Effect of Cholesterol and Curcumin on Ordering of DMPC Bilayers
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In this work, we compared the effects of curcumin and cholesterol directly competing to insert into the DMPC lipid bilayer during bilayer formation from an initially non-ordered state. 2H and 14N nuclear magnetic resonance spectroscopy showed that curcumin is not embedded deep in the lipid bilayer and interacts mainly with the head group of the lipid. In a more complex system of DMPC/CHOL/CUR, curcumin amplifies the effect of cholesterol on the ordering of lipid acyl chains.
Cholesterol (CHOL) is an essential component of mammalian cell membranes. It is embedded in the hydrophobic part of the membrane with its hydroxyl group interacting with the polar head groups of the membrane phospholipids and sphingolipids [1, 2]. One of the most significant biological properties of CHOL is its ability to increase membrane packing and ordering in the liquid crystalline phase [3, 4, 5]. According to measurements of lateral diffusion coefficients of phospholipids and sphingolipids, it has been determined that membrane fluidity decreases as the concentration of CHOL increases [6, 7]. Fluorescent microscopy and 2H nuclear magnetic resonance (NMR) have shown that CHOL also introduces the liquid ordered phase [8, 9, 10].
Curcumin (CUR) is a natural component of the spice Curcuma longa. It is used in the kitchen and actively used in traditional Indian medicine [11, 12]. CUR and its derivatives have anti-oxidant, anti-carcinogenic, anti-mutagenic, anti-inflammatory and anti-fungal properties [13, 14, 15]. CUR has shown prophylactic and therapeutic capacity for neurodegenerative diseases such as Alzheimer´s and Parkinson´s, which has been attributed to the compound’s interaction with amyloid fibrils [16, 17, 18], as well as oligomers and protofibrils . Because of its wide spectrum of pharmacological properties, CUR, such as cholesterol, has been proposed to act on a rather basic biological level, such as on biomembranes. It has been proposed that CUR can regulate the action of membrane proteins indirectly by changing the physical properties of the membrane rather than by directly binding to the proteins .
Similarities of CUR and CHOL activity on biomembranes have been suggested in a number of biochemical studies. CUR molecules initially bind to the membrane interface and then at higher concentrations gradually partition to insert into the hydrocarbon region, which has been detected by the binding of CUR with giant unilamellar vesicles . X-ray diffraction experiments have shown that the thickness of DOPC bilayers decreases as a function of the lipid/CUR ratio . Solid-state NMR on magnetically aligned bicelles of DMPC and DSC on bilayers of DMPC and DPPC have shown CUR has a strong effect on membrane structure at low concentrations . CUR inserts deep into the membrane in a transbilayer orientation, anchored through the hydrogen bonding of –OH groups of phenolic rings of CUR to the phosphate group of lipids and the hydrophobic interaction of the aromatic rings of CUR with phospholipid acyl chains in a manner analogous to that of cholesterol. Like CHOL, CUR binds DMPC in a transbilayer orientation at low concentrations and induces segmental ordering in the lipid membrane . CUR forms higher order oligomeric structures in the membrane that likely span through the bilayer, detected by analysis of the concentration dependence of the order parameter profile derived from NMR results. The apparent abolition of the pretransition with increase in CUR concentration in DMPC and DPPC implies that CUR interacts with the PC head groups .
Recently, lateral diffusion on planar lipid multibilayers and ordering of lipids on multibilayered vesicles have been studied by 1H and 2H NMR of DOPC and DMPC at concentrations of CUR varied in the range of 0–40 mol% . It has been shown that the presence of CUR in low concentrations (~ 8 mol%) leads to a moderate decrease in the lateral diffusion coefficients of both saturated and unsaturated lipids, while a further increase in CUR concentration has no effect because of the low solubility of CUR in lipid bilayers. The effect of CUR on 2H NMR quadrupolar splitting is also very small in comparison with that of CHOL. While CUR has a strong effect on the lateral diffusion of DMPC, the same as in DMPC/CHOL membranes, there is no visible effect of CUR in the presence of CHOL. Therefore, in direct competition to insert into the DMPC lipid bilayer during formation of the bilayer from an initially homogeneous mixture, CHOL overwhelmingly wins and consequently inhibits insertion of curcumin .
Despite wide interest in CUR–membrane interactions, studies of the effect of CUR on cholesterol-containing membranes are lacking. In this work, we studied the combined effect of both CUR and CHOL on a lipid bilayer, using 2H and 14N NMR as methods to study ordering of lipid membranes.
2 Materials and Methods
2.1 Sample Preparation
To prepare a vesicular sample, the lipid, cholesterol and curcumin were dissolved in a sufficient amount of ethanol. The ethanol was then evaporated under a constant flow of dry nitrogen, and then the sample was vacuum-pumped overnight to remove solvent traces. The resulting lipid film was hydrated using deionized water at a 5:1 ratio. Five freeze–thaw cycles were applied using liquid nitrogen and warm (323 K) water, according to Traikia et al., which resulted in the formation of a homogeneous vesicular sample .
2.1.1 2H and 14N NMR Spectroscopy
The NMR spectra of DMPC vesicles were recorded in a Bruker AVANCE III (Bruker BioSpin AG, Fällanden, Switzerland) operating at a proton frequency of 400.27 MHz applying solid-echo pulse sequence  at 308 K. The 2H NMR frequency was 61.422 MHz, relaxation delay was 0.25 s, rf-pulse duration was 25 µs, sweep width was 39 kHz and acquisition time was 0.21 s; a total of 16 k signal transients were collected. 14N NMR frequency was 28.904 MHz; a total of 25 k signal transients were collected.
De-Pake-ing is a method of numerical transformation of unoriented static 2H spectra into a 0°-oriented spectrum . In this case, the segmental order parameters, SCD, of lipid chains can be calculated [29, 30]. The 2H spectra were processed by the program NMR-Depaker to generate de-Paked spectra (https://launchpad.net/).
2.1.2 2H NMR
The increase (decrease) value of SCD for a C–D bond in a lipid bilayer system with axial symmetry is associated with trans-gauche rotational isomerizations of the acyl groups, molecular motions and ordering of lipids in the system [28, 29, 30, 33].
3 Results and Discussion
3.1 Lipid Phases and Ordering
The 2H spectrum of the control sample of DMPC(d54) vesicles shows three distinct resonances. The central resonance line (close to 0 Hz) is related to deuterons of water. The two other resonances close to the center of the spectrum refer to the –CD3 terminal groups of the lipid tails. The quadrupole splitting of the methyl group of DMPC is comparable with the data presented in the literature and is equal to 3.2 kHz at 308 K . Other resonances with the larger splitting correspond to –CD2– groups in the tail section, situated close to the “heads” of lipids.
The presence of curcumin at concentrations of 10 mol% increases the quadrupole splitting of the DMPC 2H NMR spectra by ca. 0.25 kHz, whereas the concentration of cholesterol in the presence of 15 mol% increases the quadrupole splitting of the DMPC 2H NMR spectra by a significantly larger value (by ca. 3.73 kHz). The mixture of 15 mol% CHOL and 10 mol% CUR together increases the quadrupole splitting by ca 4.36 kHz. The greatest effect on the quadrupole splitting of the spectra occurs in the mixture, which is caused by the presence of both, cholesterol and curcumin.
Order parameters SCD for the methyl group of the acyl chains of the lipid DMPC in the presence of curcumin (CUR) and cholesterol (CHOL)
+ CHOL + CUR (mol%)
The difference in the order parameters is probably due to the different areas of interaction of curcumin and cholesterol with lipid molecules in the bilayer. It was shown that the ordering effect of curcumin occurs in the glycerol region of the membrane with the hydrophobic core of the membrane affected to a somewhat lesser degree and this ordering effect is absent at the terminus of the acyl chain . In contrast, profiles of segmental order parameters as a function of acyl chain position indicate that cholesterol inserts deeply into the bilayer and affects all acyl chains .
3.2 14N NMR
The 14N NMR spectra show a small but detectable decrease in the quadrupole splitting, which is indicative of an electrostatic interaction of curcumin with the choline-phosphate hydrophilic part of the DMPC. Interactions of curcumin with the phosphate group oppose the electrostatic interactions between the positively charged choline groups and the negatively charged phosphate groups, which reduce the tetrahedral symmetry for lipids in a membrane and decrease the 14N quadrupolar splitting .
3.3 Effects of Cholesterol, Curcumin and Both Compounds on DMPC Bilayers
The effect of cholesterol on biomembranes has been thoroughly studied; therefore, it is reasonable to compare the effects of drug molecules (particularly those with polyphenol groups in their structure) just with CHOL. Experimental studies and simulations aiming to study the effect of curcumin have been performed on different biomembrane systems and their results are not completely in agreement with each other. The primary place of interaction of CUR with the membrane is the membrane interface (lipid–water interface) near the lipid head group (glycerol) region [37, 38, 39], while at higher concentrations, CUR can be located in the lipid membrane hydrophobic region [20, 21]. The insertion of CUR in the membrane core is a higher energetic process in comparison with surface adsorption , therefore, it can be suggested that a heterogeneous and curved membrane interface may promote insertion of CUR in the membrane hydrophobic core. Inhomogeneity of the water–lipid interface of both vesicles and planar bilayers may also depend on the homogeneity in acyl chain length and type of lipid used to construct the membrane. A recent communication by Tsukamoto et al.  showed that CUR localized at the domain’s boundary, i.e., the region of a high heterogeneity of the bilayer. Taking these factors into consideration, a higher loading capacity of CUR is expected for vesicles prepared from lipid extracts, while the lowest is expected for planar bilayers. Indeed, vesicles of egg yolk lecithin demonstrated a loading capacity of up to 84 w/w% , but planar bilayers of DOPC and DMPC did not take more than 10 mol% . Insertion of CUR in the bilayer hydrophobic core is also easier to observe in vesicles of lipid extracts [28, 41, 42] and synthetic lipids , but in the case of planar bilayers of synthetic lipids, CUR at higher concentrations tends to aggregate near the hydrophobic interface .
The overwhelming effect of CHOL over CUR probably has biological relevance. Indeed, if CUR and other polyphenolic drugs just slightly modulate the biological membrane surface (or just properties of surface of the membrane), this may not disturb the structure and essential functions of living cells. In addition, there is a possibility that the curcumin increases binding of cholesterol in the cellular membrane, thereby preventing cholesterol release into the blood stream. This could explain the lowering effect of curcumin against hypercholesterolemia . Further study of interaction between CUR and CHOL embedded in lipid bilayers might shed some light on this aspect.
We conclude according to 2H and 14N NMR spectroscopy that curcumin is not embedded deep within the lipid bilayer and interacts mainly with the head group of the lipid. In a more complex system of DMPC/CHOL/CUR, curcumin increases the effect of cholesterol on the ordering of lipid acyl chains.
We are grateful to the Kempe Foundation in memory of J.C. and Seth M. Kempe and LTU laboratory fund, which provided a few grants that funded the purchase of the NMR equipment. NMR measurements were also partly carried out on the equipment of the Federal Centre of Collective Facilities of Kazan Federal University.
- 18.K. Liu, T.L. Guo, J. Chojnacki, H.G. Lee, X. Wang, S.L. Siedlak, W. Rao, X. Zhu, S. Zhang, A.C.S. Chem, Neurosci. 3, 141–146 (2012)Google Scholar
- 31.J. Seelig, Q. Rev, Biophys. 10, 353–418 (1977)Google Scholar
- 33.M. F. Brown, S. Lope-Piedrafita, G. V Martinez, H. I. Petrache, in: Modern Magnetic Resonance, G.A. Webb (ed.) (Springer, Dordrecht, 2006) pp. 249–260Google Scholar
- 43.S.A. Hussein, Y.A. El-Senosi, M.R. Ragab, M.M.F. Hammad, Benha Veterinary. Med. J. 27, 277–289 (2014)Google Scholar
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