14934
J. Phys. Chem. B 2010, 114, 14934–14940
1-Naphthol as a Sensitive Fluorescent Molecular Probe for Monitoring the Interaction of Submicellar Concentration of Bile Salt with a Bilayer Membrane of DPPC, a Lung Surfactant Monalisa Mohapatra and Ashok K. Mishra* Department of Chemistry, Indian Institute of Technology Madras, Chennai 600 036, India ReceiVed: April 28, 2010; ReVised Manuscript ReceiVed: September 27, 2010
In this study, 1-naphthol has been used as a sensitive ESPT fluorescent molecular probe to investigate the interaction of submicellar concentrations of two physiologically important bile salts, sodium deoxycholate and sodium cholate, with dipalmitoylphosphatidylcholine small unilamellar vesicles in solid gel and liquid crystalline phases. Steady-state and time-resolved fluorescence of the two excited state prototropic forms of 1-naphthol indicate that the incorporation of monomeric bile salt molecules in the lipid bilayer membrane induces appreciable wetting of the bilayer up to the hydrocarbon core region, even at very low (e1 mM) concentrations of the bile salts. Introduction Among the vesicle forming phospholipids in biological systems, dipalmitoylphosphatidylcholine (DPPC), a disaturated phospholipid, is the most important one. It makes up to about one-third of total phospholipids present in the body and also accounts for 10-20% of the phosphatidylcholine content of brain myelin and erythrocyte membranes. This is also the major constituent of the important group of lung surfactants that are the indispensable components of respiratory mechanics.1,2 Together with DPPC, phosphatidylcholine constitutes ∼80% of total lung surfactant phospholipids. Modification of the properties of DPPC vesicle by additives has been well studied.3,4 The present work is focused on the interaction of DPPC vesicles and submicellar concentrations of naturally occurring biosurfactants (bile salts). Bile salts are a family of steroids possessing a hydrophilic moiety with two or three hydroxyl groups and a rigid, large, hydrophobic nucleus. Unlike conventional surfactants, which contain a clear polarity gradient due to their head and tail groups, the facial polarity of these biosurfactants is due to the location of the hydroxyl and methylene groups on the opposite faces (Figure 1). Due to their unique molecular structure, the surfactant behaviors of bile salts have been extensively studied to understand many important biological processes, such as solubilization of lipids, cholesterol, bilirubin, lecithin, and fat-soluble vitamins in living organisms.5,6 During several physiological processes, various types of interactions take place between bile salts and phospholipid bilayers.7,8 Below the critical micellar concentration (CMC) of the bile salt, they form mixed vesicles, bile salt monomer systems in which the bile salt molecules are mostly incorporated into the phospholipid vesicles without disrupting the membranes. Above the CMC, they usually form mixed vesicle-mixed micelle systems. With a further increase in the bile salt concentration, all mixed vesicles become solubilized.7-9 These mixed micelles act as reservoirs for cholesterol, fatty acids, and lysophospholipid monomers and facilitate the transfer of various molecules to the surface of the * To whom correspondence should be addressed. Phone: (+91) 4422574207. Fax: (+91) 44-22574202. E-mail:
[email protected].
small intestine by a solubilization process.10-13 In other words, bile salts act as shuttle carriers for phospholipids between mixed bile salt/phospholipid micelles, phospholipid vesicles, and the enterocyte membranes in the small intestine.10 In addition, during liposomal drug delivery in hepatobiliary systems, liposomes remain exposed to bile salts, and in that case, study of bile saltliposome interaction is essential. Because concentration is one of the most significant parameters, there is a need to study the bile salt-liposome interaction at variable concentrations. Among many studies, most of them have focused on the interactions at higher concentrations of bile salt (near CMC), where mostly the solubilization effect has been emphasized.7,14-16 However, study of bile salt-liposome interaction at lower concentration (submicellar concentration) of bile salts is very important, since at low concentrations, bile salts bind to membranes efficiently to enhance the spontaneous rate of the intervesicular phospholipid transfer process.17,18 Submicellar concentrations of bile salts are also present in the hepatic portal vein of humans.19,20 Bile salts in water are known to be present with multiple aggregation equilibriums at various concentrations. The lowest concentration range at which aggregation has been reported is around 3 mM.21 The submicellar concentration range in which the present study has been carried out (0.05 to 1 mM) is well below the reported concentration for the onset of aggregation. Thus, it is expected that the unaggregated monomeric form is the predominant form under the conditions of a present study. We have attempted to make use of excited state proton transfer (ESPT) as a simple steady-state technique to probe the interaction of a submicellar concentration of bile salts and phospholipid membranes. Fluorescent molecular probe-based techniques, due to their higher sensitivity and multiparametric nature, are considered suitable methods for studying biological interactions. Fluorescence is especially useful when the concentrations of biological molecules are so small that other spectroscopic and nonspectroscopic techniques cannot be used. Fluorescence molecular probes and sensors based on ESPT,22-24 such as 1-naphthol, are very useful to obtain much structural and dynamical information on a variety of organized and aggregate systems, such as micelles, lipid bilayer membranes, polymeric gels, cyclodextrin cavity, etc.25,26 Thus, the use of simple probelike
10.1021/jp103855q 2010 American Chemical Society Published on Web 11/01/2010
1-Naphthol as a Fluorescent Molecular Probe
J. Phys. Chem. B, Vol. 114, No. 46, 2010 14935
Figure 1. Molecular structures of sodium cholate (NaC) and sodium deoxycholate (NaDC).
1-naphthol at a very low concentration, which causes minimum perturbation to the system, further makes this technique more sensitive and versatile. The purpose of this present work is to study the effect of two physiologically important bile salts, NaDC and NaC on DPPC vesicles (small unilamellar vesicles), in solid gel (SG) and liquid crystalline (LC) phases. The study is carried out at a bile salt concentration range of 0.05-1 mM, using 1-naphthol fluorescence as sensor, by monitoring three fluorescence parameters: intensity, transition maximum, and lifetime.
tion of the residuals. The average fluorescence lifetime values were obtained by the following equation,28
τavg )
(
n
)( ) n
∑ Riτi2 / ∑ Riτi i)1
i)1
where τi is the individual lifetime with corresponding amplitude Ri. Results and Discussion
Material and Methods Materials. 1-Naphthol purchased from SRL, India was sublimed and used after checking its purity. NaDC and NaC, purchased from SRL, India and DPPC, purchased from Sigma Chemical Co. (Bangalore, India) were 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.27 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). The percentage of ethanol in the solution was
