ARTICLE pubs.acs.org/Langmuir
Effect of Submicellar Concentrations of Conjugated and Unconjugated Bile Salts on the Lipid Bilayer Membrane Monalisa Mohapatra and Ashok K. Mishra* Department of Chemistry, Indian Institute of Technology Madras, Chennai 600 036, India
bS Supporting Information ABSTRACT: The interaction of submicellar concentrations of various physiologically important unconjugated [sodium deoxycholate (NaDC), sodium cholate (NaC)] and conjugated [sodium glycodeoxycholate (NaGDC), sodium glycocholate (NaGC), sodium taurodeoxycholate (NaTDC), sodium taurocholate (NaTC)] bile salts with dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) small unilamellar vesicles in solid gel (SG) and liquid crystalline (LC) phases was investigated using the excited-state prototropism of 1-naphthol. Steady-state and timeresolved fluorescence of the two excited-state prototropic forms of 1-naphthol indicate that submicellar bile salt concentration induces hydration of the lipid bilayer membrane into the core region. This hydration effect is a general phenomenon of the bile salts studied. The bilayer hydration efficiency of the bile salt follows the order NaDC > NaC > NaGDC > NaTDC > NaGC > NaTC for both DPPC and DMPC vesicles in their SG and LC phases.
’ INTRODUCTION Naturally occurring biosurfactants, bile salts, possess a hydrophobic steroidal backbone on one face and two or three hydrophilic hydroxyl groups on the opposite face which provide facial polarity to these molecules (Figure 1). Surfactant behavior of these biologically important compounds have been well-studied to understand many complicated biological systems like drug absorption within the small intestine, dissolution of dietary lipids and cholesterol, efficient fat digestion in the gut, understanding of drug absorption within the small intestine, and determination of the influence of lipid formulations on this process.1,2 Their unusual properties are due to their chemical structure, which does not correspond to the classical headtail moiety as found for other surfactants. Cholates and deoxycholates are among the most important bile salts studied for biological model systems. Under normal physiological conditions, they are conjugated with the amino acid taurine or glycine to form their corresponding cholates and deoxycholates, thus gaining a longer and more polar hydrophilic peptide chain, and are then stored in gall bladder.3 The present work is focused on the interaction of submicellar concentrations of various unconjugated and conjugated bile salts with phospholipid bilayer. A number of interactions between bile salts and phospholipid bilayers are known to occur,4,5 e.g., liposomes remain exposed to bile salts during liposomal drug delivery in hepatobiliary systems. Hence, the study of bile saltliposome interaction is essential to understand the related phenomena. Among the number of studies in the literature regarding the interactions of bile salt [gcritical micellar concentration (CMC)] with liposomes, progressive changes in membrane morphology leading to micellization of lipid and solubilization of bilayer membrane with r 2011 American Chemical Society
increasing bile salt concentration have been reported.4,69 Transbilayer movement of submicellar concentration of bile salts in liposome membrane has important biological significance, and it increases with an increase in intramembrane bile salt concentration and hydrophobicity.10,11 Bile salts at their submicellar concentration are known to bind efficiently with the membranes, thereby enhancing the rate of intervesicular phospholipid transfer process.12,13 These are also present in submicellar concentrations in hepatic portal vein systems of human body.14,15 Hence, it is important to study the bile saltliposome interaction at lower concentration of bile salts. In this context, recent study of bile saltliposome interaction at submicellar concentration of bile salts indicates bile salt induced hydration of lipid bilayer membrane up to the core region of the bilayer.16 This was studied using the prototropic equilibrium of the ESPT (excited state proton transfer) molecular probe, 1-naphthol, which is sensitive to the water molecules around its microenvironment.17 1-Naphthol with pKa = 9.2 and pKa* = 0.417 remains exclusively in two emitting forms, neutral (NpOH*) or anionic (NpO*), which are in equilibrium with one another. The light-emitting form in the aqueous medium at neutral pH is the NpO* (λex = 290 nm, λem = 470 nm), whereas in liposome medium, the emissions occur from both NpOH* (λex = 290 nm, λem = 370 nm) and NpO* (λex = 290 nm, λem = 465 nm). It is known that the characteristic emissions from NpOH* and NpO* mostly arise from the 1-naphthol molecules located in the water-inaccessible hydrophobic core and water-accessible hydrophilic interfacial surface of the lipid bilayer, respectively.18,19 For an Received: August 3, 2011 Revised: October 2, 2011 Published: October 06, 2011 13461
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in their solid gel (SG) and liquid crystalline (LC) phases. Using excited-state proton transfer rates of 1-naphthol, the order for the efficiency of varying bile salt induced bilayer hydration can be deduced. The study was carried out using different fluorescence parameters of 1-naphthol with the bile salt concentrations of 0.05 to 1 mM.
’ MATERIALS AND METHODS
Figure 1. Structures of different unconjugated and conjugated bile salts.
ESPT to occur, a cluster of water molecules acts as a base which is responsible for accepting the dissociated proton.17 Since the cluster of water molecules is the proton acceptor, any change in the hydration of membrane is reflected in the changes in neutralform fluorescence. As 1-naphthol is known to be distributed among the core as well as interface, it can sense the extent of hydration across the membrane. The fluorescence decay dynamics of 1-naphthol shows biexponential characteristics. By using the amplitudes and lifetimes of different components of 1-naphthol, it was shown that bile saltliposome interaction at submicellar concentrations of bile salt results in wetting of the lipid bilayer up to the hydrocarbon core region at both phase states of the membrane. It was observed that with increase in bile salt concentration a decrease in neutral form fluorescence intensity as well as lifetime occurs. From a detailed fluorescence lifetime study and measurement of proton transfer rate constants, it was concluded that the observed spectral changes were because of the permeation of water up to the core of the bilayer membrane.16 Hence, 1-naphthol was found to be an effective sensor to study the bile salt induced lipid bilayer hydration. Since various unconjugated and conjugated bile salts have extensive biological significance, to generalize the bile saltliposome interaction model, in this work the interaction of various bile salts with lipid bilayers of different chain length has been studied, using 1-naphthol. The bilayer membranes made up of DMPC lipids with 14carbon chains are less densely packed than DPPC lipids with 16carbon chains. Since the packing efficiency of both lipid bilayers is different, the degree of hydration across the bilayer is expected to be different. Given the bile salt induced bilayer hydration, it was felt important to evaluate the efficiency of various physiologically important unconjugated (NaDC, NaC) and conjugated (NaGDC, NaGC, NaTDC, NaTC) bile salts found in the body, toward the extent of hydration in DPPC and DMPC lipid bilayer membranes,
Materials. 1-Naphthol (GR grade) purchased from SRL, India, was purified by sublimation and used after checking its purity. NaDC and NaC, purchased from SRL, India, and NaTDC, NaTC, NaGDC, and NaGC, purchased from Sigma Chemical Co. (Bangalore, India) were used as received. DPPC and DMPC were purchased from Sigma Chemical Co. (Bangalore, India) and used as received. All the solvents used were of spectral grade. Triple-distilled water, prepared using alkaline permanganate solution, was used for the experiments. Liposome Preparation. For this work, small unilamellar vesicles were prepared by the ethanol injection method.20,21 The stock solution of the lipid was prepared in ethanol. The desired amount of ethanolic solution of lipid was injected rapidly into the aqueous solution of 1-naphthol and equilibrated for 30 min at 55 °C (above phase transition temperature of DPPC and DMPC). The percentage of ethanol in the solution was less than 1% (v/v). Throughout the experiments, the 1-naphthol to lipid molar ratio was kept constant at 1:100 (4 μM 1-naphthol, 0.4 mM lipid). All experiments were performed with freshly prepared solutions of both 1-naphthol and liposome. Incorporation of Bile Salts. Stock solutions of bile salts were prepared in triple-distilled water at neutral pH (7.0 ( 0.1). The solutions were prepared by adding the desired volume of bile salt stock of appropriate concentration to the liposome solutions at 55 °C (LC phase) in order to maintain the final concentration (0 to 1 mM) of bile salts. The solutions were equilibrated for 2 h before analysis. All the experiments were performed with freshly prepared solutions of bile salts. The pH of the liposomebile salt solutions was measured before and after the experiment. It has been observed that there is almost no change in the pH of the solutions. Fluorescence Measurements. Fluorescence measurements were carried out with a Hitachi F-4500 spectrofluorometer. The emission spectra were recorded with slit widths of 5/5 nm, by fixing excitation wavelength at 290 nm. Temperature was controlled by circulating water through a jacketed cuvette holder from a refrigerated bath (JULABO, Germany). Fluorescence Lifetime Measurements. Fluorescence lifetime measurements were carried out using a Horiba Jobin Yvon TCSPC lifetime instrument in a time-correlated single-photon counting arrangement. A 295 nm nano-LED was used as the light source. The pulse repetition rate was set to 1 MHz, and the instrumental full width halfmaximum of the 295 nm LED, including the detector response was ca. 800 ps. The instrument response function was collected using a scatterer (Ludox AS40 colloidal silica). The decay data were analyzed using IBH software. A value of χ2, 0.99 e χ2 e 1.2, was considered a good fit, which was further judged by the symmetrical distribution of the residuals. The average fluorescence lifetime values were obtained by the following equation22 τavg ¼
n
∑
i¼1
! αi τ2i
=
n
∑ αi τi
!
i¼1
where τi is the individual fluorescence lifetime with corresponding amplitude αi. 13462
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Figure 2. Response of the fluorescence intensity of 1-naphthol to the changes induced by bile salts: (A) NaDC (from ref 16), (B) NaC (from ref 16), (C) NaGDC, (D) NaTDC, (E) NaGC, and (F) NaTC at 15 °C (SG phase) in DMPC vesicles. (λex = 290 nm, λem = 370 and 465 nm). [DMPC] = 0.4 mM, [1-naphthol] = 4 μM. [Without (black line) and with (red dashed line) 1 mM of bile salt].
’ RESULTS Fluorescence Intensity Studies. Figure 2 shows the emission spectra of 1-naphthol (λex = 290 nm, λem = 370 and 465 nm) without and with different bile salts (1 mM) in DMPC vesicles at 15 °C (SG phase). Figure S1 in the Supporting Information (SI) shows the same in DPPC vesicles at 30 °C (SG phase). The appearance of emission peak at ∼370 nm due to NpOH* shows the presence of membrane-bound 1-naphthol.16,18,19 This was further confirmed from the absence of NpOH* emission in the aqueous bile salt media without vesicles (Figure S2 in the SI). In the DMPC bilayer, the NpOH* intensity is observed to be more as compared to DPPC bilayer. Bile salts at their submicellar concentration range (up to 1 mM) are known to get inserted into the phospholipid molecules without disrupting the membranes.4,23 At 1 mM concentration of bile salt in liposome medium, a noticeable decrease in intensity of NpOH* and almost negligible change in intensity of NpO* can be observed in Figure 2 and SI Figure S1. However, the drop in NpOH* intensity is more pronounced in DMPC as compared to the DPPC bilayer, in SG phase. The emission of NpO* is known to arise not only from the 1-naphthol present in the membrane interface, but also from the unpartitioned 1-naphthol in the bulk water. Hence, the characteristic fluorescence of NpO* does not reflect the bile salt induced changes in the membrane. On the other hand, the emission of NpOH* originates exclusively from the 1-naphthol present in the membrane; hence, the changes in the emission of NpOH* can be directly related to the bile salt induced changes in the membrane. Figure 2 and SI Figure S1 show that, in the presence of different bile salts at their submicellar concentration (fixed at 1 mM) in vesicles, the fluorescence intensities of the respective NpOH* are distinctly altered. In the presence of both unconjugated (NaDC, NaC) and conjugated (NaGDC, NaGC, NaTDC, NaTC) bile salts, the NpOH* emission intensity is found to decrease. However, the drops in intensity in the presence of different bile salts are different. The quantitative decrease in emission intensities of NpOH* is in the order NaDC > NaC > NaGDC > NaTDC > NaGC > NaTC, for both DPPC and
Figure 3. Plots of variation in NpOH* fluorescence intensity with increase in bile salt concentration in the range of 0.05 to 1 mM at (A) 30 °C (SG phase) and (B) 54 °C (LC phase) in DPPC vesicles; (C) 15 °C (SG phase) and (D) 35 °C (LC phase) in DMPC vesicles. (λex = 290 nm, λem = 370 nm). [Lipid] = 0.4 mM, [1-naphthol] = 4 μM. [NaDC (black circle) (from ref 16), NaC (red triangle) (from ref 16), NaGDC (blue/white circle), NaTDC (purple triangle), NaGC (brown star), NaTC (green box)].
DMPC bilayers. This is further clear from the plots of variation in NpOH* fluorescence intensity with different bile salts in the concentration range of 0.05 to 1 mM at SG as well as LC phases of DPPC and DMPC vesicles, shown in Figure 3. Table 1 shows the percentage drop in fluorescence intensity of NpOH* in the presence of 1 mM different bile salts in DPPC, as well as DMPC vesicles at both SG and LC phases of membrane, as compared to vesicles without bile salts. Fluorescence Lifetime Studies. In the two different phase states (SG and LC) of lipid bilayer membrane, fluorescence lifetime studies were carried out for NpOH* (λem = 370 nm) and NpO* (λem = 465 nm) of 1-naphthol in the presence of 13463
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Table 1. Percentage Drop in Fluorescence Intensity of NpOH* (λex = 295 nm, λem = 370 nm) in DPPC as Well as DMPC Vesicles in the Presence of 1 mM Bile Salt as Compared to Vesicles without Bile Salts, at Both SG and LC Phasesa
Table 2. Percentage Drop in Fluorescence Lifetime Values of Longer (τl) and Shorter (τs) Lifetime Components of NpOH* (λex = 295 nm, λem = 370 nm) in DPPC and DMPC Vesicles in the Presence of Bile Salts (1 mM) as Compared to Vesicles without Bile Salts, at SG and LC Phasesa
percentage drop in fluorescence intensity of NpOH* DPPC
percentage drop in fluorescence lifetime values of NpOH*
DMPC
DPPC
SG (30 °C)
LC (54 °C)
SG (15 °C)
LC (35 °C)
NaDC
34.90
62.63
54.72
54.63
bile salt
NaC NaGDC
27.62 23.39
41.88 32.68
43.47 38.03
39.92 27.55
NaDC
bile salt
DMPC
SG (30 °C)
LC (54 °C)
SG (15 °C)
LC (35 °C)
τl
τs
τl
τs
τl
τs
τl
τs
8.77
19.93
10.33
13.41
10.84
24.92
10.91
20.77
9.70 7.90
6.56 5.33
9.47 9.09
9.19 7.53
19.29 16.83
9.76 6.98
18.14 16.07
NaTDC
19.73
26.36
30.75
12.31
NaGC
13.34
19.63
23.40
10.95
NaC 5.69 NaGDC 2.87
NaTC
9.10
17.01
19.96
9.12
NaTDC 2.59
6.76
4.81
8.68
6.83
15.81
6.03
15.66
NaGC
2.05
5.41
4.12
7.31
5.49
13.50
5.07
12.76
NaTC
1.91
3.10
3.44
4.16
5.06
11.00
4.55
12.14
[Lipid] = 0.4 mM, [1-naphthol] = 4 μM. The error in the data is within (5%.
a
different conjugated and unconjugated bile salts at various concentrations. The fluorescence decay dynamics of NpOH* shows biexponential characteristics due to its distribution at the hydrocarbon core as well as the interface of the lipid bilayer membrane.16,18 The emission of NpOH* from 1-naphthol in the hydrocarbon core region possesses a longer lifetime component (τl = ∼7.3 ns) and NpOH* at the interfacial region has a shorter lifetime component (τs = ∼3 ns) in the lipid bilayer membrane. Similarly, the biexponential decay dynamics of NpO* possess τl of ∼20 ns and τs of ∼8 ns in lipid bilayer membranes. The fluorescence lifetime values of NpOH* and NpO* in the presence of different concentrations of conjugated and unconjugated bile salts, at both phases of DPPC and DMPC vesicles, are presented in the SI (Tables S1 to S8 in the SI). The fluorescence lifetime of NpO* emission in water is observed to be monoexponential with a value of ∼8 ns (Table S9 and Figure S3 in the SI), which matches very closely with the τs of NpO* in the lipid bilayer. Hence, τs of NpO* can be assigned to originate from the unpartitioned 1-naphthol present in bulk water. The longer decay component τl of ∼20 ns can be assigned to NpO* present in the membrane water interfacial region in which the nonradiative decay processes are anticipated to be slower than that in water.16 It has been observed that, in the presence of various bile salts at different concentrations, the fluorescence lifetime of NpO* in aqueous solution remains almost the same (Table S9 and Figure S3 in the SI). This indicates the absence of appreciable interaction between 1-naphthol and aqueous solution of bile salts. However, in both phases of DPPC and DMPC vesicles, the values for both shorter and longer lifetime components of NpOH* as well as NpO* decrease with increasing concentration of different bile salts (up to 1 mM) (Tables S1 to S8 in the SI). Also, there is almost no change in the amplitudes of corresponding lifetime components (τl and τs) of NpOH* as well as NpO* with increasing concentration of various bile salts in both DPPC and DMPC vesicles at both phase states. The percentage drops in fluorescence lifetime values of NpOH* in the presence of 1 mM of various bile salts in DPPC, as well as DMPC vesicles at both SG and LC phases of membrane, as compared to vesicles without bile salts, are shown in Table 2. Figure 4 represents the plots of variation in ESPT rate constant of NpOH* with bile salt concentration ranging from 0.05 to
[Lipid] = 0.4 mM, [1-naphthol] = 4 μM. The error in the data is within (5%.
a
Figure 4. Plots of variation in ESPT rate constant (kpt) of NpOH* with increase in bile salt concentration from 0.05 to 1 mM for longer lifetime components in DPPC vesicles at (A) 30 °C (SG phase) and (B) 54 °C (LC phase) and in DMPC vesicles at (C) 15 °C (SG phase) and (D) 35 °C (LC phase). [Lipid] = 0.4 mM, [1-naphthol] = 4 μM. The error in the data is within (5%. [NaDC (black circle) (from ref 16), NaC (red triangle) (from ref 16), NaGDC (blue/white circle), NaTDC (purple triangle), NaGC (brown star), NaTC (green box)].
1 mM for longer lifetime components at SG and LC phase states of both DPPC as well as DMPC vesicles. The 1-naphthol dissociation rate constants are calculated using decay times as16 kpt ¼
1 1 τ τ0
assuming krpt , kpt in the excited state, where kpt is the ESPT rate constant, krpt is the rate constant for excited state protonation and τ0 is the fluorescence lifetime of 1-naphthol in the absence of ESPT. The value of τ0 for a particular membrane phase (SG or LC) would be equal to the long component fluorescence lifetime 13464
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Langmuir originating from the dry membrane core in the absence of bile salts. The small difference in the τ0 values for the SG and LC phases could be due to the differences in knr (nonradiative decay constant) for the two phases. There is an increase in kpt values with increase in various bile salt concentrations. The proton transfer rate constants are found to be in the order NaDC > NaC > NaGDC > NaTDC > NaGC > NaTC which is similar with the drop in fluorescence intensity and lifetime values of NpOH*, at both phase states of DPPC and DMPC vesicles.
’ DISCUSSION The results indicate that prototropic equilibrium of 1-naphthol is quite responsive to the submicellar concentration bile salt induced changes in the lipid bilayer membrane at both phase states of DPPC as well as DMPC. It is well-known that fluorescence lifetime of 1-naphthol is sensitive to the hydration; it decreases with increase in hydration.16,24 In previous work, it has been shown that the decrease in fluorescence lifetime values and no change in the amplitudes of corresponding lifetime components with addition of various bile salts (Tables S1 to S8 in the SI) is neither expulsion of 1-naphthol from membrane to the solution nor population redistribution of 1-naphthol within the membrane.16 Rather, the result of decrease in fluorescence lifetime values of 1-naphthol with increase in concentrations of various conjugated and unconjugated bile salts in lipid bilayer is due to the bile salt induced membrane hydration. This is further confirmed from the proton transfer rate constant of 1-naphthol which is strongly dependent on the availability of water around it. Since the core region of the lipid bilayer membrane is reasonably dry,25 any increase in the proton transfer rate constant relating to the long component of neutral form fluorescence decay [τl (NpOH*)] would reflect the overall increase in the hydration level of the membrane. In the present study, the efficiency of bile salt induced membrane hydration is reflected from enhanced kpt values with increase in various bile salt concentrations (Figure 4). From the fluorescence lifetime and proton transfer rate constant data, the efficiency of various bile salt induced hydration of the lipid bilayer follows the order NaDC > NaC > NaGDC > NaTDC > NaGC > NaTC in DPPC as well as DMPC vesicles at both SG and LC phase states. The hydrophilicity of conjugated bile salts is known to be more as compared to their unconjugated counterparts due to the longer and more polar side chains of conjugated bile salts.3 Among the taurine and glycine conjugates of bile salts, the hydrophilicity of taurine conjugates is more as compared to that of glycine conjugates; hence, the hydrophilicity order of bile salts can be presented as taurine conjugates > glycine conjugates > unconjugated bile salts.26 The hydrophobicity of the bile salts controls the extent of its interaction with the liposome membrane. The dihydroxy bile salts, being more hydrophobic, have a higher degree of incorporation into the lipid bilayer membranes than the trihydroxy bile salts.16,27 Hydrophobicity indices of several bile salts have been reported in the literature, which has been obtained by using reverse-phase high-performance liquid chromatography.28,29 Using these indices, the order of hydrophobicity of the bile salts under study is seen as NaDC (0.72) > NaGDC (0.65) > NaTDC (0.59) > NaC (0.13) > NaGC (0.07) > NaTC (0.00), the hydrophobicity indices being given in the brackets. The incorporation of bile salts into the membrane is known to result in the hydration of the lipid bilayer, which
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causes fast deprotonation of membrane-bound 1-naphthol and is reflected in an increase in kpt values.16 If hydrophobicity of the bile salts were the only reason for facile bile salt incorporation into the membrane, then the extent of their incorporation, efficiency of membrane hydration, and the resulting increase in the kpt values is expected to follow the hydrophobicity order of bile salts. However, from Figure 4 and Table 2 it is seen that, in the presence of different bile salts in DPPC as well as in DMPC lipid bilayer, the order of increase in kpt values and the decrease in fluorescence lifetime of NpOH* do not follow the hydrophobicity order of the bile salts. The anomaly in the hydrophobicity order and increase in kpt is observed in the case of NaC. A possible explanation for this lack of correspondence in the order of hydrophobicity and the extent of hydration of bilayer by bile salts could be as follows. Since the side chains of conjugated bile salts are longer than their unconjugated counterparts, steric effects may affect the extent of their incorporation into the bilayer membrane. Although NaC is less hydrophobic than NaGDC and NaTDC, it is less sterically hindered than the latter bile salts, which promotes better incorporation of NaC into the bilayer membrane as compared to NaGDC and NaTDC. This is reflected in the higher increase in the kpt values of NpOH* in the presence of NaC as compared to NaGDC and NaTDC in both SG and LC phases of bilayer membrane. The extent of ionization of the conjugated and unconjugated bile salts in the membrane environment could also affect their incorporation and result in the lack of correspondence with the hydrophobicity order.10,11 A better insight into this anomaly would require a more detailed investigation involving more bile salts of different hydrophobicity like lithocholic acid and its coujugates. The percentage drop in fluorescence lifetime values of NpOH* (Table 2), in the presence of bile salts (1 mM), can also be correlated with the extent of hydration of DMPC and DPPC bilayers. In both SG and LC phases of DPPC and in the SG phase of DMPC, considerable changes in bile salt induced bilayer hydration levels are indicated. In contrast, for the LC phase of DMPC bilayer, bile salt induced percentage drops in NpOH* fluorescence lifetime values are seen to be less pronounced, although the same trend is maintained. With an increase in the fluidity in the carbon chains, the lipid molecules are known to occupy larger area, which leads to greater exposure of the lipid head groups and increased hydration.30 The lipid bilayer with a lower phase transition temperature remains more hydrated as compared to the liposome with a higher transition temperature.31 The DMPC bilayer is more hydrated as compared to DPPC bilayer, since the SG to LC phase transition temperature of pure DMPC liposome is 23 °C, which is much lower as compared to DPPC liposomes (42 °C).20,32 Therefore, it is possible that the bile salt induced increase in the hydration level of the prehydrated LC phase of DMPC bilayer is rather small. It is possible that bilayer curvature also can affect the hydration of lipid bilayer membrane, but the liposome prepared in the present study is expected to have similar average size distribution (∼25 nm).20 The fluorescence parameters of 1-naphthol as a membrane-bound probe reflects these average membrane properties. Bile salt induced changes of these average membrane properties are sensed by 1-naphthol fluorescence. For the same concentration of 1-naphthol, the NpOH* fluorescence intensity is observed to be more in DMPC bilayers as compared to DPPC bilayers (Figure 2 and SI Figure S1). The 13465
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Langmuir DMPC lipid bilayer is less densely packed as compared to the DPPC lipid bilayer, which is reflected from their phase transition temperatures.20,30 The high intensity of NpOH* in DMPC bilayer may be due to the higher partitioning efficiency of 1-naphthol into the less densely packed DMPC bilayer as compared to DPPC bilayer. The drop in fluorescence intensity of NpOH* in DMPC and DPPC bilayers in the presence of various bile salts can also be assigned to the increase in hydration of the lipid bilayer. The drop in intensity follows the same order as observed in the case of drop in fluorescence lifetime and increase in kpt values for various bile salt induced hydration of the bilayer. Like fluorescence lifetime, the fluorescence intensity of NpOH* was found to be sensitive to the bile salt induced bilayer hydration in both SG and LC phases of DPPC and in the SG phase of DMPC vesicles. The same trend in the percentage drop in fluorescence intensity of NpOH* is maintained in both SG and LC phases of DPPC as well as DMPC (Figure 3 and Table 1). For DPPC vesicles, there is a difference in the drop in fluorescence intensity (more in the LC phase as compared to the SG phase); however, for DMPC vesicles, in the LC phase the drop in fluorescence intensity in the presence of bile salts is seen to be less distinct as compared to its SG phase.
’ CONCLUSIONS The efficiency of submicellar concentrations of (0.05 to 1 mM) various physiologically important unconjugated (NaDC, NaC) and conjugated (NaGDC, NaGC, NaTDC, NaTC) bile salts, toward the extent of hydration of DPPC and DMPC lipid bilayer membranes has been studied in their solid gel (SG) and liquid crystalline (LC) phases. It has been observed that submicellar bile salt induced hydration of the lipid bilayer membrane up to the core region is a general phenomenon. The bile salt induced bilayer hydration efficiency follows the order NaDC > NaC > NaGDC > NaTDC > NaGC > NaTC in DPPC and DMPC vesicles in both SG and LC membrane phases. This relationship does not follow exactly the hydrophobicity index order, which is NaDC > NaGDC > NaTDC > NaC > NaGC > NaTC. Due to water sensing ability of 1-naphthol, the fluorescence intensity and decay parameters of 1-naphthol are found to be very sensitive to the bile salt induced bilayer hydration of both DPPC as well as DMPC in the SG phase. However, in the LC phase it is found to be sensitive to the bile salt induced hydration effect in DPPC liposomes, but not in DMPC liposomes. All these experimental results indicate that bile salt induced bilayer membrane hydration exists even at submicellar concentrations of various bile salts, which might have further implications in many biological studies. ’ ASSOCIATED CONTENT
bS
Supporting Information. Figures showing the response of the fluorescence intensity of 1-naphthol to the changes induced by bile salts in DPPC vesicle (Figure S1), response of the fluorescence intensity of 1-naphthol in aqueous bile salt solutions without liposome (Figure S2), fluorescence lifetime decay of 1-naphthol in bile salt solutions without liposome (Figure S3), fluorescence lifetime decay of NpO* without and with various bile salts in vesicles (Figure S4). This also includes tables containing fluorescence lifetime data of NpOH* and NpO* with the increase in various bile salt concentrations in DPPC and DMPC vesicles at both phases (Table S1 to
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Table S8) as well as fluorescence lifetime data of NpO* in aqueous bile salt solutions without vesicles (Table S9). This material is available free of charge via the Internet at http:// pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Phone: (+91) 44-22574207, Fax: (+91) 44-22574202, E-mail:
[email protected].
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