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Microfluidic assembly of liposomes dual-loaded with catechin and curcumin for enhancing bioavailability
Colloids and Surfaces A 594 (2020) 124670
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Colloids and Surfaces A journal homepage: www.elsevier.com/locate/colsurfa
Microfluidic assembly of liposomes dual-loaded with catechin and curcumin for enhancing bioavailability
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Sung-Chul Honga,b,1, Kyung-Min Parkc,1, Chi Rac Hongd, Jin-Chul Kimb, Seung-Hoon Yange, Hyung-Seok Yuh, Hyun-Dong Paikh, Cheol-Ho Panb, Pahn-Shick Changa,f,g,* a
Department of Agricultural Biotechnology, Seoul National University, Seoul, 08826, Republic of Korea Natural Product Informatics Research Center, Korea Institute of Science and Technology, Gangneung, 25451, Republic of Korea c Department of Food Science and Biotechnology, Wonkwang University, Iksan, 54538, Republic of Korea d Smart Farm Research Center, Korea Institute of Science and Technology, Gangneung, 25451, Republic of Korea e Department of Medical Biotechnology, College of Life Science and Biotechnology, Dongguk University, Seoul, 04620, Republic of Korea f Center for Food and Bioconvergence, Seoul National University, Seoul, 08826, Republic of Korea g Research Institute of Agriculture and Life Sciences, Seoul Nation University, Seoul, 08826, Republic of Korea h Department of Food Science and Biotechnology of Animal Resources, Konkuk University, Seoul, 05029, Republic of Korea b
G R A P H I C A L A B S T R A C T
Schematic diagram of the microfluidic assembly method for producing dual-loading liposome.
A R T I C LE I N FO
A B S T R A C T
Keywords: Liposomes Microfluidic device Curcumin Catechin Anti-cancer effect
Curcumin and catechin have inhibitory effects on tumor cell growth. However, the biological activity of curcumin is limited by its low solubility in water. Liposomes are useful carriers of bioactive compounds given their structural similarity to the cellular membrane, but conventional methods for dual-loading liposome preparation are limited to scaled-up applications. Herein, we first applied a microfluidic technique to produce dual-loaded liposomes containing curcumin and catechin by using dipalmitoylphosphatidylcholine as a building block. The dual-loading liposomes were monodispersed (PdI < 0.2) spherical vesicles, less than 200 nm in size and the encapsulation efficiencies of curcumin and catechin on the liposome were 100 % and 16.77 %, respectively. Although all anti-proliferation activities were dose-dependently increased in colon cancer cells by catechin,
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Corresponding author at: Department of Agricultural Biotechnology, Seoul National University, Seoul, 08826, Republic of Korea. E-mail address: [email protected] (P.-S. Chang). 1 These authors contributed equally to this work as first authors. https://doi.org/10.1016/j.colsurfa.2020.124670 Received 2 December 2019; Received in revised form 20 February 2020; Accepted 4 March 2020 Available online 05 March 2020 0927-7757/ © 2020 Elsevier B.V. All rights reserved.
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curcumin, and liposomes, only the dual-loaded liposomes exhibited significantly higher inhibition activity (p < 0.05). These results demonstrate the potential of dual encapsulation of bioactive compounds for enhancing bioavailability and advance the dual-loading technique using microfluidic assembly in the field of liposomal encapsulation of drugs or functional compounds.
1. Introduction
orally ingested to exploit the beneficial health properties of bioactive compounds and materials with low solubility.
Curcumin is a substance present in Indian curry, a yellow curry spice with a long history of use in traditional Indian diets and as an herbal medicine [1,2]. Particularly, curcumin inhibits the growth of tumor cells, has strong antioxidant activity, and prevents dementia [3–5]. Moreover, curcumin shows antimicrobial activity, and is prescribed to enhance lung and liver health [6–8]. Despite these reported health benefits, the oral intake of curcumin is far below its recommended intake because of its poor solubility and short half-life in the body. Curcumin has a high rate of digestion and metabolism at ingestion, resulting in destruction of the compound before it can exert its biological activities [9]. Accordingly, numerous studies have been performed to increase the bioavailability of curcumin, including studies of drug delivery systems, solid nanoparticles, microemulsions, and liposomes. Liposomes can improve bioavailability as a physiologically active substance transporter [10,11]. Additionally, liposomes can increase the stability, biodegradation, and transport of a target molecule, allowing for low-soluble curcumin to be transported and retained with high efficiency [12,13]. The leading manufacturing technology for liposomes is based on the Bangham method, which is a thin membrane-hydration technique [14]. However, this method cannot be used to adjust lamellarity and produces liposomes at the micro-scale. These liposomes have relatively low encapsulation efficiency, thus limiting their broader applications in biomedicine and supplement development [15,16]. Recently, different methods have been developed to produce liposomes such as extrusion, ultrasonication, homogenization, and freezethawing applications. These methods are applicable for producing uniform liposomes with a smaller size which show high invasive efficiency but are not suitable for use in the food or supplement industry. Additionally, these approaches are not cost-effective for continuous mass production [17,18]. We previously reported a microfluidic hydrodynamic concept to overcome the disadvantages of the liposome manufacturing process [19]. Moreover, to improve the ability of liposomes to simultaneously encapsulate hydrophilic and hydrophobic substances, catechin was used as a hydrophilic core in liposome preparation, which is thought to act synergistically with curcumin to enhance its anti-cancer effect. Catechin is abundant in green tea extract as a polyphenol-based material [20,21]. Catechins have also been reported to contain various bioactive compounds with beneficial health properties, including anticancer and anti-oxidation effects. When catechin is combined with curcumin, their biological activities and effects are synergistically increased [22–25]. These synergistic effects are caused by differences in the mechanism of inhibition for each substance. Curcumin suppresses cell growth through miR-21 gene regulation in the G2/M phase of the cell cycle [26]. In contrast, catechin has been reported to inhibit nuclear factor-κB signaling or inhibit cell metabolism and synthesis, thereby producing an anticancer effect by suppressing cell growth and metabolism. It is thought that these different mechanisms have synergistic effects [27]. In this study, we optimized and characterized a microfluidic hydrodynamic technique for producing uniform and nano-sized liposomes dual-loaded with catechin and curcumin. Further, the anti-cancer effect in terms of cytotoxicity was investigated in the human colon cancer cell lines HT-29 and Caco-2 with manufactured liposomes. This method may be useful for the design of dual-loading liposomes that can be
2. Materials and methods 2.1. Materials Curcumin (from Curcuma longa (Turmeric)), ( ± )-catechin hydrate, cholesterol (> 99 % purity), hexadecylamine (HDA), isopropanol (IPA), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Roswell park memorial institute (RPMI) 1640 medium, minimum essential medium (MEM), fetal bovine serum (FBS), antibiotics, phosphate-buffered saline (PBS), trypsin-EDTA, and water were purchased from Hyclone Laboratories (South Logan, UT, USA). N-2Hydroxylethylpiperazine-N’-2-ethanesulfonic acid (HEPES) was obtained from Thermo Fisher Scientific (Waltham, MA, USA). All other chemicals were of analytical grade.
2.2. Preparation of dual-loaded liposomes 2.2.1. Microfluidic device The basic principle and structure of the microfluidic device were described previously [28]. The device consists of two vertically combined polydimethylsiloxane layers. A microchannel of 200–1000 μm was carved on one side of the polydimethylsiloxanes layer. Two inlet lines (inlets 1 and 2) and the outlet were directly connected with the microchannel: inlet 1 was used as the water phase solution, whereas inlet 2 was used for oil phase injection. Moreover, a stencil was attached between the microchannel and end of inlet 2, consisting of numerous tiny pores (around 5 m diameter) through which the lipid molecules could pass. The liposomes were ultimately formed by multihydrodynamic focusing between the shear flow of the water phase from inlet 1 and lipid molecules from inlet 2.
2.2.2. Dual-loading liposome production Catechin dissolved in HEPES buffer (pH 7.4) was used for the water phase (inlet 1), and a lipid mixture consisting of curcumin (turmeric, C. longa), dipalmitoylphosphatidylcholine (DPPC), and cholesterol, dissolved in isopropanol (IPA) was used as the lipid phase (inlet 2). A hypodermic syringe (10 mL) filled with the water phase was connected to inlet 1 of the microfluidic device, and the flow rate of the syringe pump was set to 60 mL/h. A plastic syringe (1 mL) filled with the lipid phase was connected to inlet 2 of the device, and the flow rate was set to 6 mL/h. The syringe pumps were operated at the same time when the temperature of the lipid phase syringe was maintained at over 50 °C. The liposomes generated from the outlet were collected in an ice container after discarding the first 1 mL of sample. The lid of the sample container was kept open and stored at 4 °C for at least 8 h for stabilization and removal of IPA from the liposomes. Finally, the liposomes were filtered through a 0.45-μm filter and stored at 4 °C until further use. For an HDA incorporation into liposomes to enhance positively charge, HDA was dissolved in IPA together with building block lipids (DPPC and cholesterol) to make the lipid phase and mixed with simultaneously the water phase in the microfluidic channel. The lipid bilayer incorporated with HDA was spontaneously generated inside the microfluidic channel (Supplementary Data Fig. S1). 2
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2.3. Characterization of dual-loading liposomes 2.3.1. Analysis of particle size by dynamic light scattering (DLS) The size distribution and polydiversity index (PdI) of the lipid nanovesicles were measured using DLS on a Zetasizer nano ZS system (DTS1070, Malvern Instruments Ltd., Malvern, UK). A sample volume of 1.0 mL was applied to a disposable plastic cuvette, and each measurement was conducted in triplicate. The sample measurement conditions were as follows: refractive index (1.330), viscosity (0.8872 cP), equilibration time (1 min), measurement temperature (25 °C), and measurement angle (173° backscattering). 2.3.2. Analysis of particle stability based on zeta-potential The physicochemical effect of ionic surfactant incorporation was quantitatively and qualitatively evaluated by measuring the zeta potentials of the dual-loaded liposomes. The sample was applied into a disposable folded capillary cell with no bubbles inside (DTS1070, Malvern Instruments). The measurement was conducted in triplicate at 25 °C. 2.3.3. Morphological analysis of dual-loaded liposomes by transmission electron microscopy (TEM) For TEM visualization, uranyl acetate was used as a negative staining reagent for the dual-loaded liposomes. Briefly, the vesicle sample (10 μL) was dropped onto a Formvar-coated silicon monoxide grid (200 mesh). After 1 min, uranyl acetate solution (2 %, w/v) was loaded onto the grid and incubated for 1 min, followed by direct washing of the grid with double-distilled water. The grid was entirely dried at ambient temperature before the vesicles were visualized by TEM (120 keV; JEOL Ltd., Tokyo, Japan). 2.4. Purification of dual-loaded liposomes Fig. 1. DLS and zeta-potential measurements for dual-loading liposomes with different molar ratios of curcumin (A), and DLS and zeta-potential measurement for liposomes incorporated with different molar ratios of hexadecylamine (B).
The dual-loaded liposomes were purified using a Sephadex G-25 pre-packed column (PD-10, GE Healthcare, Little Chalfont, UK). Before use, the column was equilibrated with HEPES buffer (5 mM, pH 7.4) as the eluent. Two milliliters of the retentate was then loaded onto the column, and the samples were collected in 0.5-mL aliquots. The first 2.0-mL samples were discarded, and the next 3.5 mL was collected for measuring the encapsulation efficiency.
Encapsulation efficiency (EE ) = 100 ×
Total material − Free material Total material
Where total material is the sum of the material in the retentate and filtrate and free material was the amount of material in the filtrate.
2.5. Calculation of catechin and curcumin contents in dual-loaded liposomes
2.6. Anti-proliferation activity of dual-loaded liposomes in human colon cells
2.5.1. Quantification of catechin and curcumin by high-performance liquid chromatography (HPLC) Curcumin and catechin were simultaneously quantified using a Waters 486 HPLC system (Waters, Milford, MA, USA) which was controlled by Clarity software (DataApex, Petrzilkova, Prague, Czech Republic) equipped with an autosampler and a silica-based C18-AR-II column (5 μm, 4.6 × 250 mm; Cosmosil, Kyoto, Japan). The mobile phase for detection of catechin and curcumin was acetonitrile, water, and trifluoroacetic acid (90:10:0.5, volume fraction rate), which was eluted isocratically at 1.0 mL/min. Catechin and curcumin were detected at 268 and 340 nm, respectively, at a column temperature of 30 °C for 60 min.
2.6.1. Cell line culture conditions Human colon adenocarcinoma cell lines (HT-29 and Caco-2) were obtained from the Korean Cell Line Bank (Seoul, Korea). HT-29 and Caco-2 cells were respectively cultured in RPMI medium and minimal essential medium containing 10 % fetal bovine serum and 1 % penicillin/streptomycin (all from Hyclone Laboratories). Both cell lines were incubated at 37 °C in a 5 % CO2 incubator (MCO-18AIC, SANYO, Osaka, Japan). The cells were sub-cultured until 80–90 % confluence. 2.6.2. Determination of anti-proliferation activity The anti-proliferation activity of samples on intestinal adenocarcinoma epithelial cells was determined using the MTT assay according to the manufacturer’s instructions (Sigma-Aldrich) with some modifications. In detail, HT-29 and Caco-2 cells were seeded into 96-well plates (5.0 × 104 cells/well) and incubated for 48 h. Next, the cells were treated with different concentrations of samples and incubated for an additional 24 h. After washing the cells with PBS, MTT solution (2.5 mg/mL in RPMI or MEM) was added to each well and incubated for 4 h. The supernatants were removed, and 200 μL of dimethyl sulfoxide
2.5.2. Encapsulation efficiency determination The sample was loaded into an ultrafiltration tube, which was centrifuged at 4000 ×g for 10 min. The retentates and filtrates were collected, and each volume was measured. In addition, the material concentration for the retentate and filtrate was analyzed by HPLC after treatment with Triton-X 100 as described above. The encapsulation efficiency (%) was then calculated using the following equation: 3
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Table 1 Cancer cell cytotoxicity of HDA-incorporated catechin and curcumin dualloaded liposomes against the human adenocarcinoma epithelial colon cell lines HT-29 and Caco-2. Cancer cell cytotoxicity (%) Cell line
Sample
Ca, Cu (250 μM, 5 μM)
Ca, Cu (500 μM, 10 μM)
HT-29
Vehicle Ca CaL Cu CuL Ca + Cu Ca + CuL CaL + Cu CaL + CuL DL
3.61 ± 1.51 18.40 ± 1.07b 41.70 ± 1.67d 12.37 ± 1.14a 18.79 ± 1.64b 31.96 ± 1.11c 75.40 ± 0.71g 49.78 ± 1.77e 67.64 ± 1.79f 78.57 ± 0.35h
29.29 60.97 32.86 41.52 49.03 80.09 80.41 80.30 84.67
Vehicle Ca CaL Cu CuL Ca + Cu Ca + CuL CaL + Cu CaL + CuL DL
4.45 ± 0.45 2.75 ± 0.31a 16.72 ± 1.33c 9.76 ± 0.93b 20.13 ± 2.01d 10.13 ± 0.68b 41.61 ± 0.68e 21.17 ± 0.67d 40.88 ± 1.52e 43.14 ± 1.00e
5.92 ± 0.26A 25.38 ± 2.09C,D 18.79 ± 1.36B 27.09 ± 0.96D 23.19 ± 0.75C 54.61 ± 1.76F 38.38 ± 0.23E 53.94 ± 0.52F 66.50 ± 1.52G
Caco-2
± ± ± ± ± ± ± ± ±
0.98A 2.70 E 0.64B 0.71C 0.47D 0.90F 0.61F 0.62F 0.27G
Ca, Cu (1000 μM, 20 μM)
41.20 75.97 43.76 61.18 69.97 82.94 84.81 84.70 91.48
± ± ± ± ± ± ± ± ±
1.68α 0.58 ε 0.36β 1.92 γ 0.50 δ 0.45ζ 0.44ζ 0.36ζ 0.79η
7.75 ± 0.40α 41.12 ± 0.46δ 26.91 ± 1.67β 37.95 ± 1.22γ 37.28 ± 3.51γ,δ 62.90 ± 1.60ζ 52.17 ± 0.52ε 65.22 ± 0.93ζ 81.72 ± 1.02η
All values are represented as mean ± S.D. of triplicate experiments. Ca, catechin; CaL, catechin-encapsulated liposome; Cu, curcumin; CuL, curcumin-incorporated liposome; DL, catechin and curcumin dual-loaded liposome. Values that have a different superscript letter (a, b, c, d, e, f, or h; A, B, C, D, E, F, or G; or α, β, γ, δ, ε, ζ, or η) differ significantly from each other (p < 0.05, Duncan’s multiple range test).
was added to each well to dissolve the converted formazan deposits. The absorbance of each well was determined at 570 nm with a microplate spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). Anti-proliferation activity, indicating cytotoxicity, was calculated using the following equation: Cytotoxicity (%) = [1 – (absorbance of the sample/absorbance of the control)] × 100 3. Results and discussion 3.1. Optimization of the microfluidic device
Fig. 2. TEM images of catechin and curcumin dual-loaded liposomes (A) and HDA-modified dual-loaded liposomes (B).
The flow rate of the water and lipid phase of the microfluidic device was adjusted to produce lipid nano-vesicles with a monodispersed size distribution. The flow rate of the lipid phase was varied from 1.5 to 12.0 mL/h and kept constant for water. DPPC was used for lipid nanovesicle production. DPPC liposomes with a lipid phase flow rate of 6.0 mL/h showed a monodispersed 100 nm size distribution. The size distribution and PdI of the lipid nano-vesicles important in drug delivery systems because the cellular uptake mode to the nucleus is highly dependent on the vesicle size [29,30] (Supplementary Data Fig. S2). For microfluidic assembly, IPA with building-block lipids was homogeneously mixed in the water phase, and the lipid bilayer was spontaneously formed inside the channel. All processes were driven by the flow rate and ratio of the water and lipid phase of the microfluidic device. Based on the results, a flow rate below 6.0 mL/h may not be a sufficient driving force for assembling monodispersed lipid nano-vesicles. Furthermore, a faster flow rate for the lipid phase of the device may lead to an overall increase of the alcohol content in the final vesicle sample, potentially disintegrating the lipid bilayer and leading to overall disruption of the lipid nano-vesicles. For the water phase, a slower flow rate may not confer an adequate shear force to facilitate
Fig. 3. DLS measurements of the diameter and catechin quantification of gel permeation chromatography-fractionated liposomes. 4
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Fig. 4. Optical microscope observation of the effect of catechin and curcumin alone or encapsulated in liposomes on the morphology and viability of the human colon cancer cell line HT-29. (A) Control, (B) catechin, (C) curcumin, (D) catechin-encapsulated liposomes, (E) curcumin-incorporated with liposomes, and (F) positively charged catechin and curcumin dual-loaded liposomes.
Fig. 5. Optical microscopic observation of the effect of catechin and curcumin alone or encapsulated with or without liposomes on the morphology and viability of the human colon cancer cell line Caco-2. (A) Control, (B) catechin, (C) curcumin, (D) catechin-encapsulated liposomes, (E) curcumin-incorporated with liposomes, and (F) positively charged catechin and curcumin dual-loaded liposomes.
vesicle assembly, whereas a faster flow rate can disrupt the microfluidic device because of excessive pressure on the overall assembly line. Therefore, 60.0 mL/h for the water phase and 6.0 mL/h for the lipid phase flow rate were determined as optimal conditions for producing evenly distributed lipid nano-vesicles (Supplementary Data Fig. S3).
catechin which is dissolved in water phase and showing low encapsulation efficiency was excluded from the independent variables for the optimization of the colloidal stability of dual-loaded liposomes. Additionally, the surface charge of liposome could be more negative as curcumin is added within 5 molar percent during liposome production. In microfluid assembly, curcumin is spontaneously incorporated into the lipid bilayer of liposomes because it is a very hydrophobic compound. This curcumin whose pKa value is about 8 exhibits a slight negative charge in the liposome conditions (pH 7.4); hence, the zetapotential of the liposomes decreased. However, when curcumin exceeds 5 molar percent, curcumin in the lipid bilayer is saturated and selfassembly of liposomes could be hindered by curcumin remnants which cannot be incorporated in liposomes. Hence, it seems that an increase of zeta-potential of liposomes in Fig. 1A was due to the decrease of the absolute quantity of negatively charged liposomes by self-assembly hindrance of curcumin remnants.
3.2. Preparation of catechin and curcumin-loaded liposomes Catechin was dissolved in a water solution, and curcumin was dissolved in an IPA solution with cholesterol. Cholesterol directly influences the incorporation efficiency, release pattern, and stability of bioactive materials [31,32]. Although it does not form a bilayer, it easily intercalates into the lipid bilayer, providing essential rigidity to the lipid bilayer, particularly under severe mechanical stress [33,34]. From 1 to 10 molar percent of curcumin was incorporated into the lipid vesicles with cholesterol and DPPC, followed by vesicle size distribution and zeta potential measurements. The results showed that 5 molar percent curcumin in liposomes with 2 molar percent cholesterol was the most suitable condition for lipid nano-vesicle formation, resulting in a monodispersed distribution of approximately 200 nm sized liposomes based on DLS measurements (Fig. 1A). However, the molar ratio of
3.3. Surface modification of dual-loaded liposomes The dual-loaded liposomes encapsulated with catechin and curcumin showed a zeta potential of ―30 mV. To enhance the cell 5
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liposomes have disadvantage of low encapsulation efficiency because those are based on self-assembly in solution state and cause damage to the liposomes directly. However, using microfluidic assembly in this study, the encapsulation efficiency of curcumin could dramatically increase without any loss of curcumin (about 100 %) because curcumin is fully dissolved in isopropyl alcohol (IPA) and easily incorporated into the lipid bilayer through microfluidic assembly.
membrane bioavailability of the liposomes, the surface charge must be adjusted. Based on an extensive literature review, we attempted ionic surfactant incorporation, which resulted in an overall increase of the absolute zeta potential of the liposomes [35]. For this, we eventually selected HDA for this study because the US FDA approved HDA as drug and cosmetics substance though HDA is known to show cytotoxicity slightly. Moreover, the cytotoxicity of HDA-incorporated liposomes was not worth considering in our liposome conditions (Table 1). However, a continuous increase in the ionic surfactant concentration can destabilize the lipid bilayer. Therefore, we determined the minimum zeta-potential at which the liposomes become colloidally stable conditions. Consequently, 4 molar percent of HDA incorporation with 8 molar percent DPPC, 2 molar percent cholesterol, and 5 molar percent curcumin in liposomes were selected as the most suitable conditions, resulting in an even size distribution with colloidal stability and a positive surface charge (Fig. 1B). Finally, the optimized HDA-incorporated liposomes were used for further experiments.
3.7. In vitro cytotoxicity of dual-loaded liposomes against cancer cell To investigate the enhancement of bioavailability, we examined the inhibition effect of catechin, curcumin, catechin-encapsulated liposomes, curcumin-incorporated liposomes, and catechin and curcumin dual-loaded liposomes on the growth of HT-29 and Caco-2 colon cancer cells by performing an MTT assay (Table 1, Supplementary Data Figs. S4 and S5). The result showed that compared to the group treated with only catechin, catechin-encapsulated liposomes resulted in 24 %, 32 %, and 35 % cytotoxicity, indicating dose-dependent anti-proliferation effects on HT-29 cells. Similarly, catechin-encapsulated liposomes exhibited enhanced cytotoxicity towards Caco-2 cells by showing 14 %, 20 %, and 25 % dose-dependent anti-proliferation effects. Although curcumin-incorporated liposomes also showed a dose-dependent enhanced killing effect compared to curcumin alone, the increase was less extensive than that of catechin-encapsulated liposomes at 6 %, 8 %, and 20 % in HT-29 cells and 12 %, 9%, and 10 % in Caco-2 cells. These results confirmed that the cytotoxicity of catechin or curcumin against cancer cells is enhanced when these agents are encapsulated in liposomes. In addition, vehicles which are empty liposomes exhibited no significant effect (below 5 % cytotoxicity) at each cell lines in experimental conditions. Moreover, the cytotoxicity of HDA was not worth considering in our liposome conditions as aforementioned. Several studies have revealed a synergistic effect of catechin and curcumin on cell death of colon cancer cells [37–40]. Thus, we predicted that this synergistic effect would be further increased by entrapping the two substances in liposomes. Indeed, the cytotoxic effect of dual-loaded liposomes on HT-29 and Caco-2 cells was higher than that of the two single compounds in a concentration-dependent manner. Moreover, the dual-loaded liposomes positively charged by HDA incorporation showed a more significant synergistic effect than the group treated by mixing the liposomes individually (Figs. 4 and 5). In HT-29 cells, catechin and curcumin dual-loaded liposomes with 0.25 mM catechin and 5 μM curcumin showed the most significant cytotoxicity, with a 47 % increase compared to catechin or curcumin treatment alone. Additionally, catechin and curcumin dual-loaded liposomes with 0.5 mM catechin and 10 μM curcumin or 1 mM catechin and 20 μM curcumin showed an increased cytotoxicity of 35 % and 21 %, respectively, compared to the single treatments. Similarly, catechin and curcumin dual-loaded liposomes with 0.25 mM catechin and 5 μM curcumin, 0.5 mM catechin and 10 μM curcumin, and 1 mM catechin and 20 μM curcumin showed a 33 %, 43 %, and 44 % increased cytotoxic effect against Caco-2 cells compared to each single treatment, respectively.
3.4. Morphology of dual-loaded liposomes TEM observations showed that both the dual-loaded liposomes and positively charged dual-loaded liposomes with HDA incorporation had an evenly distributed morphology and spherical shape with a diameter of approximately under 200 nm (Fig. 2A and B). The size of each liposomes was already determined by DLS analysis, and TEM was conducted additionally to observe the morphology of the liposomes and validate the size from DLS analysis. During TEM analysis, the contraction phenomenon of the liposomes could occur in general [36]; hence, the liposomes of 200 nm could appear to be liposomes of several tens of nanometers in the TEM image appeared in Fig. 2. 3.5. Purification of dual-loaded liposomes Gel permeation chromatography was conducted to separate the unencapsulated catechin and curcumin from the catechin and curcumin dual-loaded liposomes. The dual-loaded liposomes were passed through a PD-10 column, and DLS and HPLC analysis were conducted for each elution volume. Catechin and curcumin dual-loaded liposomes with no ionic surfactant incorporation were used to assess the purification effects at each step. DLS showed that the 3.0–5.5 mL elution volume had a lower PdI, confirming the presence of dual-loaded liposomes in the sample. In addition, HPLC analysis confirmed that catechin and curcumin were eluted between 5.5 and 10.0 mL, indicating that dualloaded liposomes were present in the elution volume below 5.5 mL (Fig. 3). 3.6. Encapsulation efficiency Process for the purification of the liposomes was nearly perfect as expected; however, it was difficult to elute free materials from Sephadex G-25 and evaluate the encapsulation efficiency. Therefore, independent methodology based on ultracentrifugation and HPLC analysis was applied to evaluate the encapsulation efficiency. The encapsulation efficiency of the dual-loaded liposomes was measured using standard curves for catechin and curcumin. The samples from the retentate and filtrate were used for HPLC analysis (n = 3), and the average peak area was used to calculate the catechin and curcumin concentrations. The efficiencies of the dual-loaded liposomes with the encapsulation of catechin and curcumin were 16.8 % and 100 %, respectively. Similarly, catechin and curcumin were encapsulated in the HDA dual-loaded liposomes showed efficiencies of 16.2 % and 100 %, respectively. Thus, the encapsulation efficiency of curcumin was clearly higher than that of catechin, which can be explained by the fact that curcumin is a hydrophobically bioactive compound and can serve as a building block in lipid vesicle like DPPC and cholesterol. Traditional methodologies for encapsulation of curcumin into
4. Conclusion We successfully produced mono-dispersed 200 nm catechin and curcumin dual-loaded liposomes by microfluidic assembly based on hydrodynamic-focusing principle. Particularly, the production of dualloaded liposomes applying microfluidic device was newly reported in this study. To increase the colloidal stability and increase intracellular functionality of catechin and curcumin, the production conditions of dual-loaded liposomes were optimized controlling the molar ratio of building-block lipids together with HDA incorporation. Moreover, the cytotoxicity of dual-loaded liposomes was evaluated resulting in highly increased anti-cancer effects by dual-loading. Based on the results from 6
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this study, further studies focusing on the validation of anti-cancer activity at in vivo models could advance the dual-loading technique using microfluidic assembly in the field of liposomal encapsulation of drugs or functional compounds.
emulsion, Food Chem. 173 (2015) 7–13. [11] N. Aditya, M. Shim, I. Lee, Y. Lee, M.-H. Im, S. Ko, Curcumin and genistein coloaded nanostructured lipid carriers: in vitro digestion and antiprostate cancer activity, J. Agr. Food Chem. 61 (2013) 1878–1883. [12] Bernard F. Gibbs, Selim Kermasha, Inteaz Alli, Catherine N. Mulligan, Encapsulation in the food industry: a review, Int. J. Food Sci. Nutr. 50 (1999) 213–224. [13] L.S. Jackson, K. Lee, Microencapsulation and the food industry, Lebensm. Wiss. Technol. 24 (1991) 289–297. [14] A.D. Bangham, M.W. Hill, N. Miller, Preparation and Use of Liposomes As Models of Biological Membranes, Methods in Membrane Biology, Springer, 1974, pp. 1–68. [15] A. Akbarzadeh, R. Rezaei-Sadabady, S. Davaran, S.W. Joo, N. Zarghami, Y. Hanifehpour, M. Samiei, M. Kouhi, K. Nejati-Koshki, Liposome: classification, preparation, and applications, Nanoscale Res. Lett. 8 (2013) 102. [16] J. Dua, A. Rana, A. Bhandari, Liposome: methods of preparation and applications, Int. J. Pharm. Stud. Res. 3 (2012) 14–20. [17] C.R. Hong, G.W. Lee, H.-D. Paik, P.-S. Chang, S.J. Choi, Influence of biopolymers on the solubility of branched-chain amino acids and stability of their solutions, Food Chem. 239 (2018) 872–878. [18] C. Lipinski, Poor aqueous solubility—an industry wide problem in drug discovery, Am. Pharm. Rev. 5 (2002) 82–85. [19] M. Jo, K.-M. Park, J.-Y. Park, H. Yu, S.J. Choi, P.-S. Chang, Microfluidic assembly of mono-dispersed liposome and its surface modification for enhancing the colloidal stability, Colloid Surf. A physicochem. Eng. Asp. 586 (2020) 124202–124210. [20] R. Amarowicz, F. Shahidi, A rapid chromatographic method for separation of individual catechins from green tea, Food Res. Int. 29 (1996) 71–76. [21] J.J. Dalluge, B.C. Nelson, J.B. Thomas, L.C. Sander, Selection of column and gradient elution system for the separation of catechins in green tea using high-performance liquid chromatography, J. Chromatogr. A 793 (1998) 265–274. [22] N. Li, X. Chen, J. Liao, G. Yang, S. Wang, Y. Josephson, C. Han, J. Chen, M.T. Huang, C.S. Yang, Inhibition of 7, 12-dimethylbenz [a] anthracene (DMBA)-induced oral carcinogenesis in hamsters by tea and curcumin, Carcinogenesis 23 (2002) 1307–1313. [23] R. Manikandan, M. Beulaja, C. Arulvasu, S. Sellamuthu, D. Dinesh, D. Prabhu, G. Babu, B. Vaseeharan, N. Prabhu, Synergistic anticancer activity of curcumin and catechin: an in vitro study using human cancer cell lines, Microsc. Res. Tech. 75 (2012) 112–116. [24] T.J. Somers‐Edgar, M.J. Scandlyn, E.C. Stuart, M.J. Le Nedelec, S.P. Valentine, R.J. Rosengren, The combination of epigallocatechin gallate and curcumin suppresses ERα‐breast cancer cell growth in vitro and in vivo, Int. J. Cancer 122 (2008) 1966–1971. [25] G. Xu, G. Ren, X. Xu, H. Yuan, Z. Wang, L. Kang, W. Yu, K. Tian, Combination of curcumin and green tea catechins prevents dimethylhydrazine-induced colon carcinogenesis, Food Chem. Toxicol. 48 (2010) 390–395. [26] M.A. Tomeh, R. Hadianamrei, X. Zhao, A review of curcumin and its derivatives as anticancer agents, Int. J. Mol. Sci. 20 (2019) 1033–1059. [27] K.-j. Min, T.K. Kwon, Anticancer effects and molecular mechanisms of epigallocatechin-3-gallate, Integr. Med. Res. 3 (2014) 16–24. [28] H. Cho, J. Kim, K. Suga, T. Ishigami, H. Park, J.W. Bang, S. Seo, M. Choi, P.S. Chang, H. Umakoshi, Microfluidic platforms with monolithically integrated hierarchical apertures for the facile and rapid formation of cargo-carrying vesicles, Lab Chip 15 (2015) 373–377. [29] E. Blanco, H. Shen, M. Ferrari, Principles of nanoparticle design for overcoming biological barriers to drug delivery, Nat. Biotechnol. 33 (2015) 941–951. [30] R. Singh, J.W. Lillard Jr., Nanoparticle-based targeted drug delivery, Exp. Mol. Pathol. 86 (2009) 215–223. [31] M.-L. Briuglia, C. Rotella, A. McFarlane, D.A. Lamprou, Influence of cholesterol on liposome stability and on in vitro drug release, Drug Deliv. Transl. Res. 5 (2015) 231–242. [32] J. Lopez-Pinto, M. Gonzalez-Rodriguez, A. Rabasco, Effect of cholesterol and ethanol on dermal delivery from DPPC liposomes, Int. J. Pharm. 298 (2005) 1–12. [33] F.J. Byfield, H. Aranda-Espinoza, V.G. Romanenko, G.H. Rothblat, I. Levitan, Cholesterol depletion increases membrane stiffness of aortic endothelial cells, Biophys. J. 87 (2004) 3336–3343. [34] K. Melzak, S. Melzak, E. Gizeli, J. Toca-Herrera, Cholesterol organization in phosphatidylcholine liposomes: a surface plasmon resonance study, Materials 5 (2012) 2306–2325. [35] C. Freitas, R.H. Müller, Effect of light and temperature on zeta potential and physical stability in solid lipid nanoparticle (SLN™) dispersions, Int. J. Pharm. 168 (1998) 221–229. [36] J. Asadi, S. Ferguson, H. Raja, C. Hacker, P. Marius, R. Ward, C. Pliotas, J. Naismith, J. Lucocq, Enhanced imaging of lipid rich nanoparticles embedded in methylcellulose films for transmission electron microscopy using mixtures of heavy metals, Micron. 99 (2017) 40–48. [37] A. Goel, C.R. Boland, D.P. Chauhan, Specific inhibition of cyclooxygenase-2 (COX2) expression by dietary curcumin in HT-29 human colon cancer cells, Cancer Lett. 172 (2001) 111–118. [38] T. Sergent, S. Garsou, A. Schaut, S. De Saeger, L. Pussemier, C. Van Peteghem, Y. Larondelle, Y.-J. Schneider, Differential modulation of ochratoxin a absorption across caco-2 cells by dietary polyphenols, used at realistic intestinal concentrations, Toxicol. Lett. 159 (2005) 60–70. [39] J.-T. Hwang, J. Ha, I.-J. Park, S.-K. Lee, H.W. Baik, Y.M. Kim, O.J. Park, Apoptotic effect of EGCG in HT-29 colon cancer cells via AMPK signal pathway, Cancer Lett. 247 (2007) 115–121. [40] B. Wahlang, Y.B. Pawar, A.K. Bansal, Identification of permeability-related hurdles in oral delivery of curcumin using the Caco-2 cell model, Eur. J. Pharm. Biopharm. 77 (2011) 275–282.
CRediT authorship contribution statement Sung-Chul Hong: Conceptualization, Methodology, Validation, Investigation, Writing - original draft, Writing - review & editing. Kyung-Min Park: Conceptualization, Methodology, Validation, Investigation, Writing - original draft, Writing - review & editing. Chi Rac Hong: Conceptualization, Formal analysis, Visualization, Writing original draft, Writing - review & editing. Jin-Chul Kim: Validation, Writing - original draft, Writing - review & editing, Funding acquisition. Seung-Hoon Yang: Validation, Formal analysis, Investigation, Writing - original draft. Hyung-Seok Yu: Investigation, Formal analysis, Validation, Visualization. Hyun-Dong Paik: Resources, Supervision. Cheol-Ho Pan: Resources, Writing - review & editing, Supervision, Funding acquisition. Pahn-Shick Chang: Resources, Writing - review & editing, Supervision, Project administration, Funding acquisition. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgement This study was supported by the project titled “Development of Advanced Process for the Production of Eye Health Materials Using Tetraselmis chuii,” funded by the Ministry of Ocean and Fisheries in Republic of Korea, and in part by “Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ01488802)”, Rural Development Administration, Republic of Korea. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.colsurfa.2020.124670. References [1] A. Das, V. Rajkumar, D. Dwivedi, Antioxidant effect of curry leaf (Murraya koenigii) powder on quality of ground and cooked goat meat, Int. Food Res. J. 18 (2011) 563–569. [2] U. Iyer, U. Mani, Studies on the effect of curry leaves supplementation (Murraya koenigi) on lipid profile, glycated proteins and amino acids in non-insulin-dependent diabetic patients, Plant Foods Hum. Nutr. 40 (1990) 275–282. [3] J. Fang, J. Lu, A. Holmgren, Thioredoxin reductase is irreversibly modified by curcumin a novel molecular mechanism for its anticancer activity, J. Biol. Chem. 280 (2005) 25284–25290. [4] P. Negi, G. Jayaprakasha, L. Jagan Mohan Rao, K. Sakariah, Antibacterial activity of turmeric oil: a byproduct from curcumin manufacture, J. Agr. Food Chem. 47 (1999) 4297–4300. [5] O. Sharma, Antioxidant activity of curcumin and related compounds, Biochem. Pharmacol. 25 (1976) 1811–1812. [6] B.B. Aggarwal, S. Shishodia, Y. Takada, S. Banerjee, R.A. Newman, C.E. BuesoRamos, J.E. Price, Curcumin suppresses the paclitaxel-induced nuclear factor-κB pathway in breast cancer cells and inhibits lung metastasis of human breast cancer in nude mice, Clin. Cancer Res. 11 (2005) 7490–7498. [7] A.A. Nanji, K. Jokelainen, G.L. Tipoe, A. Rahemtulla, P. Thomas, A.J. Dannenberg, Curcumin prevents alcohol-induced liver disease in rats by inhibiting the expression of NF-κB-dependent genes, Am. J. Physiol. Gastr. L. 284 (2003) G321–G327. [8] S. Shishodia, P. Potdar, C.G. Gairola, B.B. Aggarwal, Curcumin (diferuloylmethane) down-regulates cigarette smoke-induced NF-κB activation through inhibition of IκBα kinase in human lung epithelial cells: correlation with suppression of COX-2, MMP-9 and cyclin D1, Carcinogenesis 24 (2003) 1269–1279. [9] P. Anand, A.B. Kunnumakkara, R.A. Newman, B.B. Aggarwal, Bioavailability of curcumin: problems and promises, Mol. Pharm. 4 (2007) 807–818. [10] N. Aditya, S. Aditya, H. Yang, H.W. Kim, S.O. Park, S. Ko, Co-delivery of hydrophobic curcumin and hydrophilic catechin by a water-in-oil-in-water double
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Contents lists available at ScienceDirect
Colloids and Surfaces A journal homepage: www.elsevier.com/locate/colsurfa
Microfluidic assembly of liposomes dual-loaded with catechin and curcumin for enhancing bioavailability
T
Sung-Chul Honga,b,1, Kyung-Min Parkc,1, Chi Rac Hongd, Jin-Chul Kimb, Seung-Hoon Yange, Hyung-Seok Yuh, Hyun-Dong Paikh, Cheol-Ho Panb, Pahn-Shick Changa,f,g,* a
Department of Agricultural Biotechnology, Seoul National University, Seoul, 08826, Republic of Korea Natural Product Informatics Research Center, Korea Institute of Science and Technology, Gangneung, 25451, Republic of Korea c Department of Food Science and Biotechnology, Wonkwang University, Iksan, 54538, Republic of Korea d Smart Farm Research Center, Korea Institute of Science and Technology, Gangneung, 25451, Republic of Korea e Department of Medical Biotechnology, College of Life Science and Biotechnology, Dongguk University, Seoul, 04620, Republic of Korea f Center for Food and Bioconvergence, Seoul National University, Seoul, 08826, Republic of Korea g Research Institute of Agriculture and Life Sciences, Seoul Nation University, Seoul, 08826, Republic of Korea h Department of Food Science and Biotechnology of Animal Resources, Konkuk University, Seoul, 05029, Republic of Korea b
G R A P H I C A L A B S T R A C T
Schematic diagram of the microfluidic assembly method for producing dual-loading liposome.
A R T I C LE I N FO
A B S T R A C T
Keywords: Liposomes Microfluidic device Curcumin Catechin Anti-cancer effect
Curcumin and catechin have inhibitory effects on tumor cell growth. However, the biological activity of curcumin is limited by its low solubility in water. Liposomes are useful carriers of bioactive compounds given their structural similarity to the cellular membrane, but conventional methods for dual-loading liposome preparation are limited to scaled-up applications. Herein, we first applied a microfluidic technique to produce dual-loaded liposomes containing curcumin and catechin by using dipalmitoylphosphatidylcholine as a building block. The dual-loading liposomes were monodispersed (PdI < 0.2) spherical vesicles, less than 200 nm in size and the encapsulation efficiencies of curcumin and catechin on the liposome were 100 % and 16.77 %, respectively. Although all anti-proliferation activities were dose-dependently increased in colon cancer cells by catechin,
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Corresponding author at: Department of Agricultural Biotechnology, Seoul National University, Seoul, 08826, Republic of Korea. E-mail address: [email protected] (P.-S. Chang). 1 These authors contributed equally to this work as first authors. https://doi.org/10.1016/j.colsurfa.2020.124670 Received 2 December 2019; Received in revised form 20 February 2020; Accepted 4 March 2020 Available online 05 March 2020 0927-7757/ © 2020 Elsevier B.V. All rights reserved.
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curcumin, and liposomes, only the dual-loaded liposomes exhibited significantly higher inhibition activity (p < 0.05). These results demonstrate the potential of dual encapsulation of bioactive compounds for enhancing bioavailability and advance the dual-loading technique using microfluidic assembly in the field of liposomal encapsulation of drugs or functional compounds.
1. Introduction
orally ingested to exploit the beneficial health properties of bioactive compounds and materials with low solubility.
Curcumin is a substance present in Indian curry, a yellow curry spice with a long history of use in traditional Indian diets and as an herbal medicine [1,2]. Particularly, curcumin inhibits the growth of tumor cells, has strong antioxidant activity, and prevents dementia [3–5]. Moreover, curcumin shows antimicrobial activity, and is prescribed to enhance lung and liver health [6–8]. Despite these reported health benefits, the oral intake of curcumin is far below its recommended intake because of its poor solubility and short half-life in the body. Curcumin has a high rate of digestion and metabolism at ingestion, resulting in destruction of the compound before it can exert its biological activities [9]. Accordingly, numerous studies have been performed to increase the bioavailability of curcumin, including studies of drug delivery systems, solid nanoparticles, microemulsions, and liposomes. Liposomes can improve bioavailability as a physiologically active substance transporter [10,11]. Additionally, liposomes can increase the stability, biodegradation, and transport of a target molecule, allowing for low-soluble curcumin to be transported and retained with high efficiency [12,13]. The leading manufacturing technology for liposomes is based on the Bangham method, which is a thin membrane-hydration technique [14]. However, this method cannot be used to adjust lamellarity and produces liposomes at the micro-scale. These liposomes have relatively low encapsulation efficiency, thus limiting their broader applications in biomedicine and supplement development [15,16]. Recently, different methods have been developed to produce liposomes such as extrusion, ultrasonication, homogenization, and freezethawing applications. These methods are applicable for producing uniform liposomes with a smaller size which show high invasive efficiency but are not suitable for use in the food or supplement industry. Additionally, these approaches are not cost-effective for continuous mass production [17,18]. We previously reported a microfluidic hydrodynamic concept to overcome the disadvantages of the liposome manufacturing process [19]. Moreover, to improve the ability of liposomes to simultaneously encapsulate hydrophilic and hydrophobic substances, catechin was used as a hydrophilic core in liposome preparation, which is thought to act synergistically with curcumin to enhance its anti-cancer effect. Catechin is abundant in green tea extract as a polyphenol-based material [20,21]. Catechins have also been reported to contain various bioactive compounds with beneficial health properties, including anticancer and anti-oxidation effects. When catechin is combined with curcumin, their biological activities and effects are synergistically increased [22–25]. These synergistic effects are caused by differences in the mechanism of inhibition for each substance. Curcumin suppresses cell growth through miR-21 gene regulation in the G2/M phase of the cell cycle [26]. In contrast, catechin has been reported to inhibit nuclear factor-κB signaling or inhibit cell metabolism and synthesis, thereby producing an anticancer effect by suppressing cell growth and metabolism. It is thought that these different mechanisms have synergistic effects [27]. In this study, we optimized and characterized a microfluidic hydrodynamic technique for producing uniform and nano-sized liposomes dual-loaded with catechin and curcumin. Further, the anti-cancer effect in terms of cytotoxicity was investigated in the human colon cancer cell lines HT-29 and Caco-2 with manufactured liposomes. This method may be useful for the design of dual-loading liposomes that can be
2. Materials and methods 2.1. Materials Curcumin (from Curcuma longa (Turmeric)), ( ± )-catechin hydrate, cholesterol (> 99 % purity), hexadecylamine (HDA), isopropanol (IPA), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Roswell park memorial institute (RPMI) 1640 medium, minimum essential medium (MEM), fetal bovine serum (FBS), antibiotics, phosphate-buffered saline (PBS), trypsin-EDTA, and water were purchased from Hyclone Laboratories (South Logan, UT, USA). N-2Hydroxylethylpiperazine-N’-2-ethanesulfonic acid (HEPES) was obtained from Thermo Fisher Scientific (Waltham, MA, USA). All other chemicals were of analytical grade.
2.2. Preparation of dual-loaded liposomes 2.2.1. Microfluidic device The basic principle and structure of the microfluidic device were described previously [28]. The device consists of two vertically combined polydimethylsiloxane layers. A microchannel of 200–1000 μm was carved on one side of the polydimethylsiloxanes layer. Two inlet lines (inlets 1 and 2) and the outlet were directly connected with the microchannel: inlet 1 was used as the water phase solution, whereas inlet 2 was used for oil phase injection. Moreover, a stencil was attached between the microchannel and end of inlet 2, consisting of numerous tiny pores (around 5 m diameter) through which the lipid molecules could pass. The liposomes were ultimately formed by multihydrodynamic focusing between the shear flow of the water phase from inlet 1 and lipid molecules from inlet 2.
2.2.2. Dual-loading liposome production Catechin dissolved in HEPES buffer (pH 7.4) was used for the water phase (inlet 1), and a lipid mixture consisting of curcumin (turmeric, C. longa), dipalmitoylphosphatidylcholine (DPPC), and cholesterol, dissolved in isopropanol (IPA) was used as the lipid phase (inlet 2). A hypodermic syringe (10 mL) filled with the water phase was connected to inlet 1 of the microfluidic device, and the flow rate of the syringe pump was set to 60 mL/h. A plastic syringe (1 mL) filled with the lipid phase was connected to inlet 2 of the device, and the flow rate was set to 6 mL/h. The syringe pumps were operated at the same time when the temperature of the lipid phase syringe was maintained at over 50 °C. The liposomes generated from the outlet were collected in an ice container after discarding the first 1 mL of sample. The lid of the sample container was kept open and stored at 4 °C for at least 8 h for stabilization and removal of IPA from the liposomes. Finally, the liposomes were filtered through a 0.45-μm filter and stored at 4 °C until further use. For an HDA incorporation into liposomes to enhance positively charge, HDA was dissolved in IPA together with building block lipids (DPPC and cholesterol) to make the lipid phase and mixed with simultaneously the water phase in the microfluidic channel. The lipid bilayer incorporated with HDA was spontaneously generated inside the microfluidic channel (Supplementary Data Fig. S1). 2
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2.3. Characterization of dual-loading liposomes 2.3.1. Analysis of particle size by dynamic light scattering (DLS) The size distribution and polydiversity index (PdI) of the lipid nanovesicles were measured using DLS on a Zetasizer nano ZS system (DTS1070, Malvern Instruments Ltd., Malvern, UK). A sample volume of 1.0 mL was applied to a disposable plastic cuvette, and each measurement was conducted in triplicate. The sample measurement conditions were as follows: refractive index (1.330), viscosity (0.8872 cP), equilibration time (1 min), measurement temperature (25 °C), and measurement angle (173° backscattering). 2.3.2. Analysis of particle stability based on zeta-potential The physicochemical effect of ionic surfactant incorporation was quantitatively and qualitatively evaluated by measuring the zeta potentials of the dual-loaded liposomes. The sample was applied into a disposable folded capillary cell with no bubbles inside (DTS1070, Malvern Instruments). The measurement was conducted in triplicate at 25 °C. 2.3.3. Morphological analysis of dual-loaded liposomes by transmission electron microscopy (TEM) For TEM visualization, uranyl acetate was used as a negative staining reagent for the dual-loaded liposomes. Briefly, the vesicle sample (10 μL) was dropped onto a Formvar-coated silicon monoxide grid (200 mesh). After 1 min, uranyl acetate solution (2 %, w/v) was loaded onto the grid and incubated for 1 min, followed by direct washing of the grid with double-distilled water. The grid was entirely dried at ambient temperature before the vesicles were visualized by TEM (120 keV; JEOL Ltd., Tokyo, Japan). 2.4. Purification of dual-loaded liposomes Fig. 1. DLS and zeta-potential measurements for dual-loading liposomes with different molar ratios of curcumin (A), and DLS and zeta-potential measurement for liposomes incorporated with different molar ratios of hexadecylamine (B).
The dual-loaded liposomes were purified using a Sephadex G-25 pre-packed column (PD-10, GE Healthcare, Little Chalfont, UK). Before use, the column was equilibrated with HEPES buffer (5 mM, pH 7.4) as the eluent. Two milliliters of the retentate was then loaded onto the column, and the samples were collected in 0.5-mL aliquots. The first 2.0-mL samples were discarded, and the next 3.5 mL was collected for measuring the encapsulation efficiency.
Encapsulation efficiency (EE ) = 100 ×
Total material − Free material Total material
Where total material is the sum of the material in the retentate and filtrate and free material was the amount of material in the filtrate.
2.5. Calculation of catechin and curcumin contents in dual-loaded liposomes
2.6. Anti-proliferation activity of dual-loaded liposomes in human colon cells
2.5.1. Quantification of catechin and curcumin by high-performance liquid chromatography (HPLC) Curcumin and catechin were simultaneously quantified using a Waters 486 HPLC system (Waters, Milford, MA, USA) which was controlled by Clarity software (DataApex, Petrzilkova, Prague, Czech Republic) equipped with an autosampler and a silica-based C18-AR-II column (5 μm, 4.6 × 250 mm; Cosmosil, Kyoto, Japan). The mobile phase for detection of catechin and curcumin was acetonitrile, water, and trifluoroacetic acid (90:10:0.5, volume fraction rate), which was eluted isocratically at 1.0 mL/min. Catechin and curcumin were detected at 268 and 340 nm, respectively, at a column temperature of 30 °C for 60 min.
2.6.1. Cell line culture conditions Human colon adenocarcinoma cell lines (HT-29 and Caco-2) were obtained from the Korean Cell Line Bank (Seoul, Korea). HT-29 and Caco-2 cells were respectively cultured in RPMI medium and minimal essential medium containing 10 % fetal bovine serum and 1 % penicillin/streptomycin (all from Hyclone Laboratories). Both cell lines were incubated at 37 °C in a 5 % CO2 incubator (MCO-18AIC, SANYO, Osaka, Japan). The cells were sub-cultured until 80–90 % confluence. 2.6.2. Determination of anti-proliferation activity The anti-proliferation activity of samples on intestinal adenocarcinoma epithelial cells was determined using the MTT assay according to the manufacturer’s instructions (Sigma-Aldrich) with some modifications. In detail, HT-29 and Caco-2 cells were seeded into 96-well plates (5.0 × 104 cells/well) and incubated for 48 h. Next, the cells were treated with different concentrations of samples and incubated for an additional 24 h. After washing the cells with PBS, MTT solution (2.5 mg/mL in RPMI or MEM) was added to each well and incubated for 4 h. The supernatants were removed, and 200 μL of dimethyl sulfoxide
2.5.2. Encapsulation efficiency determination The sample was loaded into an ultrafiltration tube, which was centrifuged at 4000 ×g for 10 min. The retentates and filtrates were collected, and each volume was measured. In addition, the material concentration for the retentate and filtrate was analyzed by HPLC after treatment with Triton-X 100 as described above. The encapsulation efficiency (%) was then calculated using the following equation: 3
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Table 1 Cancer cell cytotoxicity of HDA-incorporated catechin and curcumin dualloaded liposomes against the human adenocarcinoma epithelial colon cell lines HT-29 and Caco-2. Cancer cell cytotoxicity (%) Cell line
Sample
Ca, Cu (250 μM, 5 μM)
Ca, Cu (500 μM, 10 μM)
HT-29
Vehicle Ca CaL Cu CuL Ca + Cu Ca + CuL CaL + Cu CaL + CuL DL
3.61 ± 1.51 18.40 ± 1.07b 41.70 ± 1.67d 12.37 ± 1.14a 18.79 ± 1.64b 31.96 ± 1.11c 75.40 ± 0.71g 49.78 ± 1.77e 67.64 ± 1.79f 78.57 ± 0.35h
29.29 60.97 32.86 41.52 49.03 80.09 80.41 80.30 84.67
Vehicle Ca CaL Cu CuL Ca + Cu Ca + CuL CaL + Cu CaL + CuL DL
4.45 ± 0.45 2.75 ± 0.31a 16.72 ± 1.33c 9.76 ± 0.93b 20.13 ± 2.01d 10.13 ± 0.68b 41.61 ± 0.68e 21.17 ± 0.67d 40.88 ± 1.52e 43.14 ± 1.00e
5.92 ± 0.26A 25.38 ± 2.09C,D 18.79 ± 1.36B 27.09 ± 0.96D 23.19 ± 0.75C 54.61 ± 1.76F 38.38 ± 0.23E 53.94 ± 0.52F 66.50 ± 1.52G
Caco-2
± ± ± ± ± ± ± ± ±
0.98A 2.70 E 0.64B 0.71C 0.47D 0.90F 0.61F 0.62F 0.27G
Ca, Cu (1000 μM, 20 μM)
41.20 75.97 43.76 61.18 69.97 82.94 84.81 84.70 91.48
± ± ± ± ± ± ± ± ±
1.68α 0.58 ε 0.36β 1.92 γ 0.50 δ 0.45ζ 0.44ζ 0.36ζ 0.79η
7.75 ± 0.40α 41.12 ± 0.46δ 26.91 ± 1.67β 37.95 ± 1.22γ 37.28 ± 3.51γ,δ 62.90 ± 1.60ζ 52.17 ± 0.52ε 65.22 ± 0.93ζ 81.72 ± 1.02η
All values are represented as mean ± S.D. of triplicate experiments. Ca, catechin; CaL, catechin-encapsulated liposome; Cu, curcumin; CuL, curcumin-incorporated liposome; DL, catechin and curcumin dual-loaded liposome. Values that have a different superscript letter (a, b, c, d, e, f, or h; A, B, C, D, E, F, or G; or α, β, γ, δ, ε, ζ, or η) differ significantly from each other (p < 0.05, Duncan’s multiple range test).
was added to each well to dissolve the converted formazan deposits. The absorbance of each well was determined at 570 nm with a microplate spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). Anti-proliferation activity, indicating cytotoxicity, was calculated using the following equation: Cytotoxicity (%) = [1 – (absorbance of the sample/absorbance of the control)] × 100 3. Results and discussion 3.1. Optimization of the microfluidic device
Fig. 2. TEM images of catechin and curcumin dual-loaded liposomes (A) and HDA-modified dual-loaded liposomes (B).
The flow rate of the water and lipid phase of the microfluidic device was adjusted to produce lipid nano-vesicles with a monodispersed size distribution. The flow rate of the lipid phase was varied from 1.5 to 12.0 mL/h and kept constant for water. DPPC was used for lipid nanovesicle production. DPPC liposomes with a lipid phase flow rate of 6.0 mL/h showed a monodispersed 100 nm size distribution. The size distribution and PdI of the lipid nano-vesicles important in drug delivery systems because the cellular uptake mode to the nucleus is highly dependent on the vesicle size [29,30] (Supplementary Data Fig. S2). For microfluidic assembly, IPA with building-block lipids was homogeneously mixed in the water phase, and the lipid bilayer was spontaneously formed inside the channel. All processes were driven by the flow rate and ratio of the water and lipid phase of the microfluidic device. Based on the results, a flow rate below 6.0 mL/h may not be a sufficient driving force for assembling monodispersed lipid nano-vesicles. Furthermore, a faster flow rate for the lipid phase of the device may lead to an overall increase of the alcohol content in the final vesicle sample, potentially disintegrating the lipid bilayer and leading to overall disruption of the lipid nano-vesicles. For the water phase, a slower flow rate may not confer an adequate shear force to facilitate
Fig. 3. DLS measurements of the diameter and catechin quantification of gel permeation chromatography-fractionated liposomes. 4
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Fig. 4. Optical microscope observation of the effect of catechin and curcumin alone or encapsulated in liposomes on the morphology and viability of the human colon cancer cell line HT-29. (A) Control, (B) catechin, (C) curcumin, (D) catechin-encapsulated liposomes, (E) curcumin-incorporated with liposomes, and (F) positively charged catechin and curcumin dual-loaded liposomes.
Fig. 5. Optical microscopic observation of the effect of catechin and curcumin alone or encapsulated with or without liposomes on the morphology and viability of the human colon cancer cell line Caco-2. (A) Control, (B) catechin, (C) curcumin, (D) catechin-encapsulated liposomes, (E) curcumin-incorporated with liposomes, and (F) positively charged catechin and curcumin dual-loaded liposomes.
vesicle assembly, whereas a faster flow rate can disrupt the microfluidic device because of excessive pressure on the overall assembly line. Therefore, 60.0 mL/h for the water phase and 6.0 mL/h for the lipid phase flow rate were determined as optimal conditions for producing evenly distributed lipid nano-vesicles (Supplementary Data Fig. S3).
catechin which is dissolved in water phase and showing low encapsulation efficiency was excluded from the independent variables for the optimization of the colloidal stability of dual-loaded liposomes. Additionally, the surface charge of liposome could be more negative as curcumin is added within 5 molar percent during liposome production. In microfluid assembly, curcumin is spontaneously incorporated into the lipid bilayer of liposomes because it is a very hydrophobic compound. This curcumin whose pKa value is about 8 exhibits a slight negative charge in the liposome conditions (pH 7.4); hence, the zetapotential of the liposomes decreased. However, when curcumin exceeds 5 molar percent, curcumin in the lipid bilayer is saturated and selfassembly of liposomes could be hindered by curcumin remnants which cannot be incorporated in liposomes. Hence, it seems that an increase of zeta-potential of liposomes in Fig. 1A was due to the decrease of the absolute quantity of negatively charged liposomes by self-assembly hindrance of curcumin remnants.
3.2. Preparation of catechin and curcumin-loaded liposomes Catechin was dissolved in a water solution, and curcumin was dissolved in an IPA solution with cholesterol. Cholesterol directly influences the incorporation efficiency, release pattern, and stability of bioactive materials [31,32]. Although it does not form a bilayer, it easily intercalates into the lipid bilayer, providing essential rigidity to the lipid bilayer, particularly under severe mechanical stress [33,34]. From 1 to 10 molar percent of curcumin was incorporated into the lipid vesicles with cholesterol and DPPC, followed by vesicle size distribution and zeta potential measurements. The results showed that 5 molar percent curcumin in liposomes with 2 molar percent cholesterol was the most suitable condition for lipid nano-vesicle formation, resulting in a monodispersed distribution of approximately 200 nm sized liposomes based on DLS measurements (Fig. 1A). However, the molar ratio of
3.3. Surface modification of dual-loaded liposomes The dual-loaded liposomes encapsulated with catechin and curcumin showed a zeta potential of ―30 mV. To enhance the cell 5
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liposomes have disadvantage of low encapsulation efficiency because those are based on self-assembly in solution state and cause damage to the liposomes directly. However, using microfluidic assembly in this study, the encapsulation efficiency of curcumin could dramatically increase without any loss of curcumin (about 100 %) because curcumin is fully dissolved in isopropyl alcohol (IPA) and easily incorporated into the lipid bilayer through microfluidic assembly.
membrane bioavailability of the liposomes, the surface charge must be adjusted. Based on an extensive literature review, we attempted ionic surfactant incorporation, which resulted in an overall increase of the absolute zeta potential of the liposomes [35]. For this, we eventually selected HDA for this study because the US FDA approved HDA as drug and cosmetics substance though HDA is known to show cytotoxicity slightly. Moreover, the cytotoxicity of HDA-incorporated liposomes was not worth considering in our liposome conditions (Table 1). However, a continuous increase in the ionic surfactant concentration can destabilize the lipid bilayer. Therefore, we determined the minimum zeta-potential at which the liposomes become colloidally stable conditions. Consequently, 4 molar percent of HDA incorporation with 8 molar percent DPPC, 2 molar percent cholesterol, and 5 molar percent curcumin in liposomes were selected as the most suitable conditions, resulting in an even size distribution with colloidal stability and a positive surface charge (Fig. 1B). Finally, the optimized HDA-incorporated liposomes were used for further experiments.
3.7. In vitro cytotoxicity of dual-loaded liposomes against cancer cell To investigate the enhancement of bioavailability, we examined the inhibition effect of catechin, curcumin, catechin-encapsulated liposomes, curcumin-incorporated liposomes, and catechin and curcumin dual-loaded liposomes on the growth of HT-29 and Caco-2 colon cancer cells by performing an MTT assay (Table 1, Supplementary Data Figs. S4 and S5). The result showed that compared to the group treated with only catechin, catechin-encapsulated liposomes resulted in 24 %, 32 %, and 35 % cytotoxicity, indicating dose-dependent anti-proliferation effects on HT-29 cells. Similarly, catechin-encapsulated liposomes exhibited enhanced cytotoxicity towards Caco-2 cells by showing 14 %, 20 %, and 25 % dose-dependent anti-proliferation effects. Although curcumin-incorporated liposomes also showed a dose-dependent enhanced killing effect compared to curcumin alone, the increase was less extensive than that of catechin-encapsulated liposomes at 6 %, 8 %, and 20 % in HT-29 cells and 12 %, 9%, and 10 % in Caco-2 cells. These results confirmed that the cytotoxicity of catechin or curcumin against cancer cells is enhanced when these agents are encapsulated in liposomes. In addition, vehicles which are empty liposomes exhibited no significant effect (below 5 % cytotoxicity) at each cell lines in experimental conditions. Moreover, the cytotoxicity of HDA was not worth considering in our liposome conditions as aforementioned. Several studies have revealed a synergistic effect of catechin and curcumin on cell death of colon cancer cells [37–40]. Thus, we predicted that this synergistic effect would be further increased by entrapping the two substances in liposomes. Indeed, the cytotoxic effect of dual-loaded liposomes on HT-29 and Caco-2 cells was higher than that of the two single compounds in a concentration-dependent manner. Moreover, the dual-loaded liposomes positively charged by HDA incorporation showed a more significant synergistic effect than the group treated by mixing the liposomes individually (Figs. 4 and 5). In HT-29 cells, catechin and curcumin dual-loaded liposomes with 0.25 mM catechin and 5 μM curcumin showed the most significant cytotoxicity, with a 47 % increase compared to catechin or curcumin treatment alone. Additionally, catechin and curcumin dual-loaded liposomes with 0.5 mM catechin and 10 μM curcumin or 1 mM catechin and 20 μM curcumin showed an increased cytotoxicity of 35 % and 21 %, respectively, compared to the single treatments. Similarly, catechin and curcumin dual-loaded liposomes with 0.25 mM catechin and 5 μM curcumin, 0.5 mM catechin and 10 μM curcumin, and 1 mM catechin and 20 μM curcumin showed a 33 %, 43 %, and 44 % increased cytotoxic effect against Caco-2 cells compared to each single treatment, respectively.
3.4. Morphology of dual-loaded liposomes TEM observations showed that both the dual-loaded liposomes and positively charged dual-loaded liposomes with HDA incorporation had an evenly distributed morphology and spherical shape with a diameter of approximately under 200 nm (Fig. 2A and B). The size of each liposomes was already determined by DLS analysis, and TEM was conducted additionally to observe the morphology of the liposomes and validate the size from DLS analysis. During TEM analysis, the contraction phenomenon of the liposomes could occur in general [36]; hence, the liposomes of 200 nm could appear to be liposomes of several tens of nanometers in the TEM image appeared in Fig. 2. 3.5. Purification of dual-loaded liposomes Gel permeation chromatography was conducted to separate the unencapsulated catechin and curcumin from the catechin and curcumin dual-loaded liposomes. The dual-loaded liposomes were passed through a PD-10 column, and DLS and HPLC analysis were conducted for each elution volume. Catechin and curcumin dual-loaded liposomes with no ionic surfactant incorporation were used to assess the purification effects at each step. DLS showed that the 3.0–5.5 mL elution volume had a lower PdI, confirming the presence of dual-loaded liposomes in the sample. In addition, HPLC analysis confirmed that catechin and curcumin were eluted between 5.5 and 10.0 mL, indicating that dualloaded liposomes were present in the elution volume below 5.5 mL (Fig. 3). 3.6. Encapsulation efficiency Process for the purification of the liposomes was nearly perfect as expected; however, it was difficult to elute free materials from Sephadex G-25 and evaluate the encapsulation efficiency. Therefore, independent methodology based on ultracentrifugation and HPLC analysis was applied to evaluate the encapsulation efficiency. The encapsulation efficiency of the dual-loaded liposomes was measured using standard curves for catechin and curcumin. The samples from the retentate and filtrate were used for HPLC analysis (n = 3), and the average peak area was used to calculate the catechin and curcumin concentrations. The efficiencies of the dual-loaded liposomes with the encapsulation of catechin and curcumin were 16.8 % and 100 %, respectively. Similarly, catechin and curcumin were encapsulated in the HDA dual-loaded liposomes showed efficiencies of 16.2 % and 100 %, respectively. Thus, the encapsulation efficiency of curcumin was clearly higher than that of catechin, which can be explained by the fact that curcumin is a hydrophobically bioactive compound and can serve as a building block in lipid vesicle like DPPC and cholesterol. Traditional methodologies for encapsulation of curcumin into
4. Conclusion We successfully produced mono-dispersed 200 nm catechin and curcumin dual-loaded liposomes by microfluidic assembly based on hydrodynamic-focusing principle. Particularly, the production of dualloaded liposomes applying microfluidic device was newly reported in this study. To increase the colloidal stability and increase intracellular functionality of catechin and curcumin, the production conditions of dual-loaded liposomes were optimized controlling the molar ratio of building-block lipids together with HDA incorporation. Moreover, the cytotoxicity of dual-loaded liposomes was evaluated resulting in highly increased anti-cancer effects by dual-loading. Based on the results from 6
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this study, further studies focusing on the validation of anti-cancer activity at in vivo models could advance the dual-loading technique using microfluidic assembly in the field of liposomal encapsulation of drugs or functional compounds.
emulsion, Food Chem. 173 (2015) 7–13. [11] N. Aditya, M. Shim, I. Lee, Y. Lee, M.-H. Im, S. Ko, Curcumin and genistein coloaded nanostructured lipid carriers: in vitro digestion and antiprostate cancer activity, J. Agr. Food Chem. 61 (2013) 1878–1883. [12] Bernard F. Gibbs, Selim Kermasha, Inteaz Alli, Catherine N. Mulligan, Encapsulation in the food industry: a review, Int. J. Food Sci. Nutr. 50 (1999) 213–224. [13] L.S. Jackson, K. Lee, Microencapsulation and the food industry, Lebensm. Wiss. Technol. 24 (1991) 289–297. [14] A.D. Bangham, M.W. Hill, N. Miller, Preparation and Use of Liposomes As Models of Biological Membranes, Methods in Membrane Biology, Springer, 1974, pp. 1–68. [15] A. Akbarzadeh, R. Rezaei-Sadabady, S. Davaran, S.W. Joo, N. Zarghami, Y. Hanifehpour, M. Samiei, M. Kouhi, K. Nejati-Koshki, Liposome: classification, preparation, and applications, Nanoscale Res. Lett. 8 (2013) 102. [16] J. Dua, A. Rana, A. Bhandari, Liposome: methods of preparation and applications, Int. J. Pharm. Stud. Res. 3 (2012) 14–20. [17] C.R. Hong, G.W. Lee, H.-D. Paik, P.-S. Chang, S.J. Choi, Influence of biopolymers on the solubility of branched-chain amino acids and stability of their solutions, Food Chem. 239 (2018) 872–878. [18] C. Lipinski, Poor aqueous solubility—an industry wide problem in drug discovery, Am. Pharm. Rev. 5 (2002) 82–85. [19] M. Jo, K.-M. Park, J.-Y. Park, H. Yu, S.J. Choi, P.-S. Chang, Microfluidic assembly of mono-dispersed liposome and its surface modification for enhancing the colloidal stability, Colloid Surf. A physicochem. Eng. Asp. 586 (2020) 124202–124210. [20] R. Amarowicz, F. Shahidi, A rapid chromatographic method for separation of individual catechins from green tea, Food Res. Int. 29 (1996) 71–76. [21] J.J. Dalluge, B.C. Nelson, J.B. Thomas, L.C. Sander, Selection of column and gradient elution system for the separation of catechins in green tea using high-performance liquid chromatography, J. Chromatogr. A 793 (1998) 265–274. [22] N. Li, X. Chen, J. Liao, G. Yang, S. Wang, Y. Josephson, C. Han, J. Chen, M.T. Huang, C.S. Yang, Inhibition of 7, 12-dimethylbenz [a] anthracene (DMBA)-induced oral carcinogenesis in hamsters by tea and curcumin, Carcinogenesis 23 (2002) 1307–1313. [23] R. Manikandan, M. Beulaja, C. Arulvasu, S. Sellamuthu, D. Dinesh, D. Prabhu, G. Babu, B. Vaseeharan, N. Prabhu, Synergistic anticancer activity of curcumin and catechin: an in vitro study using human cancer cell lines, Microsc. Res. Tech. 75 (2012) 112–116. [24] T.J. Somers‐Edgar, M.J. Scandlyn, E.C. Stuart, M.J. Le Nedelec, S.P. Valentine, R.J. Rosengren, The combination of epigallocatechin gallate and curcumin suppresses ERα‐breast cancer cell growth in vitro and in vivo, Int. J. Cancer 122 (2008) 1966–1971. [25] G. Xu, G. Ren, X. Xu, H. Yuan, Z. Wang, L. Kang, W. Yu, K. Tian, Combination of curcumin and green tea catechins prevents dimethylhydrazine-induced colon carcinogenesis, Food Chem. Toxicol. 48 (2010) 390–395. [26] M.A. Tomeh, R. Hadianamrei, X. Zhao, A review of curcumin and its derivatives as anticancer agents, Int. J. Mol. Sci. 20 (2019) 1033–1059. [27] K.-j. Min, T.K. Kwon, Anticancer effects and molecular mechanisms of epigallocatechin-3-gallate, Integr. Med. Res. 3 (2014) 16–24. [28] H. Cho, J. Kim, K. Suga, T. Ishigami, H. Park, J.W. Bang, S. Seo, M. Choi, P.S. Chang, H. Umakoshi, Microfluidic platforms with monolithically integrated hierarchical apertures for the facile and rapid formation of cargo-carrying vesicles, Lab Chip 15 (2015) 373–377. [29] E. Blanco, H. Shen, M. Ferrari, Principles of nanoparticle design for overcoming biological barriers to drug delivery, Nat. Biotechnol. 33 (2015) 941–951. [30] R. Singh, J.W. Lillard Jr., Nanoparticle-based targeted drug delivery, Exp. Mol. Pathol. 86 (2009) 215–223. [31] M.-L. Briuglia, C. Rotella, A. McFarlane, D.A. Lamprou, Influence of cholesterol on liposome stability and on in vitro drug release, Drug Deliv. Transl. Res. 5 (2015) 231–242. [32] J. Lopez-Pinto, M. Gonzalez-Rodriguez, A. Rabasco, Effect of cholesterol and ethanol on dermal delivery from DPPC liposomes, Int. J. Pharm. 298 (2005) 1–12. [33] F.J. Byfield, H. Aranda-Espinoza, V.G. Romanenko, G.H. Rothblat, I. Levitan, Cholesterol depletion increases membrane stiffness of aortic endothelial cells, Biophys. J. 87 (2004) 3336–3343. [34] K. Melzak, S. Melzak, E. Gizeli, J. Toca-Herrera, Cholesterol organization in phosphatidylcholine liposomes: a surface plasmon resonance study, Materials 5 (2012) 2306–2325. [35] C. Freitas, R.H. Müller, Effect of light and temperature on zeta potential and physical stability in solid lipid nanoparticle (SLN™) dispersions, Int. J. Pharm. 168 (1998) 221–229. [36] J. Asadi, S. Ferguson, H. Raja, C. Hacker, P. Marius, R. Ward, C. Pliotas, J. Naismith, J. Lucocq, Enhanced imaging of lipid rich nanoparticles embedded in methylcellulose films for transmission electron microscopy using mixtures of heavy metals, Micron. 99 (2017) 40–48. [37] A. Goel, C.R. Boland, D.P. Chauhan, Specific inhibition of cyclooxygenase-2 (COX2) expression by dietary curcumin in HT-29 human colon cancer cells, Cancer Lett. 172 (2001) 111–118. [38] T. Sergent, S. Garsou, A. Schaut, S. De Saeger, L. Pussemier, C. Van Peteghem, Y. Larondelle, Y.-J. Schneider, Differential modulation of ochratoxin a absorption across caco-2 cells by dietary polyphenols, used at realistic intestinal concentrations, Toxicol. Lett. 159 (2005) 60–70. [39] J.-T. Hwang, J. Ha, I.-J. Park, S.-K. Lee, H.W. Baik, Y.M. Kim, O.J. Park, Apoptotic effect of EGCG in HT-29 colon cancer cells via AMPK signal pathway, Cancer Lett. 247 (2007) 115–121. [40] B. Wahlang, Y.B. Pawar, A.K. Bansal, Identification of permeability-related hurdles in oral delivery of curcumin using the Caco-2 cell model, Eur. J. Pharm. Biopharm. 77 (2011) 275–282.
CRediT authorship contribution statement Sung-Chul Hong: Conceptualization, Methodology, Validation, Investigation, Writing - original draft, Writing - review & editing. Kyung-Min Park: Conceptualization, Methodology, Validation, Investigation, Writing - original draft, Writing - review & editing. Chi Rac Hong: Conceptualization, Formal analysis, Visualization, Writing original draft, Writing - review & editing. Jin-Chul Kim: Validation, Writing - original draft, Writing - review & editing, Funding acquisition. Seung-Hoon Yang: Validation, Formal analysis, Investigation, Writing - original draft. Hyung-Seok Yu: Investigation, Formal analysis, Validation, Visualization. Hyun-Dong Paik: Resources, Supervision. Cheol-Ho Pan: Resources, Writing - review & editing, Supervision, Funding acquisition. Pahn-Shick Chang: Resources, Writing - review & editing, Supervision, Project administration, Funding acquisition. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgement This study was supported by the project titled “Development of Advanced Process for the Production of Eye Health Materials Using Tetraselmis chuii,” funded by the Ministry of Ocean and Fisheries in Republic of Korea, and in part by “Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ01488802)”, Rural Development Administration, Republic of Korea. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.colsurfa.2020.124670. References [1] A. Das, V. Rajkumar, D. Dwivedi, Antioxidant effect of curry leaf (Murraya koenigii) powder on quality of ground and cooked goat meat, Int. Food Res. J. 18 (2011) 563–569. [2] U. Iyer, U. Mani, Studies on the effect of curry leaves supplementation (Murraya koenigi) on lipid profile, glycated proteins and amino acids in non-insulin-dependent diabetic patients, Plant Foods Hum. Nutr. 40 (1990) 275–282. [3] J. Fang, J. Lu, A. Holmgren, Thioredoxin reductase is irreversibly modified by curcumin a novel molecular mechanism for its anticancer activity, J. Biol. Chem. 280 (2005) 25284–25290. [4] P. Negi, G. Jayaprakasha, L. Jagan Mohan Rao, K. Sakariah, Antibacterial activity of turmeric oil: a byproduct from curcumin manufacture, J. Agr. Food Chem. 47 (1999) 4297–4300. [5] O. Sharma, Antioxidant activity of curcumin and related compounds, Biochem. Pharmacol. 25 (1976) 1811–1812. [6] B.B. Aggarwal, S. Shishodia, Y. Takada, S. Banerjee, R.A. Newman, C.E. BuesoRamos, J.E. Price, Curcumin suppresses the paclitaxel-induced nuclear factor-κB pathway in breast cancer cells and inhibits lung metastasis of human breast cancer in nude mice, Clin. Cancer Res. 11 (2005) 7490–7498. [7] A.A. Nanji, K. Jokelainen, G.L. Tipoe, A. Rahemtulla, P. Thomas, A.J. Dannenberg, Curcumin prevents alcohol-induced liver disease in rats by inhibiting the expression of NF-κB-dependent genes, Am. J. Physiol. Gastr. L. 284 (2003) G321–G327. [8] S. Shishodia, P. Potdar, C.G. Gairola, B.B. Aggarwal, Curcumin (diferuloylmethane) down-regulates cigarette smoke-induced NF-κB activation through inhibition of IκBα kinase in human lung epithelial cells: correlation with suppression of COX-2, MMP-9 and cyclin D1, Carcinogenesis 24 (2003) 1269–1279. [9] P. Anand, A.B. Kunnumakkara, R.A. Newman, B.B. Aggarwal, Bioavailability of curcumin: problems and promises, Mol. Pharm. 4 (2007) 807–818. [10] N. Aditya, S. Aditya, H. Yang, H.W. Kim, S.O. Park, S. Ko, Co-delivery of hydrophobic curcumin and hydrophilic catechin by a water-in-oil-in-water double
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