J. Phys. Chem. 1993,97, 9064-9072
9064
Diffusion Ordered 2D NMR Spectroscopy of Phospholipid Vesicles: Determination of Vesicle Size Distributions Denise P. Hinton and Charles S. Johnson, Jr.' Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599-3290 Received: May 6, 1993
Diffusion ordered 2D N M R (DOSY) has been used to characterize large unilamellar phospholipid (LUV) vesicles. Entrapped sucrose served as an N M R active label for the vesicles and permitted diffusion coefficients to be measured for vesicle preparations with average diameters ranging from 30 to 100 nm and total lipid concentrations ranging from 1 to 30 mM. Distributions of diffusion coefficients were obtained by analysis with the program CONTIN. Average diameters and distributions of diameters were then obtained from the vesicle diffusion coefficients after appropriate corrections by means of the StokeEinstein equation. Electron microscopy and dynamic light scattering measurements were also performed on the LUV's, and the results were found to be consistent with those obtained with DOSY. The apparent vesicle diffusion coefficients obtained with the DOSY experiment from phospholipids in the bilayers were up to 2 times larger than for the entrapped sucrose. This difference is a feature of polydispersity that results from size-dependent inhomogeneous magnetic dipole broadening of the resonances for the phospholipids but not the entrapped sucrose. The difference vanishes for small sonicated vesicles. DOSY has major advantages for the study of complex mixtures since molecules and assemblies of all sizes can be observed. For example, in this study both entrapped and free sucrose were resolved at the same chemical shifts. Thus, chemical equilibria involving molecules with different diffusion rates are accessible to study by DOSY.
I. Introduction Phospholipid vesicles serve as models of the biological cell and are clinically important as in vivo drug delivery vehicles. It has been argued that large unilamellar vesicles (LUV's) with diameters of at least 100 nm provide "more faithful" model membrane systems than other types of liposomes, e.g. multilamellar or small unilamellar vesicles, since they, of course, have a single bilayer, are able to entrap appreciablevolumes,and avoid problems associated with small, highly curved systems.' LUV's have not previously been characterized by means of pulsed field gradient (PFG-NMR) diffusion measurements, though studies of small sonicated vesicles (- 30 nm in diameter) have been reported.2~~ The LUV's present an interesting challenge for lH NMR diffusion measurements because of their small diffusion coefficients (- 10-8cm*s-l) and the low amplitudes of the signals from phospholipids in the bilayer. Recent improvements in PFG-NMR instrumentation and methods that greatly extend the range of accessible diffusion coefficients now permit the study of LUV's. Also, the recently developed diffusion ordered 2D NMR spectroscopy (DOSY) in principle permits the distribution of diffusion coefficients for polydispersevesicle systems to be determined.4.5 Here we report a systematic DOSY study of small, medium, and large unilamellar phospholipid vesicles. These polydisperse vesicle systems are ideal for the application of DOSY since they consist of spherical particles whose size distributions in some cases can be determined by other means. The problem of signal intensity can largely be overcome through the use of entrapped saccharides, e.g. sucrose, that serve as NMR active labels with narrow lines and long nuclear relaxation times that are independent of vesicle size. Our initial PFG-NMR diffusion measurements for LUV's gave diffusion coefficientsthat depended on the particular signal being analyzed.6 Typically, the effective diffusion rate reported by the phospholipid head group signal was found to be much higher than that for the entrapped sucrose signals. Since the minimum displacement detected in our diffusion experiment is much larger To whom correspondence should be addressed. 0022-365419312097-9064$04.00/0
than the diameter of a vesicle, all molecules whether in the bilayer or entrapped in the aqueous compartment should appear to have the same tracer diffusion coefficient. Accordingly, the first part of this study was concerned with reconciling the disparatediffusion rates. The answer lies in the polydispersity of the sample and the associated heterogeneous line broadening in lH NMR. The signals are weighted by both the number of spins of each type contributing for each size of vesicle and nuclear relaxation factors that may depend on the size of the vesicle. Experiments reported here that bear on this problem are (1) the determination of the vesicle size distribution by means of electron microscopy, (2) the determinationof effectivediffusioncoefficientsfrom phospholipid peak intensities versus signal areas, and (3) the measurement of the difference between apparent sucrose and phospholipid diffusion coefficients as a function of vesicle radius. The second and final phase of this work focused on the determination of size distributions for LUV's by means of DOSY. The signals from entrapped sucrose provided volume weighted distributions of tracer diffusion coefficients. Size distributions were then calculated from the DOSY data and compared with distributions measured by electron microscopy (EM) and calculatedfromdynamiclight scattering (DLS) data. Theconclusion is that DOSY provides a reliable method for determining vesicle size distributions over a wide range of concentrations. DLS provides similar results for vesicles but is extremely sensitive to particulate matter, e.g. dust, and suffers from multiple scattering effects at high concentrations. In contrast, DOSY is relatively insensitive to particulate solids and can be used at high concentrations. Furthermore, DOSY simultaneously reports on all solute and solvent components present and indicates approximate molecular sizes and states of aggregation. The primary limitation of DOSY is the limitation of NMR, namely low sensitivity. 11. The Diffusion Ordered 2D NMR Method
DOSY experiments on mixtures provide 2D NMR spectra in which the conventional 1D NMR spectrum is displayed in one dimension and distributionsof diffusioncoefficientsare displayed 0 1993 American Chemical Society
Diffusion Ordered 2D NMR of Phospholipid Vesicles
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90, 9o-x RF Pulses
The Journal of Physical Chemistry. Vol. 97,No. 35, 1993 9065 for determiningthe “best”distribution function. CONTIN makes use of a linear least-squares procedure augmented with prior knowledge about the solutions and the principle of parsimony. Here we specify kernels of the formf(X,sk) = exp(-hsk) and require the decay constants X and the function g(X) to be nonnegative. Parsimonyimplies selection oft he simplest solution, and here this means that the smoothest solution for g(X),consistent with the data, is selected.
90, go.,
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i
+A+
time
Gradient Pulses
+I+Signal -I.
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Figure 1. LED pulse sequence. Gradient prepulses, homospoil pulses during Te,and the phase cycling sequence are not shown.
in the other dimension. Data sets for DOSY analysesare obtained with the longitudinal eddy current delay (LED) pulse sequence shown in Figure 1.7 This sequences makes use of a stimulated echo and thus ensures that nuclear relaxation during the diffusion time, i.e. between the gradient pulses, depends on T1 rather than T2. It, also, minimizes signal distortions resulting from eddy currents by providing the settling time Te and severely limits transverse evolution of magnetization prior to signal acquisition by limiting the time interval T . The latter feature prevents J modulation effects and guarantees positive signal amplitudes. Nonnegative signal amplitudes turn out to be an important criterion for DOSY signal analysis. With these improvements in instrumentationand procedures, accurate diffusion coefficients can be measured for vesicles as large as several hundred nanometers. The free induction decay (FID) indicatedby the signalin Figure 1 is Fourier transformed with respect to t2 to obtain an NMR spectrum that can be represented by
I(K,v) = z A n ( v )exp[-D,(A - S/3)K2]
(1)
n
where K = yg6 is the area of the gradient pulse in cm-l, y is the gyromagnetic ratio, and g and 6 are the amplitude and duration of the gradient pulses, respectively. Here A,(v) is the 1D NMR spectrum of the nth diffusingspecies in the limit that g = 0 taking into account nuclear relaxation, and D, is the tracer diffusion coefficient of the nth species. Therefore, at each point in the NMR spectrum a set of intensities versus K‘ is obtained. Typically, 30-50 values of K‘ are selected with a minimum value equal to approximately 0.1 /(DmaxA)and a maximum value greater than 2*3/(DminA), where Dmix and Dman are the minimum and maximum expected diffusion coefficients, respectively. The spacing increases with increasing K‘ and ideally is exponential. The DOSY experiment generates a 2D data set of NMR intensities versus chemical shifts in one dimension and intensities versus s = K‘ in the other. The analysisin the s dimension consists of a determination of the contributions from each of the exponential components at each chemical shift. When discrete monodisperse components are known to be present, the analysis can be conveniently performed with inversion programs such as DISCRETE819and SPLMODlO along with prior knowledge of characteristics of the solutionsand limitations of the method.5In the presence of polydispersity the summation in eq 1 must be replaced with an integral, and A, becomes thedistribution function g(X) where X = D(A - 6/3). The inversion of the data set to obtain g(X) is conveniently performed with the program CONTIN. This program fits the data set, I k and sk,to functions of the form1
where NL and the forms off(X,sk) and L j ( S k ) are known, and g(X) and are to be determined.11-13 The determination of the distributiong(h) is an ill-posed problem. Uniquenessof solutions is not guaranteed, and fitting the data is not a sufficient condition
Experimental Considerations
Materials. 1-Palmitoyl-2-oleoylphosphatidylcholine (POPC) was obtained from Avanti Polar Lipids, Inc., and was used without further purification. Sucrose (99%) and the relaxation agent, manganese chloride, were supplied by Aldrich Chemical Co., and D20 (99.9%) was obtained from Cambridge Isotope Laboratories. Vesicle Preparation. Large unilamellar vesicles were prepared accordingto the procedure of Hope et a1.l This lipid is convenient for NMR studies since the gel to liquid crystal phase transition of the bilayer occurs below room temperature. The lipids were received in chloroform solutions, and volumes containing the desired masses of lipids were measured out with a glass syringe. Chloroform was blown off with a stream of argon gas, and the remaining lipid was resuspended in benzene and then frozen in a dry ice and acetone bath. The sample was lyophilized overnight to obtain the lipid as a white powder. For the preparation of medium unilamellar vesicles, MUV’s (diameter 50-100 nm), and LUV’s (diameter > 100nm), solutions of sucrose (0-250 mM) and NaCl (0-100 mM) in D20 were added to the POPC lipid powder to obtain 1-30 mM total lipid concentration. The solution was gently vortexed and allowed to stand at room temperature for 30 min. The sample was then subjected to five freeze (dry ice and acetone bath) and thaw (water bath at 35 “C) cycles, a procedure that has been shown to increase the fraction of sucrose entrapped by the ~esic1es.l~ At this point the sample was a suspension of large multilamellar vesicles (MLV’s). MUV’s and LUV’s were prepared by highpressure extrusion via argon gas (200-800 psi) through 25-mm Nucleopore polycarbonate filters (Costar Corp.) with defined pore sizes of 0.05 and 0.1 pm, respectively. Single and doubly stacked filters were used, and 10-1 5 extrusions were employed. The pH’s of the vesicle samples were in the range 6.8-7.2. Small (diameter
