment (pH, digestion enzymes and long transit time) (de Vos, Faas, Spasojevic, & Sikkema, 2010; Palzer, 2009). To overcome these challenges, a wide range of different methods have been utilized for encapsulation and delivery of bioactive ingredients, such as production of film-coated specific-release microparticles (Pu et al., 2011; Situ, Chen, Wang, & Li, 2014; Situ, Li, Liu, & Chen, 2015; Wang et al., 2011), emulsification (McClements, Decker, Park, & Weiss, 2009; McClements & Li, 2010), and ⇑ Corresponding author. E-mail addresses: [email protected] (W. Situ), [email protected] (X. Song), [email protected] (S. Luo), [email protected] (Y. Liang). http://dx.doi.org/10.1016/j.foodchem.2017.01.053 0308-8146/Ó 2017 Elsevier Ltd. All rights reserved.

microcapsulation (Augustin & Hemar, 2009; Zvonar, Berginc, Kristl, & Gasperlin, 2010). Nano-delivery systems using encapsulated bioactive ingredients are invaluable in pharmaceutics, cosmetics as well as food industry, and can be divided into two types: polymer- and lipidbased systems (Joye & McClements, 2016; Tamjidi, Shahedi, Varshosaz, & Nasirpour, 2013). Compared to polymer-based systems, lipid-based systems have some advantages for nano-scale production, including having fewer residual organic solvents, lower toxicity, better solubility for hydrophobic bioactive ingredients and higher absorption of bioactive ingredients in the small intestine (Akbarzadeh et al., 2013). In general, nano-scale lipidbased systems include liposomes, nanoemulsions, microemulsions, solid–lipid nanoparticles and nanostructured lipid carriers (Chen, Tsai, Huang, & Fang, 2010; Fathi, Mozafari, & Mohebbi, 2012; Tamjidi et al., 2013). Conventional methods for producing liposomes include thinfilm hydration (TFH) (Liau, Hook, Prestidge, & Barnes, 2015), ethanol injection (Schubert & Muller-Goymann, 2003), reverse-phase evaporation (Akbarzadeh et al., 2013; Otake et al., 2006), highpressure homogenization (Chung, Shin, Jung, Hwang, & Park,

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2014; Saheki, Seki, Nakanishi, & Tamai, 2012) and use of supercritical fluid (SCF) (Zhao, Temelli, Curtis, & Chen, 2015; de Paz et al., 2012). Methods using SCF have been attracting attention for encapsulation and delivery of bioactive ingredients over the last 10 years. Carbon dioxide (CO2), at suitable temperature and pressure (Tc 31.06 °C, Pc 7.39 MPa) in the supercritical region, is one of the most widely used fluids. The well-known advantages of using supercritical carbon dioxide (SC-CO2) are non-toxic, nonflammable and inexpensive. In addition, it has high diffusivity, low interfacial tension and low viscosity (Milad Fathi, Martin, & McClements, 2014). The SC-CO2 method has been used to produce dexamethasone phosphate nanoparticles (Thote & Gupta, 2005) and to prepare soy lecithin liposomes (Zhao & Temelli, 2015). The use of SC-CO2 to produce liposomes has overcome some limitations related to controlling the dimensions and distribution of liposomes and their low encapsulation efficiencies (Campardelli et al., 2016). Moreover, nano-scale particles produced using SCCO2 improve the absorption of the encapsulated bioactive ingredients in the small intestine (Akbarzadeh et al., 2013; Allen & Cullis, 2013). Given this background, an attempt was made in the present study to develop liposomes as a nano-delivery system for bioactive ingredient using SC-CO2. The different structures of liposomes containing bioactive ingredients were analyzed by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Moreover, the release properties, storage stability and residual organic solvent of the liposomes were investigated in an in vitro experiment and using gas chromatography–mass spectrometry (GC–MS). For the comparison, a commercial melatonin (MLT) tablet and MLT liposomes created using different methods were used in the experiment. Finally, the relationship among the release behaviors of the liposomes, the structure of the nano-delivery system and the methods to produce the liposomes were examined. 2. Materials and methods 2.1. Materials Melatonin was purchased from Dehuitang Bioscience & Technology Co., Ltd. (Shangqiu, China). Phosphatidylcholine (PC) was purchased from Yuanju Bioscience & Technology Co., Ltd. (Shanghai, China). Cholesterol (CHOL) was obtained from Yuancheng Medicinal & Chemical Co., Ltd. (Zhuhai, China). Pepsin and pancreatin were purchased from Genebase Bioscience Co., Ltd. (Guangzhou, China) and were of pharmaceutical grade. The other chemical reagents were of analytical grade. 2.2. Production of MLT liposomes using SC-CO2

2.2.1. Production of MLT liposomes Cholesterol, MLT and PC (15:10:1) were dispersed in ethanol (cosolvent) at concentrations of 7.5%. The phospholipid suspension was sealed inside the high-pressure vessel and flushed with CO2 to remove air. To reach the set temperature and pressure (10– 22 MPa), the MLT liposome was incubated at 50 °C for 35 min. After depressurization, CO2 was released into the gas state, and the liposomes were collected in pH 7.4 phosphate-buffered saline (PBS). The liposome suspension was obtained by rotary evaporation to remove the ethanol.

the blank control, the absorbance of the suspension at 278 nm was measured. The total MLT weight of the liposome suspension (Wtotal) was calculated according to the equation for the standard curve. One milliliter of the MLT liposome suspension and 1 mL of 10 mg/mL protamine solution were mixed in a tube. After sitting for 3 min, the mixture was diluted with 9 mL pH 7.4 PBS and centrifuged at 10,500g for 20 min. Exactly 1 mL of the supernatant was then diluted in 10 mL of pH 7.4 PBS. The free MLT weight of the liposome suspension (Wfree) processed identically to those of Wtotal. The encapsulation efficiency of MLT liposomes (EE/%) was calculated according to:

EE ¼

W total  W free  100% W total

ð1Þ

2.2.3. Storage stability of liposomes After storage of the liposome suspension for 1–2 months at 4 °C, the absorbance of the liquid supernatant was measured at 278 nm, and the amount of MLT leakage was calculated. The stability of the liposomes was presented by the difference of MLT leakage amount before and after storage. The stability constant of the liposomes (KE) was calculated according to the following equation:

KE ¼ ðAo  Ai Þ=Ao

ð2Þ

where Ao and Ai are the amount of MLT leakage at 278 nm before storage and storing for 1–2 months, respectively. 2.3. Production of MLT liposomes using TFH Phosphatidylcholine (PC) and CHOL were solubilized in 30 mL chloroform–ethanol solution (v:v, 100:1). Melatonin was added to the solution with continuous agitation for 5 min. During this process, the ratio of CHOL, MLT and PC was 10:25:1. Chloroform was slowly removed by evaporation at 0.1 MPa to form a thin film on the round bottom of the flask. Subsequently, 50 mL PBS (pH 7.4) was added for hydration of the thin film at 50 °C. To form the liposomes, the vesicle suspension was held at 0 °C in a water bath for ultrasound at 100 W for 10 min. The final concentration was filtered through 220-nm membranes (Millipore Corporation, Bedford, MA, USA). 2.4. Particle size of liposomes The mean size and size distribution of the liposomes were measured with a laser particle analyzer (LA-950, HORIBA, Kyoto, Japan) using DLS. The liposome suspension was diluted 5 times in PBS and measured at 25 °C for 3–5 min. The sample was measured using a laser wavelength of 650 nm. 2.5. Liposome morphology The liposome morphology was evaluated by TEM. The sample was diluted in pH 7.4 PBS, and then pipetted onto a 300-mesh formvar carbon-coated copper grid, blot dried and negatively stained by phosphotungstic acid at 3% (w/v) for 2–3 min. After blotting dry, the liposomes were examined using a transition electron microscope (JEM-2010HR, JEOL, Tokyo, Japan) at 80 kV. 2.6. In vitro release rate study

2.2.2. MLT loading of liposomes Exactly 1 mL of the MLT liposome suspension was added to 10 mL of methanol. After demulsification, 1 mL of the suspension was added to 10 mL of pH 7.4 PBS. Using MLT-free liposomes as

For tests examining the stability of the liposomes in a simulated gastrointestinal environment, the releasing media included simulated gastric fluid (SGF) (0.1 M HCl containing 0.32% w/v pepsin)

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and simulated intestinal fluid (SIF) (0.2 M phosphate buffer containing 1% w/w pancreatin). Two milliliters of the liposomes was suspended in 100 mL releasing media at 37 °C in an incubator and stirred at 100 rpm. At each of the pre-determined time points, 5 mL of the test sample was withdrawn and centrifuged at 10,500g for 10 min. The MLT content of the supernatant was analyzed using a UV spectrophotometer (Unico, UV-3802, Shanghai, China). 2.7. Analysis of residual organic solvent in the liposomes The residual organic solvent in the liposomes was fractionated using GC–MS. The samples were extracted using a solid phase extraction (SPE) method and analyzed using a chromatograph system coupled to a mass spectrometer (Agilent 5973, Agilent Technologies Inc, Santa Clara, CA, USA). Separation was performed on a DB-1 capillary column (30 m  0.25 mm  0.25 lm). The carrier gas was helium at a flow rate of 0.8 mL/min. The oven temperature was set at 60 °C for 5 min, then increased to 280 °C at 2 °C/min, and held at 280 °C for 20 min. Detection proceeded with an electron ionization (70 eV) source at 200 °C. The ions were monitored from 29 to 450 m/z. 3. Results and discussion 3.1. Particle size analysis 3.1.1. Effect of CO2 pressure The effect of pressure on the diameter and the polydispersity index (PdI) of the liposomes was investigated. With increasing pressure from 10 to 14 MPa, the particle size of the liposomes decreased from 88.74 to 66.19 nm (Table 1). The particle size then increased (from 66.19 to 147.3 nm) as CO2 pressure increased (14– 22 MPa). Table 1 The mean diameter of liposomes at different pressures using SC-CO2. SC-CO2 Pressure (MPa) Particle Size (nm) PdI

10 88.74 0.11

14 66.19 0.267

18 126.3 0.393

22 137.3 0.659

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Pressure had a significant effect on the liposomes and their size uniformity, which was evaluated in terms of the PdI. Values of the PdI lower than 0.3 are attributed to a narrow size distribution and formation of a more homogeneous colloidal system, while those greater than 0.7 are considered to indicate high polydispersity (Johnson, Ambe, & Wang, 2014). As CO2 pressure increased, the PdI of the liposomes gradually increased from 0.11 to 0.659. At high pressure, the amount of CO2 accumulated within the phospholipid bilayers increased, and led to breakage of the phospholipid bilayers into discrete molecules. When the broken microcapsule self-aggregated, its size could not be controlled, and the PdI of the liposomes increased as CO2 pressure increased. 3.1.2. Particle size of liposomes produced using different methods Using the SC-CO2 method, the mean diameter of the liposomes was 66.19 nm and the PdI was 0.267, which indicates a narrow size distribution. The distribution of MLT liposomes produced using SCCO2 was reflected as two peaks (Fig. 1). The main peak (Peak 1, 98.3%) indicates a liposome diameter of 88.61 nm, while the second peak indicates a diameter of 2209 nm (1.7%). The nano-scale liposomes produced using SC-CO2 were very uniform in size. Using TFH, the mean diameter of the liposomes was 85.62 nm and the PdI was 0.382. Three peaks are shown in Fig. 1. The first peak, indicating a diameter of 20 nm, was at 3.6%. The second peak, indicating a diameter of 102 nm, was the main peak (86%). There also existed a third peak, indicating a huge diameter of 1838 nm. These results show that controlling the dimensions and distribution of liposomes is easier using SC-CO2. 3.2. Morphology of liposomes The morphology of the liposomes produced using SC-CO2 is shown in Fig. 2(a, b). Morphology is a key characteristic to demonstrate the shape, size and uniformity of liposome vesicles. As shown in the images, the liposomes were spherical and did not leak during production. The surface of the liposomes resembled that of unilamellar vesicles. Moreover, the intensively distributed liposomes were uniform and similarly sized. Only a few parts of these did not reach the nano-scale size (>100 nm). As the result of the high depressurization rate of SC-CO2, the

Fig. 1. Size distribution of liposomes produced using different methods (SC-CO2: supercritical carbon dioxide; TFH: thin-film hydration). SC-CO2: 75% ethanol solution, 50 °C, 35 min and at 14 MPa SC-CO2 pressure; TFH: 40 °C and the ratio of CHOL, MLT and PC (10:25:1).

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Fig. 2. Morphology of liposomes produced using supercritical carbon dioxide method (a, b) and thin-film hydration (c, d).

liposomes displayed homogeneity, and a few residue lipid micelles were observed on the TEM image. As shown in Fig. 2(c, d), the liposomes produced using TFH were not uniform. The large liposomes shown on the image might be large unilamellar vesicles or multilamellar vesicles, which were much larger than nano-sized particles (>100 nm). Considering the absorption of bioactive ingredients, the ideal liposome should be small, uniform and spherical without any deformation or leakage on the surface for better encapsulation. Based on this, the liposomes produced using SC-CO2 were more appropriate than those produced using TFH. 3.3. In vitro release study of MLT liposomes Melatonin, a substance found in animals, plants and bacteria, has effects on physiological functions, including sleep timing and blood pressure regulation, as well as antioxidant effects. Melatonin easily degrades in an environment with low pH and enzymes, such as the gastrointestinal environment. Moreover, the biological halflife of MLT is 35–50 min. After oral administration, the MLT concentration in the blood quickly decreases to normal levels. Melatonin is rapidly cleared through the liver, but its half-life is improved by encapsulation in small vesicles (Allen & Cullis, 2013). Developing the capsulation of MLT is an effective way to avoid its inactivation. Melatonin liposomes produced using SCCO2 were released in SGF (pH 1.2) for 1–8 h. As shown in Fig. 3, the release percentage of MLT increased over time. At 2 h in SGF, 5% of MLT was released. After 8 h, the release percentage increased to 21%. Compared to the MLT liposomes, the commercial MLT

tablet had a low release rate (<15%, 8 h). The release properties of MLT liposomes produced using SC-CO2 and those produced using THF were similar. Considering the retention time of nutrition in the stomach is 1–2 h, 5% of MLT released during this period was acceptable. To improve the absorption and utilization of MLT, enhancing the MLT release in the small intestine at constant rate should be considered. Three types of MLT carriers were used in the in vitro release rate study in SIF (pH 6.8, pancreatin) for 8 h. The release rate for the commercial MLT tablet was fast during the first 4 h (53%), after which it obviously decreased. Until 8 h, the MLT release percentage was 65%. For liposomes produced using SC-CO2, the MLT release percentage in SIF was 43% at 4 h and 89% at 8 h, which indicates enteric solubility of the MLT liposomes. The small intestine is a critical organ for absorption of nutrients. Moreover, as mentioned early, MLT has a short half-life. A constant MLT release rate would benefit the consumer regarding utilization of MLT. The controlled-release properties of MLT liposomes produced using SC-CO2 were better than those of a commercial MLT tablet. As time progressed, the liposomal membrane was totally dissolved and MLT was released. MLT bioavailability was improved during this process. The release properties of MLT liposomes produced using SC-CO2 and those produced using TFH were also similar in SIF. To summarize the results of the in vitro release experiments, MLT liposomes were resistant to degradation by gastric acid and enzymes, and exhibited improved bioavailability via controlled release in a simulated small intestine environment.

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Fig. 3. Melatonin (MLT) release from liposomes in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF).

3.4. Stability of liposomes The stability of the bioactive ingredients is required for effective biological performance and promotion of health. Two experiments (storage stability and residual organic solvent analysis) were conducted to test the stability of the liposomes. 3.4.1. Storage stability The storage stability of the liposomes is reflected by their KE value. MLT liposomes were stored at 4 °C. Leakage of MLT from the liposomes was detected after storage for one or two months. After one month, the KE values of the liposomes were 0.068 and 0.148 for SC-CO2- and TFH-produced liposomes (Table 2), respectively. After two months, these values increased to 0.091 and 0.375 for SC-CO2- and TFH-produced liposomes, respectively.

Table 2 Storage stability of liposomes by different methods. Method

KE (1 month)

KE (2 months)

TFH SC-CO2

0.148 0.068

0.375 0.091

Compared to TFH, the liposomes produced by SC-CO2 were more stable, as shown by their lower KE value. Although the liposomes produced by SC-CO2 and TFH had similar release properties, the storage stability of these liposomes were significantly different. With the results of particle size and morphology, the physical structure of liposomes using SC-CO2 is more stable than those using TFH. The unique physical structure of liposomes would have

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Fig. 4. Chromatograms of organic solvent residues in liposomes produced by different methods (SC-CO2: supercritical carbon dioxide; TFH: thin-film hydration).

contributed to its physical stability. During long-term storage, a lipophilic bioactive ingredient would separate from liposomes. The physical stability would effect the amount of bioactive compound leaking from the liposomes. Furthermore, to avoid the oxidation reaction of PC, the SC-CO2 method uses CO2 throughout the entire process, which eliminates any contact of the liposome with oxygen, reduces the chance of PC oxidation and retains the storage stability of the liposomes.

using SC-CO2 has the advantage of producing a non-toxic organic solvent residue. The storage stability and residual organic solvent safety of liposomes are affected by the method used to produce liposomes. Regarding the safety of liposomes, using SC-CO2 is a better choice to manufacture liposomes for storage and delivery of bioactive ingredients. 4. Conclusion

3.4.2. Residue analysis of organic solvent To investigate the safety of the liposomes, the residual organic solvent was analyzed via SPE followed by GC–MS. As shown in the chromatograms (Fig. 4), a peak at 1.79 min was detected in the sample of liposomes produced using SC-CO2. According to the electron ionization data of the Wiley Library, this peak indicates the presence of ethyl alcohol. The trace amount of residue ethyl alcohol could be removed by rotary evaporation. Compared to the liposomes produced using TFH, which had residual chloroform,

A nano lipid-based system for encapsulation of bioactive ingredients using SC-CO2 was investigated in the present study. The suitable particle size of liposomes was obtained in supercritical CO2 at 14 MPa. Results from examination of the morphology and particle size indicate that liposomes produced using SC-CO2 are suitable for nano-size encapsulation. Results from in vitro release experiments showed that the MLT liposomes were resistant to degradation in a simulated gastric environment and improved

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the bioavailability of MLT via controlled release in a simulated small intestine environment. Considering the storage stability and residual organic solvent, using SC-CO2 is a more effective way to produce liposomes. Notes The authors declare no competing interest. Acknowledgements The authors from SCAU, China, would like to acknowledge the National Natural Science Funds of China (No. 31601422). References Akbarzadeh, A., Rezaei-Sadabady, R., Davaran, S., Joo, S. W., Zarghami, N., Hanifehpour, Y., ... Nejati-Koshki, K. (2013). Liposome: Classification, preparation, and applications. Nanoscale Research Letters, 8, 9. Allen, T. M., & Cullis, P. R. (2013). Liposomal drug delivery systems: From concept to clinical applications. Advanced Drug Delivery Reviews, 65(1), 36–48. Augustin, M. A., & Hemar, Y. (2009). Nano- and micro-structured assemblies for encapsulation of food ingredients. Chemical Society Reviews, 38(4), 902–912. Campardelli, R., Santo, I. E., Albuquerque, E. C., de Melo, S. V., Della Porta, G., & Reverchon, E. (2016). Efficient encapsulation of proteins in submicro liposomes using a supercritical fluid assisted continuous process. Journal of Supercritical Fluids, 107, 163–169. Chen, C.-C., Tsai, T.-H., Huang, Z.-R., & Fang, J.-Y. (2010). Effects of lipophilic emulsifiers on the oral administration of lovastatin from nanostructured lipid carriers: Physicochemical characterization and pharmacokinetics. European Journal of Pharmaceutics and Biopharmaceutics, 74(3), 474–482. Chung, S. K., Shin, G. H., Jung, M. K., Hwang, I. C., & Park, H. J. (2014). Factors influencing the physicochemical characteristics of cationic polymer-coated liposomes prepared by high-pressure homogenization. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 454, 8–15. de Paz, E., Martin, A., Estrella, A., Rodriguez-Rojo, S., Matias, A. A., Duarte, C. M. M., & Jose Cocero, M. (2012). Formulation of beta-carotene by precipitation from pressurized ethyl acetate-on-water emulsions for application as natural colorant. Food Hydrocolloids, 26(1), 17–27. de Vos, P., Faas, M. M., Spasojevic, M., & Sikkema, J. (2010). Encapsulation for preservation of functionality and targeted delivery of bioactive food components. International Dairy Journal, 20(4), 292–302. Fathi, M., Martin, A., & McClements, D. J. (2014). Nanoencapsulation of food ingredients using carbohydrate based delivery systems. Trends in Food Science & Technology, 39(1), 18–39. Fathi, M., Mozafari, M. R., & Mohebbi, M. (2012). Nanoencapsulation of food ingredients using lipid based delivery systems. Trends in Food Science & Technology, 23(1), 13–27. Johnson, N. R., Ambe, T., & Wang, Y. (2014). Lysine-based polycation:heparin coacervate for controlled protein delivery. Acta Biomaterialia, 10(1), 40–46.

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