http://informahealthcare.com/lpr ISSN: 0898-2104 (print), 1532-2394 (electronic) J Liposome Res, Early Online: 1–9 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/08982104.2014.928888
RESEARCH ARTICLE
Kinetic and equilibrium studies of bile salt–liposome interactions Lin Yang, Feifei Feng, J. Paul Fawcett, and Ian G. Tucker
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School of Pharmacy, University of Otago, Dunedin, New Zealand
Abstract
Keywords
Research has suggested that exposure to sub-micellar concentrations of bile salts (BS) increases the permeability of lipid bilayers in a time-dependent manner. In this study, incubation of soy phosphatidylcholine small unilamellar vesicles (liposomes) with sub-micellar concentrations of cholate (C), deoxycholate (DC), 12-monoketocholate (MKC) or taurocholate (TC) in pH 7.2 buffer increased membrane fluidity and negative zeta potential in the order of increasing BS liposome-pH 7.2 buffer distribution coefficients (MKC5C & TC5DC). In liposomes labeled with the dithionite-sensitive fluorescent lipid N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine (NBD-PE) in both leaflets and equilibrated with sub-micellar concentrations of BS, fluorescence decline during continuous exposure to dithionite was biphasic involving a rapid initial phase followed by a slower second phase. Membrane permeability to dithionite as measured by the rate of the second phase increased in the order control5MKC5TC C5DC. In liposomes labeled with NBD-PE in the inner leaflet only and incubated with the same concentrations of C, DC and MKC, membrane permeability to dithionite initially increased very rapidly in the order MKC5C5DC before impermeability to dithionite was restored after which fluorescence decline was consistent with NBD-PE flip-flop. For liposomes incubated with TC, membrane permeability to dithionite was only slightly increased and the decline in fluorescence was mainly the result of NBD-PE flip-flop. These results provide evidence that BS interact with lipid bilayers in a time-dependent manner that is different for conjugated and unconjugated BS. MKC appears to cause least disturbance to liposomal membranes but, when the actual MKC concentration in liposomes is taken into account, MKC is actually the most disruptive.
Bile salts, distribution coefficient, dithionite, liposomes, membrane fluidity, membrane permeability
Introduction Bile acids are facially amphiphilic compounds in which 1–3 hydroxyl groups and an acidic moiety form the hydrophilic face and the steroidal backbone the hydrophobic face. They are present in vivo in the form of bile salts (BS) in which the carboxylic acid group is either unconjugated or conjugated with glycine or taurine. BS are frequently used in drug formulations either to increase the solubility and dissolution rate of poorly water-soluble drugs or to increase the permeability of drugs across biological membranes (Gordon et al., 1985; Morimoto et al., 1987). Usual explanations for this increased membrane permeability include the ability of BS to break down mucous and, at higher concentrations, to enhance paracellular transport by opening tight junctions between cells (Chen et al., 2011; Raimondi et al., 2008). Less well appreciated is the fact that BS at concentrations below their critical micelle concentration (CMC) (i.e. at submicellar concentrations) can enhance transcellular transport by disrupting the phospholipid cell membrane and fluidizing
Address for correspondence: J. Paul Fawcett, School of Pharmacy, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand. Tel: +64 3 479 5119. Fax: +64 3 479 7034. E-mail: [email protected]
History Received 12 March 2014 Revised 13 May 2014 Accepted 25 May 2014 Published online 24 June 2014
it through changing the lipid packing density (Mikov et al., 2006). Natural BS have been applied as permeation enhancers despite the fact that the more hydrophobic BS like deoxycholate (DC) are relatively cytotoxic at low concentrations (Naveen et al., 2011). In contrast, semi-synthetic ketocholate derivatives are less cytotoxic to the extent that, in rat, triketocholate (also called dehydrocholate) has been shown to open the blood brain barrier (BBB) in vivo (Spigelman et al., 1983) and 12-monoketocholate (MKC) has been shown to increase the analgesic effect of morphine and the brain uptake of quinine with minimal cytotoxicity (Mikov et al., 2004). We have previously compared some physicochemical and biological properties of MKC with those of cholate (C), DC and taurocholate (TC) and shown that MKC has a much higher CMC value than the other BS and causes less disruption to erythrocytes and Caco-2 cell monolayers (Yang et al., 2009). Liposomes are a useful model of the lipid bilayer of cells and as such are useful to study passive diffusion of drugs through the cell membrane. Understanding how they are solubilized by surfactants is also important in order to facilitate the development of liposomal drug delivery systems. Of particular relevance to the current study is the fact that
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incorporation of BS into the lipid bilayer of liposomes has been shown to stabilize them against membrane damage and solubilization by extrinsic BS in the gastrointestinal tract (Hu et al., 2013 and references therein). These so-called bilosomes have been used to improve the delivery of orally administered vaccines and more recently for the oral, transdermal and ocular delivery of drugs (Dai et al., 2013 and references therein). The focus of the present article was to increase understanding of BS-induced changes in the permeability of liposomes and to provide a basis for use of BS to change cellular permeability. Generally, it is believed that surfactants disrupt and eventually solubilize a lipid bilayer in a three-stage process leading to formation of mixed micelles (Lichtenberg et al., 2013; Lin et al., 2011). In the first stage, detergents rapidly penetrate the outer leaflet and, because of their curvophilic nature, increase its surface area and produce a stressed membrane. In the second stage, they act as either fast (strong) solubilizers (Group A) or slow (weak) solubilizers (Group B) depending on whether they flip-flop rapidly (Group A) or slowly (Group B) into the inner leaflet to reduce the membrane stress. What happens in the third stage depends on surfactant concentration as discussed by Annesini et al. (2000). Above a certain critical concentration, Csat, micelles pinch off the bilayer and, in the case of liposomes, result in a mixture of smaller liposomes and mixed micelles; above a second critical concentration, Csol, complete vesicle bursting occurs with formation of mixed micelles. Surfactants like C, DC and sodium dodecyl sulfate belong to Group B and their slow rate of flip-flop means liposomes exposed to BS are solubilized relatively slowly. Understanding time-dependent BS-induced changes in liposome permeability is therefore a matter of understanding permeability changes induced by submicellar BS concentrations during the first and second stages of the interaction. Studies of changes in the permeability of liposomes induced by surfactants have employed techniques based on the self-quenching of carboxyfluorescein (CF) (Albalak et al., 1996) or calcein (Annesini et al., 2000; Memoli et al., 1999), the dithionite quenching of N-(7-nitrobenz-2oxa-1,3-diazol-4-yl) (NBD)-labeled lipids (Angeletti & Wylie Nichols, 1998; Matsuzaki et al., 1996; Moreno et al., 2006) and the release of inulin (Schubert & Schmidt, 1988). These studies have shown that exposure of liposomes to submicellar concentrations of surfactants in the external media causes the two-stage changes described above whereas similar exposure of liposomes incorporating surfactant during preparation does not (Annesini et al., 2000; Gubernator et al., 1999). The initial rapid increase in permeability has been ascribed to the formation of transient pores in the membrane due to the asymmetry stress produced by surfactant in the outer leaflet (Heerklotz, 2001; Schubert & Schmidt, 1988) and the subsequent reduction in permeability to the annealing of the membrane as the curvature stress is relieved by molecules translocating to the inner leaflet (Elsayed & Cevc, 2011; Hu et al., 2013). In a previous study by our group of CF release from CF-loaded small unilamellar vesicles (SUVs) exposed to BS (Hinow et al., 2012), MKC produced a more prolonged release of CF than C and DC interpreted as arising from its slower rate of flip-flop. TC also increased membrane
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permeability to CF but in a way that appeared to be different from the unconjugated BS. In the present study, we examined the interaction between these four BS and small unilamellar liposomes through determination of BS liposome/pH 7.2 buffer distribution coefficients (Dlipo/buffer) and the effect of BS on vesicle size (VS), zeta potential and membrane fluidity. Dlipo/buffer values give an indication of the relative ability of the BS to bind with and/or insert into liposomes at close to physiological pH. Knowing the concentrations of BS causing sharp reductions in VS ensures sub-micellar concentrations were used in subsequent experiments. We also carried out studies of the effect of BS on the permeability of liposomes to dithionite both during incubation and after equilibration with BS. This used the method first developed by McIntyre & Sleight (1991) which involves fluorescence quenching of liposomal NBD-1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (NBD-PE) by dithionite reduction and, in principle, allows simultaneous determination of liposomal NBD-PE flip-flop and permeability to dithionite.
Materials and methods Materials C, DC and TC (purities 99%, 97% and 97%, respectively) and sodium dithionite were purchased from Sigma–Aldrich (New Zealand). MKC (purity 96.5% with 3.1% C as the major impurity) was a gift from Professor Ksenija Kuhajda (University of Novi Sad, Serbia). Soy phosphatidylcholine (SPC) was donated by Lipoid (GmbH, Ludwigshafen, Germany). Other chemicals [suppliers] were as follows: Diphenylhexatriene (DPH) (97.5%), ammonium thiocyanate (laboratory grade) and trimethylammonium-diphenylhexatriene (TMA-DPH) (98%) [Fluka, Buchs, Switzerland]; Ringer’s buffer (10 mM D-glucose, 0.23 mM MgCl2, 0.45 mM KCl, 120 mM NaCl, 0.70 mM Na2HPO4, 1.5 mM NaH2PO4, pH 7.2), cholesterol (95%) and tetrahydrofuran (THF) (LR grade499%) [Sigma–Aldrich, Milwaukee, WI]; chloroform (HPLC grade) and methanol (analytical grade) [Merck, KGrA 64271 Darmstadt, Germany]; sodium azide (laboratory grade) and ferric chloride hexahydrate (analytical grade) [BDH Chemicals Ltd, Poole, UK]; NBD-PE triethylammonium salt [Invitrogen, New Zealand]; Nucleopore Track-Etch membranes (50, 100 and 200 nm pore size) [Whatman (UK)]. Milli-Q water was used in all studies. Liposome preparation SPC liposomes were prepared by the thin film hydration method and extruded to decrease particle size and produce SUVs with a narrow size distribution. Briefly, SPC (100 mg) and cholesterol (25 mg) (molar ratio 2:1) were dissolved in 5 ml chloroform-methanol (3:1, v/v) in a round-bottomed flask. The organic solvent was removed by rotary evaporation (Rotavapor R110, Bu¨chi) at 35 C for 30 min and the residue kept under vacuum overnight to remove traces of organic solvent. The lipid film was then hydrated with 5 ml Ringer’s buffer in the presence of 0.5 g glass beads at room temperature for 30 min. After hydration, the liposomal suspension was sonicated (bath sonicator, RK100H, Bandelin Electronic,
DOI: 10.3109/08982104.2014.928888
Kinetic and equilibrium studies of bile salt–liposome interactions
Berlin, Germany) for 10 min and then extruded 10 times sequentially through 200, 100 and 50 nm Nucleopore TrackEtch membranes using a Lipex Extruder at room temperature (Northern Lipids, Canada).
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Phospholipid assay SPC concentration was determined using the Stewart assay (O’Connor et al., 1985) after confirming that BS and Ringer’s buffer do not influence color development. Ammonium ferrothiocyanate reagent (0.1 M) was prepared by dissolving 27.03 g ferric chloride hexahydrate and 30.4 g ammonium thiocyanate in 1 l distilled water. A liposome suspension (0.05 ml) was mixed with 2 ml chloroform in a centrifuge tube and 2 ml 0.1 M ammonium ferrothiocyanate reagent added. After vigorous vortexing for 30 s, the sample was centrifuged (Eppendorf 5810R) for 10 min at 2000 rpm. The chloroform phase was then transferred to a quartz cuvette and the optical density measured at 485 nm using a Cary 1 UV-visible spectrophotometer. A calibration curve of SPC in the range 0.005–0.2 mg/ml was shown to be linear (R2 ¼ 0.9997). Molar concentrations of SPC were calculated using a molecular weight of 775. Dlipo/buffer Dlipo/buffer values of BS were determined in triplicate by equilibrium dialysis (Equilibrium Dialyzer with a 12–14 000 Da dialysis membrane, Spectrum Laboratories, Rancho Dominguez, CA). Preliminary studies showed dialysis for 5 h was sufficient to reach equilibrium with no binding of BS to the dialysis cell surface or dialysis membrane. An aliquot (volume V, 0.7 ml) of blank liposomal suspension (20 mM SPC) prepared in Ringer’s buffer was loaded into the receiver compartment and dialyzed against an equal volume (0.7 ml) of BS solution (concentration C0, 10 mM) in the same buffer in the donor compartment. After 5 h rotation at 3 rpm and room temperature (21 C), equilibrium was achieved and an aliquot (100 ml) was withdrawn from the donor compartment and analyzed for BS by liquid chromatography tandem mass spectrometry (Yang et al., 2009). Samples from the donor compartment were also analyzed for SPC in order to confirm the integrity of the dialysis membrane. Dlipo/buffer was calculated using the following equation: Dlipo=buffer ¼
C0 V Caq ð2V VL Þ VL Caq
ð1Þ
where Caq is the measured concentration of BS in the aqueous phase and VL is the volume of the phospholipid phase calculated from the SPC concentration assuming a density of 1.0 g/ml (Kra¨mer et al., 1997). VS and zeta potential VS (Z-average size), dispersity (polydispersity index) and zeta potential of extruded SPC liposomes (0.5 mM SPC) incubated with and without BS (0–10 mM except for DC 0–2 mM) for 2 h in Ringer’s buffer at 25 C were determined by dynamic light scattering (DLS) using a Zetasizer (NanoZS90, Malvern Instruments, Malvern, UK). The values of viscosity and
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refractive index of the dispersion medium were taken as 1.02 cP and 1.330, respectively. Membrane fluidity Liposomal membrane fluidity was studied by steady-state fluorescence polarization (FP) (In’t Veld et al., 1991). Fluorescence probes (DPH and TMA-DPH) were dissolved in THF and added to suspensions of liposomes in Ringer’s buffer (1 mM SPC) with gentle swirling. The lipid:probe molar ratio was 500:1 to avoid changing the structure of the liposome membrane and to allow for complete incorporation of the probes into the lipid bilayer. Suspensions of liposomes were then incubated at room temperature (21 C) for 30 min in the dark after which a BS at concentrations up to 10 mM was added and the suspensions incubated for another 30 min. FP was then measured using a POLARstar microplate fluorometer (BMG Laboratories, Germany) at excitation and emission wavelengths of 350 and 420 nm, respectively. FP [in millipolarization units (mP)] was calculated using the following equation: FP ¼
III I? III þ I?
ð2Þ
where III is the fluorescence intensity parallel to the excitation plane and I? is the fluorescence intensity perpendicular to the excitation plane. A decrease in FP is indicative of an increase in membrane fluidity. Studies with NBD-PE labeled liposomes Symmetric liposomes equilibrated with BS (dithionite permeability) Liposomes with NBD-PE homogeneously distributed in the inner and outer leaflets of the bilayer (symmetric liposomes) were prepared by incorporating NBD-PE (1% of total lipids) with the SPC and cholesterol. They were then equilibrated with sub-micellar concentrations of BS by incubating them in BS solution (C, TC and MKC at 1 mM; DC at 0.2 mM) at room temperature (21 C) for 24 h. Control symmetric liposomes were incubated with buffer. Fluorescence of suspensions (1.0 ml) of these liposomes in Ringer’s buffer (1.0 mM SPC) was monitored continuously using excitation and emission wavelengths of 467 and 530 nm, respectively. Fluorescence was first monitored for 2 min to establish baseline after which 20 ml sodium dithionite solution (20 mM) (freshly prepared in Ringer’s buffer and kept on ice under nitrogen to prevent oxidation) was added and fluorescence monitored continuously for another 5 min. The decline in fluorescence was shown to be biphasic, phase 1 being very rapid due to dithionite reduction of the NBD-PE in the outer leaflet of the membrane and corresponding to quenching of 50% of the initial fluorescence (Armstrong et al., 2003; Matsuzaki et al., 1996). The subsequent phase 2 of the decline is much slower and corresponds to fluorescence quenching of NBD-PE in the inner leaflet by dithionite that penetrates the membrane. Armstrong et al. (2003) showed that the rate of dithionite permeability of LUVs is much faster than the rate of
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phospholipid flip-flop so that phase 2, to a good approximation, is a measure of dithionite permeability.
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Asymmetric liposomes (NBD-PE flip-flop and dithionite permeability) Liposomes with NBD-PE residing exclusively in the inner leaflet (asymmetric liposomes) were prepared by vortexing an aliquot (1 ml) of a suspension of symmetric liposomes in Ringer’s buffer (20 mM SPC) with 20 ml freshly prepared sodium dithionite solution (1 mM) and allowing to stand at room temperature for 2 min. Excess dithionite was removed by passing the liposome suspension through a Sephadex G-50 column and eluting with Ringer’s buffer. A suspension of these asymmetric liposomes (2 mM SPC) was then mixed with a BS solution (C, TC and MKC at 2 mM; DC at 0.4 mM) in a 1:1 volume ratio and incubated at room temperature (21 C). Control liposomes were mixed with buffer. At various times up to 9 h, aliquots (1.0 ml) were removed and, after adding 20 ml sodium dithionite solution (1 mM), allowed to stand for 2 min. Fluorescence intensity was then measured using the same excitation and emission wavelengths as in the continuous monitoring study described above. The only difference between our method and that of Armstrong et al. (2003) is that they added 1.0 M dithionite in Tris buffer (pH 10) to samples and monitored the decline in fluorescence continuously over 10 min to observe both phase 1 and phase 2 of the decline. They were then able to calculate the rates of NBD-PE flip-flop and dithionite permeability of the membrane through non-linear least squares fitting of the data for all samples collected over 2.5 h or more. Matsuzaki et al. (1996) measured fluorescence remaining in samples after exposure to dithionite for only 3 min (a duration short enough in their study to exclude any of the phase 2 decline) and determined the rate of NBD-PE flip-flop from the exponential decline in fluorescence remaining in samples against time of sampling. We measured fluorescence remaining in samples after exposure to dithionite for 2 min with the intention of detecting NBD-PE flip-flop (phase 1 quenching) and any marked increase in dithionite permeability of the lipid bilayer caused by exposure to BS (i.e. the increase in the amount of phase 2 quenching occurring in 2 min). We anticipated the decline in fluorescence remaining would be exponential in control liposomes (NBD-PE flip-flop only) but not in liposomes made more permeable to dithionite by exposure to BS. Statistical analysis Data were analyzed using SPSS 11.0 (Systat Software Inc., San Jose, CA). One-way ANOVA with Tukey’s or Dunnett’s post hoc tests was carried out depending on the results of homogeneity of variance tests for multiple comparisons. Differences for which p50.05 were considered significant.
J Liposome Res, Early Online: 1–9
Table 1. Liposome/pH 7.2 buffer distribution coefficients (log Dlipo/ buffer) (means ± SD, n ¼ 3) of bile salts at 21 C along with their previously determined log Doctanol/buffer and CMC values (Yang et al., 2009). Bile salt DC C MKC TC
Log Dlipo/buffer
Log Doctanol/buffer
CMC (mM)
3.42 ± 0.02 2.50 ± 0.02 0.75 ± 0.06 2.49 ± 0.01
1.46 0.01 0.41 1.50
1.69 4.09 13.4 3.56
MKC5TC & C5DC and are higher than corresponding log Doctanol/buffer values indicating that BS, which are largely ionized at pH 7.2, partition more strongly into liposomes than predicted by their log Doctanol/buffer values. This is consistent with previous work showing that ionized species partition more strongly into lipid membranes than into octanol (Avdeef et al., 1998) and, as expected, is particularly true for the strongly acidic TC. The log Dlipo/buffer values show a reasonable inverse correlation with the BS CMC values as previously observed by Heerklotz & Seelig (2000) for neutral detergents. They also correlate with BS cytotoxicity to rat brain endothelial (RBE4) cells with the exception of TC whose low cytotoxicity was ascribed to the existence of a specific efflux transport system (Yang et al., 2012). Vesicle size The change in VS and dispersity of liposomes after incubation with increasing concentrations of BS for 2 h at 25 C is shown in Figure 1. VS of liposomes in the absence of BS was 58 nm and remained unchanged at low BS concentrations. Dispersity was 0.1 at low BS concentrations indicative of monodisperse samples and similarly remained unchanged at BS51 mM. As concentration increased, C, DC and TC caused marked decreases in VS (DC4TC4C) with concomitant increases in dispersity. DC caused a decrease in VS at a concentration as low as 0.5 mM but MKC had little effect on VS over the whole concentration range (0–10 mM). Zeta potential In the absence of BS, the zeta potential of liposomes was slightly negative (5 mV). Zeta potential after incubation with BS for 2 h at 25 C (Figure 2) became more negative indicating BS are inserting into the lipid bilayer of the liposomes or into the electrical double layer surrounding them (Hunter, 2001). At low BS concentration (50.5 mM), the increase in negativity is in the order MKC5C & TC5DC consistent with the log Dlipo/buffer values. At higher BS concentration, TC caused a more rapid increase in negativity than the other BS possibly because TC accumulates in the Stern layer rather than in the lipid bilayer where its negative form is relatively more stable than those of the other BS which accumulate in the lipid bilayer.
Results Log Dlipo/buffer values
Membrane fluidity
These are given in Table 1 along with previously determined log Doctanol/buffer and CMC values (Yang et al., 2009). The results show that log Dlipo/buffer values increase in the order
Figure 3 shows the effect of incubation with BS for 30 min on the FP of DPH and TMA-DPH in SPC liposomes. FP of the two probes without BS was 200 mP consistent with a
Kinetic and equilibrium studies of bile salt–liposome interactions
DOI: 10.3109/08982104.2014.928888
(A)
(B) 0.6
70
0.5
50 40
C DC MKC TC
30
0.3 0.2
20
0.1
0.1
1
10
0.0 0.01
0.1
1
10
Bile salt concentration (mM)
Bile salt concentration (mM)
Figure 1. Effect of incubation with increasing concentrations of bile salts for 2 h at 25 C on (A) vesicle (Z-average) size and (B) dispersity of SPC liposomes (0.5 mM SPC) (data are means ± SD, n ¼ 3).
0
(A) 210
−5
205 DPH Polarization (mP)
Zeta potential (mV)
−10 −15 −20 −25 −30 0.01
C DC MKC TC
0.1
200 195 190 185 180
1
175 0.01
10
Bile salt concentration (mM)
Figure 2. Zeta potential of SPC liposomes (0.5 mM SPC) after incubation with increasing concentrations of bile salts for 2 h at 25 C (data are means ± SD, n ¼ 3).
previous study of dilinoleoylphosphatidylcholine multilamellar liposomes with a phospholipid:cholesterol ratio of 2:1 (van Blitterswijk et al., 1987). All BS decreased FP of both DPH and TMA-DPH in a concentration-dependent manner indicating BS cause an increase in membrane fluidity at submicellar concentrations in the order MKC5C5TC5DC. This is also the order of the effect of the four BS on membrane fluidity of RBE4 cells as indicated by the FP of TMA-DPH (Yang et al., 2012). NBD-PE flip-flop and dithionite permeability Symmetric liposomes equilibrated with BS Addition of dithionite to symmetric liposomes caused the expected two-phase decline in fluorescence intensity (Figure 4). In phase 1, corresponding to a drop of 50% of the initial fluorescence, the decline was slower for liposomes equilibrated with TC suggesting TC interferes with dithionite reduction of NBD-PE in the outer leaflet of the bilayer.
C DC MKC TC
0.1
1
10
Bile salt concentration (mM) (B) 220 TMA-DPH Polarization (mP)
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C DC MKC TC
0.4 Dispersity
Z-average size (nm)
60
10 0.01
5
C DC MKC TC
210 200 190 180 170 160 150 140 0.01
0.1
1
10
Bile salt concentration (mM)
Figure 3. Membrane fluidity of SPC liposomes (1 mM SPC) as determined by steady-state fluorescence polarization of (A) DPH and (B) TMA-DPH in liposomes incubated with increasing concentrations of bile salts for 30 min at room temperature (21 C) (data are means ± SD, n ¼ 4).
In phase 2, corresponding to quenching of inner leaflet NBD-PE by dithionite that penetrates the membrane and, to a minor extent, to NBD-PE that flips from the inner to the outer leaflet in the short duration of the experiment, dithionite
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J Liposome Res, Early Online: 1–9
Fluorescence intensity remaining (%)
120
Fluorescence %
100 Control 1 mM C 1 mM TC 0.2 mM DC 1 mM MKC
80 60 40 20 50
100
150
200
250
300
350
400
450
Figure 4. Representative plot showing the decrease in fluorescence after addition of 20 ml sodium dithionite solution (20 mM) (at arrow) to 1.0 ml of NBD-PE symmetric liposomes (1 mM SPC) previously equilibrated with bile salts for 24 h. 0.10
80 60 40 20 0
100
200
300
400
500
600
Figure 6. Effect of duration of incubation with bile salts at room temperature (21 C) on the fluorescence of asymmetric liposomes (1 mM SPC) labeled with NBD-PE in the inner leaflet. Each point is the fluorescence of a sample (1 ml) after removal from the incubation and treatment with 20 ml dithionite (1.0 mM) for 2 min, i.e. each point indicates the NBD-PE remaining in the inner leaflet (data are means ± SD, n ¼ 3).
0.08
0.06
0.04
0.02
KC M m 1
2
m
M
M
D
TC 0.
1
m
M
C 1
m
M
tro on C
C
0.00 l
Dithionite permeation rate constant (% s−1)
100
Time (min)
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1mM C 0.2 mM DC 1 mM MKC 1 mM TC Control
Figure 5. Dithionite permeability zero-order rate constants (based on phase 2 of the decline in fluorescence shown in Figure 4) for NBD-PE symmetric liposomes (1 mM SPC) previously equilibrated with bile salts (data are means ± SD, n ¼ 3).
permeability rate constants (zero order) were determined from the slopes over the period 240–420 s. The values shown in Figure 5 indicate that liposomes equilibrated with BS are more permeable to dithionite than control liposomes (ANOVA, p50.05) with MKC having the smallest effect followed by C and TC and finally DC (at 0.2 mM compared to 1 mM for the other BS) which has the largest effect. Surprisingly, despite TC equilibrated liposomes showing a slower rate of NBD-PE fluorescence quenching in phase 1 (outer leaflet), NBD-PE fluorescence quenching of TC equilibrated liposomes in phase 2 (inner leaflet) was not slower than in C and DC equilibrated liposomes Asymmetric liposomes In the case of control liposomes, NBD-PE remaining in the inner leaflet in samples removed at various times and treated with dithionite for 2 min declined slowly and exponentially with a rate of 0.0035 min1 (5.8 105 s1) (Figure 6). This decline is due to NBD-PE flip-flop from the inner to the outer leaflet and is 15 times slower than the rate of
dithionite permeability of control liposomes shown in Figure 5 (0.04% s1 equivalent to a first order rate constant of 0.0008 s1). In comparison, Armstrong et al. (2003) found flip-flop was 200 times slower than dithionite permeability in NBD-PE labeled LUVs. If NBD-PE flip-flop is the only process that contributes to fluorescence quenching, the decline in fluorescence should continue to a limit of 50% of the initial value. Matsuzaki et al. (1996) showed this to be the case using LUVs where the rate constant for NBD-PE flip-flop at 30 C was 0.012 min1. In the case of liposomes incubated with C, DC or MKC, the decrease in fluorescence remaining in samples was dramatic (Figure 6). Thus, fluorescence remaining at 30 min in liposomes incubated with DC, C or MKC was 33%, 46% and 68%, respectively, of the initial value compared with 90% and 88%, respectively, for liposomes incubated with TC or buffer (control). The fact that for DC fluorescence remaining in samples was well below 50% of the initial value (i.e. below the level attainable by NBD-PE flip-flop alone) and that for C, DC and MKC fluorescence remaining in samples was higher over the subsequent 200–300 min than at 30 min, together indicate that the rapid initial decrease in fluorescence remaining in samples is due primarily to the high permeability of the bilayer to dithionite when BS is asymmetrically distributed (BSouter leaflet4BSinner leaflet). The subsequent recovery of fluorescence remaining is then due to a decrease in permeability of the bilayer as BS attain equilibrium in the two leaflets via BS flip-flop. Thus, fluorescence remaining in samples at 300 min for liposomes incubated with DC, C, MKC, TC or control was 53%, 59%, 72%, 67% and 71%, respectively, of the initial value. These decreases are due to flip-flop of NBD-PE over 300 min (plus a small contribution due to dithionite permeability of the liposomes during the 2-min treatment of the samples) the rate of which increases in the order control & MKC5TC5C5DC. The decrease in fluorescence should continue to a limit of 50% when NBD-PE reaches equilibrium between the two leaflets.
Kinetic and equilibrium studies of bile salt–liposome interactions
For liposomes incubated with TC, fluorescence remaining in samples declined slowly with a rate only slightly faster than in samples of control liposomes but appeared to plateau after 180 min. This difference in behavior of TC compared with the other BS is consistent with our previous report on the effect of the four BS on the release of CF from CF-loaded SUV liposomes (Hinow et al., 2012). The behavior implies that TC increases liposome permeability to dithionite to only a small extent and the decline in fluorescence remaining is primarily due to NBD-PE flip-flop.
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Discussion BS-induced changes in lipid bilayers at sub-micellar concentrations result from partitioning of BS into the lipid bilayer in accordance with their log Dlipo/buffer values. DC with two hydroxyl groups has the highest value followed by C and TC (trihydroxy BS) with similar values. MKC has the lowest log Dlipo/buffer indicating that replacement of the hydroxyl group in C by a keto group significantly reduces the ability to partition into membranes. The log Dlipo/buffer values of the four BS follow the same trend as the log Doctanol/buffer values with the exception of TC which has a markedly positive log Dlipo/buffer compared with its negative log Doctanol/ buffer. Presumably the relatively high concentration of the ionized TC that results from its much lower pKa gives it a much greater affinity for the phospholipid bilayer. Determination of VS is a direct method to monitor BSinduced solubilization of liposomes and was used here to ensure sub-micellar concentrations of BS were used in subsequent studies. DC and TC caused significant reductions in VS at concentrations below CMC consistent with a mechanism in which their insertion into the membrane leads to formation of lipid-BS mixed micelles. The process can be seen as the result of an increase in curvature of the membrane arising from insertion of curvophilic BS into the outer leaflet with subsequent shedding of the bilayer surface. MKC had no effect on VS at sub-micellar concentrations suggesting it is much less curvophilic than the other BS. In terms of zeta potential, incorporation of BS increased the negativity of liposomes consistent with the fact that BS are mainly in the negatively charged form at pH 7.2. In terms of membrane fluidity, many studies have shown that BS interact with liposomes to increase their flexibility presumably because the excess area resulting from BS binding leads to softening of the bilayer. Increased membrane fluidity was observed here with DC causing the greatest effect and MKC the least effect on membrane fluidity of both polar and non-polar regions of the lipid bilayer. MKC appeared to have a greater effect on polar regions as previously suggested by the results of our study in Caco-2 cells where MKC had a lower effect than C on the FP of DPH but a much greater effect than C on the FP of TMA-DPH (Chen et al., 2012). However, this indication that MKC is mainly located in the polar regions of the lipid bilayer is based on the premise that the two probes respond equally to changes in lipid packing, and, although they have the same fluorophore, this cannot be guaranteed. In the study of membrane permeability using NBD-PE labeled symmetric liposomes, liposomes equilibrated with BS
(B) Permeability of liposome membrane
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Bile salt
(C) (A)
Time
Figure 7. Schematic showing the time-dependence of the effect of a bile salt on the permeability of a liposome. Initial exposure causes a dramatic increase in permeability as the BS penetrates the outer leaflet of the lipid bilayer (B versus A). Prolonged incubation allows the BS to flip-flop until equilibrium is reached when the liposome is only slightly more permeable than before exposure to BS (C versus A).
were more permeable to dithionite than control (Figures 4 and 5). Some researchers have postulated that this increase in permeability is the result of BS inserting longitudinally into the membrane and aggregating as dimers or larger structures to form membrane pores (Carey, 1985) but others have shown this is not the case, at least for transport of small non-ionic solutes (Albalak et al., 1996). In fact, it has been suggested that, in interacting with lipid bilayers, BS orientate themselves parallel to the interface rather than inserting into the bilayer longitudinally (Fahey et al., 1995). Disturbance of the lipid packing in the membrane is then presumed to cause its increased permeability. Although membranes equilibrated with MKC were less permeable to dithionite than membranes equilibrated with the other BS (Figure 5), the MKC concentration in the membrane is lower based on its lower log Dlipo/ buffer suggesting MKC is more disruptive to the membrane than the other BS. In the study of NBD-PE labeled asymmetric liposomes, the changes in fluorescence during incubation with C, DC or MKC are consistent with a bilayer made initially permeable to dithionite by BS incorporation into the outer leaflet resulting in a ‘‘stressed’’ membrane followed by a return to a less permeable membrane as BS flip-flop into the inner leaflet of the bilayer until an equilibrium between BS in the inner and outer leaflets is achieved (Donovan & Jackson, 1997; Luk et al., 1997). It is reported that the half-life of flip-flop of C and DC in liposomal membranes in the liquid crystalline state at 60 C is 4–5 min (Hildebrand et al., 2002). Given an activation energy of flip-flop of 79 kJ mol1 (Liu & Conboy, 2005), the Arrhenius equation predicts a half-life of BS flip-flop at room temperature (21 C) of 180 min. The fact that this is similar to the time required for membrane impermeability to be restored during incubation with BS supports the hypothesis that an unequal distribution of BS in the two leaflets of the lipid bilayer leads to increased permeability. The time-dependence of the effect of BS on the permeability of a liposome is shown schematically in Figure 7. The reason the initial rapid increase in membrane permeability did not occur for liposomes incubated with TC may be
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because the rate of TC insertion into the outer leaflet is similar to its rate of flip-flop to the inner leaflet. This would mean the concentration of TC in the outer leaflet never exceeds that in the inner leaflet (i.e. liposomes would never be asymmetric with respect to TC), the liposomes remain impermeable to dithionite and the decline in fluorescence remaining is due to NBD-PE flip-flop only. This hypothesis is consistent with the fact that TC has been shown to undergo very slow flip-flop in egg phosphatidylcholine LUVs (Donovan & Jackson, 1997) and SUVs (Kamp & Hamilton, 1993) and would suggest that the rate of insertion of TC into the outer leaflet of liposomes is slower than that of the other BS. Interestingly, the fact that incubation of liposomes with 1 mM TC for 30 min (the time of maximum permeability to dithionite) increases their membrane fluidity more than incubation with 1 mM C or MKC (Figure 3), suggests BS induce increases in membrane fluidity irrespective of whether they are symmetrically or asymmetrically distributed in the lipid bilayer. The time-dependent changes in permeability of the lipid bilayer to dithionite are consistent with the fact that bilosomes are resistant to physical destruction by extrinsic BS (Rowland & Woodley, 1980; Schubert et al., 1983). Although incorporation of BS into the lipid bilayer increases its permeability to a small extent (Figure 7, C versus A), bilosomes are resistant to the rapid increase in permeability on first exposure to BS (Figure 7, B versus A). Further research is needed to determine how the time-dependence of permeabilization depends on BS structure, what level of BS provides optimum stabilization of the membrane to the initial shock of BS exposure and whether bilosomes incorporating one BS are stable to subsequent exposure to another. Studies are also required to establish whether bilosomes give rise to greater transport of encapsulated drugs across the BBB than liposomes, i.e. whether the BS present in the liposome membrane acts as a permeation enhancer at the BBB.
Conclusion The time-dependence of the interaction of sub-micellar concentrations of conjugated and unconjugated BS with SPC liposomal membranes appears to be different. The unconjugated BS, C, DC and MKC, insert into the membrane in the order of their liposome/pH 7.2 buffer partition coefficients MKC5C5DC. Insertion into the outer leaflet occurs rapidly and produces a stressed membrane with increased permeability while subsequent flip-flop of BS to the inner leaflet occurs slowly and is accompanied by an increase in the rate of phospholipid flip-flop that together relieve the strain and restore membrane impermeability. The conjugated BS, TC, inserts more slowly into the outer leaflet and undergoes flip-flop into the inner leaflet at an equal rate which, together with slow phospholipid flip-flop, avoids formation of a stressed membrane and avoids the highly permeable transient stage. MKC appears to cause the least disturbance to liposomal membranes but only because it has the lowest distribution coefficient. This information may be useful in the design of drug delivery systems that create transient increases in the permeability of biological barriers.
J Liposome Res, Early Online: 1–9
Acknowledgements L.Y. thanks the University of Otago for providing a Postgraduate Publishing Bursary.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
References Albalak A, Zeidel ML, Zucker SD, et al. (1996). Effects of submicellar bile salt concentrations on biological membrane permeability to low molecular weight non-ionic solutes. Biochemistry 35:7936–45. Angeletti C, Wylie Nichols J. (1998). Dithionite quenching rate measurement of the inside-outside membrane bilayer distribution of 7-nitrobenz-2-oxa-1,3-diazol-4-yl-labeled phospholipid. Biochemistry 37:15114–19. Annesini MC, Memoli A, Petralito S. (2000). Kinetics of surfactantinduced release from liposomes: a time-dependent permeability model. J Membr Sci 180:121–31. Armstrong VT, Brzustowicz MR, Wassall SR, et al. (2003). Rapid flipflop in polyunsaturated (docosahexaenoate) phospholipid membranes. Arch Biochem Biophys 414:74–82. Avdeef A, Box KJ, Comer JEA, et al. (1998). pH-Metric logP. Determination of liposomal membrane-water partition coefficients of ionizable drugs. Pharm Res 15:209–15. Carey MC. (1985). Physico-chemical properties of bile acids and their salts. In: Danielsson H, Sjo¨vall J, eds. Sterols and bile acids. 1st ed. Amsterdam: Elsevier, 345–403. Chen G, Yang L, Zhang H, et al. (2012). Effect of ketocholate derivatives on methotrexate uptake in Caco-2 cell monolayers. Int J Pharm 433: 89–93. Chen X, Oshima T, Tomita T, et al. (2011). Acidic bile salts modulate the squamous epithelial barrier function by modulating tight junction proteins. Am J Physiol Gastrointest Liver Physiol 301:G203–9. Dai Y, Zhou R, Liu L, et al. (2013). Liposomes containing bile salts as novel ocular delivery systems for tacrolimus (FK506): in vitro characterization and improved corneal permeation. Int J Nanomed 8:1921–33. Donovan JM, Jackson AA. (1997). Transbilayer movement of fully ionized taurine-conjugated bile salts depends upon bile salt concentration, hydrophobicity, and membrane cholesterol content. Biochemistry 36:11444–51. Elsayed MMA, Cevc G. (2011). The vesicle-to-micelle transformation of phospholipid-cholate mixed aggregates: a state of the art analysis including membrane curvature effects. Biochim Biophys Acta 1808: 140–53. Fahey DA, Carey MC, Donovan JM. (1995). Bile acid/phosphatidylcholine interactions in mixed monomolecular layers: differences in condensation effects but not interfacial orientation between hydrophobic and hydrophilic bile acid species. Biochemistry 34:10886–97. Gordon GS, Moses AC, Silver RD, et al. (1985). Nasal absorption of insulin: enhancement by hydrophobic bile salts. Proc Natl Acad Sci USA 82:7419–23. Gubernator J, Stasiuk M, Kosubek A. (1999). Dual effect of alkylresorcinols, natural amphiphilic compounds, upon liposomal permeability. Biochim Biophys Acta 1418:253–60. Heerklotz H. (2001). Membrane stress and permeabilization induced by asymmetric incorporation of compounds. Biophys J 81: 184–95. Heerklotz H, Seelig J. (2000). Correlation of the membrane/water partition coefficients of detergents with the critical micelle concentration. Biophys J 78:2435–40. Hildebrand A, Neubert R, Garidel P, Blume A. (2002). Bile salt induced solubilization of synthetic phosphatidylcholine vesicles studied by isothermal titration calorimetry. Langmuir 18:2836–47. Hinow P, Radunskaya A, Tucker I, Yang L. (2012). Kinetics of bile salt binding to liposomes revealed by carboxyfluorescein release and mathematical modeling. J Liposome Res 22:237–44. Hu S, Niu M, Hu F, et al. (2013). Integrity and stability of oral liposomes containing bile salts studied in simulated and ex vivo gastrointestinal media. Int J Pharm 441:693–700.
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Hunter RJ. (2001). Foundations of colloid science. 2nd ed. Chapter 7. Oxford, UK:Oxford University Press. In’t Veld G, Driessen AJ, Op den Kamp JA, Konings WN. (1991). Hydrophobic membrane thickness and lipid-protein interactions of the leucine transport system of Lactococcus lactis. Biochim Biophys Acta 1065:203–12. Kamp F, Hamilton JA. (1993). Movement of fatty acids, fatty acid analogues, and bile acids across phospholipid bilayers. Biochemistry 32:11074–86. Kra¨mer SD, Jakits-Deiser C, Wunderli-Allenspach H. (1997). Free fatty acids cause pH-dependent changes in drug-lipid membrane interactions around physiological pH. Pharm Res 14:827–32. Lichtenberg D, Ahyayauch H, Gon˜i FM. (2013). The mechanism of detergent solubilization of lipid bilayers. Biophys J 105:289–99. Lin CM, Chang GP, Tsao HK, Sheng YJ. (2011). Solubilization mechanism of vesicles by surfactants: effect of hydrophobicity. J Chem Phys 135:045102 (1–10). Liu J, Conboy JC. (2005). 1,2-Diacyl-phosphatidylcholine flip-flop measured directly by sum-frequency vibrational spectroscopy. Biophys J 89:2522–32. Luk AS, Kaler EW, Lee SP. (1997). Structural mechanisms of bile saltinduced growth of small unilamellar cholesterol-lecithin vesicles. Biochemistry 36:5633–44. Matsuzaki K, Murase O, Fujii ND, Miyajima K. (1996). An antimicrobial peptide, magainin 2, induced rapid flip-flop of phospholipids coupled with pore formation and peptide translocation. Biochemistry 35:11361–8. McIntyre JC, Sleight RG. (1991). Fluorescence assay for phospholipid membrane asymmetry. Biochemistry 30:11819–27. Memoli A, Annesini MC, Petralito S. (1999). Surfactant-induced leakage from liposomes: a comparison among different lecithin vesicles. Int J Pharm 184:227–35. Mikov M, Fawcett JP, Kuhajda K, Kevresan S. (2006). Pharmacology of bile acids and their derivatives. Eur J Drug Metab Pharmacokinet 31: 237–516. Mikov M, Kevresan S, Kuhajda K, et al. (2004). 3Alpha,7alphadihydroxy-12-oxo-5beta-cholanate as blood-brain barrier permeator. Pol J Pharmacol 56:367–71.
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Moreno MJ, Estronca LMBB, Vaz WLC. (2006). Translocation of phospholipids and dithionite permeability in liquid-ordered and liquid-disordered membranes. Biophys J 91:873–81. Morimoto K, Nakai T, Morisaka K. (1987). Evaluation of permeability enhancement of hydrophilic compounds and macromolecular compounds by bile salts through rabbit corneas in-vitro. J Pharm Pharmacol 39:124–6. Naveen C, Kiran Kumar Y, Venkateshwar Rao P, Rama Rao T. (2011). Chemical enhancers in buccal and sublingual delivery. Int J Pharm Sci Nanotech 4:1307–19. O’Connor CJ, Wallace RG, Iwamoto K. (1985). Bile salt damage of egg phosphatidylcholine liposomes. Biochim Biophys Acta 817:95–102. Raimondi F, Santori P, Barone MV, et al. (2008). Bile acids modulate tight junction structure and barrier function of Caco-2 monolayers. Am J Physiol Gastrointest Liver Physiol 294:G906–13. Rowland RN, Woodley JF. (1980). The stability of liposomes in vitro to pH, bile salts and pancreatic lipase. Biochim Biophys Acta 620:400–9. Schubert R, Jaroni H, Schoelmerich J, Schmidt KH. (1983). Studies on the mechanism of bile salt-induced liposomal membrane damage. Digestion 28:181–90. Schubert R, Schmidt KH. (1988). Structural changes in vesicle membranes and mixed micelles of various lipid compositions after binding of different bile salts. Biochemistry 27:8787–94. Spigelman MK, Zappula RA, Malis LI, et al. (1983). Intracarotid dehydrocholate infusion: a new method for prolonged reversible blood brain barrier disruption. Neurosurgery 12:606–12. van Blitterswijk WJ, van der Meer BW, Hilkmann H. (1987). Quantitative contributions of cholesterol and the individual classes of phospholipids and their degree of fatty acyl (un)saturation to membrane fluidity measured by fluorescence polarization. Biochemistry 26:1746–56. Yang L, Fawcett JP, Østergaard J, et al. (2012). Mechanistic studies of the effect of bile salts on rhodamine 123 uptake into RBE4 cells. Mol Pharm 9:29–36. Yang L, Zhang H, Mikov M, Tucker IG. (2009). Physicochemical and biological characterization of monoketocholic acid, a novel permeability enhancer. Mol Pharm 6:448–56.
RESEARCH ARTICLE
Kinetic and equilibrium studies of bile salt–liposome interactions Lin Yang, Feifei Feng, J. Paul Fawcett, and Ian G. Tucker
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School of Pharmacy, University of Otago, Dunedin, New Zealand
Abstract
Keywords
Research has suggested that exposure to sub-micellar concentrations of bile salts (BS) increases the permeability of lipid bilayers in a time-dependent manner. In this study, incubation of soy phosphatidylcholine small unilamellar vesicles (liposomes) with sub-micellar concentrations of cholate (C), deoxycholate (DC), 12-monoketocholate (MKC) or taurocholate (TC) in pH 7.2 buffer increased membrane fluidity and negative zeta potential in the order of increasing BS liposome-pH 7.2 buffer distribution coefficients (MKC5C & TC5DC). In liposomes labeled with the dithionite-sensitive fluorescent lipid N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine (NBD-PE) in both leaflets and equilibrated with sub-micellar concentrations of BS, fluorescence decline during continuous exposure to dithionite was biphasic involving a rapid initial phase followed by a slower second phase. Membrane permeability to dithionite as measured by the rate of the second phase increased in the order control5MKC5TC C5DC. In liposomes labeled with NBD-PE in the inner leaflet only and incubated with the same concentrations of C, DC and MKC, membrane permeability to dithionite initially increased very rapidly in the order MKC5C5DC before impermeability to dithionite was restored after which fluorescence decline was consistent with NBD-PE flip-flop. For liposomes incubated with TC, membrane permeability to dithionite was only slightly increased and the decline in fluorescence was mainly the result of NBD-PE flip-flop. These results provide evidence that BS interact with lipid bilayers in a time-dependent manner that is different for conjugated and unconjugated BS. MKC appears to cause least disturbance to liposomal membranes but, when the actual MKC concentration in liposomes is taken into account, MKC is actually the most disruptive.
Bile salts, distribution coefficient, dithionite, liposomes, membrane fluidity, membrane permeability
Introduction Bile acids are facially amphiphilic compounds in which 1–3 hydroxyl groups and an acidic moiety form the hydrophilic face and the steroidal backbone the hydrophobic face. They are present in vivo in the form of bile salts (BS) in which the carboxylic acid group is either unconjugated or conjugated with glycine or taurine. BS are frequently used in drug formulations either to increase the solubility and dissolution rate of poorly water-soluble drugs or to increase the permeability of drugs across biological membranes (Gordon et al., 1985; Morimoto et al., 1987). Usual explanations for this increased membrane permeability include the ability of BS to break down mucous and, at higher concentrations, to enhance paracellular transport by opening tight junctions between cells (Chen et al., 2011; Raimondi et al., 2008). Less well appreciated is the fact that BS at concentrations below their critical micelle concentration (CMC) (i.e. at submicellar concentrations) can enhance transcellular transport by disrupting the phospholipid cell membrane and fluidizing
Address for correspondence: J. Paul Fawcett, School of Pharmacy, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand. Tel: +64 3 479 5119. Fax: +64 3 479 7034. E-mail: [email protected]
History Received 12 March 2014 Revised 13 May 2014 Accepted 25 May 2014 Published online 24 June 2014
it through changing the lipid packing density (Mikov et al., 2006). Natural BS have been applied as permeation enhancers despite the fact that the more hydrophobic BS like deoxycholate (DC) are relatively cytotoxic at low concentrations (Naveen et al., 2011). In contrast, semi-synthetic ketocholate derivatives are less cytotoxic to the extent that, in rat, triketocholate (also called dehydrocholate) has been shown to open the blood brain barrier (BBB) in vivo (Spigelman et al., 1983) and 12-monoketocholate (MKC) has been shown to increase the analgesic effect of morphine and the brain uptake of quinine with minimal cytotoxicity (Mikov et al., 2004). We have previously compared some physicochemical and biological properties of MKC with those of cholate (C), DC and taurocholate (TC) and shown that MKC has a much higher CMC value than the other BS and causes less disruption to erythrocytes and Caco-2 cell monolayers (Yang et al., 2009). Liposomes are a useful model of the lipid bilayer of cells and as such are useful to study passive diffusion of drugs through the cell membrane. Understanding how they are solubilized by surfactants is also important in order to facilitate the development of liposomal drug delivery systems. Of particular relevance to the current study is the fact that
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incorporation of BS into the lipid bilayer of liposomes has been shown to stabilize them against membrane damage and solubilization by extrinsic BS in the gastrointestinal tract (Hu et al., 2013 and references therein). These so-called bilosomes have been used to improve the delivery of orally administered vaccines and more recently for the oral, transdermal and ocular delivery of drugs (Dai et al., 2013 and references therein). The focus of the present article was to increase understanding of BS-induced changes in the permeability of liposomes and to provide a basis for use of BS to change cellular permeability. Generally, it is believed that surfactants disrupt and eventually solubilize a lipid bilayer in a three-stage process leading to formation of mixed micelles (Lichtenberg et al., 2013; Lin et al., 2011). In the first stage, detergents rapidly penetrate the outer leaflet and, because of their curvophilic nature, increase its surface area and produce a stressed membrane. In the second stage, they act as either fast (strong) solubilizers (Group A) or slow (weak) solubilizers (Group B) depending on whether they flip-flop rapidly (Group A) or slowly (Group B) into the inner leaflet to reduce the membrane stress. What happens in the third stage depends on surfactant concentration as discussed by Annesini et al. (2000). Above a certain critical concentration, Csat, micelles pinch off the bilayer and, in the case of liposomes, result in a mixture of smaller liposomes and mixed micelles; above a second critical concentration, Csol, complete vesicle bursting occurs with formation of mixed micelles. Surfactants like C, DC and sodium dodecyl sulfate belong to Group B and their slow rate of flip-flop means liposomes exposed to BS are solubilized relatively slowly. Understanding time-dependent BS-induced changes in liposome permeability is therefore a matter of understanding permeability changes induced by submicellar BS concentrations during the first and second stages of the interaction. Studies of changes in the permeability of liposomes induced by surfactants have employed techniques based on the self-quenching of carboxyfluorescein (CF) (Albalak et al., 1996) or calcein (Annesini et al., 2000; Memoli et al., 1999), the dithionite quenching of N-(7-nitrobenz-2oxa-1,3-diazol-4-yl) (NBD)-labeled lipids (Angeletti & Wylie Nichols, 1998; Matsuzaki et al., 1996; Moreno et al., 2006) and the release of inulin (Schubert & Schmidt, 1988). These studies have shown that exposure of liposomes to submicellar concentrations of surfactants in the external media causes the two-stage changes described above whereas similar exposure of liposomes incorporating surfactant during preparation does not (Annesini et al., 2000; Gubernator et al., 1999). The initial rapid increase in permeability has been ascribed to the formation of transient pores in the membrane due to the asymmetry stress produced by surfactant in the outer leaflet (Heerklotz, 2001; Schubert & Schmidt, 1988) and the subsequent reduction in permeability to the annealing of the membrane as the curvature stress is relieved by molecules translocating to the inner leaflet (Elsayed & Cevc, 2011; Hu et al., 2013). In a previous study by our group of CF release from CF-loaded small unilamellar vesicles (SUVs) exposed to BS (Hinow et al., 2012), MKC produced a more prolonged release of CF than C and DC interpreted as arising from its slower rate of flip-flop. TC also increased membrane
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permeability to CF but in a way that appeared to be different from the unconjugated BS. In the present study, we examined the interaction between these four BS and small unilamellar liposomes through determination of BS liposome/pH 7.2 buffer distribution coefficients (Dlipo/buffer) and the effect of BS on vesicle size (VS), zeta potential and membrane fluidity. Dlipo/buffer values give an indication of the relative ability of the BS to bind with and/or insert into liposomes at close to physiological pH. Knowing the concentrations of BS causing sharp reductions in VS ensures sub-micellar concentrations were used in subsequent experiments. We also carried out studies of the effect of BS on the permeability of liposomes to dithionite both during incubation and after equilibration with BS. This used the method first developed by McIntyre & Sleight (1991) which involves fluorescence quenching of liposomal NBD-1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (NBD-PE) by dithionite reduction and, in principle, allows simultaneous determination of liposomal NBD-PE flip-flop and permeability to dithionite.
Materials and methods Materials C, DC and TC (purities 99%, 97% and 97%, respectively) and sodium dithionite were purchased from Sigma–Aldrich (New Zealand). MKC (purity 96.5% with 3.1% C as the major impurity) was a gift from Professor Ksenija Kuhajda (University of Novi Sad, Serbia). Soy phosphatidylcholine (SPC) was donated by Lipoid (GmbH, Ludwigshafen, Germany). Other chemicals [suppliers] were as follows: Diphenylhexatriene (DPH) (97.5%), ammonium thiocyanate (laboratory grade) and trimethylammonium-diphenylhexatriene (TMA-DPH) (98%) [Fluka, Buchs, Switzerland]; Ringer’s buffer (10 mM D-glucose, 0.23 mM MgCl2, 0.45 mM KCl, 120 mM NaCl, 0.70 mM Na2HPO4, 1.5 mM NaH2PO4, pH 7.2), cholesterol (95%) and tetrahydrofuran (THF) (LR grade499%) [Sigma–Aldrich, Milwaukee, WI]; chloroform (HPLC grade) and methanol (analytical grade) [Merck, KGrA 64271 Darmstadt, Germany]; sodium azide (laboratory grade) and ferric chloride hexahydrate (analytical grade) [BDH Chemicals Ltd, Poole, UK]; NBD-PE triethylammonium salt [Invitrogen, New Zealand]; Nucleopore Track-Etch membranes (50, 100 and 200 nm pore size) [Whatman (UK)]. Milli-Q water was used in all studies. Liposome preparation SPC liposomes were prepared by the thin film hydration method and extruded to decrease particle size and produce SUVs with a narrow size distribution. Briefly, SPC (100 mg) and cholesterol (25 mg) (molar ratio 2:1) were dissolved in 5 ml chloroform-methanol (3:1, v/v) in a round-bottomed flask. The organic solvent was removed by rotary evaporation (Rotavapor R110, Bu¨chi) at 35 C for 30 min and the residue kept under vacuum overnight to remove traces of organic solvent. The lipid film was then hydrated with 5 ml Ringer’s buffer in the presence of 0.5 g glass beads at room temperature for 30 min. After hydration, the liposomal suspension was sonicated (bath sonicator, RK100H, Bandelin Electronic,
DOI: 10.3109/08982104.2014.928888
Kinetic and equilibrium studies of bile salt–liposome interactions
Berlin, Germany) for 10 min and then extruded 10 times sequentially through 200, 100 and 50 nm Nucleopore TrackEtch membranes using a Lipex Extruder at room temperature (Northern Lipids, Canada).
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Phospholipid assay SPC concentration was determined using the Stewart assay (O’Connor et al., 1985) after confirming that BS and Ringer’s buffer do not influence color development. Ammonium ferrothiocyanate reagent (0.1 M) was prepared by dissolving 27.03 g ferric chloride hexahydrate and 30.4 g ammonium thiocyanate in 1 l distilled water. A liposome suspension (0.05 ml) was mixed with 2 ml chloroform in a centrifuge tube and 2 ml 0.1 M ammonium ferrothiocyanate reagent added. After vigorous vortexing for 30 s, the sample was centrifuged (Eppendorf 5810R) for 10 min at 2000 rpm. The chloroform phase was then transferred to a quartz cuvette and the optical density measured at 485 nm using a Cary 1 UV-visible spectrophotometer. A calibration curve of SPC in the range 0.005–0.2 mg/ml was shown to be linear (R2 ¼ 0.9997). Molar concentrations of SPC were calculated using a molecular weight of 775. Dlipo/buffer Dlipo/buffer values of BS were determined in triplicate by equilibrium dialysis (Equilibrium Dialyzer with a 12–14 000 Da dialysis membrane, Spectrum Laboratories, Rancho Dominguez, CA). Preliminary studies showed dialysis for 5 h was sufficient to reach equilibrium with no binding of BS to the dialysis cell surface or dialysis membrane. An aliquot (volume V, 0.7 ml) of blank liposomal suspension (20 mM SPC) prepared in Ringer’s buffer was loaded into the receiver compartment and dialyzed against an equal volume (0.7 ml) of BS solution (concentration C0, 10 mM) in the same buffer in the donor compartment. After 5 h rotation at 3 rpm and room temperature (21 C), equilibrium was achieved and an aliquot (100 ml) was withdrawn from the donor compartment and analyzed for BS by liquid chromatography tandem mass spectrometry (Yang et al., 2009). Samples from the donor compartment were also analyzed for SPC in order to confirm the integrity of the dialysis membrane. Dlipo/buffer was calculated using the following equation: Dlipo=buffer ¼
C0 V Caq ð2V VL Þ VL Caq
ð1Þ
where Caq is the measured concentration of BS in the aqueous phase and VL is the volume of the phospholipid phase calculated from the SPC concentration assuming a density of 1.0 g/ml (Kra¨mer et al., 1997). VS and zeta potential VS (Z-average size), dispersity (polydispersity index) and zeta potential of extruded SPC liposomes (0.5 mM SPC) incubated with and without BS (0–10 mM except for DC 0–2 mM) for 2 h in Ringer’s buffer at 25 C were determined by dynamic light scattering (DLS) using a Zetasizer (NanoZS90, Malvern Instruments, Malvern, UK). The values of viscosity and
3
refractive index of the dispersion medium were taken as 1.02 cP and 1.330, respectively. Membrane fluidity Liposomal membrane fluidity was studied by steady-state fluorescence polarization (FP) (In’t Veld et al., 1991). Fluorescence probes (DPH and TMA-DPH) were dissolved in THF and added to suspensions of liposomes in Ringer’s buffer (1 mM SPC) with gentle swirling. The lipid:probe molar ratio was 500:1 to avoid changing the structure of the liposome membrane and to allow for complete incorporation of the probes into the lipid bilayer. Suspensions of liposomes were then incubated at room temperature (21 C) for 30 min in the dark after which a BS at concentrations up to 10 mM was added and the suspensions incubated for another 30 min. FP was then measured using a POLARstar microplate fluorometer (BMG Laboratories, Germany) at excitation and emission wavelengths of 350 and 420 nm, respectively. FP [in millipolarization units (mP)] was calculated using the following equation: FP ¼
III I? III þ I?
ð2Þ
where III is the fluorescence intensity parallel to the excitation plane and I? is the fluorescence intensity perpendicular to the excitation plane. A decrease in FP is indicative of an increase in membrane fluidity. Studies with NBD-PE labeled liposomes Symmetric liposomes equilibrated with BS (dithionite permeability) Liposomes with NBD-PE homogeneously distributed in the inner and outer leaflets of the bilayer (symmetric liposomes) were prepared by incorporating NBD-PE (1% of total lipids) with the SPC and cholesterol. They were then equilibrated with sub-micellar concentrations of BS by incubating them in BS solution (C, TC and MKC at 1 mM; DC at 0.2 mM) at room temperature (21 C) for 24 h. Control symmetric liposomes were incubated with buffer. Fluorescence of suspensions (1.0 ml) of these liposomes in Ringer’s buffer (1.0 mM SPC) was monitored continuously using excitation and emission wavelengths of 467 and 530 nm, respectively. Fluorescence was first monitored for 2 min to establish baseline after which 20 ml sodium dithionite solution (20 mM) (freshly prepared in Ringer’s buffer and kept on ice under nitrogen to prevent oxidation) was added and fluorescence monitored continuously for another 5 min. The decline in fluorescence was shown to be biphasic, phase 1 being very rapid due to dithionite reduction of the NBD-PE in the outer leaflet of the membrane and corresponding to quenching of 50% of the initial fluorescence (Armstrong et al., 2003; Matsuzaki et al., 1996). The subsequent phase 2 of the decline is much slower and corresponds to fluorescence quenching of NBD-PE in the inner leaflet by dithionite that penetrates the membrane. Armstrong et al. (2003) showed that the rate of dithionite permeability of LUVs is much faster than the rate of
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phospholipid flip-flop so that phase 2, to a good approximation, is a measure of dithionite permeability.
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Asymmetric liposomes (NBD-PE flip-flop and dithionite permeability) Liposomes with NBD-PE residing exclusively in the inner leaflet (asymmetric liposomes) were prepared by vortexing an aliquot (1 ml) of a suspension of symmetric liposomes in Ringer’s buffer (20 mM SPC) with 20 ml freshly prepared sodium dithionite solution (1 mM) and allowing to stand at room temperature for 2 min. Excess dithionite was removed by passing the liposome suspension through a Sephadex G-50 column and eluting with Ringer’s buffer. A suspension of these asymmetric liposomes (2 mM SPC) was then mixed with a BS solution (C, TC and MKC at 2 mM; DC at 0.4 mM) in a 1:1 volume ratio and incubated at room temperature (21 C). Control liposomes were mixed with buffer. At various times up to 9 h, aliquots (1.0 ml) were removed and, after adding 20 ml sodium dithionite solution (1 mM), allowed to stand for 2 min. Fluorescence intensity was then measured using the same excitation and emission wavelengths as in the continuous monitoring study described above. The only difference between our method and that of Armstrong et al. (2003) is that they added 1.0 M dithionite in Tris buffer (pH 10) to samples and monitored the decline in fluorescence continuously over 10 min to observe both phase 1 and phase 2 of the decline. They were then able to calculate the rates of NBD-PE flip-flop and dithionite permeability of the membrane through non-linear least squares fitting of the data for all samples collected over 2.5 h or more. Matsuzaki et al. (1996) measured fluorescence remaining in samples after exposure to dithionite for only 3 min (a duration short enough in their study to exclude any of the phase 2 decline) and determined the rate of NBD-PE flip-flop from the exponential decline in fluorescence remaining in samples against time of sampling. We measured fluorescence remaining in samples after exposure to dithionite for 2 min with the intention of detecting NBD-PE flip-flop (phase 1 quenching) and any marked increase in dithionite permeability of the lipid bilayer caused by exposure to BS (i.e. the increase in the amount of phase 2 quenching occurring in 2 min). We anticipated the decline in fluorescence remaining would be exponential in control liposomes (NBD-PE flip-flop only) but not in liposomes made more permeable to dithionite by exposure to BS. Statistical analysis Data were analyzed using SPSS 11.0 (Systat Software Inc., San Jose, CA). One-way ANOVA with Tukey’s or Dunnett’s post hoc tests was carried out depending on the results of homogeneity of variance tests for multiple comparisons. Differences for which p50.05 were considered significant.
J Liposome Res, Early Online: 1–9
Table 1. Liposome/pH 7.2 buffer distribution coefficients (log Dlipo/ buffer) (means ± SD, n ¼ 3) of bile salts at 21 C along with their previously determined log Doctanol/buffer and CMC values (Yang et al., 2009). Bile salt DC C MKC TC
Log Dlipo/buffer
Log Doctanol/buffer
CMC (mM)
3.42 ± 0.02 2.50 ± 0.02 0.75 ± 0.06 2.49 ± 0.01
1.46 0.01 0.41 1.50
1.69 4.09 13.4 3.56
MKC5TC & C5DC and are higher than corresponding log Doctanol/buffer values indicating that BS, which are largely ionized at pH 7.2, partition more strongly into liposomes than predicted by their log Doctanol/buffer values. This is consistent with previous work showing that ionized species partition more strongly into lipid membranes than into octanol (Avdeef et al., 1998) and, as expected, is particularly true for the strongly acidic TC. The log Dlipo/buffer values show a reasonable inverse correlation with the BS CMC values as previously observed by Heerklotz & Seelig (2000) for neutral detergents. They also correlate with BS cytotoxicity to rat brain endothelial (RBE4) cells with the exception of TC whose low cytotoxicity was ascribed to the existence of a specific efflux transport system (Yang et al., 2012). Vesicle size The change in VS and dispersity of liposomes after incubation with increasing concentrations of BS for 2 h at 25 C is shown in Figure 1. VS of liposomes in the absence of BS was 58 nm and remained unchanged at low BS concentrations. Dispersity was 0.1 at low BS concentrations indicative of monodisperse samples and similarly remained unchanged at BS51 mM. As concentration increased, C, DC and TC caused marked decreases in VS (DC4TC4C) with concomitant increases in dispersity. DC caused a decrease in VS at a concentration as low as 0.5 mM but MKC had little effect on VS over the whole concentration range (0–10 mM). Zeta potential In the absence of BS, the zeta potential of liposomes was slightly negative (5 mV). Zeta potential after incubation with BS for 2 h at 25 C (Figure 2) became more negative indicating BS are inserting into the lipid bilayer of the liposomes or into the electrical double layer surrounding them (Hunter, 2001). At low BS concentration (50.5 mM), the increase in negativity is in the order MKC5C & TC5DC consistent with the log Dlipo/buffer values. At higher BS concentration, TC caused a more rapid increase in negativity than the other BS possibly because TC accumulates in the Stern layer rather than in the lipid bilayer where its negative form is relatively more stable than those of the other BS which accumulate in the lipid bilayer.
Results Log Dlipo/buffer values
Membrane fluidity
These are given in Table 1 along with previously determined log Doctanol/buffer and CMC values (Yang et al., 2009). The results show that log Dlipo/buffer values increase in the order
Figure 3 shows the effect of incubation with BS for 30 min on the FP of DPH and TMA-DPH in SPC liposomes. FP of the two probes without BS was 200 mP consistent with a
Kinetic and equilibrium studies of bile salt–liposome interactions
DOI: 10.3109/08982104.2014.928888
(A)
(B) 0.6
70
0.5
50 40
C DC MKC TC
30
0.3 0.2
20
0.1
0.1
1
10
0.0 0.01
0.1
1
10
Bile salt concentration (mM)
Bile salt concentration (mM)
Figure 1. Effect of incubation with increasing concentrations of bile salts for 2 h at 25 C on (A) vesicle (Z-average) size and (B) dispersity of SPC liposomes (0.5 mM SPC) (data are means ± SD, n ¼ 3).
0
(A) 210
−5
205 DPH Polarization (mP)
Zeta potential (mV)
−10 −15 −20 −25 −30 0.01
C DC MKC TC
0.1
200 195 190 185 180
1
175 0.01
10
Bile salt concentration (mM)
Figure 2. Zeta potential of SPC liposomes (0.5 mM SPC) after incubation with increasing concentrations of bile salts for 2 h at 25 C (data are means ± SD, n ¼ 3).
previous study of dilinoleoylphosphatidylcholine multilamellar liposomes with a phospholipid:cholesterol ratio of 2:1 (van Blitterswijk et al., 1987). All BS decreased FP of both DPH and TMA-DPH in a concentration-dependent manner indicating BS cause an increase in membrane fluidity at submicellar concentrations in the order MKC5C5TC5DC. This is also the order of the effect of the four BS on membrane fluidity of RBE4 cells as indicated by the FP of TMA-DPH (Yang et al., 2012). NBD-PE flip-flop and dithionite permeability Symmetric liposomes equilibrated with BS Addition of dithionite to symmetric liposomes caused the expected two-phase decline in fluorescence intensity (Figure 4). In phase 1, corresponding to a drop of 50% of the initial fluorescence, the decline was slower for liposomes equilibrated with TC suggesting TC interferes with dithionite reduction of NBD-PE in the outer leaflet of the bilayer.
C DC MKC TC
0.1
1
10
Bile salt concentration (mM) (B) 220 TMA-DPH Polarization (mP)
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C DC MKC TC
0.4 Dispersity
Z-average size (nm)
60
10 0.01
5
C DC MKC TC
210 200 190 180 170 160 150 140 0.01
0.1
1
10
Bile salt concentration (mM)
Figure 3. Membrane fluidity of SPC liposomes (1 mM SPC) as determined by steady-state fluorescence polarization of (A) DPH and (B) TMA-DPH in liposomes incubated with increasing concentrations of bile salts for 30 min at room temperature (21 C) (data are means ± SD, n ¼ 4).
In phase 2, corresponding to quenching of inner leaflet NBD-PE by dithionite that penetrates the membrane and, to a minor extent, to NBD-PE that flips from the inner to the outer leaflet in the short duration of the experiment, dithionite
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J Liposome Res, Early Online: 1–9
Fluorescence intensity remaining (%)
120
Fluorescence %
100 Control 1 mM C 1 mM TC 0.2 mM DC 1 mM MKC
80 60 40 20 50
100
150
200
250
300
350
400
450
Figure 4. Representative plot showing the decrease in fluorescence after addition of 20 ml sodium dithionite solution (20 mM) (at arrow) to 1.0 ml of NBD-PE symmetric liposomes (1 mM SPC) previously equilibrated with bile salts for 24 h. 0.10
80 60 40 20 0
100
200
300
400
500
600
Figure 6. Effect of duration of incubation with bile salts at room temperature (21 C) on the fluorescence of asymmetric liposomes (1 mM SPC) labeled with NBD-PE in the inner leaflet. Each point is the fluorescence of a sample (1 ml) after removal from the incubation and treatment with 20 ml dithionite (1.0 mM) for 2 min, i.e. each point indicates the NBD-PE remaining in the inner leaflet (data are means ± SD, n ¼ 3).
0.08
0.06
0.04
0.02
KC M m 1
2
m
M
M
D
TC 0.
1
m
M
C 1
m
M
tro on C
C
0.00 l
Dithionite permeation rate constant (% s−1)
100
Time (min)
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1mM C 0.2 mM DC 1 mM MKC 1 mM TC Control
Figure 5. Dithionite permeability zero-order rate constants (based on phase 2 of the decline in fluorescence shown in Figure 4) for NBD-PE symmetric liposomes (1 mM SPC) previously equilibrated with bile salts (data are means ± SD, n ¼ 3).
permeability rate constants (zero order) were determined from the slopes over the period 240–420 s. The values shown in Figure 5 indicate that liposomes equilibrated with BS are more permeable to dithionite than control liposomes (ANOVA, p50.05) with MKC having the smallest effect followed by C and TC and finally DC (at 0.2 mM compared to 1 mM for the other BS) which has the largest effect. Surprisingly, despite TC equilibrated liposomes showing a slower rate of NBD-PE fluorescence quenching in phase 1 (outer leaflet), NBD-PE fluorescence quenching of TC equilibrated liposomes in phase 2 (inner leaflet) was not slower than in C and DC equilibrated liposomes Asymmetric liposomes In the case of control liposomes, NBD-PE remaining in the inner leaflet in samples removed at various times and treated with dithionite for 2 min declined slowly and exponentially with a rate of 0.0035 min1 (5.8 105 s1) (Figure 6). This decline is due to NBD-PE flip-flop from the inner to the outer leaflet and is 15 times slower than the rate of
dithionite permeability of control liposomes shown in Figure 5 (0.04% s1 equivalent to a first order rate constant of 0.0008 s1). In comparison, Armstrong et al. (2003) found flip-flop was 200 times slower than dithionite permeability in NBD-PE labeled LUVs. If NBD-PE flip-flop is the only process that contributes to fluorescence quenching, the decline in fluorescence should continue to a limit of 50% of the initial value. Matsuzaki et al. (1996) showed this to be the case using LUVs where the rate constant for NBD-PE flip-flop at 30 C was 0.012 min1. In the case of liposomes incubated with C, DC or MKC, the decrease in fluorescence remaining in samples was dramatic (Figure 6). Thus, fluorescence remaining at 30 min in liposomes incubated with DC, C or MKC was 33%, 46% and 68%, respectively, of the initial value compared with 90% and 88%, respectively, for liposomes incubated with TC or buffer (control). The fact that for DC fluorescence remaining in samples was well below 50% of the initial value (i.e. below the level attainable by NBD-PE flip-flop alone) and that for C, DC and MKC fluorescence remaining in samples was higher over the subsequent 200–300 min than at 30 min, together indicate that the rapid initial decrease in fluorescence remaining in samples is due primarily to the high permeability of the bilayer to dithionite when BS is asymmetrically distributed (BSouter leaflet4BSinner leaflet). The subsequent recovery of fluorescence remaining is then due to a decrease in permeability of the bilayer as BS attain equilibrium in the two leaflets via BS flip-flop. Thus, fluorescence remaining in samples at 300 min for liposomes incubated with DC, C, MKC, TC or control was 53%, 59%, 72%, 67% and 71%, respectively, of the initial value. These decreases are due to flip-flop of NBD-PE over 300 min (plus a small contribution due to dithionite permeability of the liposomes during the 2-min treatment of the samples) the rate of which increases in the order control & MKC5TC5C5DC. The decrease in fluorescence should continue to a limit of 50% when NBD-PE reaches equilibrium between the two leaflets.
Kinetic and equilibrium studies of bile salt–liposome interactions
For liposomes incubated with TC, fluorescence remaining in samples declined slowly with a rate only slightly faster than in samples of control liposomes but appeared to plateau after 180 min. This difference in behavior of TC compared with the other BS is consistent with our previous report on the effect of the four BS on the release of CF from CF-loaded SUV liposomes (Hinow et al., 2012). The behavior implies that TC increases liposome permeability to dithionite to only a small extent and the decline in fluorescence remaining is primarily due to NBD-PE flip-flop.
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Discussion BS-induced changes in lipid bilayers at sub-micellar concentrations result from partitioning of BS into the lipid bilayer in accordance with their log Dlipo/buffer values. DC with two hydroxyl groups has the highest value followed by C and TC (trihydroxy BS) with similar values. MKC has the lowest log Dlipo/buffer indicating that replacement of the hydroxyl group in C by a keto group significantly reduces the ability to partition into membranes. The log Dlipo/buffer values of the four BS follow the same trend as the log Doctanol/buffer values with the exception of TC which has a markedly positive log Dlipo/buffer compared with its negative log Doctanol/ buffer. Presumably the relatively high concentration of the ionized TC that results from its much lower pKa gives it a much greater affinity for the phospholipid bilayer. Determination of VS is a direct method to monitor BSinduced solubilization of liposomes and was used here to ensure sub-micellar concentrations of BS were used in subsequent studies. DC and TC caused significant reductions in VS at concentrations below CMC consistent with a mechanism in which their insertion into the membrane leads to formation of lipid-BS mixed micelles. The process can be seen as the result of an increase in curvature of the membrane arising from insertion of curvophilic BS into the outer leaflet with subsequent shedding of the bilayer surface. MKC had no effect on VS at sub-micellar concentrations suggesting it is much less curvophilic than the other BS. In terms of zeta potential, incorporation of BS increased the negativity of liposomes consistent with the fact that BS are mainly in the negatively charged form at pH 7.2. In terms of membrane fluidity, many studies have shown that BS interact with liposomes to increase their flexibility presumably because the excess area resulting from BS binding leads to softening of the bilayer. Increased membrane fluidity was observed here with DC causing the greatest effect and MKC the least effect on membrane fluidity of both polar and non-polar regions of the lipid bilayer. MKC appeared to have a greater effect on polar regions as previously suggested by the results of our study in Caco-2 cells where MKC had a lower effect than C on the FP of DPH but a much greater effect than C on the FP of TMA-DPH (Chen et al., 2012). However, this indication that MKC is mainly located in the polar regions of the lipid bilayer is based on the premise that the two probes respond equally to changes in lipid packing, and, although they have the same fluorophore, this cannot be guaranteed. In the study of membrane permeability using NBD-PE labeled symmetric liposomes, liposomes equilibrated with BS
(B) Permeability of liposome membrane
DOI: 10.3109/08982104.2014.928888
7
Bile salt
(C) (A)
Time
Figure 7. Schematic showing the time-dependence of the effect of a bile salt on the permeability of a liposome. Initial exposure causes a dramatic increase in permeability as the BS penetrates the outer leaflet of the lipid bilayer (B versus A). Prolonged incubation allows the BS to flip-flop until equilibrium is reached when the liposome is only slightly more permeable than before exposure to BS (C versus A).
were more permeable to dithionite than control (Figures 4 and 5). Some researchers have postulated that this increase in permeability is the result of BS inserting longitudinally into the membrane and aggregating as dimers or larger structures to form membrane pores (Carey, 1985) but others have shown this is not the case, at least for transport of small non-ionic solutes (Albalak et al., 1996). In fact, it has been suggested that, in interacting with lipid bilayers, BS orientate themselves parallel to the interface rather than inserting into the bilayer longitudinally (Fahey et al., 1995). Disturbance of the lipid packing in the membrane is then presumed to cause its increased permeability. Although membranes equilibrated with MKC were less permeable to dithionite than membranes equilibrated with the other BS (Figure 5), the MKC concentration in the membrane is lower based on its lower log Dlipo/ buffer suggesting MKC is more disruptive to the membrane than the other BS. In the study of NBD-PE labeled asymmetric liposomes, the changes in fluorescence during incubation with C, DC or MKC are consistent with a bilayer made initially permeable to dithionite by BS incorporation into the outer leaflet resulting in a ‘‘stressed’’ membrane followed by a return to a less permeable membrane as BS flip-flop into the inner leaflet of the bilayer until an equilibrium between BS in the inner and outer leaflets is achieved (Donovan & Jackson, 1997; Luk et al., 1997). It is reported that the half-life of flip-flop of C and DC in liposomal membranes in the liquid crystalline state at 60 C is 4–5 min (Hildebrand et al., 2002). Given an activation energy of flip-flop of 79 kJ mol1 (Liu & Conboy, 2005), the Arrhenius equation predicts a half-life of BS flip-flop at room temperature (21 C) of 180 min. The fact that this is similar to the time required for membrane impermeability to be restored during incubation with BS supports the hypothesis that an unequal distribution of BS in the two leaflets of the lipid bilayer leads to increased permeability. The time-dependence of the effect of BS on the permeability of a liposome is shown schematically in Figure 7. The reason the initial rapid increase in membrane permeability did not occur for liposomes incubated with TC may be
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L. Yang et al.
because the rate of TC insertion into the outer leaflet is similar to its rate of flip-flop to the inner leaflet. This would mean the concentration of TC in the outer leaflet never exceeds that in the inner leaflet (i.e. liposomes would never be asymmetric with respect to TC), the liposomes remain impermeable to dithionite and the decline in fluorescence remaining is due to NBD-PE flip-flop only. This hypothesis is consistent with the fact that TC has been shown to undergo very slow flip-flop in egg phosphatidylcholine LUVs (Donovan & Jackson, 1997) and SUVs (Kamp & Hamilton, 1993) and would suggest that the rate of insertion of TC into the outer leaflet of liposomes is slower than that of the other BS. Interestingly, the fact that incubation of liposomes with 1 mM TC for 30 min (the time of maximum permeability to dithionite) increases their membrane fluidity more than incubation with 1 mM C or MKC (Figure 3), suggests BS induce increases in membrane fluidity irrespective of whether they are symmetrically or asymmetrically distributed in the lipid bilayer. The time-dependent changes in permeability of the lipid bilayer to dithionite are consistent with the fact that bilosomes are resistant to physical destruction by extrinsic BS (Rowland & Woodley, 1980; Schubert et al., 1983). Although incorporation of BS into the lipid bilayer increases its permeability to a small extent (Figure 7, C versus A), bilosomes are resistant to the rapid increase in permeability on first exposure to BS (Figure 7, B versus A). Further research is needed to determine how the time-dependence of permeabilization depends on BS structure, what level of BS provides optimum stabilization of the membrane to the initial shock of BS exposure and whether bilosomes incorporating one BS are stable to subsequent exposure to another. Studies are also required to establish whether bilosomes give rise to greater transport of encapsulated drugs across the BBB than liposomes, i.e. whether the BS present in the liposome membrane acts as a permeation enhancer at the BBB.
Conclusion The time-dependence of the interaction of sub-micellar concentrations of conjugated and unconjugated BS with SPC liposomal membranes appears to be different. The unconjugated BS, C, DC and MKC, insert into the membrane in the order of their liposome/pH 7.2 buffer partition coefficients MKC5C5DC. Insertion into the outer leaflet occurs rapidly and produces a stressed membrane with increased permeability while subsequent flip-flop of BS to the inner leaflet occurs slowly and is accompanied by an increase in the rate of phospholipid flip-flop that together relieve the strain and restore membrane impermeability. The conjugated BS, TC, inserts more slowly into the outer leaflet and undergoes flip-flop into the inner leaflet at an equal rate which, together with slow phospholipid flip-flop, avoids formation of a stressed membrane and avoids the highly permeable transient stage. MKC appears to cause the least disturbance to liposomal membranes but only because it has the lowest distribution coefficient. This information may be useful in the design of drug delivery systems that create transient increases in the permeability of biological barriers.
J Liposome Res, Early Online: 1–9
Acknowledgements L.Y. thanks the University of Otago for providing a Postgraduate Publishing Bursary.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
References Albalak A, Zeidel ML, Zucker SD, et al. (1996). Effects of submicellar bile salt concentrations on biological membrane permeability to low molecular weight non-ionic solutes. Biochemistry 35:7936–45. Angeletti C, Wylie Nichols J. (1998). Dithionite quenching rate measurement of the inside-outside membrane bilayer distribution of 7-nitrobenz-2-oxa-1,3-diazol-4-yl-labeled phospholipid. Biochemistry 37:15114–19. Annesini MC, Memoli A, Petralito S. (2000). Kinetics of surfactantinduced release from liposomes: a time-dependent permeability model. J Membr Sci 180:121–31. Armstrong VT, Brzustowicz MR, Wassall SR, et al. (2003). Rapid flipflop in polyunsaturated (docosahexaenoate) phospholipid membranes. Arch Biochem Biophys 414:74–82. Avdeef A, Box KJ, Comer JEA, et al. (1998). pH-Metric logP. Determination of liposomal membrane-water partition coefficients of ionizable drugs. Pharm Res 15:209–15. Carey MC. (1985). Physico-chemical properties of bile acids and their salts. In: Danielsson H, Sjo¨vall J, eds. Sterols and bile acids. 1st ed. Amsterdam: Elsevier, 345–403. Chen G, Yang L, Zhang H, et al. (2012). Effect of ketocholate derivatives on methotrexate uptake in Caco-2 cell monolayers. Int J Pharm 433: 89–93. Chen X, Oshima T, Tomita T, et al. (2011). Acidic bile salts modulate the squamous epithelial barrier function by modulating tight junction proteins. Am J Physiol Gastrointest Liver Physiol 301:G203–9. Dai Y, Zhou R, Liu L, et al. (2013). Liposomes containing bile salts as novel ocular delivery systems for tacrolimus (FK506): in vitro characterization and improved corneal permeation. Int J Nanomed 8:1921–33. Donovan JM, Jackson AA. (1997). Transbilayer movement of fully ionized taurine-conjugated bile salts depends upon bile salt concentration, hydrophobicity, and membrane cholesterol content. Biochemistry 36:11444–51. Elsayed MMA, Cevc G. (2011). The vesicle-to-micelle transformation of phospholipid-cholate mixed aggregates: a state of the art analysis including membrane curvature effects. Biochim Biophys Acta 1808: 140–53. Fahey DA, Carey MC, Donovan JM. (1995). Bile acid/phosphatidylcholine interactions in mixed monomolecular layers: differences in condensation effects but not interfacial orientation between hydrophobic and hydrophilic bile acid species. Biochemistry 34:10886–97. Gordon GS, Moses AC, Silver RD, et al. (1985). Nasal absorption of insulin: enhancement by hydrophobic bile salts. Proc Natl Acad Sci USA 82:7419–23. Gubernator J, Stasiuk M, Kosubek A. (1999). Dual effect of alkylresorcinols, natural amphiphilic compounds, upon liposomal permeability. Biochim Biophys Acta 1418:253–60. Heerklotz H. (2001). Membrane stress and permeabilization induced by asymmetric incorporation of compounds. Biophys J 81: 184–95. Heerklotz H, Seelig J. (2000). Correlation of the membrane/water partition coefficients of detergents with the critical micelle concentration. Biophys J 78:2435–40. Hildebrand A, Neubert R, Garidel P, Blume A. (2002). Bile salt induced solubilization of synthetic phosphatidylcholine vesicles studied by isothermal titration calorimetry. Langmuir 18:2836–47. Hinow P, Radunskaya A, Tucker I, Yang L. (2012). Kinetics of bile salt binding to liposomes revealed by carboxyfluorescein release and mathematical modeling. J Liposome Res 22:237–44. Hu S, Niu M, Hu F, et al. (2013). Integrity and stability of oral liposomes containing bile salts studied in simulated and ex vivo gastrointestinal media. Int J Pharm 441:693–700.
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Hunter RJ. (2001). Foundations of colloid science. 2nd ed. Chapter 7. Oxford, UK:Oxford University Press. In’t Veld G, Driessen AJ, Op den Kamp JA, Konings WN. (1991). Hydrophobic membrane thickness and lipid-protein interactions of the leucine transport system of Lactococcus lactis. Biochim Biophys Acta 1065:203–12. Kamp F, Hamilton JA. (1993). Movement of fatty acids, fatty acid analogues, and bile acids across phospholipid bilayers. Biochemistry 32:11074–86. Kra¨mer SD, Jakits-Deiser C, Wunderli-Allenspach H. (1997). Free fatty acids cause pH-dependent changes in drug-lipid membrane interactions around physiological pH. Pharm Res 14:827–32. Lichtenberg D, Ahyayauch H, Gon˜i FM. (2013). The mechanism of detergent solubilization of lipid bilayers. Biophys J 105:289–99. Lin CM, Chang GP, Tsao HK, Sheng YJ. (2011). Solubilization mechanism of vesicles by surfactants: effect of hydrophobicity. J Chem Phys 135:045102 (1–10). Liu J, Conboy JC. (2005). 1,2-Diacyl-phosphatidylcholine flip-flop measured directly by sum-frequency vibrational spectroscopy. Biophys J 89:2522–32. Luk AS, Kaler EW, Lee SP. (1997). Structural mechanisms of bile saltinduced growth of small unilamellar cholesterol-lecithin vesicles. Biochemistry 36:5633–44. Matsuzaki K, Murase O, Fujii ND, Miyajima K. (1996). An antimicrobial peptide, magainin 2, induced rapid flip-flop of phospholipids coupled with pore formation and peptide translocation. Biochemistry 35:11361–8. McIntyre JC, Sleight RG. (1991). Fluorescence assay for phospholipid membrane asymmetry. Biochemistry 30:11819–27. Memoli A, Annesini MC, Petralito S. (1999). Surfactant-induced leakage from liposomes: a comparison among different lecithin vesicles. Int J Pharm 184:227–35. Mikov M, Fawcett JP, Kuhajda K, Kevresan S. (2006). Pharmacology of bile acids and their derivatives. Eur J Drug Metab Pharmacokinet 31: 237–516. Mikov M, Kevresan S, Kuhajda K, et al. (2004). 3Alpha,7alphadihydroxy-12-oxo-5beta-cholanate as blood-brain barrier permeator. Pol J Pharmacol 56:367–71.
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Moreno MJ, Estronca LMBB, Vaz WLC. (2006). Translocation of phospholipids and dithionite permeability in liquid-ordered and liquid-disordered membranes. Biophys J 91:873–81. Morimoto K, Nakai T, Morisaka K. (1987). Evaluation of permeability enhancement of hydrophilic compounds and macromolecular compounds by bile salts through rabbit corneas in-vitro. J Pharm Pharmacol 39:124–6. Naveen C, Kiran Kumar Y, Venkateshwar Rao P, Rama Rao T. (2011). Chemical enhancers in buccal and sublingual delivery. Int J Pharm Sci Nanotech 4:1307–19. O’Connor CJ, Wallace RG, Iwamoto K. (1985). Bile salt damage of egg phosphatidylcholine liposomes. Biochim Biophys Acta 817:95–102. Raimondi F, Santori P, Barone MV, et al. (2008). Bile acids modulate tight junction structure and barrier function of Caco-2 monolayers. Am J Physiol Gastrointest Liver Physiol 294:G906–13. Rowland RN, Woodley JF. (1980). The stability of liposomes in vitro to pH, bile salts and pancreatic lipase. Biochim Biophys Acta 620:400–9. Schubert R, Jaroni H, Schoelmerich J, Schmidt KH. (1983). Studies on the mechanism of bile salt-induced liposomal membrane damage. Digestion 28:181–90. Schubert R, Schmidt KH. (1988). Structural changes in vesicle membranes and mixed micelles of various lipid compositions after binding of different bile salts. Biochemistry 27:8787–94. Spigelman MK, Zappula RA, Malis LI, et al. (1983). Intracarotid dehydrocholate infusion: a new method for prolonged reversible blood brain barrier disruption. Neurosurgery 12:606–12. van Blitterswijk WJ, van der Meer BW, Hilkmann H. (1987). Quantitative contributions of cholesterol and the individual classes of phospholipids and their degree of fatty acyl (un)saturation to membrane fluidity measured by fluorescence polarization. Biochemistry 26:1746–56. Yang L, Fawcett JP, Østergaard J, et al. (2012). Mechanistic studies of the effect of bile salts on rhodamine 123 uptake into RBE4 cells. Mol Pharm 9:29–36. Yang L, Zhang H, Mikov M, Tucker IG. (2009). Physicochemical and biological characterization of monoketocholic acid, a novel permeability enhancer. Mol Pharm 6:448–56.
