J. Phys. Chem. B 2007, 111, 3665-3673
3665
Fluorescence Study of the Fluidity and Cooperativity of the Phase Transitions of Zwitterionic and Anionic Liposomes Confined in Sol-Gel Glasses Rocı´o Esquembre,† Marı´a L. Ferrer,‡ Marı´a C. Gutie´ rrez,‡ Ricardo Mallavia,† and C. Reyes Mateo*,† Instituto de Biologı´a Molecular y Celular, UniVersidad Miguel Herna´ ndez, 03202-Elche, Spain, and Instituto de Ciencia de Materiales de Madrid-ICMM, Consejo Superior de InVestigaciones Cientı´ficas-CSIC, Campus de Cantoblanco, 28049-Madrid, Spain ReceiVed: December 18, 2006; In Final Form: February 16, 2007
The current work makes use of different fluorescent reporter molecules and fluorescent spectroscopic techniques to characterize the thermotropic, physical, and dynamical properties of large unilamellar liposomes formed from either 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1,2-dimyristoyl-sn-glycero-3-[phosphorac-glycerol] (DMPG) encapsulated in sol-gel matrixes. In particular, cooperativity of the phase transition is analyzed from steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH), the interfacial properties are studied by measuring the spectral shift of Laurdan, and the structural organization (heterogeneity) of the lipid bilayer is determined from the fluorescence lifetime of trans-parinaric acid (t-PnA). In addition, information regarding order and dynamical properties in the bulk hydrophobic core is obtained from timeresolved fluorescence anisotropy of t-PnA and 3-(4-(6-phenyl)-1,3,5-hexatrienyl)-phenylpropionic acid (PADPH). The spectroscopic study reveals that upon encapsulation, the basic thermodynamic properties as well as the fluidity of the lipid bilayer practically remain intact for DMPG liposomes but not for DMPC liposomes, whose lipid bilayer exhibits large gel-fluid heterogeneity. On the basis of these experimental results, electrostatic interactions between phospholipid polar heads and the porous surface of the host matrix seem to play a capital role for the preservation of the structural integrity of encapsulated bilayer.
Introduction Liposomes are spherical closed vesicles of phospholipid bilayers with an entrapped aqueous phase, and may consist of one (large unilamellar vesicles) or more bilayers (multilamellar vesicles) ranging in size from 20 to 500 nm, and occasionally as large as 10 µm.1 Phospholipid bilayers are known to undergo a characteristic gel to fluid phase transition at a temperature range (Tm) which can be modulated by the degree of saturation, and the length of the acyl chains, and/or the electrostatic charge interactions between the head groups and the surrounding aqueous environment (e.g., pH, ionic strength, ...).1 Liposomes, as models of biological membranes, are of fundamental importance by serving as selective barriers for transport, as fluid matrixes for biosynthetic transformations, and as boundaries for the transfer of energy and information. The performance of any of these biomimetic applications strongly depends on the membrane fluidity and, on occasion, on the sharpness of the gel-fluid phase transition as is the case for thermal trigger liposomes.2 For instance, the interest in using phospholipid vesicles as nanocapsules and vehicles to entrap, deliver, and control release of functional substances (e.g., proteins, enzymes, and drugs)3,4 resides in two main features. First, the bilayer can behave as a barrier and provide protection to the molecules encapsulated in the interior aqueous phase from the aggression of external denaturizing agents, and second (but principal), entrapped molecules can be control released in response to a * Corresponding author. Fax: +34 966 658 758. E-mail:
[email protected]. † Instituto de Biologı´a Molecular y Celular, Universidad Miguel Herna´ndez. ‡ Instituto de Ciencia de Materiales de Madrid-ICMM, Consejo Superior de Investigaciones Cientı´ficas-CSIC.
variety of physical and chemical stimuli (including temperature, pressure, light, pH, and ions) that, as mentioned above, determine the bilayer fluidity and, thus, its permeability.2,5 For practical applications (biosensors, microarrays, screening platforms, etc.), liposomes have been immobilized on different solid surfaces given the tremendous potential of the resulting materials to develop novel devices.6-10 However, fixation of liposomes to solid surfaces easily disrupts the hydrophobic interactions that create lipid bilayers and modify the natural dynamic motions of the membrane and its gel-fluid phase transition temperature (Tm), producing unstable immobilized structures and, in some cases, lysis of the bilayer.11-19 Different strategies have been developed to overcome these difficulties.20-22 Among them, use of sol-gel routes, involving the hydrolysis and condensation of alkoxysilane precursors, seems to be an interesting alternative to immobilize liposomes and proteoliposomes in silica and hybrid matrixes without the need for tethering the lipids to a solid surface.23-32 The preservation of the bilayer structure upon sol-gel encapsulation requires the use of alcohol-free routes.23,28,29,33,34 Otherwise, the alcohol resulting as a byproduct of the chemical reactions involved in the formation process of the silica matrix causes the disruption of the lipid bilayer structure. Nevertheless, it has been recently reported that for pure zwitterionic liposomes, (i.e., 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC)) even the use of these alcohol-free routes produces a broadening of the lipid phase transition during aging,23-25 suggesting irreversible alterations of the bilayer fluidity which prevent the use of these systems for practical applications such as controlled release. This is not the case for
10.1021/jp068685y CCC: $37.00 © 2007 American Chemical Society Published on Web 03/22/2007
3666 J. Phys. Chem. B, Vol. 111, No. 14, 2007 anionic liposomes (i.e., DMPA), which preserve their structural integrity for more than 15 days after encapsulation.29 Here in and in order to understand the underlying mechanism impeding the long-term stability of zwitterionic liposomes, we have analyzed the effects of sol-gel encapsulation on the fluidity of DMPC (zwiterionic) and 1,2-dimyristoyl-sn-glycero3-[phospho-rac-glycerol] (DMPG) (anionic) lipid bilayers below, during and above the transition phase. Quantification of membrane fluidity is not trivial.35-37 Although this term refers strictly to the dynamical properties of the membrane components, it is often used as a parameter which combines the effects of both lipid dynamics and acyl chain order. In addition, if the membrane shows some kind of lateral lipid heterogeneity, fluidity reflects the order and dynamics of phospholipid acyl chains in the specific membrane domains, as well as the fraction of each domain in the membrane. Determination of this parameter is, consequently, complex and requires the use of complementary methodologies. In this work, we combine different fluorescent reporter molecules with steady-state and time-resolved fluorescence techniques to reach this goal. In particular, the effects of sol-gel encapsulation on the interfacial properties and the structural organization (heterogeneity) of the lipid bilayer have been studied by measuring the spectral shift of the fluorescent probe laurdan and by determining the fluorescence lifetime of trans-parinaric acid (t-PnA). In addition, information regarding order and dynamics in the bulk hydrophobic core has been obtained from time-resolved fluorescence anisotropy of t-PnA and 3-(4-(6-phenyl)-1,3,5-hexatrienyl)phenylpropionic acid. Last, the cooperativity of the gel-fluid lipid-phase transition was also analyzed from steady-state fluorescence anisotropy of DPH. Materials and Methods Reagents. Tetramethyl orthosilicate (TMOS) was purchased from Aldrich. The synthetic phospholipids 1,2-dimyristoyl-snglycero-3-[phospho-rac-glycerol] (DMPG) and 1,2-dimyristoylsn-glycero-3-phosphocholine (DMPC) were from Avanti Polar Lipids (Birmingham, AL) and used as received. The fluorescent probes trans-parinaric acid (t-PnA), 2-dimethylamino-6-lauroylnaphtalene (laurdan), 1,6-diphenyl-1,3,5-hexatriene (DPH), and 3-(4-(6-phenyl)-1,3,5-hexatrienyl)-phenylpropionic acid (PA-DPH) were from Molecular Probes (Eugene, OR). Water was twice distilled in all-glass apparatus and deionized using Milli-Q equipment (Millipore, Madrid). N,N′-Dimethylformamide (DMF) and ethanol (spectroscopic grade) were from MERCK. All other compounds were of analytical grade. Liposome Formation. Chloroform/methanol solutions containing 3 mg of total phospholipid (DMPC or DMPG) were dried first by evaporation under dry nitrogen gas stream and subsequently under vacuum for 3 h. Multilamellar vesicles (MLVs) were formed by resuspending the dried phospholipid in buffer (Tris-HCl 50 mM, NaCl 250 mM, pH 7.4) to a final concentration of 1 mM. The vesicle suspension was then heated at a temperature above the phase transition of the phospholipid and vortexed several times. Large unilamellar vesicles (LUVs) with a mean diameter of 90 nm were prepared from these MLVs by pressure extrusion through 0.1 µm polycarbonate filters (Nucleopore, Cambridge, MA). Labeling of Liposomes. A few microliters from stock solutions of the fluorescent probes DPH and PA-DPH in DMF or of t-PnA and laurdan in ethanol were added to the LUVs suspension and allowed to stabilize for several minutes well above the lipid phase transition temperature before fluorescent measurements. The ethanol or DMF final concentration was
Esquembre et al. always less than 2%. The lipid-to-probe ratio, in molar terms, was 1:400 in the case of DPH, 1:200 for PA-DPH, 1:500 for laurdan, and 1:100 for t-PnA. Immobilization of Liposomes in Sol-Gel Matrixes. Liposomes were encapsulated in pure silica matrixes using an alcohol-free route recently developed by members of our group.33 Briefly, 4.41 mL of TMOS, 2.16 mL of H2O, and 0.06 mL of HCl (0.62 M) were mixed under vigorous stirring at 4 °C in a closed vessel. After 50 min, 1 mL of the resulting sol was mixed with 1 mL of deionized water, and the mixture was submitted to rotaevaporation for a weight loss of ∼0.6 g (i.e., 0.6 g is approximately the alcohol mass resulting from alkoxyde hydrolysis). The aqueous sol was mixed with 1 mL of a diluted buffered suspension of probe-doped liposomes in a disposable cuvette of poly(methyl methacrylate). Gelation occurs readily after mixing. After gelation, monoliths were wet aged in a Tris buffer (50 mM, pH 7.5) and NaCl (250 mM) solution at 4 °C before use. Absorption and Steady-State Fluorescence Spectroscopy. Absorption spectra were registered in a Shimadzu spectrophotometer (UV-1603, Tokyo, Japan). Fluorescence anisotropy measurements were carried out in a Cary Eclipse spectrofluorometer (Varian) interfaced with a Peltier cell and fitted with thin film polarizers. The steady-state anisotropy 〈r〉, defined by
〈r〉 )
(IVV - GIVH) (IVV + 2GIVH)
(1)
was obtained as a function of temperature by measuring the vertical and horizontal components of the fluorescence emission with excitation polarized vertically. The G factor (G ) IHV/ IHH) corrects for the transmissivity bias introduced by the detection system. Samples were excited at 360 nm (5 nm slit width), and the polarized emission was detected at 430 nm (5 nm slit width). Background intensities due to the sol-gel matrix were always checked and were subtracted from the sample when it was necessary. Steady-state fluorescence excitation and emission spectra of laurdan were obtained as a function of temperature with a PTIQuantaMaster spectrofluorometer interfaced with a Peltier cell. Excitation and emission spectra were recorded at 10 and 45 °C fixing wavelength at 440 nm (3 nm slit width) and 350 nm (3 nm slit width), respectively. The spectral changes of the emission spectrum of the probe were also quantified by the socalled generalized polarization (GPex)38,39 which is defined as
GPex )
I440 - I490 I440 + I490
(2)
where I440 and I490 are the fluorescence intensities recorded after subtraction of background intensity, at the characteristic emission wavelengths of the gel phase (440 nm) and of the fluid phase (490 nm), respectively. GPex values were obtained at one excitation wavelength (350 nm) and in a range of excitation wavelengths (325-410 nm), at different temperatures above and below the gel-fluid transition of the lipid. Time-Resolved Fluorescence Spectroscopy. The decay of the total fluorescence intensity, and those of the parallel and perpendicular components, was recorded using a PTI model C-720 fluorescence lifetime instrument (Photon Technology International Inc., Lawrenceville, NJ) utilizing a proprietary stroboscopic detection technique.40 The system employs a PTI GL-330 pulsed nitrogen laser pumping a PTI GL-330 highresolution laser. The dye laser output, at 600 nm for t-PnA
Liposomes in Sol-Gel Glasses
J. Phys. Chem. B, Vol. 111, No. 14, 2007 3667
excitation and 678 nm for PA-DPH, was frequency-doubled to 300 and 339 nm, respectively, with a GL-103 frequency doubler coupled to an MP-1 sample compartment via fiber optics. The emission was observed at 90° relative to the excitation via an M-101 emission monochromator and a stroboscopic detector equipped with a Hamamatsu 1527 photomultiplier. Analysis of the Time-Resolved Florescence Experiments. For lifetime measurements, data were analyzed with the Felix 32 analysis package using the lifetime distribution algorithm based on the Exponential Series Method.41 The package uses a sum of up to 200 exponential functions with fixed logarithmically spaced lifetimes, with the pre-exponential factors recovered by the least-squares minimization procedure. The anisotropy decay parameters (rotational correlation times, φi, amplitudes, βi, and residual anisotropy, r∞) were determined using a nonlinear least-squares global analysis method described in Mateo et al.,42 by simultaneously fitting the vertically and horizontally polarized emission components to a sum of n exponentials and a constant term43,44 n
r(t) ) (r(0) - r∞)[
βi exp(-t/φi)] + r∞ ∑ i)1
(3)
where n
βi ) 1 ∑ i)1
According to the model, 〈φ〉 is a function of D⊥ and of r∞/r0.50,51 The experimental D⊥ value for the lipid probe can therefore be obtained from this expression
D⊥ )
0.1674 - 0.1066(r∞/r0) - 0.062(r∞/r0)2 〈φ〉
(7)
D⊥ may be converted to microviscosity η, which represents the dynamics friction against the rotational diffusion of the probe, by assuming the Debye-Stokes-Einstein equation
η)
kBT 6D⊥Veff
(8)
where kB is the Boltzmann constant and Veff the “effective” volume of the probe, which is 360 Å3 for PA-DPH.52 The anisotropy decay of t-PnA in lipid systems where gel and fluid environments coexist was also fit by the “associated” model detailed in Mateo et al.,53 in which the lifetime parameters of the total intensity decay are specifically associated with individual anisotropy parameters. The corresponding model accounts for the possibility of a probe being localized in two different environments G and F. The corresponding species have independent fluorescence, In(t) and anisotropy, rn(t), decays (with n ) G or F). The total fluorescence I(t) is given by a linear combination of the kinetic parameters of the two emitting species
and n ) 1-3
I(t) )
∑
anIn(t)
(9)
n)G,F
In both cases, the fits tabulated here represent the minimum set of adjustable parameters that satisfy the usual statistical criteria, namely, a reduced χ2 value of
