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Inhibiting the vascular smooth muscle cells proliferation by EPC and DPPC liposomes encapsulated magnolol
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Journal of the Chinese Institute of Chemical Engineers 39 (2008) 407–411 www.elsevier.com/locate/jcice
Inhibiting the vascular smooth muscle cells proliferation by EPC and DPPC liposomes encapsulated magnolol Calvin Yu-Chian Chen * Department of Biological Science and Technology, China Medical University, Taichung 404, Taiwan Received 19 January 2008; received in revised form 11 April 2008; accepted 14 April 2008
Abstract Percutaneous transluminal coronary angioplasty (PTCA) is a non-surgical modality for treating stennosis. However, the recurrence of restenosis in 30–50% patients within 6 months is the major drawback of PTCA. The major reason of restenosis is the proliferation of the vascular smooth muscle cells (VSMCs). Magnolol, a pure compound extracted from Magnolia officinalis, encapsulated by liposome was investigated for inhibiting the VSMCs proliferation leading to restenosis by PTCA. 1,2-Diacyl-Sn-glycero-3-phosphocholine (EPC) and 1,2dipalmitoyl-Sn-glycero-3-phosphocholine (DPPC) liposomes were utilized to encapsulate the magnolol. EPC liposome obtained the higher encapsulation efficiency than DPPC lipsomes from UV–vis spectroscopy study. The inhibiting efficiency of EPC and DPPC liposomes encapsulated magnolol was higher than pure magnonol. Magnolol encapsulated by EPC liposomes had better efficiency on inhibiting VSMCs than DPPC liposome. Addition of cholesterol in liposomes could slightly enhance the encapsulation efficiency. The particles sizer analysis revealed the average particles size of EPC and DPPC liposomes encapsulated magnolol became larger than pure EPC or DPPC liposomes. From the transmission electron microscopy (TEM) analysis, the magnolol seems to interfere with EPC and DPPC liposomes to form a homogeneous lipid bilayer. # 2008 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved. Keywords: Vascular smooth muscle cells; 1,2-Dipalmitoyl-Sn-glycero-3-phosphocholine; 1,2-Diacyl-Sn-glycero-3-phosphocholine; Magnolol; Liposome
1. Introduction Atherosclerosis is a progressing disease (Lusis, 2000). The etiology of this disease is mainly due to the high plasma level of cholesterol especially low-density lipoprotein (Walsh and Isner, 2000), with subsequent oxidization resulting in atheroma formation on the aorta. The pathological process of atherosclerosis consists of chronic inflammation, and excessive formation of foam cells in the subendothelium space (Schwartz et al., 1993), resulting in outgrowth and abnormal migration of the smooth muscle cell in the blood vessel (Song et al., 2004), as well as fibrosis and calcification of the blood vessel (Chew, 2005; Lafont et al., 1995). According to the statistics, about 30– 50% of atherosclerotic patients suffered from restenosis in half year after PTCA treatment (Currier and Faxon, 1995; Farb et al., 1999; Rogers et al., 1996).
* Tel.: +886 4 22053366x2506. E-mail address: [email protected].
Magnolol is a commonly used Chinese herbal medicine known as Hou Po (Allen et al., 2006). Magnolia is a prescribed Chinese medicine isolated from Magnolia officinalis for many treatments (Lee et al., 2001; Wu et al., 2007). Magnolol had been reported to possess two kinds of different functions (Kuntz and Baim, 1993; Serruys et al., 1993). It could inhibit the cell growth at lower concentration, whereas induce cell apoptosis at higher concentration (Chen et al., 2003; Hong et al., 1996). Therefore, magnolol was expected as a pharmacological approach for restenosis by inhibiting vascular smooth muscle cells (VSMCs) proliferation (Wu et al., 2002). Liposome is used for drug delivery system due to its unique structure properties (Kuo and Chung, 2005). Liposome can carry both the hydrophobic and hydrophilic drug (Liu et al., 2006). Therefore, liposome as a drug carrier can indiscriminately deliver drugs through the cell membrane (Lin et al., 2007; Liu et al., 2000). In this study, liposome-encapsulated magnolol, especially EPC liposome, might be more efficient than bare magnolol in preventing proliferation of VSMCs, a key contributor to restenosis induced by PTCA.
0368-1653/$ – see front matter # 2008 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jcice.2008.04.005
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2. Materials and methods 2.1. Cell culture A10 vascular smooth muscle cells derived from rat thoracic aorta were obtained from Food Industry Research and Development Institute (Hsinchu, Taiwan). The cells were cultured in 10-cm dishes (TPP, Switzerland) containing Dulbecco’s modified eagle’s medium (DMEM) (Sigma, USA) supplemented with 10% FBS (Gibco, Carlsbad, CA, USA), 1.5 g/L NaHCO3 (Sigma, USA), 1.028 g/L N-acetyl L-alanyl-L-glutamine (Biochrom, Berlin), 1% Na-pyruvate (Sigma, USA), 4.5 g/L D-glucose (Sigma, USA), 100 units/ mL penicillin G (Sigma, USA), and 100 mg/mL streptomycin sulfates (Gibco,Carlsbad, CA, USA). Cells were grown in a humidified incubator with 5% CO2 at 37 8C. The culture medium was replaced every 2–3 days and the cells were passaged every week. In the last passage, the same numbers of cells were subcultured to 96-well plates. The cells that became at least 80% confluent were starved for 24 h in DMEM containing 0.5% FBS followed by a treatment with 0.01 and 0.075 mg/mL of magnolol (95% (HPLC), Sigma, USA) in DMEM containing 15% FBS for 24 h. 2.2. Preparation of liposome Magnolol (M3445, >98%, HPLC), a phenolic compound isolated from a Chinese herbal drug, M. officinalis, was purchased from Sigma Chemical Company. 1,2-DipalmitoylSn-glycero-3-phosphocholine (DPPC) (>99%pure) and 1,2diacyl-Sn-glycero-3-phosphocholine from egg yolk (EPC) (99% pure) were purchased from Sigma Chemical Company, USA and were used as-received. Ethanol (99.5% pure) was supplied by Seoul Chemical Industry Co. Ltd., Korea, and HPLC grade hexane (>99% pure) was obtained from Ferak Laborat GmbH, Germany. Cholesterol was supplied by Sigma Chemical Company, USA. The mixed liposomes (DPPC/magnolol, DPPC/magnolol/cholesterol, EPC/magnolol, EPC/magnolol/cholesterol) were dissolved in ethanol/ hexane (1:9, v/v) solvent stored at 4 8C in a round-bottomed flask. All the lipid/cholesterol ratios of liposomes were prepared with molar ratio 1:1. Before use, all glassware was exhaustively rinsed with purified water. The flask was then taken to dryness under the rotary evaporator to ensure all the organic solvent had evaporated. The mixed liposome was dispersed in 15% FBS medium magnolol and then added into the round-bottomed flask. To form the nano-scale liposome, the flask was sonicated by ultrasonic facilitation (DC-150H, Delta1, Taiwan) for 2 h to encapsulate the magnolol. 2.3. MTT assay In the MTT assay, the cells were seeded on 24-well plates at the concentration of approximately 5000 cells/cm2 before the drug treatment. The MTT assay was implemented 24 h after the addition of liposome-encapsulated magnolol. Cells were seeded in 96-well plates at a density of 1 104 cells per well
in 200 mL of DMEM/10% FBS and incubated for 1 day. Afterwards, cells were treated with 0.01 and 0.075 mg/mL of magnolol added in DMEM with 15% FBS. After 1 day of drug treatment, the effect on cell growth was examined by the MTT assay. After adding 130 mL of 0.5 mg/mL MTT (3-[4,5dimethyl thiazol-2-yl]-2,5-diphenyl tetrazolium bromide) (Sigma Chemical Co., St. Louis, MO) to each well, the mixtures were incubated for 4 h at 37 8C. The supernatant was aspirated and the MTT–formazan crystals formed by metabolically viable cells were dissolved in 130 mL of DMSO (DMSO; dimethylsulphoxide, Sigma, USA). Finally, the absorbance was measured by a microplate reader at a wavelength of 590 nm. The statistical significance of the data was evaluated with Student’s t-test. P-value of 0.05 and 0.01 were considered significant. 2.4. Drug encapsulation efficiency The content of magnolol in liposomes was measured by UV– vis spectroscopy (Bio-RAD 31, USA) at a fixed wavelength of 292 nm. The precipitated lipid was separated by centrifugation (19,000 g for 60 min). The absorbance of supernatant was then measured at 292 nm. Non-encapsulation drug was compared with the standard curve of magnolol, which was achieved from magnolol solutions in PBS with concentration between 0.0001 and 0.08 mg/mL. 2.5. Size analysis of liposome encapsulated magnolol EPC-encapsulated 0.01 and 0.075 mg/mL magnolol with cholesterol addition was measured by particle size analyzer (Coulter Beckman, N4 Submicron Particle Size Analyzer, USA). The results of the studies were plotted in a typical broken-line graph. 2.5.1. Preparation for TEM imaging Negative-staining of 2% uranyl acetate (Sigma, USA) was dissolved in distilled water. The ratio of the liposome with the stain was 1:1. The samples on copper grids were stained with uranyl acetate in dark at room temperature over night, which were then subjected to transmission electron microscopy (TEM) (JEM-2010, JEOL Co. Ltd., Japan) analysis. 3. Results and discussion 3.1. Standard curve of magnolol The calibration curve for magnolol was measured by UV– vis absorbance at a wavelength of 292 nm. A (absorbance) and C (magnolol concentration) gives an equation as follows: A ¼ 1:3573C þ 0:0006;
R2 ¼ 0:9841
This calibration curve was used to determine the extent and rate of drug encapsulation efficiency.
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Table 1 Encapsulated efficiency of with and without cholesterol liposomes (means S.D., n = 3)
EPC EPC+ cholesterol DPPC DPPC+ cholesterol
Absorbance at 292 nm
Free magnolol concentration (mg/mL)
Encapsulated efficiency (%)
0.027 0.002 0.025 0.004 0.037 0.003 0.033 0.002
19.4 1.47 18.0 2.95 26.8 2.21 23.9 1.47
74.13 1.97 76.03 3.93 64.26 2.92 68.17 1.97
3.2. Drug encapsulation efficiency Drug encapsulation efficiency was found to change depending on composition. Magnolol is easily encapsulated into liposomes with EPC and DPPC as shown in Table 1. Our current measurement demonstrated that the encapsulation efficiency was approximately over 60%. EPC liposome showed the higher encapsulation efficiency than DPPC liposomes. Moreover, the addition of cholesterol to both liposomes would slightly increase in encapsulation efficiency. 3.3. Effects of magnolol with or without liposome encapsulation on VSMCs The MTT assay demonstrated that pure DPPC and EPC did not affect the A10 cell viability as compared to 15% control. While pure magnolol significantly suppressed the cell viability, DPPC- and EPC-encapsulated magnolol showed a more potent inhibition on cell proliferation (Fig. 1). Our results further indicated that the inhibitory effects of the EPC-encapsulated magnolol in preventing VSMCs proliferation were stronger than magnolol encapsulated with DPPC. In our study, EPC- and DPPC-encapsulated magnolol added with cholesterol had worse inhibiting effect than those without cholesterol. However, addition of cholesterol in liposomes seems could not enhance the inhibiting effect. 3.4. Size of the EPC- and DPPC-encapsulated magnolol The TEM images showed the different sizes between EPCand DPPC-encapsulated magnolol (Fig. 2). The diameter in average of the pure EPC liposome was around 50 nm
Fig. 1. Inhibitory smooth muscle cells proliferation effect of magnolol encapsulated by EPC and DPPC liposomes. The cells were incubated in Dulbecco’s modified Eagle’s medium (DMEM) with 15% FBS. Each value represents the mean S.D. (n = 7). *P < 0.01 as compared to 15% FBS control group.
(Fig. 2(a)). The empty EPC liposome showed small unilamellar vesicles (SUVs) structure and was more homogeneous than the liposomes containing the magnolol. The particle size distribution of different components of liposomes was revealed in Fig. 3. The particle size of the EPC liposome containing 0.075 mg/mL magnolol showed a larger diameter than that of DPPC liposome containing 0.075 mg/mL magnolol. With the addition of the cholesterol, the particle size would be smaller than EPC- and DPPC-encapsulated magnolol. This study investigated the magnolol which encapsulated by EPC and DPPC liposomes could increase the inhibition of the proliferation of VSMCs than pure magnolol. Furthermore, the EPC liposomes-encapsulated magnolol had better inhibiting effect than DPPC lipsomes-encapsulated magnolol. According to our previous study, hexadecanol could enhance the flexibility of DPPC liposome. We suggested that the addition of cholesterol to EPC and DPPC also might enhance the flexibility of liposomes and reduce the leakage of magnolol. Therefore, EPC and DPPC added with cholesterol had worse inhibiting effect on VSMCs than pure liposomes. Addition of cholesterol could reduce the diameter of liposomes from our size distribution study. The inhibitory effect on the VSMCs was EPC encapsulated magnolol > DPPC encapsulated magnolol > magnolol. Interestingly, among the size distribution analysis, we found that the addition of cholesterol to EPC and DPPC had smaller mean size. According to these data, we suggested that the cholesterol could enhance the flexibility of the liposomes and could reduce the leakage of magnolol. The morphology of EPC and DPPC liposomes was homogeneous (Fig. 2). After adding magnolol to EPC and DPPC liposomes, the morphology of both complexes was changed heterogeneously. With the addition of cholesterol, the structure would also change from SUV to multi-lamellar liposomes (MLV). We presumed that MLV would encapsulate more magnolol in the lipid bilayer, hence the encapsulation efficiency of liposomes with cholesterol got higher than pure liposomes (Table 1). From our previous study, liposome was composed of saturated lipids would lead to higher transition temperatures (Tc) and more stable. In addition, Tc above body temperature would decrease the permeability of liposomes to cell membrane and the rate of leakage. With regard to inhibiting effect on VSMCs of liposomes Tc is a quite important evidence to explain the leakage of magnolol. In our study, DPPC had higher Tc than EPC and the leakage of magnolol would decrease. Consequently, the inhibiting effect on VSMCs of EPC liposome was better than DPPC liposome.
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Fig. 2. Microscopic analyses of various liposome treated with 2% uranyl acetate. (a) EPC liposomes, scale bar = 20 nm, (b) EPC/magnolol liposomes, scale bar = 100 nm, (c) EPC/magnolol/cholesterol liposome, scale bar = 100 nm, (d) DPPC liposomes, scale bar = 200 nm, (e) DPPC/magnolol liposomes, scale bar = 20 nm and (f) DPPC/magnolol/cholesterol liposome, scale bar = 100 nm.
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Fig. 3. Size distributions of EPC and DPPC liposomes measured by particle size analyzer.
4. Conclusion Overall, this study showed that the inhibitory effect of magnolol encapsulated by liposome was better than magnolol being treated alone. Furthermore, between these two liposomes of EPC and DPPC, EPC-encapsulated magnolol was more efficient than DPPC in our drug delivery system. According to our results, EPC not only had superior encapsulation efficiency to DPPC but also possessed better inhibitory effect on proliferation of smooth muscle cells. In conclusion, this study provides a novel drug delivery system for pharmacological intervention of restenosis after PTCA.
Acknowledgements This research was supported by grants from the National Science Council of China (NSC 94-2213-E-039-002) and China Medical University (CMU96-239, CMU96-178).
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Journal of the Chinese Institute of Chemical Engineers 39 (2008) 407–411 www.elsevier.com/locate/jcice
Inhibiting the vascular smooth muscle cells proliferation by EPC and DPPC liposomes encapsulated magnolol Calvin Yu-Chian Chen * Department of Biological Science and Technology, China Medical University, Taichung 404, Taiwan Received 19 January 2008; received in revised form 11 April 2008; accepted 14 April 2008
Abstract Percutaneous transluminal coronary angioplasty (PTCA) is a non-surgical modality for treating stennosis. However, the recurrence of restenosis in 30–50% patients within 6 months is the major drawback of PTCA. The major reason of restenosis is the proliferation of the vascular smooth muscle cells (VSMCs). Magnolol, a pure compound extracted from Magnolia officinalis, encapsulated by liposome was investigated for inhibiting the VSMCs proliferation leading to restenosis by PTCA. 1,2-Diacyl-Sn-glycero-3-phosphocholine (EPC) and 1,2dipalmitoyl-Sn-glycero-3-phosphocholine (DPPC) liposomes were utilized to encapsulate the magnolol. EPC liposome obtained the higher encapsulation efficiency than DPPC lipsomes from UV–vis spectroscopy study. The inhibiting efficiency of EPC and DPPC liposomes encapsulated magnolol was higher than pure magnonol. Magnolol encapsulated by EPC liposomes had better efficiency on inhibiting VSMCs than DPPC liposome. Addition of cholesterol in liposomes could slightly enhance the encapsulation efficiency. The particles sizer analysis revealed the average particles size of EPC and DPPC liposomes encapsulated magnolol became larger than pure EPC or DPPC liposomes. From the transmission electron microscopy (TEM) analysis, the magnolol seems to interfere with EPC and DPPC liposomes to form a homogeneous lipid bilayer. # 2008 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved. Keywords: Vascular smooth muscle cells; 1,2-Dipalmitoyl-Sn-glycero-3-phosphocholine; 1,2-Diacyl-Sn-glycero-3-phosphocholine; Magnolol; Liposome
1. Introduction Atherosclerosis is a progressing disease (Lusis, 2000). The etiology of this disease is mainly due to the high plasma level of cholesterol especially low-density lipoprotein (Walsh and Isner, 2000), with subsequent oxidization resulting in atheroma formation on the aorta. The pathological process of atherosclerosis consists of chronic inflammation, and excessive formation of foam cells in the subendothelium space (Schwartz et al., 1993), resulting in outgrowth and abnormal migration of the smooth muscle cell in the blood vessel (Song et al., 2004), as well as fibrosis and calcification of the blood vessel (Chew, 2005; Lafont et al., 1995). According to the statistics, about 30– 50% of atherosclerotic patients suffered from restenosis in half year after PTCA treatment (Currier and Faxon, 1995; Farb et al., 1999; Rogers et al., 1996).
* Tel.: +886 4 22053366x2506. E-mail address: [email protected].
Magnolol is a commonly used Chinese herbal medicine known as Hou Po (Allen et al., 2006). Magnolia is a prescribed Chinese medicine isolated from Magnolia officinalis for many treatments (Lee et al., 2001; Wu et al., 2007). Magnolol had been reported to possess two kinds of different functions (Kuntz and Baim, 1993; Serruys et al., 1993). It could inhibit the cell growth at lower concentration, whereas induce cell apoptosis at higher concentration (Chen et al., 2003; Hong et al., 1996). Therefore, magnolol was expected as a pharmacological approach for restenosis by inhibiting vascular smooth muscle cells (VSMCs) proliferation (Wu et al., 2002). Liposome is used for drug delivery system due to its unique structure properties (Kuo and Chung, 2005). Liposome can carry both the hydrophobic and hydrophilic drug (Liu et al., 2006). Therefore, liposome as a drug carrier can indiscriminately deliver drugs through the cell membrane (Lin et al., 2007; Liu et al., 2000). In this study, liposome-encapsulated magnolol, especially EPC liposome, might be more efficient than bare magnolol in preventing proliferation of VSMCs, a key contributor to restenosis induced by PTCA.
0368-1653/$ – see front matter # 2008 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jcice.2008.04.005
408
C.Y.-C. Chen / Journal of the Chinese Institute of Chemical Engineers 39 (2008) 407–411
2. Materials and methods 2.1. Cell culture A10 vascular smooth muscle cells derived from rat thoracic aorta were obtained from Food Industry Research and Development Institute (Hsinchu, Taiwan). The cells were cultured in 10-cm dishes (TPP, Switzerland) containing Dulbecco’s modified eagle’s medium (DMEM) (Sigma, USA) supplemented with 10% FBS (Gibco, Carlsbad, CA, USA), 1.5 g/L NaHCO3 (Sigma, USA), 1.028 g/L N-acetyl L-alanyl-L-glutamine (Biochrom, Berlin), 1% Na-pyruvate (Sigma, USA), 4.5 g/L D-glucose (Sigma, USA), 100 units/ mL penicillin G (Sigma, USA), and 100 mg/mL streptomycin sulfates (Gibco,Carlsbad, CA, USA). Cells were grown in a humidified incubator with 5% CO2 at 37 8C. The culture medium was replaced every 2–3 days and the cells were passaged every week. In the last passage, the same numbers of cells were subcultured to 96-well plates. The cells that became at least 80% confluent were starved for 24 h in DMEM containing 0.5% FBS followed by a treatment with 0.01 and 0.075 mg/mL of magnolol (95% (HPLC), Sigma, USA) in DMEM containing 15% FBS for 24 h. 2.2. Preparation of liposome Magnolol (M3445, >98%, HPLC), a phenolic compound isolated from a Chinese herbal drug, M. officinalis, was purchased from Sigma Chemical Company. 1,2-DipalmitoylSn-glycero-3-phosphocholine (DPPC) (>99%pure) and 1,2diacyl-Sn-glycero-3-phosphocholine from egg yolk (EPC) (99% pure) were purchased from Sigma Chemical Company, USA and were used as-received. Ethanol (99.5% pure) was supplied by Seoul Chemical Industry Co. Ltd., Korea, and HPLC grade hexane (>99% pure) was obtained from Ferak Laborat GmbH, Germany. Cholesterol was supplied by Sigma Chemical Company, USA. The mixed liposomes (DPPC/magnolol, DPPC/magnolol/cholesterol, EPC/magnolol, EPC/magnolol/cholesterol) were dissolved in ethanol/ hexane (1:9, v/v) solvent stored at 4 8C in a round-bottomed flask. All the lipid/cholesterol ratios of liposomes were prepared with molar ratio 1:1. Before use, all glassware was exhaustively rinsed with purified water. The flask was then taken to dryness under the rotary evaporator to ensure all the organic solvent had evaporated. The mixed liposome was dispersed in 15% FBS medium magnolol and then added into the round-bottomed flask. To form the nano-scale liposome, the flask was sonicated by ultrasonic facilitation (DC-150H, Delta1, Taiwan) for 2 h to encapsulate the magnolol. 2.3. MTT assay In the MTT assay, the cells were seeded on 24-well plates at the concentration of approximately 5000 cells/cm2 before the drug treatment. The MTT assay was implemented 24 h after the addition of liposome-encapsulated magnolol. Cells were seeded in 96-well plates at a density of 1 104 cells per well
in 200 mL of DMEM/10% FBS and incubated for 1 day. Afterwards, cells were treated with 0.01 and 0.075 mg/mL of magnolol added in DMEM with 15% FBS. After 1 day of drug treatment, the effect on cell growth was examined by the MTT assay. After adding 130 mL of 0.5 mg/mL MTT (3-[4,5dimethyl thiazol-2-yl]-2,5-diphenyl tetrazolium bromide) (Sigma Chemical Co., St. Louis, MO) to each well, the mixtures were incubated for 4 h at 37 8C. The supernatant was aspirated and the MTT–formazan crystals formed by metabolically viable cells were dissolved in 130 mL of DMSO (DMSO; dimethylsulphoxide, Sigma, USA). Finally, the absorbance was measured by a microplate reader at a wavelength of 590 nm. The statistical significance of the data was evaluated with Student’s t-test. P-value of 0.05 and 0.01 were considered significant. 2.4. Drug encapsulation efficiency The content of magnolol in liposomes was measured by UV– vis spectroscopy (Bio-RAD 31, USA) at a fixed wavelength of 292 nm. The precipitated lipid was separated by centrifugation (19,000 g for 60 min). The absorbance of supernatant was then measured at 292 nm. Non-encapsulation drug was compared with the standard curve of magnolol, which was achieved from magnolol solutions in PBS with concentration between 0.0001 and 0.08 mg/mL. 2.5. Size analysis of liposome encapsulated magnolol EPC-encapsulated 0.01 and 0.075 mg/mL magnolol with cholesterol addition was measured by particle size analyzer (Coulter Beckman, N4 Submicron Particle Size Analyzer, USA). The results of the studies were plotted in a typical broken-line graph. 2.5.1. Preparation for TEM imaging Negative-staining of 2% uranyl acetate (Sigma, USA) was dissolved in distilled water. The ratio of the liposome with the stain was 1:1. The samples on copper grids were stained with uranyl acetate in dark at room temperature over night, which were then subjected to transmission electron microscopy (TEM) (JEM-2010, JEOL Co. Ltd., Japan) analysis. 3. Results and discussion 3.1. Standard curve of magnolol The calibration curve for magnolol was measured by UV– vis absorbance at a wavelength of 292 nm. A (absorbance) and C (magnolol concentration) gives an equation as follows: A ¼ 1:3573C þ 0:0006;
R2 ¼ 0:9841
This calibration curve was used to determine the extent and rate of drug encapsulation efficiency.
C.Y.-C. Chen / Journal of the Chinese Institute of Chemical Engineers 39 (2008) 407–411
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Table 1 Encapsulated efficiency of with and without cholesterol liposomes (means S.D., n = 3)
EPC EPC+ cholesterol DPPC DPPC+ cholesterol
Absorbance at 292 nm
Free magnolol concentration (mg/mL)
Encapsulated efficiency (%)
0.027 0.002 0.025 0.004 0.037 0.003 0.033 0.002
19.4 1.47 18.0 2.95 26.8 2.21 23.9 1.47
74.13 1.97 76.03 3.93 64.26 2.92 68.17 1.97
3.2. Drug encapsulation efficiency Drug encapsulation efficiency was found to change depending on composition. Magnolol is easily encapsulated into liposomes with EPC and DPPC as shown in Table 1. Our current measurement demonstrated that the encapsulation efficiency was approximately over 60%. EPC liposome showed the higher encapsulation efficiency than DPPC liposomes. Moreover, the addition of cholesterol to both liposomes would slightly increase in encapsulation efficiency. 3.3. Effects of magnolol with or without liposome encapsulation on VSMCs The MTT assay demonstrated that pure DPPC and EPC did not affect the A10 cell viability as compared to 15% control. While pure magnolol significantly suppressed the cell viability, DPPC- and EPC-encapsulated magnolol showed a more potent inhibition on cell proliferation (Fig. 1). Our results further indicated that the inhibitory effects of the EPC-encapsulated magnolol in preventing VSMCs proliferation were stronger than magnolol encapsulated with DPPC. In our study, EPC- and DPPC-encapsulated magnolol added with cholesterol had worse inhibiting effect than those without cholesterol. However, addition of cholesterol in liposomes seems could not enhance the inhibiting effect. 3.4. Size of the EPC- and DPPC-encapsulated magnolol The TEM images showed the different sizes between EPCand DPPC-encapsulated magnolol (Fig. 2). The diameter in average of the pure EPC liposome was around 50 nm
Fig. 1. Inhibitory smooth muscle cells proliferation effect of magnolol encapsulated by EPC and DPPC liposomes. The cells were incubated in Dulbecco’s modified Eagle’s medium (DMEM) with 15% FBS. Each value represents the mean S.D. (n = 7). *P < 0.01 as compared to 15% FBS control group.
(Fig. 2(a)). The empty EPC liposome showed small unilamellar vesicles (SUVs) structure and was more homogeneous than the liposomes containing the magnolol. The particle size distribution of different components of liposomes was revealed in Fig. 3. The particle size of the EPC liposome containing 0.075 mg/mL magnolol showed a larger diameter than that of DPPC liposome containing 0.075 mg/mL magnolol. With the addition of the cholesterol, the particle size would be smaller than EPC- and DPPC-encapsulated magnolol. This study investigated the magnolol which encapsulated by EPC and DPPC liposomes could increase the inhibition of the proliferation of VSMCs than pure magnolol. Furthermore, the EPC liposomes-encapsulated magnolol had better inhibiting effect than DPPC lipsomes-encapsulated magnolol. According to our previous study, hexadecanol could enhance the flexibility of DPPC liposome. We suggested that the addition of cholesterol to EPC and DPPC also might enhance the flexibility of liposomes and reduce the leakage of magnolol. Therefore, EPC and DPPC added with cholesterol had worse inhibiting effect on VSMCs than pure liposomes. Addition of cholesterol could reduce the diameter of liposomes from our size distribution study. The inhibitory effect on the VSMCs was EPC encapsulated magnolol > DPPC encapsulated magnolol > magnolol. Interestingly, among the size distribution analysis, we found that the addition of cholesterol to EPC and DPPC had smaller mean size. According to these data, we suggested that the cholesterol could enhance the flexibility of the liposomes and could reduce the leakage of magnolol. The morphology of EPC and DPPC liposomes was homogeneous (Fig. 2). After adding magnolol to EPC and DPPC liposomes, the morphology of both complexes was changed heterogeneously. With the addition of cholesterol, the structure would also change from SUV to multi-lamellar liposomes (MLV). We presumed that MLV would encapsulate more magnolol in the lipid bilayer, hence the encapsulation efficiency of liposomes with cholesterol got higher than pure liposomes (Table 1). From our previous study, liposome was composed of saturated lipids would lead to higher transition temperatures (Tc) and more stable. In addition, Tc above body temperature would decrease the permeability of liposomes to cell membrane and the rate of leakage. With regard to inhibiting effect on VSMCs of liposomes Tc is a quite important evidence to explain the leakage of magnolol. In our study, DPPC had higher Tc than EPC and the leakage of magnolol would decrease. Consequently, the inhibiting effect on VSMCs of EPC liposome was better than DPPC liposome.
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Fig. 2. Microscopic analyses of various liposome treated with 2% uranyl acetate. (a) EPC liposomes, scale bar = 20 nm, (b) EPC/magnolol liposomes, scale bar = 100 nm, (c) EPC/magnolol/cholesterol liposome, scale bar = 100 nm, (d) DPPC liposomes, scale bar = 200 nm, (e) DPPC/magnolol liposomes, scale bar = 20 nm and (f) DPPC/magnolol/cholesterol liposome, scale bar = 100 nm.
References
Fig. 3. Size distributions of EPC and DPPC liposomes measured by particle size analyzer.
4. Conclusion Overall, this study showed that the inhibitory effect of magnolol encapsulated by liposome was better than magnolol being treated alone. Furthermore, between these two liposomes of EPC and DPPC, EPC-encapsulated magnolol was more efficient than DPPC in our drug delivery system. According to our results, EPC not only had superior encapsulation efficiency to DPPC but also possessed better inhibitory effect on proliferation of smooth muscle cells. In conclusion, this study provides a novel drug delivery system for pharmacological intervention of restenosis after PTCA.
Acknowledgements This research was supported by grants from the National Science Council of China (NSC 94-2213-E-039-002) and China Medical University (CMU96-239, CMU96-178).
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