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Effects of cyclodextrins on the structure of LDL and its susceptibility to copper-induced oxidation
PHASCI-03563; No of Pages 7 European Journal of Pharmaceutical Sciences xxx (2016) xxx–xxx
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Effects of cyclodextrins on the structure of LDL and its susceptibility to copper-induced oxidation Meiying Ao a,b,c, Chaoye Gan a,c, Wenxiang Shao d, Xing Zhou a,c, Yong Chen a,c,⁎ a
Nanoscale Science and Technology Laboratory, Institute for Advanced Study, Nanchang University, Nanchang, Jiangxi 330031, PR China Department of Pharmacy, Science and Technology College, Jiangxi University of Traditional Chinese Medicine, Nanchang, Jiangxi 330025, PR China College of Life Sciences, Nanchang University, Nanchang, Jiangxi 330031, PR China d School of Basic Medical Sciences, Jiangxi University of Traditional Chinese Medicine, Nanchang, Jiangxi 330025, PR China b c
a r t i c l e
i n f o
Article history: Received 8 December 2015 Received in revised form 28 April 2016 Accepted 28 April 2016 Available online xxxx Keywords: Cyclodextrin Atherosclerosis Low-density lipoprotein (LDL) Atomic force microscopy (AFM) LDL oxidation
a b s t r a c t Cyclodextrins (CDs) have long been widely used as drug/food carriers and were recently developed as drugs for the treatment of diseases (e.g. Niemann-Pick C1 and cancers). It is unknown whether cyclodextrins may influence the structure of low-density lipoprotein (LDL), its susceptibility to oxidation, and atherogenesis. In this study, four widely used cyclodextrins including α-CD, γ-CD, and two derivatives of β-CD (HPβCD and MβCD) were recruited. Interestingly, agarose gel electrophoresis (staining lipid and protein components of LDL with Sudan Black B and Coomassie brilliant blue, respectively but simultaneously) shows that cyclodextrins at relatively high concentrations caused disappearance of the LDL band and/or appearance of an additional protein-free lipid band, implying that cyclodextrins at relatively high concentrations can induce significant electrophoresisdetectable lipid depletion of LDL. Atomic force microscopy (AFM) detected that MβCD (as a representative of cyclodextrins) induced size decrease of LDL particles in a dose-dependent manner, further confirming the lipid depletion effects of cyclodextrins. Moreover, the data from agarose gel electrophoresis, conjugated diene formation, MDA production, and amino group blockage of copper-oxidized LDL show that cyclodextrins can impair LDL susceptibility to oxidation. It implies that cyclodextrins probably help to inhibit atherogenesis by lowering LDL oxidation. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Cyclodextrins (CDs) are a family of cyclic oligosaccharides, consisting of six (α-cyclodextrin or α-CD), seven (β-cyclodextrin or β-CD), eight (γ-cyclodextrin or γ-CD) or more α-(1,4)-linked glucopyranose subunits. Due to their specific structure of a lipophilic internal cavity surrounded by hydrophilic outer surface, cyclodextrins can be used as potent carriers by forming water-soluble complexes with many water-insoluble compounds, and have been intensively studied and widely applied in pharmaceuticals (Loftsson et al., 2005; Loftsson and Duchene, 2007; Zhang and Ma, 2013; Lakkakula and Krause, 2014), foods (Astray et al., 2009), and others (Singh et al., 2002; Del Valle, 2004). Although when cyclodextrin-containing pharmaceutical products are administered orally only small amounts of cyclodextrins can permeate gastrointestinal mucosa, cyclodextrins or their derivatives in many marketed parenteral formulations (Loftsson and Duchene, 2007) can enter the blood circulation or specific organs at relatively high ⁎ Corresponding author at: 999 Xuefu Ave., Honggutan District, Nanchang, Jiangxi 330031, PR China. E-mail address: [email protected] (Y. Chen).
concentrations. Cyclodextrins will be available to some blood components after delivered drugs are released. On the other hand, 2hydroxypropyl-β-cyclodextrin (2-HPβCD) (Davidson et al., 2009; Ramirez et al., 2010; Taylor et al., 2012; Crumling et al., 2012; Lopez et al., 2014) and methyl-β-cyclodextrin (MβCD) (Mohammad et al., 2014; Gotoh et al., 2014), two derivatives of β-CD, have been applied to treat some diseases (e.g. Niemann-Pick C1 and cancers) by direct injection into animal bloodstream. Therefore, it will be interesting and meaningful to know the effects of cyclodextrins on blood components, such as low-density lipoproteins (LDL), a major group of plasma lipoproteins transporting fats (mainly cholesterol) to cells. It has been widely accepted that the oxidation of LDL plays a vital role in the pathogenesis of atherosclerosis (Steinberg et al., 1989; Witztum and Steinberg, 1991; Aviram, 1993) and oxidized LDL (oxLDL) in circulation is an important risk marker for human cardiovascular diseases (Toshima et al., 2000; Holvoet et al., 2001; Mertens et al., 2001). Many factors may influence LDL susceptibility to oxidation including antioxidants, total lipid composition, fatty acids, and cholesterol (Kontush et al., 1996; Frei and Gaziano, 1993; Mosinger, 1995; Spranger et al., 1998; Mosinger, 1999; Balkan et al., 2004). Considering this, as well as the ability of cyclodextrins to form water-soluble complexes with lipids and the lipid-enriched structure of LDL, we hypothesize
http://dx.doi.org/10.1016/j.ejps.2016.04.037 0928-0987/© 2016 Elsevier B.V. All rights reserved.
Please cite this article as: Ao, M., et al., Effects of cyclodextrins on the structure of LDL and its susceptibility to copper-induced oxidation, European Journal of Pharmaceutical Sciences (2016), http://dx.doi.org/10.1016/j.ejps.2016.04.037
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Before oxidation, EDTA was removed from the LDL solutions (Cat. No. YB-001; Yiyuan Biotechnologies, Guangzhou, China; isolated from human plasma). For cyclodextrin treatments, 0.2 mg/ml LDL solutions were incubated with different concentrations of cyclodextrins (SigmaAldrich; the catalog numbers of α-CD, γ-CD, MβCD, and HPβCD are C4642, 779660, C4555, and H107, respectively) in PBS for 1 h at 37 °C. For LDL oxidation, native LDL and LDL treated with cyclodextrins were directly mixed with copper sulphate (99.0% purity; Jinshanting New Chemical Reagent Factory, Shanghai, China) at a final copper concentration of 5 μM in PBS and incubated at 37 °C. For the oxidation of phosphotidylcholine (PC), PC (Lanji Science and Technology Development Ltd., Shanghai, China) at a final concentration of 1.6 mg/ml in PBS was used. The MβCD treatment and copper oxidation of PC were similar to LDL samples. 2.2. Agarose gel electrophoresis
Fig. 1. Cyclodextrins causes depletion of lipids from native LDL detected by agarose gel electrophoresis. Left panel: the gels stained with Sudan Black B; right panel: the gels loading the same samples but stained with Coomassie brilliant blue. (A, B) The samples treated with or without γ-CD (9 and 18 mM, respectively) or α-CD (7.5 and 15 mM, respectively). (C, D) The samples treated with HPβCD (10, 40, 100, and 200 mM, respectively). (E, F) The samples treated with MβCD (10, 20, 40, and 100 mM, respectively). The first lanes (marked with “—”) of all gels represent the native LDL samples without cyclodextrin treatment.
that cyclodextrins can deplete lipids from native LDL and affect LDL susceptibility to oxidation. In this study, the effects of four widely used cyclodextrins including α-CD, γ-CD, HPβCD, and MβCD on the structure of native LDL and the susceptibility of LDL to copper-induced oxidation were evaluated to test the abovementioned hypothesis.
2. Materials and methods 2.1. Cyclodextrin treatment and LDL oxidation The water solubility of cyclodextrins is approximately 2%, 13%, and 26% weight by weight for β-CD, α-CD, and γ-CD, respectively (Davis and Brewster, 2004). Under our experimental conditions, 15 mM αCD and 18 mM γ-CD reached the highest solubility, respectively. Since β-CD is hard to be dissolved, methylated and 2-hydroxypropylated derivatives (i.e. HPβCD and MβCD, respectively) with significantly improved water solubility were utilized. The highest final concentration of HPβCD/MβCD used in this study (200 and 100 mM, respectively) did not reach their highest solubility yet.
Agarose gel electrophoresis was performed on 0.5% agarose gels in sodium barbital buffer using the electrophoretic system. Samples were electrophoresed in 0.075 M sodium barbital buffer at 55 V for 1 h. Relative electrophoretic mobility (REM) was calculated as the ratio of the migration distance of oxidized LDL to that of native LDL. To simultaneously determine the lipid and protein components of native LDL treated with or without cyclodextrins, two gels loading the same samples were run in the same electrophoresis chamber but stained with Sudan Black B for lipids and Coomassie brilliant blue R-250 for proteins, respectively. Agarose gel electrophoresis of cholesterol (Sigma-Aldrich) or PC treated with or without MβCD/copper was performed similarly. 2.3. Isolation and measurement of depleted lipids LDL was treated with MβCD at different concentrations. The Amicon Ultra-0.5 ml, 10 kDa Centrifugal Filter Unit (Merck Millipore) was utilized to remove the LDL remnants and obtain the depleted lipids according to the user's manual. The depleted cholesterols including free and esterified cholesterols were measured via LDL-C Kit (Biosino BioTechnology and Science Inc., Beijing, China) according to the user's manual. The absorbance of cholesterols or phospholipids was measured at 550 and 240 nm, respectively, by using a UV-5100 spectrophotometer (Metash Instruments, Shanghai, China). 2.4. Atomic force microscopy (AFM) The mica functionalized with aminopropyltriethoxysilane (APTES) and glutaraldehyde (GA) and the LDL samples treated with or without MβCD were prepared and imaged by an Asylum MFP-3D-SA AFM (Asylum Research, USA) in tapping mode in PBS as previously described (Gan et al., 2015). The silicon nitride tips (AppNano, USA) with an end radius of 10 nm and a spring constant of ~0.04 N/m were utilized. The full width at half maximum (FWHM; the radius r is half of the measured
Fig. 2. MβCD causes the concentration-dependent increases in the lipids drawn from LDL. After MβCD treatments, the LDL remnants were filtered by a 10 kDa Centrifugal Filter Unit, and the contents of the depleted lipids were measured. The percentage of (A) phospholipids or (B) cholesterols (including free and esterified cholesterols) drawn from LDL significantly increased with the increase of MβCD concentration. (C) Agarose gel electrophoresis of pure lipids. Lanes 1–4: LDL, free cholesterol + phosphotidylcholine (PC) + MβCD, free cholesterol + MβCD, and PC alone, respectively.
Please cite this article as: Ao, M., et al., Effects of cyclodextrins on the structure of LDL and its susceptibility to copper-induced oxidation, European Journal of Pharmaceutical Sciences (2016), http://dx.doi.org/10.1016/j.ejps.2016.04.037
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Fig. 3. MβCD induces the size decrease of LDL particles in a concentration-dependent manner detected by AFM. (A–F) AFM topographical images of bare mica, modified mica, native LDL particles, and LDL particles treated with MβCD (10, 40, and 100 mM, respectively), respectively. Scan size: 1 μm × 1 μm. (G) Height profiles of horizontal cross sections randomly across the topographical images in A–F. (H) Average volumes of LDL particles treated with or without MβCD (*, P b 0.05; ***, P b 0.001).
FWHM) and height (h) of LDL particles were measured to calculate the volume (V) of a single LDL particle using the following equation (Schneider et al., 1995): 2 V ¼ ðπ h=6Þ 3r 2 þ h :
2.6. Statistical analysis Student t-test was performed to determine the significance between different groups. All data were from at least three independent experiments. A value of P b 0.05 was considered statistically significant (*, P b 0.05; **, P b 0.01; ***, P b 0.001 in graphs).
3. Results and discussion 2.5. Measurements of conjugated dienes, malondialdehyde (MDA), and free amino groups
3.1. Cyclodextrins induce lipid depletion of native LDL
During LDL oxidation, conjugated diene formation was measured at 20-min intervals for 220 min at 37 °C. MDA production and remaining free amino groups at 18 h after oxidation were measured by TBARS Assay Kit and TNBSA Assay Kit, respectively according to the user's manuals. The blockage of amino groups was calculated by comparing the remaining free amino groups in the oxidized product to those of the un-oxidized control. The absorbance of each sample was measured at 234, 532, and 335 nm, respectively, by using the UV-5100 spectrophotometer for conjugated dienes, MDA, and free amino groups, respectively.
Prior to investigating the effects of cyclodextrins on LDL susceptibility to oxidation, the effects of cyclodextrins on the structure of native LDL were studied firstly by agarose gel electrophoresis. To simultaneously determine the lipid and protein components of native LDL treated with or without cyclodextrins, Sudan Black B (for lipids; left panel of Fig. 1) and Coomassie brilliant blue R-250 (for proteins; right panel of Fig. 1) were utilized to stain two gels loading the same samples and running in the same electrophoresis chamber. In Fig. 1A, γ-CD (both 9 and 18 mM) and 7.5 mM α-CD did not significantly influence the electrophoretic mobility of native LDL. However,
Please cite this article as: Ao, M., et al., Effects of cyclodextrins on the structure of LDL and its susceptibility to copper-induced oxidation, European Journal of Pharmaceutical Sciences (2016), http://dx.doi.org/10.1016/j.ejps.2016.04.037
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Fig. 4. Cyclodextrins inhibit the electrophoretic mobility of copper-oxidized LDL in a concentration-dependent manner. (A–C) The LDL samples treated with or without γ-CD/α-CD were oxidized by copper for 2, 10, and 18 h, respectively. (D–F) The LDL samples treated with or without HPβCD were oxidized by copper for 2, 8, and 18 h, respectively. (G–I) The LDL samples treated with or without MβCD were oxidized by copper for 2, 4, and 18 h, respectively. The first and second Lanes in A–I are native LDL and copper-oxidized LDL without cyclodextrin treatment, respectively. The gels in A–I were stained with Sudan Black B (The insets in E and H are stained with Coomassie brilliant blue). The arrows in E and H show the additional protein-free bands. (J–L) Calculated relative electrophoretic mobility (REM) of native or oxidized LDL treated with or without cyclodextrins. Treatments from left to right: γ-CD/α-CD, HPβCD, and MβCD, respectively.
15 mM α-CD caused a faint lipid band of native LDL (indicated by the arrow). Fig. 1B shows that 15 mM α-CD induced the disappearance of the protein band at the location of native LDL (indicated by the arrow) but the appearance of a protein band at the location of the origin (indicated by the arrowhead). It implies that 15 mM α-CD caused a significant separation between lipid and protein components of native LDL. Fig. 1C and Fig. 1E show that HPβCD and MβCD induced the broadening of the lipid band of native LDL and that at relatively high concentrations they (200 mM HPβCD and 40 mM MβCD) caused an additional lipid band (indicated by the arrowheads) which was protein-free as indicated by the arrowheads in Fig. 1D and Fig. 1F. MβCD at much higher concentrations (e.g. 100 mM) even caused the same effect as 15 mM α-CD (i.e. faint lipid and protein bands at the location of native LDL as indicated by the arrows in Fig. 1E, 1F). These data imply that both
HPβCD and MβCD induced a concentration-dependent separation between lipid and protein components of native LDL. To confirm the implication, the changes in the content of the depleted lipids from LDL were monitored. We selected MβCD as a representative since the effects of MβCD on the electrophoretic mobility of native LDL cover all effects of the other cyclodextrins as mentioned above. As showed in Fig. 2A,B, the percentage of the depleted lipids (e.g. phospholipid and cholesterol) increased significantly with the increase of MβCD concentration whereas no depleted proteins were detected (data not shown). Agarose gel electrophoresis (Fig. 2C) showed that both cholesterol alone (unable to be stained by Sudan Black B; not shown) and cholesterol- cyclodextrin complex (lane 3) were retained in the loading tank without electrophoretic mobility due to no net charges of both
Please cite this article as: Ao, M., et al., Effects of cyclodextrins on the structure of LDL and its susceptibility to copper-induced oxidation, European Journal of Pharmaceutical Sciences (2016), http://dx.doi.org/10.1016/j.ejps.2016.04.037
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cholesterol and MβCD whereas phosphotidylcholine (PC) alone (lane 4) could be stained faintly and formed a continuous band, a part of which even has a faster electrophoretic mobility than native LDL (lane 1). Interestingly, the cholesterol-PC-MβCD mixture (lane 2) has a slower electrophoretic band than LDL which is similar to the additional proteinfree lipid band of LDL treated by MβCD or HPβCD. Probably, two or more MβCD molecules formed complexes simultaneously with both cholesterol and PC. Then, we still selected MβCD as a representative to investigate the effects of cyclodextrins on the particle size of native LDL by using atomic force microscopy (AFM). AFM imaged the very smooth surfaces of the bare mica (Fig. 3A) and the mica modified with APTES plus glutaraldehyde (Fig. 3B). When native LDL was immobilized onto the smooth surfaces of modified mica, many nano-sized particles were imaged in PBS by AFM (Fig. 3C). After treated with 10, 40, and 100 mM MβCD, respectively, LDL particles became smaller gradually (Fig. 3D–G). Statistical analysis shows that 10, 40, and 100 mM MβCD induced ~10%, ~60%, and ~80% decreases in average volume of individual LDL particles, respectively (Fig. 3H). Therefore, cyclodextrins at a relatively high concentration caused the disappearance of native LDL band and/or the appearance of an additional protein-free band during agarose gel electrophoresis (Fig. 1), obviously implying that cyclodextrins at a relatively high concentration depleted lipids from native LDL. The concentration-dependent decrease in particle size of native LDL as detected by AFM (Fig. 3) further confirms the lipid-depleting effects of cyclodextrins. Cyclodextrins at lower concentrations probably also caused lipid depletion of native LDL since the concentration-dependent increase in percentage of the lipids depleted by cyclodextrins from LDL were detected. Moreover, AFM detected ~ 10% decrease in average volume of individual LDL particles treated with MβCD at a low concentration (10 mM) although an additional protein-free lipid band was undetectable by agarose gel electrophoresis due to relatively small amounts of depleted lipids. Taken together, the agarose gel electrophoretic data and the AFM data imply that cyclodextrins can induce lipid depletion and size decrease of native LDL in a concentration-dependent manner. It can also be supported by the facts that the three types of cyclodextrins are efficient in extracting cholesterol and/or phospholipids from cellular and model membrane (Ohtani et al., 1989; Ohvo and Slotte, 1996; Leventis and Silvius, 2001; Zidovetzki and Levitan, 2007; Wu et al., 2014; Mahammad and Parmryd, 2015) and that LDL lipids are mainly consisted of cholesterol and phospholipids. For the first time, we provide the evidence of the cyclodextrin effects on the structure of native LDL. 3.2. Cyclodextrins reduce LDL susceptibility to copper-induced oxidation Next, we investigated the effects of cyclodextrins on LDL susceptibility to copper-induced oxidation firstly via agarose gel electrophoresis (Fig. 4). Fig. 4A–C, Fig. 4D–F, and Fig. 4G–I display the effects of α-CD/ γ-CD, HPβCD, and MβCD on LDL oxidation, respectively. Obviously, γCD, HPβCD, and MβCD inhibited the electrophoretic mobility of copper-oxidized LDL in a concentration-dependent manner. The quantitative analyses of the relative electrophoretic mobility (Fig. 4J–L) further confirmed the results (the 15 mM α-CD group in Fig. 4J and the 100 mM MβCD group in Fig. 4L were not quantified due to the faintness of their LDL bands and the difficulty of accurate measurement). The insets in Fig. 4E and Fig. 4H present the corresponding gels stained by Coomassie brilliant blue showing the additional protein-free lipid bands of oxidized LDL (indicated by the arrows) caused by relatively high concentrations of HPβCD/MβCD. Subsequently, the dynamic accumulation of conjugated dienes during a period of 220 min (Fig. 5) was evaluated to determine the effects of cyclodextrins on the copper-induced oxidation of LDL lipids. Without cyclodextrin treatment copper alone induced a fast and dramatic increase in conjugated diene formation (the dark lines in Fig. 5A–C) as
Fig. 5. Cyclodextrins lower conjugated diene formation of LDL during copper-induced oxidation in a concentration-dependent manner. (A) γ-CD or α-CD treatments. (B) HPβCD treatments. (C) MβCD treatments. The samples were measured at 20-min intervals for 220 min from the beginning of oxidation.
usual. In a concentration-dependent manner the four cyclodextrins (Fig. 5), particularly HPβCD (Fig. 5B) and MβCD (Fig. 5C), caused lower conjugated dienes when reaching the plateau although the process of conjugated diene formation was faster. The cyclodextrin-induced lipid depletion might cause a temporal loosening of LDL structure under which condition copper could more easily access the targets therefore causing a faster process of conjugated diene formation. On the other hand, the cyclodextrin-induced lipid depletion decreased oxidizable lipid sources, therefore causing a lower final conjugated diene formation. Finally, malondialdehyde (MDA) production (Fig. 6) and blocked amino groups (Fig. 7) were measured to further determine the effects of cyclodextrins on the copper-induced oxidation of LDL lipids and
Please cite this article as: Ao, M., et al., Effects of cyclodextrins on the structure of LDL and its susceptibility to copper-induced oxidation, European Journal of Pharmaceutical Sciences (2016), http://dx.doi.org/10.1016/j.ejps.2016.04.037
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Fig. 6. Cyclodextrins impair malondialdehyde (MDA) production of LDL during copper-induced oxidation in a concentration-dependent manner. From left to right: γ-CD/α-CD, HPβCD, and MβCD treatments, respectively. The samples at the 18 h time point of oxidation were measured (*, P b 0.05; **, P b 0.01; ***, P b 0.001).
Fig. 7. Cyclodextrins decrease amino group blockage of LDL proteins during copper-induced oxidation in a concentration-dependent manner. From left to right: γ-CD/α-CD, HPβCD, and MβCD treatments, respectively. The samples at the 18 h time point of oxidation were measured (*, P b 0.05; **, P b 0.01; ***, P b 0.001).
proteins, respectively. Without cyclodextrin treatment copper alone induced a dramatic increase in both MDA production and blocked amino groups of LDL 18 h later after oxidation. Significantly, each of the four cyclodextrins inhibited MDA production and amino group blockage of the copper-oxidized LDL in a concentration-dependent manner. Interestingly, 10 mM HPβCD induced a statistically significant decrease in MDA production but not amino group blockage whereas 10 mM MβCD induced a statistically significant decrease in amino group blockage but not MDA production. It implies that HPβCD and MβCD might influence LDL oxidation potentially by different mechanisms which are currently unclear. The data from agarose gel electrophoresis (Fig. 8A) and TBARS assay (Fig. 8B) showed the effects of MβCD on the oxidation of pure PC molecules. Clearly or significantly, copper induced the increase in
electrophoretic mobility and MDA production of PC whereas MβCD treatments have no significant effects on PC oxidation. The data implies that the cyclodextrin-bound lipids depleted from LDL could not be one of the factors responsible for the inhibitory effects of cyclodextrins on LDL oxidation. Taken together, all results from agarose gel electrophoresis, conjugated diene formation, MDA production, and amino group blockage indicate that the four types of cyclodextrins can reduce LDL susceptibility to copper-induced oxidation in a concentration-dependent manner. The cyclodextrin-induced concentration-dependent lipid depletion of native LDL should be responsible since it is well known that total lipid composition, fatty acids, cholesterol and others can significantly influence LDL susceptibility to oxidation (Kontush et al., 1996; Frei and Gaziano, 1993; Mosinger, 1995; Spranger et al., 1998; Mosinger, 1999). Cyclodextrins
Fig. 8. MβCD has no significant effects on the copper-induced oxidation of pure PCs. (A) Agarose gel electrophoresis. PC alone was stained faintly by Sudan Black B whereas PC-MβCD complexes can be stained relatively strongly. (B) TBARS assay detects MDA production of PCs. The oxidized samples were oxidized by copper for 18 h.
Please cite this article as: Ao, M., et al., Effects of cyclodextrins on the structure of LDL and its susceptibility to copper-induced oxidation, European Journal of Pharmaceutical Sciences (2016), http://dx.doi.org/10.1016/j.ejps.2016.04.037
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caused significantly inhibitory effects on LDL oxidation probably due to the lipid depletion-caused loss of lipid hydroperoxide (LOOH) which is involved in the copper-induced LDL oxidation (Patel et al., 1997; Burkitt, 2001) since cholesteryl ester and phospholipids (major lipids of native LDL) are the major sources of LOOH. In summary, we determine for the first time that in a concentrationdependent manner cyclodextrins can induce lipid depletion of native LDL and therefore impair LDL susceptibility to oxidation at least in vitro. It implies that cyclodextrins probably help to inhibit the initiation and/or progression of atherosclerosis by lowering LDL oxidation although more in-depth studies will be needed. Author contributions YC conceived and supervised the study; YC and MA designed experiments; MA, CG, WS and XZ performed experiments; YC, MA and CG analyzed data; YC wrote the manuscript. Acknowledgments This study was supported by the National Natural Science Foundation of China (31260205 and 81560083) and the Natural Science Foundation of Jiangxi Province (20151BAB205005). References Astray, G., Gonzalez-Barreiro, C., Mejuto, J.C., Rial-Otero, R., Simal-Gandara, J., 2009. A review on the use of cyclodextrins in foods. Food Hydrocoll. 23, 1631–1640. Aviram, M., 1993. Modified forms of low-density-lipoprotein and atherosclerosis. Atherosclerosis 98, 1–9. Balkan, J., Dogru-Abbasoglu, S., Aykac-Toker, G., Uysal, M., 2004. Serum pro-oxidantantioxidant balance and low-density lipoprotein oxidation in healthy subjects with different cholesterol levels. Clin. Exp. Med. 3, 237–242. Burkitt, M.J., 2001. A critical overview of the chemistry of copper-dependent low density lipoprotein oxidation: roles of lipid hydroperoxides, alpha-tocopherol, thiols, and ceruloplasmin. Arch. Biochem. Biophys. 394, 117–135. Crumling, M.A., Liu, L.Q., Thomas, P.V., Benson, J., Kanicki, A., Kabara, L., Halsey, K., Dolan, D., Duncan, R.K., 2012. Hearing loss and hair cell death in mice given the cholesterolchelating agent hydroxypropyl-β-cyclodextrin. Plos One 7. Davidson, C.D., Ali, N.F., Micsenyi, M.C., Stephney, G., Renault, S., Dobrenis, K., Ory, D.S., Vanier, M.T., Walkley, S.U., 2009. Chronic cyclodextrin treatment of murine Niemann-Pick C disease ameliorates neuronal cholesterol and glycosphingolipid storage and disease progression. Plos One 4. Davis, M.E., Brewster, M.E., 2004. Cyclodextrin-based pharmaceutics: past, present and future. Nat. Rev. Drug Discov. 3, 1023–1035. Del Valle, E.M.M., 2004. Cyclodextrins and their uses: a review. Process Biochem. 39, 1033–1046. Frei, B., Gaziano, J.M., 1993. Content of antioxidants, preformed lipid hydroperoxides, and cholesterol as predictors of the susceptibility of human LDL to metal ion-dependent and ion-independent oxidation. J. Lipid Res. 34, 2135–2145. Gan, C., Ao, M., Liu, Z., Chen, Y., 2015. Imaging and force measurement of LDL and HDL by AFM in air and liquid. FEBS Open Bio 5, 276–282. Gotoh, K., Kariya, R., Alam, M.M., Matsuda, K., Hattori, S., Maeda, Y., Motoyama, K., Kojima, A., Arima, H., Okada, S., 2014. The antitumor effects of methyl-β-cyclodextrin against primary effusion lymphoma via the depletion of cholesterol from lipid rafts. Biochem. Biophys. Res. Commun. 455, 285–289. Holvoet, P., Mertens, A., Verhamme, P., Bogaerts, K., Beyens, G., Verhaeghe, R., Collen, D., Muls, E., Van de Werf, F., 2001. Circulating oxidized LDL is a useful marker for identifying patients with coronary artery disease. Arterioscler. Thromb. Vasc. Biol. 21, 844–848. Kontush, A., Hubner, C., Finckh, B., Kohlschutter, A., Beisiegel, U., 1996. How different constituents of low density lipoprotein determine its oxidizability by copper: a correlational approach. Free Radic. Res. 24, 135–147.
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Lakkakula, J.R., Krause, R.W.M., 2014. A vision for cyclodextrin nanoparticles in drug delivery systems and pharmaceutical applications. Nanomedicine 9, 877–894. Leventis, R., Silvius, J.R., 2001. Use of cyclodextrins to monitor transbilayer movement and differential lipid affinities of cholesterol. Biophys. J. 81, 2257–2267. Loftsson, T., Duchene, D., 2007. Cyclodextrins and their pharmaceutical applications. Int. J. Pharm. 329, 1–11. Loftsson, T., Hreinsdottir, D., Masson, M., 2005. Evaluation of cyclodextrin solubilization of drugs. Int. J. Pharm. 302, 18–28. Lopez, A.M., Terpack, S.J., Posey, K.S., Liu, B., Ramirez, C.M., Turley, S.D., 2014. Systemic administration of 2-hydroxypropyl-β-cyclodextrin to symptomatic Npc1-deficient mice slows cholesterol sequestration in the major organs and improves liver function. Clin. Exp. Pharmacol. Physiol. 41, 780–787. Mahammad, S., Parmryd, I., 2015. Cholesterol depletion using methyl-β-cyclodextrin. Methods Mol. Biol. 1232, 91–102. Mertens, A., Verhamme, P.P., Verhaeghe, R., Muls, E., Collen, D., Van de Werf, F., Holvoet, P., 2001. Circulating oxidized LDL is a useful marker for cardiovascular risk assessment. Circulation 103, 1350. Mohammad, N., Malvi, P., Meena, A.S., Singh, S.V., Chaube, B., Vannuruswamy, G., Kulkarni, M.J., Bhat, M.K., 2014. Cholesterol depletion by methyl-β-cyclodextrin augments tamoxifen induced cell death by enhancing its uptake in melanoma. Mol. Cancer 13. Mosinger, B.J., 1995. Copper-induced and photosensitive oxidation of serum low-densitylipoprotein - the relation to cholesterol level and interspecies differences. Biochimica Et Biophysica Acta-Molecular Basis of Disease 1270, 73–80. Mosinger, B.J., 1999. Higher cholesterol in human LDL is associated with the increase of oxidation susceptibility and the decrease of antioxidant defence: experimental and simulation data. Biochimica Et Biophysica Acta-Molecular Basis of Disease 1453, 180–184. Ohtani, Y., Irie, T., Uekama, K., Fukunaga, K., Pitha, J., 1989. Differential-effects of α-cyclodextrins. Beta-Cyclodextrins and Gamma-Cyclodextrins on Human-Erythrocytes, European Journal of Biochemistry 186, 17–22. Ohvo, H., Slotte, J.P., 1996. Cyclodextrin-mediated removal of sterols from monolayers: effects of sterol structure and phospholipids on desorption rate. Biochemistry 35, 8018–8024. Patel, R.P., Svistunenko, D., Wilson, M.T., DarleyUsmar, V.M., 1997. Reduction of Cu(II) by lipid hydroperoxides: implications for the copper-dependent oxidation of lowdensity lipoprotein. Biochem. J. 322, 425–433. Ramirez, C.M., Liu, B., Taylor, A.M., Repa, J.J., Burns, D.K., Weinberg, A.G., Turley, S.D., Dietschy, J.M., 2010. Weekly cyclodextrin administration normalizes cholesterol metabolism in nearly every organ of the Niemann-Pick type C1 mouse and markedly prolongs life. Pediatr. Res. 68, 309–315. Schneider, S., Folprecht, G., Krohne, G., Oberleithner, H., 1995. Immunolocalization of lamins and nuclear-pore complex proteins by atomic-force microscopy. Pflugers Arch. - Eur. J. Physiol. 430, 795–801. Singh, M., Sharma, R., Banerjee, U.C., 2002. Biotechnological applications of cyclodextrins. Biotechnol. Adv. 20, 341–359. Spranger, T., Finckh, B., Fingerhut, R., Kohlschutter, A., Beisiegel, U., Kontush, A., 1998. How different constituents of human plasma and low density lipoprotein determine plasma oxidizability by copper. Chem. Phys. Lipids 91, 39–52. Steinberg, D., Parthasarathy, S., Carew, T.E., Khoo, J.C., Witztum, J.L., 1989. Beyond cholesterol - modifications of low-density lipoprotein that increase its atherogenicity. N. Engl. J. Med. 320, 915–924. Taylor, A.M., Liu, B., Mari, Y., Liu, B., Repa, J.J., 2012. Cyclodextrin mediates rapid changes in lipid balance in Npc1(−/−) mice without carrying cholesterol through the bloodstream. J. Lipid Res. 53, 2331–2342. Toshima, S., Hasegawa, A., Kurabayashi, M., Itabe, H., Takano, T., Sugano, J., Shimamura, K., Kimura, J., Michishita, I., Suzuki, T., Nagai, R., 2000. Circulating oxidized low density lipoprotein levels - a biochemical risk marker for coronary heart disease. Arterioscler. Thromb. Vasc. Biol. 20, 2243–2247. Witztum, J.L., Steinberg, D., 1991. Role of oxidized low-density-lipoprotein in atherogenesis. J. Clin. Investig. 88, 1785–1792. Wu, L., Huang, J., Yu, X.X., Zhou, X.Q., Gan, C.Y., Li, M., Chen, Y., 2014. AFM of the ultrastructural and mechanical properties of lipid-raft-disrupted and/or cold-treated endothelial cells. J. Membr. Biol. 247, 189–200. Zhang, J.X., Ma, P.X., 2013. Cyclodextrin-based supramolecular systems for drug delivery: recent progress and future perspective. Adv. Drug Deliv. Rev. 65, 1215–1233. Zidovetzki, R., Levitan, I., 2007. Use of cyclodextrins to manipulate plasma membrane cholesterol content: evidence, misconceptions and control strategies. Biochimica Et Biophysica Acta-Biomembranes 1768, 1311–1324.
Please cite this article as: Ao, M., et al., Effects of cyclodextrins on the structure of LDL and its susceptibility to copper-induced oxidation, European Journal of Pharmaceutical Sciences (2016), http://dx.doi.org/10.1016/j.ejps.2016.04.037
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Effects of cyclodextrins on the structure of LDL and its susceptibility to copper-induced oxidation Meiying Ao a,b,c, Chaoye Gan a,c, Wenxiang Shao d, Xing Zhou a,c, Yong Chen a,c,⁎ a
Nanoscale Science and Technology Laboratory, Institute for Advanced Study, Nanchang University, Nanchang, Jiangxi 330031, PR China Department of Pharmacy, Science and Technology College, Jiangxi University of Traditional Chinese Medicine, Nanchang, Jiangxi 330025, PR China College of Life Sciences, Nanchang University, Nanchang, Jiangxi 330031, PR China d School of Basic Medical Sciences, Jiangxi University of Traditional Chinese Medicine, Nanchang, Jiangxi 330025, PR China b c
a r t i c l e
i n f o
Article history: Received 8 December 2015 Received in revised form 28 April 2016 Accepted 28 April 2016 Available online xxxx Keywords: Cyclodextrin Atherosclerosis Low-density lipoprotein (LDL) Atomic force microscopy (AFM) LDL oxidation
a b s t r a c t Cyclodextrins (CDs) have long been widely used as drug/food carriers and were recently developed as drugs for the treatment of diseases (e.g. Niemann-Pick C1 and cancers). It is unknown whether cyclodextrins may influence the structure of low-density lipoprotein (LDL), its susceptibility to oxidation, and atherogenesis. In this study, four widely used cyclodextrins including α-CD, γ-CD, and two derivatives of β-CD (HPβCD and MβCD) were recruited. Interestingly, agarose gel electrophoresis (staining lipid and protein components of LDL with Sudan Black B and Coomassie brilliant blue, respectively but simultaneously) shows that cyclodextrins at relatively high concentrations caused disappearance of the LDL band and/or appearance of an additional protein-free lipid band, implying that cyclodextrins at relatively high concentrations can induce significant electrophoresisdetectable lipid depletion of LDL. Atomic force microscopy (AFM) detected that MβCD (as a representative of cyclodextrins) induced size decrease of LDL particles in a dose-dependent manner, further confirming the lipid depletion effects of cyclodextrins. Moreover, the data from agarose gel electrophoresis, conjugated diene formation, MDA production, and amino group blockage of copper-oxidized LDL show that cyclodextrins can impair LDL susceptibility to oxidation. It implies that cyclodextrins probably help to inhibit atherogenesis by lowering LDL oxidation. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Cyclodextrins (CDs) are a family of cyclic oligosaccharides, consisting of six (α-cyclodextrin or α-CD), seven (β-cyclodextrin or β-CD), eight (γ-cyclodextrin or γ-CD) or more α-(1,4)-linked glucopyranose subunits. Due to their specific structure of a lipophilic internal cavity surrounded by hydrophilic outer surface, cyclodextrins can be used as potent carriers by forming water-soluble complexes with many water-insoluble compounds, and have been intensively studied and widely applied in pharmaceuticals (Loftsson et al., 2005; Loftsson and Duchene, 2007; Zhang and Ma, 2013; Lakkakula and Krause, 2014), foods (Astray et al., 2009), and others (Singh et al., 2002; Del Valle, 2004). Although when cyclodextrin-containing pharmaceutical products are administered orally only small amounts of cyclodextrins can permeate gastrointestinal mucosa, cyclodextrins or their derivatives in many marketed parenteral formulations (Loftsson and Duchene, 2007) can enter the blood circulation or specific organs at relatively high ⁎ Corresponding author at: 999 Xuefu Ave., Honggutan District, Nanchang, Jiangxi 330031, PR China. E-mail address: [email protected] (Y. Chen).
concentrations. Cyclodextrins will be available to some blood components after delivered drugs are released. On the other hand, 2hydroxypropyl-β-cyclodextrin (2-HPβCD) (Davidson et al., 2009; Ramirez et al., 2010; Taylor et al., 2012; Crumling et al., 2012; Lopez et al., 2014) and methyl-β-cyclodextrin (MβCD) (Mohammad et al., 2014; Gotoh et al., 2014), two derivatives of β-CD, have been applied to treat some diseases (e.g. Niemann-Pick C1 and cancers) by direct injection into animal bloodstream. Therefore, it will be interesting and meaningful to know the effects of cyclodextrins on blood components, such as low-density lipoproteins (LDL), a major group of plasma lipoproteins transporting fats (mainly cholesterol) to cells. It has been widely accepted that the oxidation of LDL plays a vital role in the pathogenesis of atherosclerosis (Steinberg et al., 1989; Witztum and Steinberg, 1991; Aviram, 1993) and oxidized LDL (oxLDL) in circulation is an important risk marker for human cardiovascular diseases (Toshima et al., 2000; Holvoet et al., 2001; Mertens et al., 2001). Many factors may influence LDL susceptibility to oxidation including antioxidants, total lipid composition, fatty acids, and cholesterol (Kontush et al., 1996; Frei and Gaziano, 1993; Mosinger, 1995; Spranger et al., 1998; Mosinger, 1999; Balkan et al., 2004). Considering this, as well as the ability of cyclodextrins to form water-soluble complexes with lipids and the lipid-enriched structure of LDL, we hypothesize
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Please cite this article as: Ao, M., et al., Effects of cyclodextrins on the structure of LDL and its susceptibility to copper-induced oxidation, European Journal of Pharmaceutical Sciences (2016), http://dx.doi.org/10.1016/j.ejps.2016.04.037
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Before oxidation, EDTA was removed from the LDL solutions (Cat. No. YB-001; Yiyuan Biotechnologies, Guangzhou, China; isolated from human plasma). For cyclodextrin treatments, 0.2 mg/ml LDL solutions were incubated with different concentrations of cyclodextrins (SigmaAldrich; the catalog numbers of α-CD, γ-CD, MβCD, and HPβCD are C4642, 779660, C4555, and H107, respectively) in PBS for 1 h at 37 °C. For LDL oxidation, native LDL and LDL treated with cyclodextrins were directly mixed with copper sulphate (99.0% purity; Jinshanting New Chemical Reagent Factory, Shanghai, China) at a final copper concentration of 5 μM in PBS and incubated at 37 °C. For the oxidation of phosphotidylcholine (PC), PC (Lanji Science and Technology Development Ltd., Shanghai, China) at a final concentration of 1.6 mg/ml in PBS was used. The MβCD treatment and copper oxidation of PC were similar to LDL samples. 2.2. Agarose gel electrophoresis
Fig. 1. Cyclodextrins causes depletion of lipids from native LDL detected by agarose gel electrophoresis. Left panel: the gels stained with Sudan Black B; right panel: the gels loading the same samples but stained with Coomassie brilliant blue. (A, B) The samples treated with or without γ-CD (9 and 18 mM, respectively) or α-CD (7.5 and 15 mM, respectively). (C, D) The samples treated with HPβCD (10, 40, 100, and 200 mM, respectively). (E, F) The samples treated with MβCD (10, 20, 40, and 100 mM, respectively). The first lanes (marked with “—”) of all gels represent the native LDL samples without cyclodextrin treatment.
that cyclodextrins can deplete lipids from native LDL and affect LDL susceptibility to oxidation. In this study, the effects of four widely used cyclodextrins including α-CD, γ-CD, HPβCD, and MβCD on the structure of native LDL and the susceptibility of LDL to copper-induced oxidation were evaluated to test the abovementioned hypothesis.
2. Materials and methods 2.1. Cyclodextrin treatment and LDL oxidation The water solubility of cyclodextrins is approximately 2%, 13%, and 26% weight by weight for β-CD, α-CD, and γ-CD, respectively (Davis and Brewster, 2004). Under our experimental conditions, 15 mM αCD and 18 mM γ-CD reached the highest solubility, respectively. Since β-CD is hard to be dissolved, methylated and 2-hydroxypropylated derivatives (i.e. HPβCD and MβCD, respectively) with significantly improved water solubility were utilized. The highest final concentration of HPβCD/MβCD used in this study (200 and 100 mM, respectively) did not reach their highest solubility yet.
Agarose gel electrophoresis was performed on 0.5% agarose gels in sodium barbital buffer using the electrophoretic system. Samples were electrophoresed in 0.075 M sodium barbital buffer at 55 V for 1 h. Relative electrophoretic mobility (REM) was calculated as the ratio of the migration distance of oxidized LDL to that of native LDL. To simultaneously determine the lipid and protein components of native LDL treated with or without cyclodextrins, two gels loading the same samples were run in the same electrophoresis chamber but stained with Sudan Black B for lipids and Coomassie brilliant blue R-250 for proteins, respectively. Agarose gel electrophoresis of cholesterol (Sigma-Aldrich) or PC treated with or without MβCD/copper was performed similarly. 2.3. Isolation and measurement of depleted lipids LDL was treated with MβCD at different concentrations. The Amicon Ultra-0.5 ml, 10 kDa Centrifugal Filter Unit (Merck Millipore) was utilized to remove the LDL remnants and obtain the depleted lipids according to the user's manual. The depleted cholesterols including free and esterified cholesterols were measured via LDL-C Kit (Biosino BioTechnology and Science Inc., Beijing, China) according to the user's manual. The absorbance of cholesterols or phospholipids was measured at 550 and 240 nm, respectively, by using a UV-5100 spectrophotometer (Metash Instruments, Shanghai, China). 2.4. Atomic force microscopy (AFM) The mica functionalized with aminopropyltriethoxysilane (APTES) and glutaraldehyde (GA) and the LDL samples treated with or without MβCD were prepared and imaged by an Asylum MFP-3D-SA AFM (Asylum Research, USA) in tapping mode in PBS as previously described (Gan et al., 2015). The silicon nitride tips (AppNano, USA) with an end radius of 10 nm and a spring constant of ~0.04 N/m were utilized. The full width at half maximum (FWHM; the radius r is half of the measured
Fig. 2. MβCD causes the concentration-dependent increases in the lipids drawn from LDL. After MβCD treatments, the LDL remnants were filtered by a 10 kDa Centrifugal Filter Unit, and the contents of the depleted lipids were measured. The percentage of (A) phospholipids or (B) cholesterols (including free and esterified cholesterols) drawn from LDL significantly increased with the increase of MβCD concentration. (C) Agarose gel electrophoresis of pure lipids. Lanes 1–4: LDL, free cholesterol + phosphotidylcholine (PC) + MβCD, free cholesterol + MβCD, and PC alone, respectively.
Please cite this article as: Ao, M., et al., Effects of cyclodextrins on the structure of LDL and its susceptibility to copper-induced oxidation, European Journal of Pharmaceutical Sciences (2016), http://dx.doi.org/10.1016/j.ejps.2016.04.037
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Fig. 3. MβCD induces the size decrease of LDL particles in a concentration-dependent manner detected by AFM. (A–F) AFM topographical images of bare mica, modified mica, native LDL particles, and LDL particles treated with MβCD (10, 40, and 100 mM, respectively), respectively. Scan size: 1 μm × 1 μm. (G) Height profiles of horizontal cross sections randomly across the topographical images in A–F. (H) Average volumes of LDL particles treated with or without MβCD (*, P b 0.05; ***, P b 0.001).
FWHM) and height (h) of LDL particles were measured to calculate the volume (V) of a single LDL particle using the following equation (Schneider et al., 1995): 2 V ¼ ðπ h=6Þ 3r 2 þ h :
2.6. Statistical analysis Student t-test was performed to determine the significance between different groups. All data were from at least three independent experiments. A value of P b 0.05 was considered statistically significant (*, P b 0.05; **, P b 0.01; ***, P b 0.001 in graphs).
3. Results and discussion 2.5. Measurements of conjugated dienes, malondialdehyde (MDA), and free amino groups
3.1. Cyclodextrins induce lipid depletion of native LDL
During LDL oxidation, conjugated diene formation was measured at 20-min intervals for 220 min at 37 °C. MDA production and remaining free amino groups at 18 h after oxidation were measured by TBARS Assay Kit and TNBSA Assay Kit, respectively according to the user's manuals. The blockage of amino groups was calculated by comparing the remaining free amino groups in the oxidized product to those of the un-oxidized control. The absorbance of each sample was measured at 234, 532, and 335 nm, respectively, by using the UV-5100 spectrophotometer for conjugated dienes, MDA, and free amino groups, respectively.
Prior to investigating the effects of cyclodextrins on LDL susceptibility to oxidation, the effects of cyclodextrins on the structure of native LDL were studied firstly by agarose gel electrophoresis. To simultaneously determine the lipid and protein components of native LDL treated with or without cyclodextrins, Sudan Black B (for lipids; left panel of Fig. 1) and Coomassie brilliant blue R-250 (for proteins; right panel of Fig. 1) were utilized to stain two gels loading the same samples and running in the same electrophoresis chamber. In Fig. 1A, γ-CD (both 9 and 18 mM) and 7.5 mM α-CD did not significantly influence the electrophoretic mobility of native LDL. However,
Please cite this article as: Ao, M., et al., Effects of cyclodextrins on the structure of LDL and its susceptibility to copper-induced oxidation, European Journal of Pharmaceutical Sciences (2016), http://dx.doi.org/10.1016/j.ejps.2016.04.037
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Fig. 4. Cyclodextrins inhibit the electrophoretic mobility of copper-oxidized LDL in a concentration-dependent manner. (A–C) The LDL samples treated with or without γ-CD/α-CD were oxidized by copper for 2, 10, and 18 h, respectively. (D–F) The LDL samples treated with or without HPβCD were oxidized by copper for 2, 8, and 18 h, respectively. (G–I) The LDL samples treated with or without MβCD were oxidized by copper for 2, 4, and 18 h, respectively. The first and second Lanes in A–I are native LDL and copper-oxidized LDL without cyclodextrin treatment, respectively. The gels in A–I were stained with Sudan Black B (The insets in E and H are stained with Coomassie brilliant blue). The arrows in E and H show the additional protein-free bands. (J–L) Calculated relative electrophoretic mobility (REM) of native or oxidized LDL treated with or without cyclodextrins. Treatments from left to right: γ-CD/α-CD, HPβCD, and MβCD, respectively.
15 mM α-CD caused a faint lipid band of native LDL (indicated by the arrow). Fig. 1B shows that 15 mM α-CD induced the disappearance of the protein band at the location of native LDL (indicated by the arrow) but the appearance of a protein band at the location of the origin (indicated by the arrowhead). It implies that 15 mM α-CD caused a significant separation between lipid and protein components of native LDL. Fig. 1C and Fig. 1E show that HPβCD and MβCD induced the broadening of the lipid band of native LDL and that at relatively high concentrations they (200 mM HPβCD and 40 mM MβCD) caused an additional lipid band (indicated by the arrowheads) which was protein-free as indicated by the arrowheads in Fig. 1D and Fig. 1F. MβCD at much higher concentrations (e.g. 100 mM) even caused the same effect as 15 mM α-CD (i.e. faint lipid and protein bands at the location of native LDL as indicated by the arrows in Fig. 1E, 1F). These data imply that both
HPβCD and MβCD induced a concentration-dependent separation between lipid and protein components of native LDL. To confirm the implication, the changes in the content of the depleted lipids from LDL were monitored. We selected MβCD as a representative since the effects of MβCD on the electrophoretic mobility of native LDL cover all effects of the other cyclodextrins as mentioned above. As showed in Fig. 2A,B, the percentage of the depleted lipids (e.g. phospholipid and cholesterol) increased significantly with the increase of MβCD concentration whereas no depleted proteins were detected (data not shown). Agarose gel electrophoresis (Fig. 2C) showed that both cholesterol alone (unable to be stained by Sudan Black B; not shown) and cholesterol- cyclodextrin complex (lane 3) were retained in the loading tank without electrophoretic mobility due to no net charges of both
Please cite this article as: Ao, M., et al., Effects of cyclodextrins on the structure of LDL and its susceptibility to copper-induced oxidation, European Journal of Pharmaceutical Sciences (2016), http://dx.doi.org/10.1016/j.ejps.2016.04.037
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cholesterol and MβCD whereas phosphotidylcholine (PC) alone (lane 4) could be stained faintly and formed a continuous band, a part of which even has a faster electrophoretic mobility than native LDL (lane 1). Interestingly, the cholesterol-PC-MβCD mixture (lane 2) has a slower electrophoretic band than LDL which is similar to the additional proteinfree lipid band of LDL treated by MβCD or HPβCD. Probably, two or more MβCD molecules formed complexes simultaneously with both cholesterol and PC. Then, we still selected MβCD as a representative to investigate the effects of cyclodextrins on the particle size of native LDL by using atomic force microscopy (AFM). AFM imaged the very smooth surfaces of the bare mica (Fig. 3A) and the mica modified with APTES plus glutaraldehyde (Fig. 3B). When native LDL was immobilized onto the smooth surfaces of modified mica, many nano-sized particles were imaged in PBS by AFM (Fig. 3C). After treated with 10, 40, and 100 mM MβCD, respectively, LDL particles became smaller gradually (Fig. 3D–G). Statistical analysis shows that 10, 40, and 100 mM MβCD induced ~10%, ~60%, and ~80% decreases in average volume of individual LDL particles, respectively (Fig. 3H). Therefore, cyclodextrins at a relatively high concentration caused the disappearance of native LDL band and/or the appearance of an additional protein-free band during agarose gel electrophoresis (Fig. 1), obviously implying that cyclodextrins at a relatively high concentration depleted lipids from native LDL. The concentration-dependent decrease in particle size of native LDL as detected by AFM (Fig. 3) further confirms the lipid-depleting effects of cyclodextrins. Cyclodextrins at lower concentrations probably also caused lipid depletion of native LDL since the concentration-dependent increase in percentage of the lipids depleted by cyclodextrins from LDL were detected. Moreover, AFM detected ~ 10% decrease in average volume of individual LDL particles treated with MβCD at a low concentration (10 mM) although an additional protein-free lipid band was undetectable by agarose gel electrophoresis due to relatively small amounts of depleted lipids. Taken together, the agarose gel electrophoretic data and the AFM data imply that cyclodextrins can induce lipid depletion and size decrease of native LDL in a concentration-dependent manner. It can also be supported by the facts that the three types of cyclodextrins are efficient in extracting cholesterol and/or phospholipids from cellular and model membrane (Ohtani et al., 1989; Ohvo and Slotte, 1996; Leventis and Silvius, 2001; Zidovetzki and Levitan, 2007; Wu et al., 2014; Mahammad and Parmryd, 2015) and that LDL lipids are mainly consisted of cholesterol and phospholipids. For the first time, we provide the evidence of the cyclodextrin effects on the structure of native LDL. 3.2. Cyclodextrins reduce LDL susceptibility to copper-induced oxidation Next, we investigated the effects of cyclodextrins on LDL susceptibility to copper-induced oxidation firstly via agarose gel electrophoresis (Fig. 4). Fig. 4A–C, Fig. 4D–F, and Fig. 4G–I display the effects of α-CD/ γ-CD, HPβCD, and MβCD on LDL oxidation, respectively. Obviously, γCD, HPβCD, and MβCD inhibited the electrophoretic mobility of copper-oxidized LDL in a concentration-dependent manner. The quantitative analyses of the relative electrophoretic mobility (Fig. 4J–L) further confirmed the results (the 15 mM α-CD group in Fig. 4J and the 100 mM MβCD group in Fig. 4L were not quantified due to the faintness of their LDL bands and the difficulty of accurate measurement). The insets in Fig. 4E and Fig. 4H present the corresponding gels stained by Coomassie brilliant blue showing the additional protein-free lipid bands of oxidized LDL (indicated by the arrows) caused by relatively high concentrations of HPβCD/MβCD. Subsequently, the dynamic accumulation of conjugated dienes during a period of 220 min (Fig. 5) was evaluated to determine the effects of cyclodextrins on the copper-induced oxidation of LDL lipids. Without cyclodextrin treatment copper alone induced a fast and dramatic increase in conjugated diene formation (the dark lines in Fig. 5A–C) as
Fig. 5. Cyclodextrins lower conjugated diene formation of LDL during copper-induced oxidation in a concentration-dependent manner. (A) γ-CD or α-CD treatments. (B) HPβCD treatments. (C) MβCD treatments. The samples were measured at 20-min intervals for 220 min from the beginning of oxidation.
usual. In a concentration-dependent manner the four cyclodextrins (Fig. 5), particularly HPβCD (Fig. 5B) and MβCD (Fig. 5C), caused lower conjugated dienes when reaching the plateau although the process of conjugated diene formation was faster. The cyclodextrin-induced lipid depletion might cause a temporal loosening of LDL structure under which condition copper could more easily access the targets therefore causing a faster process of conjugated diene formation. On the other hand, the cyclodextrin-induced lipid depletion decreased oxidizable lipid sources, therefore causing a lower final conjugated diene formation. Finally, malondialdehyde (MDA) production (Fig. 6) and blocked amino groups (Fig. 7) were measured to further determine the effects of cyclodextrins on the copper-induced oxidation of LDL lipids and
Please cite this article as: Ao, M., et al., Effects of cyclodextrins on the structure of LDL and its susceptibility to copper-induced oxidation, European Journal of Pharmaceutical Sciences (2016), http://dx.doi.org/10.1016/j.ejps.2016.04.037
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Fig. 6. Cyclodextrins impair malondialdehyde (MDA) production of LDL during copper-induced oxidation in a concentration-dependent manner. From left to right: γ-CD/α-CD, HPβCD, and MβCD treatments, respectively. The samples at the 18 h time point of oxidation were measured (*, P b 0.05; **, P b 0.01; ***, P b 0.001).
Fig. 7. Cyclodextrins decrease amino group blockage of LDL proteins during copper-induced oxidation in a concentration-dependent manner. From left to right: γ-CD/α-CD, HPβCD, and MβCD treatments, respectively. The samples at the 18 h time point of oxidation were measured (*, P b 0.05; **, P b 0.01; ***, P b 0.001).
proteins, respectively. Without cyclodextrin treatment copper alone induced a dramatic increase in both MDA production and blocked amino groups of LDL 18 h later after oxidation. Significantly, each of the four cyclodextrins inhibited MDA production and amino group blockage of the copper-oxidized LDL in a concentration-dependent manner. Interestingly, 10 mM HPβCD induced a statistically significant decrease in MDA production but not amino group blockage whereas 10 mM MβCD induced a statistically significant decrease in amino group blockage but not MDA production. It implies that HPβCD and MβCD might influence LDL oxidation potentially by different mechanisms which are currently unclear. The data from agarose gel electrophoresis (Fig. 8A) and TBARS assay (Fig. 8B) showed the effects of MβCD on the oxidation of pure PC molecules. Clearly or significantly, copper induced the increase in
electrophoretic mobility and MDA production of PC whereas MβCD treatments have no significant effects on PC oxidation. The data implies that the cyclodextrin-bound lipids depleted from LDL could not be one of the factors responsible for the inhibitory effects of cyclodextrins on LDL oxidation. Taken together, all results from agarose gel electrophoresis, conjugated diene formation, MDA production, and amino group blockage indicate that the four types of cyclodextrins can reduce LDL susceptibility to copper-induced oxidation in a concentration-dependent manner. The cyclodextrin-induced concentration-dependent lipid depletion of native LDL should be responsible since it is well known that total lipid composition, fatty acids, cholesterol and others can significantly influence LDL susceptibility to oxidation (Kontush et al., 1996; Frei and Gaziano, 1993; Mosinger, 1995; Spranger et al., 1998; Mosinger, 1999). Cyclodextrins
Fig. 8. MβCD has no significant effects on the copper-induced oxidation of pure PCs. (A) Agarose gel electrophoresis. PC alone was stained faintly by Sudan Black B whereas PC-MβCD complexes can be stained relatively strongly. (B) TBARS assay detects MDA production of PCs. The oxidized samples were oxidized by copper for 18 h.
Please cite this article as: Ao, M., et al., Effects of cyclodextrins on the structure of LDL and its susceptibility to copper-induced oxidation, European Journal of Pharmaceutical Sciences (2016), http://dx.doi.org/10.1016/j.ejps.2016.04.037
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Please cite this article as: Ao, M., et al., Effects of cyclodextrins on the structure of LDL and its susceptibility to copper-induced oxidation, European Journal of Pharmaceutical Sciences (2016), http://dx.doi.org/10.1016/j.ejps.2016.04.037